Oxidative stress is produced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. The aim of this study was to examine the involvement of oxidative stress in the progression of pancreatic beta-cell dysfunction in type 2 diabetes and to evaluate the potential usefulness of antioxidants in the treatment of type 2 diabetes. We used diabetic C57BL/KsJ-db/db mice, in whom antioxidant treatment (N-acetyl-L-cysteine [NAC], vitamins C plus E, or both) was started at 6 weeks of age; its effects were evaluated at 10 and 16 weeks of age. According to an intraperitoneal glucose tolerance test, the treatment with NAC retained glucose-stimulated insulin secretion and moderately decreased blood glucose levels. Vitamins C and E were not effective when used alone but slightly effective when used in combination with NAC. No effect on insulin secretion was observed when the same set of antioxidants was given to nondiabetic control mice. Histologic analyses of the pancreases revealed that the beta-cell mass was significantly larger in the diabetic mice treated with the antioxidants than in the untreated mice. As a possible cause, the antioxidant treatment suppressed apoptosis in beta-cells without changing the rate of beta-cell proliferation, supporting the hypothesis that in chronic hyperglycemia, apoptosis induced by oxidative stress causes reduction of beta-cell mass. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. Furthermore, expression of pancreatic and duodenal homeobox factor-1 (PDX-1), a beta-cell-specific transcription factor, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. In conclusion, our observations indicate that antioxidant treatment can exert beneficial effects in diabetes, with preservation of in vivo beta-cell function. This finding suggests a potential usefulness of antioxidants for treating diabetes and provides further support for the implication of oxidative stress in beta-cell dysfunction in diabetes.

Type 2 diabetes is one of the most prevalent and serious metabolic diseases in the world, and insulin resistance and pancreatic ␤-cell dysfunction are the hallmarks of the disease. In this study, we have shown that endoplasmic reticulum (ER) stress, which is provoked under diabetic conditions, plays a crucial role in the insulin resistance found in diabetes by modifying the expression of oxygen-regulated protein 150 (ORP150), a molecular chaperone that protects cells from ER stress. Sense ORP overexpression in the liver of obese diabetic mice significantly improved insulin resistance and markedly ameliorated glucose tolerance. Conversely, expression of antisense ORP150 in the liver of normal mice decreased insulin sensitivity. The phosphorylation state of IRS-1 and Akt, which are key molecules for insulin signaling, and the expression levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, key enzymes of gluconeogenesis, were also altered by ORP150 overexpression. This is the first report showing that ER stress plays a crucial role in the insulin resistance found in diabetes and thus could be a potential therapeutic target for diabetes.

The JNK pathway is known to be activated in several tissues in the diabetic state, and is possibly involved in the development of insulin resistance and suppression of insulin biosynthesis. Here we show a potential new therapy for diabetes using cell-permeable JNK-inhibitory peptide. Intraperitoneal administration of the peptide led to its transduction into various tissues in vivo, and this treatment markedly improved insulin resistance and ameliorated glucose tolerance in diabetic mice. These data indicate that the JNK pathway is critically involved in diabetes and that the cell-permeable JNK-inhibitory peptide may have promise as a new therapeutic agent for diabetes.

Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic ␤ cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the socalled glucose toxicity to pancreatic ␤ cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D -ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control ␤ -actin gene promoter; ‫ف‬ 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D -ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycationinduced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D -ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic ␤ cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the ␤ cell glucose toxicity. ( J. Clin. Invest. 1997. 99:144-150.)

It has been shown that oxidative stress and activation of the c-Jun N-terminal kinase (JNK) pathway induce the nucleocytoplasmic translocation of the pancreatic transcription factor PDX-1, which leads to pancreatic ␤-cell dysfunction. In this study, we have shown that the forkhead transcription factor Foxo1/FKHR plays a role as a mediator between the JNK pathway and PDX-1. Under oxidative stress conditions, Foxo1 changed its intracellular localization from the cytoplasm to the nucleus in the pancreatic ␤-cell line HIT-T15. The overexpression of JNK also induced the nuclear localization of Foxo1, but in contrast, suppression of JNK reduced the oxidative stress-induced nuclear localization of Foxo1, suggesting the involvement of the JNK pathway in Foxo1 translocation. In addition, oxidative stress or activation of the JNK pathway decreased the activity of Akt in HIT cells, leading to the decreased phosphorylation of Foxo1 following nuclear localization. Furthermore, adenovirus-mediated Foxo1 overexpression reduced the nuclear expression of PDX-1, whereas repression of Foxo1 by Foxo1-specific small interfering RNA retained the nuclear expression of PDX-1 under oxidative stress conditions. Taken together, Foxo1 is involved in the nucleocytoplasmic translocation of PDX-1 by oxidative stress and the JNK pathway.

Oxidative stress is induced in pancreatic beta-cells under diabetic conditions and causes beta-cell dysfunction. Antioxidant treatment of diabetic animals leads to recovery of insulin biosynthesis and increases the expression of its controlling transcription factor, pancreatic duodenal homeobox-1 (PDX-1), in pancreatic beta-cells. Here, we show that PDX-1 is translocated from the nuclei to the cytoplasm of pancreatic beta-cells in response to oxidative stress. When oxidative stress was charged upon beta-cell-derived HIT-T15 cells, both endogenous PDX-1 and exogenously introduced green fluorescent protein-tagged PDX-1 moved from the nuclei to the cytoplasm. The addition of a dominant negative form of c-Jun NH(2)-terminal kinase (JNK) inhibited oxidative stress-induced PDX-1 translocation, suggesting an essential role of JNK in mediating this phenomenon. Whereas the nuclear localization signal (NLS) in PDX-1 was not affected by oxidative stress, leptomycin B, a specific inhibitor of the classical leucine-rich nuclear export signal (NES), inhibited nucleo-cytoplasmic translocation of PDX-1 induced by oxidative stress. Moreover, we identified an NES at position 82-94 of the mouse PDX-1 protein. Thus, our present results revealed a novel mechanism that negatively regulates PDX-1 function. The identification of the NES, which overrides the function of the NLS in an oxidative stress-responsive, JNK-dependent manner, supports the complicated regulation of PDX-1 function in vivo and may further the understanding of beta-cell pathophysiology in diabetes.

Diabetes is the most prevalent and serious metabolic disease, and the number of diabetic patients worldwide is increasing. The reduction of insulin biosynthesis in pancreatic ␤-cells is closely associated with the onset and progression of diabetes, and thus it is important to search for ways to induce insulin-producing cells in non؊␤-cells. In this study, we showed that a modified form of the pancreatic and duodenal homeobox factor 1 (PDX- 1

Several reducing sugars brought about apoptosis in isolated rat pancreatic islet cells and in the pancreatic beta-cell-derived cell line HIT. This apoptosis was characterized biochemically by inter-nucleosomal DNA cleavage and morphologically by nuclear shrinkage, chromatin condensation and apoptotic body formation. N-Acetyl-L-cysteine, an antioxidant, and aminoguanidine, an inhibitor of the glycation reaction, inhibited this apoptosis. We also showed directly that proteins in beta-cells were actually glycated by using an antibody which can specifically recognize proteins glycated by fructose, but not by glucose. Furthermore, fluorescence-activated cell sorting analysis using dichlorofluorescein diacetate showed that reducing sugars increased intracellular peroxide levels prior to the induction of apoptosis. Levels of carbonyl, an index of oxidative modification, and of malondialdehyde, a lipid peroxidation product, were also increased. Taken together, these results suggest that reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress mainly through the glycation reaction, which may explain the deterioration of beta-cells under conditions of diabetes.