The relentless decline in ␤-cell function frequently observed in type 2 diabetic patients, despite optimal drug management, has variously been attributed to glucose toxicity and lipotoxicity. The former theory posits hyperglycemia, an outcome of the disease, as a secondary force that further damages ␤-cells. The latter theory suggests that the often-associated defect of hyperlipidemia is a primary cause of ␤-cell dysfunction. We review evidence that patients with type 2 diabetes continually undergo oxidative stress, that elevated glucose concentrations increase levels of reactive oxygen species in ␤-cells, that islets have intrinsically low antioxidant enzyme defenses, that antioxidant drugs and overexpression of antioxidant enzymes protect ␤-cells from glucose toxicity, and that lipotoxicity, to the extent it can be attributable to hyperlipidemia, occurs only in the context of preexisting hyperglycemia, whereas glucose toxicity can occur in the absence of hyperlipidemia. Diabetes 53 (Suppl. 1):S119 -S124, 2004

Chronic exposure to hyperglycemia can lead to cellular dysfunction that may become irreversible over time, a process that is termed glucose toxicity. Our perspective about glucose toxicity as it pertains to the pancreatic ␤-cell is that the characteristic decreases in insulin synthesis and secretion are caused by decreased insulin gene expression. The responsible metabolic lesion appears to involve a posttranscriptional defect in pancreas duodenum homeobox-1 (PDX-1) mRNA maturation. PDX-1 is a critically important transcription factor for the insulin promoter, is absent in glucotoxic islets, and, when transfected into glucotoxic ␤-cells, improves insulin promoter activity. Because reactive oxygen species are produced via oxidative phosphorylation during anaerobic glycolysis, via the Schiff reaction during glycation, via glucose autoxidation, and via hexosamine metabolism under supraphysiological glucose concentrations, we hypothesize that chronic oxidative stress is an important mechanism for glucose toxicity. Support for this hypothesis is found in the observations that high glucose concentrations increase intraislet peroxide levels, that islets contain very low levels of antioxidant enzyme activities, and that adenoviral overexpression of antioxidant enzymes in vitro in islets, as well as exogenous treatment with antioxidants in vivo in animals, protect the islet from the toxic effects of excessive glucose levels. Clinically, consideration of antioxidants as adjunct therapy in type 2 diabetes is warranted because of the many reports of elevated markers of oxidative stress in patients with this disease, which is characterized by imperfect management of glycemia, consequent chronic hyperglycemia, and relentless deterioration of ␤-cell function. Diabetes 52: 581-587, 2003

Type 2 diabetes (T2DM) commonly arises from islet β cell failure and insulin resistance. Here, we examined the sensitivity of key islet-enriched transcription factors to oxidative stress, a condition associated with β cell dysfunction in both type 1 diabetes (T1DM) and T2DM. Hydrogen peroxide treatment of β cell lines induced cytoplasmic translocation of MAFA and NKX6.1. In parallel, the ability of nuclear PDX1 to bind endogenous target gene promoters was also dramatically reduced, whereas the activity of other key β cell transcriptional regulators was unaffected. MAFA levels were reduced, followed by a reduction in NKX6.1 upon development of hyperglycemia in db/db mice, a T2DM model. Transgenic expression of the glutathione peroxidase-1 antioxidant enzyme (GPX1) in db/db islet β cells restored nuclear MAFA, nuclear NKX6.1, and β cell function in vivo. Notably, the selective decrease in MAFA, NKX6.1, and PDX1 expression was found in human T2DM islets. MAFB, a MAFA-related transcription factor expressed in human β cells, was also severely compromised. We propose that MAFA, MAFB, NKX6.1, and PDX1 activity provides a gauge of islet β cell function, with loss of MAFA (and/or MAFB) representing an early indicator of β cell inactivity and the subsequent deficit of more impactful NKX6.1 (and/or PDX1) resulting in overt dysfunction associated with T2DM.

Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes adverse alterations in ␤ cell function, a phenomenon termed glucose toxicity and one that may play a secondary pathogenic role in type 2 diabetes. However, no mechanism of action has been definitively identified for glucose toxicity in ␤ cells. To ascertain whether chronic oxidative stress might play a role, we chronically cultured the ␤ cell line, HIT-T15, in medium containing 11.1 mM glucose with and without the antioxidants, N-acetyl-L-cysteine (NAC) or aminoguanidine (AG). Addition of NAC or AG to the culture medium at least partially prevented decreases in insulin mRNA, insulin gene promoter activity, DNA binding of two important insulin promoter transcription factors (PDX-1͞STF-1 and RIPE-3b1 activator), insulin content, and glucose-induced insulin secretion. These findings suggested that one mechanism of glucose toxicity in the ␤ cell may be chronic exposure to reactive oxygen species, i.e., chronic oxidative stress. To ascertain the effects of these drugs on diabetes, NAC or AG was given to Zucker diabetic fatty rats, a laboratory model of type 2 diabetes, from 6 through 12 weeks of age. Both drugs prevented a rise in blood oxidative stress markers (8-hydroxy-2-deoxyguanosine and malondialdehyde ؉ 4-hydroxy-2-nonenal), and partially prevented hyperglycemia, glucose intolerance, defective insulin secretion as well as decrements in ␤ cell insulin content, insulin gene expression, and PDX-1 (STF-1) binding to the insulin gene promoter. We conclude that chronic oxidative stress may play a role in glucose toxicity, which in turn may worsen the severity of type 2 diabetes.

Antioxidant drugs have been reported to protect pancreatic islets from the adverse effects of chronic exposure to supraphysiological glucose concentrations. However, glucose has not been shown to increase intracellular oxidant load in islets, nor have the effects of increasing or inhibiting glutathione peroxidase (GPx) activity on islet resistance to sugar-induced oxidant stress been studied. We observed that high glucose concentrations increased intracellular peroxide levels in human islets and the pancreatic ␤ cell line, HIT-T15. Inhibition of ␥-glutamylcysteine synthetase (␥GCS) by buthionine sulfoximine augmented the increase in islet peroxide and decrease in insulin mRNA levels, content, and secretion in islets and HIT-T15 cells induced by ribose. Adenoviral overexpression of GPx increased GPx activity and protected islets against adverse effects of ribose. These results demonstrate that glucose and ribose increase islet peroxide accumulation and that the adverse consequences of ribose-induced oxidative stress on insulin mRNA, content, and secretion can be augmented by a glutathione synthesis inhibitor and prevented by increasing islet GPx activity. These observations support the hypothesis that oxidative stress is one mechanism for glucose toxicity in pancreatic islets. T ype 2 diabetes mellitus is polygenic in origin and usually begins in adulthood, although specific genes for subtypes of this disease that occur earlier in life, referred to collectively as maturity-onset diabetes of the young (MODY), have been identified (1). The onset of type 2 diabetes is insidious, thus hyperglycemia develops gradually and often goes untreated for years until symptoms become clinically obvious. Consequent chronic exposure of tissues to supraphysiologic levels of blood glucose can lead to adverse intracellular outcomes, a process known as glucose toxicity (2-5). Possible mechanisms of action for glucose toxicity include the formation of advanced glycosylation end products and glucosamine, increased protein kinase C activity with c-myc induction, autooxidation of glucose, and increased levels of reactive glycolytic intermediates such as glyceraldehyde-3-phosphate or dihydroxyacetone phosphate (6 -13). All these processes usually are accompanied by the formation of reactive oxygen species (ROS), setting up the potential for oxidative stress. Many weeks of exposure to high concentrations of glucose are necessary before glucotoxic effects are expressed by islet ␤ cells in vitro and in vivo (14 -20). Because isolated islets do not reliably survive in culture greater than 2 weeks, the use of short-term exposure to ribose, a sugar that generates ROS more potently than glucose, has become an accepted model for studying islet glucose toxicity (21,22). The use of ribose as a prooxidant sugar is especially valuable in adenoviral overexpression systems, because the overexpression effect is short-lived, lasting only days.In the pancreatic ␤ cell, glucose and ribose toxicity cause decreased insulin mRNA levels (14-22), one mechanism ...

Prolonged exposure of isolated islets to supraphysiologic concentrations of palmitate decreases insulin gene expression in the presence of elevated glucose levels. This study was designed to determine whether or not this phenomenon is associated with a glucosedependent increase in esterification of fatty acids into neutral lipids. Gene expression of sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes of lipid metabolism, was detected in isolated rat islets. Their levels of expression were not affected after a 72-h exposure to elevated glucose and palmitate. To determine the effects of glucose on palmitate-induced neutral lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts of [ 14 C]palmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of [14 C]palmitate into complex lipids. Addition of exogenous palmitate directed lipid metabolism toward neutral lipid synthesis. As a result, neutral lipid mass was increased upon prolonged incubation with elevated palmitate only in the presence of high glucose. The ability of palmitate to increase neutral lipid synthesis in the presence of high glucose was concentration-dependent in HIT cells and was inversely correlated to insulin mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial ␤-oxidation, reproduced the inhibitory effect of palmitate on insulin mRNA levels. In contrast, palmitate methyl ester, which is not metabolized, and the medium-chain fatty acid octanoate, which is readily oxidized, did not affect insulin gene expression, suggesting that fatty-acid inhibition of insulin gene expression requires activation of the esterification pathway. These results demonstrate that inhibition of insulin gene expression upon prolonged exposure of islets to palmitate is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids. Diabetes 50:315-321, 2001A ccording to the lipotoxicity hypothesis, chronic exposure to elevated lipid levels impairs pancreatic ␤-cell function in type 2 diabetic patients (1,2). We (3) and others (4,5) have previously shown that prolonged (>1 day) culture of normal islets in the presence of supraphysiologic concentrations of palmitate decreases insulin content and impairs insulin gene expression only in the presence of elevated glucose levels. This occurs, at least in part, via decreased insulin gene promoter activity in HIT-T15 cells (3) and decreased binding of the transcription factor pancreas-duodenum homeobox-1 (PDX-1) to the insulin gene in islets (4). In Zucker diabetic fatty (ZDF) rats, it has been postulated that ␤-cell dysfunction is due to increased triacylglycerol (TAG) content in islets (6-8), which leads to increased production of nitric oxide (9) and ceramide synthesis (10). However, the ZDF rat is an extremely obese genetic model of type 2 diabetes bearing a mutation in the leptin receptor gene. It remains...

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After the onset of type 2 diabetes, chronic hyperglycemia causes glucotoxic changes in many tissues (1, 2). Glucose toxicity in the pancreatic islet beta cell secondarily leads to further defects in beta cell function, including decreases in insulin reporter activity, gene expression, content, and secretion (3). Antioxidants have been shown to prevent these adverse changes in experimental models (4, 5). We previously observed that loss of insulin gene expression is accompanied by decreased binding to the promoter region of two important transcription factors, PDX-1 1 (STF-1, IDX-1, IPF-1) and RIPE-3b1 activator (6 -8). PDX-1 is required for pancreatic development and is a key regulator of insulin gene expression. Mutations within the RIPE-3b1/C1 element of the insulin promoter markedly reduce glucose-responsive insulin gene expression (7). We also reported that loss of RIPE-3b1 binding precedes the loss of PDX-1 binding to the insulin promoter as glucotoxicity develops (8). The chronology of these losses is likely to be important in light of a recent report that RIPE-3b1/MafA directly activates PDX-1 transcription (9).The work in this study features the use of HIT-T15 cells, a glucose-responsive beta cell line that has proven over the past decade to reproduce the molecular changes in gene expression that are caused by glucose toxicity in vivo in animal models. Since isolated islets cannot be chronically cultured to study the adverse effects of high glucose concentrations over many months, this cell line is a valuable surrogate. In our previous work, reconstitution of late passage glucotoxic HIT-T15 cells by transient transfection with PDX-1 partially restored insulin promoter activity (8). Since RIPE-3b1 had not yet been cloned, we were unable to extend our studies with this transcription factor but hypothesized that reconstitution of the cells with both PDX-1 and RIPE-3b1 activator would lead to greater recovery of insulin promoter activity. With the recent cloning and identification of RIPE-3b1 activator as MafA (10 -12), we have been able to perform new studies to examine 1) whether levels of MafA mRNA and protein are decreased in glucotoxic beta cells; 2) the mechanism of MafA protein degradation; 3) whether the antioxidant, N-acetylcysteine, can prevent glucotoxicity-induced loss of MafA protein and binding to the insulin promoter; and 4) whether overexpression of MafA and PDX-1 together can restore insulin promoter activity and mRNA levels more fully than PDX-1 alone. MATERIALS AND METHODSCell Culture-HIT-T15 cells were maintained in RPMI 1640 media containing 10% fetal bovine serum and 11.1 mM glucose as described previously (13). Cells were categorized as early passage (p71-75) or late passage (p123-128), with each passage occurring weekly. Chronic culturing of HIT-T15 cells with N-acetyl-L-cysteine (Sigma; 0.5, 1, or 5 mM) added to the media was begun at p70.