Kirsten Capito

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells.

Insulin secretion is controlled by the β cell′s metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P2-binding protein Ca2+-dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca2+- and PI(4,5)P2-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P2, represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules.

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.

Pancreatic islet insulin secretion and metabolism in adult rats malnourished during neonatal life.

Pancreatic islets were isolated from rats that had been nursed by dams fed with a control or an 8.7% protein diet during the first 12 d of the lactation period. Glucose-induced insulin secretion from islets in the 8.7% protein group was reduced 50%. The islet insulin and DNA content were similar, whereas the pancreatic insulin content was reduced by 30 % in the rats fed 8.7 % protein. In order to elucidate the mechanism responsible for the attenuation of insulin secretion, measurements were performed of the activity of several islet enzymes that had previously been supposed to be involved in the coupling of glucose stimulation to insulin secretion. Islet glucose oxidation was unaffected, but glucose-stimulated hydrolysis of phosphatidylinositol was reduced by one-third in the islets of rats fed 8.7% protein. The activity of mitochondrial glycerophosphate dehydrogenase was similar in islets of rats fed the 8.7% protein diet and those fed the control diet. The activity of Ca-independent phospholipase A2 was increased fourfold in the islets of rats fed 8.7% protein. It is concluded that impairment of glucose-induced insulin secretion in rats fed a low-protein diet may be caused by attenuation of islet phosphatidylinositol hydrolysis, and it is tentatively suggested that the increased activity of Ca-independent phospholipase A2 in islets of rats fed a low-protein diet may participate in the stimulation of apoptosis.

Differential mechanisms of glucose and palmitate in augmentation of insulin secretion in mouse pancreatic islets

Aims/hypothesis. To assess the possible importance of saturated fatty acids in glucose amplification of K+ATP channel-independent insulin secretion. Methods. Insulin release from perifused pancreatic islets of NMRI mice was determined by radioimmunoassay. Results. In the presence of K+ (20 mmol/l) and diazoxide (250 μmol/l), which stimulates Ca2+ influx and opens K+ATP channels, palmitate (165 μmol/l total; 1.2 μmol/l free) increased insulin secretion at 3.3, 10 and 16.7 mmol/l glucose while glucose (10; 16.7 mmol/l) did not increase insulin secretion. In the presence of K+ (60 mmol/l) and diazoxide (250 μmol/l), glucose (10; 16.7 mmol/l) stimulation of K+ATP channel-independent insulin secretion increased, whereas the effectiveness of palmitate (165 μmol/l total; 1.2 μmol/l free) on insulin secretion at both 3.3, 10 or 16.7 mmol/l glucose was reduced. Palmitate thereby mimicked the stimulatory pattern of the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l), which also failed to increase insulin secretion at maximum depolarising concentrations of K+ (60 mmol/l). Furthermore, the protein kinase C inhibitor calphostin C (1 μmol/l), led to a complete suppression of the effects of both palmitate (165 μmol/l total; 1.2 μmol/l free) and myristate (165 μmol/l total; 2.4 μmol/l free) stimulation of glucose (16.7 mmol/l)-induced insulin secretion. Calphostin C (1 μmol/l), however, failed to affect insulin secretion induced by glucose (16.7 mmol/l). Conclusion/interpretation. These data suggest that glucose could increase insulin secretion independently of saturated fatty acids like palmitate and myristate, which amplify glucose-induced insulin secretion by activation of protein kinase C. [Diabetologia (2001) 44: 738–746]

Inhibition of glucose-induced insulin secretion by the diacylglycerol lipase inhibitor RHC 80267 and the phospholipase A2 inhibitor ACA through stimulation of K+ permeability without diminution by exogenous arachidonic acid

The effects of the diacylglycerol lipase inhibitor 1,6-bis-(cyclohexyloximinocarbonyl-amino)-hexane (RHC 80267) and the phospholipase A2 inhibitor N-(p-amylcinnamoyl)anthranilic acid (ACA) on insulin secretion and 86Rb+ efflux in mouse pancreatic islets were studied. RHC 80267 (35 microM) and ACA (100 microM) inhibited glucose (16.7 mM)-induced insulin secretion, but did not inhibit insulin secretion induced by K+ (40 mM) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM). K+ (40 mM) or TPA (0.16 microM) potentiated glucose (16.7 mM)-induced insulin secretion, and prevented inhibition of glucose (16.7 mM)-induced insulin secretion by RHC 80267 and ACA. In comparison, potentiation of glucose-induced insulin secretion by albumin-bound arachidonic acid (AA; 200 microM total; 10 microM free unbound) failed to counteract inhibition of glucose-induced insulin secretion by RHC 80267 or ACA, suggesting that inhibition of insulin secretion by these agents was not mediated by a decrease in AA accumulation in islets. Determination of 86Rb+ efflux, a marker of K+ channel activity, revealed that both RHC 80267 and ACA stimulated K+ efflux from islets. These effects of RHC 80267 and ACA were observed at both 3.3 and 16.7 mM glucose and persisted in Ca2+-free medium, suggesting that they may represent an opening of ATP-sensitive K+ channels. RHC 80267-mediated stimulation of 86Rb+ efflux was not mimicked by the diacylglycerol analog TPA (0.16 microM) and was not prevented by the diacylglycerol kinase inhibitor R 59022 (50 microM), suggesting that stimulation of 86Rb+ efflux did not reflect a conditional increase in diacylglycerol or in phosphatidic acid upon inhibition of diacylglycerol lipase. In contrast, TPA (0.16 microM) attenuated RHC 80267 and ACA stimulation of 86Rb+ efflux. Addition of AA (200 microM total; 10 microM free unbound) stimulated 86Rb+ efflux, suggesting that stimulation of 86Rb+ efflux by RHC 80267 and ACA was not due to a decrease in AA accumulation. This stimulation by AA was not dependent on AA metabolism because it persisted in the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 microM) or the cyclooxygenase inhibitor indomethacin (50 microM). In contrast to RHC 80267 and ACA, AA stimulation of 86Rb+ efflux was attenuated in Ca2+-free medium, probably implicating Ca2+-sensitive K+ channels in AA regulation of 86Rb+ efflux. Parallel experiments with diazoxide (100 microM) revealed that RHC 80267 and ACA mimicked the effects of diazoxide, a specific activator of ATP-sensitive K+ channels in islets, on both insulin secretion and 86Rb+ efflux. In conclusion, it is suggested that RHC 80267 and ACA, independently of their action on AA release, may inhibit glucose-induced insulin secretion by the opening of ATP-sensitive K+ channels in islets.

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.

In vitro and in vivo synthesis of long-chain fatty acids from [1-14C]acetate in the renal papillae of rats

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.

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.