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Context: Total pancreatectomy followed by intrahepatic islet autotransplantation (TP/IAT) is performed to alleviate severe, unrelenting abdominal pain caused by chronic pancreatitis, to improve quality of life, and to prevent diabetes., Objective: To determine the cause of exercise-induced hypoglycemia that is a common complaint in TP/IAT recipients., Design: Participants completed 1 hour of steady-state exercise., Setting: Hospital research unit., Patients and Other Participants: We studied 14 TP/IAT recipients and 10 age- and body mass index-matched control subjects., Interventions: Peak oxygen uptake (VO2) was determined via a symptom-limited maximal cycle ergometer test. Fasted subjects then returned for a primed [6,6-2H2]-glucose infusion to measure endogenous glucose production while completing 1 hour of bicycle exercise at either 40% or 70% peak VO2., Main Outcome Measures: Blood samples were obtained to measure glucose metabolism and counterregulatory hormones before, during, and after exercise., Results: Although the Borg Rating of Perceived Exertion did not differ between recipients and control subjects, aerobic capacity was significantly higher in controls than in recipients (40.4 ± 2.0 vs 27.2 ± 1.4 mL/kg per minute; P < 0.001). This difference resulted in workload differences between control subjects and recipients to reach steady-state exercise at 40% peak VO2 (P = 0.003). Control subjects significantly increased their endogenous glucose production from 12.0 ± 1.0 to 15.2 ± 1.0 µmol/kg per minute during moderate exercise (P = 0.01). Recipients did not increase endogenous glucose production during moderate exercise (40% peak VO2) but succeeded during heavy exercise, from 10.1 ± 0.4 to 14.8 ± 2.0 µmol/kg per minute (70% peak VO2; P = 0.001)., Conclusions: Failure to increase endogenous glucose production during moderate exercise may be a key contributor to the hypoglycemia TP/IAT recipients experience., (Copyright © 2017 Endocrine Society)

Mainstays of therapy for type 2 diabetes involve drugs that are insulin-centric, i.e., they are designed to increase insulin secretion and decrease insulin resistance. The usual clinical course for people so treated is to have initially improved glycemic control but over time a need for intensification of drug-based treatment of hyperglycemia. The mechanism for this unrelenting deterioration of beta-cell function is related to chronic oxidative stress. This suggests that drug discovery should not exclusively focus on insulin-centric targets, but also include glucose-centric strategies, such as antioxidant protection of the beta-cell. This may facilitate repair of beta-cells undergoing damage by oxidative stress secondary to chronic hyperglycemia.

Objective: We examined the reproducibility of the oral glucose tolerance test (OGTT) in overweight children and evaluated distinguishing characteristics between those with concordant vs. discordant results., Design: Sixty overweight youth (8-17 yr old) completed two OGTTs (interval between tests 1-25 d). Insulin sensitivity was assessed by the surrogate measures of fasting glucose to insulin ratio, whole-body insulin sensitivity index, and homeostasis model assessment of insulin resistance, and insulin secretion by the insulinogenic index with calculation of the glucose disposition index (GDI)., Results: Of the 10 subjects with impaired glucose tolerance (IGT) during the first OGTT only three (30%) had IGT during the second OGTT. The percent positive agreement between the first and second OGTT was low for both impaired fasting glucose and IGT (22.2 and 27.3%, respectively). Fasting blood glucose had higher reproducibility, compared with the 2-h glucose. Youth with discordant OGTTs, compared with those with concordant results, were more insulin resistant (glucose/insulin 2.7+/-1.4 vs. 4.1+/-1.8, P=0.006, whole-body insulin sensitivity index of 1.3+/-0.6 vs. 2.2+/-1.1, P=0.003, and homeostasis model assessment of insulin resistance 10.6+/-8.1 vs. 5.7+/-2.8, P=0.001), had a lower GDI (0.45+/-0.58 vs. 1.02+/-1.0, P=0.03), and had higher low-density lipoprotein cholesterol (117.7+/-36.6 vs. 89.9+/-20.1, P=0.0005) without differences in physical characteristics., Conclusions: Our results show poor reproducibility of the OGTT in obese youth, in particular for the 2-h plasma glucose. Obese youth who have discordant OGTT results are more insulin resistant with higher risk of developing type 2 diabetes mellitus, as evidenced by a lower GDI. The implications of this remain to be determined in clinical and research settings.

Objective: Glucagon responses to hypoglycemia from islets transplanted in the liver are defective. To determine whether this defect is related to intrahepatic glycogen, islets from inbred Lewis rats were transplanted into the hepatic sinus (H group), peritoneal cavity (P group), omentum (O group), and kidney capsule (K group) of recipient Lewis rats previously rendered diabetic with streptozotocin (STZ)., Research Design and Methods: Glucagon responses to hypoglycemia were obtained before and after transplantation under fed conditions and after fasting for 16 h and 48 h to deplete liver glycogen., Results: Glucagon (area under the curve) responses to hypoglycemia in the H group (8,839 +/- 1,988 pg/ml per 90 min) were significantly less than in normal rats (40,777 +/- 8,192; P < 0.01). Fasting significantly decreased hepatic glycogen levels. Glucagon responses in the H group were significantly larger after fasting (fed 8,839 +/- 1,988 vs. 16-h fasting 24,715 +/- 5,210 and 48-h fasting 29,639 +/- 4,550; P < 0.01). Glucagon response in the H group decreased after refeeding (48-h fasting 29,639 +/- 4,550 vs. refed 10,276 +/- 2,750; P < 0.01). There was no difference in glucagon response to hypoglycemia between the H and the normal control group after fasting for 48 h (H 29,639 +/- 4,550 vs. control 37,632 +/- 5,335; P = NS). No intragroup differences were observed in the P, O, and K groups, or normal control and STZ groups, when comparing fed or fasting states., Conclusions: These data suggest that defective glucagon responses to hypoglycemia by intrahepatic islet alpha-cells is due to dominance of a suppressive signal caused by increased glucose flux and glucose levels within the liver secondary to increased glycogenolysis caused by systemic hypoglycemia.

Glucotoxicity, lipotoxicity, and glucolipotoxicity are secondary phenomena that are proposed to play a role in all forms of type 2 diabetes. The underlying concept is that once the primary pathogenesis of diabetes is established, probably involving both genetic and environmental forces, hyperglycemia and very commonly hyperlipidemia ensue and thereafter exert additional damaging or toxic effects on the beta-cell. In addition to their contribution to the deterioration of beta-cell function after the onset of the disease, elevations of plasma fatty acid levels that often accompany insulin resistance may, as glucose levels begin to rise outside of the normal range, also play a pathogenic role in the early stages of the disease. Because hyperglycemia is a prerequisite for lipotoxicity to occur, the term glucolipotoxicity, rather than lipotoxicity, is more appropriate to describe deleterious effects of lipids on beta-cell function. In vitro and in vivo evidence supporting the concept of glucotoxicity is presented first, as well as a description of the underlying mechanisms with an emphasis on the role of oxidative stress. Second, we discuss the functional manifestations of glucolipotoxicity on insulin secretion, insulin gene expression, and beta-cell death, and the role of glucose in the mechanisms of glucolipotoxicity. Finally, we attempt to define the role of these phenomena in the natural history of beta-cell compensation, decompensation, and failure during the course of type 2 diabetes.

Type 2 diabetes is characterized by a relentless decline in pancreatic islet beta cell function and worsening hyperglycemia despite optimal medical treatment. Our central hypothesis is that residual hyperglycemia, especially after meals, generates reactive oxygen species (ROS), which in turn causes chronic oxidative stress on the beta cell. This hypothesis is supported by several observations. Exposure of isolated islets to high glucose concentrations induces increases in intracellular peroxide levels. The beta cell has very low intrinsic levels of antioxidant proteins and activities and thus is very vulnerable to ROS. Treatment with antioxidants protects animal models of type 2 diabetes against complete development of phenotypic hyperglycemia. The molecular mechanisms responsible for the glucose toxic effect on beta cell function involves disappearance of two important regulators of insulin promoter activity, PDX-1 and MafA. Antioxidant treatment in vitro prevents disappearance of these two transcription factors and normalizes insulin gene expression. These observations suggest that the ancillary treatment with antioxidants may improve outcomes of standard therapy of type 2 diabetes in humans.

Diabetes is commonly referred to in terms of type 1 and type 2. Both forms involve pancreatic islet beta-cell abnormalities, characterized by death in type 1 and accelerated apoptosis in type 2. The resultant chronic hyperglycemia leads to chronic oxidative stress for all tissues because glucose in abnormally high concentrations forms reactive oxygen species. It has been repeatedly emphasized that this can lead to oxidative damage in the classical secondary targets of diabetes, such as eyes, kidneys, nerves, and blood vessels. However, it has been much less appreciated that the beta cell itself is also a prime target, a case of double jeopardy. This situation is all the more pernicious because islets contain among the lowest levels of antioxidant enzyme activities compared to other tissues. This adverse effect of high glucose concentrations is referred to as glucose toxicity. A major manifestation of glucose toxicity in the beta cell is defective insulin gene expression, diminished insulin content, and defective insulin secretion. The molecular mechanisms involve the development of decreased levels of two very important insulin promoter transcription factors, PDX-1 and MafA. Studies with animal models of type 2 diabetes have established that pharmacologic protection against oxidative stress ameliorates the severity of diabetes progression. Translational research with humans is now under way to ascertain whether this protection can be provided to patients experiencing inadequate glycemic control.

The insulin gene is expressed almost exclusively in pancreatic beta-cells. Metabolic regulation of insulin gene expression enables the beta-cell to maintain adequate stores of intracellular insulin to sustain the secretory demand. Glucose is the major physiologic regulator of insulin gene expression; it coordinately controls the recruitment of transcription factors [e.g., pancreatic/duodenal homeobox-1 (PDX-1), mammalian homologue of avian MafA/L-Maf (MafA), Beta2/Neuro D (B2), the rate of transcription, and the stability of insulin mRNA. However, chronically elevated levels of glucose (glucotoxicity) and lipids (lipotoxicity) also contribute to the worsening of beta-cell function in type 2 diabetes, in part via inhibition of insulin gene expression. The mechanisms of glucotoxicity, which involve decreased binding activities of PDX-1 and MafA and increased activity of C/EBPbeta, are mediated by high-glucose-induced generation of oxidative stress. On the other hand, lipotoxicity is mediated by de novo ceramide synthesis and involves inhibition of PDX-1 nuclear translocation and MafA gene expression. Glucotoxicity and lipotoxicity have common targets, which makes their combination particularly harmful to insulin gene expression and beta-cell function in type 2 diabetes.

In this study, we investigated the use of a novel oxygen biosensor system to detect changes in oxygen consumption rates (OCRs) by islets in response to glucose. Islets from non-human primate and human pancreata were seeded into an oxygen biosensor system microplate and exposed to basal (2.8 or 5.6 mM) or high (16.7 or 33.3 mM) glucose over either a long-term or a short-term culture. Our data clearly demonstrated that non-human primate islets cultured in high glucose conditions exhibited significant increases in OCRs over a 168 h extended culture period (P<0.05), which indicates an accelerated rate of beta-cell metabolism triggered by glucose over time. Significant increases in OCRs (P<0.01) were also attained in both non-human primate and human islets exposed to high glucose conditions in a 120 min short-term incubation period. OCRs exhibited by human islets exposed to different glucose concentrations correlated with insulin secretion (r(2)=0.7681, P<0.01). Moreover, the OCR stimulation index (i.e. OCR at high glucose/OCR at basal glucose) was significantly greater in human islets displaying high viabilities as opposed to islets exhibiting low viabilities (P<0.05). Together these data demonstrate that this novel oxygen biosensor system documents significant increases in islet oxygen consumption upon acute and chronic exposure to high glucose concentrations. Importantly, this methodology rapidly and robustly detects changes in OCRs by islets in response to high glucose stimulation that correlate well with the metabolic activities and functional viability of islets and clearly delineates significant differences in OCR stimulation index between high and low viability human islets, and therefore may prove to be an effective approach for quickly assessing the functional viability of islets prior to transplantation.

Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. Recently, RIPE-3b1 activator was cloned and identified as the mammalian homologue of avian MafA/Maf-L (MafA). This enabled us to carry out more extensive studies of the role of MafA in glucotoxicity than were hitherto possible. Northern analysis of glucotoxic HIT-T15 cells revealed normal amounts of MafA mRNA, but Western analysis demonstrated a 97 +/- 1% reduction in MafA protein (p < 0.0001). The proteasome is a likely site for MafA degradation as lactacystin, an irreversible proteasome inhibitor, caused an accumulation of MafA protein. Antioxidants have previously been shown to prevent the adverse effects of glucose toxicity on beta cell function both in vivo and in vitro. In the current study, chronic culturing of HIT-T15 cells with the antioxidant N-acetylcysteine (NAC) prevented loss of MafA protein (late passage = 18.9 +/- 10.4% of early passage, p < 0.001; late passage with NAC = 68.7 +/- 19.7% of early passage, p = not significant) and loss of DNA binding (late passage = 63.7 +/- 9% of early passage, p < 0.02; late passage with NAC = 116 +/- 10% of early passage, p = not significant). Additionally, transient transfection of PDX-1 or MafA cDNA into glucotoxic cells increased PDX-1 and MafA protein levels and individually increased insulin promoter activity (untreated = 34%, PDX-1 = 70%, MafA = 78%; percentage of activity of early passage cells), whereas the combined transfection of MafA and PDX-1 completely restored insulin promoter activity. This recovery of promoter activity following transient transfection had no effect on endogenous insulin mRNA. However, adenoviral infection of MafA and PDX-1 significantly increased endogenous insulin mRNA levels by 93% (121 +/- 9 versus 233 +/- 18 density light units; n = 5, p < 0.001). We conclude that the absence of MafA protein from beta cells via chronic oxidative stress contributes importantly to the loss of endogenous insulin gene expression as glucose toxicity develops.

The "switch-off" hypothesis to explain beta-cell regulation of alpha-cell function during hypoglycemia has not been assessed previously in isolated islets, largely because they characteristically do not respond to glucose deprivation by secreting glucagon. We examined this hypothesis using normal human and Wistar rat islets, as well as islets from streptozotocin (STZ)-administered beta-cell-deficient Wistar rats. As expected, islets perifused with glucose and 3-isobutryl-1-methylxanthine did not respond to glucose deprivation by increasing glucagon secretion. However, if normal rat islets were first perifused with 16.7 mmol/l glucose to increase endogenous insulin secretion, followed by discontinuation of the glucose perifusate, a glucagon response to glucose deprivation was observed (peak change within 10 min after switch off = 61 +/- 15 pg/ml [mean +/- SE], n = 6, P < 0.01). A glucagon response from normal human islets using the same experimental design was also observed. A glucagon response (peak change within 7 min after switch off = 31 +/- 1 pg/ml, n = 3, P < 0.01) was observed from beta-cell-depleted, STZ-induced diabetic rats whose islets still secreted small amounts of insulin. However, when these islets were first perifused with both exogenous insulin and 16.7 mmol/l glucose, followed by switching off both the insulin and glucose perifusate, a significantly larger (P < 0.05) glucagon response was observed (peak change within 7 min after switch off = 71 +/- 11 pg/ml, n = 4, P < 0.01). This response was not observed if the insulin perifusion was not switched off when the islets were deprived of glucose or when insulin was switched off without glucose deprivation. These data uniquely demonstrate that both normal, isolated islets and islets from STZ-administered rats can respond to glucose deprivation by releasing glucagon if they are first provided with increased endogenous or exogenous insulin. These results fully support the beta-cell switch-off hypothesis as a key mechanism for the alpha-cell response to hypoglycemia.

Various brief metabolic tests have been proposed as surrogate measures of insulin secretory reserve, which is normally determined by the more complicated and labor-intensive method of glucose potentiation of arginine-induced insulin secretion (GPAIS). This article provides correlations between insulin responses to intravenous arginine (AIRarg) and intravenous glucose (AIRgluc) in 39 normal control subjects in whom insulin secretory reserve was measured by GPAIS. The correlation coefficients were.78 and.77, respectively, both of which were highly statistically significant (P <.001). These results indicate that either AIRarg or AIRgluc, tests that can be performed in less than 15 minutes, can be used as reliable predictors of insulin secretory reserve in normal controls and, presumably, in recipients of islet and pancreas transplantation.

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 beta-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 beta-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 beta-cell function.

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 beta cell line, HIT-T15. Inhibition of gamma-glutamylcysteine synthetase (gammaGCS) 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.

HIT-T15 cells, a clonal beta-cell line, were cultured and passaged weekly for 6 mo in RPMI 1640 media containing various concentrations of glucose. Insulin content decreased in the intermediate- and late-passage cells as a continuous rather than a threshold glucose concentration effect. In a second series of experiments, cells were grown in media containing either 0.8 or 16.0 mM glucose from passages 76 through 105. Subcultures of passages 86, 92, and 99 that had been grown in media containing 16.0 mM glucose were switched to media containing 0.8 mM glucose and also carried forward to passage 105. Dramatic increases in insulin content and secretion and insulin gene expression were observed when the switches were made at passages 86 and 92 but not when the switch was made at passage 99. These findings suggest that glucose toxicity of insulin-secreting cells is a continuous rather than a threshold function of glucose concentration and that the shorter the period of antecedent glucose toxicity, the more likely that full recovery of cell function will occur.

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.

Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes adverse alterations in beta 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 beta cells. To ascertain whether chronic oxidative stress might play a role, we chronically cultured the beta 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-1/STF-1 and RIPE-3b1 activator), insulin content, and glucose-induced insulin secretion. These findings suggested that one mechanism of glucose toxicity in the beta 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 beta 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.

Refractoriness of the pancreatic beta-cell to glucose stimulation plays a role in the secretory defect of NIDDM, but the mechanisms underlying this refractoriness are not clear. The purpose of this study was to determine whether the HIT-T15 pancreatic beta-cell line can be used as an investigative model for refractoriness of glucose-induced insulin secretion and, if so, whether the mechanism for this refractoriness involves alteration in stimulus-secretion coupling (desensitization) or results from exhaustion of insulin stores. In perifusion experiments, acute insulin responses (AIRs) in HIT-T15 cells progressively diminished during consecutive 5-min glucose (11.1 mmol/l) pulses (G) given every 20 min (G1=9.2+/-1.3, G2=4.1+/-1.0, G3=2.7+/-0.7, G4=2.5+/-1.1 microU/ml). To determine whether this refractoriness for glucose extended to the potentiating effects of glucose on nonglucose secretagogues, cells were challenged with arginine after desensitization with glucose. In HIT-T15 cells, the response to the arginine pulse (16.7+/-5.2 microU/ml) after three glucose pulses was significantly less (P < 0.01) than the response to a control arginine pulse (29.6+/-12.1 microU/ml) preceded by an infusion of buffer in the absence of glucose pulses. Variable rest periods after desensitization allowed recovery of the AIR in HIT-T15 cells; responses 30, 60, 90, and 120 min after the desensitization procedure increased in a stepwise fashion (3.8+/-2.7, 4.5+/-2.7, 7.8+/-5.2, and 9.7+/-5.3 microU/ml, respectively). To differentiate desensitization from exhaustion of insulin stores, studies were performed in the presence of epinephrine, a potent inhibitor of insulin secretion. In HIT-T15 cells, after three pulses of glucose during the epinephrine infusion, epinephrine was discontinued and the insulin response to a fourth pulse was assessed. The G4 AIR (1.9+/-0.6 microU/ml) was markedly less than a control G4 AIR (5.4+/-1.2 microU/ml) that followed an epinephrine infusion alone with no concurrent glucose pulses. Beta-cell refractoriness was also induced in the HIT-T15 cell using 45-min steady-state infusions of glucose. Cells were exposed to a 45-min infusion of either 3.7 or 11.1 mmol/l glucose, rested for 20 min in the absence of glucose, and then challenged with a 5-min, 11.1 mmol/l glucose pulse. In both cases, the AIR to the 5-min pulse (10.2+/-5.1 and 2.9+/-1.4 microU/ml after the 3.7 and 11.1 mmol/l infusion, respectively) was lower than the AIR to a control pulse (27.4+/-5.9 microU/ml) not preceded by glucose infusion. These studies demonstrated that the HIT-T15 cell line is an appropriate model for studying mechanisms of beta-cell refractoriness to glucose signaling. The short-term intensive glucose stimulation paradigms used in our studies induced an abnormality in insulin secretion that is consistent with desensitization but not beta-cell exhaustion.

Chronic exposure of HIT-T15 cells to supraphysiologic glucose concentration diminishes insulin gene expression and decreased binding of two critical insulin gene transcription factors, STF-1 and RIPE-3b1 activator. To distinguish whether these changes are caused by glucose toxicity or beta cell exhaustion, HIT-T15 cells grown from passage 75 through 99 in media containing 11.1 mM glucose were switched to 0.8 mM glucose at passage 100. They regained binding of STF-1 and RIPE-3b1 activator and had a partial but minimal return of insulin mRNA expression. In a second study, inclusion of somatostatin in the media-containing 11.1 mM glucose inhibited insulin secretion; however, despite this protection against beta cell exhaustion, dramatic decreases in insulin gene expression, STF-1 and RIPE-3b1 binding, and insulin gene promoter activity still occurred. These data indicate that the glucotoxic effects caused by chronic exposure to supraphysiologic concentration of glucose are only minimally reversible and that they are not due simply to beta cell exhaustion. These observations carry with them the clinical implication that Type II diabetic patients who remain hyperglycemic for prolonged periods may have secondary glucose toxic effects on the beta cell that could lead to defective insulin gene expression and worsening of hyperglycemia.

Adenine nucleotide metabolism was characterized in intact insulin secreting HIT-T15 cells during the transition from non-stimulated (i. e. 0.2 mM glucose) to the glucose-stimulated secretory state. Metabolic dynamics were monitored by assessing rates of appearance of 18O-labeled phosphoryls of endogenous nucleotides in cells incubated in medium enriched in [18O]water. Most prominent of the metabolic alterations associated with stimulated insulin secretion was the suppression in the rate of adenylate kinase (AK)-catalyzed phosphorylation of AMP by ATP. This was manifest as a graded decrease of up to 50% in the rate of appearance of beta-18O-labeled species of ADP and ATP and corresponded to the magnitude of the secretory response elicited over a range of stimulatory glucose concentrations. The only nucleotide exhibiting a significant concentration change associated with suppression of AK activity was AMP, which decreased by about 50%, irrespective of the glucose concentration. Leucine-stimulated secretion also decreased the rate of AK-catalyzed phosphotransfer. This secretory stimulus-related suppression of AK-catalyzed phosphotransfer occurs within 45 s of glucose addition, precedes insulin secretion, depends on the internalization and metabolism of glucose, and is independent of membrane depolarization and the influx of extracellular calcium. The secretory stimulus-induced decrease in AK-catalyzed phosphotransfer, therefore occurs prior to or at the time of KATP+ channel closure but it is not associated with or a consequence of events occurring subsequent to KATP+ channel closure. These results indicate that AK-catalyzed phosphotransfer may be a determinant of ATP to ADP conversion rates in the KATP+ channel microenvironment; secretory stimuli-linked decreased rates of AK-catalyzed ADP generation from ATP (and AMP) would translate into an increased probability of ATP-liganded and, therefore, closed state of the channel.

We have shown previously that chronic exposure of HIT-T15 cells to supraphysiologic glucose concentrations causes decreased insulin gene transcription and decreased binding activities of two beta-cell specific transcription factors, STF-1 and the RIPE3b1 activator, and have suggested that these events may provide a mechanism for glucose toxicity on beta-cell function. However, this contention can be criticized because it is not clear whether these observations are unique to the HIT-T15 cell or generalizable to other beta-cell lines and the islet. Therefore, we cultured betaTC-6 cells for up to 41 wk in either 11.1 or 0.8 mM glucose. We observed a passage-dependent decrease in insulin content and insulin mRNA levels in betaTC-6 cells chronically cultured in 11.1 mM glucose. Cells chronically cultured in 0.8 mM glucose had higher insulin mRNA levels than cells chronically cultured in 11.1 mM glucose. The relative activity of a chloramphenicol acetyl transferase (CAT) reporter gene controlled by the 5' regulatory region of the human insulin gene was decreased in late passage betaTC-6 cells chronically cultured in 11.1 mM glucose, but was preserved in late passages of cells chronically cultured in 0.8 mM glucose. Electromobility shift assays demonstrated that binding of a specific nuclear protein that recognizes the RIPE3b1 binding site of the insulin gene was markedly diminished in late passage cells chronically exposed to 11.1 mM glucose, whereas binding activities of STF-1 and ICE activators were unchanged. RIPE3b1 binding activity was preserved in late passage cells chronically exposed to 0.8 mM glucose. Mutation of the RIPE3b1 binding site almost completely abolished insulin gene transcription as well as binding activity. We conclude that chronic exposure of betaTC-6 cells to high glucose concentrations paradoxically decreases insulin gene transcription, in part, by decreasing activity of the trans-activating factor which binds to the RIPE3b1 sequence. This study uniquely demonstrates that altered binding to the RIPE3b1 sequence mediates glucose toxicity in betaTC-6 cells, thus reinforcing the importance of this cis-acting element in the regulation of insulin gene transcription. We conclude that the phenomenon of glucose toxicity decreasing binding of transcription factors and thereby reducing insulin gene expression is not a feature solely of HIT-T15 cells and may be demonstrable generally in beta-cell lines.

The most important regulator of insulin gene expression in pancreatic beta- cells is glucose, which affects gene transcription, mRNA translation, and secretion. Insulin gene transcription is both positively and negatively regulated by glucose. Recently, we have shown that the inhibition of insulin gene transcription caused by passaging HIT T-15 beta-cells, in the presence of high glucose, was due, in part, to reduced expression of a key regulator of insulin enhancer-mediated expression, somatostatin transcription factor-1 (STF-1). In this study, we have examined whether the activity of the other essential transcription regulators of insulin gene expression, the RIPE3b1 and insulin control element (ICE) activators, were also influenced in these HIT T-15 cells. The results show that the binding and trans-activation functions of the RIPE3b1 activator are reduced in parallel with the loss in STF-1 and insulin gene expression. In contrast, the regulatory properties of the ICE activator are unaffected. Our studies indicate that insulin gene transcription is inhibited by glucose through a mechanism involving reduced expression of both the RIPE3b1 and STF-1 activators in HIT T-15 cells but is independent of the ICE activator.

Healthy humans undergoing hemipancreatectomy for the purpose of donation to a family member with IDDM have previously been demonstrated to maintain serum glucose values equal to matched control subjects during short-term glucose infusion despite significant decrements in glucose- and arginine-induced insulin secretion. In order to determine whether humans compensate for hemipancreatectomy by increasing insulin- or glucose-mediated glucose uptake, we measured glucose turnover and insulin sensitivity by three protocols. Insulin-mediated glucose uptake was measured during sequential infusions of insulin at rates of 0.25, 1.0, and 10.0 mU.kg-1.min-1 in 12 donor subjects and 12 matched control subjects maintained at euglycaemia. Both groups displayed similar increases in rates of glucose disappearance and similar decreases in rates of hepatic glucose production. Glucose-mediated uptake was calculated as the difference between the rates of glucose disappearance measured during a hyperglycaemic clamp performed at identical rates of insulin infusion and was also found to be similar in both donor subjects and control subjects. Both groups also had indistinguishable measures of insulin sensitivity and glucose effectiveness as determined by the minimal model technique. Therefore, donor subjects appear to compensate for diminished insulin secretion following hemipancreatectomy by an unidentified mechanism since neither insulin- nor glucose-mediated glucose uptake are increased.

Our perspective is that the concepts of glucose toxicity and glucose desensitization should be differentiated because they carry very different connotations. The term glucose desensitization most properly refers to a pharmacological event involving a temporary, readily induced, physiological and reversible state of cellular refractoriness because of repeated or prolonged exposure to high concentrations of glucose. The term glucose toxicity should be reserved for nonphysiological, irreversible alterations in cellular function caused by chronic exposure to high glucose concentrations. With regard to the pancreatic islet beta-cell, the mechanism of action for glucose desensitization seems most likely to be expressed at the level of the insulin exocytotic apparatus or insulin stores within the beta-cell, whereas the mechanism of action for glucose toxicity may be at the level of insulin gene transcription. This differentiation raises the possibility that exposure of patients to chronic hyperglycemia may cause glucose toxic effects on the process of insulin gene transcription and/or expression that are irreversible. If so, this may contribute to so-called secondary drug failure and, in any event, reemphasizes the need to intensify therapeutic efforts to better regulate glycemia in type II diabetes.

We observed in the HIT cell, a clonal insulin-secreting cell line, that epinephrine and somatostatin lower insulin mRNA levels and intracellular insulin content in addition to the well-recognized effect of these hormones to inhibit insulin secretion. To determine whether these inhibitory hormones might regulate insulin synthesis at the level of insulin gene transcription, we studied HIT cell expression of a human insulin-chloramphenicol acetyl transferase (CAT) reporter gene in the presence of glucose, epinephrine, and somatostatin. HIT cell expression of this human insulin-CAT reporter gene was responsive to glucose in a concentration-dependent manner, increasing threefold as the glucose concentration increased from 0.4 to 11 mM. Epinephrine significantly inhibited insulin-CAT reporter gene expression (61 +/- 5% of control), an effect mediated specifically by the human insulin gene promoter/enhancer sequence. Somatostatin significantly inhibited expression of the human insulin-CAT reporter gene (65 +/- 4% of control) and, to a lesser extent, expression of a control reporter gene, pRSVCAT (78 +/- 4% of control). Thus, somatostatin may inhibit insulin gene transcription by insulin gene-specific effects as well as more general effects on gene expression. Both epinephrine and somatostatin inhibited expression of the human insulin-CAT reporter gene in a concentration-dependent manner that paralleled inhibition of insulin secretion. These studies indicate that epinephrine and somatostatin lower HIT cell insulin mRNA levels in part by inhibiting insulin gene transcription. Thus, hormonal inhibition of insulin secretion may be coupled with inhibition of insulin synthesis, thereby allowing the beta-cell to match insulin supply to secretory demand.

Chronically culturing HIT-T15 cells in media containing high glucose concentrations leads to decreased insulin mRNA levels, insulin content, and insulin secretion. These changes can be prevented by culturing the cells in media containing lower glucose levels (Robertson, R. P., H.-J. Zhang, K. L. Pyzdrowski, and T. F. Walseth. 1992. J. Clin. Invest. 90:320-325). The mechanism of this seemingly paradoxical phenomenon was examined by transiently transfecting HIT cells with a chloramphenicol acetyl transferase (CAT) reporter gene controlled by the 5'-regulatory domain of the human insulin gene (INSCAT). Early passages of HIT cells readily expressed INSCAT, whereas late passages of cells chronically cultured in 11.1 mM glucose expressed only 28.7 +/- 2.3% (mean +/- SEM) of the CAT activity expressed in early passages. In contrast, late passages of HIT cells chronically cultured in 0.8 mM glucose retained the ability to express the INSCAT reporter gene to 69.6 +/- 10.0% of the CAT activity observed in early passages. The decrease in INSCAT expression in late passages of cells serially cultured in 11.1 mM glucose was associated with the inability to form a specific nuclear protein-DNA complex with the CT motifs of the human insulin promoter. Formation of this specific protein-DNA complex was preserved in late passages of HIT cells when serially cultured in 0.8 mM glucose. Mutations of the CT motifs caused markedly diminished CAT activity in all passages examined. These data indicate that chronic exposure of the beta cell to high glucose concentrations can paradoxically decrease insulin gene transcription, in part, by altering the ability of a regulatory protein (GSTF) to interact with the insulin gene promoter. This provides a potential mechanism for glucotoxic effects on the beta cell at the level of the insulin gene.

G-proteins are important mediators of hormonal inhibition of insulin secretion. To characterize the pertussis toxin-sensitive substrates present in HIT cell membranes, we performed immunoblots with specific antisera and found evidence for the presence of Gi alpha 1, Gi alpha 2, Gi alpha 3, and three forms of Go alpha. We observed that pertussis toxin-sensitive substrates mediate all of the effects of SRIF, and a major portion of the effects of EPI, on insulin secretion from rat islets during static incubations. These results agree with our previously reported studies examining phasic glucose-induced insulin secretion from HIT cells. To ascertain whether inhibition of adenylate cyclase, presumably involving coupling of the catalytic subunit to Gi, may be a common mechanism for both hormones, we studied the effects of 8-bromo-cyclic AMP and found that this agent partially prevented the inhibitory effects of both hormones. We also observed that the inhibitory effects of SRIF and EPI on insulin were nonadditive, that both hormones were additive to nickel chloride during inhibition of insulin release, and that they noncompetitively inhibited glipizide-induced insulin secretion through pertussis toxin-sensitive mechanisms. Together, these results suggest that both hormones exert their effects on insulin secretion at multiple G-protein-regulated sites including adenylate cyclase and sites distal to the glipizide-binding site on the KATP channel.

Glucose toxicity of the pancreatic beta cell is considered to play a secondary role in the pathogenesis of type II diabetes mellitus. To gain insights into possible mechanisms of action of glucose toxicity, we designed studies to assess whether the loss of insulin secretion associated with serial passages of HIT-T15 cells might be caused by chronic exposure to high glucose levels since these cells are routinely cultured in media containing supramaximal stimulatory concentrations of glucose. We found that late passages of HIT cells serially cultured in media containing 11.1 mM glucose lost insulin responsivity and had greatly diminished levels of insulin content and insulin mRNA. In marked contrast, late passages of HIT cells cultured serially in media containing 0.8 mM glucose retained insulin mRNA, insulin content, and insulin responsivity to glucose in static incubations and during perifusion with glucose. No insulin gene mutation or alteration of levels of GLUT-2 were found in late passages of HIT cells cultured with media containing 11.1 mM glucose. These data uniquely indicate that loss of beta cell function in HIT cells passed serially under high glucose conditions is caused by loss of insulin mRNA, insulin content, and insulin secretion and is preventable by culturing HIT cells under low glucose conditions. This strongly suggests potential genetic mechanisms of action for glucose toxicity of beta cells.

Messenger RNA from intact rat pancreatic islets, or from transformed hamster beta (HIT) cells, hybridized with the cDNA probe for type I (but not type II) phospholipase A2. The levels of phospholipase A2 mRNA increased in islets from fasted rats; they decreased in islets cultured in a high glucose concentration (control values at 5.5 mM glucose = 150 +/- 6% of those at 22 mM) which impaired subsequent insulin secretion (reduction in second-phase release = 70 +/- 11%). These studies uniquely demonstrate that type I phospholipase A2 is expressed specifically in beta cells and that nutrient availability modulates transcript levels, an effect which could that nutrient availability modulates transcript levels, an effect which could contribute to the detrimental influence of prolonged hyperglycemia on islet function.

Noninsulin dependent diabetics have insulin responses to nonglucose secretagogues that are subnormal for their plasma glucose levels. Since endogenous prostaglandins have been implicated in the abnormal insulin responses to glucose in diabetics, the present study was performed to explore whether prostaglandins might also play a role in the defective insulin responses to nonglucose stimuli. We examined the effects of infusions of either prostaglandin E2 (PGE2) or sodium salicylate (SS), a PG synthesis inhibitor, on the acute insulin responses (AIR's) to arginine and isoproterenol and on the glucose potentiation of the insulin response to arginine in both normal and diabetic subjects. The AIR to arginine was augmented by SS in diabetics (SS = 61 +/- 12 microunits/ml, control = 37 +/- 5 microunits/ml, n = 11, p less than .01). SS, however, had no effect on the AIR to arginine in normal subjects (SS = 39 +/- 4 microunits/ml. control = 34 +/- 4 microunits/ml, n = 6, p = ns). Similarly, SS augmented the AIR to an isoproterenol pulse in diabetics (SS = 38 +/- 9 microunits/ml, control = 18 +/- 3, n = 9, p less than .05) but not in normal subjects (SS = 19 +/- 4 microunits/ml, control = 21 +/- 4 microunits/ml, n = 8, p = ns), suggesting a SS-sensitive defect in the insulin response to these nonglucose stimuli in diabetics. Conversely, PGE2 inhibited the AIR to arginine in diabetics (PGE = 28 +/- 5 microunits/ml, control = 39 +/- 7 microunits/ml, n = 7, p less than .05), but not in normal subjects (PGE = 74 +/- 7 microunits/ml, control = 80 +/- 14 microunits/ml, n = 5, p = ns). The effect of SS on glucose potentiation of the AIR to arginine was studied by measuring the AIR to arginine at two different levels of plasma glucose, one before and one after an insulin infusion, with glucose potentiation defined as the ratio delta AIR/delta prestimulus glucose. Glucose potentiation was significantly less in diabetics than in normals and SS significantly improved glucose potentiation toward normal values in diabetics but did not change glucose potentiation in normals. These findings suggest that endogenous PG's may play a role in the defective glucose potentiation of the AIR to nonglucose secretagogues in diabetics resulting in impaired insulin responses to these stimuli. This defect is partially reversible by an inhibitor of PG synthesis.

Although the cyclo-oxygenase pathway of arachidonic acid (AA) metabolism inhibits glucose-stimulated insulin release through synthesis of prostaglandins, very little attention has been given to the effects of lipoxygenase pathway products on beta cell function. We have examined the effects of two structurally-dissimilar lipoxygenase inhibitors on insulin release from monolayer-cultured rat islet cells. Both nordihydroguaiaretic acid (NDGA, 20-50 microM) and BW755c (100-250 microM) caused a dose-responsive inhibition of glucose-induced insulin release. This inhibitory effect occurred despite concomitant inhibition of prostaglandin E synthesis. Lipoxygenase inhibitors also impeded cyclic AMP accumulation. Insulin and cyclic AMP release induced by glucagon were also blunted. These studies suggest the hypothesis that AA released in or near the beta cell is metabolized to lipoxygenase product(s) which have feed-forward properties important to glucose- and glucagon-stimulated cyclic nucleotide accumulation and insulin release.

Although prostaglandin E2 (PGE2) is known to inhibit glucose-induced insulin secretion, it is uncertain whether PGE2 actions on the beta-cell are direct, whether they are equipotent for both phases of hormone secretion, and whether the same mechanism of action prevails throughout. Study of the HIT cell, a clonal line of pancreatic beta-cells, provides answers to these questions because perifusion with glucose and 3-isobutyl-1-methylxanthine stimulates biphasic insulin secretion. Perifusion with PGE2 decreased both the first and second phases of glucose-induced insulin release to 47 +/- 4% of controls. Pretreatment with pertussis toxin partly prevented PGE2 inhibition to 80 +/- 4% of controls for first phase and 79 +/- 4% of controls for second phase. To evaluate whether the partial prevention of PGE2 inhibition seen with pertussis toxin pretreatment was caused by Gi heterotrimer association between the preincubation period and the end of perifusion, PGE2 actions were also examined during continuous treatment with pertussis toxin. Under these conditions, PGE2 inhibition of both phases was totally prevented. However, no difference was observed in membrane protein ADP ribosylation when cells were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pretreatment or continuous treatment with pertussis toxin. Cyclic AMP (cAMP) accumulation was inhibited by PGE2 (from 3263 +/- 153 to 1549 +/- 158 fmol/10(6) cells) but less so after pretreatment with pertussis toxin (correlation between insulin release and cAMP accumulation during perifusion; n = 18, r = .85, P less than .001). Thus, PGE2 equally inhibits both phases of glucose-induced insulin secretion and cAMP generation through a pertussis toxin-sensitive G protein-mediated direct effect on the pancreatic beta-cell.

Monolayer cultures of neonatal rat pancreatic cells were examined to ascertain whether they synthesize prostaglandin E (PGE) and to determine the effects on insulin secretion caused by PGE and drugs that inhibit its synthesis. PGE release into the medium was observed. Sodium salicylate and ibuprofen (at drug concentrations similar to those achieved therapeutically in humans in vivo) inhibited PGE synthesis in a dose-responsive fashion to a maximum of 70-80% inhibition. Inhibition of PGE synthesis was accompanied by augmented insulin secretion. Both PGE synthesis inhibitors shifted the glucose dose-insulin response curves to the left at low glucose concentrations and augmented maximal insulin release at high glucose concentrations. Increments in glucose-induced insulin secretion induced by sodium salicylate correlated well (r = 0.89) with inhibition of PGE synthesis and addition of exogenous PGE1 to the cultures reversed the augmenting effects of the drug on insulin secretion. It is concluded that cultures of pancreatic cells synthesize PGE and that a function of PGE in these cultures appears to be a tonic negative modulation of glucose-induced insulin secretion.

The glucoreceptor hypothesis proposes that diabetics have a selective defect in both pancreatic and extra-pancreatic tissues in the recognition of, and hormonal responses to glucose. Many of these defective or even paradoxical responses to hyperglycemia and hypoglycemia can be mimicked by known actions of prostaglandins, particularly PGE, in normal subjects. Inhibition of PGE synthesis in diabetics partially reverses several abnormal responses. We propose that excessive production of, or sensitivity to, prostaglandins may play a role in some of the metabolic abnormalities associated with defective glucose recognition in diabetes mellitus.

Adult-onset diabetics have markedly diminished or absent acute insulin responses to glucose that can be partially restored by sodium salicylate infusion. To determine whether this restoration of the acute insulin response is glucose dose dependent and whether complete restoration can be achieved, adult-onset diabetics with a mean fasting plasma glucose value of 216 +/- 20 mg. per deciliter (x +/- S.E.) were stimulated with various doses of intravenous glucose. Restoration occurred in a glucose dose-dependent manner. Complete restoration could not be achieved with the maximal tolerable glucose dose (80 gm.). Second phase insulin secretion also improved in a glucose dose-dependent manner. These findings are compatible with the hypothesis that defective insulin secretion in adult-onset, hyperglycemic diabetics is not due to absolute deficiency of insulin but may be a result of defective recognition of glucose signals by pancreatic B-cells--a defect that can be partially reversed by sodium salicylate.

Prostaglandin E(2) (PGE(2)) infusion in normal humans inhibited acute insulin responses to a glucose (5 g i.v.) pulse (response before PGE(2) = 593 +/- 104%; during PGE(2) = 312+/-55%; mean+/-SE, mean change 3-5 min insulin,% basal, P < 0.005). This effect was associated with a decrease in glucose disappearance rates (K(G) before PGE(2) = 0.73+/-0.07; during PGE(2) = 0.49+/-0.06%/min, P < 0.025). Acute insulin responses to arginine (2 g i.v.) were not affected by PGE(2) (response before PGE(2) = 592+/-164%; during PGE(2) = 590+/-118%; P = NS). Infusion of sodium salicylate (SS), an inhibitor of endogenous prostaglandin synthesis, augmented acute insulin responses to glucose in normals (response before SS = 313+/-62%; during SS = 660+/-86%; P < 0.001). In adult-onset diabetes with fasting hyperglycemia, SS restored absent acute insulin responses to glucose (20 g i.v.) pulses (response before SS = 5+/-6%; during SS = 97+/-24%; P < 0.005). This was accompanied by a fourfold augmentation in second phase insulin secretion (second phase before SS = 1,696+/-430%; during SS = 5,176+/-682%; change 10-60 min insulin, muU/ml.min,% basal, P < 0.001) and by acceleration of glucose disappearance rates (K(G) before SS = 0.56+/-0.06; during SS = 1.02+/-0.17%/min, P < 0.005). These findings uniquely demonstrate that (a) PGE(2) inhibits glucose-induced acute insulin responses and decreases glucose disposal in nondiabetic humans and (b) SS restores acute insulin responses, augments second phase insulin secretion, and accelerates glucose disposal in hyperglycemic, adultonset diabetics. It is hypothesized that endogenous PGE synthesis may play a role in defective insulin secretion and glucose intolerance in diabetes mellitus.

A universal finding in hyperglycemic patients with type II (non-insulin-dependent) diabetes mellitus is that all share a common defect in glucose recognition resulting in abnormal insulin secretion by pancreatic islet beta-cells. This defect is 1) specific for glucose signals rather than global, 2) related to chronic hyperglycemia, and 3) partially reversible after brief treatment with insulin to induce normoglycemia and through use of other pharmacological agents without normalizing glucose levels. My perspective is that an essential component of this defect is secondary and may represent a state of homologous desensitization of the beta-cell secretory apparatus to glucose. Elucidation of the biochemical mechanism(s) of defective recognition of glucose signals by beta-cells--or glucose "non-sense"--in these patients will provide key insights into the pathogenesis of type II diabetes mellitus.

Objective: Standardized, reproducible, and feasible quantification of β-cell function (BCF) is necessary for the evaluation of interventions to improve insulin secretion and important for comparison across studies. We therefore characterized the responses to, and reproducibility of, standardized methods of in vivo BCF across different glucose tolerance states.Research Design and Methods: Participants classified as having normal glucose tolerance (NGT; n = 23), prediabetes (PDM; n = 17), and type 2 diabetes mellitus (T2DM; n = 22) underwent two standardized mixed-meal tolerance tests (MMTT) and two standardized arginine stimulation tests (AST) in a test-retest paradigm and one frequently sampled intravenous glucose tolerance test (FSIGT).Results: From the MMTT, insulin secretion in T2DM was >86% lower compared with NGT or PDM (P < 0.001). Insulin sensitivity (Si) decreased from NGT to PDM (∼50%) to T2DM (93% lower [P < 0.001]). In the AST, insulin secretory response to arginine at basal glucose and during hyperglycemia was lower in T2DM compared with NGT and PDM (>58%; all P < 0.001). FSIGT showed decreases in both insulin secretion and Si across populations (P < 0.001), although Si did not differ significantly between PDM and T2DM populations. Reproducibility was generally good for the MMTT, with intraclass correlation coefficients (ICCs) ranging from ∼0.3 to ∼0.8 depending on population and variable. Reproducibility for the AST was very good, with ICC values >0.8 across all variables and populations.Conclusions: Standardized MMTT and AST provide reproducible and complementary measures of BCF with characteristics favorable for longitudinal interventional trials use. [ABSTRACT FROM AUTHOR]

Patients with chronic pancreatitis who undergo total pancreas resection inevitably become diabetic unless their islets are autotransplanted to prevent diabetes. We studied patients who underwent this procedure to assess its long-term efficacy in providing stable glucose regulation. Six patients were followed for up to 13 (6.2 +/- 1.7) years after intrahepatic islet autotransplantation. From 290,000 to 678,000 islets were transplanted and no patients received drugs to control glucose levels postoperatively. Islet function was assessed by measurements of fasting plasma glucose (FPG), intravenous glucose disappearance rate (KG), HbA1c, insulin responses to intravenous glucose and to arginine, and insulin secretory reserve. Patients were studied two to four times each to obtain longitudinal data. Five of six patients remained free of insulin treatment and maintained FPG <126 mg/dl and HbA1c levels <6.5%. As a group, they maintained stable insulin secretory reserve, but insulin responses to glucose tended to decrease over time in three patients. KG values correlated significantly with the number of islets originally transplanted. These data indicate that intrahepatic autoislet transplantation can successfully maintain stable beta-cell function and normal levels of blood glucose and HbA1c for up to 13 years after total pancreatectomy as treatment for chronic painful pancreatitis. This usually overlooked procedure of intrahepatic islet transplantation designed to prevent diabetes in patients undergoing pancreatectomy for chronic pancreatitis should be considered more often. [ABSTRACT FROM AUTHOR]

Signalling by intraislet β-cells to neighbouring α-cells was recognized almost 40 years ago, leading to the hypothesis that this is an essential mechanism to regulate the glucagon counterregulatory response to hypoglycaemia. The thesis was that during normoglycaemia or hyperglycaemia insulin secretion from β-cells would enter the islet periportal circulation and travel downstream to α-cells to dampen glucagon secretion. As a corollary, during hypoglycaemia β-cells would stop secreting insulin, which would permit α-cells to release glucagon into the hepatic portal circulation so it could travel to the liver to increase glucose production and thereby correct hypoglycaemia. This mini-review briefly mentions the early work that established this hypothesis and more extensively examines more recent work that has provided direct evidence supporting the hypothesis. A new twist has been introduced based on the fact that zinc is bound to insulin within β-cells and co-secreted with insulin. Zinc is released from insulin when it reaches the higher pH of blood, and zinc has recently been shown to negatively regulate α-cell secretion. It is now suggested that a switch-off signal provided by a sudden cessation of zinc secretion from β-cells during hypoglycaemia may play a critical role in stimulating glucagon secretion that is independent of the effect of insulin. [ABSTRACT FROM AUTHOR]

Background: Overexpression of antioxidant enzymes has been reported to protect rodent beta cells from oxidative stress. However, very little is known about protein expression and activity of antioxidant enzymes in human islets. Method/Results: Human islet protein levels by Western analysis and enzymatic activity for the key antioxidant enzymes superoxide dismutases (SODs), catalase, and glutathione peroxidase-1 (GPx) were examined. Enzyme protein expression and activity were in the order SODs > catalase > GPx. Human islet GPx protein expression was significantly less than that found for catalase (p < 0.0001) and levels of GPx activity were virtually undetectable. As glucose and estrogens have been proposed to alter antioxidant enzyme levels, we examined islet data from male and female donors separately and under varying glucose concentrations. We found significantly less (p < 0.001) GPx protein expression in islets from females compared to males, but no significant regulation by glucose in either gender. Conclusions: Human islets have very low protein and activity levels for GPx, the essential enzyme for protection against excessive levels of intracellular lipid peroxides. GPx mimetics may be especially valuable in providing human islets with the broadest spectrum of protection against oxidative stress during isolation and transplantation. [ABSTRACT FROM AUTHOR]