Sphingosine kinase 1 knockdown reduces insulin synthesis and secretion in a rat insulinoma cell line.

To evaluate the role of sphingosine kinase 1 (SphK1) in insulin secretion, we used stable transfection to knock down the expression of the Sphk1 gene in the rat insulinoma INS-1 832/13 cell line. Cell lines with lowered Sphk1 mRNA expression and SphK1 enzyme activity (SK11 and SK14) exhibited lowered glucose- and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine-stimulated insulin release and low insulin content associated with decreases in the mRNA of the insulin 1 gene. Overexpression of the rat or human Sphk1 cDNA restored insulin secretion and total insulin content in the SK11 cell line, but not in the SK14 cell line. The Sphk1 cDNA-transfected SK14 cell line expressed significantly less SphK1 activity than the Sphk1 cDNA-transfected SK11 cells suggesting that the shRNA targeting SK14 was more effective in silencing the exogenous rat Sphk1 mRNA. The results indicate that SphK1 activity is important for insulin synthesis and secretion.

ATP-independent glucose stimulation of sphingosine kinase in rat pancreatic islets.

Sphingosine kinase (SPHK) catalyzes sphingosine 1-phosphate production, promoting cell survival and reducing apoptosis in isolated rat pancreatic islets. Glucose, the primary islet beta-cell growth factor and insulin secretagogue, increased islet SPHK activity by 3- to 5-fold following acute (1 h) or prolonged (7 days) stimulation. Prolonged stimulation of islets with glucose induced SPHK1a and SPHK2 mRNA levels; there were no changes in SPHK protein expression. To isolate the metabolic effects of glucose on SPHK activation, islets were stimulated with glucose analogs or metabolites. 2-deoxy-D-glucose (2-DG), an analog phosphorylated by glucokinase but not an effective energy source, activated SPHK similarly to glucose. In contrast, 3-o-methylglucose (3-oMeG), which is transported but neither phosphorylated nor metabolized, did not increase islet SPHK activity. Glyceraldehyde and alpha-ketoisocaproic acid (KIC), metabolites that stimulate glycolysis and the citric acid cycle, respectively, did not activate islet SPHK. Moreover, inorganic phosphate blocked glucose-induced SPHK activation. A role for SPHK activity in beta-cell growth was confirmed when small interfering (si)SPHK2 RNA transfection reduced rat insulinoma INS-1e cell SPHK levels and activity and cell growth. Glucose induced an early and sustained increase in islet SPHK activity that was dependent on glucose phosphorylation, but independent of ATP generation or new protein biosynthesis. Glucose-supported beta-cell growth appears to be in part mediated by SPHK activity.

Ultrastructural and biochemical evidence for a steroid-containing secretory organelle in the perfused cat adrenal gland.

A correlative study of the ultrastructural and biochemical effects of ACTH on fasciculata cells was carried out on the isolated cat adrenal gland perfused in situ with Locke's solution. The outstanding morphologic feature of cortical cells exposed to microunit ACTH concentrations for 40 min was the abundance of electron-dense granules (0.2-0.4 mum). These organelles were observed in small groups in close proximity to the Golgi region and to the cell membrane. Morphometric and biochemical analysis of control and ACTH-treated glands demonstrated that ACTH stimulation was associated with a fourfold increase in the number of these granules and a comparable increase in the corticosteroid content of the gland. By contrast, ACTH failed to augment cortical lysosomal enzyme activity. These findings, which link steroid release to the appearance of intracellular granules, extend further the parallels between the mechanism of release of newly synthesized steroid and the release of preformed hormones stored in secretory organelles. These results also lend support to the concept that a process related to exocytosis may be the underlying mechanism for extruding steroid from the cortical cell.

Metabolic programming: Role of nutrition in the immediate postnatal life.

Although genes and dietary habits are generally implicated in the aetiology of the prevailing obesity epidemic, the steep increase in the incidence of obesity within a relatively short span of time suggests that other contributing factors may be at play. The role of nutritional experience during the very early periods of life is increasingly being recognized as contributing to growth and metabolic changes in later life. Epidemiological data and studies from animal models have established a strong correlation between an aberrant intrauterine environment and adult-onset disorders in offspring. The nutritional experience in the immediate postnatal life is another independent factor contributing to the development of metabolic diseases in adulthood. Although studies on the small-litter rat model have shown that overnourishment during the suckling period results in adult-onset metabolic disorders, our studies have shown that a change in the quality of calories-specifically, increased carbohydrate intake by newborn rat pups in the immediate postnatal period-results in chronic hyperinsulinaemia and adult-onset obesity. Several functional alterations in islets and in the hypothalamic energy homeostatic mechanism appear to support this phenotype. Remarkably, female rats that underwent the high-carbohydrate dietary modification as neonates spontaneously transmitted the obesity phenotype to their offspring, thus establishing a vicious generational effect. The high-carbohydrate diet-fed rat model has particular relevance in the context of the current human infant feeding practices: reduction in breast feeding and increase in formula feeding for infants, accompanied by early introduction of carbohydrate-enriched baby foods.

The effect of corticotropin on phospholipid metabolism in isolated adrenocortical cells.

Trypsin-dispersed cat adrenocortical cells were incubated at 37 degrees C in modified Eagle's medium containing [14C]arachidonic acid of sodium [14C]-acetate and then in non-radioactive medium. Radioactive incorporation was obtained in all phospholipids, with the greatest amount of radioactivity in phosphatidylcholine, followed by phosphatidylethanolamine, phosphatidyl-serine, and phosphatidylinositol. Concentrations of individual phospholipids generally paralleled the relative amounts of corresponding radiolabeled phospholipids, although the percentage of phosphatidylinositol was considerably lower than its radioactive counterpart, resulting in a high specific activity of this particular phospholipid. Although a potently steroidogenic concentration of corticotropin failed to enhance release of label from any particular phospholipid, analysis of specific activity showed that corticotropin stimulation was accompanied by an increased turnover of phosphatidylinositol and phosphatidic acid. These studies demonstrate that isolated cortical cells have the capacity to synthesize phospholipids from radioactive precursors. The finding that the acute effects of corticotropin are associated with changes in specific phospholipids, including phosphatidylinositol and phosphatidic acid, conforms to the general pattern observed in other secretory systems.

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells.

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.

Characterization of phospholipase A2 and acyltransferase activities in purified zymogen granule membranes.

Phospholipase A2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules. In homogenate and granule membranes, phospholipase activity was linearly related to protein concentration and was Ca2(+)-dependent with an alkaline pH optimum. The Ca2+ sensitivity was observed over the range of concentrations through which intracellular ionic Ca2+ is elevated by physiological stimuli in intact cells. Intact zymogen granules and granule membranes also demonstrated reacylating activity in the presence and absence of an exogenous acceptor. Reacylating activity was related to the concentration of lyosphospholipid added and was optimally activated at alkaline pH. A more rapid rate of reacylation was observed when [14C]arachidonoyl CoA was employed as the donor molecule rather than [3H]arachidonate (plus coenzyme A); this suggests the absence of acyl-CoA synthetase in the purified granule membranes. We conclude that granule membrane phospholipase A2 and acyltransferases may be involved in arachidonic acid turnover in exocrine pancreas and perhaps in membrane fusion events associated with exocytosis.

Fatty acid incorporation into phospholipids of isolated pancreatic islets of the rat. Relationship to insulin release.

Fatty acid incorporation into specific phospholipids of isolated islets of the rat was investigated using unsaturated [14C]arachidonic acid. Glucose (25 mM) stimulated the incorporation of arachidonic acid into phosphatidylinositol (PI) and phosphatidylcholine (PC) in a time-related manner correlated with two phases of insulin release. Arachidonate incorporation was inhibited by calcium deprivation. The sulfonylurea tolbutamide stimulated an early monophasic release of insulin that was accompanied by increased [14C]arachidonate incorporation into PI and PC. The cholinergic agonist and insulin secretagogue, carbamylcholine, also promoted the incorporation of [14C]arachidonate into PI/phosphatidylserine (PS) and PC fractions. 2-Deoxy-D-glucose, which does not support insulin release, did not enhance arachidonate incorporation into phospholipids. However, phenylephrine, an inhibitor of glucose-induced insulin secretion, stimulated arachidonate turnover in PI. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, markedly depressed both glucose-stimulated arachidonate incorporation into phospholipids and insulin release. The stimulated release of arachidonate from endogenous radiolabeled phospholipids provided additional evidence that phospholipase A2 mediates glucose stimulation. However, since glucose also promoted the incorporation of saturated [14C]palmitic acid into PE (phosphatidylethanolamine) and PI/PS fractions, a phospholipase A1 may also mediate the glucose response. Thus, fatty acid incorporation into islet phospholipids mediates the effects of various secretagogues on insulin release. However, the ability of phenylephrine to stimulate arachidonyl PI turnover suggests that fatty acid turnover is not a sufficient stimulus for release. Augmented levels of unsaturated fatty acids in islet cell membranes may promote fusion or activate enzymes important for hormone release.

Insulin secretion, myo-inositol transport, and Na(+)-K(+)-ATPase in glucose-desensitized rat islets.

Glucose-induced insulin secretion is desensitized during long-term exposure of pancreatic islet beta-cells to elevated glucose levels. This study characterizes an in vitro model of glucose-induced desensitization in cultured isolated islets of the rat. Insulin secretion in desensitized islets cultured with 11 mM glucose for 4-7 days was progressively reduced compared with the normal freshly isolated (fresh) islets. When desensitized islets were returned to a basal concentration of glucose (5.5 mM) for up to 2 h, the glucose sensitivity of insulin secretion was restored to normal (recovered islets). Carbachol and L-arginine also reversed the effects of desensitization. However, basal insulin release was elevated in desensitized and recovered islets. Sodium-dependent myo-inositol uptake was reduced during desensitization by up to 49% within 4 days. myo-Inositol uptake was restored to normal in a time-dependent manner during recovery of islets at 5.5 mM glucose. The recovery of myo-inositol uptake paralleled that of insulin release. The apparent transport constant for myo-inositol uptake was significantly increased during desensitization, whereas the maximum uptake was not changed. myo-Inositol supplementation (35 or 250 microM) during islet culture did not alter myo-inositol uptake or insulin secretion in desensitized islets. Na(+)-K(+)-ATPase activity, but not 5'-nucleotidase activity, in desensitized islets was also inhibited by 65 and 47% when compared with fresh islet and recovered islet Na(+)-K(+)-ATPase activity, respectively. Thus, cultured islets represent an appropriate model to study biochemical parameters associated with the onset and reversibility of glucose desensitization of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C and calcium regulation of adenylyl cyclase in isolated rat pancreatic islets.

Rat islets express several isoforms of adenylyl cyclase (AC), and the regulation of AC activity in isolated islets by Ca(2+) and protein kinase C (PKC) was investigated. At basal 2.8 mmol/l glucose, the muscarinic receptor agonist carbamylcholine chloride (CCh) evoked a concentration-dependent increase in cAMP generation with a maximum increase at least 4.5-fold above control. In contrast, forskolin and glucagon-like peptide 1 fragment 7-36 amide increased cAMP accumulation 23-fold and almost 10-fold, respectively. Cholecystokinin 26-33 sulfated amide (CCK) also stimulated cAMP production by up to eightfold, as did the phorbol ester, phorbol 12,13-dibutyrate (PDBu). PDBu and CCh or CCK responses were not additive. The effects of phorbol ester, CCh, and CCK were inhibited by as much as 75% by the PKC inhibitors GF 109203X and Ro-32-0432 and after PKC downregulation. In the absence of extracellular Ca(2+), PDBu-, CCh-, and CCK-induced cAMP production was inhibited by approximately 50% in each case. Chelation of intracellular Ca(2+) with 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM) inhibited CCh- and CCK-stimulated cAMP generation by approximately 50% but did not inhibit the stimulatory effect of PDBu. Stringent Ca(2+) depletion by removal of extracellular Ca(2+) and inclusion of BAPTA/AM allowed for increased cAMP production in response to CCh and CCK; PKC inhibitors and PKC downregulation prevented this stimulation. Glucose stimulation also increased islet cAMP production, but PDBu did not potentiate the glucose response. The results suggest that Ca(2+) influx, Ca(2+) mobilization, and PKC activation play important roles in the modulation of AC activity in pancreatic islets.

Choline turnover in phosphatidylcholine of pancreatic islets. Implications for CDP-choline pathway.

The CDP-choline pathway is the major route of phosphatidylcholine (PC) biosynthesis in mammalian cells. The incorporation of [14C]choline into PC of isolated pancreatic islets of the rat was time dependent, glucose stimulable, and inhibited by mannoheptulose. Removal of extracellular Ca2+ enhanced glucose-stimulated choline incorporation without affecting basal levels. Glucose stimulated PC synthesis in islets labeled to equilibrium with 32PO4 in the presence or absence of extracellular Ca2+. The water-soluble intermediates of the CDP-choline pathway, phosphorylcholine and CDP-choline, accumulated to a lesser extent under Ca2+-free conditions; however, glucose enhanced the levels of these intermediates in the presence and absence of Ca2+. Thus, glucose stimulates CDP-choline-pathway activity. Ca2+-free conditions may promote flux of choline intermediates through the pathway and retard the hydrolysis of PC. The phospholipase A2-activating agents delta-9-tetrahydrocannabinol and melittin enhanced [3H]choline incorporation into PC and potentiated incorporation in response to a submaximal secretagogic concentration of glucose (8.5 mM); insulin release paralleled the changes in PC. p-Bromophenacyl bromide and mepacrine reduced islet glucose utilization and glucose-stimulated [3H]choline levels in PC. An inhibitor of CTP: phosphorylcholine cytidylyltransferase, 5'-deoxy-5'-isobutylthioadenosine, reduced glucose-stimulated [14C]choline incorporation into PC; insulin release was inhibited in a parallel fashion. Thus, islet PC turnover and CDP-choline pathway activity appear to be modulated by glucose metabolism and membrane phospholipid hydrolysis. PC turnover and insulin release appear to be related.

Atrial natriuretic peptide and cyclic nucleotides affect glucose-induced Ca2+ responses in single pancreatic islet beta-cells: correlation with (Ca[2+] + Mg2+)-ATPase activity.

Glucose stimulation of pancreatic islets is characterized by an initial decline in intracellular Ca2+ concentration ([Ca2+]i) (phase 0), followed by an increase in peak [Ca2+]i (phase 1). The effect of atrial natriuretic peptide (ANP) and cyclic nucleotides on the glucose-induced phase 0 [Ca2+]i was investigated by Fura-2 fluorescent imaging in single beta-cells from isolated islets of rats maintained at 1.67 mmol/l glucose. ANP (1 nmol/l to 1 micromol/l) inhibited the glucose (8.2 mmol/l)-induced phase 0 [Ca2+], in a concentration-dependent manner. Forskolin, 8-bromo-cyclic AMP (8BrcAMP), and 8-bromo-cyclic guanosine monophosphate (8BrcGMP) also inhibited the glucose-induced phase 0 [Ca2+]i. The Ca2+ channel blocker, D 600, prevented the response to 8BrcAMP but not to ANP or 8BrcGMP on phase 0 [Ca2+]i. Thapsigargin (TG) also inhibited phase 0 [Ca2+]i by 90%. ANP, 8BrcGMP, and TG also reduced the time required for glucose to initiate the phase 1 increase in [Ca2+]i, and each of these agents potentiated the effect of glucose on peak [Ca2+]i. Furthermore, sarco(endo)-plasmic reticulum (Ca[2+] + Mg2+)-ATPase (SERCA) activity in RINm5F insulinoma cells was inhibited by 8BrcGMP and TG, but not 8BrcAMP. Thus, ANP and cGMP modulate [Ca2+]i regulation in pancreatic beta-cells perhaps through mechanisms involving changes in SERCA activity and Ca2+ influx.

Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose.

Phospholipase A2 activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from E. coli membranes. Phospholipase A2 activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A2 activity which was assayable in islet homogenates. Glucose stimulated phospholipase A2 in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A2 activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A2 activity measured in the presence of high calcium concentrations. Phospholipase A2 activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A2 activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.

Effects of hydroxyeicosatetraenoic acids on fatty acid esterification in phospholipids and insulin secretion in pancreatic islets.

The ability of lipoxygenase products to become incorporated into islet cell phospholipids and to affect fatty acid mobilization was investigated. Isolated intact islets or homogenized islets were incubated with tritiated 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, the leukotrienes C4 and D4, or prostaglandin E2. Tritiated 5-HETE and 12-HETE were largely esterified into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of intact islet membranes. Glucose stimulation increased [3H]5-HETE esterification to islet PC and PE. In islet homogenates, tritiated 5-HETE, 12-HETE, 15-HETE and arachidonic acid (AA) were also esterified into endogenous PC and PE, with less incorporation in phosphatidylinositol (PI) or phosphatidylserine. Addition of exogenous lysophospholipid acceptors potentiated the esterification of [3H]5-HETE to PC especially; [3H]AA was uniformly esterified to exogenous lysophospholipids. In addition, unlabeled 5-HETE (40 nM to 8 microM) affected the incorporation of [3H]AA into PC and PE of homogenates in a biphasic manner, whereas unlabeled AA inhibited [3H]AA incorporation into phospholipids in a concentration-dependent manner. Glucose (8.5 mM) stimulated the loss of labeled AA from prelabeled islet PC and PI. On the other hand, 5-HETE (40 nM) increased AA recovery in PC, PI, and PE of prelabeled islets, and HETE antagonized the glucose-stimulated release of AA from PC and PE. A 100-fold higher concentration of 5-HETE increased the glucose-stimulated loss of AA from phospholipids in prelabeled islets. Nanomolar concentrations of 5-HETE elicited a rapid and transient increase in insulin release, which was additive to the release response to a submaximal stimulatory concentration of glucose, whereas micromolar 5-HETE did not affect insulin release. Thus, pancreatic islets not only esterify HETE into phospholipids, but HETE also alters the turnover of AA in membrane phospholipids. HETE-induced changes in islet membrane fatty acid composition and/or AA mobilization may modulate stimulus-secretion coupling.

Effects of guanosine 3',5'-monophosphate on glucose utilization in isolated islets of Langerhans.

Dynamic changes in total glucose utilization in isolated islets of Langerhans of the rat were determined by quantitation of the formation of 3H2O from D-[5-3H]glucose. The addition of 8-bromo-cGMP (8-Br-cGMP) or monobutyryl cGMP to the islets during a linear phase of glucose utilization resulted in concentration- and time-dependent increases in glucose utilization. Effects of the analogs of cGMP on glucose utilization were noted as early as 5 min after the onset of stimulation in the presence of 10 mM glucose. 8-Br-cGMP also increased the utilization of 1 mM glucose within 20 min. Stimulatory effects of 8-Br-cGMP were observed in the presence of cycloheximide or N-acetylglucosamine. Neither 8-bromo-cAMP (8-Br-cAMP) nor monobutyryl cAMP induced significant changes in glucose utilization at 1 or 10 mM glucose. In the presence of 3-isobutyl-1-methylxanthine (IBMX), 8-Br-cGMP, but not 8-Br-cAMP, induced a rapid change in glucose utilization. N-Methyl-N'-nitro-N-nitrosoguanidine, which activates guanylate cyclase, also stimulated glucose utilization in the presence of IBMX by 3-fold. IBMX alone did not change glucose utilization. In contrast, 8-Br-5'-GMP reduced glucose utilization, whereas 8-bromoinosine 3',5'-monophosphate and 8-bromoguanosine did not change glucose utilization. Sodium bromide did not affect glucose utilization. Glucose-stimulated insulin release was potentiated by 8-Br-cGMP, whereas insulin release from islets incubated in the absence of glucose or the presence of glyceraldehyde or 2-ketoisocaproic acid was not altered by 8-Br-cGMP. Thus, glucose utilization in pancreatic islets is modulated by cGMP, and the secretory response to 8-Br-cGMP is glucose dependent.

Regulation of inositol trisphosphate receptor isoform expression in glucose-desensitized rat pancreatic islets: role of cyclic adenosine 3',5'-monophosphate and calcium.

The regulation of inositol 1,4,5-trisphosphate receptor (IP3R) messenger RNA (mRNA) and protein expression was investigated in glucose-desensitized rat isolated pancreatic islets. Islets were cultured for 4 days with glucose (11 mM; G-treated) to induce desensitization; IP3R-I mRNA levels were similar to basal (5.5 mM glucose) values, whereas IP3R-II mRNA levels were increased and IP3R-III levels were reduced compared with basal levels. Somatostatin increased the expression of IP3R-II mRNA and reduced the expression of IP3R-III mRNA compared with basal values, but did not significantly affect G-treated islet IP3R expression. When forskolin (FSK), 8-bromo-cAMP, and glucagon-like peptide 1-(7-36) amide were added to G-treated islets after 4 days of culture, IP3R-II mRNA levels were reduced, whereas IP3R-III mRNA levels increased, to levels observed in control islets, within 3 h. The levels of IP3R-I mRNA were unaffected by either somatostatin or FSK. The protein kinase A inhibitor. H-89, and actinomycin D prevented the effects of FSK. A Ca2+ ionophore mimicked the effects of FSK on IP3R mRNA expression, whereas blockade of voltage-dependent Ca2+ channels or chelation of intracellular Ca2+ inhibited the actions of FSK. cAMP also increased IP3R-III mRNA in insulinoma cells. In G-treated islets, FSK slowed IP3R-III mRNA degradation. FSK, but not glucose, stimulated protein kinase A activation in G-treated islets. Thus, cAMP mediates changes in IP3R-II and -III mRNA transcription and stability in glucose-desensitized islets. The regulated expression of IP3R-II and -III mRNA is mediated in part by intracellular Ca2+ availability.

Epiandrosterone and dehydroepiandrosterone affect glucose oxidation and interleukin-1 beta effects in pancreatic islets.

Isolated rat islets or RINm5F insulinoma cells treated with interleukin-1 beta (IL-1 beta) for 18 h show reduced glucose-sensitive insulin release and increased nitrite formation as a result of nitric oxide synthase induction. Although a phosphodiesterase inhibitor, isobutylmethylxanthine, potentiated insulin release in response to glucose stimulation, the secretory response was not restored to normal in IL-1 beta-treated islets. Islets that were cultured for 18 h in the presence of IL-1 beta and epiandrosterone (EA) or dehydroepiandrosterone (DHEA) and then washed responded with a concentration-dependent reversal of the effects of IL-1 beta on insulin release in the presence of a glucose or glucose plus isobutylmethylxanthine stimulus. In contrast, when EA and DHEA were not washed from the islets before determination of insulin release, the presence of EA or DHEA inhibited insulin release in both freshly isolated and cultured islets. Nitrite formation in islets and RINm5F cells in response to IL-1 beta was also significantly reduced during culture with EA or DHEA, although nitrite levels were still elevated above control values. Neither steroid affected cell growth or DNA or protein content. Pyrrolidine dithiocarbamate also reduced IL-1 beta-induced nitrite formation. EA and DHEA inhibited [U-14C]glucose oxidation in islets and RINm5F cells. Comparison of [1-14C]glucose and [6-14C]glucose oxidation in islets and RINm5F cells when EA was present during culture and metabolic determination indicated that EA inhibited glycolysis and the pentose shunt contribution to glucose utilization. Neither IL-1 beta in islets nor DHEA in RINm5F cells inhibited pentose shunt activity, although total glucose oxidation and utilization were inhibited. The effects of DHEA and EA on glucose oxidation were rapidly reversible. EA and DHEA reduced glucose-6-phosphate dehydrogenase activity only when added directly to tissue homogenates. Thus, EA and DHEA antagonize the effects of IL-1 beta on beta-cells. Inhibition of glucose metabolism and pentose shunt activity may protect the cells from nitric oxide synthase activation and related toxicities.

A protective role for heme oxygenase expression in pancreatic islets exposed to interleukin-1beta.

Heme oxygenase (HO)-1 expression was investigated in rat isolated pancreatic islets. Freshly isolated islets showed no evidence of HO-1 expression. After a 20-h culture, there was a small increase in HO-1 in control islets, and interleukin-1beta (IL-1beta) induced HO-1 expression above control levels. N(G)-monomethyl-L-arginine inhibited the IL-1beta-induced increase in HO-1. Sodium nitroprusside-generated nitric oxide also increased HO-1 expression. CoCl2 induced a concentration- and time-dependent increase in HO-1, but not heat shock protein 70, expression. Cobalt chloride (CoCl2) protected islets from the inhibitory effects of IL-1beta on glucose-stimulated insulin release and glucose oxidation. Nickel chloride did not mimic the effects of CoCl2. An inhibitor of HO-1 activity, zinc-protoporphyrin IX (ZnPP), prevented the protective effect of CoCl2 on insulin release with IL-1beta but did not affect HO-1 expression or the inhibitory response to IL-1beta alone. ZnPP also inhibited the protective effect of hemin in IL-1beta-treated islets. CoCl2 inhibited the marked increase in islet nitrite production in response to IL-1beta. Cobalt-protoporphyrin IX (CoPP), which increased HO expression and activity, also protected islets from the inhibitory effects of IL-1beta, even though IL-1beta largely blocked the CoPP-induced increase in HO-1 expression. In betaHC9 cells, CoCl2 increased HO-1 expression and HO activity, whereas CoPP directly activated HO. ZnPP inhibited basal and CoCl2-stimulated HO activity. Thus, increased HO-1 expression and/or HO activity in response to CoCl2, CoPP, and hemin, seems to mediate protective responses of pancreatic islets against IL-1beta. HO-1 may be protective of beta-cells because of the scavenging of free heme, the antioxidant effects of the end-product bilirubin, or the generation of carbon monoxide, which might have insulin secretion-promoting effects and inhibitory effects on nitric oxide synthase.

Further studies on the mechanisms controlling prostaglandin biosynthesis in the cat adrenal cortex: the role of calcium and cyclic AMP.

In light of previous studies which have implicated prostaglandin (PG) formation as a link in ACTH-induced steroid production by isolated cat adrenocortical cells, experiments were carried out to provide additional information regarding the role of PGs in adrenal steroidogenesis and their interactions with calcium and cyclic AMP. Perfusion of cat adrenal glands with Locke's solution plus beta(1-24)-ACTH resulted in an immediate increase in PGF2alpha release, which rapidly declined to basal levels after the stimulus was withdrawn. In contrast, maximal rates of steroid release were manifest some 30 min after removal of ACTH. ACTH and its onitrophenyl sulfenyl derivative (NPS-ACTH) increased PG (PGF2alpha and PGE2) and steroid release by trypsin-dispersed cat cortical cells, but NPS-ACTH, unlike ACTH, did not augment cortical cyclic AMP levels. In this same preparation, indomethacin completely blocked ACTH and NPS-ACTH facilitated PGF2alpha and PGE2 release but failed to suppress steroid release markedly. Calcium-deprivation blocked PG and steroid release evoked by these two polypeptides, and depressed PG release elicited by monobutyryl cyclic AMP (bcAMP) without affecting steroid release. These experiments offer additional evidence to support the concept that PGs play a role in the mode of action of ACTH; however, they do not appear to be obligatory intermediates in the steroidogenic process. The importance of calcium in regulating PG formation is discussed with special regard for the idea that this cation has a direct action on the enzyme systems which control PG synthesis.

Proteasomal activation mediates down-regulation of inositol 1,4,5-trisphosphate receptor and calcium mobilization in rat pancreatic islets.

Inositol 1,4,5-trisphosphate receptor (IP3R) protein levels in isolated rat pancreatic islets were investigated in response to carbachol (CCh) and sulfated cholecystokinin 26-33 amide stimulation. Within 2 h, CCh reduced IP3R-I protein levels by 22% and IP3R-II and -III levels to 65% or more below basal. Sulfated cholecystokinin 26-33 amide decreased the levels of IP3R-I, -II, and -III by 34%, 60%, and 66% below basal, respectively. The effect of CCh was concentration- and time-dependent, with a persistent decline in IP3R levels for up to 6 h after the onset of stimulation. CCh-pretreated islets also showed an inhibition of glucose-stimulated insulin secretion. Proteasome inhibition completely blocked the down-regulatory effects of CCh on IP3Rs and significantly increased the insulin secretory response to glucose stimulation in the presence of CCH: Islet stimulation by glucose, alpha-ketoisocaproic acid, and tolbutamide completely protected IP3Rs against the down-regulatory effects of CCH: 2-deoxyglucose and 3-O-methyl glucose failed to affect CCh-induced IP3R down-regulation. The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca(2+) channel-blocking agent nimodipine. Intracellular Ca(2+) ([Ca(2+)](i)) levels in Fura-2 (fluorescent Ca(2+) indicator)-loaded islets, in the absence of extracellular Ca(2+), increased in response to glucose stimulation; but in islets pretreated with CCh, glucose did not increase [Ca(2+)](i) above basal levels. However, in islets pretreated with CCh and the proteasomal inhibitor MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinyl-H), the glucose-stimulated increase in [Ca(2+)](i) was significantly higher than the change observed for glucose-stimulated [Ca(2+)](i) in the absence of MG-132. The results suggest that muscarinic receptor stimulation modulates IP3R protein levels in islets through a proteasomal activation pathway, and that down-regulation of IP3Rs has a profound effect on Ca(2+) mobilization in islets that may relate to insulin secretory responsiveness.