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.

Background:Optineurin is a polyubiquitin-binding protein of unknown function. Result: Macrophages from mice expressing a polyubiquitin-binding defective mutant of optineurin show reduced activation of TANK-binding kinase 1 (TBK1) and reduced production of interferon ␤. Conclusion:The binding of polyubiquitin to optineurin is required for optimal activation and function of TBK1. Significance: This study identifies a new physiological role for optineurin.

The AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic ␤-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of ␤-cell function. Here, we show that inhibition of AMPK in ␤-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the ␤-cell.The ␤-cell is unique compared with other mammalian cell types in that its primary function to synthesize and secrete insulin is tightly coupled to its metabolic rate. Glucose is the most potent nutrient in stimulating insulin release. Upon entry into the ␤-cell, glucose is rapidly metabolized, resulting in the generation of mitochondria-derived metabolic intermediates including ATP. This increase in ATP leads to closure of ATPsensitive K ϩ (K ATP )-channels, depolarization of the plasma membrane and opening of voltage-gated L-type Ca 2ϩ channels. The subsequent increase in intracellular Ca 2ϩ concentration [Ca 2ϩ ] i triggers insulin exocytosis (1). The ␤-cell also utilizes certain key amino acids that, via mitochondrial metabolism, can further generate coupling factors that elicit an insulin secretory response (2, 3). In addition to their role as insulin secretagogues, glucose and other nutrients stimulate protein translation and ␤-cell growth and proliferation (4, 5). While much is known regarding how the ␤-cell couples glucose metabolism to insulin secretion, the mechanisms by which ␤-cells sense metabolism of other fuels, such as amino acids, and augment glucose-stimulated insulin release are less clear. Further, it is unclear how the ␤-cell coordinates nutrient abundance with enhanced protein translation and cell growth. This aspect of ␤-cell function is particularly important under conditions of increased insulin demand, such as obesity a...

Previous work has suggested that functional interrelationships may exist between inhibition of insulin secretion by interleukin (IL)-1␤ and the endogenous synthesis of prostaglandin E 2 (PGE 2 ) in the pancreatic islet. These studies were performed to ascertain the relative abundance of E prostaglandin (EP) receptor mRNAs in tissues that are major targets, or major degradative sites, of insulin; to identify which EP receptor type mediates PGE 2 inhibition of insulin secretion in pancreatic islets; and to examine possible sites of action through which sodium salicylate might affect IL-1␤/PGE 2 interactions. Real-time fluorescence-based RT-PCR indicated that EP3 is the most abundant EP receptor type in islets, liver, kidney, and epididymal fat. EP3 mRNA is the least, whereas EP2 mRNA is the most, abundant type in skeletal muscle. Misoprostol, an EP3 agonist, inhibited glucose-induced insulin secretion from islets, an event that was prevented by preincubation with pertussis toxin, by decreasing cAMP. Electromobility shift assays demonstrated that sodium salicylate inhibits IL-1␤-induced nuclear factor-B (NF-B) activation. Sodium salicylate also prevented IL-1␤ from inducing EP3 and cyclooxygenase (COX)-2 gene expression in islets and thereby prevented IL-1␤ from inhibiting glucose-induced insulin secretion. These findings indicate that the sites of action through which sodium salicylate inhibits these negative effects of IL-1␤ on ␤-cell function include activation of NF-B as well as generation of PGE 2 by COX-2.

Interleukin-1␤ (IL-1␤) and prostaglandin E 2 (PGE 2 ), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic ␤ cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1␤ effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1␤ induces cyclooxygenase-2 (COX-2) gene expression and PGE 2 synthesis in islet ␤ cells, we have reassessed the possibility that PGE 2 mediates IL-1␤ effects on ␤ function. By using two cell lines (HIT-T15 and ␤HC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1␤ inhibition of insulin secretion. Both drugs prevented IL-1␤ from inducing PGE 2 synthesis and inhibiting insulin secretion; adding back exogenous PGE 2 re-established inhibition of insulin secretion in the presence of IL-1␤. We also found that EP3, the PGE 2 receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE 2 mediates the inhibitory effects of exogenous IL-1␤ on ␤ cell function. Prostaglandin E 2 (PGE 2 )1 is known to be an inhibitor of glucose-induced insulin secretion from studies in a ␤ cell line (1, 2) and isolated and neonatal islets of Langerhans (3-6) as well as in vivo in both animal (7,8) and human (9 -11) studies. These findings have been reinforced by studies in which inhibitors of cyclooxygenase, hence PGE 2 synthesis, have augmented glucose-induced insulin secretion. The only discordant result in the latter category of studies has been observed when indomethacin was used as the cyclooxygenase inhibitor. This discrepant result can be attributed to other effects of indomethacin that would be expected to inhibit insulin secretion through adverse effects on exocytosis that are unrelated to its effects on prostaglandin synthesis (12).Interleukin-1␤ (IL-1␤) has been reported to have major inhibitory effects on ␤ cell function, especially under conditions of high glucose concentrations and prolonged exposure to this cytokine (13)(14)(15)(16). This is an especially relevant observation because many reports suggest that IL-1␤ is an important force in the pathogenesis of diabetes mellitus (17,18). Previously, studies have concluded that endogenous PGE 2 does not play a participatory role in the adverse effects of IL-1␤ on ␤ cell function (5, 6). Ironically, however, the drug that was chosen to test this hypothesis and found not to reverse IL-1␤ inhibitory effects on insulin secretion was indomethacin, which itself has independent inhibitory actions on ␤ cell exocytosis (12).Recently, it has been appreciated that the pancreatic is...

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Type 2 diabetes is caused by a combination of ␤-cell dysfunction and insulin resistance. Over time, hyperglycemia worsens, a phenomenon that has been attributed to deleterious effects of chronic hyperglycemia (glucotoxicity) or chronic hyperlipidemia (lipotoxicity) on ␤-cell function and is often accompanied by increased islet triacylglycerol (TAG) content and decreased insulin gene expression. To examine these two potentially pathogenic forces, we studied Zucker rats (leptin receptor wild type, ؉/؉; heterozygous, ؉/؊; and mutant, ؊/؊). First, ؉/؉ and ؉/؊ Zucker rats were compared metabolically. At 6 weeks of age, the ؉/؊ rats had a lower level of islet insulin mRNA compared with ؉/؉. At 12 weeks of age, differences were found in body weight and islet TAG content; however, levels of insulin mRNA were equivalent. Second, we examined whether worsening of the diabetic state in the homozygous mutant (؊/؊) Zucker diabetic fatty (ZDF) rat is related more to chronic hyperglycemia or to hyperlipidemia. The ZDF rats were treated for 6 weeks with either bezafibrate, a lipid-lowering drug that does not affect plasma glucose levels, or phlorizin, a drug that reduces plasma glucose without lowering lipid levels. Bezafibrate treatment lessened the rise in plasma TAG observed in nontreated rats (239 ؎ 16 vs. 388 ؎ 36 mg/dl, treated versus nontreated; P < 0.0001) but did not prevent the rise in fasting plasma glucose. Despite lowering plasma TAG, bezafibrate was not effective in preventing an increased islet TAG content and did not prevent the associated decrease in insulin mRNA levels. Phlorizin treatment prevented hyperglycemia (61 ؎ 2 vs. 145 ؎ 7 mg/dl, treated versus nontreated; P < 0.0001) and lowered islet TAG content (32.7 ؎ 0.7 vs. 47.8 ؎ 2.7 ng/islet, treated versus nontreated; P < 0.0001) and preserved insulin mRNA levels without preventing hypertriglyceridemia. Plasma free fatty acid level did not correlate with changes in islet TAG or insulin mRNA levels. We conclude that antecedent elevated plasma glucose levels, not plasma lipid levels, are associated with elevated islet TAG content and decreased insulin mRNA levels in ZDF animals.

Chronic exposure of pancreatic islet beta-cell lines to supraphysiologic glucose concentrations causes defects in insulin gene expression and insulin secretion. To determine whether these in vitro phenomena have pathophysiologic relevance in vivo, we studied the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. The ZDF animals had relatively higher levels of glycemia and islet insulin mRNA at 6 weeks of age than age-matched Zucker lean control (ZLC) rats. As glycemia increased in 12- and 16-week-old ZDF rats, we observed decrements in glucose-induced insulin secretion during static incubations of pancreatic islets and in insulin mRNA levels, PDX-1 mRNA levels, and PDX-1 protein binding to the insulin promoter compared with age-matched ZLC rats and 6-week-old ZDF rats. To determine whether normalization of blood glucose levels would prevent these defects, ZDF rats were treated with troglitazone beginning at 6 weeks of age. Troglitazone prevented ZDF rats from becoming hyperglycemic and preserved glucose-induced insulin responses. Furthermore, troglitazone-treated ZDF animals had greater levels of insulin and PDX-1 mRNAs compared with untreated ZDF animals of the same ages at 12 and 16 weeks. Our results demonstrate that chronic and progressive hyperglycemia resulting from type 2 diabetes in ZDF rats is associated with loss of insulin and PDX-1 mRNAs and loss of glucose-stimulated insulin secretion. Prevention of hyperglycemia prevented the associated defects in insulin and PDX-1 gene expression and improved insulin secretion. These findings provide the first in vivo evidence that prevention of progressive hyperglycemia in a model of type 2 diabetes preserves insulin and PDX-1 gene expression.