OBJECTIVEWe tested whether an elevation in the serum proinsulin–to–C-peptide ratio (PI:C), a biomarker of β-cell endoplasmic reticulum (ER) dysfunction, was associated with progression to type 1 diabetes.RESEARCH DESIGN AND METHODSFasting total PI and C levels were measured in banked serum samples obtained from TrialNet Pathway to Prevention (PTP) participants, a cohort of autoantibody-positive relatives without diabetes of individuals with type 1 diabetes. Samples were obtained ∼12 months before diabetes onset from PTP progressors in whom diabetes developed (n = 60), and were compared with age-, sex-, and BMI-matched nonprogressors who remained normoglycemic (n = 58). PI:C ratios were calculated as molar ratios and were multiplied by 100% to obtain PI levels as a percentage of C levels.RESULTSAlthough absolute PI levels did not differ between groups, PI:C ratios were significantly increased in antibody-positive subjects in whom there was progression to diabetes compared with nonprogressors (median 1.81% vs. 1.17%, P = 0.03). The difference between groups was most pronounced in subjects who were ≤10 years old, where the median progressor PI:C ratio was nearly triple that of nonprogressors; 90.0% of subjects in this age group within the upper PI:C quartile progressed to the development of diabetes. Logistic regression analysis, adjusted for age and BMI, demonstrated increased odds of progression for higher natural log PI:C ratio values (odds ratio 1.44, 95% CI 1.02, 2.05).CONCLUSIONSThese data suggest that β-cell ER dysfunction precedes type 1 diabetes onset, especially in younger children. Elevations in the serum PI:C ratio may have utility in predicting the onset of type 1 diabetes in the presymptomatic phase.

Streptozotocin (STZ) is a selective pancreatic β cell toxin used to generate experimental hyperglycemia in rodent models. Several laboratory animal protocols suggest that STZ be administered to fasted rodents to minimize competition between STZ and glucose for low affinity GLUT2 transporters on β cells. However, whether the diabetogenic effects of multiple low dose (MLD)-STZ administration are enhanced by fasting has not been addressed. Given that repeated bouts of fasting can cause undue metabolic stress in mice, we compared the efficacy of MLD-STZ injections (50 mg/kg body weight daily for 5 days) to induce experimental hyperglycemia in both NOD/SCID/γchainnull and C57BL/6J mice that were either ad libitum fed (STZ-Fed) or that had been fasted for 6 h (STZ-Fasted) prior to the time of STZ administration. Both STZ-Fed and STZ-Fasted mice had significantly worse glucose tolerance than vehicle-treated control mice 10 days after initiation of the MLD-STZ regimen. In C57BL/6J mice, fasting glucose levels, serum insulin levels, β cell mass, and glucose disposal during intraperitoneal glucose tolerance tests (IPGTTs) were indistinguishable between STZ-Fed and STZ-Fasted mice 20 days after MLD-STZ. The glucose intolerant phenotypes persisted for 20 weeks thereafter, irrespective of whether C57BL/6J mice were fed or fasted at the time of STZ injections. However, STZ-Fasted C57BL/6J mice experienced significant weight loss during the repeated bouts of fasting/re-feeding that were required to complete the MLD-STZ protocol. In summary, induction of experimental hyperglycemia can be achieved using the MLD-STZ protocol without repeated bouts of fasting, which have the potential to cause metabolic stress in laboratory mice.

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ObjectivesEpidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic β-cell function using rodent and in vitro models.MethodsBeginning at 8 weeks of age, C57BL/6J mice were concurrently fed high fat-diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD exposure. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured β-cells (INS-1) and primary islets were exposed ex vivo to CS extract (CSE), and β-cell function and viability were tested. Since CS increases ceramide in lungs cells and these bioactive sphingolipids have been implicated in pancreatic β-cell dysfunction in diabetes, islet and β-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry.ResultsCompared to HFD-fed ambient air-exposed mice, HFD-fed and CS- exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and a significantly greater increase in glucose intolerance compared to non-smoking control mice. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of β-cell dysfunction, significantly lower β-cell mass (p=0.02), and reduced β-cell proliferation (p=0.006), and increased islet ceramide accumulation. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide accumulation, reduce insulin gene expression and glucose-stimulated insulin secretion, and increase β-cell oxidative and ER stress. Treatment with the antioxidant N-acetylcysteine, markedly attenuated the effects of CSE on ceramide levels, restored β-cell function and survival, and increased cyclin D2 expression, while also reducing activation of β-cell ER and oxidative stress.ConclusionsOur results indicate that CS exposure inhibits insulin production, processing, and secretion and reduced β-cell viability and proliferation. These effects were linked to increased β-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of mechanisms by which CS exposure impairs β-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.