Impaired FAD-glycerophosphate dehydrogenase activity in islet and liver homogenates of fa/fa rats

The mitochondrial FAD-linked enzyme glycerophosphate dehydrogenase plays a key role in the pancreatic B-cell glucose sensing device. In the present study, the activity of this enzyme was examined in islets of fa/fa rats in which inherited diabetes mellitus is associated with obesity, hyperinsulinism and severe insulin resistance. The specific activity of both FAD-linked glycerophosphate dehydrogenase and glutamate dehydrogenase were decreased in islet and liver homogenates prepared from fa/fa, as compared to Fa/Fa, rats, this coinciding with a low ratio between glutamateoxalacetate and glutamate-pyruvate transaminase activity in both islet and liver extracts, islet hyperplasia, hyperinsulinemia and hepatic steatosis in the hyperglycemic fa/fa rats. It is speculated that a low activity of FAD-linked glycerophosphate dehydrogenase in the pancreatic B-cell may participate to the perturbation of glucose homeostasis in fa/fa rats, like in other animal models of non-insulin-dependent diabetes mellitus.     

Metabolic and secretory effects of methylamine in pancreatic islets

The metabolic and secretory effects of methylamine in rat pancreatic islets were investigated. Methylamine accumulated in islet cells, was incorporated into endogenous islet proteins, and inhibited the incorporation of [2,5-³H] histamine into either N,N-dimethylcasein or endogenous islet proteins. Methylamine (2 mM ) did not affect the oxidation of glucose or endogenous nutrients or the intracellular pH in islet cells. Glucose did not affect the activity of transglutaminase in islet homogenates, the uptake of ¹⁴C-methylamine by intact islets or its incorporation into endogenous islet proteins. Methylamine inhibited insulin release evoked by glucose, other nutrient secretagogues, and non-nutrient insulinotropic agents such as L -arginine or gliclazide. The inhibitory effect of methylamine upon insulin release was diminished in the presence of cytochalasin B or at low extracellular pH. Methylamine retarded the conversion of proinsulin to insulin. Trimethylamine (0.7 mM ) was more efficiently taken up by islet cells than methylamine (2.0 mM ), and yet caused only a modest inhibition of insulin release. These findings suggest that methylamine interferes with a late step in the secretory sequence, possibly by inhibiting the access of secretory granules to their exocytotic site.     

Hexose metabolism in pancreatic islets

Summary In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-¹⁴C]glucose than utilization of D-[5-³H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-¹⁴C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-¹⁴C]glucose oxidation, was impaired in the absence of extracellular Ca²⁺, and failed to be affected by NH₄ ⁺. Although the ratio between D-[6-¹⁴C]glucose oxidation and, D-[5-³H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca²⁺, as attributable not solely to stimulation of Ca²⁺ inflow into the islet cells but also to an increase in ATP availability.     

Fasting-induced dissociation of cationic and secretory events in pancreatic islets

In pancreatic islets removed from 48 h-fasted rats, as distinct from fed animals, the release of insulin evoked by D-glucose is more severely impaired than that evoked by 2-ketoisocaproate. This decreased secretory response to D-glucose contrasts with an unimpaired cationic response to the sugar in terms of the glucose-induced decrease in both 86Rb and 45Ca outflow from pre-labelled islets. Likewise, fasting only causes a modest decrease of the secondary rise in 45Ca outflow evoked by D-glucose in islets perifused at normal Ca2+ concentration. The latter decrease appears more marked, however, if the cationic response to glucose is expressed relative to that evoked by 2-ketoisocaproate in islets removed from rats in the same nutritional state. It is concluded that, in the process of nutrient-stimulated insulin release, neither the decrease in K+ conductance (inhibition of 86Rb outflow) nor the sequestration of Ca2+ by intracellular organelles and/or direct inhibition of Ca2+ outward transport (decrease in 45Ca outflow) represent the sole determinant(s) of the subsequent gating of Ca2+ channels (secondary rise in 45Ca efflux).     

Coxsackie B4 virus induces short-term changes in the metabolic functions of mouse pancreatic islets in vitro

Mouse pancreatic islets cultured in vitro were infected with a tissue culture-adapted or a mouse pancreas-adapted strain of Coxsackie B4 (CB4) virus. The effects of the viruses on the islets were assessed by examination of their biochemical functions. It was found that the mouse pancreas-adapted strain of CB4 induced a 'leakage' of insulin from islets incubated at a basal (2 mmol l-1) glucose concentration, both at two and four days following infection. However, at a stimulatory concentration of glucose (20 mmol l-1) the rate of insulin secretion appeared to be normal in these islets. At two days the rate of total protein synthesis in islets infected with mouse pancreas-adapted CB4, incubated at high glucose concentration, was reduced; at four days the degree of inhibition was more severe, the rate at basal glucose concentration falling to half that of the control islets and at the stimulatory glucose concentration to a quarter of the control islets. (Pro)insulin biosynthesis was also inhibited, the rate being reduced to less than half the mean control value in islets infected with mouse pancreas-adapted CB4 virus at 20 mmol l-1 glucose at two days; at four days the rate was greatly reduced at both 2 and 20 mmol l-1 glucose. It is concluded from this study that only certain strains of CB4 virus can infect mouse pancreatic islets in vitro and that infection with strains of virus tropic for the islets leads to an impairment of metabolic functions of the B-cells, and is not necessarily lytic.     

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 © 1998 John Wiley & Sons, Ltd.     

Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is associated with altered pancreatic islets redox status

In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H₂O₂), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H₂O₂ and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H₂O₂ production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H₂O₂ (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine.     

Effect of dehydroepiandrosterone in hereditarily diabetic rats

Goto-Kakizaki rats (GK rats) were given access for 4 weeks to a diet enriched with dehydroepiandrosterone (DHEA, 0·2 per cent, w/w). The incorporation of DHEA in the food failed to affect significantly body growth, plasma D -glucose and insulin concentrations, pancreatic islet insulin content or the activity of both mitochondrial glycerophosphate dehydrogenase (mGDH) and NADP-malate dehydrogenase (malic enzme) in islet homogenates. DHEA however, increased the activity of mGDH and, at least in male rates, that of the malic enzyme also in the liver. It lowered the abnormally high basal insulin release otherwise found in the islets from diabetic rats, and, as judged from the ratio of insulin output at 16·7 mM /2·8 mM D -glucose, improved the cell responsiveness to the hexose. This coincided with a decreased plasma insulin/D -glucose ratio, suggesting that the major effect of DHEA was to increase the sensitivity to insulin of extrapancreatic targets, thus resulting in a secondary improvement of cell secretory behaviour. © 1997 John Wiley & Sons, Ltd.     

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: Enzymatic,metabolic and cationic determinants

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.     

Stimulation of insulin release from pancreatic islets by quinones

Coenzyme Q (CoQ₀) and other quinones were shown to be potent insulin secretagogues in the isolated pancreatic islet. The order of potency was CoQ₀benzoquinonehydroquinonemenadione. CoQ₆ and CoQ₁₀ (ubiquinone), duroquinone and durohydroquinone did not stimulate insulin release. CoQ₀'s insulinotropism was enhanced in calcium-free medium and CoQ₀ appeared to stimulate only the second phase of insulin release. CoQ₀ inhibited inositol mono-, bis- and trisphosphate formation. Inhibitors of mitochondrial respiration (rotenone, antimycin A, FCCP and cyanide) and the calcium channel blocker verapamil, did not inhibit CoQ₀-induced insulin release. Dicumarol, an inhibitor of quinone reductase, did not inhibit CoQ₀-induced insulin release, but it did inhibit glucose-induced insulin release suggesting that the enzyme and quinones play a role in glucose-induced insulin release. Quinones may stimulate insulin release by mimicking physiologically-occuring quinones, such as CoQ₁₀, by acting on the plasma membrane or in the cytosol. Exogenous quinones may bypass the quinone reductase reaction, as well as many reactions important for exocytosis.     

Effect of streptozotocin and nicotinamide upon FAD-glycerophosphate dehydrogenase activity and insulin release in purified pancreatic B-cells

Purified rat pancreatic insulin-producing B-cells, which display a 12-fold higher activity of FAD-linked glycerophosphate dehydrogenase than other islet endocrine cells, were exposed for 30 min to 2 mM streptozotocin and subsequently cultured for 2 days in the absence or presence of 2 mM nicotinamide. Streptozotocin decreased by 54% the number of B-cells and, in surviving cells, lowered by 75% the activity of FAD-linked glycerophosphate dehydrogenase, whilst failing to affect that of glutamate dehydrogenase. This coincided with a 42–51% reduction of insulin secretion, when expressed relative to either the DNA or hormonal content of surviving cells. After exposure to streptozotocin, the presence of nicotinamide in the culture medium reduced cell death by 44% and also reduced the deleterious effects of streptozotocin upon both the enzymic and secretory activities of surviving cells. These findings indicate that the decreased activity of FAD-linked glycerophosphate dehydrogenase previously documented in pancreatic islets from streptozotocin-injected rats, as well as the protective effect of nicotinamide thereupon, are not attributable solely to changes in the number of B-cells but also to an altered enzymic activity in surviving B-cells. The latter anomaly may account, in part at least, for an impaired B-cell secretory response to D-glucose. (Mol Cell Biochem120: 135–140, 1993)     

Hexose metabolism in pancreatic islets: Succinate dehydrogenase activity in islet homogenates

Succinate dehydrogenase activity was measured in rat pancreatic islet homogenates incubated in the presence of [1,4-¹⁴C]succinate, the reaction velocity being judged through the generation of ¹⁴CO₂ in the auxiliary reactions catalysed by pig heart fumarase and chicken liver NADP-malate dehydrogenase. In the presence of 1·0 mM succinate, the reaction velocity averaged 5·53 ± 0·44 pmol min^?1 μg^?1 islet protein. The K_m for succinate was close to 0·4 mM and the enzymic activity was restricted to mitochondria. These kinetic results indicate that, under the present experimental conditions, the activity of succinate dehydrogenase does not vastly exceed that of either NAD-isocitrate dehydrogenase or the 2-ketoglutarate dehydrogenase complex, at least when the latter enzymes are activated by ADP and/or Ca²⁺. Nevertheless, the activity of succinate dehydrogenase is sufficient to account for the increase in O₂ uptake evoked in intact islets by the monomethyl ester of succinic acid. It could become a rate-limiting step of the Krebs cycle in models of B-cell dysfunction.     

Hexose metabolism in pancreatic islets: The glucose-6-phosphatase riddle

Summary Glucose-6-phosphatase activity was measured in rat liver or pancreatic islet crude homogenates and microsomes. The data recorded in the liver were comparable to those reported in prior studies. However, in the islets, the hydrolysis of D-glucose 6-phosphate by disrupted microsomes represented, when expressed relative to the protein content, less than 2% of the value recorded in liver microsomes. Moreover, no phosphotransferase activity was detected in the islets. These findings impose reservation on both the presence of glucose-6-phosphatase in rat islets and its participation to stimulus-secretion coupling.     

Cell swelling induced secretion of TRH by posterior pituitary,hypothalamic paraventricular nucleus and pancreatic islets: effect of L-canavanine

The aims of this study were to test if ethanol induces thyrotropin-releasing hormone (TRH) secretion in vitro from the posterior pituitary and hypothalamic explants by a mechanism involving cell swelling, and to characterize the pathway of stimulated secretion. Ethanol, at a concentration of 80 mM, stimulated the release of TRH from the posterior pituitary, the hypothalamic paraventricular nucleus, the median eminence, and the brain septum, when administered only in isosmolar but not in hyperosmolar medium. This indicates the involvement of a cell swelling-inducing mechanism. L-canavanine in a concentration of 3 mM, increased the basal and hyposmosis-induced TRH secretion from the posterior pituitary and the paraventricular nucleus, and both basal and ethanol-induced TRH secretion from isolated pancreatic islets. This indicates the presence of both constitutive and regulatory secretory pathways. Our results suggest that cell swelling induces exocytosis from clathrin coated granules.     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

Leptin suppresses basal insulin secretion from rat pancreatic islets

The effects of leptin on insulin secretion from pancreatic islets of Sprague–Dawley rats were examined in vitro. In a basal glucose medium (5.5 mM), insulin secretion from isolated islets was significantly decreased after addition of a recombinant leptin (80 nM) (3.20±0.14 nmol/10 islets/h) compared with that before the addition (4.41±0.30 nmol/10 islets/h). Although significant leptin suppression of insulin secretion was not observed under a glucose-stimulated (11.1 mM) condition, these results suggest that a negative feedback system may exist between leptin and insulin, which increases the production of leptin from adipose tissues.     

In vitro reconstitution of pancreatic islets

The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from β cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.     

In vitro reconstitution of pancreatic islets

The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from β cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.     

L-glutamine and palmitate catabolism in pancreatic islets from rats depleted in long-chain polyunsaturated omega 3 fatty acids

The catabolism of D-glucose was recently found to be impaired in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids. The specificity of this alteration was now investigated by characterizing the oxidative fate of endogenous nutrients in islets preincubated with either L-[U-14C]glutamine or [U-14C]palmitate and then incubated variously in the absence of D-glucose, presence of the hexose or presence of metabolic poisons. Relative to their radioactive content after preincubation, the production of 14CO2 by islets prelabelled with [U-14C]glutamine was higher in omega3-depleted rats than control animals. The enhancing action of D-glucose upon such production was impaired, however, in the omega3-depleted rats. The net uptake of 14C-palmitate and absolute value for 14CO2 output were both increased in omega3-depleted rats, whilst the ratio between 14CO2 output and islet radioactive content was decreased in the same animals. The inhibition of 14CO2 production by metabolic poisons was comparable in all cases. These results are consistent with recent findings on such items as the availability of endogenous amino acids and uptake of unesterified fatty acids in extrapancreatic sites of the omega3-depleted rats. They also support the view that the alteration of D-glucose metabolism in the islets of the latter animals may be attributable, in part at least, to alteration of glucokinase kinetics by high intracellular acyl-CoA levels.