Metformin Ameliorates Dysfunctional Traits of Glibenclamide- and Glucose-Induced Insulin Secretion by Suppression of Imposed Overactivity of the Islet Nitric Oxide Synthase-NO System

Metformin lowers diabetic blood glucose primarily by reducing hepatic gluconeogenesis and increasing peripheral glucose uptake. However, possible effects by metformin on beta-cell function are incompletely understood. We speculated that metformin might positively influence insulin secretion through impacting the beta-cell nitric oxide synthase (NOS)-NO system, a negative modulator of glucose-stimulated insulin release. In short-time incubations with isolated murine islets either glibenclamide or high glucose augmented insulin release associated with increased NO production from both neural and inducible NOS. Metformin addition suppressed the augmented NO generation coinciding with amplified insulin release. Islet culturing with glibenclamide or high glucose revealed pronounced fluorescence of inducible NOS in the beta-cells being abolished by metformin co-culturing. These findings were reflected in medium nitrite-nitrate levels. A glucose challenge following islet culturing with glibenclamide or high glucose revealed markedly impaired insulin response. Metformin co-culturing restored this response. Culturing murine islets and human islets from controls and type 2 diabetics with high glucose or high glucose + glibenclamide induced a pronounced decrease of cell viability being remarkably restored by metformin co-culturing. We show here, that imposed overactivity of the beta-cell NOS-NO system by glibenclamide or high glucose leads to insulin secretory dysfunction and reduced cell viability and also, importantly, that these effects are relieved by metformin inhibiting beta-cell NO overproduction from both neural and inducible NOS thus ameliorating a concealed negative influence by NO induced by sulfonylurea treatment and/or high glucose levels. This double-edged effect of glibenclamide on the beta-cellsuggests sulfonylurea monotherapy in type 2 diabetes being avoided.     

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.     

Characterization of sulfonylurea receptors in isolated human pancreatic islets

Current information on pancreatic islet sulfonylurea receptors has been obtained with laboratory animal pancreatic β cells or stable β-cell lines. In the present study, we evaluated the properties of sulfonylurea receptors of human islets of Langherans, prepared by collagenase digestion and density-gradient purification. The binding characterisitics of labeled glibenclamide to pancreatic islet membrane preparations were analyzed, displacement studies with several oral hypoglycemic agents were performed, and these latter compounds were tested as for their insulinotropic action on intact human islets. [³H]glibenclamide saturable binding was shown to be linear at ≤0.25 mg/ml protein; it was both temperature and time dependent. Scatchard analysis of the equilibrium binding data at 25°C indicated the presence of a single class of saturable, high-affinity binding sites with a K_d value of 1.0 ± 0.07 nM and a Bmax value of 657 ± 48 fmol/mg of proteins. The displacement experiments showed the following rank order of potency of the oral hypoglycemic agents we tested: glibenclamide = glimepiride > tolbutamide > chlorpropamide ≫ metformin. This binding potency order was parallel with the insulinotropic potency of the evaluated compounds. J. Cell. Biochem. 71:182–188, 1998. © 1998 Wiley-Liss, Inc.     

Glibenclamide inhibits islet carnitine palmitoyltransferase 1 activity,leading to PKC-dependent insulin exocytosis

Hypoglycemic sulfonylureas such as glibenclamide have been widely used to treat type 2 diabetic patients for 40 yr, but controversy remains about their mode of action. The widely held view is that they promote rapid insulin exocytosis by binding to and blocking pancreatic beta-cell ATP-dependent K+ (KATP) channels in the plasma membrane. This event stimulates Ca2+ influx and sets in motion the exocytotic release of insulin. However, recent reports show that >90% of glibenclamide-binding sites are localized intracellularly and that the drug can stimulate insulin release independently of changes in KATP channels and cytoplasmic free Ca2+. Also, glibenclamide specifically and progressively accumulates in islets in association with secretory granules and mitochondria and causes long-lasting insulin secretion. It has been proposed that nutrient insulin secretagogues stimulate insulin release by increasing formation of malonyl-CoA, which, by blocking carnitine palmitoyltransferase 1 (CPT-1), switches fatty acid (FA) catabolism to synthesis of PKC-activating lipids. We show that glibenclamide dose-dependently inhibits beta-cell CPT-1 activity, consequently suppressing FA oxidation to the same extent as glucose in cultured fetal rat islets. This is associated with enhanced diacylglycerol (DAG) formation, PKC activation, and KATP-independent glibenclamide-stimulated insulin exocytosis. The fat oxidation inhibitor etomoxir stimulated KATP-independent insulin secretion to the same extent as glibenclamide, and the action of both drugs was not additive. We propose a mechanism in which inhibition of CPT-1 activity by glibenclamide switches beta-cell FA metabolism to DAG synthesis and subsequent PKC-dependent and KATP-independent insulin exocytosis. We suggest that chronic CPT inhibition, through the progressive islet accumulation of glibenclamide, may explain the prolonged stimulation of insulin secretion in some diabetic patients even after drug removal that contributes to the sustained hypoglycemia of the sulfonylurea.     

Determination of the specific interaction between sulfonylurea-incorporated polymer and rat islets

A SU derivative, mimicking glibenclamide in chemical structure, was synthesized to incorporate it into a water-soluble polymeric backbone as a biospecific and stimulating polymer for insulin secretion. The ability of insulin secretion was examined with different glucose concentrations (3.3 and 11.6 mM). Although the vinylated SU did not exhibit significant activity compared to the control, the SU-incorporated copolymer could enhance insulin secretion as much as or more than glibenclamide did. In this study, a polymer fluorescence-labeled with rodamine-B isothiocyanate was used to visualize the interactions and we found that the labeled polymer was strongly absorbed to rat islets, probably due to its specific interaction mediated by SU receptors on the cell membrane. To verify the specific interaction between the SU (K+ channel closer)-incorporated copolymer and rat islets, cells were pretreated with diazoxide, an agonist of ATP-sensitive K+ channels (K+ channel opener), before adding the incorporated polymer to the cell culture medium. This treatment suppressed the action of SUs on rat islets. A confocal laser microscopic study further confirmed this interaction. The results of this study provided evidence that the SU-incorporated copolymer stimulates insulin secretion through specific interactions of SU moieties in the polymer with rat islets.     

Synthesis and evaluation of a glibenclamide glucose-conjugate: a potential new lead compound for substituted glibenclamide derivatives as islet imaging agents

The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands.     

Nutrient-induced changes in the pH of pancreatic islet cells

Fluorescein rapidly accumulates in rat pancreatic islets exposed to fluorescein diacetate. The influence of environmental agents upon cellular pH was examined in fluorescein-labelled islets by recording their fluorescence intensity at 520 nm after excitation at 490 and 435 nm, respectively. Glucose caused a rapid, sustained and dose-related increase in cellular pH. Another nutrient secretagogue, 2-ketoisocaproic acid, also increased cellular pH. The stimulation of islet cells by non-nutrient secretagogues, e.g. by glibenclamide or in response to an increase in extracellular K+ concentration, decreased cellular pH, indicating that the nutrient-induced increase in cellular pH is not merely a consequence of stimulated Ca2+ inflow and/or insulin release. In either the presence of amiloride or absence of bicarbonate, glucose decreased cellular pH. These results strongly suggest that the acidification of islet cells which can be expected from the increased metabolism of glucose in glucose-stimulated islets is normally masked and overcome by stimulation of such processes as Na+/H+ and HCO3-/Cl- exchange.     

Enhanced GLP-1- and sulfonylurea-induced insulin secretion in islets lacking leptin signaling

We have previously reported that the absence of leptin signaling in β-cells enhances glucose-stimulated insulin secretion and improves glucose tolerance in vivo. To investigate the relevance of β-cell leptin signaling in the context of postprandial or therapeutic insulin secretion, we examined the cross talk between leptin and glucagon-like peptide (GLP)-1 and sulfonylurea actions. Single and size-matched islets isolated from control or pancreas-specific leptin receptor knockout (pancreas-ObR-KO) mice were treated either with GLP-1 or with glibenclamide. Leptin suppressed GLP-1-stimulated intracellular Ca(2+) concentrations ([Ca(2+)](i)) increase that paralleled the decrease in insulin secretion in controls. In contrast, and as expected, the ObR-KO islets were nonresponsive to leptin, and instead, showed a 2.8-fold greater GLP-1-stimulated [Ca(2+)](i) increase and a 1.7-fold greater insulin secretion. Phosphorylation of cAMP-responsive element binding protein was enhanced, and phosphodiesterase enzymatic activity was suppressed in MIN6 β-cells with ObR knockdown compared with controls. The ObR-KO islets also showed significantly higher glibenclamide-induced insulin secretion compared with control islets, whereas [Ca(2+)](i) was similar to the controls. These data support enhanced insulinotropic effects of glucose, GLP-1, and sulfonylureas in the islets lacking leptin signaling with potential therapeutic implications.     

Effects of glucose on 45Ca2+ outflow, cytosolic Ca2+ concentration and insulin release from freshly isolated and cultured adult rat islets

The present study aimed at comparing the effects of glucose on ionic and secretory events in freshly isolated and 5-7 day cultured rat pancreatic islets. The capacity of glucose to provoke insulin release was severely reduced in islets maintained in culture. Whether in freshly isolated or cultured islets, glucose provoked a marked and sustained decrease in 45Ca2+ outflow from islets deprived of extracellular Ca2+. In the presence of extracellular Ca2+ throughout, the magnitude of the glucose-induced secondary rise in 45Ca2+ outflow was reduced in cultured islets. Glucose provoked a weaker increase in [Ca2+]i in islet cells obtained from cultured islets than in islet cells dissociated from freshly isolated pancreatic islets. On the other hand, the stimulatory effect of carbamylcholine on 45Ca2+ outflow was unaffected by tissue culture. Lastly, in islet cells obtained from cultured islets, the increase in [Ca2+]i evoked by K+ depolarization averaged half of that observed in control experiments. These results indicate that the reduced secretory potential of glucose in cultured pancreatic islets can be ascribed to the inability of the nutrient secretagogue to provoke a suitable increase in Ca2+ influx.     

The receptor for antidiabetic sulfonylureas controls the activity of the ATP-modulated K+ channel in insulin-secreting cells

Sulfonylureas are powerful hypoglycemic drugs that have been used for decades to treat diabetic patients. This paper describes a 86Rb+ flux technique that permits one to study easily the properties of ATP-modulated K+ channels in RINm5F insulinoma cells. Sulfonylureas inhibit this type of K+ channel under conditions of intracellular ATP depletion. The most potent sulfonylureas (glibenclamide, glipizide, and gliquidone) are acting in the nanomolar range of concentration. Inhibition of the single ATP-modulated K+ channels by low concentrations of sulfonylureas was also observed using the patch-clamp technique. The sulfonylurea receptor has been biochemically identified with [3H]glibenclamide. For 10 different sulfonylureas (or sulfonylurea analogs) there was an excellent correlation between efficacy of blockade of ATP-modulated K+ channels and efficacy of binding to the sulfonylurea receptors using the 3H-ligand.     

SREBP1 is required for the induction by glucose of pancreatic beta-cell genes involved in glucose sensing

Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have reexamined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/l glucose led to enhanced secretion at both basal (3 mmol/l) and stimulatory (17 mmol/l) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K(+) channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/l glucose, SREBP1-deficient (SREBP1(-/-)) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1(-/-) mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.     

TGF-beta i promotes islet beta-cell function and regeneration

TGF-βi is a secreted protein and is capable of binding to both extracellular matrix (ECM) and cells. It thus acts as a bifunctional molecule enhancing ECM and cell interactions, a lack of which results in dysfunction of many cell types. In this study, we investigated the role of TGF-βi in the function and survival of islets. Based on DNA microarray followed by quantitative PCR confirmation, TGFβi gene showed drastic increase in expression in islets after culture. We demonstrated that recombinant TGF-βi could preserve the integrity and enhance the function of cultured islets. Such a beneficial effect was mediated via signaling through FAK. Exogenous TGF-βi was capable of sustaining high-level FAK phosphorylation in isolated islets, and FAK knockdown by small interfering RNA in islets resulted in compromised islet function. TGF-βi transgenic (Tg) islets showed better integrity and insulin release after in vitro culture. In vivo, β-cell proliferation was detectable in Tg but not wild-type pancreata. At age above 12 mo, Tg pancreata contained giant islets. Tg mice displayed better glucose tolerance than that of the controls. Tg islets were more potent in lowering blood glucose when transplanted into syngeneic mice with streptozotocin-induced diabetes, and these transplanted islets also underwent regeneration. Our results indicate that TGF-βi is a vital trophic factor promoting islet survival, function, and regeneration. At least some of its beneficial effect was mediated by signaling through FAK.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

Cysteamine exerts multiple effects on the endocrine cells of the rat pancreas

We have studied the effects by cysteamine in vitro and in vivo on hormone production and islet cell metabolism in isolated pancreatic islets and perfused pancreas of the rat. In isolated islets, cysteamine dose-dependently depleted somatostatin immunoreactivity by 50% after 60 min exposure to 1 mmol/l of the compound. This effect appeared to be independent of interaction of the drug with secretion of somatostatin from the pancreatic D-cells. Cysteamine, however, interacted acutely not only with the D-cells, but also markedly suppressed glucose-induced insulin release. Moreover, cysteamine inhibited islet glucose oxidation, an effect which reflects interference with the metabolism mainly of the B-cells. The effect of cysteamine on glucose-induced insulin release was prolonged, since it was still observed in the isolated rat pancreas perfused 24 h after in vivo treatment with cysteamine. In contrast to the effects on glucose-induced insulin release, the response to glibenclamide remained unaffected by a previous exposure to cysteamine in vivo. However, both glucose- and glibenclamide-induced somatostatin secretion was reduced by 50%, whereas basal glucagon secretion was significantly enhanced in pancreata from cysteamine-treated rats vs. control rats. We conclude that (1) cysteamine does not specifically affect the D-cells of the islets, and (2) the multiple effects by cysteamine on islet cell function, particularly on B-cell metabolism and secretion, renders the compound unsuitable for the study of paracrine interactions in the islets.     

ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells.     

Hexose metabolism in pancreatic islets. The phosphorylation of fructose

Fructose, like glucose, rapidly equilibrates across the plasma membrane of pancreatic islet cells, but is poorly metabolized and is a weak insulin secretagogue in rat pancreatic islets. A possible explanation for such a situation was sought by investigating the modality of fructose phosphorylation in islet homogenates. Several findings indicated that the phosphorylation of fructose is catalyzed by hexokinase, but not fructokinase. First, at variance with the situation found in liver homogenates, the phosphorylation of fructose in the islet homogenate was unaffected by K+ and inhibited by glucose, mannose, glucose 6-phosphate or glucose 1,6-bisphosphate. Second, the Km for fructose was much higher in islets than in liver. Third, in islet homogenates the Km and Vmax for fructose were much higher than those for glucose or mannose phosphorylation, at low aldohexose concentrations, in good agreement with the properties of purified hexokinase. In intact islets fructose augmented the islet content in glucose 6-phosphate sufficiently to cause marked inhibition of its own rate of phosphorylation. These findings may account, in part at least, for the low rate of fructose utilization by rat pancreatic islets.     

Effect of thioloxidant diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) on 45Ca2+ net uptake into rat pancreatic islets

The effect of DIP (an oxidant of glutathione) on 45Ca2+ net uptake induced by a variety of stimulators of insulin secretion was studied in rat pancreatic islets. In addition the effect of exogenous glutathione (GSH) on 45Ca2+ net uptake in response to glucose was tested. DIP (0.1 mM) inhibited the increase of 45Ca2+ net uptake in the presence of glucose (16.7 mM) and glyceraldehyde (10 mM). A similar inhibitory effect could be demonstrated, when 45Ca2+ net uptake was enhanced by tolbutamide (100 micrograms/ml), glibenclamide (0.5 micrograms/ml), b-BCH (20 mM), 2-ketoisocaproate (20 mM), arginine (20 mM) in the presence of 3 mM glucose or by high extracellular potassium (20 mM). The increase of 45Ca2+ net uptake stimulated by leucine (20 mM) plus glucose (3 mM) was further augmented by DIP. Exogenous GSH did not affect 45Ca2+ net uptake in the presence of (5.6-16.7 mM) glucose. It is suggested that 45Ca2+ net uptake of pancreatic islets depends on the redox state of islet thiols regardless of whether uptake is promoted via inhibition of potassium efflux (nutrients, sulfonylureas) or by high potassium and arginine. The voltage sensitive calcium-channel is the site of action of critical thiols. It is possible that these thiols are localized at the inner side of the plasma membrane.     

Studies of the Ca2+ requirements for glucose- and carbachol-induced augmentation of inositol trisphosphate and inositol tetrakisphosphate accumulation in digitonin-permeabilized islets. Evidence for a glucose recognition site in insulin secretion

The metabolism of D-glucose is believed to initiate and regulate insulin secretion by islet beta-cells, although the identity of the metabolite which couples glucose metabolism to the cellular events involved in insulin secretion is unknown. An alternative hypothesis involves the presence of a glucoreceptor for which there has been no biochemical evidence. We have investigated whether glucose recognition by the beta-cell is coupled to phospholipase C. We have used digitonin-permeabilized, [3H]inositol-prelabeled islets to study glucose and carbachol activation of phospholipase C. In this model, carbachol recognition by its muscarinic receptor was coupled to phospholipase C activation. D-Glucose (but not L-glucose) also stimulated phospholipase C activity in these permeabilized islets. This effect was not due to glucose metabolism since glucose 6-phosphate did not affect phospholipase C activity and since phosphorylation of [3H]glucose was not detectable in digitonin-permeabilized islets. Glucose had no effect on the myo-inositol-1,4,5-trisphosphate-5-phosphatase or 3-kinase activities. In the absence of agonist, free Ca2+ concentrations between 0.1 and 1 microM (as determined with a Ca2+-specific electrode) did not influence phospholipase C activity. Stimulation of phospholipase C activity by either carbachol or glucose required Ca2+ in the submicromolar range and was optimal at 0.5 microM free Ca2+.myo-Inositol-1,3,4,5-tetrakisphosphate production from permeabilized islets was synergistically augmented by Ca2+ (0.5-10 microM) and glucose. Phospholipase C activity in islets is therefore not directly activated by free Ca2+ concentrations in the submicromolar range. Furthermore, glucose per se activates phospholipase C activity independently of glucose metabolism. A working hypothesis based on these findings is that glucose is recognized by a site which is coupled to phospholipase C in islets.     

Hexose metabolism in pancreatic islets. Inhibition of hexokinase.

In islet homogenates, hexokinase-like activity (Km 0.05 mM; Vmax. 1.5 pmol/min per islet) accounts for the major fraction of glucose phosphorylation. Yet the rate of glycolysis in intact islets incubated at low glucose concentrations (e.g. 1.7 mM) sufficient to saturate hexokinase only represents a minor fraction of the glycolytic rate observed at higher glucose concentrations. This apparent discrepancy between enzymic and metabolic data may be attributable, in part at least, to inhibition of hexokinase in intact islets. Hexokinase, which is present in both islet and purified B-cell homogenates, is indeed inhibited by glucose 6-phosphate (Ki 0.13 mM) and glucose 1,6-bisphosphate (Ki approx. 0.2 mM), but not by fructose 2,6-bisphosphate. In intact islets, the steady-state content of glucose 6-phosphate (0.26-0.79 pmol/islet) and glucose 1,6-bisphosphate (5-48 fmol/islet) increases, in a biphasic manner, at increasing concentrations of extracellular glucose (up to 27.8 mM). From these measurements and the intracellular space of the islets, it was estimated that the rate of glucose phosphorylation as catalysed by hexokinase represents, in intact islets, no more than 12-24% of its value in islet homogenates.     

Photoaffinity Labeling of the Cerebral Sulfonylurea Receptor Using a Novel Radioiodinated Azidoglibenclamide Analogue

Abstract: In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145–150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [³H]glibenclamide or a ¹²⁵I-labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4-azido-5-iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high-affinity binding of this novel probe to the particulate ( K _D = 0.13 n M ) and solubilized ( K _D = 0.56 n M ) sulfonylurea receptor from cerebral cortex. The novel probe showed >100-fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175-kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a K _D value of 0.38 n M . Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed K _D values for these sulfonylureas of 0.06 n M , 1.6 n M , and 1.2 µ M , respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175-kDa protein represents the cerebral sulfonylurea receptor.