C. J. Hedeskov

Fat-induced changes in mouse pancreatic islet insulin secretion, insulin biosynthesis and glucose metabolism

Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with α-ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice. This was associated with a reduction of approximately 50% in islet glucose oxidation. Islet insulin secretion stimulated by the non-carbohydrate secretagogues tended to be higher in the fat-fed mice, but a statistically significant effect was not observed. Pancreatic insulin content was reduced by 50%, whereas the islet insulin and DNA content was unchanged after fat feeding. Proinsulin mRNA was reduced by 35% in islets from fat-fed mice, and was associated with a reduction of approximately 50% in glucose-stimulated (pro)insulin biosynthesis. It is concluded that the insulin secretory response of islets isolated from fat-fed mice is similar to the secretory pattern known from human type 2, non-insulin-dependent diabetics, and that a defect in islet glucose recognition, resulting in decreased glucose oxidation, may be responsible for the observed insulin secretory and biosynthetic defects seen after glucose stimulation.

The effect of mannoheptulose on glucose- and pyruvate-stimulated oxygen uptake in normal mouse pancreatic islets

1. 1. Cartesian divers of the ampulla type were used in measurements of the oxygen uptake of individual islets of Langerhans separated from pancreas of normal mice by a collagenase method. 2. 2. Incubated in a phosphate buffer supplemented with glucose the islets respired at a constant rate for several hours. The endogenous respiratory rate was constant for about 1.5 but then declined rather rapidly. In the first hour it amounted to 14.2·10−4 μl/h per islet or 94 μmoles/h per g dry weight. Mannoheptulose did not inhibit endogenous respiration. 3. 3. 16.7 mM glucose stimulated islet oxygen uptake by 154% and this stimulation was completed abolished by 28.6 mM mannoheptulose. 4. 4. 10 mM pyruvate stimulated islet oxygen uptake by 55% and this stimulation was also abolished by mannoheptulose. The decarboxylation of [I-14C]pyruvate by islets incubated in a bicarbonate buffer was inhibited 71% by mannoheptulose. 5. 5. Raising the extracellular concentration of Ca2+ from 0.9 to 2.5 mM did not affect the oxygen uptake in the presence of high glucose concentrations. 6. 6. The dry weight of islets of the size used in these experiments was 0.67 ± 0.02 μg/islet (mean ± S.E.). DNA constituted 2.4% of the dry weight and it was calculated that one medium-sized islet contains approximately 2200 cells. 7. 7. The results are discussed with reference to the diabetogenic action of mannoheptulose on whole animals and the interaction of mannoheptulose with stimulus-secretion coupling in the β-cells of pancreatic islets.

Time course studies of glucose-induced changes in glucose-6-phosphate and fructose-1, 6-diphosphate content of mouse and rat pancreatic islets

SummaryThe concentrations of glucose 6-phosphate (G6P) and fructose 1–6-diphosphate plus triose-phosphates (FDP + TPs) were measured in isolated islets of Langerhans from mice and rats after a sudden increase in extracellular glucose concentration from 0.5 to 3.4 mg/ml. In mouse islets, the contents of G6P and (FDP + TPs) were both raised after a two minute incubation at the high glucose concentration and remained elevated for at least 30 min. In rat islets, the G6P but not the (FDP + TPs) content was increased after a 5 min exposure to high glucose. After a 30 min incubation, both G6P and (FDP + TPs) contents were higher than at low glucose concentration. The (G6P)/(FDP + TPs) ratio was some tenfold higher in mouse islets than in rat islets. Increasing extracellular glucose concentration was associated with an increase in the (G6P)/(FDP + TPs) ratio. The results are consistent with the increased glycolytic rate in response to a raised extracellular glucose concentration arising primarily from an increase in the rate of phosphorylation of glucose, and with the hypothesis that the insulin secretory response to glucose may be mediated by a metabolite of the sugar.

Long-term fat-feeding-induced insulin resistance in normal NMRI mice: postreceptor changes of liver, muscle and adipose tissue metabolism resembling those of type 2 diabetes.

Postreceptor insulin resistance was studied in liver, muscle and adipose tissue from NMRI mice of both sexes made diabetic by long-term fat-feeding. Intravenous glucose tolerance tests showed a combination of impaired glucose tolerance and increased plasma insulin concentrations consistent with insulin resistance and reduced peripheral and hepatic uptake of glucose. In the morning, the fat-fed mice were normoinsulinaemic and hyperglycaemic. Liver glucokinase activity and glycogen content were reduced whereas lactate dehydrogenase activity was enhanced. Fatty acid synthase activity was decreased but glucose 6-phosphate dehydrogenase and the rate limiting enzyme in fatty acid synthesis, acetyl CoA carboxylase, were both unaffected. In muscle, the proportion of glycogen synthase in the active I-form was decreased. Total glycogen synthase activity was not affected. In isolated adipocytes, basal and insulin-stimulated glucose oxidation, as well as basal and insulin-stimulated lipogenesis from glucose were all severely inhibited, oxidation more so than lipogenesis. It is concluded that insulin resistance and postreceptor metabolic disorders in liver, muscle and adipose tissue from mice made diabetic by long-term fat-feeding are very similar to those demonstrated in human type 2 diabetics and may be studied in more detail and with more ease in this particular animal model.

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumulation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5--1 microgram/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 microgram/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 microgram/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 microgram/ml). Somatostatin (1 microgram/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated. The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.

Pancreatic islet metabolism and redox state during stimulation of insulin secretion with glucose and fructose

SummaryThe mechanism of potentiation of insulin secretion by fructose was investigated. Twenty mM fructose + 3 mM glucose stimulated insulin secretion in a biphasic manner similar to what is found during stimulation with 20 mM glucose, whereas 20 mM fructose alone did not affect secretion. Fructose utilization was measured as formation of tritiated water from 5-3H-fructose. At 27.8 mM fructose the utilization rate was 258 pmol/2 h/10 islets, which is less than the utilization rate of 2.8 mM glucose. 20 mM glucose increased the islet NADH/NAD+ and NADPH/-NADP+ redox ratios as well as islet concentration of ATP and PEP. 20 mM fructose + 3 mM glucose did not affect the concentration of ATP and PEP or the NADH/NAD+ redox ratio. The NADPH/NADP+ ratio was significantly decreased (60%) after 2.5 min incubation with 20 mM fructose + 3 mM glucose. It is concluded that fructose potentiation of insulin secretion is not primarily dependent on fructose metabolism and that any conceivable effect on plasma membrane ion fluxes as caused by a reduction of plasma membrane disulfides, may be caused by mechanisms other than a mere increase in the pyridine nucleotide substrates for the transhydrogenation process.

Polyamine-enhanced casein kinase 11 in mouse pancreatic islets

SummaryThe occurrence of polyamine-stimulated protein kinase (casein kinase II) in cytosol of mouse pancreatic islets was investigated. Islet protein phosphorylation was enhanced by spermidine, spermine, lysine-rich histone and polylysine; the major endogenous substrates in the cytosol were three proteins of Mr 50000, 55000 and 100000. Cadaverine and putrescine were without effects. A Mr 100 000 protein is a major substrate for Ca2+-calmodulin-dependent protein kinase, and Mr 50 000 and 55 000 proteins are substrates for cyclic adenosine 3′,5′-cyclic monophosphate (AMP) dependent protein kinase in mouse islets. However, neither cyclic-AMP-dependent protein kinase inhibitor nor trifluoperazine inhibited polyamine-enhanced protein phosphorylation. Both basal and polyamine-enhanced protein phosphorylation patterns were identical when either [γ-32P] adenosine 5′-triphosphate (ATP) or [γ-32P] guanosine 5′-triphosphate (GTP) was used as phosphate donors, indicative of the presence of a polyaminestimulated casein kinase 11 in pancreatic islets. It is suggested that polyamines and polyamine-enhanced casein kinase II activity may have an important role in regulation of protein phosphorylation in pancreatic islets.

Phosphoenol pyruvate carboxykinase in mouse pancreatic islets ATP-induced changes in sensitivity to Mn2+ activation

The presence of high phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity in mouse islet cytosol has been demonstrated. The enzyme was activated by Mn2+ with a Ka of 100 X 10(-6) mol/l. The mean total activity of the Mn2+-stimulated phosphoenolpyruvate carboxykinase in islet cytosol estimated at 22 degrees C with saturating concentrations of the substrates oxaloacetate and ITP was 146 pmol/min per micrograms DNA. Km was calculated to be 6 X 10(-6) mol/l for oxaloacetate and 140 X 10(-6) mol/l for ITP. The islet phosphoenolpyruvate carboxykinase activity was not increased after starvation of the animals for 48 h. Preincubation of the cytosol at 4 degrees C with Fe2+, quinolinate, ATP, Pi, glucose 6-phosphate, fructose 1,6-bisphosphate, NAD+, NADH, oxaloacetate, ITP, cyclic AMP and Ca2+ had no effect on the enzyme activity. However, preincubation of the cytosol at 37 degrees C with ATP-Mg inhibited the Mn2+-stimulated phosphoenolpyruvate carboxykinase activity progressively with time and in a concentration-dependent manner. A similar but weaker inhibitory effect was observed with p[NH]ppA, whereas p[CH2]ppA, ADP, AMP, adenosine and Pi had no effect. It is tentatively suggested that ATP and p[NH]ppA either by adenylation or otherwise affect the interaction between islet phosphoenolpyruvate carboxykinase and the recently discovered Mr = 29000 protein modulator of the enzyme in such a way - perhaps by causing a dissociation between them - that phosphoenolpyruvate carboxykinase loses its sensitivity to Mn2+ activation.

Effect of diacylglycerol lipase inhibitor RHC 80267 on pancreatic mouse islet metabolism and insulin secretion

SummaryThe effect of interference with diacylglycerol metabolism was investigated in pancreatic mouse islets. In the presence of the diacylglycerol lipase inhibitor RHC 80267, glucose-induced insulin secretion was reduced 50–60%; whereas carbacholin-induced insulin secretion was unaffected. Addition of the diacylglycerol kinase inhibitor R 59022 did not change glucose-stimulated insulin secretion but abolished the inhibition seen in the presence of RHC 80267. RHC 80267 increased islet glucose utilisation, measured as formation of tritiated water from 5-[3H]-glucose, 3-fold but did not affect glucose oxidation to CO2, lactate production or islet ATP levels. Glucose utilisation in leucocytes and hepatocytes was not increased by addition of RHC 80267. Islet lipid production from glucose was augmented 4-fold in the presence of RHC 80267 but only accounted for about 5% of the increase in glucose utilisation. The activity of adenylate cyclase and phosphoinositide-specific phospholipase C was unaffected by RHC 80267. Concentrations of RHC 80267 below 35 μmol/l did not alter the activity of phospholipase A2; whereas higher concentrations of the drug inhibited phospholipase A2 activity approx 25%. The data support the hypothesis that production of arachidonic acid from diacylglycerol may be involved in regulation of insulin secretion.

Characteristics of an adenylate cyclase enhancing factor from mouse pancreatic islet cytosol

SummaryThe role of cytosolic components in the regulation of mouse pancreatic islet adenylate cyclase activity was studied. Addition of mouse islet cytosol (27000 g supernatant of mouse islet sonicate), devoid of adenylate cyclase activity itself, increased adenylate cyclase activity by 93±17% (n = 9) in the 27000 g total particulate fraction of mouse islets. Addition of GTP stimulated adenylate cyclase activity by 91±11% (n = 13) or to the same degree as cytosol. Like GTP, the substance causing the enhancing activity of the cytosol was found to be dialysable, resistant to heat, sensitive to charcoal treatment and alkaline phosphatase and insensitive to digestion with trypsin. However, in contrast to the stimulation by GTP, the stimulation by cytosol was not inhibited by guanosine 5′-0-(2-thiodiphosphate), and furthermore, the effects of cytosol and GTP were additive. Neither NAD nor phosphoenolpyruvate stimulated adenylate cyclase activity. The cytosolic factor did not confer sensitivity towards glucose, Ca2+ or Ca2+-calmodulin on adenylate cyclase. The results demonstrate that mouse pancreatic islets contain a phosphocompound (or several compounds) distinct from GTP and capable of markedly stimulating adenylate cyclase. The identity of the compound and its physiological significance remain to be established.