Leucine-induced Insulin Release Research Articles

Preliminary report: Leucine supplementation enhances glutamate dehydrogenase expression and restores glucose-induced insulin secretion in protein-malnourished rats

Low-protein diet impairs insulin secretion in response to nutrients and may induce several metabolic disorders including diabetes, obesity, and cardiovascular disease. In the present study, the influence of leucine supplementation on glutamate dehydrogenase (GDH) expression and glucose-induced insulin secretion (GIIS) was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal-protein diet (17%) without or with leucine supplementation or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine (1.5%) was supplied in the drinking water. Western blotting analysis revealed reduced GDH expression in LP, whereas LPL displayed improved GDH expression, similar to control. The GIIS and leucine-induced insulin release were also enhanced in LPL compared with LP and similar to those observed in rats fed a normal-protein diet without leucine supplementation. In addition, GDH allosteric activators produced an increased insulin secretion in LPL. These findings indicate that leucine supplementation was able to increase GDH expression leading to GIIS restoration, probably by improved leucine metabolic pathways.

Inhibition of insulin release by alpha- and beta-D-galactose pentaacetate.

Several monosaccharide polyacetate esters display higher biological efficiency than the corresponding unesterified carbohydrates, this being attributable to their capacity to cross the plasma membrane without requiring the intervention of a specific carrier system and to their subsequent intracellular esterase-catalyzed hydrolysis. Some of these esters, however, also exert a direct effect upon a receptor system apparently displaying analogies with that involved in the identification of bitter compounds by taste buds. For instance, under suitable experimental conditions, esters of non-nutrient monosaccharides, such as L-glucose, D-mannoheptulose or 2-deoxy-D-glucose, unexpectedly stimulate insulin release. The present work reveals that alpha-D-galactose pentaacetate and, to a lesser extent, beta-D-galactose pentaacetate both inhibit leucine-induced insulin release in rat pancreatic islets. This indicates that the postulated receptor system displays anomeric specificity and may participate in a negative modulation of insulin secretion. It is proposed that advantage could be taken of the negative insulinotropic action of D-galactose pentaacetate esters to prevent excessive insulin release in pathological situations.

Glucose regulates leucine-induced insulin release and the expression of the branched chain ketoacid dehydrogenase E1 alpha subunit gene in pancreatic islets.

Much evidence has accumulated to support the idea that leucine can stimulate insulin release by allosterically activating glutamate dehydrogenase thus enhancing glutamate metabolism. It is less clear how the metabolism of leucine itself contributes to the signal for insulin release. We recently found that culturing pancreatic islets for 1 day at low glucose (1 mM) suppressed glucose-induced insulin release, but preserved leucine-induced insulin release. When islets were cultured at high glucose (20 mM), glucose-induced insulin release was preserved, but leucine-induced insulin release was suppressed (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1990) Am. J. Physiol., 259, E548-E554). The suppression of leucine-induced insulin release can be explained by glucose's suppression of the synthesis of the enzyme that catalyzes the first committed step of leucine metabolism, branched chain ketoacid dehydrogenase complex (BCKDH). High glucose suppressed the enzyme activity of the E1 component of the BCKDH complex, as well as the total activity of the BCKDH complex, to usually negligible levels in islets and decreased by an average of 90% the mRNA which encodes E1 alpha, the catalytic subunit of the E1 component of BCKDH, in islets and rat insulinoma cells. Time course studies showed that about 24 h in culture was required to maximally induce or suppress the expression of BCKDH E1 alpha. Culture at high glutamine with or without leucine mimicked to a lesser and more variable degree the effects of high glucose on leucine-induced insulin release and BCKDH E1 alpha mRNA. Leucine-plus-glutamine-induced insulin release was present after culture of islets with glucose and with or without any other secretagogue. Also, glutamate dehydrogenase transcripts and enzyme activity were not significantly altered by varying the concentration of glucose in the culture medium. Thus, leucine's insulinotropism via activation of glutamate dehydrogenase is constitutive. Preproinsulin mRNA levels were markedly increased at high glucose and glyceraldehyde phosphate dehydrogenase transcripts were either unaffected or slightly increased by glucose. Glutamine did not significantly effect the expression of genes other than BCKDH E1 alpha, and leucine had little or no effect on the expression of any of the four genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Elusive Proximal Signals of β-Cells for Insulin Secretion

The beta-cell is unique because its major agonists, i.e., insulin secretagogues, undergo metabolism instead of interacting with a receptor. This perspectives presents the hypothesis that the first part of a metabolic signal of a secretagogue is specific to the secretagogue and the beta-cell and can be envisioned as proximal. The second part, which occurs after transduction to more universal signaling mechanisms, is viewed as distal. Distal signaling and exocytosis in the beta-cell operate the same as in other cells. Aerobic glycolysis is required for glucose-induced insulin release. Because glyceraldehyde, which enters metabolism at the triose phosphates in the glycolytic pathway, is a potent insulin secretagogue but pyruvate, which is metabolized in the mitochondrion, is not an insulin secretagogue, the proximal signal for glucose-induced insulin release originates with an interaction between the central part of the glycolytic pathway and mitochondrial metabolism. The proximal message in leucine-induced insulin release originates with leucine allosterically activating glutamate dehydrogenase, which activates endogenous glutamate metabolism, and by the metabolism of leucine itself. The methyl ester of succinate is a potent experimental insulin secretagogue. It is puzzling why the glucose signal requires the interplay of glycolysis and mitochondrial metabolism, whereas the signals from leucine and succinate originate entirely from within the mitochondrion. Leucine-induced insulin release is suppressed and glucose-induced insulin release is activated in islets cultured at a high concentration of glucose. Conversely, leucine-induced insulin release is activated and glucose-induced insulin release is suppressed in islets cultured at low glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of insulin release by factors that also modify glutamate dehydrogenase.

Leucine and monomethyl succinate initiate insulin release, and glutamine potentiates leucine-induced insulin release. Alanine enhances and malate inhibits leucine plus glutamine-induced insulin release. The insulinotropic effect of leucine is at least in part secondary to its ability to activate glutamate oxidation by glutamate dehydrogenase (Sener, A., Malaisse-Lagae, F., and Malaisse, W. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5460-5464). The effect of these other amino acids or Krebs cycle intermediates on insulin release also correlates with their effects on glutamate dehydrogenase and their ability to regulate inhibition of this enzyme by alpha-ketoglutarate. For example, glutamine enhances insulin release and islet glutamate dehydrogenase activity only in the presence of leucine. This could be because leucine, especially in the presence of alpha-ketoglutarate, increases the Km of glutamate and converts alpha-ketoglutarate from a noncompetitive to a competitive inhibitor of glutamate. Thus, in the presence of leucine, this enzyme is more responsive to high levels of glutamate and less responsive to inhibition by alpha-ketoglutarate. Malate could decrease and alanine could increase insulin release because malate increases the generation of alpha-ketoglutarate in islet mitochondria via the combined malate dehydrogenase-aspartate aminotransferase reaction, and alanine could decrease the level of alpha-ketoglutarate via the alanine transaminase reaction. Monomethyl succinate alone is as stimulatory of insulin release as leucine alone, and glutamine enhances the action of both. Succinyl coenzyme A, leucine, and GTP are all bound in the same region on glutamate dehydrogenase, where GTP is a potent inhibitor and succinyl coenzyme A and leucine are comparable activators. Thus, the insulinotropic properties of monomethyl succinate could result from it increasing the level of succinyl coenzyme A and decreasing the level of GTP via the succinate thiokinase reaction.

Generalized diminution in the response to nutrients as insulin-releasing agents during the early neonatal period in the rat.

Glucose-induced insulin secretion is diminished during the fetal and neonatal period. To investigate whether the “defect” in B-cell secretion is specific for glucose and linked to glucose metabolism, the insulin-releasing effect of three nutrient secretagogues, glucose, glyceraldehyde, and leucine, were compared at different ages of the rat in vivo (1- and 7-day neo-nates) and in vitro (1- and 7-day neonates and adult animals). Intraperitoneal injections produced a marked insulin response to glucose, glyceraldehyde, and leucine in 7-day animals but only small increments that were not significant in 1-day neonates. The accumulation of insulin from isolated islets of 1-day neonates during 60-min incubations was slightly but significantly enhanced by 11.1-34.4 m M glucose or 40 mM leucine. No response was seen with 1-16 mM glyceraldehyde. By contrast, all three secretagogues markedly stimulated insulin secretion from 7-day and adult islets. However, quantitative relationships differed. Thus, the response from 7-day islets to the highest concentration of glucose was 20%, to glyceraldehyde 10%, and to leucine 50% of adult values. A “basal” concentration of glucose (4.4 mM) amplified glyceraldehyde- or leucine-induced insulin release, but did not influence the age-related differences in secretory response. The nutrient secregogues induced a small percent stimulation of (32P)-orthophosphate efflux from prelabeled islets of 1-day neonates. This stimulation increased progressively with age. However, in 1- and 7-day islets glucose and leucine stimulated the efflux of 32P more than glyceraldehyde; in adult islets the nutrients were equipotent. Glucose, glyceraldehyde, and leucine increased the accumulation of (3H) cyclic AMP in prelabeled adult but not in 7- or 1-day islets. The foregoing indicates that the obtunded insulin secretion at birth in the rat may apply to all nutrient secretagogues. Because of the difference in metabolic disposition of tested secretagogues, the secretory limitation cannot be ascribed to faulty glycolysis but must involve some communally shared, probably early, step in stimulus-secretion coupling. Continued maturation includes preferential development of the secretory responsiveness to some secretagogues.

Evidence of specialization among microvilli of rat hepatocytes

This chapter describes the development and biochemistry of antidiabetics. Diabetes is characterized by a deficiency of effective insulin that could result from the defective synthesis or release of insulin by the pancreatic B-cells, an increased hepatic degradation of insulin by glutathione-insulin transhydrogenase, or the binding of insulin to a larger protein in the bloodstream. Several amino acids and proteins also stimulate insulin release. The mechanism by which the various amino acids cause insulin release is apparently different as diazoxide inhibits the insulin releasing effect of leucine but not of arginine. Leucine-induced insulin release is magnified by pretreatment with sulfonylureas, whereas this is not the case with other amino acids. It has been postulated that the synalbumin insulin antagonist might actually be the B chain of insulin. The antagonist has been separated from albumin and has been shown to be similar to insulin B chain in many of its physicochemical properties. The finding that albumin bound B chain antagonizes insulin in vitro and in vivo adds credence to the hypothesis that synalbumin and insulin B chain could be identical. The detection of immunoassayable B chain in human plasma further supports the B chain-synalbmin theory.

Onset and reversibility of changes in secretory function and composition of isolated rat pancreatic islets following long-term administration of high or low tolbutamide doses

Chronic administration of a high tolbutamide dose to rats induces islet hypertrophy associated with a decreased insulin content per islet and with a diminished insulin release in response to a glucose or leucine stimulus. These changes are reversible after discontinuation of tolbutamide. Chronic administration of a low tolbutamide dose (effective dose 30%) has no effect on islet size, on insulin content per islet, or on leucine-induced insulin release. In contrast, the glucose-induced insulin release is impaired. However, glucose-induced insulin release is normal in the presence of glucagon (5 μg/ml) or theophylline (5 m M). Since islet hypertrophy occurs following admin-istration of high tolbutamide doses only and is associated with hypofunction rather than with hyperfunction, it seems hardly conceivable that the therapeutic principle of tolbutamide is based on a beta-cytotrophic effect. B-cell hypofunction seems to be due to at least three factors: the decrease in the insulin content per islet, an impairment in secretory signal recognition, and an interference with the process of signal transmission.

Cytochalasin B-induced impariment of glucose metabolism in islets of Langerhans.

Cytochalasin B (10 mug/ml) facilitated glucose-, glyceraldehyde-, and leucine-induced insulin release. It inhibited glucose uptake, utilization, and oxidation, as well as lactate output in islets exposed to glucose (16.7 mM). However, it failed to affect lactate output in the presence of glyceraldehyde, and leucine oxidation. Cytochalasin B also caused a partial inhibition of 45calcium uptake and proinsulin synthesis evoked by glucose in low concentration (5.6 mM), these findings being compatible with a modest impairment of the process of glucose recognition by the beta-cell. At a higher glucose level (16.7 mM), cytochalasin B failed to affect proinsulin synthesis, the immediate inhibitory effect of glucose upon 45calcium efflux, and the subsequent accumulation of 45calcium in the islets. In the presence of cytochalasin B, mannoheptulose further reduced glucose utilization and lactate production and suppressed glucose-induced insulin release. These data suggest that, although insulin release in the presence of cytochalasin B apparently remains dependent on a sufficient glycolytic flux, the facilitating effect of the drug upon insulin secretion cannot be ascribed to any favorable influence on glucose handling by islet tissue. It is suggested that cytochalasin B, possibly through its effect on the microfilamentous web which is part of the cell boundary, may both facilitate insulin release and inhibit glucose transport across the cell membrane, although no direct cause and effect relationship would exist between the two phenomena.

Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release

Microdissected pancreatic islets from non-inbred ob/ob-mice were cultured for 6 or 7 days in serum-free tissue culture medium 199. The insulin content of the islets decreased 60% during culture in 17 mM or 28 mM glucose and about 70% in the presence of 3.3 mM or 5.6 mM glucose. At the end of a culture period in high glucose, the sum of the insulin in the islet plus that in the culture medium was almost twice as high as the insulin content of fresh islets, indicating an active insulin biosynthesis. The maximal insulin response to glucose after culture in 17 mM or 28 mM glucose was about 40% of that in fresh islets; after culture in 3.3 mM glucose it was 10%. Half-maximal stimulation was observed at a glucose concentration of 5 mM for islets cultured with high glucose as compared to 9 mM for fresh islets. Like glucose, glibenclamide was a more effective insulin stimulator after culture with a high glucose concentration than with a low one. However, leucine-induced insulin release was not affected by the glucose concentration in the preceding culture medium. Whereas potentiation of glucose-stimulated release by arginine or dibutyryl-cAMP was independent of glucose concentration during the culture, theophylline released three times more insulin when the islets had been cultured with high glucose.

The stimulatory and inhibitory function of theophylline on amino acid-induced insulin release: studies with the perfused rat pancreas.

The effects of 5 mM theophylline on L-phenylalanine, L-alanine, and L-leucine-induced insulin secretion were studied using the isolated perfused rat pancreas, varying the sequence of the stimuli. Theophylline in the absence or presence of 3 mM D-glucose caused a small amount of insulin release with a slow onset and a slight and steady increase. Theophylline had no effect on phenylalanine (5, 10, or 20 mM)-induced insulin release independent of the sequence of stimuli. Alanine (20 mM), in the absence of glucose, had no significant insulin stimulatory action. When theophylline was added during the alanine perfusion only a small insulin release, comparable to that given by theophylline alone, could be observed. However, superimposing alanine on a theophylline perfusion led to a potentiation of insulin release. The leucine-induced insulin secretion was significantly altered by the addition of theophylline. At a low concentration of leucine (5 mM) theophylline caused potentiation of leucine-induced insulin secretion. At 10 and 20 mM leucine, theophylline led to a rapid concentration-dependent inhibitory period, followed by a potentiation in the case of 10 mM leucine, and by a restoration of the secretion rate at 20 mM leucine which did not exceed the secretion rate of 20 mM leucine alone. Subsequent removal of theophylline caused a marked "off effect." When 5 or 20 mM leucine was superimposed on a theophylline perfusion, a marked dose-dependent potentiation of the biphasic leucine-induced insulin release and no inhibitory phase could be observed. From these data it must be concluded that the effect of theophylline on the insulin secretory reposure of the beta cell to theophylline and the stimulus. Possible explanations for these phenomena are discussed.

Dynamics of insulin release and microtubular-microfilamentous system. VII. Do microfilaments provide the motive force for the translocation and extrusion of beta granules?

The active role played by beta-cell microfilamentous structures in the dynamics of insulin secretion was investigated by examining the influence of cytochalasin B upon various parameters of hormonal release by the isolated perfused rat pancreas. The view that the cytochalasin-induced changes in insulin release are due to a primary biophysical effect on microfilaments, rather than to an unrelated biochemical alteration of the beta-cell glucose-sensor device, was strengthened by the following observations: (1) the onset and disappearance of the cytochalasin B-induced facilitating action upon insulin release followed a time-course parallel to that characterizing the ultrastructural changes provoked by the drug in the distribution of beta-cell microfilamentous material; and (2) cytochalasin B facilitated leucine-induced insulin release in the presence of a very low glucose concentration. The mold metabolite was also found to transform transient secretory responses into biphasic ones and to prevent the reduction that normally affects the early response to insulinotropic agents when the pancreas is stimulated a few minutes after a prior and short exposure to glucose. The release of insulin evoked by either glucose or gliclazide was abolished in the absence of extracellular calcium, whether in the presence or absence of cytochalasin B. Theophylline and cytochalasin B exerted a synergistic effect upon glucose-induced insulin release. These data support the concept that calcium-dependent contractile events involving cytochalasin B-sensitive microfilamentous structures provide the motive force for both the intracellular translocation and exocytotic release of beta granules.