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COS-7 cells were transfected with the green fluorescent protein (GFP) ofAequorea victoria,human mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH), a mGDHwt-EGFP construct, or two mutant mGDH-proteins fused with EGFP. The site of mutation was selected to affect cationic amino acids in the peptide signal sequence currently believed to play a key role in the subcellular distribution of mitochondrial proteins. All proteins were suitably expressed in the COS-7 cells. However, an increase in mGDH enzymatic activity above the control value in non-transfected COS-7 cell homogenates was only observed in cells transfected with mGDH, indicating that the catalytic activity of mGDH was masked in fused proteins. Confocal microscopy documented that, in the cells transfected with the mGDHwt-EGFP construct, the fusion protein was located exclusively in mitochondria, this contrasting with the nuclear labelling of cells expressing the green fluorescent protein alone. The mitochondrial anchoring of the mutated mGDH fused protein was altered, this alteration being most obvious in the mGDH313233-EGFP mutant. These findings raise the idea that a conformation change of the mGDH protein, as resulting from either an inherited or acquired alteration of its amino acid sequence, may affect its subcellular distribution and, hence, modify its immunogenic potential.

A recent model concerning the ionic determinants involved in the insulinotropic action of nutrients, namely an anion channel hypothesis, is presented, and selected experimental findings in support of such a model, are reviewed.

The fate of 3H- and 14C-labelled A-4166 was examined in rat pancreatic islets. The net uptake of the meglitinide analogue by islets incubated for 60 min in the presence of 0.1 mM A-4166 and then submitted to repeated washes was close to 0.1 pmol/islet. It was significantly increased when the concentration of D-glucose in the incubation medium was raised from 2.8 to 16.7 mM. No sizeable internalization of tritiated A-4166 into insulin-producing cells could be detected by autoradiography. These findings suggest that the interaction of A-4166 with the beta-cell may be restricted to its insertion on the plasma membrane and binding to sulphonylurea receptors.

In the animal model of type-1 diabetes found in BioBreeding rats, an enteropathy characterized by morphological, biochemical and functional features precedes the autoimmune insulitis, and both lesions are modulated in their severity by dietary factors. Alterations of the intestinal mucin content, of gut permeability and of the information transfer between intestinal GLP-1 and its islet receptors, may link this enteropathy to the islet pathology.

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-14C]glucose than D-[6-14C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired 14C/3H ratio found in islets exposed to both D-[1-14C]glucose or D-[6-14C]glucose and D-[3-3H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-14C]glucose. The labelling of islet lipids by D-[6-14C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C1-C2-C3 moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C4-C5-C6 moiety of the hexose, (iii) that only a limited amount of [3-3H]glycerone 3-phosphate generated from D-[3-3H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-14C]glucose or D-[6-14C]glucose rather than D-[3-3H]glucose.

In 1995, several new molecules under study as potential insulinotropic agents for the treatment of non-insulindependent diabetes mellitus were identified as analogs of meglitinide, previously known as the non-sulfonylurea moiety of glibenclamide. Three of these molecules, namely repaglinide, nateglinide and mitiglinide are or will be soon available for administration to diabetic patients. The present report aims at reviewing both preclinical studies and clinical investigations concerning the latter three meglitinide analogs. Their insulinotropic action seems attributable, like that of hypoglycaemic sulfonylureas, to a primary effect on the ATP-sensitive K+ channels of pancreatic insulin-producing cells. These meglitinide analogs differ from one another, however, by their potency as insulinotropic agents and by the time course of their biological effects, especially in terms of the reversibility of such effects.

Glucagon-like peptide-1 (GLP-1), an incretin hormone which helps to regulate plasma glucose levels, is considered a potential agent for the treatment of type-2 diabetes mellitus, because of its insulinotropic capacity and insulinomimetic actions. In normal conditions, the beta-cell secretory response to GLP-1 is modulated by the extracellular concentration of D-glucose; however, the recognition of D-glucose by the beta-cell is often impaired in type-2 diabetes, and this could impede the full GLP-1 insulinotropic action. Non-glucidic substrates, such as the dimethyl ester of succinic acid, restore the effect of GLP-1 in the isolated perfused rat pancreas of normal or diabetic rats, in the absence of any other exogenous nutrient; likewise, the dimethyl ester of succinic or L-glutamic acid, and the monomethyl ester of pyruvic acid, potentiate the in vivo beta-cell secretory response to GLP-1 in normal and diabetic rats. Therefore, it was proposed that nutrients susceptible to bypass the site-specific defects of the diabetic beta-cell, could be used to potentiate and/or prolong the insulinotropic action of antidiabetic agents such as GLP-1. In vitro, GLP-1 insulin-like effects on glucose metabolism have been documented in normal and diabetic rat liver, and in rat and human skeletal muscle. In rat and human adipocytes, GLP-1 is lipolytic and/or lipogenic, and also stimulates parameters involved in the glucose metabolism. In liver, muscle and fat, GLP-1 seems to act through specific receptors, apparently different--at least in liver and muscle--in structure or signaling pathway from the pancreatic one. It is proposed that an inositolphosphoglycan might be a second messenger of GLP-1 action in extrapancreatic tissues.

Fructose-1-phosphate, the product of fructose phosphorylation by fructokinase, favours glucose phosphorylation by glucokinase, at the intervention of a regulatory protein. Glucose confers to glucokinase positive cooperativity, towards fructose. These reciprocal effects are operative in hepatocytes and pancreatic islets. They could optimize their glucostatic function.

Isolated hepatocytes from fed rats were exposed for 120 min to D-glucose (10 mM) and either D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]fructose (also 10 mM) in the presence of D2O. The identification and quantification of 13C-enriched D-fructose and its metabolites (D-glucose, L-lactate, L-alanine) in the incubation medium and the measurement of their deuterated isotopomers indicated, by comparison with a prior study conducted in the absence of exogenous D-glucose, that the major effects of the aldohexose were to increase the recovery of 13C-enriched D-fructose, decrease the production of 13C-enriched D-glucose, restrict the deuteration of the 13C-enriched isotopomers of D-glucose to those generated by cells exposed to D-[2-13C]fructose, and to accentuate the lesser deuteration of the C, (as compared to C5) of 13C-enriched D-glucose derived from D-[2-13C]fructose. The ratio between C2-deuterated and C2-hydrogenated L-lactate, as well as the relative amounts of the CH3-, CH2D-, CHD, and CD3- isotopomers of 13C-enriched L-lactate were not significantly different, however, in the absence or presence of exogenous D-glucose. These findings indicate that exogenous D-glucose suppressed the deuteration of the C1 of D-[I-13C]glucose generated by hepatocytes exposed to D-[1-13C]fructose or D-[6-13C]fructose, as otherwise attributable, in part at least, to gluconeogenesis from fructose-derived [3-13C]pyruvate, and apparently favoured the phosphorylation of D-fructose by hexokinase isoenzymes, probably through stimulation of D-fructose phosphorylation by glucokinase.

D-[3H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS-1 lines. We have now investigated the effects of D-mannoheptulose upon D-glucose metabolism in these two cell lines. D-mannoheptulose (1.0-10.0 mM) only caused a minor decrease of D-glucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) D-glucose concentration. A comparable situation was found in INS-1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of D-mannoheptulose (5.0 mM) efficiently inhibited D-glucose metabolism. In the INS-1 cells, the relative extent of the inhibitory action of D-mannoheptulose upon D-glucose metabolism increased from 12.4 +/- 2.6 to 38.3 +/- 3.8% as the number of passages was decreased from more than 20 to 13-15 passages, the latter percentage remaining lower, however, than that recorded in INS-I cells also examined after 13-15 passages but exposed to D-mannoheptulose hexaacetate (66.9 +/- 2.2%). These findings when compared to our recent measurements of D-[3H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on D-glucose metabolism are dictated by expression of the GLUT2 gene.

The dimethyl ester of L-glutamic acid (DMG) stimulates insulin release and was proposed as a possible insulinotropic tool in the treatment of non-insulin-dependent diabetes. In such a perspective, it was investigated whether DMG enhances the B-cell secretory response to GLP-1 in fed anaesthetized rats. The primed constant infusion of DMG (1.0 micromol and then 0.5 micromol/min, both per g body wt.) provoked a rapid and sustained increase in plasma insulin concentration and augmented the release of insulin caused by GLP-1. Thus, DMG indeed appears as a suitable tool to potentiate the insulinotropic action of GLP-1.

It was recently proposed that suitable succinic acid esters could be used to potentiate the insulinotropic action of glucagon-like peptide 1 (GLP-1) in the treatment of type-2 diabetes mellitus. In such a perspective, the present study aimed mainly at investigating whether exendin-4 (Ex-4), a peptide structurally related to GLP-1(7-36)amide, and succinic acid dimethyl ester (SAD) also act synergistically upon insulin secretion in anaesthetized rats. Despite a higher plasma insulin concentration in SAD-infused rats (5.5+/-1.1 ng/ml) than in saline-infused animals (1.9+/-0.7 ng/ml), the intravenous injection of Ex-4 augmented to a greater extent the plasma concentration of insulin in the former rats (+7.4+/-2.5 ng/ml) than in the latter animals (+2.8+/-0.6 ng/ml). These findings document that the insulinotropic actions of Ex-4 and GLP-1 display comparable nutrient dependency, being both potentiated by a non-glucidic nutrient secretagogue such as SAD.

In terms of glucose sensing by pancreatic islet beta-cells, emphasis is currently placed on both the role of glucokinase, with negligible activity of low-Km hexokinase(s), and the prevalence of the oxidative over non-oxidative modality of glycolysis, a situation tentatively attributed, in part at least, to a low activity of lactate dehydrogenase. Conflicting information is available, however, on the activity of both low-Km hexokinase(s) and lactate dehydrogenase in purified beta-cell homogenates. This issue was reinvestigated, therefore, in two populations of purified rat islet beta-cells selected on the basis of their low (betaL) or high (betaH) content in reduced pyridine nucleotides. The size and protein content of betaH cells represented about twice that of betaL cells. Such was also the case for low-Km hexokinase(s), lactate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, glutamate dehydrogenase and glutamate-alanine and glutamate-aspartate transaminases. Whether in betaH or betaL cells, the activity of low-Km hexokinase(s) was at least as high as or higher than that of glucokinase. In both betaH and betaL, the activity of lactate dehydrogenase exceeded that required to catalyze the full reduction of glucose-derived pyruvate to L-lactate, as estimated from the rate of D-glucose phosphorylation under physiological conditions. These findings thus argue against a low expression of either low-Km hexokinase(s) or lactate dehydrogenase as major determinants of the glucose-sensing device in beta-cells.

The present studies were undertaken to examine concomitant diet-induced changes in pancreatic islets and cells of the gut immune system of diabetes-prone BB rats in the period before classic insulitis. Diabetes-prone (BBdp) and control nondiabetes prone (BBc) BB rats were fed for approximately 17 days either a mainly plant-based standard laboratory rodent diet associated with high diabetes frequency, NIH-07 (NIH) or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. By about 7 weeks of age, NIH-fed BBdp rats had lower plasma insulin and insulin/glucose ratio, lower insulin content of isolated islets, lower basal levels of NO but higher responsiveness of NO production to IL-1beta in cultured islets, and higher Con A response and biosynthetic activities in mesenteric lymphocytes than control rats fed the same diet. In control rats, the HC diet caused only minor changes in most variables, except for a decrease in oxidation of L-[U-14C]glutamine in Peyer's patch (PP) cells and an increase in protein biosynthesis in mesenteric lymphocytes. In BBdp rats, however, the HC diet increased plasma insulin concentration, islet insulin/protein ratio, and tended to normalize the basal and IL-1beta-stimulated NO production by cultured islets. The HC diet decreased oxidation of L[U-14C]glutamine in BBdp pancreatic islets, whereas oxidation of L-[U-14C]glutamine in PP cells was increased, and the basal [Methyl-3H]thymidine incorporation in mesenteric lymphocytes was decreased. These findings are compatible with the view that alteration of nutrient catabolism in islet cells as well as key cells of the gut immune system, particularly changes in mitotic and biosynthetic activities in mesenteric lymphocytes, as well as basal and IL-1beta stimulated NO production, participate in the sequence of events leading to autoimmune diabetes in BB rats. Thus, the protection afforded by feeding a hydrolysed casein-based diet derives from alterations in both the target islet tissue and key cells of the gut immune system in this animal model of type 1 diabetes.

Under conditions of sustained hyperglycemia, glycogen accumulates in pancreatic islets, but not so in acinar pancreatic cells. We investigated whether advantage could be taken of such a situation in the perspective of the noninvasive imaging of the endocrine pancreas. Control rats or animals injected with streptozotocin (STZ) were infused with solutions of D-glucose mixed with a tracer amount of D-[U-14C]glucose, and the radioactive glycogen content of both liver and pancreas was then measured. After 48 h of infusion, the radioactive glycogen content of the pancreas was 30 times lower in STZ rats than in control animals, coinciding with a 50 times lower insulin content. In the control rats, a sizable labeling of pancreatic glycogen was also recorded when D-[U-14C]glucose was infused for only the last 4 h of unlabeled D-glucose infusion; such a labeling was not decreased when the animals were further infused for 1 h with only the unlabeled hexose. Moreover, a pronounced difference in the pancreatic gland and blood radioactive content of control rats was still observed when the hyperglycemic animals were killed only 40 min after the i.v. injection of D-[U-14C]glucose. In STZ rats transplanted with islets and later infused with D-[U-14C]glucose, the total radioactive content and radioactive glycogen content were both much higher in the transplanted islets than in the pancreatic gland. These results allow one to define the conditions under which the administration of either 2-deoxy-2-[18F]fluoro-D-glucose or 11C-labeled D-glucose could conceivably be used to favor the selective labeling of the endocrine, as distinct from exocrine, pancreas.

Methyl pyruvate (MP) stimulates insulin release both in vivo and in vitro. The present study aims at investigating whether MP is also able to enhance the B-cell secretory response to glucagon-like peptide 1 (GLP-1). In anaesthetized rats receiving a primed constant infusion of MP, the ester augmented plasma insulin concentration before GLP-1 injection and potentiated the insulinotropic action of the intestinal hormone. MP infusion also augmented plasma D-glucose concentration, whether in the absence or presence of GLP-1. A further rise in plasma D-glucose concentration was observed when the infusion of MP was halted, this coinciding with a fall in plasma insulin concentration. Whilst documenting that MP indeed enhances the B-cell secretory response to GLP-1, these findings do not suggest that MP is an appropriate tool for optimizing the hypoglycaemic action of the enteric hormone in the treatment of type-2 diabetes mellitus.

D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of GLUT2. In the present study, it was investigated whether advantage could be taken from such a situation to assess the contribution of insulin-producing B-cells to the total mass of isolated rat pancreatic islets. After 60 min incubation at 37 degrees C in the presence of 8.3 mM D-glucose, the intracellular distribution space of D-[3H]mannoheptulose (0.1 mM) averaged, in islets from control and streptozotocin-induced diabetic rats, respectively 49.0+/-2.3 and 6.2+/-1.5% of the corresponding intracellular 3HOH space, all values being corrected for extracellular contamination as judged from the distribution space of [U-14C]sucrose (1.0 mM). These findings indicate that the present approach indeed allows to assess the relative contribution of B-cells to total islet mass for purpose of comparison between animals with different metabolic and/or hormonal status.

The apparent distribution space of 6-deoxy-6-[125I]iodo-D-glucose, recently proposed as a tracer of D-glucose transport, was measured in rat isolated islets, acinar tissue, and pieces of pancreas. While such a space reached a steady-state value corresponding to the 3HOH volume in pancreatic islets within 5 min, it slowly increased in pieces of pancreas and, even after 60-min incubation, remained lower than the 3HOH volume. Moreover, the net uptake of 6-deoxy-6-[125I]iodo-D-glucose by pancreatic pieces was inhibited by unlabeled 6-deoxy-6-iodo-D-glucose, D-glucose, and cytochalasin B, while being less or not affected by these agents in isolated islets. A preferential labeling of the endocrine, relative to exocrine, moiety of the pancreas was documented both by comparing, after 2 min incubation, the uptake of 6-deoxy-6-[125I]iodo-D-glucose by pieces of pancreas from normal vs streptozotocin-injected rats and by comparing the radioactive content of pancreatic islets and acinar tissue obtained from normal rats injected intravenously 3 min before sacrifice with 6-deoxy-6-[125I]iodo-D-glucose. It is proposed, therefore, that advantage could conceivably be taken from the vastly different time course for the uptake of selected monosaccharides by pancreatic islets vs acinar cells in the perspective of imaging of the endocrine pancreas by a non invasive method.

The uptake of [2-(14)C]alloxan by the pancreatic gland was investigated in control and streptozotocin-induced diabetic (STZ) rats, using both in vitro and in vivo techniques. Whether after 10 to 60 min incubation of pieces of pancreas in the presence of [2-(14)C]alloxan or 60 min to 24 h after intravenous injection of [2-(14)C]alloxan to control and insulin-treated STZ rats, the radioactive content of the pancreas (dpm/mg wet weight) only represented, in the STZ rats, about two thirds of the reference value found in control animals. These findings indicate that insulin-producing islet B-cells participate to a sizeable extent to the overall uptake of [2-(14)C]- alloxan by the whole pancreatic gland, despite the fact that they account for no more than about one percent of the total pancreas mass. Hence, it should be possible to preferentially label the endocrine moiety of the pancreas, in the perspective of its imaging and quantification by a non-invasive procedure, by use of a suitable radiolabelled molecule selectively taken up by islet, as distinct from acinar, pancreatic cells.

In order to assess the respective contribution of the exocrine and endocrine moieties of the pancreas to the overall net uptake of selected monosaccharides by the pancreatic gland, the apparent distribution space of L-[1-14C]glucose, 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was measured in pieces of pancreas obtained from either control rats or animals injected with streptozotocin. Although the time course for the uptake of 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was much slower in the pieces of pancreas than that previously documented in isolated pancreatic islets, no significant difference could, as a rule, be detected between the results obtained in pancreatic pieces of control and streptozotocin rats. A comparable situation prevailed in the pancreas of animals examined 3 min after the intravenous injection of 3-O-[14C-methyl]-D-glucose. D-Glucose inhibited the uptake of 3-O-[14C-methyl]-D-glucose and that of D-[U-14C]fructose. Likewise, 3-O-methyl-D-glucose inhibited the uptake of D-[U-14C]glucose. Cytochalasin B (20 microm) also inhibited the uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose, but not that of D-[U-14C]fructose. D-Mannoheptulose hexaacetate, but not the unesterified heptose, inhibited the metabolism of tritiated and 14C-labelled D-glucose, as well as the net uptake of D-[U-14C]glucose and D-[U-14C]mannose and, to a lesser extent, that of D-[U-14C]fructose. These findings indicate that despite marked differences between endocrine and exocrine pancreatic cells in terms of both the time course for the uptake of several hexoses and the inhibition of their phosphorylation by D-mannoheptulose, little or no preferential labelling of the endocrine moiety of the pancreas by the 14C-labelled hexoses is observed, at least when judged from their distribution space in pancreatic pieces or the whole pancreatic gland. Nevertheless, the findings made with D-mannoheptulose and its hexaacetate ester raise the view that this heptose could conceivably be used to achieve a sizeable preferential labelling of the endocrine pancreas under the present experimental conditions.

Aliphatic dioic acids have been proposed as alternative nutrients in selected clinical situations. In this study, their possible insulinotropic action was investigated in isolated rat pancreatic islets prepared from fed rats. Azelaic acid, sebacic acid and tridecanedioic acids, when tested at a 10.0 mm concentration, were found to augment insulin release evoked by D-glucose (7.0 mm) in the pancreatic islets. Likewise, glycerol-1,2,3-tris(dodecanoedioate), when used at concentrations close to 1.0 mm, increased the secretory response to the hexose. It is speculated that these findings may extend to insulin-producing cells, the knowledge that aliphatic dioic acids or their esters may act as energy substrates, e.g. in parenteral nutrition.

The fate of 6-deoxy-6-[125I]iodo-D-glucose (6-DIG), injected intravenously, was compared in control rats and animals that had received streptozotocin and were then treated with insulin or not. In the control rats, the measurement of plasma radioactivity suggested that, after an initial and rapid (up to min 10) distribution phenomenon (Kvalue: 12.2 x 10(-2) min(-1)), the clearance of the iodinated hexose occurred mainly by glomerular filtration (K value: 0.2 x 10(-2) min(-1)). Three minutes after the injection of 6-DIG, the radioactive content of muscle, liver, and pancreas, relative to the paired value in blood, was lower in untreated diabetic rats than in control animals. In the case of muscle and liver, such a difference was no longer observed when the treatment of the diabetic rats by insulin resulted in restoration of normoglycemia. In the pancreas, however, the radioactive content, whether expressed relative to the paired blood or liver value, remained significantly lower in the insulin-treated diabetic rats than in the control animals. No significant difference between control and diabetic rats, in terms of pancreatic radioactivity, was observed 10 min after the injection of 6-DIG. These findings indicate that advantage can be taken from the vastly different time course for 6-DIG uptake by pancreatic acinar and islet cells, as recently documented in vitro, to label preferentially the endocrine moiety of the pancreatic gland shortly after 6-DIG injection.

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.

The metabolism of D-glucose was characterized in both normal dispersed rat islet cells and the 2-mercaptoethanol-dependent insulin-secreting cells of the INS-1 line. The normal and tumoral islet cells differed from one another by the relative magnitude, concentration dependency and hierarchy of the increase in the production of 3HOH from D-[5-(3)H]glucose and 14C-labelled CO2, acidic metabolites and amino acids from D-[U-14C]glucose at increasing concentrations of the hexose. For instance, whilst the paired ratio between D-[U-14C]glucose oxidation and D-[5-(3)H]glucose utilization augmented in a typical sigmoidal manner in normal islet cells exposed to increasing concentrations of D-glucose, it progressively decreased under the same experimental conditions in INS-1 cells. Nevertheless, the absolute values and concentration-response relationship for the increase in ATP generation rate attributable to the catabolism of D-glucose were virtually identical in normal and tumoral cells. These findings indicate that the analogy in the secretory response to D-glucose of normal and INS-1 islet cells, although coinciding with a comparable response to the hexose in terms of ATP generation, contrasts with a vastly different pattern of D-glucose metabolism in these two cell types.

This study was designed to assess the effect of the dimethyl ester of succinic acid (SAD) upon the hormonal and metabolic response to a 60-min exercise in overnight-starved Goto-Kakizaki rats. Twenty Goto-Kakizaki rats were starved overnight and then either maintained at rest or obliged to swim for 60 min. Half of the rats were injected intraperitoneally with the dimethyl ester of succinic acid (SAD, 5.0 micromol g(-1) body wt) immediately before exercise (or 60 min of rest). In the hereditarily diabetic rats, overnight starvation lowered the plasma D- glucose, insulin and lactate concentrations, while increasing that of free fatty acids and beta-hydroxybutyrate. In resting rats, the injection of SAD increased the glycogen content of liver, heart and muscle and the plasma concentration of D-glucose, insulin, glycerol and free fatty acids. In control animals, not injected with SAD, exercise increased the plasma concentration of D- glucose, lactate and glycerol, whilst lowering both that of insulin and the glycogen content of liver, heart and muscle. The injection of SAD before exercise failed to prevent and, on occasion, even accentuated the changes in both the glycogen content of liver, heart and muscle and the plasma concentration of D-glucose, insulin, glycerol and free fatty acids, whilst minimizing the increase in lactate concentration otherwise caused by exercise. Nevertheless, the comparison between resting and exercising rats, both injected with SAD, suggested that the ester abolished the exercise-induced rise in D-glucose, glycerol and fatty acid concentrations. By comparison with comparable experiments conducted in overnight-starved normal rats, these findings emphasize both the difference between normal and diabetic rats in their metabolic response to exercise, especially in terms of changes in glycemia, and the usefulness of SAD to compensate for the increased consumption of endogenous nutrients during exercise.

Human pancreatic islets were cultured for 63 hr at 2.8 or 16.7 mM D-glucose in the absence or presence of dexamethasone. In the 1.0 to 10 microM range, dexamethasone caused a concentration-related decrease in the FAD (flavin adenine dinucleotide)-linked mitochondrial glycerophosphate dehydrogenase (mGDH) mRNA content of the islets, and decreased both the mGDH content of the islets and the catalytic activity of the enzyme in islet homogenates, these effects being often more marked in islets cultured at 16.7 mM, rather than 2.8 mM, D-glucose. Even after culture in the presence of no more than 10 nM dexamethasone, namely under conditions in which the mGDH mRNA content and activity were both virtually unaffected, the corticosteroid restored the capacity of the beta-cells to display an increase in insulin output in response to a rise in D-glucose concentration in islets first cultured at 2.8 mM D-glucose but suppressed the insulinotropic action of the hexose in islets first cultured at 16.7 mM D-glucose. Whilst revealing an untoward effect of high concentrations of dexamethasone upon mGDH mRNA, content and activity in human islets, these findings also document a dual effect of a low concentration of the corticosteroid (10 nM) upon the secretory responsiveness of human islets to D-glucose, independently of any significant change in mGDH gene expression. It is proposed that such a dual action may account, in part at least, for both the well known increase in insulin output found in hypercorticism and the more recently discovered unfavourable direct effect of corticosteroid hormones on the secretory activity of islet beta-cells.

The metabolism of D-glucose and/or D-fructose was investigated in both pancreatic islets and parotid cells of control and hereditarily diabetic Goto-Kakizaki (GK) rats. In the islets from GK rats, a preferential alteration of the oxidative response to D-glucose coincided with an impaired secretory response to the aldohexose. Such a metabolic alteration was not found in the parotid cells of GK rats. Whether in islet or parotid cells, D-fructose little affected the catabolism of glucose in either control or GK rats. The metabolism of D-fructose and the effect of D-glucose thereupon were essentially comparable in control and GK rats in both pancreatic islets and parotid cells. In both cell types, the comparison between the metabolism of D-glucose and D-fructose in cells simultaneously exposed to the two hexoses suggested a far from negligible contribution of fructokinase to the phosphorylation of D-fructose. Although the catabolism of the ketohexose and its modulation by D-glucose were closely comparable in islets from control and GK rats, the insulinotropic action of the ketohexose, relative to that of the aldohexose, was severely impaired in the GK rats. The present work thus emphasizes the specificity of the alteration in D-glucose metabolism in islets, as opposed to extrapancreatic cells, of GK rats. It also reveals in the islets of GK rats a further secretory anomaly apparently not attributable to the impairment of nutrient catabolism in the islet cells of these diabetic animals.

The binding of tritiated S21403, a novel insulin-releasing agent of the meglitinide family, to multilamellar liposomes formed of egg yolk phosphatidylcholine was compared to that of tritiated glibenclamide. The binding of [3H]S21403 reached equilibrium within 5 min of incubation at 37 degrees C, was virtually proportional to its concentration (2.8 to 56.0 nM) and failed to be inhibited by a much higher concentration (0.1 mM) of unlabelled S21403. It was much lower than that of [3H]glibenclamide tested at a comparable concentration (14.8 nM) and only slightly decreased by 0.1 mM unlabelled glibenclamide. The latter sulfonylurea inhibited more severely the binding of [3H]glibenclamide, which was unaffected, however, by 0.1 mM unlabelled S21403. These findings suggest a much higher affinity of glibenclamide than S21403 for the artificial phospholipid bilayer, this coinciding with a higher biological potency, as insulin secretagogue, of the hypoglycemic sulfonylurea as compared to meglitinide analog. It is proposed, therefore, that the insertion of these antidiabetic agents in the phospholipid domain of the B-cell membrane may condition their access to the ATP-responsive K+ channels known to represent the main target for their insulinotropic action.

The metabolism of glycerol-1,2,3-trimethylsuccinate ester was investigated in rat hepatocytes. The ester displayed a greater nutritional value than D-glucose, as a precursor of either CO2 or glycogen. In terms of 14CO2 production, the value calculated from experiments conducted in the presence of 1.9 mM [U-14C] glycerol-1,2,3-trimethylsuccinate, glycerol-1,2,3-trimethyl[1,4-14C] succinate and glycerol- 1,2,3-trimethyl[2,3-14C] succinate represented about 50 times that found in cells incubated with 1.0 mM D-[U-14C] glucose. For glycogen synthesis, the results found with the ester were approximately 7-8 times higher than those found with the hexose. A further advantage of the ester over D-glucose consisted in the fact that, at increasing concentrations of these nutrients, a maximal metabolic response may be reached at lower levels of glycerol- 1,2,3-trimethylsuccinate than D-glucose. By comparison with previous data obtained in the same experimental model, glycerol-1,2,3-trimethylsuccinate was also found to display a higher nutritional value than the dimethyl ester of succinic acid. It is proposed, therefore, that glycerol-1,2,3-trimethylsuccinate could be used to support ATP generation in cells endangered by an imbalance between the rate of synthesis and hydrolysis of this adenine nucleotide.

In the light of recent findings on the effect of D-glucose upon D-fructose phosphorylation by human B-cell glucokinase, the influence of the aldohexose upon the metabolism of the ketohexose was investigated in rat pancreatic islets. D-glucose, although slightly decreasing D-[5-(3)H]fructose utilization, augmented the oxidation of the ketohexose, indicating that the aldohexose stimulates preferentially the oxidative, as distinct from anaerobic, modality of glycolysis. Such was not the case in parotid cells, taken as representative of functionally nonglucose-responsive cells. In the islets exposed to D-fructose, D-glucose also decreased the fractional contribution of the pentose shunt to the generation of CO2 and D-glyceraldehyde 3-phosphate from the ketohexose, and increased the inflow into the Krebs cycle of dicarboxylic metabolites relative to that of fructose-derived acetyl-CoA. This glucose-induced remodeling of D-fructose metabolism may optimize the insulin secretory response of islet cells to these hexoses, e.g. after food intake.

The effect of the meglitinide analog S21403 (10 microm) upon(86)Rb and(45)Ca outflow and insulin release was investigated in perifused rat islets exposed to a high concentration of D-glucose (16.7 mm) in order to simulate the situation found in diabetic patients. Under these conditions, S21403 provoked a rapid, sustained and rapidly reversible increase in(86)Rb outflow, (45)Ca efflux and insulin release. These effects were suppressed or reversed when the experiments were conducted in the absence of extracellular Ca2+. They support the view that S21043 could be used as a novel insulinotropic tool in the treatment of non-insulin-dependent diabetes mellitus, the cationic and secretory responses to the drug displaying a favourable time course for prompt and not unduly prolonged activation of islet B-cells.

The pentaacetate ester of alpha-D-glucose was recently introduced as a new insulin secretagogue. Its insulinotropic action appears mainly attributable to the catabolism of its hexose moiety in islet cells, but a direct effect of the ester itself upon a receptor apparently displaying analogy with that involved in the recognition of bitter agents by taste buds may also be operative. In the present study, the secretory response of rat isolated pancreatic islets to alpha-D-glucose pentaacetate (1.7 mM) was found to be preserved, except in the absence of any other exogenous nutrient, when the extracellular pH was raised from about 7.4 to 8.0. Inversely, however, when the extracellular pH was lowered to about 7.0, alpha-D-glucose pentaacetate inhibited both basal and D-glucose- or L-leucine-stimulated insulin output. These findings are interpreted to support a dual mode of action of alpha-D-glucose pentaacetate upon insulin secretion, a lowering of extracellular pH revealing a negative component of the islet B-cells functional response to such a monosaccharide ester.

Repaglinide is a new carbamoylmethyl benzoic acid derivative that is structurally related to meglitinide. In common with all active oral hypoglycaemic agents, it displays a comparable U-shaped configuration. Repaglinide exerts its effects by binding to a site on the plasma membrane of beta-cells, thereby closing ATP-sensitive potassium channels. This causes depolarization of the beta-cell and the opening of voltage-sensitive calcium channels allowing the influx of extracellular calcium ions. This increase in intracellular calcium, in turn, stimulates insulin release. The insulinotropic effect of repaglinide is not related to its ionophoretic capacity. Repaglinide does not cause insulin release in the absence of exogenous glucose, nor does it inhibit the biosynthesis of proinsulin. In vivo administration of an ester of succinic acid potentiates the insulin secretory response to concomitantly administered repaglinide. Repaglinide causes a more rapid increase in plasma insulin levels in rats than either glibenclamide or glimepiride.

In pancreatic islets prepared from fed rats and incubated at a low concentration (1.7 mM) of D-glucose, D-mannoheptulose (10.0 mM) virtually fails to affect the metabolism of the hexose. Likewise, in islets from starved rats, the relative extent of the inhibitory action of D-mannoheptulose upon D-glucose metabolism is much more marked at high (16.7 mM) than low (1.7 mM) hexose concentration. Nevertheless, despite decreasing the metabolism of D-glucose, starvation augments the sensitivity to D-mannoheptulose in the islets incubated at a low concentration of the hexose, D-galactose, but not D-fructose, also augments the inhibitory action of D-mannoheptulose upon D-glucose metabolism in islets prepared from fed rats and exposed to the low concentration of D-glucose. A comparable situation prevails in islets exposed to 2-ketoisocaproate. Forskolin, however, which decreases D-glucose catabolism in the islets from fed rats exposed to 1.7 mM D-glucose, fails to affect significantly the inhibitory action of D-mannoheptulose on D-glucose metabolism. It is proposed that hexoses transported by the same carrier as D-glucose and non-glucidic nutrient secretagogues somehow increase D-mannoheptulose uptake by the islet cells. The latter two conditions may be operative in islets exposed to a high concentration of D-glucose, this accounting for the exquisite sensitivity to D-mannoheptulose of glucose-stimulated islets.

The hypoglycemic sulfonylurea gliquidone, used at a 10 microM concentration, failed to affect the metabolism of D-glucose in rat pancreatic islets incubated in the presence of 5.6 mM, 8.3 mM or 16.7 mM D-glucose. However, at 2.8 mM D-glucose, gliquidone increased D-[U-14C]glucose oxidation while decreasing the utilization of D-[5-3H]glucose and generation of radioactive acidic metabolites and amino acids from D-[U-14C]glucose. These dissociated effects could conceivably be attributable, respectively, to activation of FAD-linked glycerophosphate dehydrogenase as a result of an increase in cytosolic Ca2+ concentration and to a subsequent inhibition of phosphofructokinase as a result of an increase in cytosolic ATP concentration. The effect of gliquidone on the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was indeed duplicated by repaglinide and suppressed in the absence of extracellular Ca2+ or at low temperature. The present findings thus provide a further illustration of the often contrasting effects of pharmacological and physiological insulinotropic agents on selected metabolic, cationic and functional variables in pancreatic islet cells.

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

The esterification of several monosaccharides, such as D-glucose, D-mannoheptulose and 2-deoxy-D-glucose was recently reported to increase their biological efficiency as either nutrient or antimetabolic agent. In the present study, however, the tetraacetate ester of streptozotocin was unexpectedly found to be less potent than unesterified streptozotocin in inhibiting D-glucose metabolism and insulinotropic action in isolated rat pancreatic islets. This coincided with a much lower rate for the hydrolysis of streptozotocin tetraacetate than D-glucose pentaacetate in islet homogenates. These findings document that the esterification of single sugars is not always a successful procedure to enhance their biological potency, for instance because of too low a rate for the intracellular hydrolysis of the ester. To the extent that the activity of the concerned esterase(s) may differ in distinct cell types, as suggested by a prior observation, advantage could be taken of such a situation to target selected esters towards specific, e.g. tumoural cells.

D-mannoheptulose is currently used as a tool to inhibit, in a competitive manner, D-glucose phosphorylation, metabolism and functional effects in the pancreatic islet B-cell. In order to better understand the mode of action of the heptose, we have explored its effect upon D-glucose phosphorylation in liver, parotid cells and islet homogenates, this allowing to characterize the interference of the heptose with glucokinase and/or hexokinase. The effect of D-mannoheptulose upon the metabolism of D-glucose was also examined in both intact parotid cells and pancreatic islets. Last, the effect of D-mannoheptulose upon glucose-stimulated insulin release was reinvestigated over large concentration ranges of both the heptose and hexose. The experimental data revealed a mixed type of D-mannoheptulose inhibitory action upon D-glucose phosphorylation, predominantly of the non-competitive and competitive type, in liver and parotid homogenates, respectively. Despite efficient inhibition of hexose phosphorylation in both parotid cell and islet homogenates, the heptose suppressed the metabolic and functional responses to D-glucose only in pancreatic islets, whilst failing to affect adversely D-glucose catabolism in parotid cells. These findings suggest that factors such as the intracellular transport and availability of the heptose may interfere with the expression of its antagonistic action upon D-glucose metabolism.

Selected esters of succinic acid were recently proposed as novel nutrients to support ATP generation in cells endangered by an imbalance between the formation and breakdown of this adenine nucleotide. In the present study, a new ester, glycerol-1,2,3-trimethylsuccinate, was examined for its potential beneficial effect in the procedures preceding liver transplantation.The viability and metabolic behavior of hepatocytes were examined after perfusion and storage of rat livers for 20 hr at 4 degrees C with a Belzer UW-CSS solution in the absence or presence or 2 mM glycerol-1,2,3-trimethylsuccinate.Although it failed to affect significantly the release of cellular enzymes during storage and the protein or glycogen content of the liver, and was unable to prevent the storage-induced decrease in both biosynthetic activity and D-[U-14C]glucose incorporation into glycogen in isolated hepatocytes, the ester restored to a close-to-normal value the viability of the hepatocytes and opposed the starvation-like effects of liver storage upon both the conversion of D-[U-14C]glucose to 14CO2 and radioactive amino acids and the de novo generation of 14C-labeled D-glucose from [2-14C]pyruvate.Because succinic acid esters are efficiently metabolized in several cell types, the present results suggest that such esters may have a wide field of application in transplantation procedures.

Selected esters of D-glucose were recently proposed as tools to provide the sugar to cells, whilst bypassing the carrier system for hexose transport across the plasma membrane. In the present study, alpha-D-glucose pentaacetate, beta-D-glucose pentaacetate, alpha-D-mannose pentaacetate and, to a lesser extent, 6-O-acetyl-D-glucose, all tested at a 1.7 mM concentration, were found to increase lactate production above basal value in rat erythrocytes. Over 90 min incubation, the increment in lactate production ranged from about 1.2 (alpha-D-glucose pentaacetate) to 0.6 (6-O-acetyl-D-glucose) micromol/microl of erythrocytes. Little or no change in lactate production was observed in cells exposed to beta-L-glucose pentaacetate, alpha-D-glucose pentaethylsuccinate, alpha-D-galactose pentaacetate or beta-D-galactose pentaacetate. The metabolic response to alpha-D-glucose pentaacetate was resistant to 3-O-methyl-D-glucose (10-80 mM) which suppressed, however, that evoked by D-glucose. D-mannoheptulose (10 mM) virtually failed to affect the response to D-glucose and its pentaacetate ester. On the contrary, 2-deoxy-D-glucose (10.6 mM) inhibited to the same relative extent (55% decrease) lactate production in erythrocytes exposed to either unesterified D-glucose or alpha-D-glucose pentaacetate. The tetraacetic ester of 2-deoxy-D-glucose was more efficient than unesterified 2-deoxy-D-glucose in inhibiting lactate production from alpha-D-glucose pentaacetate. It is proposed that selected esters of saccharides represent useful tools to bypass defects in hexose transport, and to increase their nutritional or therapeutic efficiency.

This study aims at gaining further insight into the mode of action of repaglinide in pancreatic islet B-cells. At a 1.0 mumol/L concentration, the meglitinide analog failed to affect the metabolism of exogenous D-glucose and that of endogenous nutrients in islets prelabeled with either L-[U-14C]glutamine or [U-14C]palmitate. Likewise, repaglinide (1.0 mumol/L) failed to modify significantly the incorporation of L-[4-3H]phenylalanine into TCA-precipitable material in islets exposed to a close-to-physiological concentration of D-glucose (7.0 mmol/L). The threshold concentration for the insulinotropic action of repaglinide was close to 0.1-1.0 mumol/L and a maximal response was reached at 10.0 mumol/L in islets incubated in the presence of 5.6-8.3 mmol/L D-glucose. At a higher hexose concentration (16.7 mmol/L), however, an enhancing action of repaglinide (10 mumol/L) upon glucose-stimulated insulin release was only observed over 25 min stimulation in perifused islets, no significant increase in insulin output being detected when islets were exposed to repaglinide (0.1 mumol/L to 0.1 mmol/L) over 90 min incubation at the high D-glucose level. The increase in insulin output evoked by repaglinide in the islets perifused at 16.7 mmol/L D-glucose coincided with a modest increase in 86Rb outflow and a marked stimulation of 45Ca efflux from prelabeled islets, suggesting stimulation of Ca2+ influx into the islet cells and subsequent activation of Ca(2+)-responsive K+ channels. When the administration of repaglinide was halted, the reversibility of its cationic and secretory effects was more pronounced in islets perifused at a high (16.7 mmol/L), rather than a low (6.0 mmol/L), D-glucose concentration. These findings support the view that the primary site of action of repaglinide consists in a remodeling of cationic fluxes, and document that this drug displays favorable attributes as an insulinotropic agent for the treatment of non-insulin-dependent diabetes, such as its lack of interference with nutrient metabolism and biosynthetic activity in isolated islets, the low threshold concentration for its insulin-releasing action and its capacity to augment, at least transiently, insulin release at a high concentration of D-glucose.

The polyacetate esters of selected monosaccharides were recently found to either stimulate insulin release or inhibit glucose-stimulated insulin secretion in islets from normal rats. The present study extends such findings both to new combinations of either D-glucose or L-leucine and some polyacetate esters and to hereditarily diabetic, as distinct from normal, rats. In the normal animals, 2-deoxy-D-glucose tetraacetate (1.7 mM) increased both glucose- and leucine-stimulated insulin output. The secretory response to L-leucine was also increased by beta-D-glucose pentaacetate, but inhibited by alpha-D-galactose pentaacetate and D-mannoheptulose hexaacetate (1.7 mM) in the islets of normal rats. In the diabetic rats, the secretory response to D-glucose (8.3 mM) was increased by alpha- or beta-D-glucose pentaacetate and 2-deoxy-D-glucose tetraacetate (1.7 mM), inhibited by alpha-D-galactose pentaacetate and D-mannoheptulose hexaacetate, and unaffected by beta-L-glucose pentaacetate, all esters being tested at 1.7 mM concentration. L-Leucine-stimulated insulin release was also increased by alpha-D-glucose pentaacetate, but not significantly affected by beta-D-galactose pentaacetate, beta-L-glucose pentaacetate, 2-deoxy-D-glucose tetraacetate and D-mannoheptulose hexaacetate in the islets of diabetic animals. These findings suggest a dual mode of action of the esters in the pancreatic islet B-cell, involving both the metabolic response to their sugar moieties and a direct effect of the esters themselves upon a specific receptor system. It is proposed that selected esters could be used as insulinotropic tools in non-insulin-dependent diabetes mellitus.

99mTc-sesta-(2-methoxy-isobutyl-isonitrile)(Tc-MIBI) is currently used for imaging of several organs. In the present study, its uptake by rat pancreatic islets, rat parotid cells, and human breast adenocarcinoma cells (MCF-7 cells) was found to be grossly proportional to its concentration (up to 0.1 microM), time-related (with a fractional turnover rate close to 2-3 10(-2).min(-1)), and stimulated by D-glucose. Comparable values for the fractional turnover rate were found in prelabeled islets and MCF-7 cells, D-glucose failing to affect Tc-MIBI efflux from prelabeled islets. In the islets, the uptake of Tc-MIBI was decreased at low temperature, in the presence of mitochondrial poisons and at high extracellular K+ concentration, unaffected by the absence of extracellular Ca2+, and increased by nutrient secretagogs, such as 2-ketoisocaproate and the association of L-leucine and L-glutamine. These findings are consistent with the view that Tc-MIBI uptake is ruled by its extracellular concentration, and the polarization of both plasma and mitochondrial membranes. It is proposed that this lipophilic cation may be useful to detect alteration of nutrient metabolism in pancreatic islets deprived of any exogenous fuel.

It was recently proposed that stimulation of pancreatic islet by D-glucose results in the translocation of glucokinase from the perinuclear area to the cell periphery, where the enzyme might conceivably interact with either the glucose transporter GLUT-2 or some other proteins and, by doing so, become better able to express its full catalytic activity. To explore the possible interaction between glucokinase and the cell boundary, dispersed rat pancreatic islet cells were preincubated for 60 min at a low (2.8 mM) or high (16.7 mM) concentration of D-glucose, then exposed for 1 min to digitonin (0.5 mg/ml) and eventually centrifuged through a layer of oil for separation of the cell pellet from the supernatant fraction containing the material released by digitonin. Under these conditions, the bulk of lactate dehydrogenase and glutamate dehydrogenase activities were recovered in the supernatant fraction and cell pellet, respectively. The measurement of hexokinase isoenzyme activities in the two subcellular fractions, as conducted at low or high hexose concentrations and in either the absence or presence of exogenous hexose phosphates (3.0 mM glucose 6-phosphate and 1.0 mM fructose 1-phosphate) indicated a preferential location of the low-Km hexokinase in the cell pellet and of the high-Km glucokinase in the cytosolic fraction. Such a distribution pattern failed to be significantly affected by the concentration of D-glucose used during the initial incubation of the dispersed islet cells. These findings argue against the view that the glucose-induced translocation of glucokinase would result in any sizeable binding of the enzyme to a plasma membrane-associated protein.