Shoji Kawazu

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.

REGULATION OF CALCIUM FLUXES AND THEIR REGULATORY ROLES IN PANCREATIC ISLETS

It is today considered as crystal clear that calcium plays an essential role in the regulation of insulin release by the pancreatic B-cell. Some of the major issues concerning such a role are as follows: ( i ) what is the detailed mechanism by which secretagogues are susceptible to influence the handling of calcium in the B-cell; (ii) what is the nature and location of the critical pool of calcium that controls insulin release; (iii) what is the relative and respective contribution of calcium influx, efflux, and subcellar distribution in the regulation of such a pool; and (iv) how does calcium influence the process by which secretory granules migrate to the cell boundary and are extruded via exocytosis in the interstitial fluid.’ In the present report, for the sake of clarity, we will restrict the discussion of these questions to the process of glucose-induced insulin release, with the main emphasis on the possible significance of passive ionophoretic movements. The process by which glucose provokes insulin release can be viewed as a sequence of three major events, namely: ( i ) the recognition or identification of glucose by the B-cell; (ii) the subsequent remodelling of cationic fluxes; and (iii) the activation by calcium of an effector system controlling the migration and exocytosis of secretory granules.2 The role of calcium in insulin release is here considered within the framework of such a sequential view.

The stimulus-secretion coupling of glucose-induced insulin release. Metabolic effects of menadione in isolated islets.

Pancreatic islets contain an enzyme system which catalyzes the donation of hydrogen from NAD(P)H to menadione (2-methyl-1,4-naphthoquinone). In high concentrations (20 to 50 micrometer), menadione, in addition to lowering the concentration of reduced pyridine nucleotides in the islets, also impairs glycolysis and glucose oxidation, decreases ATP concentration, and inhibits proinsulin biosynthesis. However, at a 10 micrometer concentration, menadione fails to affect the concentration of adenine nucleotides, the utilization of glucose, the production of lactate and pyruvate, the oxidation of [6-14C]glucose and the synthesis of proinsulin; whereas the metabolism of glucose through the pentose shunt is markedly increased. The sole inhibitory effect of menadione 10 micrometer upon metabolic parameters is to reduce the concentration of both NADH and NADPH, such an effect being noticed in islets exposed to glucose 11.1 mM but not in those incubated at a higher glucose level (27.8 mM). Since, in the presence of glucose 11.1 mM, menadione 10 micrometer also severely decreases glucose-stimulated45 calcium net uptake and subsequent insulin release, it is concluded that the availability of reduced pyridine nucleotides may play an essential role in the secretory sequence by coupling metabolic to cationic events. Thus, when insulinotropic nutrients are oxidized in the B-cell, the increased availability of reduced pyridine nucleotides could modify the affinity for cations of native ionophoretic systems, eventually leading to the accumulation of calcium up to a level sufficient to trigger insulin release.

Association analyses of the polymorphisms of angiotensin-converting enzyme and angiotensinogen genes with diabetic nephropathy in Japanese non-insulin-dependent diabetics

To investigate predictive genetic markers for diabetic nephropathy, we studied the genetic polymorphisms of angiotensin-converting enzyme (ACE) and angiotensinogen (AGN) in Japanese subjects with non-insulin-dependent diabetes mellitus (NIDDM) with and without nephropathy. Genotype distributions were studied in 132 unrelated NIDDM patients of three groups with normoalbuminuria ([Normo] n = 53), microalbuminuria ([Micro] n = 54), and macroalbuminuria ([Macro] n = 25). The ACE insertion/deletion (I/D) polymorphism of intron 16 was identified by polymerase chain reaction, and the AGN M235T polymorphism was identified by restriction fragment length polymorphism analysis. There were no significant associations between AGN 235 allele or genotype and diabetic nephropathy. The D allele of ACE was significantly more frequent in the Micro (P = .003) and Macro (P = .009) group than in the Normo group. Overall frequencies of the ACE genotype did not differ significantly between the Micro and Macro groups. There were significant relationships between I/D polymorphism and plasma ACE activity; the DD genotype had the highest activity. A multiple logistic regression analysis revealed that the D allele is a strong and independent risk factor for abnormal albuminuria in NIDDM patients. These results suggested that ACE I/D polymorphism, but not AGN M235T polymorphism, is a possible genetic risk factor for diabetic nephropathy in Japanese NIDDM patients.

Hypoxia Induces Transcription of the Plasminogen Activator Inhibitor-1 Gene Through Genistein-Sensitive Tyrosine Kinase Pathways in Vascular Endothelial Cells

A decline in oxygen concentration perturbs endothelial function, which promotes local thrombosis. In this study, we determined whether hypoxia in the range of that observed in pathophysiological hypoxic states stimulates plasminogen activator inhibitor-1 (PAI-1) production in bovine aortic endothelial cells. PAI-1 production, measured by ELISA, was increased by 4.7-fold (P<0.05 versus normoxic control, n=4) at 12 hours after hypoxic stimulation. Northern blot analysis showed the progressive time-dependent increase in the steady-state level of PAI-1 mRNA expression by hypoxia, which reached a 7.5-fold increase (P<0.05 versus control, n=4) at 12 hours. Deferoxamine, which has been known to bind heme protein and to reproduce the hypoxic response, induced PAI-1 production at both the mRNA and protein levels. The half-life of PAI-1 mRNA, as determined by a standard decay assay, was not affected by hypoxia, suggesting that induction of PAI-1 mRNA was regulated mainly at the transcriptional level. Transient transfection assays of the human PAI-1 promoter-luciferase construct indicates that a hypoxia-responsive region lies between -414 and -107 relative to the transcription start site, where no putative hypoxia response element is found. The hypoxia-mediated increase in PAI-1 mRNA levels was attenuated by the tyrosine kinase inhibitors genistein (50 micromol/L) and herbimycin A (1 micromol/L), whereas PD98059 (50 micromol/L, MEK1 inhibitor), SB203580 (10 micromol/L, p38 mitogen-activated protein kinase inhibitor), and calphostin C (1 micromol/L, protein kinase C inhibitor) had no effect on the induction of PAI-1 expression by hypoxia and deferoxamine. Genistein but not daidzein blocked the production of hypoxia- and deferoxamine-induced PAI-1 protein. Thus, we conclude that hypoxia stimulates PAI-1 gene transcription and protein production through a signaling pathway involving genistein-sensitive tyrosine kinases in vascular endothelial cells.

The stimulus-secretion coupling of glucose-induced insulin release: Fuel metabolism in islets deprived of exogenous nutrient

The fuel hypothesis for insulin release postulates that the secretory response to nutrient secretagogues reflects their capacity to augment catabolic fluxes in pancreatic islet cells. Hence, both the oxidation of exogenous nutrients and their effect upon the metabolism of endogenous nutrients should be taken into consideration to account for the stimulation of insulin release. In the present work, an attempt was made to quantify the respective contribution of carbohydrates, fatty acids, and amino acids in the respiration of islets deprived of exogenous nutrient. The metabolism of glycerol, fatty acids, and amino acids was found to account for the major part of the basal respiratory rate. Glucose modestly decreased the oxidation of endogenous fatty acids, lowered the production of NH4+, but did not impair the oxidative catabolism of 2-keto acids derived from endogenous amino acids. These findings suggest that the catabolism of noncarbohydrate nutrients largely contributes to the respiration of the islets, even when the latter are exposed to circulating glucose in its physiological concentration.

Age-Related Alteration of Pancreatic β-cell Function: Increased proinsulin and proinsulin-to-insulin molar ratio in elderly, but not in obese, subjects without glucose intolerance

OBJECTIVE To determine the secretion of insulin, C-peptide, and proinsulin after oral glucose loading in healthy elderly subjects compared with middle-aged subjects with and without obesity and with NIDDM. RESEARCH DESIGN AND METHODS Subjects fell into four groups: nonobese middle-aged normal control subjects (CNT group; n = 38, 40–64 years old); obese normal subjects (OB group; n = 18, 40–64 years old); nonobese NIDDM subjects (NIDDM group; n = 28, 40–64 years old); and nonobese elderly subjects (OL group; n = 17, 65–92 years old). Insulin, C-peptide, and proinsulin were determined by radioimmunoassay in plasma samples taken at 0, 30, 60, and 120 min during a 75-g oral glucose tolerance test (OGTT). RESULTS There were no differences in plasma glucose during the OGTT among the three nondiabetic groups. Hyperinsulinemia was significant in the OB and NIDDM groups but not in the OL group. On the other hand, absolute hyperproinsulinemia was significant in the OL and NIDDM groups compared with the CNT group. Increased proinsulin was rather dominant in the OL group, especially late after glucose loading. Molar ratios of proinsulin to insulin or C-peptide thus were significantly higher in the OL and NIDDM groups. CONCLUSIONS Alteration of pancreatic β-cell function independent of that seen with NIDDM occurred in relation to aging. This may be a predisposing factor to the development of impaired glucose tolerance or NIDDM in elderly subjects, that is, independent of obesity.

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.

The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO2, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ⪢ l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K+, Ca2+) across membrane systems and, hence, regulates the net uptake of Ca2+ and subsequent release of insulin. The effect of l-lactate upon Ca2+ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.