The prevalence of diabetes mellitus (DM) is increasing worldwide, with a commensurate increase in the amount of related cardiovascular events.

DM affects approximately 100 million persons.¹ Five to ten percent have type 1 (insulin-dependent) and 90% to 95% have type 2 (non–insulin-dependent) diabetes mellitus.²

Type 2 diabetes is notable for the increased cardiovascular risk that it carries. In fact, these patients have the same risk of suffering a cardiovascular event than a non-diabetic who has already had his first acute coronary syndrome (ACS).³ DM is therefore generally regarded as a coronary equivalent, with several research studies suggesting the same targets of treatment as the patients who have suffered a previous myocardial infarction (MI). These treatment strategies involve the use of antiaggregants as part of both primary and secondary prevention therapy. Antiplatelet drug resistance has emerged as a new concept and is responsible for a significant amount of treatment failures.

Endothelial Dysfunction and Cardiovascular Disease in Type 2 Diabetes

DM produces endothelium dysfunction and increased arterial stiffness, both important predictors of cardiovascular risk.¹

Endothelium is the boundary between blood and the different tissues. Its functions include regulating cellular metabolism, vessel-tone regulation through vasoconstriction and vasodilation and the prevention of oxidative stress, thrombosis and leukocyte diapedesis. The pathogenesis of endothelial dysfunction in type 2 diabetes is multifactorial, with oxidative stress, dyslipidaemia and hyperglycaemia as some of the principal contributors.

Nitric oxide (NO) plays a central role in endothelium homeostasis. The bioavailability of NO represents a key marker of vascular health. NO causes vasodilation on subjacent vascular smooth muscle cells⁴ and protects vessels from endogenous injury. Synthesis of NO in DM is diminished, consequently predisposing to atherosclerosis and cardiovascular disease (CVD).

Other endothelial injuries that appear in diabetic patients are an increase in oxidative products due to the metabolism of the excess in blood glucose, diabetic dyslipidaemia, insulin resistance, increased synthesis of vasoconstrictor prostanoids, vascular smooth muscle dysfunction and alterations in thrombosis and coagulation.²

Hyperglycaemia produces high levels of reactive oxidant species (ROS). It also contributes to accelerated arterial stiffening by increasing the formation of advanced glycation end-products, which alter vessel wall structure and function. Another cause of endothelial dysfunction is diabetic dyslipidaemia, which is characterised by the accumulation of triglyceride-rich lipoproteins, small dense low-density lipoprotein particles, a decrease in high-density lipoprotein-cholesterol and increased postprandial free fatty acid flux. These abnormalities in lipid metabolism contribute to heightened oxidative stress and may directly inhibit endothelial NO synthase activity,¹ the enzyme that produce constitutively endothelial NO.

Insulin resistance is also a cause of endothelial dysfunction. In healthy subjects, insulin produces endothelium vasodilation; this occurs conversely in diabetics.

Endothelial cell dysfunction is characterised not only by decreased NO but also by an increase in the synthesis of vasoconstrictor prostanoids and endothelin,⁵ which promote inflammation and causes smooth muscle contraction and growth, hence constituting an important factor on the pathophysiology of vascular disease in diabetes.⁶

The impairments that appear in sympathetic nervous system function as a consequence of type 2 DM also contribute to the endothelial dysfunction⁷ through different pathways.

In brief, diabetes mellitus represents an important factor in the genesis of atherosclerosis and endothelial dysfunction, representing one of the main cardiovascular risk factors and being therefore considered a coronary equivalent.

Platelet Dysfunction in Type 2 Diabetes

Platelet function is also altered in diabetic patients, who present a hypercoagulant status caused by a dysfunction in platelet aggregation after vascular injury. This happens due to the disbalance caused by the increase in coagulant factors and the decrease in fibrinolytic factors.²

In these patients, platelets present an increased surface expression of both glycoprotein Ib and IIb/IIIa, which mediate an augmenting binding of both platelet–von Willebrand factor and platelet–fibrin interactions⁸ increasing plaquetary aggregation.

Hyperglycaemia in diabetes produces elevated intraplatelet glucose concentration, hence increasing the synthesis of ROS, which inactivate NO to form peroxynitrite.⁹ As a consequence, platelet-derived NO decreases.⁸

Hyperglycaemia also changes platelet conformation, interfering with platelet calcium homeostasis and modifying aspects of platelet activation and aggregation, thereby altering essential aspects of platelet function.¹⁰

Another issue that poses a relevant risk of thrombosis complications of plaque rupture in diabetics patients is the increment in coagulation factors such as factor VII, thrombin and lesion-based coagulants (eg, tissue factor).¹¹

Diminished synthesis of endogenous anticoagulants (eg, thrombomodulin and protein C) and increased production of plasminogen activator inhibitor-1, a fibrinolysis inhibitor, also contribute to this general procoagulant status. ^12–14

These series of abnormalities are responsible for the hypercoagulant and hyperthrombotic status in diabetic patients and the cause of the accelerated development of coronary artery disease (CAD).

OVERVIEW OF CURRENT ANTIPLATELET THERAPY

Antiplatelet agents act through different pathways. These drugs target enzymes or receptors that are critical for the synthesis or action of important mediators of the functional responses that take place in the process of platelet aggregation.¹⁵

This is a general review based on the currently available antiplatelet agents and novel agents under clinical development.¹⁶

Inhibitors of TXA2 Pathway

The main representative of this group is aspirin. Aspirin irreversibly inhibits cyclooxygenase 1 (COX-1) thereby blocking the production of TXA2, released by adherent platelets at a vascular injury for further platelet adhesion. It is produced de novo through the conversion of arachidonic acid by the enzymes COX-1 and TX synthases.^15,16 Through its irreversible inhibition of COX-1, Aspirin prevents TXA2 from being synthesised and, as a result, diminishes platelet aggregation. This inhibition persists for the whole platelets’ lifetime.

Today, aspirin remains as the foundation of antiplatelet therapy. In high-risk patients, aspirin reduces vascular death by 15% and non-fatal vascular events by 30% as evidenced by meta-analysis of over 100 randomised trials.¹⁷ Aspirin can also be used as part of a strategy of primary prevention of cardiovascular events, but the effect is more modest and its recommendation is highly debated.

Inhibitors of P2Y12 Receptor

The P2Y12 receptor is present in the platelet's surface and contributes to its recruitment and activation through its union with ADP, a soluble platelet-activating factor.¹⁸

The drugs that present an effect over this reaction are ticlopidine, clopidogrel, prasugrel, and ticagrelor. Other compounds under late-stage development are cangrelor and elinogrel.¹⁵

Ticlopidine is a first-generation thienopyridine, which was the first P2Y12 inhibitor approved. Ticlopidine showed better results than aspirin in the primary endpoint, non-fatal stroke or death, in the Ticlopidine Asprin Stroke Study (TASS),¹⁹ but it has been replaced by clopidogrel because of its side effects (neutropenia, thrombotic thrombocytopenia, purpura and rash²⁰) and its inability to induce platelet inhibition rapidly.

Clopidogrel is a second-generation thienopyridine. It is metabolised by the hepatic cytochrome P450 (CYP). Clopidogrel showed its superiority as a single agent over aspirin in patients with high risk [patients with a history of recent MI, ischaemic stroke or established peripheral artery disease (PAD)] in the Clopidogrel versus Aspirin in Patients at Risk of Ischaemic Events (CAPRIE) trial.²¹ Clopidogrel showed a significant reduction in the risk of ischaemic events of 5.32% versus 5.83% with aspirin (P=.043) with no significant differences in safety profile. The benefit of clopidogrel therapy was higher in diabetic patients (15.6% vs 17.7%; P=.042). Clopidogrel plus aspirin is currently the standard therapy recommended by most guidelines for patients with non-ST segment elevation (NSTE)-ACS, ST segment elevation MI (STEMI) and patients undergoing percutaneous coronary intervention (PCI).²² The limitation of dual antiplatelet therapy is the increased bleeding risk. In the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial, clopidogrel in addition to aspirin reduced the incidence of cardiovascular death and non-fatal MI or stroke compared with aspirin alone (9.3% vs 11.4%; RR, 0.80; 95% CI, 0.72–0.90; P<.001) in patients with NSTE-ACS associated with elevated cardiac markers or ST-segment depression on electrocardiogram or age >60 years with prior CAD history.²² However, more patients in the clopidogrel plus ASA group than in the ASA alone group presented major bleeding (3.7% vs 2.7%, respectively; P=.001), although such increase was not observed in life-threatening and fatal bleeds. The addition of clopidogrel to ASA did not show more efficacy than ASA alone in patients with clinically evident CVD or multiple risk but not presenting with an ACS or undergoing PCI in the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA).²³ In a post hoc subgroup analysis of the CHARISMA population, dual antiplatelet therapy with aspirin plus clopidogrel showed a reduction of the primary outcome (8.8% vs 7.3%; P=.01) in patients with prior MI, stroke or symptomatic PAD. Severe bleeding was not different between treatment arms. The high variability observed in clinical response is also considered as a potentially important limitation of clopidogrel, because inadequate inhibition of the ADP platelet activation pathway may leave patients at risk for thrombotic events.²⁴ Assessment of the proper dose of clopidogrel was evaluated in the CURRENT/OASIS7 trial, comparing a double dose of clopidogrel versus standard dose in patients with ACS treated with an early invasive strategy. No significant difference was observed in the high-dose regimen group in the primary endpoint (composite of cardiovascular death, MI or stroke) but in the subgroup of patients undergoing PCI, the high clopidogrel dose strategy diminished the rates of ischaemic outcomes (3.9% vs 4.5%) and reduced the risk of stent thrombosis by 30% with a modest excess in major bleeds.²⁵

Prasugrel, a third-generation thienopyridine has shown more potent inhibition of platelet aggregation, a faster onset of activity and lower interindividual variability than clopidogrel.^26,27 The Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel (TRITON) trial showed reduced rates of ischaemic events, including stent thrombosis in patients with ACS scheduled for PCI, in the group treated with prasugrel over clopidogrel. The side effect was an increased risk of major bleeding, including fatal bleeding, but overall mortality did not differ significantly between both treatments.²⁸

Prasugrel demonstrated the greatest benefit among patients with DM and those presenting with STEMI undergoing primary PCI, in whom there were no differences in major bleeding complications.^29,30

Ticagrelor is the first oral reversible P2Y12 antagonist. It achieves a more rapid and potent platelet inhibition than clopidogrel without significantly increasing major bleeding. ^31–33 Some of the side effects of ticagrelor were the occurrence of dyspnoea and ventricular pause, apparently in a dose-dependent manner.³⁴ The Platelet Inhibition and Patient Outcomes (PLATO) trial compared the effect of ticagrelor vs clopidogrel for the prevention of cardiovascular events in 18 624 patients admitted to the hospital with an ACS, with or without ST-segment elevation.³⁴ The overall trial demonstrated a significant reduction in the primary endpoint, a composite of death from vascular causes, MI or stroke at 12 months with ticagrelor, without an increase in overall major bleeding, although non-surgical bleeding was significantly increased.

Cangrelor is an intravenous reversible inhibitor of the P2Y12 receptor, which exerts its function with a rapid onset and offset.³⁵ Two large-scale randomised trials were conducted as part of the CHAMPION (Cangrelor versus standard tHerapy to Achieve optimal Management of Platelet InhibitiOn) clinical trial programme: CHAMPION-PCI and CHAMPION-PLAT-FORM; however, these studies were stopped early owing to lack of efficacy.^36,37 Cangrelor has been studied in patients undergoing cardiac surgery as a bridge for patients who could benefit from a treatment with a thienopyridine but needed to terminate treatment before surgery. Cangrelor showed a higher rate of maintenance of platelet inhibition at least 48 hours before surgery and discontinuing it 1 to 6 hours before coronary artery bypass graft surgery.³⁸

Elinogrel is a reversible, potent, competitive inhibitor of the P2Y12 receptor with fast onset and offset of action that can be administered by both oral and intravenous routes.³⁹ Elinogrel has been studied in the Phase II INNOVATE-PCI (IntraveNous and Oral administration of PRT060128 to eVAluate Tolerability and Efficacy in non-urgent PCI patients) showing better platelet inhibition at doses of 100 and 150 mg twice daily than those treated with clopidogrel, with those receiving 150 mg elinogrel without increase in major or minor bleeding.⁴⁰

PAR-1 Inhibitors

This group is composed by vorapaxar and atopaxar, which are selective inhibitors of the principal protease-activated receptor (PAR)-1 for thrombin, the most potent platelet activator. It represents a promising novel strategy to reduce ischaemic events without increasing bleeding risk.^41,42

Vorapaxar has already been studied in phase II safety and dose-ranging trials, performed in patients undergoing non-urgent PCI on standard antiplatelet therapy with aspirin and clopidogrel, showing an excellent safety profile without a significant increase in bleeding.⁴³ However, the association described between vorapaxar and hepatic toxicity as well as with intracraneal haemorrhage in patients with a history of previous stroke are important concerns.⁴⁴

These results have promoted the initiation of two large phase III trials, the TRACER (Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome) and TRA 2° P-TIMI 50 (Thrombin-Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events) to prove the potential of this new class of antiplatelets to reduce ischaemic complications with a favourable bleeding profile.^45,46

On the other hand, atopaxar, was evaluated in two phase II trials, J-LANCELOT (Japanese lessons from Antagonizing the Cellular Effects Of Thrombin) with the objective to evaluate its safety profile in patients with CAD and ACS, respectively. The results of these trials have recently been reported and show that, in overall, atopaxar was not associated with any increase in serious bleeding events in both ACS and CAD patients^47,48 although a transient dose-dependent transaminase elevation and relative QTc prolongation were observed with the highest doses.⁴⁸

Phosphodiesterase Inhibitors

The representatives of this group are dipyridamole and cilostazol. Dipyridamole is a pyrimidopyrimidine derivative with antiplatelet and vasodilator properties.⁴⁹ The study ESPS-2 (European Stroke Prevention Study) showed that aspirin plus dipyridamole was significantly more effective than aspirin alone in secondary prevention of stroke with a similar low risk of bleeding.⁵⁰ European/Australasian Stroke Prevention in Reversible Ischemia Trial (ESPRIT) demonstrated that dual therapy has a significantly lower incidence rate of the primary outcome (nonfatal MI, nonfatal stroke, vascular death, or major bleeding complication)⁵¹ but this therapy was not superior to clopidogrel alone in the PROFESS (Prevention Regimen for Effectively Avoiding Second Strokes) trial.⁵²

Cilostazol is another vasodilatory and antiplatelet drug, which exerts its function over a selective PDE type III (PDE III) inhibitor, which increases cAMP levels in platelets and endothelial and smooth muscle cells.⁵³ It is currently used for the treatment of intermittent claudication.

Recent studies have shown that the addition of cilostazol to dual antiplatelet therapy (triple antiplatelet therapy) is associated with a reduced risk of stent thrombosis, restenosis and major adverse cardiac events without increased bleeding complications, even in patients undergoing PCI with drug-eluting stents. ^54–57 The side effects include rash, gastrointestinal disturbance and headache. Cilostazol should be avoided in patients with congestive heart failure of any severity because of increased mortality risk.

Cilostazol has shown to be particularly effective in diabetic patients.⁵⁸

Glycoprotein IIb/IIIa Inhibitors

These drugs inhibit the final common pathway of platelet aggregation: its binding to the integrin GPIIb/IIIa located on the platelet's surface.

GPIIb/IIIa inhibitors administered intravenously have proved to reduce ischaemic events in patients suffering ACS undergoing PCI; however, GPIIb/IIIa inhibitors are associated with an increased risk of bleeding. Oral administration of these drugs has failed to demonstrate any benefit.⁵⁹ This group is composed by eptifibatide, tirofiban and abciximab. Eptifibatide and tirofiban reversibly and competitively bind to the GPIIb/IIIa receptor. Abciximab irreversibly binds to the GPIIb/IIIa.

ASPIRIN AND CLOPIDOGREL RESISTANCE IN TYPE 2 DM

Double antiaggregation with aspirin and clopidogrel is the base of care therapy of ACS. Although this treatment has shown considerable benefits as secondary prevention therapy in this group of patients, there are patients who continue to experience atherothrombotic complications, especially those at high-risk such as diabetic patients. Interindividual variability of platelet aggregation in response to these antiplatelet agents may be an explanation for some of these recurrent events. The prevalence of aspirin resistance (AR) described in the literature varies considerably (from 0% in some studies to >50% of patients in others). This variability depends on the definition of AR, the type of assay used, aspirin dose and patient population under consideration.⁶⁰ Aspirin efficacy is attenuated in patients suffering from DM, as demonstrated in the Antithrombotic Trialists’ Collaboraton (ATT) meta-analysis of antiplatelet therapy, with a non-significant 7% odds reduction of vascular events in patients with DM treated with aspirin compared with a 22% odds reduction observed in the overall meta-analysis cohort.¹⁷

Several studies indicate that patients with diabetes and hyperlipidaemia have higher susceptibility to AR. Patients with diabetes are typically characterised by platelet hyperreactivity. Aspirin exerts its action inhibiting COX-1 completely, but high residual platelet reactivity may persist in these patients as a result of upregulation of other signalling pathways which are not blocked by aspirin.⁶¹

Chronic hyperglycaemia is related to platelet hyperreactivity and in vivo platelet activation in patients with DM, which seems to be one of the causes of the remaining increased risk of recurrent ischaemic events in these patients. Increased glycation of platelets and protein coagulation factors may interfere with the acetylation process, thereby contributing to inadequate aspirin-induced antiplatelet effects in diabetic patients. However, it still remains undetermined whether improved glycaemic control enhances the efficacy of aspirin or whether increased doses of aspirin are beneficial in the presence of poor glycaemic control.⁶²

The dose of aspirin seems to have an effect on AR in patients with type 2 DM too. In a study of 108 diabetic patients and 67 non-diabetic subjects, using low-dose aspirin (100 mg) was a risk factor for AR status for both diabetic patients (odds ratio, 1.26;, 95% CI, 1.01–1.58; P<.05) and the overall study group (odds ratio, 1.3; 95% CI, 1.08–1.56, P<.01).⁶² This study also showed a relation between body mass index, fast blood glucose, HbA1c and AR. Similar results were obtained in relation to the dose of aspirin necessary to decrease AR in diabetics in a subanalysis of the Aspirin-Induced Platelet Effect (ASPECT) trial, which compared the pharmacodynamic effect of different doses of aspirin in patients with and without DM, showing a higher percentage of AR in the DM subgroup with the lower dose.⁶³

Other situations that could contribute to AR in diabetics are the increased TXA2 synthesis in these patients⁶⁴ and an accelerated platelet turnover.⁶⁵

In the case of clopidogrel, it shows a variable platelet inhibitory response in patients undergoing PCI, associated with increased platelet aggregation and increased expression of platelet surface receptors. The effect of clopidogrel over DM patients has been largely studied showing a high number of non-responders among this population. Angiolillo et al. studied the different platelet function profiles in diabetic and non-diabetic patients on aspirin and clopidogrel therapy. Two patient populations were included to investigate the acute effects of a 300-mg clopidogrel loading dose as well as its long-term effects at a maintenance dose on platelet function in diabetic compared to non-diabetic patients already on aspirin treatment. The results conclude that diabetic subjects had a higher number of clopidogrel non-responders (P=.04).⁶⁶

Similar research studies carried our among diabetic patients considered non-responders to clopidogrel have compared DM patients on clopidogrel and aspirin therapy with prasugrel plus aspirin. The results have shown significantly lower levels of active metabolites (AMs) of clopidogrel than those of prasugrel and better pharmacodynamic response to prasugrel over clopidogrel.⁶⁷ This relative lack of AM of clopidogrel could happen as a result of impaired absorption, impaired metabolism from prodrug to AM, increased clearance, increased platelet turnover or as due to a combination of some or all of the aforementioned.⁶⁸ Other causes of clopidogrel unresponsiveness could be genetic differences in P2Y12 receptors, leading to decreased effectiveness of clopidogrel,^69,70 increased expression of P2Y12 receptors, increased circulating ADP, increased circulating esterases and upregulation of other platelet activation pathways.^71,72

On the other hand, DM patients with poor metabolic control and those who require insulin therapy have the worst response to clopidogrel in patients on dual antiplatelet therapy.^73,74 It is known that among DM patients, the presence of moderate to severe chronic kidney disease is associated with decreased response to clopidogrel,⁷⁵ which is in line with the results of a post hoc analysis of the CHARISMA trial, which suggests that clopidogrel use might increase adverse outcomes in patients with diabetic nephropathy.⁷⁶

In summary, clopidogrel unresponsiveness in patients with diabetes is likely multifactorial in origin, with several different causes such as decreased circulating AMs in patients treated with clopidogrel, variability of genetic differences in P2Y12 receptors, poor metabolic control in DM patients and other causes like increased platelet turnover⁷⁷ and the prothrombotic conditions associated with DM.

FUTURE DIRECTIONS OF TREATMENT IN PATIENTS RESISTANT TO ASPIRIN AND CLOPIDOGREL

Despite the fact that currently available antiplatelet drugs have shown its effectiveness in reducing cardiovascular adverse events in patients with CAD, there is still a significant group of patients, particularly patients at high risk, like diabetics, who present low response to standard antiplatelet agents. This has promoted the continuous search for more potent and safe antiplatelet treatment strategies to optimise platelet inhibitory effects.

Several new agents that block multiple pathways involved in platelet adhesion, activation and aggregation are currently at different stages of clinical development or already in use.⁷⁸

Some of this drugs, which have been commented previously, are prasugrel, ticagrelor, cangrelor and elinogrel, all of them in the group of inhibitors of P2Y12 receptor. Particularly relevant for diabetic patients are prasugrel and ticagrelor. A subanalysis of the TRITON-TIMI 38 trial evaluated the effect of prasugrel versus clopidogrel among 3146 study participants with DM.²⁹ In this subset, the effect of prasugrel on the primary endpoint was statistically superior to clopidogrel and greater in magnitude than the one observed in the overall study cohort (HR, 0.74; P=.001).⁶⁸ Rates of MI were also significantly reduced in patients with DM when compared to those without (40% vs 18%, respectively). Another advantage of prasugrel over clopidogrel in this study was the absence of significantly increased bleeding risks associated with prasugrel.

Ticagrelor was studied in PLATO compared with clopidogrel, showing a significant reduction of cardiovascular death, MI or stroke (HR, 0.84; 95% CI, 0.77–0.92; P<.001), without a significant increase in major bleeding (P=.43). Similar results were found in the subset of diabetic patients.⁷⁹

Cilostazol, a phosphodiesterase inhibitor, has shown to be particularly effective in diabetic patitents.^58,80 Moreover, recent studies have shown that adjunctive cilostazol to dual antiplatelet therapy after PCI can achieve greater platelet inhibition than high maintenance dose of clopidogrel, 150 mg daily,⁸¹ even in patients with CYP2C19 mutant allele.⁸²

Another treatment option in diabetic patients has been the increased dosing of currently approved agents. The pharmacodynamic efficacy of a high (150 mg) versus standard (75 mg) maintenance dose of clopidogrel in type 2 DM was evaluated in the OPTIMUS study. A 150-mg maintenance dose of clopidogrel was associated with enhanced antiplatelet effects compared with 75 mg in high-risk type 2 DM patients, although platelet reactivity⁸³ remained high in a considerable number of patients.

CONCLUSIONS

Diabetic patients continue presenting cardiovascular events despite the use of antiaggregants after an ACS. Lack of responsiveness in this group of patients has been described and is thought to be influenced by poor metabolic control, disturbed endothelial and platelet function and differences in pharmacodynamic mechanisms. These considerations underscore the need to maximise the efficacy of antiaggregant effect in this high-risk population with more potent antiplatelet therapy. There are novel antiplatelet drugs currently under investigation, which may provide significant improvements in cardiovascular risk control in diabetic population.

Disclosure: The authors declare no conflict of interest.

REFERENCES

1. Woodman RJ, Chew GT, Watts GF. Mechanisms, significance and treatment of vascular dysfunction in type 2 diabetes mellitus: focus on lipid-regulating therapy. Drugs. 2005;65(1):31–74.
2. Creager MA, Luscher TF, Cosentino F, Beckman JA. Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: Part I. Circulation. 2003;108(12):1527–1532.
3. Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med. 1998;339(4):229–234.
4. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993;329(27):2002–2012.
5. O’Driscoll G, Green D, Maiorana A, Stanton K, Colreavy F, Taylor R. Improvement in endothelial function by angiotensin-converting enzyme inhibition in non-insulin-dependent diabetes mellitus. J Am Coll Cardiol. 1999;33(6):1506–1511.
6. Hopfner RL, Gopalakrishnan V. Endothelin: emerging role in diabetic vascular complications. Diabetologia. 1999;42(12):1383–1394.
7. McDaid EA, Monaghan B, Parker AI, Hayes JR, Allen JA. Peripheral autonomic impairment in patients newly diagnosed with type II diabetes. Diabetes Care. 1994;17(12):1422–1427.
8. Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2 diabetes. Diabetes Care. 2001;24(8):1476–1485.
9. Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature. 2000;404(6779):787–790.
10. Li Y, Woo V, Bose R. Platelet hyperactivity and abnormal Ca(2) homeostasis in diabetes mellitus. Am J Physiol Heart Circ Physiol. 2001;280(4):H1480–H1489.
11. Ceriello A, Giacomello R, Stel G, et al. Hyperglycemia-induced thrombin formation in diabetes. The possible role of oxidative stress. Diabetes. 1995;44(8):924–928.
12. Ceriello A, Giugliano D, Quatraro A, Marchi E, Barbanti M, Lefebvre P. Evidence for a hyperglycaemia-dependent decrease of antithrombin IIIthrombin complex formation in humans. Diabetologia. 1990;33(3): 163–167.
13. Ren S, Lee H, Hu L, Lu L, Shen GX. Impact of diabetes-associated lipoproteins on generation of fibrinolytic regulators from vascular endothelial cells. J Clin Endocrinol Metab. 2002;87(1):286–291.
14. Pandolfi A, Cetrullo D, Polishuck R, et al. Plasminogen activator inhibitor type 1 is increased in the arterial wall of type II diabetic subjects. Arterioscler Thromb Vasc Biol. 2001;21(8):1378–1382.
15. Angiolillo DJ, Ueno M, Goto S. Basic principles of platelet biology and clinical implications. Circ J. 2010;74(4):597–607.
16. Ueno M, Kodali M, Tello-Montoliu A, Angiolillo DJ. Role of platelets and antiplatelet therapy in cardiovascular disease. J Atheroscler Thromb. 2011;18(6):431–442.
17. Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. 2002;324(7329):71–86.
18. Offermanns S. Activation of platelet function through G protein-coupled receptors. Circ Res. 2006;99(12):1293–1304.
19. Hass WK, Easton JD, Adams HP Jr, et al. A randomized trial comparing ticlopidine hydrochloride with aspirin for the prevention of stroke in high-risk patients. Ticlopidine Aspirin Stroke Study Group. N Engl J Med. 1989;321(8):501–507.
20. Bertrand ME, Rupprecht HJ, Urban P, Gershlick AH. Double-blind study of the safety of clopidogrel with and without a loading dose in combination with aspirin compared with ticlopidine in combination with aspirin after coronary stenting: the clopidogrel aspirin stent international cooperative study (CLASSICS). Circulation. 2000;102(6):624–629.
21. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). CAPRIE Steering Committee. Lancet. 1996;348(9038):1329–1339.
22. Hamm CW, Bassand JP, Agewall S, et al. ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: the Task Force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC). Eur Heart J. 2011;32(23):2999–3054.
23. Bhatt DL, Flather MD, Hacke W, et al. Patients with prior myocardial infarction, stroke, or symptomatic peripheral arterial disease in the CHARISMA trial. J Am Coll Cardiol. 2007;49(19):1982–1988.
24. Ferreiro JL, Angiolillo DJ. Clopidogrel response variability: current status and future directions. Thromb Haemost. 2009;102(1):7–14.
25. Mehta SR, Bassand JP, Chrolavicius S, et al. Design and rationale of CURRENT-OASIS 7: a randomized, 2 ( 2 factorial trial evaluating optimal dosing strategies for clopidogrel and aspirin in patients with ST and non- ST-elevation acute coronary syndromes managed with an early invasive strategy. Am Heart J. 2008;156(6):1080–1088 e1081.
26. Brandt JT, Payne CD, Wiviott SD, et al. A comparison of prasugrel and clopidogrel loading doses on platelet function: magnitude of platelet inhibition is related to active metabolite formation. Am Heart J. 2007;153(1):66.e9–66.e16.
27. Wiviott SD, Trenk D, Frelinger AL, et al. Prasugrel compared with high loading- and maintenance-dose clopidogrel in patients with planned percutaneous coronary intervention: the Prasugrel in Comparison to Clopidogrel for Inhibition of Platelet Activation and Aggregation- Thrombolysis in Myocardial Infarction 44 trial. Circulation. 2007;116(25):2923–2932.
28. Wiviott SD, Braunwald E, McCabe CH, et al. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2007;357(20):2001–2015.
29. Wiviott SD, Braunwald E, Angiolillo DJ, et al. Greater clinical benefit of more intensive oral antiplatelet therapy with prasugrel in patients with diabetes mellitus in the trial to assess improvement in therapeutic outcomes by optimizing platelet inhibition with prasugrel-Thrombolysis in Myocardial Infarction 38. Circulation. 2008;118(16):1626–1636.
30. Montalescot G, Wiviott SD, Braunwald E, et al. Prasugrel compared with clopidogrel in patients undergoing percutaneous coronary intervention for ST-elevation myocardial infarction (TRITON-TIMI 38): double-blind, randomised controlled trial. Lancet. 2009;373(9665):723–731.
31. Husted S, Emanuelsson H, Heptinstall S, Sandset PM, Wickens M, Peters G. Pharmacodynamics, pharmacokinetics, and safety of the oral reversible P2Y12 antagonist AZD6140 with aspirin in patients with atherosclerosis: a double-blind comparison to clopidogrel with aspirin. Eur Heart J. 2006;27(9):1038–1047.
32. Storey RF, Husted S, Harrington RA, et al. Inhibition of platelet aggregation by AZD6140, a reversible oral P2Y12 receptor antagonist, compared with clopidogrel in patients with acute coronary syndromes. J Am Coll Cardiol. 2007;50(19):1852–1856.
33. Capodanno D, Dharmashankar K, Angiolillo DJ. Mechanism of action and clinical development of ticagrelor, a novel platelet ADP P2Y12 receptor antagonist. Expert Rev Cardiovasc Ther. 2010;8(2):151–158.
34. Wallentin L, Becker RC, Budaj A, et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2009;361(11): 1045–1057.
35. Ferreiro JL, Ueno M, Angiolillo DJ. Cangrelor: a review on its mechanism of action and clinical development. Expert Rev Cardiovasc Ther. 2009;7(10):1195–1201.
36. Harrington RA, Stone GW, McNulty S, et al. Platelet inhibition with cangrelor in patients undergoing PCI. N Engl J Med. 2009;361(24): 2318–2329.
37. Bhatt DL, Lincoff AM, Gibson CM, et al. Intravenous platelet blockade with cangrelor during PCI. N Engl J Med. 2009;361(24):2330–2341.
38. Angiolillo DJ, Firstenberg MS, Price MJ, et al. Bridging antiplatelet therapy with cangrelor in patients undergoing cardiac surgery: a randomized controlled trial. JAMA. 2012;307(3):265–274.
39. Ueno M, Rao SV, Angiolillo DJ. Elinogrel: pharmacological principles, preclinical and early phase clinical testing. Future Cardiol. 2010;6(4):445– 453.
40. Leonardi S, Rao SV, Harrington RA, et al. Rationale and design of the randomized, double-blind trial testing INtraveNous and Oral administration of elinogrel, a selective and reversible P2Y(12)-receptor inhibitor, versus clopidogrel to eVAluate Tolerability and Efficacy in nonurgent Percutaneous Coronary Interventions patients (INNOVATE-PCI). Am Heart J. 2010;160(1):65–72.
41. Angiolillo DJ, Capodanno D, Goto S. Platelet thrombin receptor antagonism and atherothrombosis. Eur Heart J. 2010;31(1):17–28.
42. Ueno M, Ferreiro JL, Angiolillo DJ. Mechanism of action and clinical development of platelet thrombin receptor antagonists. Expert Rev Cardiovasc Ther. 2010;8(8):1191–1200.
43. Becker RC, Moliterno DJ, Jennings LK, et al. Safety and tolerability of SCH 530348 in patients undergoing non-urgent percutaneous coronary intervention: a randomised, double-blind, placebo-controlled phase II study. Lancet. 2009;373(9667):919–928.
44. Gurbel PA, Jeong YH, Tantry US. Vorapaxar: a novel protease-activated receptor-1 inhibitor. Expert Opin Investig Drugs. 2011;20(10):1445–1453.
45. TRA—CER Executive and Steering Committees. The Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRA—CER) trial: study design and rationale. Am Heart J. 2009;158(3): 327–334.e4.
46. Morrow DA, Scirica BM, Fox KA, et al. Evaluation of a novel antiplatelet agent for secondary prevention in patients with a history of atherosclerotic disease: design and rationale for the Thrombin-Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events (TRA 2 degrees P)-TIMI 50 trial. Am Heart J. 2009;158(3):335– 341.e3.
47. Wiviott SD, Flather MD, O’Donoghue ML, et al. Randomized trial of atopaxar in the treatment of patients with coronary artery disease: the lessons from antagonizing the cellular effect of Thrombin-Coronary Artery Disease Trial. Circulation. 2011;123(17):1854–1863.
48. O’Donoghue ML, Bhatt DL, Wiviott SD, et al. Safety and tolerability of atopaxar in the treatment of patients with acute coronary syndromes: the lessons from antagonizing the cellular effects of Thrombin-Acute Coronary Syndromes Trial. Circulation. 2011;123(17):1843–1853.
49. Aktas B, Utz A, Hoenig-Liedl P, Walter U, Geiger J. Dipyridamole enhances NO/cGMP-mediated vasodilator-stimulated phosphoprotein phosphorylation and signaling in human platelets: in vitro and in vivo/ ex vivo studies. Stroke. 2003;34(3):764–769.
50. Diener HC, Cunha L, Forbes C, Sivenius J, Smets P, Lowenthal A. European Stroke Prevention Study. 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke. J Neurol Sci. 1996;143(1–2): 1–13.
51. Halkes PH, van Gijn J, Kappelle LJ, Koudstaal PJ, Algra A. Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT): randomised controlled trial. Lancet. 2006;367(9523):1665–1673.
52. Sacco RL, Diener HC, Yusuf S, et al. Aspirin and extended-release dipyridamole versus clopidogrel for recurrent stroke. N Engl J Med. 2008;359(12):1238–1251.
53. Goto S. Cilostazol: potential mechanism of action for antithrombotic effects accompanied by a low rate of bleeding. Atheroscler Suppl. 2005;6(4):3–11.
54. Lee SW, Park SW, Hong MK, et al. Triple versus dual antiplatelet therapy after coronary stenting: impact on stent thrombosis. J Am Coll Cardiol. 2005;46(10):1833–1837.
55. Lee SW, Park SW, Yun SC, et al. Triple antiplatelet therapy reduces ischemic events after drug-eluting stent implantation: Drug-Eluting stenting followed by Cilostazol treatment REduces Adverse Serious cardiac Events (DECREASE registry). Am Heart J. 2010;159(2):284–291.e1.
56. Chen KY, Rha SW, Li YJ, et al. Triple versus dual antiplatelet therapy in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention. Circulation. 2009;119(25):3207–3214.
57. Lee SW, Park SW, Kim YH, et al. A randomized, double-blind, multicenter comparison study of triple antiplatelet therapy with dual antiplatelet therapy to reduce restenosis after drug-eluting stent implantation in long coronary lesions: results from the DECLARE-LONG II (Drug-Eluting Stenting Followed by Cilostazol Treatment Reduces Late Restenosis in Patients with Long Coronary Lesions) trial. J Am Coll Cardiol. 2011;57(11):1264–1270.
58. Angiolillo DJ, Capranzano P, Goto S, et al. A randomized study assessing the impact of cilostazol on platelet function profiles in patients with diabetes mellitus and coronary artery disease on dual antiplatelet therapy: results of the OPTIMUS-2 study. Eur Heart J. 2008;29(18):2202–2211.
59. Chew DP, Bhatt DL, Sapp S, Topol EJ. Increased mortality with oral platelet glycoprotein IIb/IIIa antagonists: a meta-analysis of phase III multicenter randomized trials. Circulation. 2001;103(2):201–206.
60. Angiolillo DJ. Antiplatelet therapy in diabetes: efficacy and limitations of current treatment strategies and future directions. Diabetes Care. 2009;32(4):531–540.
61. Gao F, Wang ZX, Men JL, Ren J, Wei MX. Effect of polymorphism and type II diabetes on aspirin resistance in patients with unstable coronary artery disease. Chin Med J (Engl). 2011;124(11):1731–1734.
62. Ertugrul DT, Tutal E, Yildiz M, et al. Aspirin resistance is associated with glycemic control, the dose of aspirin, and obesity in type 2 diabetes mellitus. J Clin Endocrinol Metab. 2010;95(6):2897–2901.
63. DiChiara J, Bliden KP, Tantry US, et al. The effect of aspirin dosing on platelet function in diabetic and nondiabetic patients: an analysis from the aspirin-induced platelet effect (ASPECT) study. Diabetes. 2007;56(12):3014–3019.
64. Davi G, Catalano I, Averna M, et al. Thromboxane biosynthesis and platelet function in type II diabetes mellitus. N Engl J Med. 1990;322(25):1769–1774.
65. Guthikonda S, Lev EI, Patel R, et al. Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin. J Thromb Haemost. 2007;5(3):490–496.
66. Angiolillo DJ, Fernandez-Ortiz A, Bernardo E, et al. Platelet function profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes. 2005;54(8): 2430–2435.
67. Erlinge D, Varenhorst C, Braun OO, et al. Patients with poor responsiveness to thienopyridine treatment or with diabetes have lower levels of circulating active metabolite, but their platelets respond normally to active metabolite added ex vivo. J Am Coll Cardiol. 2008;52(24):1968–1977.
68. Hall HM, Banerjee S, McGuire DK. Variability of clopidogrel response in patients with type 2 diabetes mellitus. Diab Vasc Dis Res. 2011;8(4): 245–253.
69. Brandt JT, Close SL, Iturria SJ, et al. Common polymorphisms of CYP2C19 and CYP2C9 affect the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel. J Thromb Haemost. 2007;5(12):2429–2436.
70. Fontana P, Hulot JS, De Moerloose P, Gaussem P. Influence of CYP2C19 and CYP3A4 gene polymorphisms on clopidogrel responsiveness in healthy subjects. J Thromb Haemost. 2007;5(10):2153–2155.
71. Mathewkutty S, McGuire DK. Platelet perturbations in diabetes: implications for cardiovascular disease risk and treatment. Expert Rev Cardiovasc Ther. 2009;7(5):541–549.
72. Ferreiro JL, Gomez-Hospital JA, Angiolillo DJ. Platelet abnormalities in diabetes mellitus. Diab Vasc Dis Res. 2010;7(4):251–259.
73. Singla A, Antonino MJ, Bliden KP, Tantry US, Gurbel PA. The relation between platelet reactivity and glycemic control in diabetic patients with cardiovascular disease on maintenance aspirin and clopidogrel therapy. Am Heart J. 2009;158(5):784.e1–784.e6.
74. Angiolillo DJ, Bernardo E, Ramirez C, et al. Insulin therapy is associated with platelet dysfunction in patients with type 2 diabetes mellitus on dual oral antiplatelet treatment. J Am Coll Cardiol. 2006;48(2):298–304.
75. Angiolillo DJ, Bernardo E, Capodanno D, et al. Impact of chronic kidney disease on platelet function profiles in diabetes mellitus patients with coronary artery disease taking dual antiplatelet therapy. J Am Coll Cardiol. 2010;55(11):1139–1146.
76. Dasgupta A, Steinhubl SR, Bhatt DL, et al. Clinical outcomes of patients with diabetic nephropathy randomized to clopidogrel plus aspirin versus aspirin alone (a post hoc analysis of the clopidogrel for high atherothrombotic risk and ischemic stabilization, management, and avoidance [CHARISMA] trial). Am J Cardiol. 2009;103(10):1359–1363.
77. Guthikonda S, Alviar CL, Vaduganathan M, et al. Role of reticulated platelets and platelet size heterogeneity on platelet activity after dual antiplatelet therapy with aspirin and clopidogrel in patients with stable coronary artery disease. J Am Coll Cardiol. 2008;52(9):743–749.
78. Ferreiro JL, Cequier AR, Angiolillo DJ. Antithrombotic therapy in patients with diabetes mellitus and coronary artery disease. Diab Vasc Dis Res. 2010;7(4):274–288.
79. James S, Angiolillo DJ, Cornel JH, et al. Ticagrelor vs. clopidogrel in patients with acute coronary syndromes and diabetes: a substudy from the PLATelet inhibition and patient Outcomes (PLATO) trial. Eur Heart J. 2010;31(24):3006–3016.
80. Lee SW, Chun KJ, Park SW, et al. Comparison of Triple antiplatelet therapy and dual antiplatelet therapy in patients at high risk of restenosis after drug-eluting stent implantation (from the DECLARE-DIABETES and -LONG Trials). Am J Cardiol. 2010;105(2):168–173.
81. Jeong YH, Lee SW, Choi BR, et al. Randomized comparison of adjunctive cilostazol versus high maintenance dose clopidogrel in patients with high post-treatment platelet reactivity: results of the ACCEL-RESISTANCE (Adjunctive Cilostazol Versus High Maintenance Dose Clopidogrel in Patients With Clopidogrel Resistance) randomized study. J Am Coll Cardiol. 2009;53(13):1101–1109.
82. Hwang SJ, Jeong YH, Kim IS, et al. Cytochrome 2C19 polymorphism and response to adjunctive cilostazol versus high maintenance-dose clopidogrel in patients undergoing percutaneous coronary intervention. Circ Cardiovasc Interv. 2010;3(5):450–459.
83. Angiolillo DJ, Shoemaker SB, Desai B, et al. Randomized comparison of a high clopidogrel maintenance dose in patients with diabetes mellitus and coronary artery disease: results of the Optimizing Antiplatelet Therapy in Diabetes Mellitus (OPTIMUS) study. Circulation. 2007;115(6):708–716.

Myocardial infarction or heart attack is the leading cause of death for both men and women all over the world. It is caused due to an interruption in blood supply to any part of the heart, resulting in death of cardiac tissue (myocardial necrosis). Isoproterenol (ISO) is a potent synthetic catecholamine that causes severe stress in the myocardium, resulting in infarct-like necrosis of the heart muscle. These changes resemble the subendocardial laminar necrosis produced by myocardial ischemia in humans and, therefore, are a suitable model system to study myocardial infarction.¹

Mitochondria are the main consumers of molecular oxygen in the cardiac cell and also act as a major source of reactive oxygen species (ROS). It plays a central role in energy generating processes within the cell and also involved in apoptosis and cardioprotection.² Excessive production of reactive free radicals causes mitochondrial damage and dysfunction resulting in the modification of lipids, proteins, and DNA in the mitochondria. These free radicals have been culpably involved in oxidative ischemic injury and are the central component of cellular damage that severely affects the myocardium.³ Thus, mitochondria play a central role in molecular events leading to tissue damage occurring in conditions of ischemia.

At present natural medicine and herbal drugs are acquiring much attention as a potential source of antioxidants. They serve as excellent candidates against ROS-induced pathologies. The N-acetylcysteine (NAC), a thiol reducing agent, is a naturally occurring compound found in several vegetables including garlic, onion,⁴ peppers, and asparagus.⁵ Previous studies have shown that it exhibits antioxidant, antiangiogenic, and anticancer activities.⁶ It also acts as a cysteine donor and maintains the intracellular levels of glutathione.^7,8 Furthermore, NAC is able to inhibit oxidative stress and DNA damage.^9,10 Since, mitochondrial dysfunction plays a vital role in the pathology of myocardial infarction, we undertook this study to know the protective effects of N-acetylcysteine on mitochondrial lipid peroxidation, antioxidants, tricarboxylic acid cycle and respiratory marker enzymes, lipids, and calcium in isoproterenol treated myocardial infarcted rats. Transmission electron microscopic study on the structure of heart mitochondria was carried out to confirm the protective effects of N-acetylcysteine. In addition to this, in vitro study on the total antioxidant activity [(2,2-azinobis- (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS⁺)] of N-acetylcysteine was determined.

2. MATERIALS AND METHODS

2.1. Experimental animals

The whole experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by the Animal Ethical Committee of Annamalai University (Approval No. 593: 15.12.2008). Male albino Wistar rats weighing 160–190 g, obtained from The Central Animal House, Rajah Muthiah Institute of Health Sciences, Annamalai University, Tamil Nadu, India were used in this study. They were housed in polypropylene cages (47 cm×34 cm×20 cm) lined with husk, renewed every 24 h under a 12:12 h light and dark cycle at around 22°C. The rats had free access to tap water and food. The rats were fed on a standard pelleted diet (Pranav Agro Industries Ltd., Maharashtra, India).

2.2. Drug and chemicals

The N-acetylcysteine was obtained from Himedia Laboratories Private Limited, Mumbai, India. Isoproterenol hydrochloride, nitroblue tetrazolium, phenazine methosulphate, butylated hydroxy toluene, 1-chloro-2,4-dinitro benzene, 2,4-dinitro phenyl hydrazine, p-phenylene diamine, dimethyl sulphoxide, reduced glutathione, and diphenyl carbazide were purchased from Sigma Chemical Co., St. Louis, MO, USA. Flavine adenine dinucleotide, thiobarbituric acid, ammonium molybdate, trichloro acetic acid, and triphenyl tetrazolium chloride were purchased from Himedia, Mumbai, India. All the other chemicals used were of analytical grade.

2.3. Induction of myocardial infarction in rats

The ISO (100 mg/kg) was dissolved in saline and subcutaneously injected to male albino Wistar rats at an interval of 24 h for 2 days. Myocardial infarction was confirmed by elevated activity of serum creatine kinase in rats.

2.4. Dose dependent effect of NAC

A preliminary study was conducted with two different doses of NAC (5 mg and 10 mg/kg) to know the dose dependent effect in ISO treated rats. It was noted that after 14 days of NAC pretreatment at doses of 5 mg and 10 mg/kg considerably (P<.05) decreased the elevated activity of serum creatine kinase (CK) in ISO treated rats. But NAC (10 mg/kg) exerted the highest significant effect in lowering serum CK, we have chosen NAC (10 mg/kg) for our further study.

2.5. Study design

The rats were randomly divided into four groups of eight rats each. Two rats from each group were used for transmission electron microscopic study. Group I: normal control rats; Group II: rats were orally treated with NAC (10 mg/kg) daily for 14 days using an intragastric tube; Group III: rats were subcutaneously injected with ISO (100 mg/kg) at an interval of 24 h for 2 days; Group IV: rats were pretreated with NAC (10 mg/kg) daily for 14 days and then subcutaneously injected with ISO (100 mg/kg) at an interval of 24 h for 2 days. Normal control and ISO control rats received saline alone for 14 days of the experimental period. The NAC was dissolved in saline and administered one ml to each rat orally using an intragastric tube daily for a period of 14 days.

At the end of the experimental period, after 12 h of a second ISO injection, all the rats were anesthetized and then sacrificed by cervical decapitation. Blood was collected and serum was separated by centrifugation. Heart tissues were excised immediately and rinsed in ice-chilled saline.

2.6. Isolation of heart mitochondria

Heart mitochondria were isolated by the standard method of Takasawa et al.¹¹

2.7. Biochemical estimations

2.7.1. Activity of serum creatine kinase

The activity of serum CK was assayed by a commercial kit (Agappe Diagnostics, Kerala, India).

2.7.2. Estimation of lipid peroxidation products

The concentration of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) in the heart mitochondrial fraction was estimated by Fraga et al.¹² and Jiang et al.¹³ method, respectively.

2.7.3. Assay of antioxidants

The activity of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and reduced glutathione (GSH) in the heart mitochondrial fraction were assayed by Kakkar et al.,¹⁴ Sinha,¹⁵ Rotruck et al (1973), and Ellman¹⁶ methods, respectively.

2.7.4. Assay of heart mitochondria enzymes

The activity of isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and α-ketoglutarate dehydrogenase (α-KDH) in the mitochondrial fraction of the heart were assayed according to the method of King,¹⁷ Slater and Borner,¹⁸ Mehler et al,¹⁹ and Reed and Mukherjee,²⁰ respectively. NADH-dehydrogenase and cytochrome-C-oxidase were assayed by the method of Minakami et al.²¹ and Pearl et al.²²

2.7.5. Estimation of protein content in the heart homogenate and heart mitochondrial fraction

Protein content in the heart tissue homogenate and heart mitochondrial fraction was estimated by the method of Lowry et al.²³

2.8. Transmission electron microscopic (TEM) study

Small pieces of heart were taken and rinsed in 0.1 M phosphate buffer (pH 7.2). Approximately 1 mm heart pieces were trimmed and immediately fixed into 3% ice-cold glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and kept at 4°C for 12 h. Then, tissues processing for TEM study were carried out. The grids containing sections were stained with 2% uranyl acetate and 0.2% lead acetate. Then the sections were examined under a transmission electron microscope (20 000×).

2.9. Statistical analysis

Statistical analysis was performed by one way analysis of variance followed by Duncan's Multiple Range Test (DMRT) using Software Package for the Social Science version 12.0. Results were expressed as mean±SD for six rats in each group. P-values<.05 were considered significant.

3. RESULTS

The activity of serum CK enzyme was significantly (P < .05) increased in ISO induced rats compared to control rats. Pretreatment with NAC (5 mg and 10 mg/kg) daily for a period of 14 days significantly (P < .05) lowered the activity of this enzyme in ISO induced rats compared to ISO control rats. The effect exerted by 10 mg/kg of NAC was better than 5 mg/kg. Hence, we have chosen 10 mg/kg of NAC for our further study (dose dependent study; Figure. 1).

Isoproterenol treated rats showed considerable (P < .05) increased levels of TBARS and LOOH in the heart mitochondria compared to normal control rats. Pretreatment wit NAC (10 mg/kg) daily for a period of 14 days decreased (P < .05) the levels of TBARS and LOOH in ISO treated rats compared to ISO alone treated rats (Figure. 2).

There was a considerable (P < .05) decrease in the activities of SOD, catalase, GPx, and the levels of GSH in the heart mitochondria of ISO treated rats. Pretreatment with NAC (10 mg/kg) considerably (P < .05) increased the activities of these antioxidants in ISO treated rats compared to ISO alone treated rats (Figure. 3 and Figure. 4).

The activities of ICDH, SDH, MDH, α-KDH, NADH-dehydrogenase (Figure. 5) and cytochrome-C-oxidase (Figure. 6) were lowered considerably (P < .05) in the heart mitochondria of ISO treated rats. Prior pretreatment with NAC (10 mg/kg) considerably (P < .05) enhanced the activities of these enzymes in ISO treated rats.

Figure. 7 shows the percentage scavenging effects of NAC on ABTS⁺ (total antioxidant activity). NAC scavenges in vitro ABTS radicals in a dose dependent manner. The scavenging activity of NAC on ABTS⁺ increases with increasing concentration. The scavenging activity of ABTS⁺ at various concentrations (15, 30, 45, 60 µM) of N-acetylcysteine were found to be 20.08, 39.75, 59.67, and 79.61%, respectively.

In the present study, the TEM images of the normal control rat heart mitochondria showed normal heart mitochondrial structure (Plate 1 a). Normal rats treated with NAC (10 mg/kg) heart mitochondria showed mitochondria with normal architecture of cristae (Plate 1 b). ISO treated rats showed swelling of heart mitochondria with loss of cristae, irregular shape, and size (Plate 1 c). Rats pretreated with NAC (10 mg/kg) to ISO treated rats showed mitochondria without swelling and normal myofibrils (Plate 1 d).

For all the biochemical parameters studied, pretreatment with NAC (10 mg/kg) showed considerable (P < .05) effects in ISO treated rats. Treatment with NAC (10 mg/kg) to normal control rats did not show any considerable effects on all the biochemical parameters studied and did not show any structural change in heart mitochondria.

4. DISCUSSION

Isoproterenol-induced myocardial necrosis is a well established model of MI in rats (Goyal et al 2009; Zhou et al 2008). The activities and capacities of antioxidant systems of the heart declined following ISO challenge leading to the gradual loss of prooxidant/antioxidant balance, which accumulates into oxidative damage of cardiacmyocyte. The present study demonstrated that NAC ameliorated the mitochondrial dysfunction and alterations in energy metabolism of myocardial infarcted rats. The results obtained provide substantive evidence in favor of cardioprotective potential of NAC. The ISO induced cardiac damage was indicated by elevated level of marker enzyme such as CK in the serum. On the contrary, pretreatment with NAC (5 and 10 mg/kg) to ISO treated rats significantly lowered the activity of CK in the serum. Our results confirmed that NAC has potential to reduce the elevated level of serum marker enzyme in myocardial infarcted rats that reflects its cardioprotective action.

The major source of energy for contraction comes from the oxidative metabolism of mitochondria in the myocardial cell. For this reason the function of mitochondria in myocardial infarction is of particular interest for recent research. Lipid peroxidation is an important pathogenic event in myocardial necrosis and its accumulation reflects damage of the cardiac constituents.²⁴ Increased lipid peroxidation damages both the structure and function of the heart mitochondria in ISO treated rats. Our experimental data showed that prior treatment with NAC to ISO treated rats decreased the levels of heart mitochondria lipid peroxidation products such as TBARS and LOOH. Earlier, studies have revealed the reducing and antioxidant properties of NAC by acting as a direct scavenger of free radicals such as hydroxyl, hydrogen peroxide radicals, and superoxide anion.^{25 26} Thus, oral administration of NAC protected the heart from myocardial damage by scavenging free radicals, thereby blocking the peroxidation of lipids in mitochondria and exhibited the protective effect on the myocardium cell membrane integrity.

The balance between the endogenous antioxidative defense system and ROS production determines the severity of ischemic injury. This defense system plays an important role in combating stress by neutralizing or scavenging free radicals. The SOD and CAT serves as two important antioxidant enzymes and GSH as nonenzymatic endogenous antioxidant. The ISO induction leads to decreased activities of mitochondrial antioxidants in myocardial infarction by increased generation of ROS such as superoxide and hydrogen peroxide, which in turn leads to inhibition of these enzymes. Mitochondrial GSH maintains cell viability through the regulation of mitochondrial inner membrane permeability by maintaining sulfhydryl groups in the reduced state. The GSH forms an important substrate for GPx, GST, and several other enzymes, which are also involved in free radical scavenging. Decreased activity of GPx enzyme may affect the heart mitochondria substrate oxidation, resulting in mitochondrial dysfunction.²⁷ Mitochondrial and cellular damage can be prevented by increasing intracellular GSH content. Cysteine donor compounds such as NAC supplementation is an effective method of restoring GSH content1998.²⁸ Pretreatment with NAC reduces oxidative stress by scavenging reactive oxygen species and prevents the decrease in antioxidant enzymes in ISO treated rats. In this context, NAC, a thiol containing radical scavenger and glutathione precursor, reduces oxidative stress.²⁹ Thus, increased levels of GSH prevented mitochondrial damage in NAC pretreated ISO induced rats.

In the present study, the activities of ICDH, SDH, MDH, and α-KDH were decreased in ISO treated rats. It is well documented that ISO can decrease the activities of TCA enzymes.³⁰ The ISO enhances the level of lipid peroxidation of mitochondrial phospholipid bilayer in which the respiratory chain is embedded. Thus, the marker enzymes of TCA cycle are affected by the free radicals produced by ISO. Increased lipid peroxidation in ISO treated rats decreases the levels of total and readily oxidizable lipid (ie, cardiolipin). The unavailability of cardiolipin decreases the activity of the respiratory chain marker enzymes, cytochrome-C-oxidase, and NADH dehydrogenase in isoproterenol treated rats.³¹ Pretreatment with NAD increased the activities of these mitochondrial marker enzymes in the heart mitochondria of ISO treated rats due to its free radical scavenging activity. Our results indicate that pretreatment with N-acetylcysteine substantially prevented the excessive impairment of these enzyme activities. Furthermore, this study suggests that NAC may restore energy status of the mitochondria, thereby maintaining membrane integrity. Thus, the improved multienzyme activities of these mitochondrial marker enzymes might be one of the reasons for improved cardiac mitochondrial function in ISO treated rats.

The TEM images of the heart mitochondria of normal control and NAC alone treated rats (10 mg/kg) showed mitochondria with normal architecture. The ISO treated rats showed swelling of heart mitochondria with loss of cristae, irregular shape, and size. These changes clearly revealed the cardiotoxic action of ISO. The swelling morphology is typical for mitochondria that have been subjected to ischemic and hypoxic conditions,³² which could be due to the accumulation of lipid peroxide products as a result of gutathione depletion.³³ NAC treated normal rat's heart mitochondria showed no pathological changes, which indicate that NAC does not possess any adverse effects under normal conditions. Prior treatment with NAC to ISO treated rats showed mitochondria with no swelling and normal myofibrils. Thus, NAC prevented swelling of mitochondria and protected the mitochondria from the cardiotoxic action of ISO and maintains the normal structure and function of cardiac mitochondria.

To know the underlying mechanism of NAC, we carried out an in vitro study. Generation of ABTS radical cation forms the basis of one of the spectrophotometric methods that have been applied to the total antioxidant activities of solutions of pure substances. In this study, NAC in vitro scavenges ABTS⁺ dose dependently. The highest percentage of scavenging effect of NAC at the concentration of 60 µM on ABTS⁺ was found to be 79.61 that exhibit its potent antioxidant activity. Thus, NAC protected cardiac mitochondria in ISO treated myocardial infarction.

5. CONCLUSION

The overall protective effects of NAC on cardiac mitochondria might be due to the ability of scavenging free radicals produced in excess by the induction of oxidative stress produced by ISO and decreasing lipid peroxidation, improving the activities/levels of antioxidants, and mitochondrial marker enzymes in ISO treated rats. The TEM study on heart mitochondrial structure also confirmed the biochemical findings of this study. In vitro study clearly revealed the potent antioxidant property of NAC. Restoration of cellular normalcy of mitochondria accredits the cardioprotective role of NAC on myocardial cells. Administration of NAC (10 mg/kg) to normal control rats had no effect on the measured biochemical parameters and no adverse effects were observed. Thus, NAC up to the concentration of 10 mg/kg is nontoxic. Our study may have significant impact on myocardial infarcted patients. A diet containing N-acetylcysteine may be beneficial to infarcted heart.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

REFERENCES

1. Thomes P, Rajendran M, Pasanban B, Rengasamy R. Cardioprotective activity of cladosiphon okamuranus fucoidan against isoproterenol induced myocardial infarction in rats. Phytomedicine. 2010;18:52–57.
2. Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med. 2000;29:222–230.
3. Sachdevaa J, Tanwara V, Golechhaa M, et al. Crocus sativus L. (saffron) attenuates isoproterenol-induced myocardial injury via preserving cardiac functions and strengthening antioxidant defense system. Exp Toxicol Pathol. 2010 (article in press).
4. Hsu CC, Huang CN, Hung YC, Yin MC. Five cysteine containing compounds have antioxidative activity in Balb/cS mice. J Nutr. 2004;134:149–152.
5. Demirkol O, Adams C, Ercal N. Biologically important thiols in various vegetables and fruits. J Agric Food Chem. 2004;52:8151–8154.
6. Wang X, Martindale JL, Holbrook NJ. Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem. 2000;275:39435–39443.
7. Carageorgiou H, Tzotzes V, Pantos C, Mourouzis C, Zarros A, Tsakiris S. In vivo and in vitro effects of cadmium on adult rat brain total antioxidant status, acetylcholinesterase, (Na+, K+)-ATPase and Mg2+-ATPase activities: protection by L-cysteine. Basic Clin Pharmacol Toxicol. 2004;94: 112–118.
8. Oh SH, Lima SC. A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation. Toxicol Appl Pharmacol. 2006;212:212–213.
9. Park JE, Yang JH, Yoon SJ, Lee JH, Yang ES, Park JW. Lipid peroxidationmediated cytotoxicity and DNA damage in U937 cells. Biochimie. 2002;84:1199–1205.
10. Reliene R, Fischer E, Schiestl RH. Effect of N-acetylcysteine on oxidative DNA damage and the frequency of DNA deletions in atm-deficient mice. Cancer Res. 2004;64:5148–5153.
11. Takasawa M, Hayakawa M, Sugiyama S, Hattori K, Ito T, Ozawa T. Ageassociated damage in mitochondrial function in rat hearts. Exp Gerontol. 1993;28:269–280.
12. Fraga CG, Leibovitz BE, Tappel AL. Lipid peroxidation measured as thiobarbituric acid reactive substances in tissue slices; characterization and comparison with homogenate and microsomes. Free Rad Biol Med. 1988;4:155–161.
13. Jiang ZY, Hunt JV, Wolff SP. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem. 1992;202:384–389.
14. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys. 1984;21:130–132.
15. Sinha AK. Colorimetric assay of catalase. Anal Biochem. 1972;47:389–394.
16. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70–77.
17. King J. Isocitrate dehydrogenase. In: King JC, Van D, eds. Practical Clinical Enzymology. London: Nostrand Co.; 1965:363.
18. Slater EC, Borner WD. The effect of fluoride on the succinic oxidase system. Biochem J. 1952;52:185–196.
19. Mehler AH, Kornberg A, Grisolia S, Ochoa S. The enzymatic mechanisms of oxidation reductions between malate or isocitrate or pyruvate. J Biol Chem. 1948;174:961–977.
20. Reed LJ, Mukherjee RB. a-Ketoglutarate dehydrogenase complex from Escherichia coli. In: Lowenstein JM, ed. Methods in Enzymology. London: Academic Press; 1969:53–61.
21. Minakami S, Ringler RL, Singer TP. Studies on the respiratory chainlinked dihydrodiphosphopyridine nucleotide dehydrogenase. I. Assay of the enzyme in particulate and in soluble preparations. J Biol Chem. 1962;237:569–576.
22. Pearl W, Cascarano FJ, Zweifach BW. Micro determination of cytochrome oxidase in rat tissues by the oxidation on N-phenyl-p-phenylene diamine or ascorbic acid. J Histochem Cytochem. 2002;11:102–104.
23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–275.
24. Devika PT, Stanely Mainzen Prince P. Epigallocatechin-gallate (EGCG) prevents mitochondrial damage in isoproterenol-induced cardiac toxicity in albino Wistar rats: a transmission electron microscopic and in vitro study. Pharmacol Res. 2008;57:351–357.
25. Aruoma OI, Halliwell B. The antioxidant activity of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide and hypochlorous acid. Free Radic Biol Med. 1989;6:593–597.
26. Benrahmoune M, Therond P, Abedinzadeh Z. The reaction of superoxide radical with N-acetylcysteine. Free Radic Biol Med. 2000;29:775–782.
27. Capetanaki Y. Desmin cytoskeleton: a potential regulator of muscle mitochondrial behavior and function. Trends Cardiovasc Med. 2002;12: 339–348.
28. Roy S, Packer L. Redox regulation of cell functions by a-lipoate: biochemical and molecular aspects. Biofactors. 1998;8:17–21.
29. Pocernich CB, Cardin AL, Racine CL, Lauderback CM, Butterfield DA. Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurogenerative disease. Neurochem Intl. 2001;39:141–149.
30. Suchalatha S, Srinivasan P, Devi CS. Effect of T. chebula on mitochondrial alterations in experimental myocardial injury. Chem Biol Interac. 2007;169:145–153.
31. Nicolay K, Vander Neut R, Fok JJ, De Kruijff B. Effects of adriamycin on lipid polymorphism in cardiolipin-containing model and mitochondrial membranes. Biochim Biophys Acta. 1985;819:55–65.
32. Chagoya De Sanchez V, Munoz RH, Barrera F, Yanez L, Suarez J. Sequential changes of energy metabolism and mitochondrial function in myocardial infarction induced by isoproterenol in rats: a long term and integrative study. Can J Physiol Pharmacol. 1997;75:1300–1311.
33. Hegstad AD, Ytrehus K, Myklebust R, Jorgensen L. Ultrastructural changes in the myocardial myocytic mitochondria: crucial step in the development of oxygen radical induced damage in isolated rat hearts. Basic Res Cardiol. 1994;89:128–138.
34. Echtay KS, Murphy MP, Smith RA, Talbot DA, Brand MD. Superoxide activates mitochondrial uncoupling protein-2 from the matrix side: studies using targeted antioxidants. J Biol Chem. 2002;277:47129–47135.
35. Suchalatha S, Shyamala Devi CS. Effect of Arogh, a polyherbal formulation on the marker enzymes in isoproterenol induced myocardial injury. Ind J Clin Biochem. 2004;19:184–189.

INTRODUCTION

Extensive research and clinical outcomes analysis have demonstrated the increased risk of coronary heart disease in patients with dyslipidemia and hyperlipidemia. Elevated low density lipoprotein cholesterol (LDL-c) has specifically been shown to increase the risk of coronary heart disease (CHD) and many recent trials have shown a significant improvement in mortality with LDL-c lowering therapy.

In 2001 the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults released its most recent guidelines. The APT III (Adult Treatment Panel III) report provides a framework for evaluating and treating dyslipidemia in adult patients. The guidelines use the Framingham risk score (smoking, hypertension, low HDL, family history of premature CHD, and age) along with the presence of CHD or its equivalent to help stratify LDL-c goals. Diabetes as well as atherosclerosis in noncoronary beds are considered CHD equivalents while a high density lipoprotein (HDL) >60 is considered a “negative” or protective risk factor.

The highest risk category consists of those with known CHD, diabetes, or two or more risk factors conferring a Framingham 10-year risk of 20% for major coronary events. The original ATP-III guidelines recommended an LDL-c goal of less than 100 mg/dl; however, a 2004 update added a consideration for an optional LDL-c goal of <70 mg/dl for those at very high risk of future CHD events.

Patients with a 10-year risk of 10–20% and multiple risk factors should be treated with a goal of LDL-c <130 mg/dl with the option of <100 mg/dl (also added with the 2004 update). The lowest risk category consists of zero to one risk factors with a 10-year risk of <10%. The current guidelines specify a goal of LDL-c <160 mg/dl that was left unchanged with the 2004 update.

The treatment of dyslipidemia and hyperlipidemia is a combined strategy consisting of lifestyle modification and the addition of lipid lowering medications if necessary. Due to the serious health risks associated with lipid disorders and the difficulty in treating some patients due to medication side effects and complex lipid disorders, multiple medications are not infrequently required. Even with our current knowledge of the significant cardiovascular risk an elevated LDL-c level imparts, many patients are not reaching their goal LDL-c reduction. A recent review of more than 10,000 patients from a large cardiology subspecialty practice found that only 79% of patients were able to achieve a goal of LDL-c <100 mg/dl and only 35% had achieved a goal of <70 mg/dl. 

HMG-COA REDUCTASE INHIBITORS (“STATINS”)

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) category of medications reduces hepatic cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. The statin medications are very effective in reducing LDL-c as well as modestly increasing HDL, have a low rate of side effects, and have become first-line agents in the treatment of dyslipidemia as evidenced by atorvastatin's rank as the number one best selling prescription drug worldwide.

This class contains simvastatin, atorvastatin, lovastatin, pravastatin, fluvastatin, and rosuvastatin. Currently, lovastatin, pravastatin, and simvastatin are available in generic form in the United States. These medications have been shown to cause a significant reduction in total mortality in both primary and secondary prevention. The Scandinavian Simvastatin Survival Study in 1994 was the first to show this reduction in secondary prevention, decreasing total mortality among patients with coronary artery disease by 30% in those who were treated with simvastatin as compared to placebo. This was followed by the West of Scotland Coronary Prevention Study (WOSCOPS) in 1995 which showed a 22% reduction in all-cause mortality over a period of 5 years in those receiving pravastatin. Several other studies in both primary and secondary prevention have since been completed, solidifying the benefits of this class of medications. No other class of lipid lowering medications has the wealth of evidence-based outcome benefits that has been demonstrated with the statins.

Each statin medication has a different starting and maximum dose and the LDL-c lowering potential varies as well (). Atorvastatin and rosuvastatin appear to be the two most potent statins and are able to reduce LDL-c by up to 60 mg/dl and 63 mg/dl, respectively, at maximum doses. On average the HMG-CoA reductase inhibitors reduce LDL-c by 20–60%, total cholesterol by 15–60%, and increase HDL by 10–25%.

Although controversial, recent research has focused on possible benefits statins may provide beyond their potent ability to lower LDL-C. Reduction of inflammation, improvement of endothelial function, and stabilization of platelets have been suggested as possible mechanisms although data is sparse. The JUPITER trial, which began in 2003, evaluated 17,802 healthy men with low or normal LDL-c levels (<130 mg/dl) and elevated high sensitivity C-reactive protein (hsCRP) levels who were assigned to either rosuvastatin 20 mg daily or placebo. In addition to reducing LDL-c by 50%, hsCRP was reduced by 37%. The study demonstrated a reduction in the combined endpoint of myocardial infarction, stroke, or death from cardiovascular causes by 53%. While received with interest, many feel that the reduction in LDL-c may in large part account for the reduction in morbidity.

Overall the statins are well tolerated and safe, however there are two well-known side effects. Elevations in liver function tests have been reported with all HMG-CoA reductase inhibitors and tend to be mild and transient. Current guidelines recommend initial liver function tests, again at 12 weeks after starting therapy and then annually. Therapy should be stopped if a greater than threefold increase in liver function tests occurs. These elevated transaminase levels often return to normal within 2 weeks, at which time a lower dose or a different HMG-CoA reductase inhibitor can be substituted.

Myalgias and Myopathy are the other well-known side effects of statin medications. Myopathy has been described for all medications of this class and although rare can be a potentially serious adverse reaction. This often presents with muscle pain, tenderness, or weakness and is diagnosed when the creatinine kinase level is found to be greater than 10 times the upper limit of normal. Due to the risk of acute renal failure the medication should be stopped at once. As with liver function tests, the creatinine kinase level often returns to normal within a few weeks. Although this is a class side effect, patients are often able to tolerate a different statin and can be rechallenged with fluvastatin or alternate day dosing of rosuvastatin.

Niacin (Nicotinic Acid)

Keywords

dyslipidemia, coronary heart disease, treatment, lipoproteins, coronary artery disease, statins

References

The prevalence of diabetes mellitus (DM) is increasing all over the world.¹ The number of cases is expected to increase from 171 million in 2000 to 366 million in 2030.² According to the latest data from the 2011 National DM Fact Sheet,³ in the United States, 25.8 million children and adults (8.3% of the US population) have DM. In 2004, DM was considered to be the cause of 44% of new cases of kidney failure in the United States and to be the leading cause for kidney failure. It is also considered the leading cause of new cases of blindness in adults aged 20–74 years. The estimated DM cost in the United States in 2007 was $174 billion.³ The tremendous increase in the prevalence of DM has been linked to multiple factors, which include the obesity epidemic, sedentary lifestyle, population aging, diabetes awareness, early diagnosis, and environmental factors. Several observational studies have linked hypovitaminosis D with the incidence of DM.^4–7

Since the industrial revolution, hypovitaminosis D has become a major health concern all over the world.^8,9 This can be related to insufficient exposure to ultraviolet (UV) radiation from sunlight due to a variety of socioeconomic and cultural-ethnic conditions such as the elderly living in senior citizen housing, individuals with a physical handicap, women covering their bodies from head to toes, individuals with black skin preventing sunlight penetration, and population living at high latitude. Several studies describe hypovitaminosis D as an unrecognized and underestimated health concern in both undeveloped and developing countries^8,10 because many apparently healthy individuals have hypovitaminosis D.

The definition of hypovitaminosis D is still an area of debate since a clearly established cut-point value for vitamin D adequacy has not yet been established. Bone health is the only objective outcome that can be used as a reliable indicator for vitamin D adequacy since the extra-skeletal outcomes of vitamin D supplementation are still inconclusive. Some studies estimate the cut-point value of vitamin D adequacy for bone health to be 75 nmol/L (30 ng/mL) while others give the value of 50 nmol/L (20 ng/mL).^11,12 The 2010 Institute of Medicine report on dietary reference intake for vitamin D states the need to establish cut-point values for vitamin D deficiency and insufficiency, since there is overestimation of vitamin D deficiency prevalence in North America due to low cut-point values and non-standardized methods for measuring vitamin D levels.¹³

In this review, current knowledge of vitamin D, parathyroid hormone (PTH), and parathyroid hormone-related peptide (PTHrP) physiology will be reviewed. Hypotheses and possible explanations about the special interaction between vitamin D, PTH, and PTHrP in diabetic subjects and how this information can provide a potential therapeutic revolution in DM management will be proposed.

SYNTHESIS AND METABOLISM OF VITAMIN D

Vitamin D is a secosteroid prohormone with multiple physiological functions. Vitamin D derives from two sources, nutritional and endogenous. Nutritional sources include vitamin D from plant origin (vitamin D2 or ergocalciferol) and vitamin D from animal origin (vitamin D3 or cholecalciferol). Endogenous D3 is synthesized in the skin under the effects of UV radiation from sunlight. Skin synthesis of D3 is considered the main and the most reliable source of vitamin D. In the liver, a P450 enzyme, 25-hydroxylase (CYP27), hydroxylates vitamin D3 and D2 at position 25 resulting in 25OHD2 and 25OHD3 (both referred to here as 25OHD). Over 88% of 25OHD2 and 25OHD3 are tightly bound to the vitamin D-binding protein, which can be considered a storage form, and the remaining 12% is loosely bound to serum albumin.¹⁴ Since hepatic CYP27 is constitutively active, the serum concentration of 25OHD is a clinical indicator of vitamin D sufficiency.

After the first hydroxylation, 25OHD can be stored bound to its binding protein or sequestered in fat tissues. A small fraction of 25OHD undergoes a second hydroxylation step by 1α-hydroxylase (CYP1-α) in the renal proximal tubules to produce the active forms 1,25(OH)2D2 and 1,25(OH)2D3 (both referred to here as 1,25(OH)2D). The renal CYP1-α, unlike the hepatic CYP27, is tightly regulated by multiple hormonal inputs, which include PTH and fibroblast growth factor (FGF) 23, and by serum calcium and phosphorus ion concentrations.

It was originally thought that the active form of vitamin D, 1,25(OH)2D, is only produced by the proximal renal tubules and that all the vitamin D target cells utilize circulating 1,25(OH)2D. However, later studies showed that 25OHD is required by some target cells, which express their own 1α-hydroxylase for hydroxylation and activation of 25OHD intracellularly. These interesting observations were seen in pancreatic beta cells, prostate, vascular smooth muscles, keratinocyte, bone, placenta, breast, prostate, colon, macrophages, T-lymphocytes, dendritic cells, adrenal medulla, and parathyroid gland.^15–22

These findings have provided us with a tremendous improvement in our understanding of vitamin D autocrine, paracrine, and intracrine physiological actions and their important role in the pathophysiology of many diseases.

MECHANISM OF ACTION OF VITAMIN D

The classical genomic effect of 1,25(OH)2D, mediated by the vitamin D receptor (VDR), is considered to be its main mechanism of action. Vitamin D target cells express VDR, a member of the steroid, thyroid, and retinoid hormones superfamily.²³ Interestingly, VDR is expressed in a large number of cells, most of which are not involved in calcium and phosphorus metabolism. VDR requires a cofactor, retinoid X receptor (RXR), for expression of its biological activity. The binding of 1,25(OH)2D to VDR induces recruitment of RXR and the formation of active VDR-RXR heterodimers that can bind to specific DNA sequences in the promoter regions of target genes, the vitamin D responsive element (VDRE). Ultimately, these interactions end by a stimulation of histone acetylase and the opening of a DNA promoter to start transcription of the target gene.²⁴ Final response to 1,25(OH)2D depends on the target gene. 1,25(OH)2D upregulates transcription of some genes, such as the calcium-binding protein gene in intestinal epithelial cells, the osteocalcin, osteopontin, and FGF23 genes in osteoblasts, and the integrin gene in macrophages, ^25–27 and downregulates others such as PTH, collagen, c-myc, 25(OH)D3 1α-hydroxylase, IL-2, vitamin D 25 hydroxylase, and PTHrP (Table 1).^26,28–32

The last three decades have witnessed a tremendous renaissance of interest in the physiological actions of vitamin D. This is based on the observation that VDR is not limited to osteoblast and intestinal epithelial cells as was thought before. VDR has been shown to be expressed in the brain, cardiovascular system, pancreas, prostate, activated T and B lymphocyte, stomach, colon, skin, placenta, parathyroid gland, and breast.^{19,21,25,33–35} Thus, vitamin D has been proposed to have essential roles in many physiological actions of which we were previously unaware, and therefore vitamin D has become an interesting topic for researchers. Many non-traditional (non-classical) physiological actions of vitamin D have emerged, and many observational studies have supported a role of vitamin D deficiency in the incidence of cardiovascular diseases, DM I and II, hypertension, hyperlipidemia, obesity, and colon, breast, and prostate malignancies.^{4,6,7,36–42} Since most of these studies are observational, prospective, randomized, double blind, and well-controlled studies are still required to establish a causality relationship between vitamin D deficiency and the incidence of these diseases.

The serum level of 1,25(OH)2D3 is regulated by PTH, PTHrP, and 1,25(OH)2D itself. Both PTH and PTHrP act as stimulators for the renal 1α-hydroxylase enzyme, whereas 1,25(OH)2D acts as a negative feedback inhibitor of 1α-hydroxylase. Transcription of the PTH and PTHrP genes is inhibited by 1,25(OH)2D3. In order to understand these interactions and the difference in diabetic subjects from non-diabetic subjects, we shall review the physiology of PTHrP.

PTHrP IS A PHYSIOLOGICAL HORMONE

PTHrP is a preprohormone, discovered in 1987 as the factor responsible for humoral hypercalcemia of malignancy.⁴³ The 13 N-terminal amino acids of PTHrP are identical to their corresponding sequences in PTH (Fig. 1); this explains their hypercalcemic effects through activation of one single receptor, the PTH/PTHrP receptor (or PTHR1). Amino acids from 35 to 81 are very different in PTH and PTHrP. PTH and PTHrP are products of different genes; the PTH gene is located on chromosome 11 whereas the PTHrP gene is located on chromosome 12.⁴⁴ The PTHrP mRNA undergoes multiple splicing activities to produce three forms of PTHrP: (1–139), (1–141), and (1–173). Before their secretion, PTHrP undergo posttranslational modifications and endoproteolytic cleavages at different sites of their polypeptide chains to produce different mature and active fragments of PTHrP.

PTHrP has been the subject of extensive studies that have explored its previously unknown endocrine, autocrine, paracrine, and intracrine physiological actions. Cells and organs that express PTHrP and PTH/PTHrP receptors include osteoblasts, chondrocytes, fibroblasts, keratinocytes, renal tubular cells, pancreatic beta cells, lymphocytes, smooth muscle cells, mammary gland, cardiovascular system, and many others.^45–49 These findings emphasize the physiological importance of PTHrP. Further, different fragments of PTHrP may have different receptors and different physiological functions. Several studies were conducted to define the exact cleavage sites of PTHrP preprohormone, different mechanisms, and physiological actions of each fragment of PTHrP (Table 2).^46,50–67

PTHrP shares many mechanisms of action with PTH with respect to mineral ion homeostasis. The N-terminal fragment of PTHrP(1–36) conducts these actions through the shared classical receptor (PTH1R) with efficiency almost equal to that of PTH. Early studies were focused on the N-terminal fragment of PTHrP. However, the recent revolution in PTHrP knowledge is related to newly explored mechanisms of action for its N-terminal, midregional, and C-terminal fragments. These fragments can act on PTH1R or a novel receptor specific for PTHrP.⁶⁸ After the description of a nuclear localization sequence in PTHrP(88–106), focus was shifted to the midregional and C-terminal fragments of PTHrP.^49,69 These fragments may give PTHrP its unique physiological actions in addition to its shared actions with PTH. One of the interesting physiological actions of PTHrP is related to its antiapoptotic activities.⁶⁹

VITAMIN D, PTH, AND PTHrP INTERACTIONS IN HEALTHY AND DIABETIC SUBJECTS

In healthy individuals, serum PTHrP levels range from undetectable to very low. PTHrP acts locally as a regulator of cell proliferation, growth, and apoptosis. On the other hand, PTH acts mainly as an endocrine hormone that is secreted by the parathyroid gland and travels in the blood to its target tissues, mainly bone and kidney. PTH is secreted in response to hypocalcemia or low 1,25(OH)2D. PTH acts on kidney and bone to increase serum concentrations of calcium and 1,25(OH)2D and to decrease serum phosphate levels. PTH increases Ca⁺² reabsorption in distal convoluted tubules. In the proximal convoluted tubules, PTH increases the expression of 1α-hydroxylase, leading to increased 1,25(OH)2D, and suppresses the transcription of Na-PO4 co-transporter, thereby inhibiting phosphate reabsorption. In turn, Ca⁺² and 1,25(OH)2D exert a negative feedback on PTH secretion. 1,25(OH)2D downregulates PTH transcription by binding to a negative VDRE on the PTH gene.⁷⁰ These interactions explain the mechanism of secondary hyperparathyroidism in hypovitaminosis D, hypocalcemia, and chronic kidney diseases.

Studies have shown that synthesized N-terminal fragment of PTHrP has many actions that are similar to that of PTH. Both stimulate cAMP production in bone and kidney and result in hypercalcemia.^48,55 Also, they stimulate the production of 1,25(OH)2D. As a negative feedback inhibition, the PTHrP gene has VDRE, which responds to 1,25(OH)2D3 by inhibiting transcription of the PTHrP gene.⁷¹

However, these interactions are found to be different in diabetic subjects compared to non-diabetic subjects. Many observational studies have shown that diabetic patients have low vitamin D and normal Ca⁺² levels in the blood.^6,72 At the same time, they have low or inappropriately normal PTH and higher PTHrP than non-diabetic subjects.^5,72–74

Thus, these observations have raised many questions regarding the interaction between vitamin D, PTH, and PTHrP in diabetic patients. What does an increased PTHrP level instead of PTH in diabetic patients with hypovitaminosis D mean? What is the main source of PTHrP in these patients? And, why is the level of 1,25(OH)2D low despite the high level of PTHrP?

We can link these observations to other studies that have shown that PTHrP is secreted by pancreatic beta cells that also have receptors for PTHrP.^59,75 Other studies have shown that PTHrP increases pancreatic islets’ mass, induces expression of insulin, and causes hypoglycemia in transgenic mice in which PTHrP are overexpressed in their pancreatic islets.^58,76,77

The progression in pancreatic islets mass seems to be related to repression of apoptosis rather than induction of proliferation.⁷⁸ In the study by Legakis and Mantouridis, which was conducted on 28 type II diabetic patients and 28 healthy subjects, a strong positive relation between PTHrP levels and fasting blood glucose levels and a positive relation between insulin and PTHrP levels in healthy subjects was shown.⁷⁴ Thus, PTHrP seems to be a potential therapeutic tool for management and prevention of DM, similar to its role in the treatment of postmenopausal osteoporosis, which is currently under clinical trials.⁷⁹ In order to reach that promise, many questions regarding vitamin D, PTH, and PTHrP interactions in type II DM need to be answered to explain and correlate all these in vitro, animal, and human studies in a logical way.

IS HYPOVITAMINOSIS D A RISK FOR, OR A CONSEQUENCE OF, DM?

Several cross-sectional studies have demonstrated a correlation between hypovitaminosis D and type I and II DM.^5,6 Studies on mice lacking VDR showed that 1,25(OH)2D3 plays an important role in synthesis and secretion of insulin and reduction of insulin resistance in peripheral tissue.^80,81 VDRE was identified in promoter of insulin receptor gene in human.²⁵ When vitamin D level is normalized, insulin secretion and sensitivity improved.⁸²

In order to see if hypovitaminosis D increases the risk of DM or if it is just a consequence of DM, a birth cohort study was done in London in which 10 366 children were assessed for their vitamin D supplementation and suspicion of rickets during the first year of life. Then, they were followed up for 31 years for type I DM incidence. Children who had a suspicion of rickets during their first year had a higher incidence of type I DM with relative risk (RR) of 3 compared to those without such a suspicion. However, children who took the recommended dose of vitamin D (2000 IU/d) regularly during the first year of life had a RR of 0.22 compared to those who received less than the recommended dose.³⁷ These results can be related directly to vitamin D or indirectly by increasing Ca⁺² mobilization into beta cells. Other prospective studies have shown that hypovitaminosis D increases the risk of DM incidence rather than just being a consequence of it.^4,7

However, there is no change in carbohydrate metabolism in non-diabetic subjects with low vitamin D level when they are given vitamin D.⁸³ Thus, the effect of vitamin D on glucose control might be related to factors that are specific for diabetics. Body weight can be one of these factors since it is negatively correlated with vitamin D level, and repletion of vitamin D reduces the body weight and subsequently increases insulin sensitivity.⁸⁴

WHAT STIMULATES SYNTHESIS OF PTHrP INSTEAD OF PTH IN DIABETIC PATIENTS WITH HYPOVITAMINOSIS D, AND WHAT IS THE MAIN SOURCE OF PTHrP IN THESE PATIENTS?

High blood glucose can be a stimulator for PTHrP. Beta cells can be stimulated by hyperglycemia to secrete both insulin and PTHrP in secretory packages.⁸⁵ This can be one of the sources of high PTHrP in diabetic patients, but it is certainly not the only source. PTHrP was undetectable in portal plasma of transgenic mice that overexpressed PTHrP in islet cells in the study by Vasavada et al.⁷⁶ Thus, it seems to be an auto/paracrine action rather than an endocrine action. No study has been conducted to show the direct effect of glucose on PTHrP secretion or synthesis in cells other than beta cells.

A second explanation for high PTHrP in diabetic patients is that many diabetic patients have an element of nephropathy as a complication of DM. In the study by Burtis et al.,⁸⁶ all 15 patients with chronic renal failure had a high level of PTHrP(109–138) and 14 of them had a normal level of PTHrP(1–74). This can be due to a defect in renal excretion of C-terminal PTHrP(109–138). The study by Ishida et al.⁷² does not support the possibility of decreased renal excretion of PTHrP fragment. The PTHrP level was measured in serum and urine of non-insulin-dependent diabetes mellitus (NIDDM) patients by radioimmunoassay using the C-terminal antibody. Both serum level and urinary excretion of PTHrP were found to be high in NIDDM patients in comparison to control subjects. Thus, it is a matter of increased production rather than decreased excretion of PTHrP. That study also showed that urinary excretion of PTHrP level increases gradually with the severity of diabetic nephropathy, and it was higher in those with microalbuminurea or macroproteinurea.⁷² Therefore, the possibility that hyperglycemia stimulates PTHrP production looks more reasonable than the possibility of decreased excretion of PTHrP(109–138) as an explanation of the high PTHrP level in diabetic patients.

A third explanation is that DM is commonly associated with hypomagnesemia that would affect PTH secretion from the parathyroid gland.⁸⁷ Thus, PTHrP increases in diabetic patients as a compensatory action to maintain mineral homeostasis. A study was conducted on 23 children with type I DM and hypomagnesemia and 20 healthy children as control. The study showed lower levels of intact PTH, 1,25(OH)2D3, and total and ionized Ca⁺² in diabetic children with hypomagnesemia compared to controls. After hypomagnesemia was corrected, PTH, 1,25(OH)2D3, and ionized and total Ca⁺² became normal. More interestingly, when those diabetic children with corrected magnesium level were exposed to a low calcium diet, they showed a normal response of increment of PTH and 1,25(OH)2D3 in a manner that was not significantly different from control.⁸⁸ Unfortunately, the level of PTHrP was not measured in this study. It would be helpful if we knew what PTHrP levels were for both groups before and after the correction of magnesium level in the diabetic children.

The last explanation for a high PTHrP level instead of PTH in diabetic patients with hypovitaminosis D is taken from the study of Li et al.,⁸⁹ which showed that 1,25(OH)2D3 suppresses the transcription of renin. Thus, a low level of 1,25(OH)2D3 in the blood stimulates the transcription of renin, which stimulates angiotensin II production. Angiotensin II upregulates PTHrP mRNA and increases PTHrP production.⁹⁰ This seems to be another logical explanation for the high PTHrP level. However, this last explanation is not specific for diabetics. Why would not the scenario be similar for non-diabetic subjects?

WHY DOES 1,25(OH)2D3 LEVEL REMAIN LOW DESPITE A HIGH LEVEL OF PTHrP IN DIABETIC PATIENTS?

A possible explanation can be taken from the study by Sebastian et al.⁹¹ on rats. Rats were divided into four groups: group 1 with sham parathyroidectomy and sham nephrectomy, group 2 with parathyroidectomy (PTX), group 3 with nephrectomy (NPX), and group 4 with both PTX and NPX. PTH1R mRNA expression in bone and kidney were measured in each group before and after treatment with vehicle (placebo), PTH(1–34), and PTH(7–34). Interestingly, NPX rats showed a highly significant reduction in PTH1R mRNA in bone and kidney compared to sham. Daily PTH(1–34) injections increased PTH1R mRNA. The reduction in PTH1R mRNA was more significant in NPX than in PTX rats.⁹¹ From this, we can presume a significant role of kidney in expression of PTH1R, at least in bone and kidney; 1,25(OH)2D3 can contribute to this activity. What supports 1,25(OH)2D3 more than any other factor as the contributor in PTH1R mRNA expression is the increment of PTH1R mRNA expression by daily injection of PTH(1–34). Unfortunately, neither 1,25(OH)2D3 nor PTHrP were measured in this study, but mostly 1,25(OH)2D3 would be expected to be low in NPX and PTX. This is a logical hypothesis because most if not all of the cells that express PTH1R also express VDR.

Going back to our question with all these observations in mind, we can say that 1,25(OH)2D3 and PTHrP have permissive actions in which 1,25(OH)2D3 is necessary for PTH1R expression in different cells. Thus, in cases of hypovitaminosis D, there would be resistance to PTH and PTHrP due to reduction in their classical receptors PTH1R in target tissues. Target tissues of PTHrP include cells that express 25(OH)D 1α-hydroxylase enzyme and pancreatic beta cells. Improvement of insulin secretion and peripheral tissue sensitivity after 1,25(OH)2D3 supplementation can be related to increased sensitivity of target tissues to PTHrP after the increment in PTH1R. The results in the study by Sneddon et al.⁹² support this hypothesis. It showed that 1,25(OH)2D3 upregulates PTH1R gene expression in distal convoluted tubules in kidney through binding to VDRE.⁹² However, the study by Errazahi et al.⁴⁵ showed that 1,25(OH)2D3 deficiency increases PTH1R expression in kidney and keratinocyte but not in fibroblast. This is not consistent with other observations that have been mentioned. It seems that upregulation and downregulation of PTH1R in response to 1,25(OH)2D3 is cell type dependent. Some cells respond to 1,25(OH)2D3 by upregulating PTH1R while others respond by downregulating them.

Another explanation of the low 1,25(OH)2D3 level despite high PTHrP is that diabetic patients may have an elevated fragment of PTHrP other than PTHrP(1–36), which is known as a stimulant of 25(OH)D 1α-hydroxylase. This fragment can be an inhibitor of 25(OH)D3 1α-hydroxylase. This may be midregional or C-terminal PTHrP that acts on specific receptors other than PTH1R. PTH(7–84) has been shown as an inhibitor of PTH(1–34)-induced 1,25(OH)2D3 production in murine renal tubules.⁹³ It can be a similar action for PTHrP(7–84) or other fragments. Osteostatin is a synthesized PTHrP(107–139) that has been shown to be an inhibitor for bone osteoclast activity.⁶⁶ Thus, the possibility of fragments other than PTHrP(1–36) as an inhibitor of 1,25(OH)2D3 production in diabetic patients can be supported by similar observations in humoral hypercalcemia of malignancy, which is usually associated with hypovitaminosis D.

The last explanation is taken from the study by Marcinkowski et al.⁹⁴ on diabetic rats that showed that hyperglycemia was associated with high intracellular Ca⁺² in proximal tubule cells, and this was associated with downregulation of mRNA of PTH and PTHrP receptors. These results were reversed by using a Ca⁺² channel blocker such as amlodipine.⁹⁴

PTHrP FROM A PARANEOPLASTIC PROTEIN TO A POTENTIAL DRUG FOR TYPE II DM

PTHrP was known as a paraneoplastic protein only. Based on the above experimental studies, PTHrP is a physiological hormone that plays a promising role in type II DM management. Observations from previous human studies support this promising role of PTHrP. The explanations postulated above need to be investigated through specifically designed experiments. A normal level of 1,25(OH)2D3 in the blood seems to be necessary for PTHrP to conduct its actions through upregulation of PTHrP receptors. PTHrP(1–36) is currently in clinical trials for treatment of osteoporosis.⁷⁹ This would shorten the time for PTHrP to be approved for other indications such as treatment or prevention for type II DM. More exploration of the exact mechanism of action of PTHrP in promoting beta cells’ functions and increasing peripheral insulin sensitivity is needed to reach that point.

Knowing that PTHrP and 25(OH)2D3 have broad effects on many tissues in the body necessitates targeting the delivery of these molecules to the pancreatic beta cells and tissues expressing the insulin receptors. Targeted drug delivery is one of the main applications of nanotechnology in medicine (nanomedicine). Nanotechnology is the study of materials (nanoparticles) at the nanometer level, equal to 10⁻⁹ m. These extremely small particles have the unique ability to cross cell membranes and interact with molecules at the cellular level. Nanoparticles can be designed to carry a specific drug and to target a specific organ in the body (targeted drug delivery). This principle helps to maximize the desired effect of a drug on its targeted cells and to reduce the undesired side effects on other cells. A nanoparticle that carries PTHrP and 1,25(OH)2D3 molecules could possibly be formulated to target the pancreatic beta cells. Other potential applications of nanotechnology in diabetes research include nanoparticles that can be administered orally and nanoparticles that can act as a subcutaneous glucose sensor along with an insulin pump that delivers insulin based on the readings from these sensors. For more information on nanotechnology applications in DM, refer to Samuel et al.⁹⁵

CONCLUSION

Despite the tremendous advances in medicine over the last decades, the incidence of DM is still increasing at an alarming rate. Currently, new promising approaches for treatment and prevention of DM are under investigation. Recent studies have shown that vitamin D and PTHrP can act in cooperation with each other to play major roles in blood sugar control. 1,25(OH)2D3 seems to have a permissive effect on PTHrP action through the upregulation of PTHrP receptors. Through these receptors, PTHrP conducts its physiological actions, which includes stimulation of insulin production, increase of peripheral sensitivity to insulin, reduction of beta cells apoptosis, and stimulation of 25(OH)D3 1α-hydroxylase. Many questions are waiting for their answers regarding the cooperation between 1,25(OH)2D3 and PTHrP and their special interaction in diabetic patients. These answers would hasten progress toward the hope of treatment and prevention of DM. Double blind and well-randomized controlled trials are required to clarify the relation between hypovitaminosis D and DM type II in humans. There is a need for human studies that are designed to demonstrate the efficiency of PTHrP(1–36) as a drug in blood sugar control similar to its anabolic role in osteoporosis treatment. If researchers direct their efforts toward this approach, we can reach our hope of reducing the current alarming incidence rates and prevalence of type II DM.

Disclosure: The authors declare no financial or other conflicts of interest.

REFERENCES

1. Adeghate E, Schattner P, Dunn E. An update on the etiology and epidemiology of diabetes mellitus. Ann NY Acad Sci. 2006;1084:1–29.
2. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 2004;27(5):1047–1053.
3. Centers for Disease Control. National Diabetes Fact Sheet, 2011. http:// www.cdc.gov/diabetes/pubs/pdf/ndfs_2011.pdf. Accessed April 24, 2012.
4. Mattila C, Knekt P, Mannisto S, et al. Serum 25-hydroxyvitamin D concentration and subsequent risk of type 2 diabetes. Diabetes Care. 2007;30(10):2569–2570.
5. Janner M, Ballinari P, Mullis PE, Flück CE. High prevalence of vitamin D deficiency in children and adolescents with type 1 diabetes. Swiss Med Wkly. 2010;140:w13091.
6. Scragg R, Sowers M, Bell C. Serum 25-hydroxyvitamin D, diabetes, and ethnicity in the Third National Health and Nutrition Examination Survey. Diabetes Care. 2004;27(12):2813–2818.
7. Knekt P, Laaksonen M, Mattila C, et al. Serum vitamin D and subsequent occurrence of type 2 diabetes. Epidemiology. 2008;19(5):666–671.
8. Arabi A, El Rassi R, El-Hajj Fuleihan G. Hypovitaminosis D in developing countries-prevalence, risk factors and outcomes. Nat Rev Endocrinol. 2010;6(10):550–561.
9. Holick MF. McCollum Award Lecture, 1994: vitamin D—new horizons for the 21st century. Am J Clin Nutr. 1994;60(4):619–630.
10. Mark S. Vitamin D status and recommendations to improve vitamin D status in Canadian youth. Appl Physiol Nutr Metab. 2010;35(5):718.
11. Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am J Clin Nutr. 2008;87(4):1080S–1086.
12. Lips P, Bouillon R, van Schoor NM, et al. Reducing fracture risk with calcium and vitamin D. Clin Endocrinol (Oxf). 2010;73(3):277–285.
13. Committee to Review Dietary Reference Intakes for Vitamin D and Calcium, Food and Nutrition Board. Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: Institute of Medicine of the National Academies; 2010. http://www.iom.edu/Reports/2010/Dietary-Reference- Intakes-for-Calcium-and-Vitamin-D.aspx. Accessed April 24, 2012.
14. Speeckaert M, Huang G, Delanghe JR, Taes YE. Biological and clinical aspects of the vitamin D binding protein (Gc-globulin) and its polymorphism. Clin Chim Acta. 2006;372(1–2):33–42.
15. Holick MF. Vitamin D: a millenium perspective. J Cell Biochem. 2003;88(2):296–307.
16. Schwartz GG, Whitlatch LW, Chen TC, Lokeshwar BL, Holick MF. Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25- hydroxyvitamin D3. Cancer Epidemiol Biomarkers Prev. 1998;7(5):391–395.
17. Somjen D, Weisman Y, Kohen F, et al. 25-hydroxyvitamin D3-1alphahydroxylase is expressed in human vascular smooth muscle cells and is upregulated by parathyroid hormone and estrogenic compounds. Circulation. 2005;111(13):1666–1671.
18. Fritsche J, Mondal K, Ehrnsperger A, Andreesen R, Kreutz M. Regulation of 25-hydroxyvitamin D3-1alpha-hydroxylase and production of 1alpha,25-dihydroxyvitamin D3 by human dendritic cells. Blood. 2003;102(9):3314–3316.
19. Ordonez-Moran P, Alvarez-Diaz S, Valle N, Larriba MJ, Bonilla F, Mun˜oz A. The effects of 1,25-dihydroxyvitamin D3 on colon cancer cells depend on RhoA-ROCK-p38MAPK-MSK signaling. J Steroid Biochem Mol Biol. 2010;121(1–2):355–361.
20. Zehnder D, Bland R, Williams MC, et al. Extrarenal expression of 25-hydroxyvitamin d(3)-1alpha-hydroxylase. J Clin Endocrinol Metab. 2001;86(2):888–894.
21. Diaz L, Sanchez I, Avila E, Halhali A, Vilchis F, Larrea F. Identification of a 25-hydroxyvitamin D3 1alpha-hydroxylase gene transcription product in cultures of human syncytiotrophoblast cells. J Clin Endocrinol Metab. 2000;85(7):2543–2549.
22. Segersten U, Correa P, Hewison M, et al. 25-hydroxyvitamin D(3)-1alphahydroxylase expression in normal and pathological parathyroid glands. J Clin Endocrinol Metab. 2002;87(6):2967–2972.
23. Haussler MR, Haussler CA, Jurutka PW, et al. The vitamin D hormone and its nuclear receptor: molecular actions and disease states. J Endocrinol. 1997;154(Suppl):S57–S73.
24. Kim S, Shevde NK, Pike JW. 1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact osteoblasts. J Bone Miner Res. 2005;20(2):305–317.
25. Maestro B, Davila N, Carranza MC, Calle C. Identification of a vitamin D response element in the human insulin receptor gene promoter. J Steroid Biochem Mol Biol. 2003;84(2–3):223–230.
26. Darwish HM, DeLuca HF. Analysis of binding of the 1,25-dihydroxyvitamin D3 receptor to positive and negative vitamin D response elements. Arch Biochem Biophys. 1996;334(2):223–234.
27. Medhora MM, Teitelbaum S, Chappel J, et al. 1alpha,25-dihydroxyvitamin D3 up-regulates expression of the osteoclast integrin alpha v beta 3. J Biol Chem. 1993;268(2):1456–1461.
28. Artaza JN, Norris KC. Vitamin D reduces the expression of collagen and key profibrotic factors by inducing an antifibrotic phenotype in mesenchymal multipotent cells. J Endocrinol. 2009;200(2):207–221.
29. Reitsma PH, Rothberg PG, Astrin SM, et al. Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite. Nature. 1983;306(5942):492–494.
30. Murayama A, Takeyama K, Kitanaka S, et al. Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alpha-hydroxylase gene by parathyroid hormone, calcitonin, and 1alpha,25(OH)2D3 in intact animals. Endocrinology. 1999;140(5):2224–2231.
31. van Etten E, Mathieu C. Immunoregulation by 1,25-dihydroxyvitamin D3: basic concepts. J Steroid Biochem Mol Biol. 2005;97(1–2):93–101.
32. Theodoropoulos C, Demers C, Mirshahi A, Gascon-Barré M. 1,25-Dihydroxyvitamin D(3) downregulates the rat intestinal vitamin D(3)-25-hydroxylase CYP27A. Am J Physiol Endocrinol Metab. 2001;281(2): E315–E325.
33. Eyles DW, Smith S, Kinobe R, Hewison M, McGrath JJ. Distribution of the vitamin D receptor and 1alpha-hydroxylase in human brain. J Chem Neuroanat. 2005;29(1):21–30.
34. Reinhart GA. Vitamin D analogs: novel therapeutic agents for cardiovascular disease? Curr Opin Investig Drugs. 2004;5(9):947–951.
35. Pospechova K, Rozehnal V, Stejskalova L, et al. Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines. Mol Cell Endocrinol. 2009;299(2):178–187.
36. Bays HE, Bazata DD, Clark NG, et al. Prevalence of self-reported diagnosis of diabetes mellitus and associated risk factors in a national survey in the US population: SHIELD (Study to Help Improve Early evaluation and management of risk factors Leading to Diabetes). BMC Public Health. 2007;7:277.
37. Hypponen E, Power C. Vitamin D status and glucose homeostasis in the 1958 British birth cohort: the role of obesity. Diabetes Care. 2006;29(10):2244–2246.
38. Anderson JL, May HT, Horne BD, et al. Relation of vitamin D deficiency to cardiovascular risk factors, disease status, and incident events in a general healthcare population. Am J Cardiol. 2010;106(7):963–968.
39. Yin L, Grandi N, Raum E, Haug U, Arndt V, Brenner H. Meta-analysis: longitudinal studies of serum vitamin D and colorectal cancer risk. Aliment Pharmacol Ther. 2009;30(2):113–125.
40. Jenab M, Bueno-de-Mesquita HB, Ferrari P, et al. Association between pre-diagnostic circulating vitamin D concentration and risk of colorectal cancer in European populations:a nested case-control study. BMJ. 2010;340:b5500.
41. John EM, Schwartz GG, Dreon DM, Koo J. Vitamin D and breast cancer risk: the NHANES I Epidemiologic follow-up study, 1971–1975 to 1992. National Health and Nutrition Examination Survey. Cancer Epidemiol Biomarkers Prev. 1999;8(5):399–406.
42. Ahonen MH, Tenkanen L, Teppo L, et al. Prostate cancer risk and prediagnostic serum 25-hydroxyvitamin D levels (Finland). Cancer Causes Control. 2000;11(9):847–852.
43. Stewart AF, Mangin M, Wu T, Hakama M, Tuohimaa P. Synthetic human parathyroid hormone-like protein stimulates bone resorption and causes hypercalcemia in rats. J Clin Invest. 1988;81(2):596–600.
44. Martin TJ, Ebeling PR. A novel parathyroid hormone-related protein: role in pathology and physiology. Prog Clin Biol Res. 1990;332:1–37.
45. Errazahi A, Lieberherr M, Bouizar Z, Rizk-Rabin M. PTH-1R responses to PTHrP and regulation by vitamin D in keratinocytes and adjacent fibroblasts. J Steroid Biochem Mol Biol. 2004;89–90(1–5):381–385.
46. Luparello C. Midregion PTHrP and human breast cancer cells. ScientificWorldJournal. 2010;10:1016–1028.
47. Monego G, Arena V, Pasquini S, et al. Ischemic injury activates PTHrP and PTH1R expression in human ventricular cardiomyocytes. Basic Res Cardiol. 2009;104(4):427–434.
48. Kemp BE, Moseley JM, Rodda CP, et al. Parathyroid hormone-related protein of malignancy: active synthetic fragments. Science. 1987;238(4833):1568–1570.
49. Watson PH, Fraher LJ, Hendy GN, et al. Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues. J Bone Miner Res. 2000;15(6): 1033–1044.
50. Burton DW, Brandt DW, Deftos LJ. Parathyroid hormone-related protein in the cardiovascular system. Endocrinology. 1994;135(1):253–261.
51. Isowa S, Shimo T, Ibaragi S, et al. PTHrP regulates angiogenesis and bone resorption via VEGF expression. Anticancer Res. 2010;30(7):2755–2767.
52. Adachi N, Yamaguchi K, Miyake Y, et al. Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes. Biochem Biophys Res Commun. 1990;166(3):1088–1094.
53. Kaiser SM, Laneuville P, Bernier SM, Rhim JS, Kremer R, Goltzman D. Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide. J Biol Chem. 1992;267(19):13623–13628.
54. Endlich K, Massfelder T, Helwig JJ, Steinhausen M. Vascular effects of parathyroid hormone and parathyroid hormone-related protein in the split hydronephrotic rat kidney. J Physiol. 1995;483(Pt 2):481–490.
55. Syed MA, Horwitz MJ, Tedesco MB, Garcý´a-Ocan˜a A, Wisniewski SR, Stewart AF. Parathyroid hormone-related protein-(1–36) stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy. J Clin Endocrinol Metab. 2001;86(4):1525–1531.
56. Stewart AF. PTHrP(1–36) as a skeletal anabolic agent for the treatment of osteoporosis. Bone. 1996;19(4):303–306.
57. Song GJ, Fiaschi-Taesch N, Bisello A. Endogenous parathyroid hormonerelated protein regulates the expression of PTH type 1 receptor and proliferation of vascular smooth muscle cells. Mol Endocrinol. 2009;23(10):1681–1690.
58. Guthalu Kondegowda N, Joshi-Gokhale S, Harb G, et al. Parathyroid hormone-related protein enhances human b-cell proliferation and function with associated induction of cyclin-dependent kinase 2 and cyclin E expression. Diabetes. 2010;59(12):3131–3138.
59. Gaich G, Orloff JJ, Atillasoy EJ, Burtis WJ, Ganz MB, Stewart AF. Aminoterminal parathyroid hormone-related protein: specific binding and cytosolic calcium responses in rat insulinoma cells. Endocrinology. 1993;132(3):1402–1409.
60. Horiuchi N, Hongo T, Clemens TL. A 7–34 analog of the parathyroid hormone-related protein has potent antagonist and partial agonist activity in vivo. Bone Miner. 1991;12(3):181–188.
61. Divieti P, John MR, Juppner H, Bringhurst FR. Human PTH-(7–84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/PTHrP receptor. Endocrinology. 2002;143(1):171–176.
62. Abou-Samra AB, Uneno S, Jueppner H, et al. Non-homologous sequences of parathyroid hormone and the parathyroid hormone related peptide bind to a common receptor on ROS 17/2.8 cells. Endocrinology. 1989;125(4):2215–2217.
63. Soifer NE, Dee KE, Insogna KL, et al. Parathyroid hormone-related protein. Evidence for secretion of a novel mid-region fragment by three different cell types. J Biol Chem. 1992;267(25):18236–18243.
64. Care AD, Abbas SK, Pickard DW, et al. Stimulation of ovine placental transport of calcium and magnesium by mid-molecule fragments of human parathyroid hormone-related protein. Exp Physiol. 1990;75(4): 605–608.
65. Philbrick WM, Wysolmerski JJ, Galbraith S, et al. Defining the roles of parathyroid hormone-related protein in normal physiology. Physiol Rev. 1996;76(1):127–173.
66. Casado-Diaz A, Santiago-Mora R, Quesada JM. The N- and C-terminal domains of parathyroid hormone-related protein affect differently the osteogenic and adipogenic potential of human mesenchymal stem cells. Exp Mol Med. 2010;42(2):87–98.
67. Wu TL, Vasavada RC, Yang K, et al. Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein. J Biol Chem. 1996;271(40):24371–24381.
68. Orloff JJ, Kats Y, Urena P, et al. Further evidence for a novel receptor for amino-terminal parathyroid hormone-related protein on keratinocytes and squamous carcinoma cell lines. Endocrinology. 1995;136(7): 3016–3023.
69. Toribio RE, Brown HA, Novince CM, et al. The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice. FASEB J. 2010;24(6):1947–1957.
70. Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM. Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA. 1992;89(17):8097–8101.
71. Nishishita T, Okazaki T, Ishikawa T, et al. A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D. J Biol Chem. 1998;273(18):10901–10907.
72. Ishida H, Suzuki K, Someya Y, et al. Possible compensatory role of parathyroid hormone-related peptide on maintenance of calcium homeostasis in patients with non-insulin-dependent diabetes mellitus. Acta Endocrinol (Copenh). 1993;129(6):519–524.
73. McNair P, Christensen MS, Madsbad S, Christiansen C, Transbøl I. Hypoparathyroidism in diabetes mellitus. Acta Endocrinol (Copenh). 1981;96(1):81–86.
74. Legakis I, Mantouridis T. Positive correlation of PTH-related peptide with glucose in type 2 diabetes. Exp Diabetes Res. 2009;2009:291027.
75. Drucker DJ, Asa SL, Henderson J, Goltzman D. The parathyroid hormone-like peptide gene is expressed in the normal and neoplastic human endocrine pancreas. Mol Endocrinol. 1989;3(10):1589–1595.
76. Vasavada RC, Cavaliere C, D’Ercole AJ, et al. Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia, hyperinsulinemia, and hypoglycemia. J Biol Chem. 1996;271(2):1200–1208.
77. Zhang B, Hosaka M, Sawada Y, et al. Parathyroid hormone-related protein induces insulin expression through activation of MAP kinase-specific phosphatase-1 that dephosphorylates c-Jun NH2-terminal kinase in pancreatic beta-cells. Diabetes. 2003;52(11):2720–2730.
78. Cebrian A, Garcý´a-Ocan˜a A, Takane KK, Sipula D, Stewart AF, Vasavada RC. Overexpression of parathyroid hormone-related protein inhibits pancreatic beta-cell death in vivo and in vitro. Diabetes. 2002;51(10): 3003–3013.
79. Horwitz MJ, Tedesco MB, Garcý´a-Ocan˜a A, et al. Parathyroid hormonerelated protein for the treatment of postmenopausal osteoporosis: defining the maximal tolerable dose. J Clin Endocrinol Metab. 2010;95(3):1279–1287.
80. Zeitz U, Weber K, Soegiarto DW, Wolf E, Balling R, Erben RG. Impaired insulin secretory capacity in mice lacking a functional vitamin D receptor. FASEB J. 2003;17(3):509–511.
81. Bourlon PM, Billaudel B, Faure-Dussert A. Influence of vitamin D3 deficiency and 1,25 dihydroxyvitamin D3 on de novo insulin biosynthesis in the islets of the rat endocrine pancreas. J Endocrinol. 1999;160(1):87–95.
82. Cade C, Norman AW. Vitamin D3 improves impaired glucose tolerance and insulin secretion in the vitamin D-deficient rat in vivo. Endocrinology. 1986;119(1):84–90.
83. Pittas AG, Harris SS, Stark PC, Dawson-Hughes B. The effects of calcium and vitamin D supplementation on blood glucose and markers of inflammation in nondiabetic adults. Diabetes Care. 2007;30(4):980–986.
84. Parikh SJ, Edelman M, Uwaifo GI, et al. The relationship between obesity and serum 1,25-dihydroxy vitamin D concentrations in healthy adults. J Clin Endocrinol Metab. 2004;89(3):1196–1199.
85. Shor R, Halabe A, Aberbuh E, et al. PTHrP and insulin levels following oral glucose and calcium administration. Eur J Intern Med. 2006;17(6):408–411.
86. Burtis WJ, Brady TG, Orloff JJ, et al. Immunochemical characterization of circulating parathyroid hormone-related protein in patients with humoral hypercalcemia of cancer. N Engl J Med. 1990;322(16):1106–1112.
87. Mooradian AD, Morley JE. Micronutrient status in diabetes mellitus. Am J Clin Nutr. 1987;45(5):877–895.
88. Saggese G, Federico G, Bertelloni S, Baroncelli GI, Calisti L. Hypomagnesemia and the parathyroid hormone-vitamin D endocrine system in children with insulin-dependent diabetes mellitus: effects of magnesium administration. J Pediatr. 1991;118(2):220–225.
89. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, Cao LP. 1,25-Dihydroxyvitamin D(3) is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest. 2002;110(2):229–238.
90. Lorenzo O, Ruiz-Ortega M, Esbrit P, et al. Angiotensin II increases parathyroid hormone-related protein (PTHrP) and the type 1 PTH/PTHrP receptor in the kidney. J Am Soc Nephrol. 2002;13(6):1595–1607.
91. Sebastian EM, Suva LJ, Friedman PA. Differential effects of intermittent PTH(1–34) and PTH(7–34) on bone microarchitecture and aortic calcification in experimental renal failure. Bone. 2008;43(6):1022–1030.
92. Sneddon WB, Barry EL, Coutermarsh BA, Gesek FA, Liu F, Friedman PA. Regulation of renal parathyroid hormone receptor expression by 1,25- dihydroxyvitamin D3 and retinoic acid. Cell Physiol Biochem. 1998;8(5): 261–277.
93. Nakajima K, Nohtomi K, Sato M, Takano K, Sato K. PTH(7–84) inhibits PTH(1–34)-induced 1,25-(OH)2D3 production in murine renal tubules. Biochem Biophys Res Commun. 2009;381(2):283–287.
94. Marcinkowski W, Zhang G, Smogorzewski M, Massry SG. Elevation of [Ca2–]i of renal proximal tubular cells and down-regulation of mRNA of PTH-PTHrP, V1a and AT1 receptors in kidney of diabetic rats. Kidney Int. 1997;51(6):1950–1955.
95. Samuel D, Bharali D, Mousa SA. The role of nanotechnology in diabetes treatment: current and future perspectives. Int J Nanotech. 2011;8(1–2): 53–65.
96. Kolek OI, Hines ER, Jones MD, et al. 1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renalgastrointestinal- skeletal axis that controls phosphate transport. Am J Physiol Gastrointest Liver Physiol. 2005;289(6):G1036–G1042.

Type 2 diabetes (T2D) is one of the metabolic disorder that affects population; its prevalence increased quickly during these last decades in all regions of the world. It is currently estimated that 190 million people suffer from diabetes mellitus, with over 330 millions predicted to have the condition by 2025 and 366 million by the year 2030.¹ The rate prevalence heterogeneity of diabetes mellitus is evident; the number of diabetic patients in the USA, India, and China or in Arab countries is not the same but its increasing year on year is similar.

Every ethnic group has a special culture and tradition but, at the same time, has heredity characters proper from the first generation to the last. It expressed according to some factors that are correlated to the environment where an individual lives, and it can began from the embryogenesis to the puberty. From this concept, a multifactorial etiology of diabetes mellitus was identified with an important contribution from environment and lifestyle together with genetic factors in predisposed individuals. ^2–4 This interaction between these two most classes factors (genetic–environment)⁵ has interested many authors in their studies, but the question that remains answered is “Which factor has a more impact in the development of diabetes mellitus: Genetic or environment?” In this article, we want to answer this question by the analysis of previously published studies.

EPIDEMIC OF T2D

T2D is one of the most common endocrine disorders affecting almost 6% of the world's population,¹ and there will be a 69% increase in the number of adults with diabetes in developing countries and 20% increase in developed countries.⁶ The prevalence of diabetes mellitus ranges from Africa 3.8%, world pacific 4.7%, south America 6.6%, Europe 6.9%, South Asia 7.4%, to North America 10.2%.⁶

In USA, the prevalence of diagnosed diabetes increased progressively from 0.9% in 1958 to 5.9% in 2006.⁷ Prevalence of diabetes in the USA in 1976–1980 was 6.6% and about half the people with diabetes did not know their condition.⁸ In Canada, age adjusted increase in prevalence increased from 4.4% in 1986 to 6.6% in 1991.⁹ Similarly, in Iceland, the prevalence in males was 2.8% in 1970–1972, 4.5% in 1979–1984, and 5.0% in 1985–1990.¹⁰ Prevalence of diabetes among adults above 20 years in urban India was around 1% in 1960, which increased steeply reaching to about 12% by 2005.¹¹ Similarly, analysis of previous trends of age-adjusted prevalence rates of diabetes in different urban areas reveals 7.7% in 1990 and 8.9% in 1995 in Hong Kong, 8.1% in 1993 in Singapore, and 11% in 1995 in Taiwan¹²; 5% in 1994 in urban Sri Lanka, 9.7% in 2004 in urban Cambodia, and 4.5% in 1997 and 8.1% in 2005 in Dhaka¹³; and 9.5% in Latinos and 13.3% in Africans in 2005 in the USA.¹⁴

In the Arab countries, the prevalence was more higher, 14.8% in Kuwait,¹⁵ 15.6% in Syria,¹⁶ 17.1% in Jordon,¹⁷ 23.7% in Saudi Arabia,¹⁸ and 25.5% in Bahrain,¹⁹ similarly, for North African countries like Algeria, the prevalence of diabetes mellitus increased rapidly from 6.8% in²⁰ 1994 to 8.2% in 1998,²¹ to 10.5% in 2007.²² In Morocco, the prevalence ranges from 6.6% in²³ 1996 to 8% in 2004.²⁴ In Tunisia, 4.4% was noted in 1994 to²⁵ 9% in 2007,²⁶ in Egypt 9.3%,²⁷ and in Libya 14.1% in 1999.²⁸

This global statistics indicate that the burden of T2D is not only restricted to the developed countries but also a problem for the developing countries, which being either part of or in close proximity with industrialized countries like the USA and Canada, mostly that received the modern technology, benefits of lifestyle factors that favor a sedentary life and obesity, but between us: Are these the only factors contributed to the development of T2D?

OBESITY, SEDENTARY LIFE, AND A RAPID TRANSITION IN NUTRITIONAL STATUS OF POPULATION

Sedentary behavior is one of the main contributory factors to increased obesity rates; a high percentage of the population have decreased physical activity at work and leisure by spending more hours per week watching TV and the using of more vehicles and activity saving appliances.

In the USA, there is progressive increase in obesity and an estimated 66% of adults were overweight or obese in 2003–2004.²⁹ Similar rises in obesity was seen in Europe.³⁰ In the USA, prevalence of diabetes has increased in accord with the increasing prevalence of obesity,³¹ and 80% of individuals with diabetes are obese. In Morocco, the rate of anthropometric status in adult individuals showed that 27.1% were overweight and 11.7% were obese.³² In Tunisia, 27.7% were obese (BMI > 30) and 36% have an android obesity,³³ about 15% of the population aged 18–69 years are inactive.³⁴ The association between physical inactivity and diabetes indicated that sedentary life appears to play a critical role in the development of body fat and may be a risk indicator for metabolic syndrome.³⁵ Despite the ongoing debate as to whether obesity should be labeled as a disease, obesity, directly and indirectly, has become the global health challenge. In a 16-year follow-up study of 84 941 women, it was documented that 3300 new cases of T2D were diagnosed from 1980 to 1996.³⁶ Among these cases, body weight was the single most important predictor of diabetes. As high, 80% of the cases of T2D could be attributed to the combined effect of inactivity and high body weight.³⁷

The changes of dietary composition, in addition to the total caloric intake, are probably another important factor that is implicated in the epidemic of T2D. For example, one important but not well-appreciated dietary change has been the substantial increase in refined carbohydrate especially simple carbohydrate from high intake of sucrose and fructose corn syrup, a common sweetener used in the food industry. High influx of carbohydrate is predicted to drive the increase of the now hepatic lipogenesis and perturbs the glucose homeostasis that appears to underlie the induction of insulin resistance.³⁸

Effect of Nutritional Transition During Fetal Life

The rapid transition in the nutritional status of population leads to dissociation in metabolic states of fetal life, associated with nutritional want, and adult life, with nutritional surfeit of people. The relative malnutrition during fetal life is associated with increased risk of insulin resistance, impaired pancreatic islet cell development, and diabetes in people who are underweight at birth but become overweight or obese in later life, ^39–41 helping to explain the greatly increased frequency of diabetes in the populations, which move rapidly from nutritional want to adequacy or surfeit.⁴²

The Relationship Between Exposure of Fetus to Maternal Hyperglycemia and Risk of Developing Diabetes in Later Life

It is independent of genetic influences.⁴³ Intrauterine exposure to hyperglycemia and hyperinsulinemia may affect development of adipose tissue and pancreatic beta cells leading to future obesity and altered glucose metabolism.⁴³ The babies of mothers who have diabetes during pregnancy were reported to have up to 45% risk of developing diabetes, compared with 8.6% in babies of mothers who develop diabetes after pregnancy or 1.4% in babies of mothers without diabetes as seen in Pima Indians.⁴⁴ Youth with T2D were diagnosed at younger ages among those exposed to hyperglycemia in utero.⁴⁵ The prevalence of T2D was 2.2% in 10–14 years old and 5.1% in 15–19 years old in 1992–1996, in Pima Indians.⁴⁶ Further, the prevalence of glucose intolerance in the children of mothers with a diabetic pregnancy increases with time, for example, from 1.2% at less than 5 years of age to 19.3% at 10–16 years of age.⁴¹

GENETIC FACTORS IN T2D

Ethnicity is considered to be an important factor in diabetes development with higher rates being reported in Asians, Hispanics, African-Americans, and indigenous peoples of the USA, Canada, Australia, and Pacific regions.³⁹ The term ethnicity appears to carry the notion of predominant genetic element, which we cannot change.

The search of human genetic factors that predispose to T2D has gone through two directions, by studying of rare mutations and by population-based gene-hunting for the primary causes.⁴⁷ Several previous familial linkage and candidate gene studies have confirmed the association T2D-single nucleotide polymorphisms (SNPs); it is widely replicated but with modest effects on disease risk (Table 1).^48,49 These variants include the E23K variation in KCNJ11, encoding the Kir6.2 subunit of the K + -ATP channel,⁵⁰ the Pro12Ala variant in PPARG,⁵¹ the -30G/A polymorphism in the β-cell specific promoter of glucokinase GCK,^{50 52} and the K121Q variant of ENPP1 encoding ectonucleotide pyrophosphatase phosphodiesterase, the inhibitor of insulin receptor.⁵³ The rs7903146 variant in the TCF7L2 locus was consistently replicated in population of various ethnic origins, among which Moroccans.⁵⁴ The frequencies of these alleles varied, depending on ethnicity and geographical location, for the ENPP1 Q121 allele, Keshavarz et al.⁵⁵ reported that, in European Caucasians, the Q121 prevalence ranged from 10% in Finns⁵⁶ to 17.8% in Sicilians.⁵⁷ In the Dominican Republic, a population with a mixed genetic background, the allele's frequency was 54.2%,⁵⁸ and an even higher Q121 allele frequency (79%) was reported in African-Americans.⁵⁹

Importance of Heredity Factor in Determining the Risk of T2D

Is heredity an important factor in determining the risk of T2D? This apparently simple question is actually very difficult to answer, because the contribution of heredity appears to differ considerably between different populations and in different environments. The best evidence that heredity plays an important role comes from the following observations:

Concordance rates for T2D and its predecessor, impaired glucose tolerance, are consistently higher in monozygotic than in dizygotic twin pairs.

Sibling recurrence rates are consistently higher than population prevalence rates; although the reported excess is modest, groups of patients labeled as having T2D include individuals suffering from unrecognized monogenic and digenic disorders and certain common SNPs appear to influence diabetes risk.⁶⁰

The influence of environment and lifestyle on risk should not be underestimated. More recent attention has focused on the possible effects of prenatal and early postnatal environment on diabetes risk.⁶¹ The correlation between low birth weight and later diabetes is consistent across many populations studied but could, of course, imply that genetic factors influence both birth weight and diabetes risk, like mutations in glucokinase gene of fetus lead to reduced intrauterine growth insulin levels and also to postnatal diabetes.⁶² When genetic factors are held constant, intrauterine factors influencing fetal growth may have long-term implications for metabolic health. For geneticists interested in this disease is that, if they ignore the effects of the pre- and postnatal environment and their capacity to interact with genetic variants, real progress in understanding diabetes may be impeded.

Discovering the Precise Genetic Basis of T2D Risk, For Why?

The great hope that inspired most efforts in disease gene discovery is that such genes will then become therapeutic targets and that drugs targeted to molecules that are fundamentally involved in disease causation will be more effective than the cruder therapies we now use. In this scenario, genetics is being used as a tool to aid the discovery of drugs. Perhaps more immediately applicable is the notion that the presence of particular genetic variants in an individual will influence his/her response to particular therapies (pharmacogenomics), as in the case of patients with MODY resulting from HNF1a mutations who preferentially respond to sulfonylurea therapy.⁶³ In this case, we are looking at genetics as a guide to better drug use in the clinic. Greater understanding of the basis of disease also allows for more accurate provision of prognostic information to patients as the risk of future complications and likely disease trajectory is more accurately determined.

Perhaps, the most exciting future for genetics in T2D is not necessarily in pharmacogenomics or provision of prognostic information but in understanding how specific genes interact with diet, exercise, and other lifestyle factors in the control of intermediary metabolism. The identification of individuals at high risk and determining which particular combination and type of diet and exercise program and perhaps pharmacotherapy can be optimally used to prevent the onset of hyperglycemia is the first goal of genetic technology that we try to develop every day with the collaboration of many researchers.

Interaction Genetic-Environment

The gene-environment interaction in, traditionally, genetic investigators have tackled as a second-order question, the neglect of environmental exposure may mean that important effects of particular alleles are not only attenuated but even obscured if the effect of genotype differs markedly according to lifestyle. The measurement of dietary factors and physical activities by epidemiological method is imprecise, and it should be critical to the ability to detect this interaction. Simple cross-sectional case-control studies of gene-lifestyle interaction will not work because of biased estimation of lifestyle in people who have been given the diagnostic label of diabetes. The optimal approach will involve case-control studies nested within major epidemiological cohort studies. These need to be large, have careful measurement of lifestyle factors at baseline, and critically amass sufficient person-years of follow-up before they are of use.

CONCLUSION

In the combination genetic-environment, it is very difficult to understanding which part has a more impact in the development of diabetes mellitus. To be careful, we say that genetic factors influence the susceptibility of T2D, but no simple approach is likely to be successful, and no gene are specify responsible for this disease without the intervention of the environmental factors for its expression. At the same time, we say that the environmental factors have more impact is not true; having a sedentary modern life style, changes in dietary habits, and being obese contribute to the development of diabetes mellitus but except in predisposed individuals. To be clear, we can summarize this problem in three equations:

Disclosure: The authors declare no conflict of interest.

REFERENCES

1. Azimi-Nezhad M, Ghayour-Mobarhan M, Parizadeh MR, et al. Prevalence of type 2 diabetes mellitus in Iran and its relationship with gender, urbanisation, education, marital status and occupation. Singapore Med J. 2008;49(7):571–576.
2. Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature. 2001;414(6865):782–787.
3. Frayling TM. Genome-wide association studies provide new insights into type 2 diabetes aetiology. Nat Rev Genet. 2007;8(9):657–662.
4. Belmokhtar F, Belmokhtar R, Dali-Sahi M, Charef M. Risk factors associated with type 2 diabetes mellitus in west region of Algeria, Maghnia. J Diabetes Metab. 2011;2:148. doi:10.4172/2155–6156.1000148.
5. McCarthy MI, Froguel P. Genetic approaches to the molecular understanding of type 2 diabetes. Am J Physiol Endocrinol Metab. 2002;283: 217–225.
6. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract. 2010;87:4–14.
7. Nathan DM, Davidson MB, DeFronzo RA, et al. Impaired fasting glucose and impaired glucose tolerance: implications for care. Diabetes Care. 2007;30(3):753–759.
8. Hadden WC, Harris MI. Prevalence of Diagnosed Diabetes, Undiagnosed Diabetes, and Impaired Glucose Tolerance in Adults 20–74 Years of Age, United States, 1976–80. National Center for Health Statistics, Vital and Health Statistics Series 11, No 237. Washington: US Government Printing Office; 1987.
9. Blanchard JF, Ludwig S, Wajda A, et al. Incidence and prevalence of diabetes in Manitoba 1986–1991. Diabetes Care. 1996;19:807–811.
10. Vilbregson S, Sigurdsson G, Sigvaldason H, Hreidarsson AB, Sigfusson N. Prevalence and incidence of NIDDM in Iceland: evidence for stable incidence among males and females 1967–1991: the Reykjavik Study. Diabet Med. 1997;14(6):491–498.
11. Gupta R, Misra A. Type 2 diabetes in India: regional disparities. Br J Diabetes Vasc Dis. 2007;7:12–16.
12. Cockram CS. The epidemiology of diabetes mellitus in the Asia-Pacific region. Hong Kong Med J. 2000;6(1):43–52.
13. Chan JCN, Malik V, Jia W, et al. Diabetes in Asia: epidemiology, risk factors, and pathophysiology. JAMA. 2009;301:2129–2140.
14. Powers AC, Fauci AS, Kasper DL, et al Harrison’s Principles of Internal Medicine. 17th ed. New York: The McGraw-Hill Companies, Inc; 2008:2275–2304.
15. Abdella N, Al Arouj M, Al Nakhi A, Al Assoussi A, Moussa M. Noninsulin- dependent diabetes in Kuwait: prevalence rates and associated risk factors. Diabetes Res Clin Pract. 1998;42(3):187–196.
16. Albache N, Al Ali R, Rastam S, Fouad FM, Mzayek F, Maziak W. Epidemiology of type 2 diabetes mellitus in Aleppo, Syria. J Diabetes. 2010;2(2):85–91.
17. Ajlouni K, Khader YS, Batieha A, Ajlouni H, El-Khateeb M. An increase in prevalence of diabetes mellitus in Jordan over 10 years. J Diabetes Complications. 2008;22(5):317–324.
18. Al-Nozha MM, Al-Maatouq MA, Al-Mazrou YY, et al. Diabetes mellitus in Saudi Arabia. Saudi Med J. 2004;25(11):1603–1610.
19. Al Zurba FI, Al Garf A. Prevalence of diabetes mellitus among Bahrainis attending primary health care centres. Eastern Mediterranean Health J. 1996;2(2):274–282.
20. Unwin N, Sobngwi E, Alberti KGMM. Type 2 diabetes: the challenge of preventing a global epidemic. Diabetes Int. 2001;11:4–8.
21. Malek R, Belateche F, Laouamri S, et al. Prevalence of type 2 diabetes mellitus and glucose intolerance in the Setif area (Algeria). Diabetes Metab. 2001;27(2 Pt 1):164–171.
22. Zaoui S, Biémont C, Meguenni K. Epidemiology of diabetes in urban and rural regions of Tlemcen (Western Algeria) (in French). Santé. 2007;17:15–21.
23. King H, Aubert RE, Herman WH. Global burden of diabetes 1995–2025. Prevalence, numerical estimates, and projections. Diabetes Care. 1998;21(9):1414–1431.
24. Ujcic-Voortman JK, Schram MT, Jacobs-van der Bruggen MA, Verhoeff AP, Baan CA. Diabetes prevalence and risk factors among ethnic minorities. Eur J Public Health. 2009;19(5):511–515.
25. Kamoun M, Abid M, Ben Abdallah N, et al. Epidémiologie du diabète en Tunisie. Etude multicentrique de la Société tunisienne d’endocrinologie Diabete 2004. Ibidem, pp. 41–46.
26. Bouguerra R, Alberti H, Salem LB, et al. The global diabetes pandemic: the Tunisian experience. Eur J Clin Nutr. 2007;61(2):160–165.
27. Herman W, Ali M, Aubert R, et al. Diabetes mellitus in Egypt: risk factors and prevalence. Diabet Med. 1995;12:1126–1131.
28. Kadiki OA, Roaeid RB. Prevalence of diabetes mellitus and impaired glucose tolerance in Benghazi Libya. Diabetes Metab. 2001;27:647–654.
29. National Center for Health Statistics. Prevalence of overweight and obesity among adults: United States, 2003–2004 (Online). April 2006. http://www.cdc.gov/nchs/products/pubs/pubd/hestats/overweight/overwg htadult_03.htm. Accessed February 23, 2009.
30. World Health Organization. Consultation on Obesity. Obesity: Preventing and Managing the Global Epidemic. Geneva: WHO; 1998.
31. World Health Organization. World Health Report 2002: Reducing Risks, Promoting Healthy Life. Geneva: WHO; 2003.
32. Direction de la statistique. Consommation et dépenses des ménages 1984/1985. Rabat, Morocco: Statistics office; 1992. p. 1, 5, 7.
33. Ghannem H, Fredj AH. Epidemiological transition and cardiovascular risk factors in Tunisia. Rev Epidemiol Sante Publique. 1997;45(4):286–92.
34. World Health Organization. Global InfoBase. Online: http:// www.who.int/ncd_surveillance/infobase/web/infobasepolicymaker/reports/ reportlistcountries.aspx 35. Rguibi M, Belahsen R. Metabolic syndrome among Moroccan Sahraoui adult women: its prevalence and characteristics. Am J Hum Biol. 2004;16:598–601.
36. Hu FB, Manson JE, Stampfer MJ, et al. Diet, lifestyle, and the risk of type 2 diabetes mellitus in women. N Engl J Med. 2001;345:790–797.
37. Stein CJ, Colditz GA. The epidemic of obesity. J Clin Endocrinol Metab. 2004;89:2522–2525.
38. Basciano H, Federico L, Adeli K. Fructose, insulin resistance, and metabolic dyslipidemia. Nutr Metab (Lond). 2005;2:5.
39. International Diabetes Federation. Diabetes atlas. Online, 2006. http:// www.eatlas.idf.org/. Accessed March 5, 2009.
40. Hales CN, Barker DJ. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia. 1992;35(7):595–601.
41. Flanagan DE, Moore VM, Godsland IF, Cockington RA, Robinson JS, Phillips DI. Fetal growth and the physiological control of glucose tolerance in adults: a minimum model analysis. Am J Physiol Endocrinol Metab. 2000;278:700–706.
42. Cohen MP, Stern E, Rusecki Y, Zeidler A. A high prevalence of diabetes in young adult Ethiopian immigrants to Israel. Diabetes. 1988;37(6):824.
43. World Health Organization. Expert Consultation. Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet. 2004;363:157–163.
44. Pettitt DJ, Aleck KA, Baird HR, Carraher MJ, Bennett PH, Knowler WC. Congenital susceptibility to NIDDM: role of intrauterine environment. Diabetes. 1988;37(5):622–628.
45. Misra A, Chowbey P, Makkar BM, et al. Consensus statement for diagnosis of obesity, abdominal obesity and the metabolic syndrome for Asian Indians and recommendations for physical activity, medical and surgical management. J Assoc Physicians India. 2009;57:163–170.
46. Fagot-Campagna A, Pettitt DJ, Engelgau MM, et al. Type 2 diabetes among North American children and adolescents: an epidemiologic review and a public health perspective. J Pediatr. 2000;136(5):664–672.
47. Barsh GS, Farooqi IS, O’Rahilly S. Genetics of body-weight regulation. Nature. 2000;404:644–651.
48. Weedon MN, McCarthy MI, Hitman G, et al. Combining information from common type 2 diabetes risk polymorphisms improves disease prediction. PLoS Med. 2006;3(10):e374.
49. Vaxillaire M, Veslot J, Dina C, et al. Impact of common type 2 diabetes risk polymorphisms in the DESIR prospective study. Diabetes. 2008;57(1):244–254.
50. Nielsen EM, Hansen L, Carstensen B, et al. The E23K variant of Kir6.2 associates with impaired post- OGTT serum insulin response and increased risk of type 2 diabetes. Diabetes. 2003;52(2):573–577.
51. Altshuler D, Hirschhorn JN, Klannemark M, et al. The common PPARgamma Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes. Nat Genet. 2000;26(1):76–80.
52. Winckler W, Weedon MN, Graham RR, et al. Evaluation of common variants in the six known maturity-onset diabetes of the young (MODY) genes for association with type 2 diabetes. Diabetes. 2007;56(3):685–693.
53. Meyre D, Bouatia-Naji N, Tounian A, et al. Variants of ENPP1 are associated with childhood and adult obesity and increase the risk of glucose intolerance and type 2 diabetes. Nat Genet. 2005;37(8):863–867.
54. Cauchi S, El Achhab Y, Choquet H, et al. TCF7L2 is reproducibly associated with type 2 diabetes in various ethnic groups: a global metaanalysis. J Mol Med. 2007;85(7):777–782.
55. Keshavarz P, Inoue H, Sakamoto Y, et al. No evidence for association of the ENPP1 (PC-1) K121Q variant with risk of type 2 diabetes in a Japanese population. J Hum Genet. 2006;51:559–566.
56. Kubaszek A, Pihlajamäki J, Karhapää P, Vauhkonen I, Laakso M. The K121Q polymorphism of the PC-1 gene is associated with insulin resistance but not with dyslipidemia. Diabetes Care. 2003;26:464–467.
57. Pizzuti A, Frittitta L, Argiolas A, et al. A polymorphism (K121Q) of the human glycoprotein PC-1 genecoding region is strongly associated with insulin resistance. Diabetes. 1999;48:1881–1884.
58. Hamaguchi K, Terao H, Kusuda Y, et al. The PC-1 Q121 allele is exceptionally prevalent in the Dominican Republic and is associated with type 2 diabetes. J Clin Endocrinol Metab. 2004;89:1359–1364.
59. Lyon HN, Florez JC, Bersaglieri T, et al. Common variants in the ENPP1 gene are not reproducibly associated with diabetes or obesity. Diabetes. 2006;55:3180–3184.
60. McCarthy MI, Froguel P. Genetic approaches to the molecular understanding of type 2 diabetes. Am J Physiol Endocrinol Metab. 2002;283:E217.
61. Hales CN, Barker DJ. The thrifty phenotype hypothesis. Br Med Bull. 2001;60:5–20.
62. Hattersley AT, Beards F, Ballantyne E, et al. Mutations in the glucokinase gene of the fetus result in reduced birth weight. Nat Genet. 1998;19: 268–270.
63. Stride A, Hattersley AT. Different genes, different diabetes: lessons from maturity-onset diabetes of the young. Ann Med. 2002;34(3):207–216.
64. Hager J, Hansen L, Vaisse C, et al. A missense mutation in the glucagon receptor gene is associated with non-insulin-dependent diabetes mellitus. Nat Genet. 1995;9:299–304.
65. Hinokio Y, Lindner TH, Mashima H, et al. Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet. 2000;26:163–175.
66. Weedon MN, Schwarz PE, Horikawa Y, et al. Meta-analysis and a large association study confirm a role for calpain-10 variation in type 2 diabetes susceptibility. Am J Hum Genet. 2003;73:1208–1212.
67. Inoue H, Ferrer J, Welling CM, et al. Sequence variants in the sulfonylurea receptor (SUR) gene are associated with NIDDM in Caucasians. Diabetes. 1996;45:825–831.
68. Hani EH, Clement K, Velho G, et al. Genetic studies of the sulfonylurea receptor gene locus in NIDDM and in morbid obesity among French Caucasians. Diabetes. 1997;46:688–694.
69. Yamagata K, Furuta H, Oda N, et al. Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young (MODY1). Nature. 1996;384:458–460.
70. Yamagata K, Oda N, Kaisaki PJ, et al. Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3). Nature. 1996;384:455–458.
71. Li SR, Baroni MG, Oelbaum RS, Stock J, Galton DJ. Association of genetic variant of the glucose transporter with non-insulindependent diabetes mellitus. Lancet. 1988;2:368–370.
72. Huxtable SJ, Saker PJ, Haddad L, et al. Analysis of parent-offspring trios provides evidence for linkage and association between the insulin gene and type 2 diabetes mediated exclusively through paternally transmitted class III variable number tandem repeat alleles. Diabetes. 2000;49:126–130.
73. Kadowaki T, Bevins CL, Cama A, et al. Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance. Science. 1988;240:787–790.
74. Taylor SI, Kadowaki T, Kadowaki H, Accili D, Cama A, McKeon C. Mutations in insulin-receptor gene in insulin-resistant patients. Diabetes Care. 1990;13:257–279.
75. Poulton J, Luan J, Macaulay V, Hennings S, Mitchell J, Wareham NJ. Type 2 diabetes is associated with a common mitochondrial variant: evidence from a population-based case-control study. Hum Mol Genet. 2002;11:1581–1583.

Early-onset neonatal hypocalcaemia (hypocalcaemia occurring within the first 48–72 hours of life)^1,2 accounts for 95% of all cases of neonatal hypocalcaemia.³ It is related to the abrupt cessation of transplacental supply of calcium from the mother to the foetus. The healthy term infant experiences a physiological nadir in serum calcium between the age of 24 and 48 hours and then stabilize and rise.^4–6 This normal pattern may be exacerbated with the nadir dropping to hypocalcaemic levels in high-risk neonates who have suffered severe birth asphyxia.^3,4,6 Tsang and Oh⁷ reported a lower total serum calcium value at the age of 8 hours prior to the actual development of hypocalcaemia at the age of 29 hours. Jain et al.⁶ recommended that early-onset neonatal hypocalcaemia should be treated with calcium supplementation for at least 72 hours, underscoring the need to check for hypocalcaemia in high-risk neonates.

The pathogenetic mechanism by which birth asphyxia causes hypocalcaemia is poorly understood. However, it has been speculated that delayed introduction of feeds, increased calcitonin production, increased endogenous phosphate load, transient functional hypoparathyroidism, target organ unresponsiveness and sodium bicarbonate therapy may play a role.^2,5,8 Of the total serum calcium, 40% is protein bound (mainly albumin). The remaining 60%, which is not protein bound, comprise 10% complexed with anions (such as citrate, sulphate, bicarbonate, phosphate and lactate) and 50% that is the free or ionized physiologically active form.^2,4 Changes in plasma protein concentration alter the total serum calcium concentration in the same direction as the protein concentration.⁴ However, available literature suggests that it is most unlikely that a significant change in the concentration of the main serum protein fraction will occur in less than 1 week.⁹ False elevations of total serum calcium values by 4%–7% may be caused by prolonged storage (>1 week) of blood and upright position for about 15 minutes.⁹ On the other hand, patients in recumbent posture may have lower serum calcium values.¹⁰

The definition of hypocalcaemia varies with age. In general, hypocalcaemia is defined as total serum calcium less than 1.75 mmol/L in all infants, <1.75 mmol/L in preterm infants, <2 mmol/L in full-term infants and <2.2 mmol/L in children and adolescents.¹¹

Information on the overall prevalence of early-onset neonatal hypocalcaemia is scarce. This paucity of information is even worse for clinical conditions known to increase its rate of occurrence. Tsang et al.^12,13 in two separate studies reported a prevalence of 37.6% for preterm infants and 14.3% for infants with birth asphyxia. In a retrospective study in Benin City, Omene and Diejomaoh¹⁴ reported a prevalence of 9.4% among their asphyxiated newborn infants. In that study, they neither defined hypocalcaemia nor stated the method used in measuring total serum calcium concentration. Early-onset neonatal hypocalcaemia is often asymptomatic.⁸ When symptoms and signs are present, they tend to be non-specific and mimic many other neonatal disorders such as hypoglycaemia, hypomagnesaemia, septicaemia, opiate withdrawal syndrome and anoxic brain injury.^15,16 From the foregoing, it is obvious that there is a need for mandatory serum calcium determination in high-risk neonates to improve their perinatal health. Indeed, Speidel et al.⁴ recommended that serum calcium concentration should be determined in all newborn infants with encephalopathy. However, serial serum calcium concentration (ideal) determination may not be feasible in our society where poverty is widespread and parents have to pay from their pockets for medical care of their children.

A report from Ilesa in Nigeria indicate that the incidence of severe birth asphyxia varied from 93.7 to 100.2 per 1000 admissions, with a case fatality rate of 19.2% to 22.4%.¹⁷ Another Nigerian study reported that 27.0% of their asphyxiated neonates were severe with a case fatality rate of 15.7% and accounted for 25.7% of their overall neonatal deaths.¹⁸ It may, therefore, be surmised that additional presence of hypocalcaemia is likely to further increase the case fatality rate of severe birth asphyxia. This study sought to determine the prevalence of early-onset neonatal hypocalcaemia and highlight its associated risk factors and predictors. The knowledge gained will promote clinical suspicion, early diagnosis and prompt treatment, thereby improving the outcome of severe birth asphyxia.

MATERIAL AND METHODS

The study population consisted of full-term neonates with severe birth asphyxia (1-minute Apgar score of 3 or less) delivered at St. Philomena Catholic Hospital (SPCH), Benin City between June 1, 2007, and May 31, 2008. The control infants were comparable but without birth asphyxia (1-minute Apgar score of 7 or more) and were delivered in the same hospital during the same period. All study infants had birthweight appropriate for gestational age. The study design was approved by the hospital authority and consent was also obtained from the mothers after explaining to them that infants found to have low serum calcium concentration will have it corrected by administration of calcium. Exclusion criteria included (i) death within 48 hours of age; (ii) infants of diabetic mother; (iii) infants who required exchange blood transfusion; (iv) infants on frusemide (lasix) and (v) admission after age of 48 hours. Infants with severe birth asphyxia were matched with control infants by sex and birthweight. Using O-cresulphthalein Complexone Method of Baginski et al.,¹⁹ serial total serum calcium concentration were determined at 12, 24 and 48 hours of age. Thus, three samples per subject was analysed for total serum calcium. To avoid venous stasis and eliminate artefactual haemoconcentration, blood sample was collected (without applying a tourniquet) using the open-ended needle method recommended by Wilkinson and Calvert.²⁰ Each of the blood samples was processed within 24 hours of collection. Infants with two sequential total serum calcium values less than 1.75 mmol/L were considered to have significant hypocalcaemia and were treated with intravenous or oral calcium gluconate. No calcium supplement was given before the first serum calcium measurement. The corresponding serum albumin concentration was determined for each blood sample used for serum calcium measurement. Maternal age and parity as well as the infants’ birthweight were recorded. Whether or not sodium bicarbonate was administered during resuscitation was noted. Infants with severe birth asphyxia were examined serially from the time of birth and compared with their counterparts who did not have birth asphyxia and the clinical manifestations in both groups were documented. Infants with 1-minute Apgar score of 6 and below were deemed to have birth asphyxia. Student's t test was used in assessing the significance of the results, which was set at P < .05.

RESULTS

One hundred and twenty-nine (9.5%) of 1364 live-births had 1-minute Apgar score of 6 or less. Of the 129 infants, 38 (29.5%) had 1-minute Apgar score of 3 or less (severe birth asphyxia) and they constituted the study population. More males (61.1%) than females (38.9%) suffered severe birth asphyxia with a ratio of 1.6:1. Seven (18.4%) of the 38 severely asphyxiated babies died within the first 48 hours of life, leaving 31 babies for further analysis. The mean maternal age of severely asphyxiated and non-asphyxiated infants was 24.2±0.9 years (95% confidence interval, CI = 23.9–24.5) versus 24.8±0.7 years (95% CI = 26.4–25.0) t-statistic = 2.93 P<.05 respectively. The mean maternal parity was 2.9±1.1 (95% CI = 2.5–3.3) for severely asphyxiated infants and 2.3±1.4 (95% CI = 1.8–2.8) for non-asphyxiated infants (t-statistic = 1.88 P>.05). Comparing total duration of labour in mothers of infants with severe birth asphyxia and those whose infants did not have birth asphyxia, it was 8.2±1.2. hours (95% CI = 7.8–8.6) versus 6.9±0.8 hours (95% CI = 6.6–7.2); t-statistic 5.02 P<0.01. Further details on maternal characteristics are shown in Table 1. More male than female study infants had hypocalcaemia in the ratio of 1.5:1. Mean birthweight of severely asphyxiated and non-asphyxiated infants was 3248±540 g versus 3304±327g t-statistic = 0.82 P>.05 respectively. At the age of 24 hours, the mean total serum calcium of nine asphyxiated infants who received sodium bicarbonate therapy compared with their counterparts who did not receive bicarbonate therapy was 1.65±0.07 mmol/L (95% CI = 1.61–1.68) and 1.73±0.06 mmol/L (95% CI = 1.70–1.75); t-statistic = 5.40 P<.01, respectively. Even at the age of 48 hours, the lower total serum calcium values persisted in the bicarbonate-treated infants (1.70±0.06 mmol/L). Although the numbers were small, comparing the frequency of hypocalcaemia in severely asphyxiated and non-asphyxiated infants, it was 22.6% (7/31) versus 9.7% (3/31) with an odd ratio of 2.7 (95% CI = 0.90–2.91). As shown in Table 2, total serum calcium levels at the ages of 12, 24 and 48 hours were very significantly lower (P < .001) in severely asphyxiated compared with their non-asphyxiated counterparts, with lowest value at age of 24 hours. Asphyxiated infants with normal total serum calcium at the age of 12 and 24 hours did not subsequently developed hypocalcaemia at 48 hours of age. The serum albumin levels were within normal range: 32.8–44.5 g/L with a mean value of 39.5±0.6 g/L.

As shown in Table 3, the leading clinical manifestations associated with hypocalcaemia in infants with birth asphyxia were convulsion, twitching of one or more extremities, high pitched cry and hypertonia. Carpopedal spasm was not a prominent sign.

DISCUSSION

In this study, the overall prevalence (22.6%) of early-onset neonatal hypocalcaemia among infants with severe birth asphyxia was three times higher than that reported by Omene and Diejomaoh¹⁴ among their asphyxiated infants. Their lower prevalence may be accounted for by the retrospective nature of their study and the fact that serum calcium was not measured in all their study population. In this regard, some case records may be missing and some infants with hypocalcaemia may also be missed, leading to the lower prevalence reported in that study.

Data from the present study showed that infants with 1-minute Apgar scores of 3 or less had significantly lower mean total serum calcium concentration than their counterparts with 1-minute Apgar scores of 7 or more. Tsang et al.¹³ have reported similar findings. This implies that birth asphyxia plays a separate role in early neonatal calcium homeostasis. It has been speculated that phosphate released from cell necrosis, transient functional hypoparathyroidism and hypercalcitoninaemia may be responsible.^2,5,8 It is also speculated that a relative hyperphosphataemia secondary to increased circulating endogenous phosphorus following postasphyxial renal impairment may contribute to the early-onset hypocalcaemia seen in infants with severe birth asphyxia.¹¹

It is intriguing to note that our asphyxiated infants with normal total serum calcium at 12 and 24 hours of age did not develop hypocalcemia at the age of 48 hours. The clinical implication is that the total serum calcium at the age of 12 hours may be useful in predicting the likelihood of subsequent development of hypocalcaemia in infants with birth asphyxia.

In the present study, as in a previous one,¹³ infants who had sodium bicarbonate therapy during resuscitation tended to have significantly lower serum calcium level compared to their counterparts who did not have sodium bicarbonate therapy. Correction of acidosis by administration of sodium bicarbonate is believed to be associated with movement of calcium from blood to bone, resulting in hypocalcaemia.²¹ The adverse effect of bicarbonate administration on serum calcium level reported in the present study is supported by the reports of several other studies, which have indicated that bicarbonate therapy during neonatal resuscitation was not only useless but also detrimental to the asphyxiated neonate.^11,22,23

In this study, the four leading signs associated with hypocalcaemia in infant with birth asphyxia were seizures, twitching of one or more extremities, high pitched cry and hypertonia. It is worthy of note that carpopedal spasm was not a prominent physical sign as is the case in hypocalcaemic infants without birth asphyxia. The clinical implication is that since the above signs are non-specific, serum calcium estimation should be carried out at the slightest suspicion and in all infants with severe birth asphyxia even when carpopedal spasm is absent.

Some limitations of the present study must be considered. Firstly, the use of Apgar score in defining birth asphyxia. The Apgar Scoring System,²⁴ though very useful in the measurement of birth asphyxia, has its shortcomings in that it does not fully define birth asphyxia.^25,26 It is known that factors (maternal medication) other than asphyxia may affect the Apgar score of an infant. However, in the review by Addy,²⁷ he noted that Apgar score was the basis of many papers on the outcome of birth asphyxia, justifying its use in the present study. Secondly, our inability to measure directly ionized serum calcium concentration and blood gases. This was due to lack of facility for their determination in our hospital. Future study will take this into consideration. Despite these limitations, the study gave an insight into the prevalence of the early-onset neonatal hypocalcaemia and factors that may predict the likelihood of its occurrence.

In conclusion, early-onset neonatal hypocalcaemia was common in neonates with severe birth asphyxia particularly among those infants who received bicarbonate therapy. Serum calcium level at the age of 12 hours predicted the likelihood of occurrence of hypocalcaemia in the first 48 hours of life. It is, therefore, suggested that measurement of serum calcium concentration at the age of 12 hours should be routinely performed for all neonates with severe birth asphyxia.

Disclosure: The authors declare no conflict of interest.

Acknowledgements: I am grateful to Dr O. Peters, Dr K. Eki-Idoko, Dr P. Omo-Idornijie and Senior Nursing Sister R. Igbinosa for their participation and contribution to the care of the patients used in this study. I also thank Mrs C. Iheagwara, the Medical Laboratory Scientist for working on the blood samples.

REFERENCES

1. Thilo EH, Rosenberg AA. The newborn infant. In: Hay WW Jr, Levin MH, Deterding RR, eds. Current Diagnosis and Treatment in Pediatrics. 18th ed. New York: McGraw Hill; 2007:1–64.
2. Sheldon RE, Venkataraman PS. Tetany. In: Nelson NM, ed. Current Therapy in Neonatal—Perinatal Medicine. Philadelphia: BC Decker Inc; 1990:364–6.
3. Rennie JM, Roberton NRC. A Manual of Neonatal Intensive Care. 4th ed. London: Arnold Publishers Limited; 2002:28–32.
4. Speidel B, Fleming P, Henderson J, et al. A Neonatal Vade Mecum. 3rd ed. London: Arnold Publishers Limited; 1998:282–3.
5. Salle BL, Delvin E, Glorieux F, David L. Human neonatal hypocalcaemia. Biol Neonate. 1990;58:22–31.
6. Jain A, Agarwal R, Sankar MJ, Deorari A, Paul VK. Hypocalcaemia in the newborn. Indian J Pediatr. 2010;77(10):1123–8.
7. Tsang RC, Oh W. Neonatal hypocalcaemia in low birth weight infants. Pediatrics. 1970;45:773–81.
8. Gowen CW Jr. Fetal and neonatal medicine. In: Kliegman RM, Marcdante KJ, Jenson HB, Behrman RE, eds. Nelson Essentials of Pediatrics. 5th ed. New Delhi: Elsevier Publishers Limited; 2006:271–335.
9. Zilva JF, Pannall PR, Mayne PD Clinical Chemistry in Diagnosis and Treatment. 5th ed. London: Edward Arnold; 1987:454–61.
10. Jennings CD. General clinical chemistry. In: Tietz NW, ed. Clinical Guide to Laboratory Tests. 2nd ed. Philadelphia: WB Saunders Company; 1990: 8–680.
11. Gomella TL, Cunningham MD, Eyal F. Calcium disorders (Hypocalcemia, Hypercalcemia). In: Gomella TL, ed. Neonatology: Management, Procedures, On-call Problems, Diseases and Drugs. 6th edn. New York: McGraw Hill; 2009:423–8.
12. Tsang RC, Light IJ, Sutherland JM, Kleiman LI. Possible pathogenetic factors in neonatal hypocalcaemia of prematurity. J Pediatr. 1973;82: 423–9.
13. Tsang RC, Chen I, Hayes W, Atkinson W, Akherton H, Edward N. Neonatal hypocalcaemia in infants with birth asphyxia. J Pediatr. 1974;84(3):428–33.
14. Omene JA, Diejomaoh FME. Analysis of 226 asphyxiated new born infants at the University of Benin Teaching Hospital, Benin City (1974– 1976). Nig J Paediatr. 1978;5:25–9.
15. Hochberg Z. Hypocalcaemia: neonatal hypocalcaemia. In: Hochberg Z, ed. Practical Alogorithms in Pediatric Endocrinology. Basel: Kerger (Publishers); 1999:68–9.
16. Minouni F, Tsang RC. Neonatal hypocalcaemia: To treat or not to treat. J Am Coll Nutr. 1994;13:408–15.
17. Ogunsi TA, Oseni SBA. Severe birth asphyxia in Wesley Guild Hospital, Ilesa: a persistent plague! Nig Med Pract. 2008;53(3):40–3.
18. Onyiriuka AN. Birth asphyxia in a mission hospital in Benin City, Nigeria. Trop J Obstet Gynaecol. 2006;23:34–9.
19. Baginski EX, Marie SS, Clark WL, Zak B. Microdetermination of serum calcium. Clin Chim Acta. 1973;46:49–54.
20. Wilkinson A, Calvert SA. Procedures. In: Rennie JM, Robertson WRC, eds. Textbook of Neonatology. 3rd ed. Edinburgh: Churchill Livingstone; 1992:1167–91.
21. Raisz LG. Physiologic-pharmacologic regulation of bone resorption. N Engl J Med. 1970;282:909–14.
22. Aschner JL, Poland R. Bicarbonate: basically useless therapy. Pediatrics. 2008;122:831–5.
23. Jajoo D, Kumar A, Shankar R, Bhargava V. Effects of birth asphyxia on serum calcium levels in neonates. Indian J Pediatr. 1995;62:455–9.
24. Apgar V. A proposal for a new method of evaluation of the newborn infant. Curr Res Anaesth Analgesia. 1953;32:260–7.
25. Ibe BC. Birth asphyxia and Apgar Score—a review. Nig Med Pract. 1990;20:111–3.
26. Sykes G, Molloy P, Johnson P. Do Apgar scores indicate asphyxia? Lancet. 1982;1:494–6.
27. Addy DP. Birth asphyxia. BMJ. 1982;284:1288–9.

Diabetic ketoacidosis (DKA) is a potentially life-threatening acute complication of type 1 diabetes mellitus (T1DM), characterized by a biochemical triad of hyperglycemia, ketonemia (ketonuria), and acidemia. The current criteria for diagnosis published by the International Society for Paediatric and Adolescent Diabetes (ISPAD) is blood glucose concentration greater than 11 mmol/L, blood pH less than 7.3 (serum bicarbonate level < 15 mmol/L), and ketonuria.¹ DKA is caused by a decrease in effective circulating insulin associated with elevations in counterregulatory hormones (glucagon, catecholamines, cortisol, growth hormone, and estrogen).^2,3 The severity of DKA is classified based on the degree of acidosis into mild, venous pH 7.2–7.3 (bicarbonate 15–18 mmol/L); moderate, pH 7.1– < 7.2 (bicarbonate 10–14 mmol/L); and severe, pH < 7.1 (bicarbonate < 10 mmol/L).^1,3 DKA, together with its major complication of cerebral edema, is the leading cause of mortality and morbidity in pediatric cases of diabetes, particularly at the time of first presentation.^2,4

About 25%–40% of children with T1DM present for the first time with DKA.⁵ Levy-Marchal et al.⁶ have reported some geographic variation of presentation at diagnosis of pediatric type 1 diabetes. In that study, they stated that, in populations with low overall prevalence of childhood type 1 diabetes, symptoms of diabetes may be less familiar to medical practitioners; consequently, patients more frequently present with DKA as the initial manifestation of diabetes. Reports from some countries suggest a higher frequency of DKA at the time of diagnosis of T1DM. For instance, it is 55.3% and 80.0% in Saudi Arabia and the United Arab Emirates, respectively.^7,8 Frequency of DKA at the time of diagnosis of T1DM is as high as 80%–88% in some African countries. ^9–11 The reason why some children present with DKA at the onset of T1DM and others do not is unclear. In this regard, whether the development of DKA is a consequence of delayed diagnosis and treatment or whether it reflects a particularly aggressive form of diabetes is not known with certainty.¹² Although the estimated mortality rates from pediatric DKA is low (range between 0.15% and 0.31%), DKA is the most frequent cause of diabetes-related death in children.^2,4

Understanding the magnitude of the problem of DKA at the new-onset of T1DM and the clinical characteristics of the patients has the potential of improving our knowledge of the disease, enhancing the development of patient-, professional-, and population-based interventions to reduce the proportion of children presenting with DKA at the time of first diagnosis of T1DM. The purpose of the present study, therefore, was to determine the frequency of DKA at the time of diagnosis of new cases of T1DM in children and adolescents and to describe the clinical characteristics of ketoacidosis among these patients seen at the University of Benin Teaching Hospital (UBTH), Benin City, Nigeria, between 1996 and 2011.

MATERIALS AND METHODS

In this retrospective study, the case records of all children and adolescents with T1DM complicated by DKA seen in the Department of Child Health, University UBTH, between 1996 and 2011 were retrieved and audited. Information extracted included age, sex, presenting features, laboratory findings, educational attainment of parents, and occupation of parents. The socioeconomic status of the patients’ parents was determined using the criteria suggested by Ogunlesi et al.¹³ This was analyzed via combining the highest educational attainment, occupation, and income of the parents (based on the mean income of each educational qualification and occupation). In this Social Classification System, Groups I and II represent high socioeconomic class, Group III represents middle socioeconomic class, while Groups IV and V represent low socioeconomic class. In this way, the subjects were categorized into high, middle, and low socioeconomic classes. Descriptive statistics such as frequencies, means, ratios, standard deviations, confidence intervals, and percentages were used to describe all the variables. The significance of differences in proportion was assessed with Z-score statistics while the differences in means was assessed with Student t test with P value set at <.05.

RESULTS

The study population came mainly from Edo State and the neighboring States of Delta, Ondo, and Kogi. During the period under review, of the 48 cases with new-onset T1DM, 37 (77.1%) [95% confidence interval (CI) = 76.2–78.0] presented with DKA at the time of initial diagnosis. Of these 37, 15 (40.5%) were males while 22 (59.5%) were females, giving a male-to-female ratio was 1:1.5. When comparing the frequency of DKA at the time of diagnosis of T1DM during the initial 8 years (1996–2003) with during the later 8 years (2004–2011), it was 81.8% (18/22) versus 73.1% (19/26), representing a decline of 8.7%; Z-statistic = 0.7268; P>.05. DKA was absent in 11 (22.9%) patients at the time of diagnosis of new-onset type 1 diabetes. The mean age at presentation in relation to gender was 11.4±3.9 years (95% CI = 9.4–13.4) for boys and 13.2±1.8 years (95% CI = 12.4–14.0) for girls; t = 1.67; P>.05. When both sexes were combined, the mean age at presentation was 12.7±2.6 years (95% CI = 11.9–13.5). The age and gender distribution of patients with DKA are displayed in Table 1. The clinical characteristics of the patients are depicted in Table 2. The serum electrolyte profile at the point of admission revealed that 27 (73.0%) of the 37 cases had serum bicarbonate level between 10 and 15 mmol/L, representing moderate DKA. The remaining 10 (27.0%) had serum bicarbonate level below 10 mmol/L, representing severe DKA. Over half (52.3%) of the families of the patients with DKA at initial presentation were of the middle social class while 11.2% were of the high social class and 36.5% were of the low social class. Twenty-four (64.9%) of the 37 patients with DKA had symptoms for 2–3 weeks before presentation with seven (29.2%) of the 24 having had at least one medical consultation during the period in a private clinic. Two (8.3%) had more than one medical consultations during the same period. The duration of symptoms before presentation was as follows: less than 2 weeks in 10.9% of cases; 2–4 weeks in 46.4% of cases; 5–7 weeks in 27.3% of cases; and above 7 weeks in 15.4% of cases. The frequency of the presenting symptoms and signs is displayed in Figure. 1. Eight (21.6%) mothers of the 37 patients who presented with DKA at onset of T1DM admitted they did not know that diabetes mellitus could occur in children. Three (8.1%) of the 37 patients with DKA had positive family history of diabetes mellitus, involving two mothers and a maternal grandmother. Two (9.1%) of the 22 girls with DKA had delayed pubertal maturation: Tanner Stage II at the ages of 15 and 16 years, respectively. None of the two has attained menarche at the ages of 16 and 17 years, respectively. Their weights and body mass indices were 29 and 30 kg and 16.0 and 17.4 kg/m², respectively. One of the two girls had vaginal candidiasis. Data were not available on number of deaths at the onset of DM.

DISCUSSION

Over three-quarter (77.1%) of the children and adolescents in the present study manifested with DKA at the time of initial diagnosis of T1DM. This is not surprising as similar prevalence rates have been reported from Iran (76.0%) and the United Arab Emirates (80.0%).^8,14 The prevalence in the present study was lower than the 88.0% reported from Ebonyi State Teaching Hospital in Abakaliki, Nigeria.¹⁵ Some other studies have reported even lower prevalence rates, ranging from 37.7% to 55.3%,^7,16 all reflecting the well-known wide geographic variation in frequency of DKA at the onset of pediatric DM.³ The differing prevalence rates might be explained by differences in study population, the background prevalence of diabetes in the given population, presence or absence of family history of T1DM, family socioeconomic status, delayed diagnosis and treatment, as well as the definition of DKA used in the particular study. All these factors have been reported to influence the prevalence of DKA at the onset of type 1 diabetes.¹⁷ It is also possible that the older age of our patients (mean age, 12.7 years) may contribute to the observed higher prevalence. This view is supported by a report from Finland, which indicated a higher prevalence among patients aged 10–14 years (26.4%) compared to those aged 5–9 years (14.8%) with an overall prevalence of 19.4%.¹⁸ Secular changes in frequency of DKA at onset of T1DM has equally been reported.¹⁹ The clinical implication of some children presenting with DKA at onset of T1DM may be viewed from two perspectives as proposed by Neu et al.¹² It is possible that the development of DKA at the time of initial diagnosis of diabetes is a reflection of a delayed diagnosis and treatment. Alternatively, it might be a reflection of a particularly aggressive form of diabetes. Either way, it represents a poor prognostic factor. In this context, studies have shown that children with DKA at the time of initial diagnosis of diabetes have a poorer glycemic control,^16,19 less residual β-cell function (up to 2 years) after diagnosis,^19,20 and lower frequency of remission.^21,22

Data from the present study indicate that, over the years, the frequency of DKA at the time of first diagnosis of T1DM has not shown any significant decline when the frequency during the initial 8 years (1996–2003) of the 16-year review was compared with the frequency in the later 8 years (2004–2011). This finding is consistent with the report of Bui et al.²³ In contrast, a study in Northern Finland reported a significant decline in frequency (18.9% vs 29.5%) of DKA at the time of initial diagnosis of T1DM over the later 10 years of a 20-year review.¹⁹ The explanation is that the prevalence of DM is high in Finland, making the recognition of the disease easier by both the public and the physician. The clinical implication is that this indicator of diabetes care has not improved in parallel with our current clinical pediatric endocrine practice. This scenario suggests that greater efforts are needed to reduce the occurrence of DKA at the time of initial diagnosis of T1DM in children and adolescents in Nigeria. One possible approach is focusing on earlier diagnosis by improving on level of public awareness of the symptoms of diabetes and greater medical alertness to the occurrence of T1DM in children and adolescents. This view is supported by two different findings in the present study. First, nearly one-third (29.2%) of the patients who had symptoms for 2–3 weeks before presentation in DKA at diagnosis of new-onset T1DM had at least one medical consultation during that period in a private clinic, suggesting missed diagnosis and a window for intervention. Second, over one-fifth (21.6%) of the mothers whose children presented in DKA at the time of diagnosis of T1DM thought that DM was a disease exclusive to the adult population, suggesting the need to increase general public awareness of the symptoms of DM. The mean age (12.7 years) at presentation in the present study was significantly higher than that reported from some countries: Spain, 7.44 years²⁴; Saudi Arabia, 6.7 years⁷; and Iran, 7.53 years,¹⁴ suggesting that T1DM in children and adolescents presents at a relatively older age in Nigeria. The youngest child in our series was 5 years old. The reason for this difference is not clear. It might be related to racial variations.

In the present study, over one-quarter (27.0%) had severe form of DKA. This is in agreement with the frequency (25.9%) of severe DKA reported from Kuwait.¹⁶ On the other hand, the frequency of severe DKA observed in the present study is four- to five-fold higher than that reported from Finland.²⁵ The reason for this difference might be that the background higher prevalence of DM in Finland makes the recognition of the disease easier by both the public and the physician, reducing delays in diagnosis and treatment, ultimately preventing severe form of DKA. The older age of our patients might be contributory, given that the frequency of severe form of DKA has been shown to be higher in patients aged 10–14 years (5.9%) compared to patients aged 5–9 years (3.1%) in Finland.¹⁸ Consistent with the report of Habib,⁷ majority of the patients with impaired consciousness at presentation in the present study had severe form of DKA. In that study, it was revealed that there was a strong correlation between degree of acidosis and depression of the central nervous system.⁷

When compared with reports from developed countries of the west, ^3–6 data from the present study revealed some differences in clinical characteristics of the patients. For instance, the prevalence of DKA at diagnosis of new-onset T1DM, the mean age at presentation, the mean duration of symptoms before presentation, and the mean blood glucose at the point of admission were all higher in the present series, representing some noteworthy features of diabetes in children and adolescents in a developing country. On the other hand, reports from the Gulf countries such as Saudi Arabia, Kuwait, United Arab Emirates, and Iran are generally in agreement with the findings of the present study.^7,8,14,16,22 This finding is not surprising, considering that other studies have reported geographic variations in the clinical presentation of DM in children and adolescents.⁶ The implication is that the clinicians should be alert to these differences, depending on where they are practicing.

Some limitations of the present study that need be considered include the relatively small sample size from one center. Future multicenter study with a larger sample size is being designed to enhance the interpretation of results and ultimately strengthen conclusions.

In conclusion, at the time of initial diagnosis of T1DM, three in four children and adolescents presented with DKA and its frequency has not shown a significant decline over a 16-year period in UBTH. To further reduce the frequency of DKA at diagnosis of new-onset type 1 diabetes, increasing public awareness of early symptoms of diabetes and greater alertness on the part of clinicians are advocated.

Acknowledgements: We wish to appreciate the invaluable care provided to some of the patients used in this study as well as its documentation by late Dr R O Amiengheme. We equally thank Dr L C Onyiriuka (for assisting in identifying the relevant case files) and all the staff in the Medical Records Department, UBTH (for assisting in retrieving the case files).

Disclosure: The authors declare no conflict of interest.

REFERENCES

1. Wolfsdorf J, Craig M, Daneman D, et al. International Society for Paediatric and Adolescent Diabetes clinical practice consensus guidelines 2009: diabetic ketoacidosis. Pediatr Diabetes. 2009;10(Suppl 12):118–133.
2. Tasker RC, McClure RJ, Acerini CL. Oxford Handbook of Paediatrics. Oxford: Oxford University Press; 2008:405–458.
3. Wolfsdort J, Glaser N, Sperling MA. Diabetic ketoacidosis in infants, children, and adolescents: a consensus statement from the American Diabetes Association. Diabetes Care. 2006;29(5):1150–1159.
4. Edge JA, Ford-Adams ME, Dunger DB. Causes of death in children with insulin-dependent diabetes 1990–1996. Arch Dis Child. 1999;81:318–323.
5. Pinkney JH, Bingley PJ, Sawtell PA, Dunger DB, Gale EA. Presentation and progress of childhood diabetes mellitus: a prospective populationbased study: the Bart’s-Oxford Study Group. Diabetologia. 1994;37:70–74.
6. Levy-Marchal C, Patterson C, Green A. Geographic variation of presentation at diagnosis of type 1 diabetes in children: the EURODIAB study. Diabetologia. 2001;44:B75–B80.
7. Habib HS. Frequency and clinical characteristics of ketoacidosis as onset of childhood type 1 diabetes mellitus in Northwest Saudi Arabia. Saudi Med J. 2005;26(12):1936–1939.
8. Punnose J, Agarwal MM, El Khadir A, Devadas K, Mugamer IT. Childhood and adolescent diabetes mellitus in Arabs residing in the United Arab Emirates. Diabetes Res Clin Pract. 2002;55(1):29–33.
9. Akanji AO. Clinical experience with adolescent diabetes in a Nigerian teaching hospital. Natl Med Assoc. 1996;88:101–105.
10. Majaliwa ES, Munubhi E, Kaushik R, et al. Survey on acute and chronic complications in children and adolescent with Type 1 diabetes at Muhimbili National Hospital in Dar es Salaam, Tanzania. Diabetes Care. 2007;30:2187–2192.
11. Monabeka HG, Mbika-Cardorelle A, Moyen G. [Ketoacidosis in children and teenagers in Congo]. Sante. 2003;13:139–141.
12. Neu A, Chehalt S, Willasch A, Kehrer M, Hub R, Ranke MB. Varying clinical presentations at onset of type 1 diabetes mellitus in children— epidemiological evidence for different subtypes of disease? Pediatr Diabetes. 2001;2:147–153.
13. Ogunlesi TA, Dedeke IOF, Kuponiyi OT. Socio-economic classification of children attending specialist paediatric centres in Ogun State, Nigeria. Nig Med Pract. 2008;54(1):21–25.
14. Razavi Z. Frequency of ketoacidosis in newly diagnosed type 1 diabetic children. Oman Med J. 2010;25(2):114–117.
15. Ibekwe MU, Ibekwe RC. Pattern of type 1 diabetes mellitus in Abakaliki, Southeastern Nigeria. Paediatric Oncall [serial online] 2011 [cited July 1]; 8, Art. No 48. Available from: http://www.pediatriconcall.com fordoctor/ Medical original article/diabetes.asp.
16. Abdul-Rasoul M, Al-Mahdi M, Al-Quttan H, et al. Ketoacidosis at presentation of type 1 diabetes in children in Kuwait: frequency and clinical characteristics. Pediatr Diabetes. 2010;11(5):351–356.
17. Mallare JT, Cordice CC, Ryan BA, Carey DE, Kreitzer PM, Frank GR. Identifying risk factors for development of diabetic ketoacidosis in new onset type 1 diabetes mellitus. Clin Pediatr (Phila). 2003;42(7):591–597.
18. Hekkala A, Reunanen A, Koski M, Knip M, Veijola R. Age-related differences in frequency of ketoacidosis at diagnosis of type 1 diabetes in children and adolescents. Diabetes Care. 2010;33:1500–1502.
19. Hekkala A, Knip M, Veijola R. Ketoacidosis at diagnosis of type 1 diabetes in children in Northern Finland: temporary changes over 20 years. Diabetes Care. 2007;30(4):861–866.
20. Fernanddez Castaner M, Montana E, Camps I, et al. Ketoacidosis at diagnosis is predictive of lower residual beta-cell function and poor metabolic control in type 1 diabetes. Dibetetes Metab. 1996;22:349–355.
21. Bowden SA, Duck MM, Hoffman RP. Young children (B5 years) and adolescents (
12 years) with type 1 diabetes mellitus have lower rate of partial remissions: DKA is an important risk factor. Pediatr Diabetes. 2008;9:197–201.
22. Abdul-Rasoul M, Habib H, Al-Khouly M. ‘‘The honeymoon phase’’ in children with type 1 diabetes mellitus: frequency, duration and influential factors. Pediatr Diabetes. 2006;7:101–107.
23. Bui TP, Werther GA, Comeron FJ. Trends in DKA in childhood and adolescence: a 15-year experience. Pediatr Diabetes. 2002;3(2):82–88.
24. Oyarzabal Iriqoyen M, Garcia Cuartero B, Barrio Castellanos R, et al. Ketoacidosis at onset of type 1 diabetes mellitus in pediatric age group in Spain and review of the literature. Pediatr Endocrinol Rev. 2012;9(3): 669–671.
25. Mooney RA, Senn J, Cameron S, et al. Suppressors of cytokine signaling-1 and -6 associate with and inhibit the insulin receptor: a potential mechanism for cytokine-mediated insulin resistance. J Biol Chem. 2001;276(28):25889–25893.

Elevated maternal body mass index (BMI) is associated with higher prevalence of pregnancy-related complications for both the mother and her fetus during gestation, parturition, and in the immediate postpartum period.¹ Recently, overweight and obese women were shown to have altered lipid profiles during pregnancy compared to their normal-weight peers, suggesting that metabolic dysregulation associated with maternal overweight and obesity could partly mediate the increased risk of adverse outcomes found in these women.²

Pregnancy-related changes in lipid metabolism³ and in gestational diabetes mellitus (GDM)⁴ have been examined by other investigators. Women with GDM have significantly higher circulating triacylglycerol (TG) concentrations in the second trimester compared to women with normal glucose tolerance (NGT), suggesting that the expected hypertriglyceridemia of pregnancy occurs earlier in this group.⁵ This is in contrast with findings of Koukkou et al.,⁴ who found that increases in low-density lipoprotein (LDL)-cholesterol are blunted during pregnancy in GDM, leading to lower total cholesterol than in normal pregnancy.

Maternal serum TG during midpregnancy in GDM may help identify women likely to give birth to large-for-gestational age newborns.⁶ Moreover, maternal hyperlipidemia in GDM actively enhances the availability of lipids to the fetus, contributing to fat accumulation.⁷ With respect to long-term maternal outcomes, the risk of developing type 2 diabetes, impaired fasting glucose, or glucose intolerance in mothers with a previous GDM is increased 7-fold compared to that of a normoglycemic pregnancy.^8,9 Qureshi et al.¹⁰ investigated changes in lipids and lipoproteins throughout pregnancy. In the postpartum period, hyperlipidemFfolda including hypercholesterolemia and elevated TG was a common finding, which may markedly increase the long-term risk of cardiovascular disease and other vascular complications. Lipids usually improve in the postpartum period but little is known to what extent and how this related to metabolic status.

To our knowledge, there are no data on the associations between lipid changes from pregnancy to early postpartum and the metabolic profiles of women with and without GDM. Therefore, the objective of the present study was to examine changes in lipid profile from GDM screening to 2 months after pregnancy in relationship with insulin sensitivity and body fatness in women with GDM versus women with NGT.

METHODS AND SUBJECTS

Subjects

This pilot study included 48 pregnant women recruited from March 2008 to September 2009 at the Centre Mère-Enfant of the Centre Hospitalier Universitaire de Québec (CHUQ). Women were screened for GDM with a 75 g-oral glucose tolerance test (OGTT) in the morning after an overnight fast before the 32nd week of gestation. The analysis included 21 women diagnosed with GDM according to the Canadian Diabetes Association (CDA) guidelines¹¹ and a control group of 27 women with NGT. The GDM group was followed up according to the guidelines provided by the CDA,¹¹ that is, with glucose monitoring with glucose targets of fasting glycemia <5.3 mmol/l, 1-h postprandial glycemia <7.8 mmol/l, and 2-h postprandial glycemia <6.7 mmol/l. In the GDM group, nutritional therapy was implemented and insulin used if needed (57%). This study was approved by the CHUQ Ethics Review Board, and all subjects provided written informed consent.

Anthropometric Measurements

Height, weight, and skinfold thickness were determined at GDM screening and 2 months postpartum following standard procedures.¹² BMI was calculated from height measured to the nearest millimeter with a stadiometer, and body weight measured to the nearest 0.1 kg on a calibrated scale. Prepregnancy BMI was calculated from self-reported prepregnancy weight and measured height. Six measures of skinfold thicknesses (suprailiac, subscapular, midaxillary, medial calf, biceps, and triceps) were obtained with a Harpenden skinfold caliper following the procedures recommended by the International Biological Program.¹³ We calculated the sum of six skinfolds as an indicator of subcutaneous adiposity.

Measures of Glucose Homeostasis and Plasma Lipid-Lipoprotein Profile

A 75-g OGTT was performed in the morning after a 12-h overnight fast both at the time of GDM screening and 2 months postpartum. Blood samples were collected in EDTA-containing tubes through a venous catheter from an antecubital vein at −15, 0, 15, 30, 45, 60, 90, and 120 min for the determination of plasma glucose and insulin. Plasma glucose was measured enzymatically, whereas plasma insulin was measured by radioimmunoassay with polyethylene glycol separation.^14,15 Glucose and insulin responses were quantified using areas under the curves of glycemia and insulinemia over 120 min after glucose load. The Matsuda and Cederholm (insulin sensitivity) indices were also calculated.^16–18 On the morning of the OGTT, blood samples were collected in the fasting state to measure a complete lipid-lipoprotein profile.¹⁹ Cholesterol and TG concentrations were determined enzymatically in plasma and lipoprotein fractions with a Technicon RA-500 analyzer (Bayer Corporation, Inc., Tarrytown, NY). Plasma lipoprotein fractions (very low-density lipoprotein and LDL) and high-density lipoprotein (HDL) were isolated by sequential ultracentrifugations.

Statistical Analyses

Statistical analyses were performed using JMP software (SAS Institute, Cary, NC). Student's t tests were computed to compare women with GDM versus those with NGT. ANOVA for repeated measures was used to assess differences in the lipid profile between GDM screening measurements and postpartum measurements. Pearson correlation coefficients were calculated between insulin sensitivity indices derived from the OGTT and blood lipid concentrations. Pearson correlations were computed to quantify the associations between blood lipid concentrations and measures of adiposity or insulin resistance. The P value for significance was set at .05.

RESULTS

Subjects’ characteristics before, during, and after pregnancy are presented in Table 1. There were significant differences in maternal age, prepregnancy BMI (P<.05 for all), and near significant difference in prepregnancy weight (P=.06) between women with NGT and GDM. Women with GDM had a shorter duration of pregnancy (P<.05), but when adjusting for this difference, infant birth weight was similar in both groups. At GDM screening, BMI was significantly higher in GDM women, and at 2 months postpartum, BMI remained significantly higher. As expected, insulin sensitivity, estimated by Cederholm and Matsuda indices, was lower in women with GDM at the time of GDM screening (P<.0005), which remains different between groups for Cederholm index 2 months after delivery (P=.0006). Blood lipid and lipoprotein values were similar between groups, except for higher TG concentrations in women with GDM at GDM screening (2.22±0.42 vs 1.90±0.64, P=.05) and at 2 months postpartum (1.52±0.77 vs 1.05±0.77, P=.01).

Changes in blood lipid profile are shown in Figure 1. There was a parallel decrease in TG and HDL-chol levels in the NGT and GDM group, although TG levels remained higher in GDM patients at both the testing time points. The chol/HDL-chol increased significantly only in women with GDM. Total cholesterol decreased in both groups although this decrease was slightly more pronounced in women with NGT. These differences in the blood lipid changes of GDM compared to women with NGT from GDM screening to 2 months postpartum show that women with GDM presented lesser improvements in total cholesterol and chol/HDL-chol after pregnancy compared to women with NGT (P < .05, Table 2).

In women with GDM, changes in TG were inversely associated with changes in insulin sensitivity estimated by the Cederholm and Matsuda indices (r=−.67 and −.59, respectively, P<.05, Figures 2 A and 2B, respectively). Associations were found between changes in chol/HDL-chol and Matsuda (r=−.51) and between HDL-chol and Matsuda (r=.57, all P<.05, Figures 2 C and 2D, respectively).

In the entire group, positive correlations were observed between changes in TG (from GDM screening to 2 months postpartum) and self-reported prepregnancy BMI (r=.45), BMI at screening (r=.39), as well as with postpartum BMI (r=.59) or postpartum sum of skinfolds (r=.34, P<.05 for all). Correlations were also present between changes in HDL-chol (from GDM screening to 2 months postpartum) and self-reported prepregnancy BMI (r=−.28), BMI at screening (r=−.32), as well as with postpartum BMI (r=−.41) or postpartum Matsuda index (r=.34, P<.05 for all).

DISCUSSION

During pregnancy, there is a steady increase in plasma lipid levels.²⁰ Significant deviation from non-pregnant levels occurs mainly in the second and third trimesters.^3,21 Results of this pilot study have shown that transition from pregnancy to the postpartum state involves a clear improvement in blood lipid levels. Indeed, both groups showed a parallel decrease in many lipid fractions, with the exception of the chol/HDL-chol ratio in the GDM group. The latter variable remained stable in the NGT group while it was increased in the GDM group. This deterioration seemed attributable to a redistribution of cholesterol in various particles, which would be increased in the GDM group. Consistent with these findings, Retnakaran et al.²² have recently showed that women with GDM have a more atherogenic lipid profile than normal women by 3 months postpartum, with increased LDL and apoB levels.

We also documented the associations between changes in the lipid profile and body weight, insulin sensitivity indices, and metabolic parameters in women with and without GDM. We made two important observations: (1) women with GDM with the smallest improvements in the lipid profile also showed the lesser improvements in insulin sensitivity and (2) women with GDM or NGT who presented smaller improvements in the lipid profile also presented higher pre-, peri-, and postpartum adiposity as evaluated by anthropometric measurements. To our knowledge, it is the first time that early postpartum changes in lipid values are related to pregnancy anthropometry indices such as BMI and sum of skinfolds in young women with or without GDM. This suggests that the evaluation of early postpartum blood lipids as well as pre-, peri-, and postpregnancy anthropometry in this group may allow identification of those at risk for later metabolic complications, even when postpartum OGTT results are in the normal range. Longer-term studies in larger cohorts of patients will be required to support this notion.

In diabetic pregnancy, exaggerated hypertriglyceridemia has been found in the first,²³ second,^23,24 and third^4,23,24 trimesters of gestation compared to normal pregnancy, although there are also studies where no change in plasma TG levels were found.^25–27 Similar to others,^4,28 we have shown that women with GDM have higher serum TG compared to women with NGT, this being partly explained by increased insulin resistance. Contrary to Koukkou et al.,⁴ our patients with GDM did not present lower LDL-chol values.

Both maternal hyperglycemia and hypertriglyceridemia are frequent in diabetic mothers in association with a deteriorated insulin resistant state, and this condition is likely to enhance substrate availability for the fetus.^29,30 The association of lipid levels and diabetic status were assessed in the present pilot study. Changes in serum lipid levels from pregnancy to postpartum were evaluated, and we found that women with GDM presented lesser improvements in total cholesterol and chol/HDL-chol ratio after pregnancy compared to NGT. Recently, changes in lipid levels during pregnancy in type 1 and 2 diabetic subjects were assessed³¹ and, consistent with our findings, smaller changes in serum lipid concentrations occurred in type 2 diabetic mothers. The lipid and lipoprotein changes that we observe may appear subtle; yet, they may increase the risk of cardiovascular disease, especially with multiple pregnancies.³² For example, Meyers-Seifer and Vohr³³ showed that even 5–6 years after pregnancy, former GDM mothers had altered lipid levels with TG and HDL cholesterol levels correlating with other cardiovascular risk factors. In women aged between 40 and 60 years, although glycemia was not related to TG levels, current hyperinsulinemia did predict higher TG concentrations.³⁴

Our results also showed that improvements in the lipid profile (TG, chol/HDL-chol, and HDL-chol) were related to improvements in insulin sensitivity estimated by the Cederholm and Matsuda indices. To our knowledge, it is the first time that short-term changes in lipid values are related to changes in insulin sensitivity in a group of recently pregnant women. Although the precise mechanism is uncertain, alterations in the hormonal milieu during pregnancy may be responsible for reduced insulin sensitivity. Changes in β cell responsiveness occur in parallel with the growth of the fetoplacental unit and its elaboration of hormones.²⁰ On the other hand, in non-pregnant individuals, higher triglyceride levels reflect excess substrate availability and impaired capacity to handle and adequately store excess dietary energy, a condition that has been closely related to insulin resistance.³⁵ This mechanism may also be operative in obese, insulin-resistant, pregnant women.

A recent study of 90 mothers who had GDM or gestational impaired glucose tolerance in pregnancy and 99 women with NGT and their daughters underwent lifestyle assessment and metabolic tests 15-years after delivery.³⁶ Among study participants that remained normoglycemic at follow-up, those with a history of GDM were more insulin resistant, with a 40% greater risk for central adiposity, a 4-fold greater risk for elevated TG levels and elevated lipid profile relative to control subjects and an elevated risk for future pancreatic β-cell fatigue. High prepregnancy BMI is a strong predictor for GDM requiring insulin treatment³⁷ and is a critical risk factor for the subsequent development of diabetes and prediabetes,³⁸ which eliminates or at least attenuates the well-known protection against ischemic heart disease in women.³⁹ We suggest that the peripartum period offers a critical time when prevention strategies could impact on these young women's future health. We should aim at lowering BMI and body fat in women, possibly through aerobic activity in order to reduce cardiovascular disease risk.³⁴

In summary, our study of the associations between changes in lipid profile from pregnancy to 2 months postpartum showed that (1) the lipid profile of both women with GDM and NGT improves after delivery, but the improvement is less pronounced in women with GDM, (2) women with GDM with the smallest improvements in the lipid profile also showed the least improvements in insulin sensitivity, which seems to be at least partly related to higher prepregnancy BMI and postpartum overweight, and (3) women with GDM or NGT who presented smaller improvements in the lipid profile also presented higher pre-, peri-, and postpartum body adiposity, suggesting blood lipid changes are related to anthropometry in this group as it is in non-pregnant women, allowing the early identification of women at higher risk for later cardiometabolic diseases. We suggest that excess adiposity and dyslipidemia should be targeted early after delivery in order to reduce long-term metabolic disease risk in women of reproductive age. However, intervention studies will be required to evaluate the impact of such an approach.

Acknowledgements: We would like to thank all participants and study staff for their great help in the realisation of this pilot study. Anne-Sophie Morisset is the recipient of a Canada Graduate Scholarships Doctoral Awards from the Canadian Institutes of Health Research. André Tchernof is the recipient of a Senior Scholarship from the Fonds de la recherche en santé du Québec. We also would like to thank the late Dr André Nadeau for funding this project.

Disclosure: The authors have no conflict of interest.

REFERENCES

1. Heslehurst N, Simpson H, Ells L, et al. The impact of maternal BMI status on pregnancy outcomes with immediate short-term obstetric resource implications: a meta-analysis. Obes Rev. 2008;9(6):635–683.
2. Vahratian A, Misra VK, Trudeau S, Misra DP. Prepregnancy body mass index and gestational age-dependent changes in lipid levels during pregnancy. Obstet Gynecol. 2010;116(1):107–113.
3. Brizzi P, Tonolo G, Esposito F, et al. Lipoprotein metabolism during normal pregnancy. Am J Obstet Gynecol. 1999;181(2):430–434.
4. Koukkou E, Watts GF, Lowy C. Serum lipid, lipoprotein and apolipoprotein changes in gestational diabetes mellitus: a cross-sectional and prospective study. J Clin Pathol. 1996;49(8):634–637.
5. Toescu V, Nuttall SL, Martin U, et al. Changes in plasma lipids and markers of oxidative stress in normal pregnancy and pregnancies complicated by diabetes. Clin Sci. 2004;106(1):93–98.
6. Son GH, Kwon JY, Kim YH, Park YW. Maternal serum triglycerides as predictive factors for large-for-gestational age newborns in women with gestational diabetes mellitus. Acta Obstet Gynecol Scand. 2010;89(5):700– 704.
7. Schaefer-Graf UM, Graf K, Kulbacka I, et al. Maternal lipids as strong determinants of fetal environment and growth in pregnancies with gestational diabetes mellitus. Diabetes Care. 2008;31(9):1858–1863.
8. Bellamy L, Casas JP, Hingorani AD, Williams D. Type 2 diabetes mellitus after gestational diabetes: a systematic review and meta-analysis. Lancet. 2009;373(9677):1773–1779.
9. Kim C, Newton KM, Knopp RH. Gestational diabetes and the incidence of type 2 diabetes: a systematic review. Diabetes Care. 2002;25(10):1862– 1868.
10. Qureshi IA, Xi XR, Limbu YR, Bin HY, Chen MI. Hyperlipidaemia during normal pregnancy, parturition and lactation. Ann Acad Med Singapore. 1999;28(2):217–221.
11. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2008;31(Suppl. 1):S55–S60.
12. Lohman TG. Research progress in validation of laboratory methods of assessing body composition. Med Sci Sports Exerc. 1984;16(6):596–605.
13. Weiner JS, Lourie J. Human Biology: A Guide to Field Methods. Oxford, UK: Blackwell Scientific Publications; 1969.
14. Desbuquois B, Aurbach GD. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocrinol Metab. 1971;33(5):732–738.
15. Richterich R, Dauwalder H. Determination of plasma glucose by hexokinase-glucose-6-phosphate dehydrogenase method. Schweiz Med Wochenschr. 1971;101(17):615–618.
16. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 1985;28(7):412–419.
17. Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care. 1999;22(9):1462–1470.
18. Piche ME, Arcand-Bosse JF, Despres JP, Perusse L, Lemieux S, Weisnagel SJ. What is a normal glucose value? Differences in indexes of plasma glucose homeostasis in subjects with normal fasting glucose. Diabetes Care. 2004;27(10):2470–2477.
19. Major GC, Piche ME, Bergeron J, Weisnagel SJ, Nadeau A, Lemieux S. Energy expenditure from physical activity and the metabolic risk profile at menopause. Med Sci Sports Exerc. 2005;37(2):204–212.
20. Butte NF. Carbohydrate and lipid metabolism in pregnancy: normal compared with gestational diabetes mellitus. Am J Clin Nutr. 2000;71 (5 Suppl):1256S–1261S.
21. Lippi G, Albiero A, Montagnana M, et al. Lipid and lipoprotein profile in physiological pregnancy. Clin Lab. 2007;53(3–4):173–177.
22. Retnakaran R, Qi Y, Connelly PW, Sermer M, Hanley AJ, Zinman B. The graded relationship between glucose tolerance status in pregnancy and postpartum levels of low-density-lipoprotein cholesterol and apolipoprotein B in young women: implications for future cardiovascular risk. J Clin Endocrinol Metab. 2010;95(9):4345–4353.
23. Sanchez-Vera I, Bonet B, Viana M, et al. Changes in plasma lipids and increased low-density lipoprotein susceptibility to oxidation in pregnancies complicated by gestational diabetes: consequences of obesity. Metabolism. 2007;56(11):1527–1533.
24. Hollingsworth DR, Grundy SM. Pregnancy-associated hypertriglyceridemia in normal and diabetic women. Differences in insulindependent, non-insulin-dependent, and gestational diabetes. Diabetes. 1982;31(12):1092–1097.
25. Montelongo A, Lasuncion MA, Pallardo LF, Herrera E. Longitudinal study of plasma lipoproteins and hormones during pregnancy in normal and diabetic women. Diabetes. 1992;41(12):1651–1659.
26. Marseille-Tremblay C, Ethier-Chiasson M, Forest JC, et al. Impact of maternal circulating cholesterol and gestational diabetes mellitus on lipid metabolism in human term placenta. Mol Reprod Dev. 2008;75(6):1054–1062.
27. Rizzo M, Berneis K, Altinova AE, et al. Atherogenic lipoprotein phenotype and LDL size and subclasses in women with gestational diabetes. Diabet Med. 2008;25(12):1406–1411.
28. Metzger BE, Phelps RL, Freinkel N, Navickas IA. Effects of gestational diabetes on diurnal profiles of plasma glucose, lipids, and individual amino acids. Diabetes Care. 1980;3(3):402–409.
29. Freinkel N. Banting lecture 1980. Of pregnancy and progeny. Diabetes. 1980;29(12):1023–1035.
30. Herrera E, Amusquivar E, Lopez-Soldado I, Ortega H. Maternal lipid metabolism and placental lipid transfer. Horm Res. 2006;65(Suppl. 3): 59–64.
31. Gobl CS, Handisurya A, Klein K, et al. Changes in serum lipid levels during pregnancy in type 1 and type 2 diabetic subjects. Diabetes Care. 2010;33(9):2071–2073.
32. Ness RB, Harris T, Cobb J, et al. Number of pregnancies and the subsequent risk of cardiovascular disease. N Engl J Med. 1993;328(21):1528–1533.
33. Meyers-Seifer CH, Vohr BR. Lipid levels in former gestational diabetic mothers. Diabetes Care. 1996;19(12):1351–1356.
34. Schubert CM, Rogers NL, Remsberg KE, et al. Lipids, lipoproteins, lifestyle, adiposity and fat-free mass during middle age: the Fels Longitudinal Study. Int J Obes. 2006;30(2):251–260.
35. Frayn KN. Adipose tissue as a buffer for daily lipid flux. Diabetologia. 2002;45(9):1201–1210.
36. Egeland GM, Meltzer SJ. Following in mother’s footsteps? Motherdaughter risks for insulin resistance and cardiovascular disease 15 years after gestational diabetes. Diabet Med. 2010;27(3):257–265.
37. Ogonowski J, Miazgowski T, Kuczynska M, et al. Pregravid body mass index as a predictor of gestational diabetes mellitus. Diabet Med. 2009;26(4):334–338.
38. Lin CH, Wen SF, Wu YH, Huang YY, Huang MJ. The postpartum metabolic outcome of women with previous gestational diabetes mellitus. Chang Gung Med J. 2005;28(11):794–800.
39. Barrett-Connor E, Wingard D. Diabetes and heart disease in women. In: Eaker E, Packard B, Wenger N, eds. Coronary Heart Diease in Women. New York: Haymarket Doyms; 1987:190–194.

Insulin-induced oedema is a recognised but uncommon complication of insulin therapy especially in the paediatric age group (16 years and below).¹ At present, the pathogenesis is poorly understood and its epidemiology is unknown. It affects mainly patients with newly diagnosed type 1 diabetes mellitus or patients with poorly controlled diabetes mellitus after starting intensive insulin therapy or grossly underweight patients on large doses of insulin.^2,3 It has also been reported in patients with diabetic ketoacidosis.^4,5 Its clinical presentation is quiet variable ranging from mild peripheral oedema to cardiac failure and massive serosal effusions.⁶ DeFronzo et al. ^7–9 have attempted to clarify its underlying pathophysiological mechanism by demonstrating insulin-induced anti-natriuresis in man. Insulin-induced oedema is usually self-limiting but may occasionally progress to overt cardiac failure and development of pleural effusion.² Sometimes, too, the oedema may be gradually replaced by fat tissue with persistent weight gain.¹⁰

A survey of available standard textbooks of Paediatrics^1,1 as well as Paediatric Endocrinology¹² revealed that there is no reference to insulin in a discussion of differential diagnosis of oedema. In addition, oedema is not mentioned in the list of complications of insulin therapy. The lack of awareness among clinicians concerning insulin therapy in the differential diagnosis of oedema was further confirmed during an Update Course in Paediatrics for Resident Doctors from various training centres in Nigeria who were shown a case of insulin-induced oedema in an adolescent girl being treated for diabeticketoacidotic coma for discussion. None of the 30 candidates mentioned insulin-induced oedema in their discussion of the differential diagnosis of the oedema in this patient.

The purpose of this report is to review the scanty existing medical literature on the subject of insulin-induced oedema and raise awareness of clinicians about its continued occurrence.

CASE REPORT

The patient is a 15-year-old adolescent Nigerian girl who was diagnosed of type 1 diabetes mellitus 4 years ago. Compliance with therapy has been poor because of lack of support from the father and some misconception on the part of the patient and her mother, resulting in a poor glycaemic control. Before she defaulted from follow-up, she was on premixed combination of human insulin 70% intermediate-acting insulin plus 30% short-acting (regular) insulin given twice daily (20 units in the morning and 10 units in the evening).

The patient presented with abdominal pain and fever of 1 day duration, difficulty in breathing of 8 hours duration and loss of consciousness of 5 hours duration. No history of jaundice, chest pain, palpitation, cyanosis or urinary symptoms was found. She has not achieved menarche.

Physical examination revealed an acutely ill-looking adolescent girl who was pyrexial (temperature 39.6°C) and dehydrated. No facial puffiness or oedema of the limb was found. She was underweight (weighed 29 kg) with a sexual maturity rating of Tanner stage 1. She was unconscious (Glassgow coma scale of 3/15) with global hypotonia and hyporeflexia. Abdominal reflexes were absent. No evidence of diabetic retinopathy. Her pulse rate was 148/minute, regular and good volume. Blood pressure was 100/60 mmHg. Heart sounds were normal. No cardiac murmur was found. She was tachypnoeic (RR 34 cycles/minute) and dyspnoeic with acidotic breathing. Lung fields were clear on auscultation. Laboratory investigations showed blood glucose 18.2 mmol/L (327 mg/dl), ketonuria 3+, and acidosis (bicarbonate 10 mmol/L), confirming diabetic ketoacidosis. Serum electrolytes values were normal except for the bicarbonate. Serum creatinine level was 0.7 mg/dl. Urinalysis did not reveal any significant proteinuria or bilirubinuria. Urine glucose was 2+ and pH was 5. Full blood count, peripheral blood film and chest radiograph were essentially normal. Blood culture yielded no growth. Dehydration was corrected with normal saline (0.9% sodium chloride). She was given 0.9% sodium chloride 20 ml/kg (bolus) over the first 1 hour, then (85 ml/kg + 100% maintenance) minus fluid bolus over the remaining 23 hours. She was treated with insulin infusion at a rate of 0.1 unit/kg/h, and the standard protocol for management of diabetic ketoacidosis was followed.^11,12 Insulin infusion was discontinued on the second day of admission and replaced with subcutaneous insulin(soluble) at 1 unit/kg/day 6 hourly. Antibiotics I.V. Ceftriaxone and I.V. Gentamycin were administered. She regained consciousness between the fifth and sixth hour of treatment and the fever subsided on the fourth day of admission. On the fifth day on admission, she developed facial swelling that was followed 2 days later by pitting ankle and pretibial oedema. The weight had increased from 29 kg on admission to 34 kg. A repeat serum creatinine level was normal and a repeat urinalysis did not reveal proteinuria or bilirubinuria. Repeat urea and electrolytes revealed normal values, including bicarbonate level. Urine output remained adequate. Reassurance was given to the patient and her mother. The oedema resolved over the next 7 days without any specific medical intervention such as administration of diuretics. She discharged from the ward after spending 14 days on admission and is currently being followed up in Paediatric Endocrine metabolic clinic of the hospital.

DISCUSSION

The diagnosis of insulin-induced oedema in this patient was based on: (1) the exclusion of all other major causes of oedema; (2) its temporal relationship to improved glycaemic control and resolution of diabetes ketoacidosis; and (3) its benign nature as evidenced by its spontaneous resolution. For instance, in this patient, there was no clinical or laboratory evidence of renal, hepatic or cardiovascular disease. Oedema appeared following improved glycaemic control with insulin infusion, and the odema resolved spontaneously without any specific medical intervention, indicating its transient/benign nature.

Some of the epidemiological observations in the present case were in tandem with those of previous reports. For instance, she is an underweight adolescent girl with type 1 diabetes mellitus with ketoacidosis.^1,4,5 The natural outcome of insulin-induced oedema was observed in our patient because no specific medical intervention was instituted before the resolution of the oedema. This observation is in keeping with several other previous reports.^6,10,13,14 In contrast, there are also reports of resolution of oedema following specific therapies such as administration of loop diuretics¹ or ephedrine.⁴ However, the clinicians did not observe spontaneous resolution. The clinical implication is that in cases where the oedema fails to resolve spontaneously, loop diuretics or ephedrine may be administered. Similarly, Suzuki et al.¹⁵ reported four cases with diabetes mellitus due to the 3243 mitochondrial tRNA mutation who developed oedema of the lower extremities following glycaemic control, and their oedema responded to the administration of Co-enzyme Q 10. In the present case, facial puffiness was noticed on the fifth day of admission. Two days later, pitting ankle and pretibial oedema developed. The oedema resolved spontaneously 7 days later. A review of the literature revealed that duration of the insulin therapy before onset of oedema as well as duration of oedema before spontaneous resolution varied widely. For instance, the duration of insulin therapy before onset of oedema ranged from 1 to 14 days,^1,6,14 whereas the duration of oedema before resolution ranged from 4 to 10 days.^1,14 In the present case, both the duration (5 days) of insulin therapy before onset of oedema and duration (7 days) of oedema before resolution were keeping with previous reports.^1,6,14

Although insulin-induced oedema has been recognised for a long time as an uncommon complication of insulin therapy, its pathogenesis remains poorly understood, even today. Some authors have suggested that it may involve vasomotor changes induced by the rapid glycaemic control or that a rapid improvement of glycaemic control might have induced hepatic re-oxygenation, resulting in the production of reactive oxygen species in the liver that ultimately contribute to cell damage.¹⁵ Other authors think that insulin induces oedema through its effect on vascular permeability and renal tubules.¹⁴

In conclusion, there is a need to raise the awareness of clinicians with regard to the existence of insulin-induced oedema, especially with the current trend towards intensive insulin therapy. Clinicians should document its occurrence and include this entity in the differential diagnosis of oedema in children and adolescents with type 1 diabetes mellitus.

Disclosure: The authors declare no conflict of interest.

REFERENCES

1. Mamoulakis D, Bitsori M, Galanakis E, Raissaki M, Kalmanti M. Insuininduced oedema in children and adolescents. J Paediatr Child Health. 2006;42(10):655–657.
2. Cheliah A, Burge MR. Insulin edema in the twenty-first century: review of existing literature. J Investig Med. 2004;52(2):104–108..
3. Juliusson PB, Bjerknes R, Sovik O, Kvistad PH. Generalized edema following insulin treatment of newly diagnosed diabetes mellitus. Tidsskr Nor Laegeforen. 2001;121(8):919–920. Abstract. [Article in Norwegian].
4. Hopkins DF, Cotton SJ, Williams G. Effective treatment of insulininduced edema using ephedrine. Diabetes Care. 1993;16(7):1026–1028.
5. Khalangot ND, Koka MA, Latypova GA, Bakhtiiarova AA. Insulin edema in patients with diabetes mellitus and recent diabetic ketoacidosis (epidemiology and case reports). Lik Sprava. 2004;8:39–43.
6. Lee P, Kinsella J, Borkman M, Carter J. Bilateral pleural effusion, ascites, and facial and peripheral oedema in a 19-year-old woman two weeks following commencement of insulin lispro and determir—an unusual presentation of insulin oedema. Diabetic Med. 2007;24(11):1282–1285.
7. DeFronzo RA, Cooke RC, Andres R, Faloona GR, Davis PJ. The effect of insulin on renal handling of sodium, potassium, calcium, and phosphate in man. J Clin Investig. 1975;55:845–846.
8. DeFronzo RA. The effect of insulin on renal sodium metabolism: a review with clinical implications. Diabetologia. 1981;21:165–171.
9. DeFronzo RA, Goldberg M, Agus ZS. The effect of glucose and insulin on renal electrolyte transport. J Clin Investig. 1976;58:83–90.
10. Hirshberg B, Muszkat M, Marom T, Shalit M. Natural course of insulin oedema. J Endocrinol Investig. 2000;23(3):187–188.
11. Kliegman RM, Behrman RE, Jenson HB, Stanton BF, eds. Nelson Textbook of Pediatrics. 18th ed. Philadelphia, PA: Saunder Elsevier; 2007:1–3147.
12. Sperling MA. Diabetes mellitus. In: Kaplan SA, ed. Clinical Pediatric Endocrinology. 2nd ed. Philadelphia, PA: WB Saunders Company; 1990: 127–164.
13. Cosmi F, Bianco C, Mollaili M, Aimi M, Corbacelli C, Ricca M. Edematogenic syndrome in diabetics treated with insulin: a little-known complication. Minerva Med. 1986;77(5–6):171–174.
14. Ran TS, Nelli P, Rai P, Kundaje GN. Insulin oedema: an uncommon complication of insulin therapy. J Assoc Physicians India. 1991;39(5): 411–412.
15. Suzuki Y, Kadowaki H, Taniyama M, et al. Insulin oedema in diabetes mellitus associated with 3243 mitochondrial tRNALeu(UUR) mutation: case reports. Diabetes Res Clin Pract. 1995;29(2):137–142.

Moderate caloric restriction elicits a myriad of metabolic alterations that positively influence the prognosis of obesity and type 2 diabetes. A common factor in these aforementioned conditions is a progressive reduction in insulin sensitivity, defined in this review as the extent to which a specified quantity of insulin is able to affect metabolic and signaling events. Although often considered to be a negative outcome, insulin resistance may alternatively be viewed as an adaptation to an environmental stressor—an overabundance of nutrients in this case. Relief from said stress via a modest reduction in energy intake (while providing sufficient macro- and micronutrients for normal cellular function) increases insulin sensitivity in a rapid and sustainable manner. ^1–4 Deciphering the underlying mechanisms that drive this phenomenon has powerful implications for the development of therapies to counter obesity and type 2 diabetes. This review focuses on changes in the molecular mediators of insulin signaling in the context of obesity and caloric restriction, with an emphasis on the kinases that translate stress and nutrient signals into cellular, and ultimately, organism-wide events. A brief overview of insulin action, signaling, and the role of serine phosphorylation of insulin receptor substrate (IRS) proteins in insulin resistance will be followed by a detailed review of current research linking increased nutrient- and stress-related serine kinase activity with insulin resistance, and reduced serine kinase activity with moderate caloric restriction and improved insulin sensitivity.

Insulin action

In higher organisms, insulin is normally secreted in response to an influx of nutrients. In skeletal muscle, insulin stimulates glucose uptake and glycogen synthesis in addition to inducing a metabolic shift from oxidation of fatty acids to glucose (reviewed in⁵). In adipose tissue, insulin promotes lipid synthesis and uptake and inhibits lipolysis.⁶ Finally, in liver, insulin suppresses glucose and lipoprotein production and output^{7, 8} while stimulating glycogen synthesis.⁹ Therefore, the principal role of the hormone is to properly partition exogenous nutrients among the three primary insulin-responsive peripheral tissues by inducing nutrient uptake/storage while inhibiting endogenous nutrient release/production. These effects are acutely mediated through cellular signaling pathways and, in the long term, are subject to genomic regulation.

Insulin signaling

The key mediators of insulin signaling—the IRS proteins— reside upstream in the pathway, proximal to the insulin receptor. When insulin binds to its receptor, the tyrosine kinase activity of the receptor phosphorylates (activates) the IRS proteins on tyrosine residues, which in turn amplifies and transduces the insulin signal downstream. Conversely, serine phosphorylation of IRS proteins by a number of serine kinases generally antagonizes this action, attenuating propagation of the insulin signal.¹⁰ The critical role of the IRS proteins with respect to insulin function has been established in a number of in vitro and in vivo models utilizing knockout ^11–14 as well as siRNA-mediated knockdown;^{15, 16} a detailed review discussing the significance of the IRS proteins, as well as their structural and regulatory aspects, can be found in.¹⁰ Although there are several IRS isoforms, in this review, we focus on the ramifications of serine phosphorylation of IRS-1, as its regulation, phosphorylation sites, and role in insulin sensitivity are most well characterized. Given the central position of the IRS proteins in insulin action, it is not surprising that their regulation is governed by upstream, downstream, and cross-pathway feedback. Two signaling nodes of particular interest are the stress (MAPK) and nutrient-sensitive (mTOR/p70S6K) kinases, due to their potent inhibitory effects on insulin signaling,^3,17–20 their strong positive association with chronic energy excess, ^21–24 and an abundance of data showing that their inhibition or ablation can reverse or prevent insulin resistance.^{20, 23, 25, 26}

SERINE PHOSPHORYLATION OF IRS PROTEINS AND INSULIN RESISTANCE

Transgenic animal models, siRNA and adenovirus-mediated knockdown studies, and the use of specific kinase inhibitors provide substantial evidence to support a mechanism in which activation of serine kinases results in insulin resistance through IRS serine phosphorylation. ^{12–16 27} The exact mechanism(s) by which serine phosphorylation of IRS proteins leads to insulin resistance is/are not completely understood. A wealth of data indicates that IRS becomes a poor substrate for the insulin receptor upon serine phosphorylation; other potential mechanisms include serine phosphorylation-induced degradation of IRS protein via the ubiquitin–proteasome pathway and translocation of IRS proteins from the plasma membrane to the cytosol (reviewed in¹⁰).

At the molecular level, hyperinsulinemia,^{28, 29}, cytokines,^{30, 31}, growth factors,^{32, 33} oxidative stress,^{34, 35} free fatty acids,^{36, 37} hyperaminoacidemia,^{38, 39} and osmotic stress⁴⁰ have all been found to induce insulin resistance and enhance serine phosphorylation of IRS proteins. Physiologically, high-fat diets/overfeeding,^{23, 41} hypertension^{32, 42} and abnormal stressors (e.g., infection,⁴³ trauma,⁴⁴ and burn injuries⁴⁵) result in insulin resistance and IRS serine phosphorylation as well. These effectors signal through diverse upstream pathways, but share in common the activation of stress- and/or nutrient-sensitive kinases, which in turn directly and/or indirectly increase serine phosphorylation of the IRS proteins.

In the context of energy flux and insulin action, the serine kinases most potently induced by chronic nutrient excess— the MAPKs and mTOR/p70S6K—act as key determinants of insulin sensitivity. The ability of these kinases to serine phosphorylate IRS-1 is well documented; within the MAPKs, Erk-1/2 phosphorylate mouse and rat IRS-1 at serine 612/632 (S616/636 in humans), whereas JNK acts at serine 302/307 in mice and rats (serine 307/312 in humans). mTOR/p70S6K have been shown to target IRS-1 at serine 302 and S632/635 in mice and rats (serine 636/639 in humans) (reviewed in¹⁰). The regulation of these kinases and the significance of their effects on IRS-1 and insulin signaling will be discussed in detail in the following sections.

STRESS SIGNALING AND INSULIN RESISTANCE

The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that mediate cellular response to extracellular stress (i.e., oxidative stress, UV radiation, inflammatory hormones, etc.), growth factors (i.e., insulin, insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), etc.), and longchain saturated fatty acids (i.e., palmitate and stearate). There are three main classes of MAPKs, all of which are phosphorylated by upstream MAPK kinases: the extracellular-regulated kinases-1 and -2, the p38 MAPKs (a, b, d, and c), and the c-Jun amino-terminal kinases-1, -2, and -3 (also known as stressactivated protein kinases, SAPK). Erk-1/2, activated upstream by MEK1, seem to be the primary mediators of growth factor signaling. JNK 1–3, whose upstream kinase is MKK7, appear to mediate general stress response. The p38 MAPK family, directly downstream of MKK3/6, generally governs differentiation and development (for an excellent review, see⁴⁶). MAPK signaling may be initiated by a variety of cytoplasmic receptors, including receptor tyrosine kinases and G protein-coupled receptors; specificity is mediated in part by different scaffolding proteins which couple upstream receptor activation to MAPKKKs and MAPKKs. Furthermore, treatments that upregulate PKC, including palmitate and the diacylglycerol analog PMA or TPA, induce Erk-1/2 (and JNK) activity and lead to serine phosphorylation of IRS-1 at the known Erk-1/2 site (S612). ^47–49

We focus on Erk-1/2 in this review because of our observation that caloric restriction results in drastic changes in the activity of Erk-1/2, but not JNK or p38 MAPK.³ The role of JNK in insulin resistance, particularly that associated with high-fat feeding, is well established, and the reader is directed to a recent review.⁵⁰ Furthermore, in the majority of studies, Erk-1/2 and JNK activation occurs in parallel, with only a few studies (in which both kinases were investigated) showing activation of one kinase but not the other.^{3, 51} The p38 MAPK appears to have a role in insulin resistance as well,^{28, 34, 52, 53} but the lack of a well-defined phenotype in the knockout models of p38 precludes its discussion in this review.

Erk-1/2 and insulin resistance

Erk-1/2 has been found to be upregulated in several in vivo models of insulin resistance related to nutrient excess. The hyperglycemic, hyperinsulinemic ob/ob mouse has severalfold higher basal hepatic Erk-1/2 phosphorylation.⁵¹ Aging, which has been shown to decrease the efficiency of nutrient partitioning,⁵⁴ is also associated with enhanced Erk-1/2 phosphorylation, which is increased in older, insulin-resistant mice compared with younger, more sensitive controls.⁵⁵ Myotubes isolated from type 2 diabetic humans have reduced insulin-stimulated PI3K activity and significantly higher basal Erk-1/2 phosphorylation,⁵⁶ and myotubes from subjects with polycystic ovary syndrome (PCOS; an endocrine disorder featuring hyperandrogenism and insulin resistance, among other abnormalities) have increased MEK1 (the upstream kinase for Erk-1/2) and Erk-1/2 phosphorylation;⁵⁷ the same pattern can be observed in adipocytes isolated from type 2 diabetics.²⁸ The fact that Erk-1/2 activity is strongly enhanced in these diverse insulin-resistant models suggests that its role may be (at least partially) causative, rather than simply correlative.

In vitro studies of insulin resistance support the in vivo observations with respect to Erk-1/2. Treatment of many different cell types with growth factors, cytokines, and free fatty acids known to antagonize insulin resistance, including angiotensin II (AT II),^{32, 58} tumor necrosis factor (TNF)a,²⁶ C-reactive protein,³¹ prostaglandin E2,⁵⁹ palmitate,^{49, 60} and (chronic) insulin enhances Erk-1/2 phosphorylation. In the majority of these studies, the serine phosphorylation of IRS-1 at the known Erk target site (S616/636 in humans and S612/632 in mice and rats) is enhanced as well. The mechanism underlying AT II-, cytokine-, and prostaglandin-induced resistance appears to be activation of cognate receptors with the resulting signaling cascade leading to Erk-1/2 (or JNK) activation; receptor transactivation may also play a role.³² Palmitate, however, appears to induce stress signaling via PKC activation secondary to MLK and MKK upregulation.⁴⁸ Additional studies support the ability of fatty acids to activate Erk-1/2; Erk-2 immunoprecipitated from PMA (a diacylglycerol analog commonly used to activate PKC)-treated cells is able to serine phosphorylate an IRS-1 peptide containing the S612 site.⁴⁷ A subsequent study using an IRS-1 S612A mutant (unphosphorylatable) confirms that activation of PKC is sufficient to induce phosphorylation of IRS-1 S612.⁴⁹ These findings show that nutrient and stress signals are potent inducers of insulin resistance in a controlled in vitro environment, enhance Erk-1/2 activity in parallel with their ability to enhance serine phosphorylation of IRS-1, and antagonize insulin signaling; furthermore, the ability of Erk to directly phosphorylate an IRS-1 fragment at a key serine site argues in favor of a mechanistic role for this kinase.

Erk-1/2 and insulin sensitivity

Just as Erk-1/2 activity positively associates with obesity, diabetes, and stress factor-induced insulin resistance, a decrease in Erk-1/2 phosphorylation strongly correlates with insulin sensitivity. Recently, it was discovered that Zucker fatty rats placed on a calorie-restricted diet have dramatically reduced hepatic Erk-1/2 activity compared with ad libitum-fed controls, with a concomitant reduction in serine phosphorylation of IRS-1 at S612 and S632, known Erk-1/2 target sites in rats.³ Likewise, caloric restriction is able to bring Erk-1/2 activity in aged mice down to the levels seen in young controls.⁵⁵ When myotubes isolated from type 2 diabetics are treated with PD98059, an inhibitor of MEK1, Erk-1/2 phosphorylation is almost completely suppressed, and IRS-1 S636 phosphorylation significantly reduced;⁵⁶ a MEK1 inhibitor was shown to have similar effects on myotubes from PCOS subjects, restoring the association of PI3K with IRS-1.⁵⁷ Finally, the Erk-1 knockout mouse, which is resistant to diet-induced obesity and insulin resistance, lends support for a mechanistic and not a merely associative role for Erk in mediating insulin sensitivity in vivo.²⁰

In vitro, insulin-stimulated Akt phosphorylation in endothelial cells made resistant with angiotensin II is restored by PD98059, and the ATII-induced IRS-1 S616 phosphorylation is prevented.^{32, 58} Twenty-four-hour TNFa treatment causes a marked decrease in insulin receptor and IRS protein in 3T3-L1 adipocytes, which can be reversed by coincubation with PD98059.²⁶ In L6 myotubes, the C-reactive protein-incited reduction in insulin-stimulated Akt and GKS3b phosphorylation, as well as glycogen synthesis, is reversed by a MEK1 inhibitor; in line with this, IRS-1 S612 phosphorylation is reduced by more than 80%.³¹ In the HepG2 cell line, prostaglandin E2 suppression of insulin-stimulated Akt phosphorylation and serine phosphorylation of S636 can be abrogated by inhibition of MEK1.⁵⁹ Palmitate activates both MEK1 and JNK, and this can be reversed by PD98059.⁶⁰ PMA treatment of 293 cells induces Erk-2 activity and IRS-1 S612 phosphorylation, which is also abrogated by a MEK1 inhibitor.⁴⁷ Hyperinsulinemia in 3T3-L1 cells augments IRS-1 S612 phosphorylation, which again can be prevented almost entirely by PD98059.²⁹ Finally, when serine 612 is mutated to alanine in an IRS-1 fragment, the resulting peptide can no longer be phosphorylated (at residue 612); however, the mutant IRS-1 is capable of being tyrosine phosphorylated by the insulin receptor and signals normally to PI3K, even in the presence of PMA.⁴⁹ Thus, experiments in cultured cells are consistent with in vivo findings: that is, suppression of Erk-1/2, whether by pharmacological or molecular means, reverses or prevents IRS-1 serine phosphorylation and insulin resistance.

In summary, obesity and type 2 diabetes, two conditions characterized by chronic nutrient excess, lead to elevated serum levels of cytokines, growth factors, free fatty acids, glucose, insulin, and hypertension-associated hormones. These factors have been shown to induce insulin resistance, with concomitant elevation in Erk-1/2 activity and, when examined, serine phosphorylation of IRS-1. Ablation of Erk-1/2 through wholebody knockout, siRNA, or inhibitors is able to prevent and/or reverse these deleterious effects on insulin action and signaling. Furthermore, treatments that prevent or reverse insulin resistance have the same abrogating effect on the known Erk-1/2 serine phosphorylation sites in IRS-1. Finally, site-directed mutation of a primary Erk-1/2 IRS-1 phosphorylation site permits normal downstream insulin signaling in the presence of a resistance-inducing diacylglycerol analog. The sum of these findings suggests that factors that activate Erk-1/2 induce insulin resistance through site-specific serine phosphorylation of IRS proteins, and imply that inhibition of said phosphorylation events might at least partially abrogate insulin resistance secondary to Erk-1/2 activation.

NUTRIENT SIGNALING AND INSULIN RESISTANCE

There are two principal intracellular ‘‘sensors’’ of energy status: the mammalian target of rapamycin, or mTOR, which exists in two complexes, mTORC-1 and mTORC-2, and the AMP-activated protein kinase (AMPK). The role of AMPK in energy sensing and insulin resistance is well established, but a comprehensive evaluation of its structure and regulation is beyond the scope of this review. Thus, AMPK will be discussed only within the context of its role in mTOR regulation; interested readers are directed to recent reviews.^{61, 62}

mTOR governs protein synthesis, cell growth, cytoskeleton organization, and cell survival and proliferation (see^{63, 64} for recent reviews) and has been proposed to be a nutrient sensor.⁶⁴ mTOR is activated by a high ratio of ATP:AMP, amino acids (in particular the branched-chain amino acids, leucine, valine, and isoleucine), and growth factors, including insulin.⁶⁴ A decrease in the cellular energy state inhibits mTOR, an effect that is almost certainly mediated by an increase in AMP leading to activation of AMPK, rather than a decrease in ATP leading to inactivation of mTOR. This results from the intracellular ratio of ATP:AMP, which is on the order of 100:1, in conjunction with the adenylate kinase reaction, which interconverts equimolar quantities of AMP and ATP to ADP and vice versa; this implies that changes in AMP are of greater relative significance than changes in ATP.⁶⁵ Thus, when cellular AMP levels rise, AMPK becomes activated and phosphorylates the TSC-1/2 complex as well as the mTORC-1 scaffolding protein Raptor, the net result of which is inhibition of the mTORC-1 complex.⁶⁶

mTOR is the catalytic component of two multiprotein complexes, mTORC-1 and mTORC-2. The mTORC-1 complex is composed of four main components: mTOR, the catalytic subunit, Raptor, a scaffolding protein, mLST8 (also known as GbL), a binding protein, and PRAS40, a regulatory protein.⁶⁶ The mTORC-2 complex also contains mTOR and mLST8, with Rictor as a scaffolding protein and mSIN1 as a regulatory protein.^{66, 67} There are several key differences between the two complexes, in terms of both regulation and downstream targets. mTORC-1 is rapamycin sensitive and is linked to S6K1 and 4EBP-1 activation, whereas mTORC-2 is rapamycin insensitive and modulates cytoskeleton organization as well as acting as the putative PDK-2 activity leading to phosphorylation of Akt on serine 473.⁶⁸ The focus of this review will be on the mTORC-1 complex, on account of its ability to phosphorylate and activate p70S6K, a downstream mediator of protein synthesis that plays a crucial role in the attenuation of insulin signaling through serine phosphorylation of IRS-1. ^17–19

mTOR/p70S6K and insulin resistance

Hypercaloric, high-fat diets are commonly used to induce insulin resistance in mice, and also lead to several-fold higher p70S6K activation and IRS-1 phosphorylation at S632/635, an mTOR target site.^{21,, 23} High-fat diets would be expected to augment not only plasma glucose (and insulin) and fatty acid concentrations, but also amino acid levels, which when elevated, have been shown to induce insulin resistance in vitro and in vivo. High-fat diet-induced hyperaminoacidemia is mediated either by increased net food intake or by the sparing effect fatty acids and/or carbohydrates have on amino acid catabolism.^{69, 70} Recently, it was reported that high-fat/ high-branched-chain amino acid feeding induces a greater degree of resistance than high-fat feeding alone.⁷¹ This regimen elevates mTOR, p70S6K, and IRS-1 S302 phosphorylation (another known mTOR/p70S6K site in mice¹⁷). In humans, amino acid infusion elevates p70S6K and IRS-1 S612 and S636 phosphorylation.⁷² In further support of diet-induced activation of the mTOR/p70S6K signaling pathway and insulin resistance, p70S6K and IRS-1 serine 632/635 (the mouse equivalent of human S636/639) phosphorylation is significantly augmented in hyperphagic mouse models of obesity²³ and type 2 diabetes,^{23, 73} even when the animals are placed on a standard chow diet. A direct role for hyperlipidemia with respect to mTOR/p70S6K-mediated resistance is less well defined, with most studies focusing rather on the role of mTOR on lipid regulatory genes. These studies emphasize the close association between overfeeding and mTOR/p70S6K activation leading to serine phosphorylation of IRS-1 at the mTOR/p70S6K target sites and insulin resistance.

In vitro, treatments that induce insulin resistance, including angiotensin II³² and hyperinsulinemia,^{32, 74} particularly hyperinsulinemia in the presence of elevated leucine,⁷¹ have effects that parallel those seen in vivo. Glucose also plays a role in mTOR/p70S6K-induced insulin resistance, possibly through suppression of AMPK, as treatment of L6 myotubes with high glucose augments IRS-1 S636/639 phosphorylation and tempers insulin-stimulated Akt S308 phosphorylation in culture.¹⁷ The effects of elevated ATP:AMP ratios and mTOR/p70S6K activation leading to insulin resistance are difficult to evaluate independently of the numerous effects that ATP-depleting treatments have on intracellular metabolism; however, studies in which TSC-1/2 (negative regulators of mTORC and targets of AMPK) have been knocked out or knocked down^{18, 75} show a significant increase in mTOR/p70S6K activity, a decrease in insulin signaling efficiency, and an elevation in IRS-1 S302 and S636/639 phosphorylation. Likewise, when Rheb (a positive effector of mTOR) is constitutively overexpressed, IRS-1 S636/639 is enhanced and insulin signaling to Akt dampened.¹⁷ The in vitro findings point towards a primary role for mTOR/p70S6K in inducing insulin resistance; whether mTOR/p70S6K activity is augmented by treatment with individual nutrients or through suppression of inhibitory mTORC regulators, the resulting effect is insulin resistance coincident with site-specific IRS-1 serine phosphorylation.

mTOR/p70S6K and insulin sensitivity

In the same vein as Erk-1/2, the inhibition of p70S6K not only correlates with improved insulin sensitivity, but can act as the primary mediator of this effect. The p70S6K knockout mouse provides compelling support for this claim; the lack of p70S6K prevents weight and fat gain in the context of a highfat diet, preserves insulin action, maintains insulin signaling, and ablates serine 632/635 phosphorylation of IRS-1.²³ In mice fed a high-fat/high-branched-chain amino acid diet, rapamycin restores glucose clearance to the level of chow diet controls.⁷¹ Oral rapamycin, an immunosuppressant and chemotherapeutic commonly used to inhibit mTOR, successfully prevents IRS-1 serine phosphorylation in the skeletal muscle of humans infused with amino acids, and modestly increases the glucose infusion rate needed to clamp plasma glucose concentrations.⁷² Finally, in insulin-resistant Zucker fatty rats, hepatic IRS-1 S632/635 phosphorylation is among one of only two sites to show significant reduction following caloric restriction (the other being the Erk-1/2 site, S612).³ These data show that inhibition or ablation of mTOR/p70S6K signaling is sufficient to prevent or reverse insulin resistance in vivo.

The in vivo findings concerning mTOR/p70S6K and insulin sensitivity are largely paralleled by in vitro studies. When p70S6K is knocked down in HeLa cells, insulin-stimulated Akt phosphorylation increases concomitant with a significant reduction in IRS-1 S636/639 (the human equivalent of murine 632/635) phosphorylation,²³ providing direct evidence for the ability of mTOR/p70S6K signaling to influence insulin signaling even in a basal state. Additional support may be found in studies utilizing rapamycin: L6 myotubes made resistant by incubation with elevated concentrations of amino acids demonstrate normal insulin-stimulated glucose uptake and IRS-1-associated PI3K activity when coincubated with rapamycin.³⁹ The hyperinsulinemia-induced increase in total IRS-1 serine phosphorylation is abrogated by rapamycin in L6 myotubes¹⁷ and rat liver;¹⁹ in both instances, insulin-stimulated glucose disposal is restored as well. In myotubes coincubated with high glucose or high glucose plus leucine, rapamycin reverses IRS-1 S632/635 phosphorylation and restores insulin stimulation of Akt;¹⁷ similar effects may be observed in white adipose tissue²⁹ and 3T3-L1 adipocytes⁷⁴ exposed to hyperinsulinemia. Rapamycin significantly reduces or completely prevents IRS-1 serine 632/635 phosphorylation and restores insulin-stimulated Akt when TSC-1 and/or TSC-2 are deleted,^{18, 75} or when the mTOR agonist, Rheb, is constitutively expressed.¹⁷ Interestingly, rapamycin is as effective as a MEK1 inhibitor in preventing angiotensin II-induced insulin resistance in epithelial cells,³² and can also prevent serine phosphorylation of IRS-1 at residue 307, a classic JNK target, in TNFatreated hepatoma cells⁷⁶ and insulin-treated 3T3-L1 adipocytes.²⁹

Although the majority of studies use rapamycin to abrogate the impact of mTOR/p70S6K on insulin signaling, a recent study provides direct evidence that increasing AMPK activity may have similarly beneficial effects. Overexpression of a constitutively active AMPK abrogates glucose-induced IRS-1 S636/639 phosphorylation and inhibition of insulin-stimulated Akt S308 in HEK 293 cells.¹⁷ Similarly, overexpression of an active upstream activator of AMPK, LKB-1, reverses the effect of Rheb overexpression on IRS-1 S636/639. Finally, metformin, a pharmacological AMPK agonist and a front-line medication in the treatment of insulin resistance, is able to prevent insulinincited IRS-1 serine phosphorylation and restore insulin signaling towards Akt. An additional finding of interest is that treating 3T3-L1 adipocytes with 2-deoxyglucose, a non-metabolizable glucose analog commonly used to mimic energy deprivation (in vitro and in vivo), activates AMPK and ablates IRS-1 phosphorylation on S632/635.

In total, these data provide compelling evidence to support a mechanism by which activation of mTOR/p70S6K signaling (by general overfeeding or treatment with excessive quantities of individual nutrients), most likely via inhibition of AMPK secondary to changes in the ATP:AMP ratio, results in IRS-1 serine phosphorylation, inhibition of downstream insulin signaling, and insulin resistance. On the other hand, suppression of mTOR/p70S6K activity, whether by treatment with rapamycin or through activation of AMPK, reduces IRS-1 serine phosphorylation on the mTOR/p70S6K target sites, normalizes insulin signaling, and ameliorates resistance.

CONCLUSION

In vivo models support the central role of Erk-1/2 and mTOR/p70S6K in mediating insulin resistance: high-fat feeding strongly upregulates the activity of these kinases, whereas caloric restriction has the opposite effect; insulin sensitivity, in turn, inversely tracks the activity of Erk-1/2 and mTOR/p70S6K. Knockout animal models provide mechanistic evidence for the ability of these kinases to control insulin sensitivity, as the absence of either Erk-1/2 or p70S6K protects against high-fat diet-induced IRS-1 serine phosphorylation and insulin resistance. These data are supported by a wealth of in vitro studies demonstrating the close inverse association between Erk-1/2 and mTOR/p70S6K activity and insulin action, with targeted knockdown and site-directed mutagenesis experiments confirming the primary role of the kinases and site-specific IRS-1 serine phosphorylation in the determination of insulin signaling.

Stress- and nutrient-related signals induce insulin resistance through augmentation of the Erk-1/2 and p70S6K/mTOR signaling pathways (Fig. 1). An increase in the activity of the serine kinases, which govern these aforementioned pathways, results in serine phosphorylation of the IRS proteins at specific residues. This attenuates propagation of the insulin signal through mechanisms that are not completely defined. Evidence suggests that serine phosphorylation of IRS proteins precludes their association with the tyrosine kinase activity of the insulin receptor; as tyrosine phosphorylation of IRS is necessary for the recruitment of immediate downstream signaling molecules (i.e., PI3K), the entire signaling chain would be compromised. Serine phosphorylation additionally appears to antagonize the binding between IRS and downstream signaling partners, induces degradation of IRS, and may alter its cellular localization. IRS signaling to PI3KRAktRFoxo1/GLUT4/ GSK3B is largely responsible for insulin inhibition of hepatic glucose output, stimulation of glucose uptake/clearance, and storage of glucose as glycogen. Thus, any impairment in insulin signaling may result in systemic insulin resistance; this effect, in turn, becomes a causative agent in the worsening of resistance, due to the ability of various nutrients to antagonize insulin signaling—an effect largely secondary to upregulation of Erk-1/2 and mTOR/p70S6K activity.

Insulin signaling is a complex network of interconnected signaling nodes and, as such, it would be presumptuous to exclude the roles of other mediating kinases, phosphatases, secondary messengers, etc. in the regulation of insulin sensitivity. However, serine phosphorylation of IRS molecules emerges as a common theme in cellular insulin resistance: further understanding of the factors that determine the extent to which IRS proteins are serine phosphorylated may provide valuable insight into targeted approaches towards the treatment and prevention of insulin resistance.

Disclosure:We declare no conflicts of interest.

Acknowledgments: We would like to thank Dr. Matthew Brady for his constructive suggestions and insight.

REFERENCES

1. Lee CK, Klopp RG, Weindruch R, Prolla TA. Gene expression profile of aging and its retardation by caloric restriction. Science. 1999;285(5432):1390–1393.
2. Wang ZQ, Floyd ZE, Qin J, et al. Modulation of skeletal muscle insulin signaling with chronic caloric restriction in cynomolgus monkeys. Diabetes. 2009;58(7):1488–1498.
3. Zheng Y, Zhang W, Pendleton E, et al. Improved insulin sensitivity by calorie restriction is associated with reduction of ERK and p70S6K activities in the liver of obese Zucker rats. J Endocrinol. 2009;203(3): 337–347.
4. Bodkin NL, Ortmeyer HK, Hansen BC. Long-term dietary restriction in older-aged rhesus monkeys: effects on insulin resistance. J Gerontol A Biol Sci Med Sci. 1995;50(3):B142–B147.
5. Corpeleijn E, Saris WH, Blaak EE. Metabolic flexibility in the development of insulin resistance and type 2 diabetes: effects of lifestyle. Obes Rev. 2009;10(2):178–193.
6. Campbell PJ, Carlson MG, Hill JO, Nurjhan N. Regulation of free fatty acid metabolism by insulin in humans: role of lipolysis and reesterification. Am J Physiol. 1992;263(6 Pt 1):E1063–E1069.
7. Basu R, Chandramouli V, Dicke B, Landau B, Rizza R. Obesity and type 2 diabetes impair insulin-induced suppression of glycogenolysis as well as gluconeogenesis. Diabetes. 2005;54(7):1942–1948.
8. Pavlic M, Xiao C, Szeto L, Patterson BW, Lewis GF. Insulin acutely inhibits intestinal lipoprotein secretion in humans in part by suppressing plasma free fatty acids. Diabetes 2010 Mar;59(3):580–7.
9. Aiston S, Coghlan MP, Agius L. Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Eur J Biochem. 2003;270(13):2773–2781.
10. Sun XJ, Liu F. Phosphorylation of IRS proteins Yin-Yang regulation of insulin signaling. Vitam Horm. 2009;80:351–387.
11. Yamauchi T, Tobe K, Tamemoto H, et al. Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice. Mol Cell Biol. 1996;16(6):3074–3084.
12. Araki E, Lipes MA, Patti ME, et al. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature. 1994;372(6502):186–190.
13. Sadagurski M, Weingarten G, Rhodes CJ, White MF, Wertheimer E. Insulin receptor substrate 2 plays diverse cell-specific roles in the regulation of glucose transport. J Biol Chem. 2005;280(15):14536–14544.
14. Tamemoto H, Kadowaki T, Tobe K, et al. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature. 1994;372(6502):182–186.
15. Bouzakri K, Zachrisson A, Al-Khalili L, et al. siRNA-based gene silencing reveals specialized roles of IRS-1/Akt2 and IRS-2/Akt1 in glucose and lipid metabolism in human skeletal muscle. Cell Metab. 2006;4(1):89–96.
16. Taniguchi CM, Ueki K, Kahn R. Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism. J Clin Invest. 2005;115(3): 718–727.
17. Tzatsos A, Kandror KV. Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTOR-mediated insulin receptor substrate 1 phosphorylation. Mol Cell Biol. 2006;26(1):63–76.
18. Shah OJ, Wang Z, Hunter T. Inappropriate activation of the TSC/Rheb/ mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr Biol. 2004;14(18):1650–1656.
19. Ueno M, Carvalheira JB, Tambascia RC, et al. Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1/2 serine phosphorylation and the mTOR/p70 S6K pathway. Diabetologia. 2005;48(3):506–518.
20. Bost F, Aouadi M, Caron L, et al. The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis. Diabetes. 2005;54(2):402–411.
21. Khamzina L, Veilleux A, Bergeron S, Marette A. Increased activation of the mammalian target of rapamycin pathway in liver and skeletal muscle of obese rats: possible involvement in obesity-linked insulin resistance. Endocrinology. 2005;146(3):1473–1481.
22. Ono H, Pocai A, Wang Y, et al. Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats. J Clin Invest. 2008;118(8):2959–2968.
23. Um SH, Frigerio F, Watanabe M, et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature. 2004;431(7005):200–205.
24. Wang J, Ma H, Tong C, Zhang H, Lawlis GB, Li Y, et al. Overnutrition and maternal obesity in sheep pregnancy alter the JNK-IRS-1 signaling cascades and cardiac function in the fetal heart. FASEB J 2010 Jun;24(6):2066–76.
25. Adochio R, Leitner JW, Hedlund R, Draznin B. Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Aktmammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3-kinase and S6K1 kinase. Endocrinology. 2009;150(3):1165–1173.
26. Fujishiro M, Gotoh Y, Katagiri H, et al. Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes. Mol Endocrinol. 2003;17(3):487–497.
27. Kubota N, Kubota T, Itoh S, et al. Dynamic functional relay between insulin receptor substrate 1 and 2 in hepatic insulin signaling during fasting and feeding. Cell Metab. 2008;8(1):49–64.
28. Carlson CJ, Koterski S, Sciotti RJ, Poccard GB, Rondinone CM. Enhanced basal activation of mitogen-activated protein kinases in adipocytes from type 2 diabetes: potential role of p38 in the downregulation of GLUT4 expression. Diabetes. 2003;52(3):634–641.
29. Gual P, Gremeaux T, Gonzalez T, Le Marchand-Brustel Y, Tanti JF. MAP kinases and mTOR mediate insulin-induced phosphorylation of insulin receptor substrate-1 on serine residues 307, 612 and 632. Diabetologia. 2003;46(11):1532–1542.
30. Plomgaard P, Bouzakri K, Krogh-Madsen R, Mittendorfer B, Zierath JR, Pedersen BK. Tumor necrosis factor-alpha induces skeletal muscle insulin resistance in healthy human subjects via inhibition of Akt substrate 160 phosphorylation. Diabetes. 2005;54(10):2939–2945.
31. D’Alessandris C, Lauro R, Presta I, Sesti G. C-reactive protein induces phosphorylation of insulin receptor substrate-1 on Ser307 and Ser 612 in L6 myocytes, thereby impairing the insulin signalling pathway that promotes glucose transport. Diabetologia. 2007;50(4):840–849.
32. Arellano-Plancarte A, Hernandez-Aranda J, Catt KJ, Olivares-Reyes JA. Angiotensin-induced EGF receptor transactivation inhibits insulin signaling in C9 hepatic cells. Biochem Pharmacol. 2010;79(5):733–745.
33. Rui L, Aguirre V, Kim JK, et al. Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest. 2001;107(2):181–189.
34. Archuleta TL, Lemieux AM, Saengsirisuwan V, et al. Oxidant stressinduced loss of IRS-1 and IRS-2 proteins in rat skeletal muscle: role of p38 MAPK. Free Radic Biol Med. 2009;47(10):1486–1493.
35. Chen B, Wei J, Wang W, et al. Identification of signaling pathways involved in aberrant production of adipokines in adipocytes undergoing oxidative stress. Arch Med Res. 2009;40(4):241–248.
36. Jove M, Planavila A, Sanchez RM, Merlos M, Laguna JC, Vazquez-Carrera M. Palmitate induces tumor necrosis factor-alpha expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation. Endocrinology. 2006;147(1):552–561.
37. Yu C, Chen Y, Cline GW, et al. Mechanism by which fatty acids inhibit insulin activation of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem. 2002;277(52):50230–50236.
38. Patti ME, Brambilla E, Luzi L, Landaker EJ, Kahn CR. Bidirectional modulation of insulin action by amino acids. J Clin Invest. 1998;101(7):1519–1529.
39. Tremblay F, Marette A. Amino acid and insulin signaling via the mTOR/ p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. J Biol Chem. 2001;276(41):38052–38060.
40. Gual P, Gonzalez T, Gremeaux T, Barres R, Le Marchand-Brustel Y, Tanti JF. Hyperosmotic stress inhibits insulin receptor substrate-1 function by distinct mechanisms in 3T3-L1 adipocytes. J Biol Chem. 2003;278(29):26550–26557.
41. Danielsson A, Fagerholm S, Ost A, et al. Short-term overeating induces insulin resistance in fat cells in lean human subjects. Mol Med. 2009;15 (7– 8):228–234.
42. Sartori M, Ceolotto G, Papparella I, et al. Effects of angiotensin II and insulin on ERK1/2 activation in fibroblasts from hypertensive patients. Am J Hypertens. 2004;17(7):604–610.
43. Agwunobi AO, Reid C, Maycock P, Little RA, Carlson GL. Insulin resistance and substrate utilization in human endotoxemia. J Clin Endocrinol Metab. 2000;85(10):3770–3778.
44. Xu J, Kim HT, Ma Y, et al. Trauma and hemorrhage-induced acute hepatic insulin resistance: dominant role of tumor necrosis factor-alpha. Endocrinology. 2008;149(5):2369–2382.
45. Li L, Messina JL. Acute insulin resistance following injury. Trends Endocrinol Metab. 2009;20(9):429–435.
46. Aouadi M, Binetruy B, Caron L, Le Marchand-Brustel Y, Bost F. Role of MAPKs in development and differentiation: lessons from knockout mice. Biochimie. 2006;88(9):1091–1098.
47. De FK, Roth RA. Modulation of insulin receptor substrate-1 tyrosine phosphorylation and function by mitogen-activated protein kinase. J Biol Chem. 1997;272(50):31400–31406.
48. Jaeschke A, Davis RJ. Metabolic stress signaling mediated by mixedlineage kinases. Mol Cell. 2007;27(3):498–508.
49. De FK, Roth RA. Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612. Biochemistry. 1997;36(42):12939–12947.
50. Yang R, Trevillyan JM. c-Jun N-terminal kinase pathways in diabetes. Int J Biochem Cell Biol. 2008;40(12):2702–2706.
51. Gum RJ, Gaede LL, Heindel MA, et al. Antisense protein tyrosine phosphatase 1B reverses activation of p38 mitogen-activated protein kinase in liver of ob/ob mice. Mol Endocrinol. 2003;17(6):1131–1143.
52. Fujishiro M, Gotoh Y, Katagiri H, et al. MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression. J Biol Chem. 2001;276(23): 19800–19806.
53. Liu Z, Cao W. p38 mitogen-activated protein kinase: a critical node linking insulin resistance and cardiovascular diseases in type 2 diabetes mellitus. Endocr Metab Immune Disord Drug Targets. 2009;9(1):38–46.
54. Park S, Komatsu T, Hayashi H, et al. Calorie restriction initiated at middle age improved glucose tolerance without affecting age-related impairments of insulin signaling in rat skeletal muscle. Exp Gerontol. 2006;41(9):837–845.
55. Kim HJ, Jung KJ, Yu BP, Cho CG, Chung HY. Influence of aging and calorie restriction on MAPKs activity in rat kidney. Exp Gerontol. 2002;37(8–9):1041–1053.
56. Bouzakri K, Roques M, Gual P, et al. Reduced activation of phosphatidylinositol- 3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes. 2003;52(6):1319–1325.
57. Corbould A, Zhao H, Mirzoeva S, Aird F, Dunaif A. Enhanced mitogenic signaling in skeletal muscle of women with polycystic ovary syndrome. Diabetes. 2006;55(3):751–759.
58. Andreozzi F, Laratta E, Sciacqua A, Perticone F, Sesti G. Angiotensin II impairs the insulin signaling pathway promoting production of nitric oxide by inducing phosphorylation of insulin receptor substrate-1 on Ser312 and Ser616 in human umbilical vein endothelial cells. Circ Res. 2004;94(9):1211–1218.
59. Henkel J, Neuschafer-Rube F, Pathe-Neuschafer-Rube A, Puschel GP. Aggravation by prostaglandin E2 of interleukin-6-dependent insulin resistance in hepatocytes. Hepatology. 2009;50(3):781–790.
60. Coll T, Jove M, Rodriguez-Calvo R, Eyre E, Palomer X, Sanchez RM, et al. Palmitate-mediated downregulation of peroxisome proliferator-activated receptor-gamma coactivator 1alpha in skeletal muscle cells involves MEK1/2 and nuclear factor-kappaB activation. Diabetes. 2006;55(10):2779– 2787.
61. Zhang BB, Zhou G, Li C. AMPK: an emerging drug target for diabetes and the metabolic syndrome. Cell Metab. 2009;9(5):407–416.
62. Viollet B, Athea Y, Mounier R, et al. AMPK: Lessons from transgenic and knockout animals. Front Biosci. 2009;14:19–44.
63. Rivas DA, Lessard SJ, Coffey VG. mTOR function in skeletal muscle: a focal point for overnutrition and exercise. Appl Physiol Nutr Metab. 2009;34(5):807–816.
64. Sarbassov DD, Ali SM, Sabatini DM. Growing roles for the mTOR pathway. Curr Opin Cell Biol. 2005;17(6):596–603.
65. Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology. 2003;144(12): 5179–5183.
66. Gwinn DM, Shackelford DB, Egan DF, et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell. 2008;30(2):214–226.
67. Sabatini DM. mTOR and cancer: insights into a complex relationship. Nature Rev Cancer. 2006;6(9):729–734.
68. Yan L, Mieulet V, Lamb RF. mTORC2 is the hydrophobic motif kinase for SGK1. Biochem J. 2008;416(3):e19–e21.
69. Krotkiewski M. Value of VLCD supplementation with medium chain triglycerides. Int J Obes Relat Metab Disord. 2001;25(9):1393–1400.
70. Hart DW, Wolf SE, Zhang XJ, et al. Efficacy of a high-carbohydrate diet in catabolic illness. Crit Care Med. 2001;29(7):1318–1324.
71. Newgard CB, An J, Bain JR, et al. A branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. Cell Metab. 2009;9(4):311–326.
72. Krebs M, Brunmair B, Brehm A, et al. The Mammalian target of rapamycin pathway regulates nutrient-sensitive glucose uptake in man. Diabetes. 2007;56(6):1600–1607.
73. Koketsu Y, Sakoda H, Fujishiro M, et al. Hepatic overexpression of a dominant negative form of raptor enhances Akt phosphorylation and restores insulin sensitivity in K/KAy mice. Am J Physiol Endocrinol Metab. 2008;294(4):E719–E725.
74. Berg CE, Lavan BE, Rondinone CM. Rapamycin partially prevents insulin resistance induced by chronic insulin treatment. Biochem Biophys Res Commun. 2002;293(3):1021–1027.
75. Harrington LS, Findlay GM, Gray A, et al. The TSC1–2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J Cell Biol. 2004;166(2):213–223.
76. Bae EJ, Yang YM, Kim JW, Kim SG. Identification of a novel class of dithiolethiones that prevent hepatic insulin resistance via the adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway. Hepatology. 2007;46(3):730–739.