OBJECTIVE-Mammalian target of rapamycin (mTOR) and its downstream target S6 kinase 1 (S6K1) mediate nutrient-induced insulin resistance by downregulating insulin receptor substrate proteins with subsequent reduced Akt phosphorylation. Therefore, mTOR/S6K1 inhibition could become a therapeutic strategy in insulin-resistant states, including type 2 diabetes. We tested this hypothesis in the Psammomys obesus (P. obesus) model of nutrition-dependent type 2 diabetes, using the mTOR inhibitor rapamycin.RESEARCH DESIGN AND METHODS-Normoglycemic and diabetic P. obesus were treated with 0.2 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 i.p. rapamycin or vehicle, and the effects on insulin signaling in muscle, liver and islets, and on different metabolic parameters were analyzed.RESULTS-Unexpectedly, rapamycin worsened hyperglycemia in diabetic P. obesus without affecting glycemia in normoglycemic controls. There was a 10-fold increase of serum insulin in diabetic P. obesus compared with controls; rapamycin completely abolished this increase. This was accompanied by weight loss and a robust increase of serum lipids and ketone bodies. Rapamycin decreased muscle insulin sensitivity paralleled by increased glycogen synthase kinase 3␤ activity. In diabetic animals, rapamycin reduced ␤-cell mass by 50% through increased apoptosis. Rapamycin increased the stress-responsive c-Jun NH 2 -terminal kinase pathway in muscle and islets, which could account for its effect on insulin resistance and ␤-cell apoptosis. Moreover, glucose-stimulated insulin secretion and biosynthesis were impaired in islets treated with rapamycin.CONCLUSIONS-Rapamycin induces fulminant diabetes by increasing insulin resistance and reducing ␤-cell function and mass. These findings emphasize the essential role of mTOR/S6K1 in orchestrating ␤-cell adaptation to hyperglycemia in type 2 diabetes. It is likely that treatments based on mTOR inhibition will cause exacerbation of diabetes. Diabetes 57:945-957, 2008

BackgroundPalmitate is a potent inducer of endoplasmic reticulum (ER) stress in β-cells. In type 2 diabetes, glucose amplifies fatty-acid toxicity for pancreatic β-cells, leading to β-cell dysfunction and death. Why glucose exacerbates β-cell lipotoxicity is largely unknown. Glucose stimulates mTORC1, an important nutrient sensor involved in the regulation of cellular stress. Our study tested the hypothesis that glucose augments lipotoxicity by stimulating mTORC1 leading to increased β-cell ER stress.Principal FindingsWe found that glucose amplifies palmitate-induced ER stress by increasing IRE1α protein levels and activating the JNK pathway, leading to increased β-cell apoptosis. Moreover, glucose increased mTORC1 activity and its inhibition by rapamycin decreased β-cell apoptosis under conditions of glucolipotoxicity. Inhibition of mTORC1 by rapamycin did not affect proinsulin and total protein synthesis in β-cells incubated at high glucose with palmitate. However, it decreased IRE1α expression and signaling and inhibited JNK pathway activation. In TSC2-deficient mouse embryonic fibroblasts, in which mTORC1 is constitutively active, mTORC1 regulated the stimulation of JNK by ER stressors, but not in response to anisomycin, which activates JNK independent of ER stress. Finally, we found that JNK inhibition decreased β-cell apoptosis under conditions of glucolipotoxicity.Conclusions/SignificanceCollectively, our findings suggest that mTORC1 mediates glucose amplification of lipotoxicity, acting through activation of ER stress and JNK. Thus, mTORC1 is an important transducer of ER stress in β-cell glucolipotoxicity. Moreover, in stressed β-cells mTORC1 inhibition decreases IRE1α protein expression and JNK activity without affecting ER protein load, suggesting that mTORC1 regulates the β-cell stress response to glucose and fatty acids by modulating the synthesis and activity of specific proteins involved in the execution of the ER stress response. This novel paradigm may have important implications for understanding β-cell failure in type 2 diabetes.

Accumulation of misfolded proinsulin in the β-cell leads to dysfunction induced by endoplasmic reticulum (ER) stress, with diabetes as a consequence. Autophagy helps cellular adaptation to stress via clearance of misfolded proteins and damaged organelles. We studied the effects of proinsulin misfolding on autophagy and the impact of stimulating autophagy on diabetes progression in Akita mice, which carry a mutation in proinsulin, leading to its severe misfolding. Treatment of female diabetic Akita mice with rapamycin improved diabetes, increased pancreatic insulin content, and prevented β-cell apoptosis. In vitro, autophagic flux was increased in Akita β-cells. Treatment with rapamycin further stimulated autophagy, evidenced by increased autophagosome formation and enhancement of autophagosome–lysosome fusion. This was associated with attenuation of cellular stress and apoptosis. The mammalian target of rapamycin (mTOR) kinase inhibitor Torin1 mimicked the rapamycin effects on autophagy and stress, indicating that the beneficial effects of rapamycin are indeed mediated via inhibition of mTOR. Finally, inhibition of autophagy exacerbated stress and abolished the anti-ER stress effects of rapamycin. In conclusion, rapamycin reduces ER stress induced by accumulation of misfolded proinsulin, thereby improving diabetes and preventing β-cell apoptosis. The beneficial effects of rapamycin in this context strictly depend on autophagy; therefore, stimulating autophagy may become a therapeutic approach for diabetes.

Experimental lipotoxicity constitutes a model for β-cell demise induced by metabolic stress in obesity and type 2 diabetes. Fatty acid excess induces endoplasmic reticulum (ER) stress, which is accompanied by ER morphological changes whose mechanisms and relevance are unknown. We found that the GTPase dynamin-related protein 1 (DRP1), a key regulator of mitochondrial fission, is an ER resident regulating ER morphology in stressed β-cells. Inhibition of DRP1 activity using a GTP hydrolysis-defective mutant (Ad-K38A) attenuated fatty acid-induced ER expansion and mitochondrial fission. Strikingly, stimulating the key energy-sensor AMP-activated protein kinase (AMPK) increased the phosphorylation at the anti-fission site Serine 637 and largely prevented the alterations in ER and mitochondrial morphology. Expression of a DRP1 mutant resistant to phosphorylation at this position partially prevented the recovery of ER and mitochondrial morphology by AMPK. Fatty acid-induced ER enlargement was associated with proinsulin retention in the ER, together with increased proinsulin/insulin ratio. Stimulation of AMPK prevented these alterations, as well as mitochondrial fragmentation and apoptosis. In summary, DRP1 regulation by AMPK delineates a novel pathway controlling ER and mitochondrial morphology, thereby modulating the response of β-cells to metabolic stress. DRP1 may thus function as a node integrating signals from stress regulators, such as AMPK, to coordinate organelle shape and function.

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Aims/hypothesis In type 2 diabetes, glucose toxicity leads to beta cell apoptosis with decreased beta cell mass as a consequence. Thioredoxin-interacting protein (TXNIP) is a critical mediator of glucose-induced beta cell apoptosis. Since hyperglycaemia leads to elevated serum insulin, we hypothesised that insulin is involved in the regulation of TXNIP protein levels in beta cells. Methods We studied the production of TXNIP in INS-1E beta cells and in islets of Psammomys obesus, an animal model of type 2 diabetes, in response to glucose and different modulators of insulin secretion. Results TXNIP production was markedly augmented in islets from diabetic P. obesus and in beta cells exposed to high glucose concentration. In contrast, adding insulin to the culture medium or stimulating insulin secretion with different secretagogues suppressed TXNIP. Inhibition of glucose and fatty acid-stimulated insulin secretion with diazoxide increased TXNIP production in beta cells. Nitric oxide (NO), a repressor of TXNIP, enhanced insulin signal transduction, whereas inhibition of NO synthase abolished its activation, suggesting that TXNIP inhibition by NO is mediated by stimulation of insulin signalling. Treatment of beta cells chronically exposed to high glucose with insulin reduced beta cell apoptosis. Txnip knockdown mimicking the effect of insulin prevented glucose-induced beta cell apoptosis. Conclusions/interpretation Insulin is a potent repressor of TXNIP, operating a negative feedback loop that restrains the stimulation of TXNIP by chronic hyperglycaemia. Repression of TXNIP by insulin is probably an important compensatory mechanism protecting beta cells from oxidative damage and apoptosis in type 2 diabetes.

Aims/hypothesis Cytokines stimulate nitric oxide production in pancreatic beta cells, leading to endoplasmic reticulum (ER) stress and apoptosis. Treatment of beta cells with glucose and NEFA induces nitric oxide synthase (NOS) as well as ER stress. However, the role of NO in glucolipotoxicity-induced ER stress in beta cells is not clear. Methods We studied the effect of high glucose and palmitate levels on NOS isoform production in rat and Psammomys obesus islets and in insulinoma-1E beta cells. The effects of neuronal NOS (nNOS) inhibition by small interfering RNA or by N ω -nitro-L-arginine methyl ester (L-NAME) on beta cell function, ER stress and apoptosis under conditions of glucolipotoxicity were investigated. Results Overnight incubation of rat and P. obesus islets at 22.2 mmol/l glucose with 0.5 mmol/l palmitate induced the production of nNOS but not inducible NOS (iNOS), in contrast with the robust stimulation of iNOS by cytokines. NOS inhibition by L-NAME did not prevent the decrease in glucose-stimulated insulin secretion and proinsulin biosynthesis or the depletion of islet insulin content observed under conditions of glucolipotoxicity. Moreover, treatment of beta cells with palmitate and L-NAME together resulted in marked activation of the IRE1α and PERK pathways of the unfolded protein response. This was associated with increased JNK phosphorylation and apoptosis in islets and beta cells. Moreover, partial nNos knockdown increased JNK phosphorylation and CHOP production, leading to apoptosis. Conclusions/interpretation In beta cells subjected to glucolipotoxic conditions, chronic inhibition of NOS exacerbates ER stress and activates JNK. Therefore, induction of nNOS is an adaptive response to glucolipotoxicity that protects beta cells from stress and apoptosis.

Fragmented islets, obtained by mild overdigestion of the adult rat pancreas with collagenase, readily formed monolayer cultures on dishes coated with extracellular matrix derived from bovine corneal endothelial cells. Contaminating fibroblasts were removed by treatment with sodium ethylmercurithiosalicylate. The cultured islets remained functional for over 6 weeks in primary culture and up to 9 weeks in secondary culture, as indicated by their substantial insulin response to an acute glucose stimulus. Insulin secretion from islet monolayers showed biphasic kinetics. The functional competence of the monolayers was further evaluated by studying glucose-stimulated insulin release in the presence of various modulators of B-cell function. The response to physiological agents such as somatostatin, epinephrine, glucagon, and arginine was retained for at least 4 weeks in culture. The sensitivity to inhibition by somatostatin and epinephrine (ID50 = 10 ng/ml) and that to stimulation by glucagon (ED50 = 3 ng/ml) were similar to or better than those for freshly isolated islets. We have thus obtained a fibroblast-free monolayer culture of pancreatic islets from adult rats containing B-cells that retain normal function for long periods. This experimental system appears ideally suited for studying chronic modulations of islet cell function under controlled in vitro conditions, which can allow the stimulation of normal and diabetic environments.