E. Olivares

Stimulus-secretion coupling of arginine-induced insulin release: comparison between the cationic amino acid and its methyl ester.

The role currently ascribed to the accumulation of l-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to l-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on d-glucose metabolism, more severe inhibition of the catabolism of endogenous l-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-meadiated entry of l-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.The role currently ascribed to the accumulation of L-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to L-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on D-glucose metabolism, more severe inhibition of the catabolism of endogenous L-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-mediated entry of L-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.

Feeding a protective hydrolysed casein diet to young diabetic-prone BB rats affects oxidation of L[U-14C]glutamine in islets and Peyer's patches, reduces abnormally high mitotic activity in mesenteric lymph nodes, enhances islet insulin and tends to normalize NO production.

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 non-diabetes prone (BBc) BB rats were fed for ~ 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-1β 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−C14]glutamine in Peyers 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-1β-stimulated NO production by cultured islets. The HC diet decreased oxidation of L-[U−C14]glutamine in BBdp pancreatic islets, whereas oxidation of L-[U−C14]glutamine in PP cells was increased, and the basal [Methyl-H3] 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-1β 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.

Immobilization of pancreatic islet cells with preserved secretory potential

Abstract Dispersed pancreatic islet cells from rats were cultured overnight in the presence of macroporous gelatin microcarriers. The cells attached to the microcarriers were then incubated for 90 min in the absence or presence of 15.0 mM d-glucose and/or 1.25 mM theophylline. The release of insulin during incubation was about three times higher in the simultaneous presence of these two secretagogues than in their absence. This procedure could thus be used for the immobilization of pancreatic islet cells with preserved secretory potential.

Insulinotropic action of β-l-glucose pentaacetate

The metabolism of β-l-glucose pentaacetate and its interference with the catabolism ofl-[U-14C]glutamine, [U-14C]palmitate,d-[U-14C]glucose, andd-[5-3H]glucose were examined in rat pancreatic islets. Likewise, attention was paid to the effects of this ester on the biosynthesis of islet peptides, the release of insulin from incubated or perifused islets, the functional behavior of individual B cells examined in a reverse hemolytic plaque assay of insulin secretion, adenylate cyclase activity in a membrane-enriched islet subcellular fraction, cAMP production by intact islets, tritiated inositol phosphate production by islets preincubated with myo-[2-3H]inositol, islet cell intracellular pH, 86Rb and 45Ca efflux from prelabeled perifused islets, and electrical activity in single isolated B cells. The results of these experiments were interpreted to indicate that the insulinotropic action of β-l-glucose pentaacetate is not attributable to any nutritional value of the ester but, instead, appears to result from a direct effect of the ester itself on a yet unidentified receptor system, resulting in a decrease in K+ conductance, plasma membrane depolarization, and induction of electrical activity.

D-glucose metabolism in normal dispersed islet cells and tumoral INS-1 cells

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-[5-3H]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-[5-3H]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.

Comparison of the effects of D-mannoheptulose and its hexaacetate ester on D-glucose metabolism and insulinotropic action in rat pancreatic islets

Summary It was recently, and surprisingly, found that D-mannoheptulose did not affect D-glucose metabolism and insulinotropic action in pancreatic islets incubated at a low concentration of D-glucose. To explain this finding, the metabolism and secretory response to the hexose were investigated in rat islets exposed to D-mannoheptulose hexaacetate, which was recently found to inhibit D-glucose catabolism in cells that are otherwise fully resistant to the heptose. At a high concentration of D-glucose (16.7 mmol/l), the utilisation of D-[5-3H]glucose and oxidation of D-[U-14C]glucose, as well as the insulinotropic action of the hexose, were affected less by D-mannoheptulose tetraacetate than by unesterified D-mannoheptulose. This coincided with a reduced uptake of the ester by intact islets and a lower rate of hydrolysis of the ester in islet homogenates compared with findings in other monosaccharide esters such as D-glucose pentaacetate. At a low concentration of D-glucose (2.8 mmol/l), D-mannoheptulose hexaacetate was slightly more efficient than the unesterified heptose in reducing D-glucose catabolism, but still failed to suppress the secretory response to the hexose. These findings do not necessarily mean that unesterified D-mannoheptulose enters beta-cells more efficiently at high than at low extracellular D-glucose concentrations, especially if possible differences in the respective contributions of distinct islet cell types to the overall catabolism of D-glucose by whole islets is allowed for. These data do not rule out the possibility that D-glucose phosphorylation is more resistant to D-mannoheptulose in beta cells incubated at a low than a high concentration, independently of any difference in the intracellular concentration of the heptose. However, the mechanism of this resistance is still not explained. [Diabetologia (1998) 41: 1109–1113]

Failure of streptozotocin tetraacetate to undergo extensive hydrolysis and to inhibit D-glucose metabolism and insulinotropic action in rat pancreatic islets

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. Copyright

Immobilization of tumoral pancreatic islet cells on a two-dimensional microsupport.

A new procedure for the immobilization of tumoral pancreatic islet cells to a two-dimensional microsupport is presented. Tumoral islet cells of the RINm5F line (0.7x10(6) cells/ml) were immobilized to two-dimensional microcarriers (16.6 cm2/ml). Within 24 h of culture, and as judged from the number of cells, their protein or insulin content, less than 10% of the cells escaped immobilization. The metabolic response of the immobilized cells to D-glucose was well preserved, the paired ratio between D-[U-14C] glucose oxidation and D-[5-3H]glucose utilization being even significantly higher in immobilized than free cells. The advantages of this novel approach in the perspective of islet cell transplantation are underlined.