Beta-cell Glucose Metabolism Research Articles

Laminin matrix regulates beta-cell FGFR5 expression to enhance glucose-stimulated metabolism

We previously showed that pancreatic beta-cells plated on laminin matrix express reduced levels of FGFR1, a receptor linked to beta-cell metabolism and differentiation. Due to recent evidence that adult beta-cells also express FGFR5, a co-receptor for FGFR1, we now aim to determine the effect of laminin on FGFR5 expression and consequent effects on beta-cell metabolism. Using a genetically encoded sensor for NADPH/NADP+ redox state (Apollo-NADP+), we show overexpression of FGFR5 enhances glucose-stimulated NADPH metabolism in beta-cell lines as well as mouse and human beta-cells. This enhanced response was accompanied by increased insulin secretion as well as increased expression of transcripts for glycolytic enzymes (Glucokinase/GCK, PKM2) and the functional maturity marker Urocortin 3 (UCN3). Culturing beta-cells on laminin matrix also stimulated upregulation of endogenous FGFR5 expression, and similarly enhanced beta-cell glucose-stimulated NADPH-metabolism as well as GCK and PKM2 transcript expression. The metabolism and transcript responses triggered by laminin were disrupted by R5ΔC, a truncated receptor isoform that inhibits the FGFR5/FGFR1 signaling complex. Collectively these data reveal that beta-cells respond to laminin by increasing FGFR5 expression to enhance beta-cell glucose metabolism.

MicroRNA-375 regulates glucose metabolism-related signaling for insulin secretion.

Enhanced beta cell glycolytic and oxidative metabolism are necessary for glucose-induced insulin secretion. While several microRNAs modulate beta cell homeostasis, miR-375 stands out as it is highly expressed in beta cells where it regulates beta cell function, proliferation and differentiation. As glucose metabolism is central in all aspects of beta cell functioning, we investigated the role of miR-375 in this process using human and rat islets; the latter being an appropriate model for in-depth investigation. We used forced expression and repression of mR-375 in rat and human primary islet cells followed by analysis of insulin secretion and metabolism. Additionally, miR-375 expression and glucose-induced insulin secretion were compared in islets from rats at different developmental ages. We found that overexpressing of miR-375 in rat and human islet cells blunted insulin secretion in response to glucose but not to α-ketoisocaproate or KCl. Further, miR-375 reduced O2 consumption related to glycolysis and pyruvate metabolism, but not in response to α-ketoisocaproate. Concomitantly, lactate production was augmented suggesting that glucose-derived pyruvate is shifted away from mitochondria. Forced miR-375 expression in rat or human islets increased mRNA levels of pyruvate dehydrogenase kinase-4, but decreased those of pyruvate carboxylase and malate dehydrogenase1. Finally, reduced miR-375 expression was associated with maturation of fetal rat beta cells and acquisition of glucose-induced insulin secretion function. Altogether our findings identify miR-375 as an efficacious regulator of beta cell glucose metabolism and of insulin secretion, and could be determinant to functional beta cell developmental maturation.

Inter-organ communication and regulation of beta cell function.

The physiologically predominant signal for pancreatic beta cells to secrete insulin is glucose. While circulating glucose levels and beta cell glucose metabolism regulate the amount of released insulin, additional signals emanating from other tissues and from neighbouring islet endocrine cells modulate beta cell function. To this end, each individual beta cell can be viewed as a sensor of a multitude of stimuli that are integrated to determine the extent of glucose-dependent insulin release. This review discusses recent advances in our understanding of inter-organ communications that regulate beta cell insulin release in response to elevated glucose levels.

Heterozygous inactivation of plasma membrane Ca(2+)-ATPase in mice increases glucose-induced insulin release and beta cell proliferation, mass and viability.

Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca(2+) extrusion--the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. Biological and morphological methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. Pmca (isoform 2) heterozygous inactivation increased intracellular Ca(2+) stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. The present data indicates that inhibition of Ca(2+) extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca(2+) extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes.

Aberrant activation of liver X receptors impairs pancreatic beta cell function through upregulation of sterol regulatory element-binding protein 1c in mouse islets and rodent cell lines

Liver X receptors (LXR) are important transcriptional regulators of lipid and glucose metabolism. Our previous report demonstrated that LXR activation inhibited pancreatic beta cell proliferation through cell cycle arrest. Here we explore the role of LXR activation in beta cell insulin secretion and the underlying mechanism that might be involved. Mouse pancreatic islets or insulin-secreting MIN6 cells were exposed to the LXR agonist, T0901317, and insulin secretion, glucose and fatty acid oxidation, and lipogenic gene expression were assessed. The unsaturated fatty acid eicosapentaenoic acid and the dominant negative sterol regulatory element binding protein 1c (SREBP1c) were used to inhibit endogenous SREBP1c and evaluate the involvement of SREBP1c in beta cell dysfunction induced by LXR activation. Treatment with the LXR agonist decreased beta cell glucose sensitivity and impaired glucose-stimulated insulin secretion in vivo and in vitro. This was accompanied by derangements of beta cell glucose oxygen consumption, glucose oxidation, ATP production and intracellular voltage-gated calcium channel flux. LXR activation also regulated the expression of lipid metabolism-related genes such as Fas, Acc (also known as Acaca) and Cpt1a, and led to intracellular lipid accumulation. Further studies revealed that inhibition of SREBP1c abolished LXR activation-induced lipid accumulation and improved beta cell glucose metabolism, ATP production and insulin secretion. Our data reveal that aberrant activation of LXR reproduced the phenomenon of beta cell dysfunction in the development of type 2 diabetes in vitro and in vivo. Upregulation of SREBP1c production and the lipotoxicity mediated by it played a central role in this process.

Linkage studies for T2D in Chop and C/EBPbeta chromosomal regions in Italians

The genes causing type 2 diabetes (T2D), a complex heterogeneous disorder, differ and/or overlap in various populations. Among others there are two loci in linkage to T2D, the chromosomes 20q12-13.1 and 12q15. These two regions harbor two genes, C/EBPbeta and CHOP, which are excellent candidate genes for T2D. In fact, C/EBPbeta protein cooperates with HNF4alpha (MODY1, monogenic form of diabetes) and 1alpha (MODY3, monogenic form of diabetes). C/EBPbeta mediates suppression of insulin gene transcription in hyperglycemia and may contribute to insulin-resistance. It interacts in a complex pathway with the CHOP protein. CHOP may play a role in altered beta-cell glucose metabolism, in beta-cell apoptosis, and in lack of beta-cell replication. Thus, both C/EBPbeta and CHOP genes may independently and interactively contribute to T2D. The chromosomal regions targeting C/EBPbeta and CHOP genes have never been previously explored in T2D. We planned to identify their potential contribution to T2D in Italians. We have genotyped a group of affected siblings/families with both late- and early-onset T2D around the C/EBPbeta and the CHOP genes. We have performed non-parametric linkage analysis in the total T2D group, in the late-onset and the early-onset group, separately. We have identified a suggestive linkage to T2D in the CHOP gene locus in the early-onset T2D group (P = 0.04). We identified the linkage to T2D in the chromosome 12q15 region in the early-onset T2D families and specifically target the CHOP gene. Our next step will be the identification of CHOP gene variants, which may contribute to the linkage to T2D in Italians.

A dominant role for glucose in β cell compensation of insulin resistance

Increased insulin secretion and expansion of pancreatic beta cell mass work together to maintain normal glucose levels when insulin resistance develops. Changes in glucose concentration have long been known to have profound effects upon the rates of insulin secretion and beta cell mass, but various other agents can also cause changes, raising questions about which mechanisms are dominant. Evidence favoring a dominant role for glucose is provided by Terauchi et al. in this issue of the JCI (see the related article beginning on page 246). Mice haploinsufficient for beta cell glucokinase (Gck) were unable to increase their beta cell mass in response to insulin resistance produced by high-fat feeding. Gck is known to be the glucose sensor for glucose metabolism in beta cells. The study also provides strong evidence that insulin receptor substrate 2 (Irs2), which is known to have major effects on beta cell growth and survival, is a key downstream mediator of the effects of glucose found in this study.

A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice

C57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes. Glucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis. C57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close KATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver. The C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.

Oxidative Stress Is a Mediator of Glucose Toxicity in Insulin-secreting Pancreatic Islet Cell Lines

Pancreatic beta cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on beta cells and results in beta cell dysfunction and ultimately beta cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to beta cells, we used two model systems where glucose metabolism was increased in beta cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3-20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in beta cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in beta cells. The level of beta cell GK may also be critical in this process.

Genetic Regulation of Metabolic Pathways in β-Cells Disrupted by Hyperglycemia

In models of type 2 diabetes the expression of beta-cell genes is altered, but these changes have not fully explained the impairment in beta-cell function. We hypothesized that changes in beta-cell phenotype and global alterations in both carbohydrate and lipid pathways are likely to contribute to secretory abnormalities. Therefore, expression of genes involved in carbohydrate and lipid metabolism were analyzed in islets 4 weeks after 85-95% partial pancreatectomy (Px) when beta-cells have impaired glucose-induced insulin secretion and ATP synthesis. Px rats after 1 week developed mild to severe hyperglycemia that was stable for the next 3 weeks, whereas neither plasma triglyceride, non-esterified fatty acid, or islet triglyceride levels were altered. Expression of peroxisome proliferator-activated receptors (PPARs), with several target genes, were reciprocally regulated; PPARalpha was markedly reduced even at low level hyperglycemia, whereas PPARgamma was progressively increased with increasing hyperglycemia. Uncoupling protein 2 (UCP-2) was increased as were other genes barely expressed in sham islets including lactate dehydrogenase-A (LDH-A), lactate (monocarboxylate) transporters, glucose-6-phosphatase, fructose-1,6-bisphosphatase, 12-lipoxygenase, and cyclooxygenase 2. On the other hand, the expression of beta-cell-associated genes, insulin, and GLUT2 were decreased. Treating Px rats with phlorizin normalized hyperglycemia without effecting plasma fatty acids and reversed the changes in gene expression implicating the importance of hyperglycemia per se in the loss of beta-cell phenotype. In addition, parallel changes were observed in beta-cell-enriched tissue dissected by laser capture microdissection from the central core of islets. In conclusion, chronic hyperglycemia leads to a critical loss of beta-cell differentiation with altered expression of genes involved in multiple metabolic pathways diversionary to normal beta-cell glucose metabolism. This global maladaptation in gene expression at the time of increased secretory demand may contribute to the beta-cell dysfunction found in diabetes.

Beta-cell glucokinase deficiency and hyperglycemia are associated with reduced islet amyloid deposition in a mouse model of type 2 diabetes.

Type 2 diabetes is characterized by impaired beta-cell function, hyperglycemia, and islet amyloid deposition. The primary constituent of islet amyloid is the 37-amino acid beta-cell product called islet amyloid polypeptide (IAPP) or amylin. To study mechanisms of islet amyloid formation, we developed a transgenic mouse model that produces and secretes the amyloidogenic human IAPP (hIAPP) molecule and have shown that 81% of male transgenic mice develop islet amyloid after 14 months on a high-fat diet. To test whether impaired beta-cell function and hyperglycemia could enhance islet amyloid formation, we cross-bred our hIAPP transgenic mice with beta-cell glucokinase-knockout mice (GKKO) that have impaired glucose-mediated insulin secretion and fasting hyperglycemia. The resulting new (hIAPPxGKKO) line of mice had higher basal plasma glucose concentrations than the hIAPP transgenic mice at 3, 6, and 12 months of age (P < 0.05), as did GKKO mice compared with hIAPP transgenic mice at 6 and 12 months of age (P < 0.05). Basal plasma immunoreactive insulin (IRI) levels were lower in hIAPP x GKKO mice than in hIAPP transgenic mice at 6 months of age (P < 0.05). The area under the glucose curve in response to an intraperitoneal glucose challenge (1 g/kg body weight) was larger in hIAPPxGKKO mice than in hIAPP transgenic mice at 3, 6, and 12 months of age (P < 0.005) and in GKKO mice compared with hIAPP transgenic mice at 6 and 12 months of age (P < 0.005). The area under the IRI curve was lower in hIAPPxGKKO mice at 6 and 12 months of age (P < 0.05) than in hIAPP transgenic mice and in GKKO mice compared with hIAPP transgenic mice at 12 months of age (P < 0.05). Despite the presence of hyperglycemia, hIAPPxGKKO mice had a lower incidence (4 of 17 vs. 12 of 19, P < 0.05) and amount (0.40 +/- 0.24 vs. 1.2 +/- 0.3 arbitrary units, P < 0.05) of islet amyloid than hIAPP transgenic mice had. As expected, no islet amyloid was observed in GKKO mice lacking the hIAPP transgene (0 of 13). There was no difference in pancreatic content of IRI and hIAPP among the three groups of mice. Thus, despite the presence of impaired islet function and hyperglycemia, hIAPPxGKKO mice had a decreased incidence and quantity of islet amyloid. Therefore, our data suggest that impaired beta-cell glucose metabolism or hyperglycemia are not likely to contribute to islet amyloid formation in diabetes. Furthermore, this finding may explain the lack of progression of glycemia in patients with maturity-onset diabetes of the young.

Preserved initiatory and potentiatory effect of alpha-ketoisocaproate on insulin release in islets of glucose intolerant rats.

Insulin responses to glucose and non-glucose secretagogues were studied in short-term cultured pancreatic islets and perfused pancreata of the glucose intolerant F1 hybrid rats of spontaneously diabetic Goto-Kakizaki and control Wistar rats. After culture at 5.5 mmol/l glucose, hybrid islet responses to 11.1, 16.7 and 27.0 mmol/l glucose were between 60 and 40% of control islet responses. A combination of 1 mmol/l isobutylmethylxanthine and 16.7 mmol/l glucose induced a pronounced insulin release, which was of similar magnitude in hybrid and control rat islets. This response was not further augmented by addition of glibenclamide and arginine. The slope of potentiation of arginine (10 mmol/l)-stimulated insulin secretion by glucose (5.5-16.7 mmol/l) was greatly impaired in hybrid islets. In contrast to glucose, alpha-ketoisocaproate (KIC), which is metabolized in Krebs cycle, dose-dependently stimulated insulin secretion to similar levels in hybrid and control islets, cultured at 5.5 mmol/l glucose. Also in hybrid islets depolarized by potassium chloride (30 mmol/l) and with adenosine triphosphate-sensitive K+-channels kept open by diazoxide, insulin responses to glucose were greatly impaired but intact to KIC. Furthermore, KIC potentiated normally the insulin response to arginine in hybrid islets. In the isolated perfused pancreas, KIC induced similar insulin responses in hybrid rats and control rats. The potentiating effect by 5.5 mmol/l glucose on the KIC-stimulated insulin responses was, however, greatly reduced in isolated islets and absent in the perfused pancreata of hybrid rats. Taken together, these findings suggest an intact capacity for insulin release, although the initiating and potentiating effect by glucose on insulin release are defective in the Goto-Kakizaki-hybrid rats. An abnormal beta-cell glucose metabolism proximal to the Krebs cycle is likely to account for the impairment of insulin release.

GLUT1 is adequate for glucose uptake in GLUT2-deficient insulin-releasing beta-cells.

GLUT2 may play an important role in pancreatic beta-cell glucose metabolism. A decrease in glucose uptake due to underexpression of GLUT2 has been considered as the cause of beta-cell dysfunction in diabetes with different pathogenesis. However, this view has been challenged by recent studies, in which the underexpression of GLUT2 was not accompanied by a decrease in glucose uptake. Our present aim is to evaluate the presumed importance of GLUT2 in maintaining the efficiency of beta-cell glucose uptake. We studied the kinetic characteristics of 3-O-methylglucose uptake in two beta-cell lines. One of these is the beta TC3 cell line which expresses GLUT1 and the other is the beta HC9 cell line which expresses both GLUT1 and GLUT2. Under equilibrium exchange conditions, 3-O-methylglucose transport in these two cell lines showed similar values of K(m) and V(max). The apparent IC50 of cytochalasin B for inhibiting 3-O-methylglucose transport in beta HC9 cells was nine times as high as in beta TC3 cells, indicating that GLUT1 is the critically important glucose transporter in the beta TC3 cell line and GLUT2 in the beta HC9 cell line. In both cell lines, the rates of glucose uptake were at least three times as fast as that of glucose phosphorylation. Our results suggest that GLUT1 is able to compensate for GLUT2 loss as it occurs in beta TC3 and maintains a commensurately high capacity of glucose uptake to sustain glucose metabolism in pancreatic beta-cells.

Insulin production: from gene to granule.

The pancreatic beta cell synthesizes and secretes appropriate amounts of insulin to allow correct regulation of blood glucose levels in non-diabetic individuals. In the short term, the beta cell releases precise quantities of insulin in response to minute-to-minute changes in blood glucose. In the long term, beta cells adapt to metabolic changes such as the insulin resistance associated with obesity by appropriately altering insulin synthesis and beta-cell mass. Defects in insulin production ‐ from its synthesis through to its secretion ‐ by the beta cell have been implicated in the pathophysiology of both insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetes mellitus. Recent investigations focussed on the biology of the beta cell have led to an explosion of new information on the pathways involved in insulin synthesis and secretion and how defects in these pathways might be implicated in the pathogenesis of diabetes. Thus, for example, during the past few years we have gained a further understanding of the regulation of insulin gene transcription and the development of normal differentiated beta-cell function; the important role of translational control in the regulation of proinsulin biosynthesis; the molecular physiology of the ion channels involved in the coupling of beta-cell glucose metabolism with insulin secretion; and the exocytotic machinery employed by beta cells to secrete insulin granules. Each of the genes encoding a protein involved in insulin production, from its biosynthesis to its release, represents a candidate susceptibility gene for diabetes; indeed, several recently identified genes associated with diabetes encode proteins that appear to play important roles in normal beta-cell function. Through improving our knowledge of the mechanism of insulin production by the beta cell, we will undoubtedly gain new insight into the pathogenesis of diabetes. We report here highlights from recent research devoted to the study of beta-cell biology.

Unequal potency of transgenic yeast hexokinase on pancreatic beta cell metabolism and secretion.

Recent reports have proposed that sequence specific interactions between glucokinase and other beta cell proteins are important to glucokinase regulation of beta cell activity. We have previously reported enhancement of beta cell function by a transgenic hexokinase derived from yeast which has only 30% amino acid sequence homology to glucokinase. To test the functional significance of the amino acid sequence of islet glucokinase we have made a quantitative study of the effect of yeast hexokinase on beta cell glucose metabolism and insulin secretion. Transgenic and normal islets were assayed for hexokinase activity, glucose usage and insulin secretion. Most parameters were measured at six glucose concentrations between 0.5 and 20 mM glucose. Transgenic islet hexokinase activity measured in islet extracts exceeded normal islet hexokinase activity by 31 to 77 percent at different glucose concentrations. At all glucose concentrations tested the percentage increase in transgenic glucose metabolism greatly exceeded the percentage increase in transgenic hexokinase activity. The increase in transgenic glucose metabolism produced a proportional reduction in the threshold for glucose stimulated insulin secretion. However, yeast hexokinase had little if any effect on the first phase of insulin secretion. The finding that metabolism was very sensitive to yeast hexokinase but first phase secretion was not, supports recent proposals that hexokinase and glucokinase may be physically and functionally separated in the beta cell.

Mathematical model of beta-cell glucose metabolism and insulin release. I. Glucokinase as glucosensor hypothesis.

To quantitatively test the theory that glucokinase controls the rate of glucose metabolism and therefore the rate of insulin secretion, a minimal mathematical model of glycolysis in the pancreatic beta-cell was developed. The model represents our current hypothesis of how the normal beta-cell transduces the glucose signal. In this report, the model was used to address questions regarding the control strength of transport, hexokinase, glucose-6-phosphatase, and phosphofructokinase in the metabolism of glucose. The hypothesis that fructose 6-phosphate and a protein regulator modulate glucokinase activity was evaluated by simulation analysis, as was the possibility that glucose-6-phosphatase, working in concert with phosphofructokinase, can modulate the glucose-sensing system. It was found that, in the absence of glucose-6-phosphatase, transport, hexokinase, and phosphofructokinase do not greatly influence the rate of glucose metabolism unless their activities are dramatically altered from the measured values. Glucose metabolism was profoundly affected by the activity of glucokinase. However, in the presence of glucose-6-phosphatase, the ratio of glucose-6-phosphatase to phosphofructokinase activities was a very important parameter, and this potential control mechanism deserves more attention. The results further support the notion that glucokinase is indeed the glucosensor of the beta-cell and that modeling the system in toto provides quantitative evaluation needed to interpret the experimental tests of hypotheses.

Nitric oxide opens ATP-sensitive K+ channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic beta cells.

Nitric oxide (NO) is known to be a potent messenger in the intracellular signal transduction system in many tissues. In pancreatic beta cells, NO has been reported to be formed from L-arginine through NO synthase. To elucidate the effect of NO on insulin secretion and to investigate the intracellular mechanism of its effect, we have used sodium nitroprusside (SNP) as a NO donor. SNP inhibited glucose-induced insulin secretion in a dose-dependent manner, and its effect was reversed by hemoglobin, a known NO scavenger. However, glyceraldehyde-induced insulin secretion was not affected by SNP. Since the closure of ATP-sensitive K+ channels (KATP channel) has been established as a key step in glucose-induced insulin secretion, we have directly assessed the effect of SNP on KATP channel activity using the patch clamp technique. The KATP channel activity reduced by glucose was found to be reversibly activated by the addition of SNP, and this activation was able to be similarly reproduced by applying S-Nitroso-N-acetyl-DL-penicillamine (SNAP), another NO generator. Furthermore, these activating effects were completely eliminated by hemoglobin, in accordance with the reversibility in inhibition of glucose-induced insulin release. However, SNP could not affect the KATP channel suppression by ATP applied to the inside of the plasma membrane. The activation of the KATP channel by NO, therefore, seems to be due to the decreased ATP production attributable to impairment of glucose metabolism in beta cells. Since SNP exhibited no effect on glyceraldehyde-induced KATP channel inhibition, NO may disturb a glycolytic step before glyceraldehyde-3-phosphate. The KATP channel activation by 2-deoxyglucose through presumable ATP consumption due to its phosphorylation by glucokinase was, however, not affected even in the presence of SNP. But in the permeabilized beta cells made by exposure to a low concentration (0.02 U/ml) of streptolysin O (open cell-attached configuration), SNP reopens KATP channels which have been eliminated by fructose-6-phosphate, while this effect was not observed in the KATP channels inhibited by fructose-1,6-bisphosphate. On the other hand, in rat ventricular myocyte KATP channels were not activated by SNP even under a low concentration of glucose. From these observations, the inhibition of phosphofructokinase activity is probably the site responsible for the impairment of glucose metabolism induced by NO in pancreatic beta cells. NO, therefore, seems to be a factor in the deterioration of glucose-induced insulin secretion from pancreatic beta cells through a unique intracellular mechanism.

Post-GLUT-2 defects in beta-cells of non-insulin-dependent diabetic obese rats

Zucker diabetic fatty (ZDF) rats develop non-insulin-dependent diabetes mellitus concomitantly with loss of glucose responsiveness and GLUT-2, the high-Michaelis constant glucose transporter of beta-cells. To determine the integrity of beta-cell glucose metabolism distal to the level of glucose transport and phosphorylation, we examined the insulin responses of isolated pancreata to 5, 10, and 20 mM D-glyceraldehyde and monomethylsuccinate, as well as to glucose. The insulin response of diabetic pancreata to glucose was 90% below the response prior to the onset of diabetes, whereas the responses to glyceraldehyde and succinate had declined to 65 and 44%, respectively, below the prediabetic responses. D-[14C]glyceraldehyde oxidation by diabetic islets was 74% below that of islets from lean nondiabetic controls. We conclude that 1) the insulin responses to glyceraldehyde and monomethylsuccinate, as well as to glucose, are impaired in the diabetes of ZDF rats and 2) the impairment of the glucose response was greater than that of the glyceraldehyde response, which was, in turn, greater than that of the monomethylsuccinate response; this decrescendo pattern of impairment is consistent with defects at multiple sites in glucose metabolism; if the defect were entirely due to a postmetabolic signaling defect, the impairment to glucose and its metabolites should be comparable.

Reduced sensitivity of dihydroxyacetone on ATP-sensitive K+ channels of pancreatic beta cells in GK rats

In the GK (Goto-Kakizaki) rat, a genetic model of non-insulin-dependent diabetes mellitus, glucose-induced insulin secretion is selectively impaired. In addition, it has been suggested by previous studies that impaired glucose metabolism in beta cells of the GK rat results in insufficient closure of ATP-sensitive K+ channels (KATP channels) and a consequent decrease in depolarization, leading to a decreased insulin release. We have recently reported that the site of disturbed glucose metabolism is probably located in the early stages of glycolysis or in the glycerol phosphate shuttle. In the present study, in order to identify the impaired metabolic step in diabetic beta cells, we have investigated insulin secretory capacity by stimulation with dihydroxyacetone (DHA), which is known to be directly converted to DHA-phosphate and to preferentially enter the glycerol phosphate shuttle. In addition, using the patch-clamp technique, we also have studied the sensitivity of DHA on the KATP channels of beta cells in GK rats. The insulin secretion in response to 5 mmol/l DHA with 2.8 mmol/l glucose was impaired, and DHA sensitivity of the KATP channels was reduced in beta cells of GK rats. From these results, we suggest that the intracellular site responsible for impaired glucose metabolism in pancreatic beta cells of GK rats is located in the glycerol phosphate shuttle.

Expression of GLUT-2 antisense RNA in beta cells of transgenic mice leads to diabetes.

An insulin response to glucose is required to correct hyperglycemia. Two proteins, the glucose transporter GLUT-2 and the glucose-phosphorylating enzyme glucokinase, have been implicated in the control of glucose metabolism in beta cells. To study the role of glucose transporter GLUT-2 in the regulation of insulin secretion and in the development of diabetes mellitus, we have obtained transgenic mice expressing high levels of GLUT-2 antisense RNA in beta cells. Western blot analysis showed an 80% reduction in GLUT-2 protein in the beta cells of these animals. Islets from transgenic mice showed impaired glucose-stimulated insulin secretion. In addition, much higher levels of blood glucose were detected in transgenic mice than in controls when glucose tolerance tests were performed. These results suggest that the reduction of GLUT-2 in the pancreas could be a crucial step in the development of diabetes mellitus.