Vincent Poitout

Minireview: Secondary β-Cell Failure in Type 2 Diabetes—A Convergence of Glucotoxicity and Lipotoxicity

Chronic hyperglycemia and hyperlipidemia can exert deleterious effects on -cell function, respectively referred to as glucotoxicity and lipotoxicity. Over time, both contribute to the progressive deterioration of glucose homeostasis characteristic of type 2 diabetes. The mechanisms of glucotoxicity involve several transcription factors and are, at least in part, mediated by generation of chronic oxidative stress. Lipotoxicity is probably mediated by accumulation of a cytosolic signal derived from the fatty acid esterification pathway. Our view that hyperglycemia is a prerequisite for lipotoxicity is supported by several recent studies performed in our laboratories. First, prolonged in vitro exposure of isolated islets to fatty acids decreases insulin gene expression in the presence of high glucose concentrations only, and glucose is ratelimiting for the incorporation of fatty acids into neutral lipids. Second, normalization of blood glucose in Zucker diabetic fatty rats prevents accumulation of triglycerides and impairment of insulin gene expression in islets, whereas normalization of plasma lipid levels is without effect. Third, high-fat feeding in Goto-Kakizaki rats significantly impairs glucoseinduced insulin secretion in vitro, whereas a similar diet has no effect in normoglycemic animals. We propose that chronic hyperglycemia, independent of hyperlipidemia, is toxic for -cell function, whereas chronic hyperlipidemia is deleterious only in the context of concomitant hyperglycemia. (Endocrinology 143: 339 –342, 2002)

A glucose monitoring system for on line estimation in man of blood glucose concentration using a miniaturized glucose sensor implanted in the subcutaneous tissue and a wearable control unit

SummaryWe have developed a miniaturized glucose sensor which has been shown previously to function adequately when implanted in the subcutaneous tissue of rats and dogs. Following a glucose load, the sensor output increases, making it possible to calculate a sensitivity coefficient to glucose in vivo, and an extrapolated background current in the absence of glucose. These parameters are used for estimating at any time the apparent subcutaneous glucose concentration from the current. In the previous studies, this calibration was performed a posteriori, on the basis of the retrospective analysis of the changes in blood glucose and in the current generated by the sensor. However, for clinical application of the system, an on line estimation of glucose concentration would be necessary. Thus, this study was undertaken in order to assess the possibility of calibrating the sensor in real time, using a novel calibration procedure and a monitoring unit which was specifically designed for this purpose. This electronic device is able to measure, to filter and to store the current. During an oral glucose challenge, when a stable current is reached, it is possible to feed the unit with two different values of blood glucose and their corresponding times. The unit calculates the in vivo parameters, transforms every single value of current into an estimation of the glucose concentration, and then displays this estimation. In this study, 11 sensors were investigated of which two did not respond to glucose. In the other nine trials, the volunteers were asked to record every 30 s what appeared on the display during the secondary decrease in blood glucose. The results were analysed by comparing these readings (approximately 220 measurements per trial) to the changes in plasma glucose, measured every 15 min. The Error Grid Analysis indicated that 84.1±3.6% of the measurements were in zone A (accurate) and 15±3.6% were in zone B (acceptable). Considering each individual trial, the differences between the displayed value and the concomitant plasma glucose concentration ranged between −1.7 and 0.69 mmol/l. These excellent results were due to the absence of any significant lag between the changes in plasma glucose concentration and the changes in the result on the display. We conclude that this glucose monitoring system, based on subcutaneous sensing of glucose, is able to provide a direct on line estimation of blood glucose concentration.

GPR40 Is Necessary but Not Sufficient for Fatty Acid Stimulation of Insulin Secretion In Vivo

Long-chain fatty acids amplify insulin secretion from the pancreatic β-cell. The G-protein–coupled receptor GPR40 is specifically expressed in β-cells and is activated by fatty acids; however, its role in acute regulation of insulin secretion in vivo remains unclear. To this aim, we generated GPR40 knockout (KO) mice and examined glucose homeostasis, insulin secretion in response to glucose and Intralipid in vivo, and insulin secretion in vitro after short- and long-term exposure to fatty acids. Our results show that GPR40 KO mice have essentially normal glucose tolerance and insulin secretion in response to glucose. Insulin secretion in response to Intralipid was reduced by ∼50%. In isolated islets, insulin secretion in response to glucose and other secretagogues was unaltered, but fatty acid potentiation of insulin release was markedly reduced. The Gαq/11 inhibitor YM-254890 dose-dependently reduced palmitate potentiation of glucose-induced insulin secretion. Islets from GPR40 KO mice were as sensitive to fatty acid inhibition of insulin secretion upon prolonged exposure as islets from wild-type animals. We conclude that GPR40 contributes approximately half of the full acute insulin secretory response to fatty acids in mice but does not play a role in the mechanisms by which fatty acids chronically impair insulin secretion.

Glucolipotoxicity of the pancreatic beta cell

The concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and fatty acid levels on pancreatic beta-cell function and survival. Significant progress has been made in recent years towards a better understanding of the cellular and molecular basis of glucolipotoxicity in the beta cell. The permissive effect of elevated glucose on the detrimental actions of fatty acids stems from the influence of glucose on intracellular fatty acid metabolism, promoting the synthesis of cellular lipids. The combination of excessive levels of fatty acids and glucose therefore leads to decreased insulin secretion, impaired insulin gene expression, and beta-cell death by apoptosis, all of which probably have distinct underlying mechanisms. Recent studies from our laboratory have identified several pathways implicated in fatty acid inhibition of insulin gene expression, including the extracellular-regulated kinase (ERK1/2) pathway, the metabolic sensor Per-Arnt-Sim kinase (PASK), and the ATF6 branch of the unfolded protein response. We have also confirmed in vivo in rats that the decrease in insulin gene expression is an early defect which precedes any detectable abnormality in insulin secretion. While the role of glucolipotoxicity in humans is still debated, the inhibitory effects of chronically elevated fatty acid levels has been clearly demonstrated in several studies, at least in individuals genetically predisposed to developing type 2 diabetes. It is therefore likely that glucolipotoxicity contributes to beta-cell failure in type 2 diabetes as well as to the decline in beta-cell function observed after the onset of the disease.

Towards continuous glucose monitoring: in vivo evaluation of a miniaturized glucose sensor implanted for several days in rat subcutaneous tissue

SummaryA miniaturized amperometric, enzymatic, glucose sensor (outer diameter 0.45 mm) was evaluated after implantation in the subcutaneous tissue of normal rats. A simple experimental procedure was designed for the long-term assessment of the sensors function which was performed by recording the current during an intraperitoneal glucose load. The sensor was calibrated by accounting for the increase in the current during the concomitant increase in plasma glucose concentration, determined in blood sampled at the tail vein. This made it possible to estimate the glucose concentration in subcutaneous tissue. During the glucose load, the change in subcutaneous glucose concentration followed that in blood with a lag time consistently shorter than 5 min. The estimations of subcutaneous glucose concentration during these tests were compared to the concomitant plasma glucose concentrations by using a grid analysis. Three days after implantation (n=6 experiments), 79 estimations were considered accurate, except for five which were in the acceptable zone. Ten days after implantation (n=5 experiments), 101 estimations were accurate, except for one value, which was still acceptable. The sensitivity was around 0.5 nA mmol−1·l−1 on day 3 and day 10. A longitudinal study on seven sensors tested on different days demonstrated a relative stability of the sensors sensitivity. Finally, histological examination of the zone around the implantation site revealed a fibrotic reaction containing neocapillaries, which could explain the fast response of the sensor to glucose observed in vivo, even on day 10. We conclude that this miniaturized glucose sensor, whose size makes it easily implanted, works for at least ten days after implantation into rat subcutaneous tissue.

Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol.

Background. Current techniques for isolating islets require that pancreata stored with University of Wisconsin solution (UW) are processed within 12 hours of cold storage. In this study, we hypothesized that the two-layer method (TLM) could extend the acceptable preservation period of pancreata before islet isolation and increase islet yields. Methods. In the first experimental set, eight pancreata were maintained for an average of 8.3±1.2 hours in UW and transferred into the TLM for an additional 14.3±1.1 hours for a total cold ischemic period of 22.6±1.6 hours (prolonged TLM). Four pancreata were maintained as a control group in UW alone for a total of 21.3±2.0 hours. In the second experimental set, six pancreata were maintained for an average of 6.4±1.8 hours in UW followed by 4.8±0.8 hours with the TLM for a total preservation time of 11.3±2.5 hours (short TLM). The control organs for the short TLM group were stored for an average of 9.5±1.3 hours in UW alone. Islets were isolated and evaluated according to the Edmonton protocol. Results. Between each group of the two experimental sets, there was no significant difference in donor-related factors (i.e. gender, age, body mass index [BMI], etc.). The TLM as compared with UW preservation resulted in a significant increase in islet yields postpurification for both short (3,353±394 islet equivalents [IE] vs. 2,027±415 IE; mean±SEM) and prolonged (2,404±503 IE vs. 514±180 IE) periods of storage. Furthermore, islet yields after prolonged storage with the TLM were not significantly different from organs maintained for only a short period with UW (P =0.17). The quality of islets as assessed by size, postculture viability, survival rates, insulin content, and insulin secretion were similar for each of the four groups. Conclusion. In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.

Inhibition of insulin gene expression by long-term exposure of pancreatic β cells to palmitate is dependent on the presence of a stimulatory glucose concentration

Long-term exposure of pancreatic beta cells to elevated levels of fatty acids (FAs) impairs glucose-induced insulin secretion. However, the effects of FAs on insulin gene expression are controversial. We hypothesized that FAs adversely affect insulin gene expression only in the presence of elevated glucose concentrations. To test this hypothesis, isolated rat islets were cultured for up to 1 week in the presence of 2.8 or 16.7 mmol/L glucose with or without 0.5 mmol/L palmitate. Insulin release, insulin content, and insulin mRNA levels were determined at the end of each culture period. Palmitate increased insulin release at each time point independently of the glucose concentration. In contrast, insulin content was unchanged in the presence of palmitate at 2.8 mmol/L glucose, but was markedly decreased in the presence of 0.5 mmol/L palmitate and 16.7 mmol/L glucose after 2, 3, and 7 days of culture. In the presence of a basal concentration of glucose, insulin mRNA levels were transiently increased by palmitate at 24 hours but were unchanged thereafter. In contrast, palmitate significantly inhibited the stimulatory effects of 16.7 mmol/L glucose on insulin mRNA levels after 2, 3, and 7 days. To determine whether the inhibitory effect of palmitate on glucose-stimulated insulin mRNA levels was associated with decreased insulin promoter activity, HIT-T15 cells were cultured for 24 hours in 11.1 mmol/L glucose in the presence or absence of palmitate, and insulin gene promoter activity was measured in transient transfection experiments using the insulin promoter-reporter construct INSLUC. INSLUC activity was decreased more than 2-fold after 24 hours of exposure to 0.5 mmol/L palmitate. We conclude that long-term exposure of pancreatic beta cells to palmitate decreases insulin gene expression only in the presence of elevated glucose concentrations, in part through inhibition of insulin gene promoter activity.

The Fatty-Acid Receptor GPR40 Plays a Role in Insulin Secretion In Vivo After High-Fat Feeding

OBJECTIVE—The G-protein–coupled receptor GPR40 is expressed in pancreatic β-cells and is activated by long-chain fatty acids. Gene deletion studies have shown that GPR40 mediates, at least in part, fatty acid–amplification of glucose-induced insulin secretion (GSIS) but is not implicated in GSIS itself. However, the role of GPR40 in the long-term effects of fatty acids on insulin secretion remains controversial. This study aimed to test the hypothesis that GPR40 plays a role in insulin secretion after high-fat feeding. RESEARCH DESIGN AND METHODS—GPR40 knockout (KO) mice on a C57BL/6 background and their wild-type (WT) littermates were fed a high-fat diet (HFD) for 11 weeks. Glucose tolerance, insulin tolerance, and insulin secretion in response to glucose and Intralipid were assessed during the course of the diet period. RESULTS—GPR40 KO mice had fasting hyperglycemia. They became as obese, glucose intolerant, and insulin resistant as their WT littermates given HFD and developed a similar degree of liver steatosis. Their fasting blood glucose levels increased earlier than those of control mice during the course of the HFD. The remarkable increase in insulin secretory responses to intravenous glucose and Intralipid seen in WT mice after HFD was of much lower magnitude in GPR40 KO mice. CONCLUSIONS—GPR40 plays a role not only in fatty acid modulation of insulin secretion, but also in GSIS after high-fat feeding. These observations raise doubts on the validity of a therapeutic approach based on GPR40 antagonism for the treatment of type 2 diabetes.

Reduction of acetaminophen interference in glucose sensors by a composite Nafion membrane: demonstration in rats and man

SummaryAmperometric glucose sensors typically monitor the production of hydrogen peroxide generated in the course of the enzymatic oxidation of glucose. At the applied potential necessary to oxidize the peroxide produced, other species are also electroactive and contribute to the signal. Interference of ascorbate or urate has been effectively eliminated, but that resulting from the widely used analgesic acetaminophen is not. The aim of this work was to reduce this interference, which was found to be possible by introducing a membrane constructed of Nafion. We compared the in vitro sensitivity to acetaminophen of five Nafion sensors with that of five non-Nafion sensors with identical glucose sensitivity (2.0±0.4 vs 1.9±0.1 nA·mmol−1· 1−1, NS): sensitivity to acetaminophen was 12.2±2.7 vs 30.8±6.3 nA·mmol−1·1−1, respectively (p<0.05). These sensors were tested in rats by implanting in each animal one Nafion and one non-Nafion sensors. The in vivo sensitivity to glucose was similar (0.33±0.09 vs 0.30±0.05 nA·mmol−1·1−1, NS). The current generated by an acetaminophen infusion (plasma acetaminophen plateau=140±10 μmol/l) was much decreased in the case of the Nafion sensor: 0.5±0.3 vs 2.0±0.7 nA, p<0.05). Five Nafion sensors were implanted in the subcutaneous tissue of normal human volunteers who were given on oral dose of 500 mg acetaminophen. No change in the sensor current was observed, although plasma acetaminophen reached a peak (35±6 μmol/l) at 60 min and decreased by 50% at 180 min. In contrast, the current increased from 2.1±0.7 to 3.9±1.2 nA (p<0.05) at 60 min of a subsequent oral glucose tolerance test when plasma glucose concentration increased from 4.8±0.3 to 8.0±0.9 mmol/l. This paper provides the evidence that the presence of a Nafion membrane is a solution to acetaminophen interference in glucose sensing.

Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line, HIT-T15.

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.