John-Charles Hutton

The stimulus-secretion coupling of glucose-induced insulin release. XXXV. The links between metabolic and cationic events.

SummaryWhen isolated rat islets were exposed to glucose, the concentrations of NADH and NADPH, and the NADH/NAD+ and NADPH/NADP+ ratios were increased. The dose-response curve resembled that characterising the glucose-induced secondary rise in45Ca efflux, displaying a sigmoidal pattern with a half-maximal value at glucose 7.5 mmol/l. The glucose-induced increase in NAD(P)H was detectable within 1 min of exposure to the sugar. Except for the fall in ATP concentration and ATP/ADP ratio found at very low glucose concentrations (zero to 1.7 mmol/l) no effect of glucose (2.8–27.8 mmol/l) upon the steady-state concentration of adenine nucleotides was observed. However, a stepwise increase in glucose concentration provoked a dramatic and transient fall in the ATP concentration, followed by a sustained increase in both O2 consumption and oxidation of exogenous + endogenous nutrients. This may be essential to meet the energy requirements in the stimulated B-cell. Although no significant effect of glucose upon intracellular pH was detected by the 5,5-dimethyloxazolidine-2,4-dione method, the net release of H+ was markedly increased by glucose, with a hyperbolic dose-response curve (half-maximal response at glucose 2.9 mmol/l) similar to that characterising the glucose-induced initial fall in45Ca efflux. It is proposed that the generation of both NAD(P)H and H+ participates in the coupling of glucose metabolism to distal events in the secretory sequence, especially the ionophoretic process of Ca2+ inward and outward transport, and that changes in these parameters occur in concert with an increased turn-over rate of high-energy phosphate intermediates.L-Glutamine enhances insulin release evoked by L-leucine in isolated rat pancreatic islets. The enhancing action of L-glutamine, which is a rapid but steadily increasing and not rapidly reversible phenomenon is not attributable to any major change in either K+ or Ca2+ outflow from the islet cells. It coincides with an apparent increase in Ca2+ inflow rate and, hence, with Ca accumulation in the islets. The initial ionic response to L-leucine is not qualitatively altered by the presence of L-glutamine. In their combined capacity to stimulate 45Ca net uptake in the islets, L-glutamine can be replaced by L-asparagine but not by L-glutamate, whereas L-leucine can be replaced by L-norvaline or L-isoleucine, but not by L-valine, glycine or L-lysine. Such a specificity is identical to that characterizing the effect of these various amino acids upon insulin release. It is postulated that the release of insulin evoked by the combination of L-leucine and L-glutamine involves essentially the same remodelling of ionic fluxes as that evoked by other nutrient secretagogues with, however, an unusual time course for the functional response to L-glutamine.

The stimulus secretion coupling of glucose-induced insulin release. XXVII. Effect of glucose on K+ fluxes in isolated islets.

The effect of glucose upon the handling of K+ by islets of Langerhans removed from normal rats was investigated by measuring both the net uptake of86Rb+ and its efflux from prelabelled islets. The inflow of K+ into islet cells is mediated, in part at least, by an ouabain-sensitive pump. Glucose fails to affect the inflow rate of K+, but it apparently decreases the permeability of islet cells plasma membrane to effluent K+. The glucose-induced change in permeability is a rapid and rapidly reversible phenomenon. Under steady-state conditions, it leads to an increase in the islet cells K+ pool and a decrease of its fractional turnover rate.

Dynamics of O2 consumption in rat pancreatic islets

SummaryThe O2 consumption of rat pancreatic islets was determined by monitoring pO2 in the perifusate from groups of 200–300 islets. Basal respiration was maintained for up to 2 h. The insulin secretagogues, glucose and 4-methyl-2-oxopentanoate, provoked an immediate (<5 s) increase in islet respiration which attained a new steady-state within 10–40 min. The respiratory changes were immediately reversible upon removal of the substrate and were parallelled by changes in insulin release and substrate oxidation. The concentration dependence of glucose-induced respiratory changes was sigmoidal with a threshold at 3 mmol/l. The concentration dependence with 4-methyl-2-oxopentanoate was characterised by a hyperbolic relationship. The weak insulin secretagogues 3-methyl-2-oxobutyrate and d,1-3-methyl-2-oxopentanoate, although stimulating islet respiration were not more effective than 4-methyl-2-oxopentanoate at non-insulinotropic concentrations. Rotenone, antimycin and oligomycin inhibited both basal O2 consumption and the ability of glucose and 4-methyl-2-oxopentanoate to increase islet respiration. 2,4-Dinitrophenol increased islet O2 consumption. The omission of Ca2+ and Mg2+ from the perifusing media, or the addition of the ionophore A23187, had little effect on respiration. The omission of K+ inhibited glucose-induced changes but had a lesser effect in the absence of substrate or in the presence of 4-methyl-2-oxopentanoate. The omission of HCO3- reduced both basal and secretagogue-induced changes in islet respiration. It is concluded that mitochondrial O2 consumption linked to oxidative phosphorylation is a major component in the respiratory response, and that some energy consuming process in the islets depends on the availability of HCO3-. Mitochondrial reactions may generate a signal initiating the secretory process.

Insulin secretory granule biogenesis and the proinsulin-processing endopeptidases.

SummaryThe insulin storage granule of the pancreatic beta cell is assembled within the trans Golgi network from around 50 or so gene products many of which are synthesized coordinately with the major component, proinsulin. An important contribution to our understanding of the regulation of this process has come from studies of the post-translational processing of proinsulin and of other proteins which are stored in the granule, particularly the processing enzymes themselves. The present review focusses on recent insights into the molecular nature of the processing machinery, and the granule Ca2+-dependent subtilisin-related endopeptidases which catalyse the initial rate-limiting step in the enzymic conversion of proinsulin.

Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells.

SummarySulphonylureas stimulate insulin secretion by binding to a receptor in the pancreatic beta-cell plasma membrane resulting in inhibition of ATP-sensitive K+ channels, membrane depolarization and thus influx of Ca2+ through voltage-dependent Ca2+ channels. Sulphonylureas can also induce hormone release at fixed membrane potentials without Ca2+ entry suggesting that these drugs may have other modes of action. We have determined whether different forms of sulphonylurea-binding proteins are present in insulin-secreting cells and their subcellular localization by density gradient centrifugation. Binding studies using [3H]-glibenclamide showed that islet and insulinoma membranes contained a single high affinity sulphonylurea binding site (Kd = 1 nmol/l). Photo-crosslinking of the drug to the membranes resulted in labelling of two proteins with apparent molecular weights of 170 and 140 kDa. The same analyses of insulinoma subcellular fractions showed that the majority (>90%) of binding proteins were localized to intracellular membranes with only minor levels (<10%) on plasma membranes. The 170 kDa sulphonylurea binding protein was present in both plasma and granule membrane fractions whereas the 140 kDa form was not present in the plasma membrane fraction. The differences in the molecular forms and subcellular distribution of the receptor are consistent with sulphonylureas having multiple sites of action in the pancreatic beta cell.

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets

1. The metabolic situation found in pancreatic islets exposed to both L-glutamine and 2-ketoisocaproate was investigated in order to assess its relevance to the synergistic effects of these nutrients upon insulin release. 2. In islet homogenates, serveral 2-keto acids could be used as partners for the transamination of L-glutamate to 2-ketoglutarate. The rate of transamination did not correlate positively with the capacity of each 2-keto acid to stimulate insulin release in the presence of L-glutamine. 3. L-Glutamine enhanced the production of L-leucine from 2-ketoisocaproate and inhibited the conversion of the 2-keto acid to acetoacetate and CO2. L-Glutamine also inhibited the oxidation of pyruvate. 4. In the presence of 2-ketoisocaproate, the rate of generation of 2-ketoglutarate from exogenous L-glutamine was increased, but the oxidative deamination of glutamate was suppressed. 5. L-Valine antagonized the effect of 2-ketoisocaproate to augment 14CO2 output from islets prelabelled with L-[U-14C]glutamine. 6. L-Glutamine did not increase the islet content of reduced pyridine nucleotides beyond the high level reached in the sole presence of 2-ketoisocaproate. 7. If allowance was made for the influence of exogenous nutrients upon the oxidation of endogenous nutrients, the insulin output evoked by L-glutamine and/or 2-keto acids tightly depended on the increment in oxidation rate attributable to these nutrients. 8. The metabolic and secretory responses to L-glutamine and 2-ketoisocaproate were best explained by a stimulation of transamination reactions between 2-ketoisocaproate and glutamate derived from exogenous glutamine.

The stimulus-secretion coupling of glucose-induced insulin release. Metabolic and functional effects of NH4+ in rat islets.

NH4+ caused a dose-related, rapid, and reversible inhibition of glucose-stimulated insulin release by isolated rat islets. It also inhibited glyceraldehyde-, Ba2+-, and sulfonylurea-stimulated insulun secretion. NH4+ failed to affect glucose utilization and oxidation, glucose-stimulated proinsulin biosynthesis, the concentration of ATP, AD, and AMP, and the intracellular pH. NH4+ also failed to affect the ability of theophylline and cytochalasin B to augment glucose-induced insulin release. However, in the presence and absence of glucose, accumulation of NH4+ in islet cells was associated with a fall in the concentration of NADH and HADPH and a concomitant alteration of 86Rb+ and 45Ca2+ (or 133Ba2+) handling. These findings suggest that reduced pyridine nucleotides, generated by the metabolism of endogenous of exogenous nutrients, may modulate ionophoretic processes in the islet cells and by doing so, affect the net uptake of Ca2+ and subsequent release of insulin.

Isolation and Characterisation of Insulin Secretory Granules from a Rat Islet Cell Tumour

SummaryDensity gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour. These procedures were readily completed within 7 h and from each animal yielded approximately 1 mg of granule protein. The isolated granules were essentially free of other subcellular organelles as evaluated by their contents of marker proteins, electron microscopy and by electrophoretic analyses. Their susceptibilities to lysis at low osmotic strength, at pH values above 7 or in media containing sodium ions were similar to those of granules partially purified from islets. Insulin comprised 50–60% of the total granule protein when determined by immunoassay or by densitometry of electrophoretic profiles. The proinsulin content was marginally higher than that of islets, as was the ratio of insulins I to II. Electrophoretic analyses revealed that the secretory granules contained 150 or more proteins besides insulin-related peptides. The majority of these had acidic isoelectric points and were located both within the granule interior and its enveloping membrane.

Insulin secretion by a transplantable rat islet cell tumour

SummaryInvestigation of the subcellular and molecular components of insulin secretion has been made difficult by the small quantities of material available. The recent development of a transplantable rat islet cell tumour of high insulin content and state of differentiation suggested a system more amenable to analysis. To validate the tumour as a model of secretion we have studied its release of insulin. In acute experiments in vitro immunoreactive insulin release was increased by leucine, glucagon, theophylline and dibutyryl cyclic AMP, though not by glucose. Leucine (20mmol/l) plus theophylline (5 mmol/l) caused an abrupt, sustained and rapidly reversible stimulation of two- to fivefold. The response was inhibited by antagonists of cellular oxidative phosphorylation (cyanide, 2,4-dinitrophenol, antimycin A), calcium flux (EGTA, verapamil, Mg2+), calmodulin (trifluoperazine), microtubules (vinblastine, colchicine) and by adrenaline and somatostatin. These findings suggest that the tumour secretes insulin by an exocytotic mechanism similar to that of normal islet tissue.

The stimulus-secretion coupling of amino acid-induced insulin release. Synergistic effects of L-glutamine and 2-keto acids upon insulin secretion.

Abstract 1. L -Glutamine markedly enhances insulin release evoked in rat pancreatic islets by 2-ketoisocaproate or 2-ketocaproate. L -Glutamine exerts a lesser enhancing action in the presence of 2-ketovalerate or 2-ketoisovalerate, which are themselves poor insulin secretagogues. L -Glutamine fails to affect insulin release in the presence of 2- ketobutyrate, pyruvate and β-hydroxybutyrate. 2. The relase of insulin evoked by the combination of L -glutamine and 2-ketoisocaproate represents a sustained phenomenon. It coincides with a stimulation of 45 Ca net uptake by the islets, and is inhibited in the absence of extracellular Ca 2+ and presence of either menadione or epinephrine. 3. L -Valine inhibits insulin releaseevoked by either 2-ketoisocaproate or 2-ketocaproate, whether in the presence or absence of L -glutamine, but does not abolish the enhancing action of L -glutamine. L -Valine fails to affect insulin release evoked by the combination of L -leucine and L -glutamine. 4. L -Isoleucine also inhibits 2-keto acid-induced insulin release. However, in contrast to L -valine, L -isoleucine fails to affect or slightly augments insulin release in the simultaneous presence of L -glutamine and either 2-ketoisocaproate or 2-ketocaproate. 5. L -Leucine causes a dose-related enhancement of insulin release evoked by the combination of 2-ketoisocaproate and L -glutamine. Likewise, in the presence of L -glutamine, L -leucine and 2-ketocaproate act synergistically upon insulin release. 6. The hypothesis is advances that the enhancing action of L -glutamine upon 2-keto acid-stimulated insulin release depends on the availability of the 2-keto acid to act as a partner in the conversion of L -glutamate derived from exogenous L -glutamine to 2-ketoglutarate by transamination reaction, rather than being attributable to activation of glutamate dehydrogenase as observed in islets exposed to both L -glutamine and L -leucine.