Interpolymer complexes and polymer compatibility.

A reliable method to decide whether two polymers A and B are miscible or incompatible would be very helpful in many ways. In this contribution we demonstrate why traditional procedures cannot work. We propose to use the intrinsic viscosities [η] of the polymer blends instead of the composition dependence of the viscosities as a criterion for polymer miscibility. Two macromolecules A and B are miscible because of sufficiently favorable interactions between the two types of polymer segments. For solutions of these polymers in a joint solvent, this Gibbs energetic preference of dissimilar intersegmental contacts should prevail upon dilution and lead to the formation of interpolymer complexes, manifesting themselves in deviations from the additivity of intrinsic viscosities.

Cell mitotic cycle synthesis of NIL hamster glycolipids including the Forssman antigen.

The synthesis of phospholipids and glycolipids during the cell mitotic cycle of an established hamster line, NIL, has been studied. Cells were synchronized with excess thymidine and mitotically harvested by shaking. Cells were radioactively labeled for 4 h with palmitate, glucosamine, or galactose. Lipids were analyzed by thin-layer chromatography. As cells progressed through the mitotic cycle, incorporation into phospholipids increased but the fraction represented by each remained constant. Similarly, ceramide monohexoside, dihexoside, and hematoside were labeled equally in all phases. Ceramide trihexoside and tetrahexoside were labeled only during G(1) and S. Ceramide pentahexoside (the Forssman antigen) shows density-dependent synthesis, accumulation, and reactivity. Ceramide pentahexoside was labeled during all phases of the mitotic cycle but the rate of incorporation decreased in S and G(2). The total amount of lipid assayed immunologically in cell extracts gradually increased. Exposure of the Forssman antigen in untreated or trypsin-treated cells was studied using binding of chemically labeled antiForssman antiserum. The amount of antigen detected in trypsinized cells increased during G(1) and early S but then remained constant. Mitotic cells exposed all detectable antigen. As cells progressed through the mitotic cycle, a large fraction of the Forssman antigen became cryptic.

Diacylglycerol synthesis de novo from glucose by pancreatic islets isolated from rats and humans.

Recent evidence has suggested that pancreatic islets isolated from rats synthesize 1,2-diacyl-sn-glycerol (DAG) de novo from glucose and that this process may constitute the long-sought link between the metabolism of glucose and the induction of insulin secretion. The cell-permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (200 microM) has been found here to amplify both the first and second phases of insulin secretion from perifused human islets. Measurements of the mass of endogenous DAG in human pancreatic islets by enzymatic and by mass spectrometric methods indicate that levels of 200 microM may be achieved under physiologic conditions. Conversion of [14C]glucose to [14C]DAG has been demonstrated here to occur within 60 s of exposure of rat and human islets to stimulatory concentrations of glucose. This process has been found to be a quantitatively minor contributor to the total islet DAG mass after acute stimulation with glucose, however, and glucose has been found not to induce a rise in total islet DAG content within 20 min of induction of insulin secretion. In contrast to the case with rodent islets, two pharmacologic inhibitors of DAG-induced activation of protein kinase C (staurosporine and sphingosine) have been found not to influence glucose-induced insulin secretion from isolated human islets. These findings indicate that de novo synthesis of DAG from glucose does not participate in acute signal-response coupling in islets.

Diacylglycerol accumulation and microvascular abnormalities induced by elevated glucose levels.

The present experiments were undertaken to examine the hypothesis that glucose-induced increased de novo synthesis of 1,2-diacyl-sn-glycerol (which has been observed in a number of different tissues, including retinal capillary endothelial cells exposed to elevated glucose levels in vitro) and associated activation of protein kinase C may play a role in mediating glucose-induced vascular functional changes. We report here that twice daily instillation of 30 mM glucose over 10 d in a rat skin chamber granulation tissue model induces approximately a 2.7-fold increase in diacylglycerol (DAG) levels (versus tissues exposed to 5 mM glucose) in association with marked increases in vascular clearance of albumin and blood flow. The glucose-induced increase in DAG levels as well as the vascular functional changes are prevented by addition of 3 mM pyruvate. Pharmacological activation of protein kinase C with the phorbol ester TPA in the presence of 5 mM glucose increases microvascular albumin clearance and blood flow, and similar effects are observed with 1-monoolein (MOG), a pharmacological inhibitor of the catabolism of endogenous DAG. A pharmacological inhibitor of protein kinase C (staurosporine) greatly attenuates the rise in microvascular albumin clearance (but not the rise in blood flow) induced by glucose or by MOG. These findings are compatible with the hypothesis that elevated concentrations of glucose increase tissue DAG content via de novo synthesis, resulting in protein kinase C activation, and that these biochemical events are among the factors that generate the increased microvascular albumin clearance.

Inhibition of phospholipase A2 and insulin secretion in pancreatic islets.

Arachidonic acid may be an important mediator of insulin secretion since (1) glucose activates phospholipase A2 thus increasing endogenous unesterified levels of arachidonic acid, (2) arachidonic acid mobilizes Ca2+ from the islet endoplasmic reticulum and (3) arachidonic acid has been proposed to regulate voltage-dependent Ca2+ channels in the beta-cell. We have used the phospholipase A2 inhibitor, (p-amylcinnamoyl)anthranilic acid (ACA), to determine whether phospholipase A2 activation is required for glucose-induced insulin secretion. ACA inhibited in a dose-dependent manner glucose-induced insulin secretion, as well as glyceraldehyde and alpha-ketoisocaproic acid-induced insulin secretion. ACA also totally abolished glucose-induced arachidonate accumulation but did not affect phospholipase C suggesting that it was specific for phospholipase A2. Furthermore, ACA did not inhibit glucose oxidation. These observations suggest that glucose-induced arachidonate increase is essential for insulin secretion.

Arachidonic acid metabolism in isolated pancreatic islets. IV. Negative ion-mass spectrometric quantitation of monooxygenase product synthesis by liver and islets.

Deuterium-labelled standards of four regionally isomeric epoxyeicosatrienoic acids (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-methane chemical ionization (MCI)-mass spectrometry (MS) as the pentafluorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectrometric assay which was linear from near the detection limit over several orders of magnitude. NADPH-dependent synthesis of both EETs and DHETs from arachidonate by hepatic microsomal cytochrome P-450-mono-oxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 microM), but not by eicosa-5,8,11,14-tetraynoic acid (ETYA, 20 microM) or by nordihydroguaiaretic acid (NDGA, 50 microM). All three compounds suppress glucose-induced insulin secretion and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by isolated pancreatic islets with similar concentration dependence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET compounds as a comparable amount of hepatic microsomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12-HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [3H]12-HETE and to [3H]prostaglandin E2 (but not to [3H]leukotriene C4, D4, or E4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C4 stimulated insulin secretion from the isolated islets or reversed the suppression of glucose-induced secretion by the lipoxygenase inhibitor BW755C. The cytochrome P-450-monooxygenase inhibitor, metyrapone (50 microM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.

Arachidonic acid metabolism in isolated pancreatic islets. V. The enantiomeric composition of 12-hydroxy-5,8,10,14-eicosatetraenoic acid indicates synthesis by a 12-lipoxygenase rather than a monooxygenase.

Recent evidence indicates that the arachidonate metabolite 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) or its precursor may act as a second messenger in stimulus-response coupling in a variety of cells including Aplysia neurons, adrenal glomerulosa cells, and pancreatic islets. The compound 12(S)-HETE is generated from the precursor 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12(S)-HPETE), which is a product of the 12-lipoxygenase enzyme. Some cells have recently been found to produce the enantiomer 12(R)-HETE, apparently via a cytochrome P-450 monooxygenase, and the biologic actions of 12(R)-HETE and 12(S)-HETE differ. We have examined the stereochemistry of 12-HETE from isolated pancreatic islets both radiochemically and by a new mass spectrometric method capable of quantitating subnanogram amounts of 12-HETE stereoisomers. Endogenous 12-HETE from islets was found to be exclusively the S-isomer. D-Glucose stimulated both insulin secretion and islet accumulation of 12(S)-HETE but not of 12(R)-HETE. Pharmacologic inhibition of islet 12-HETE biosynthesis also suppressed glucose-induced insulin secretion. These findings suggest that islet 12-HETE is a product of a 12-lipoxygenase rather than of a cytochrome P-450 monooxygenase and further implicate 12-lipoxygenase products in stimulus-secretion coupling.

Glucose-induced phospholipid hydrolysis in isolated pancreatic islets: quantitative effects on the phospholipid content of arachidonate and other fatty acids.

Our recent findings indicate that glucose-induced insulin secretion from isolated pancreatic islets is temporally associated with accumulation of substantial amounts of free arachidonic acid and that arachidonate may serve as a second messenger for intracellular calcium mobilization in islets. In an effort to determine the source of this released arachidonate, the endogenous fatty acid composition of phospholipids from islets has been determined by thin-layer chromatographic separation of the phospholipids, methanolysis to the fatty acid methyl esters, and quantitative gas chromatographic analyses. The relative abundance of phospholipids in islets as judged by their fatty acid content was phosphatidylcholine (PC), 0.63; phosphatidylethanolamine (PE), 0.23; phosphatidylinositol (PI), 0.067; phosphatidylserine (PS), 0.049. Arachidonate constituted 17% of the total islet fatty acid content, and PC contained 43% of total islet arachidonate. Islets incubated with [3H]arachidonate in the presence of 28 mM D-glucose incorporated radiolabel into PC with a considerably higher specific activity than that of PE, PS or PI. The total fatty acid content of PC from islets incubated with 28 mM glucose for 30 min was significantly lower than that of islets incubated with 3 mM glucose, and smaller effects were observed with PE, PS and PI. The molar decrement in PC arachidonate was 3.2 pmol/islet under these conditions, which is sufficient to account for the previously observed accumulation of free arachidonate (2 pmol/islet). A sensitive method involving negative ion-chemical ionization-mass spectrometric analyses of the pentafluorobenzyl esters of fatty acids derived from trace amounts of lysophosphatidylcholine (lyso-PC) was developed, and glucose-stimulation was found to reduce islet lyso-PC content by about 10-fold. These findings indicate that the insulin secretagogue D-glucose induces phospholipid hydrolysis in islets and suggest that PC may be the major source of free arachidonate which accumulates in glucose-stimulated islets.

Interleukin-1beta stimulation of c-Jun NH(2)-terminal kinase activity in insulin-secreting cells: evidence for cytoplasmic restriction.

Cytokines have been shown to have dramatic effects on pancreatic islets and insulin-secreting beta-cell lines. It is well established that cytokines such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (IFN-gamma) inhibit beta-cell function and are cytotoxic to human and rodent pancreatic islets in vitro. Despite the pleiotropic effects of cytokines on beta-cells, the specific signal transduction pathways and molecular events involved in beta-cell dysfunction remain largely unresolved. In this report, we have examined IL-1beta stimulation of c-Jun NH(2)-terminal kinase (JNK) activity in insulin-secreting clonal cell lines. We demonstrate that IL-1beta transiently activates 46- and 54-kDa isoforms of JNK in cultured RINm5F beta-cells. Furthermore, IL-1beta stimulation of JNK activity is specific, because TNF-alpha and IFN-gamma were without effect. Stable overexpression of JNK1 in RINm5F cells increased levels of activated JNK without affecting kinase activity. JNK-interacting protein (JIP) associates with endogenous as well as overexpressed JNK, suggesting that JIP may serve to regulate JNK activity. Finally, we demonstrate that activated JNK is fully retained in cytoplasmic and membrane compartments without any nuclear translocation. Together, these data indicate that IL-1beta-stimulated JNK activity may be distinctly targeted to cytoplasmic and/or membrane compartments in clonal insulin-producing cells, and that JIP may serve to localize JNK activity to specific substrates.

Activation of the sphingomyelinase/ceramide signal transduction pathway in insulin-secreting beta-cells: role in cytokine-induced beta-cell death.

Activation of the sphingomyelin/ceramide pathway may mediate interleukin-1-induced beta-cell death (Welsh, N: Interleuken-1beta-induced ceramide and diacylglycerol generation may lead to activation of the c-Jun NH2-terminal kinase and the transcription factor ATF-2 in the insulin-producing cell line RINm5F. J Biol Chem 271: 8307-8312, 1996). In this report, we have examined this pathway in more detail. Culture of beta-TC3 cells with 25 micromol/l ceramide analogs (N-acetyl- and N-hexanoylsphingosine) for 72 h did not significantly affect glucose- and carbachol-induced insulin secretion. Dihydroceramide (N-acetyl- or N-hexanoylsphinganine), a structurally similar analog, had no effect on agonist-induced secretion. However, ceramide analogs both time- and dose-dependently decreased cell viability, while the dihydroceramide analog had no effect. The ceramide effect on cell viability mimicked the effect of the cytokines TNF-alpha, IL-1beta, and IFN-gamma, reported stimulators of sphingomyelin hydrolysis. Cytokines, however, failed to stimulate sphingomyelin metabolism. Furthermore, using two different methods to quantitate ceramide, cytokines failed to cause an increase in beta-cell ceramide content versus unstimulated or time-matched vehicle controls. Taken together, these data suggest that although ceramide analogs mimic the cytotoxic effect of cytokines, activation of the sphingomyelin/ceramide signaling pathway is not involved in cytokine-induced beta-cell death.

G-protein specificity in signaling pathways that mobilize calcium in insulin-secreting beta-TC3 cells.

Fuel- and receptor-induced signal transduction pathways were investigated in beta-TC3 cells, an insulin-secreting cell line. An increase of glucose concentration from 0 to 15 mM and stimulation with 0.5 mM carbachol resulted in up to a twofold increase in insulin secretion by beta-TC3 cells, and their simultaneous addition increased insulin release eightfold. In single fura 2-loaded cells, a potentiating effect of carbachol was also observed on glucose-induced intracellular Ca2+ mobilization. Immunoblotting with antibodies raised to the COOH-terminal of G-protein alpha-subunits showed that G alpha i, G alpha o, and G alpha q are present in beta-TC3 cells in commensurable quantities. The novel technique of microinjection of anti-G alpha antibodies into single beta-cell was used to probe the functional role of these G-proteins. Microinjection of anti-G alpha i antibodies into beta-TC3 cells had no effect on glucose- and carbachol-induced Ca2+ mobilization. However, anti-G alpha q completely inhibited the Ca(2+)-mobilizing effect of carbachol, but not of glucose, within 5 min. Microinjection of anti-G alpha o antibodies had no effect on carbachol-induced Ca2+ mobilization. Microinjection of anti-G alpha i and anti-G alpha q antibodies had a minimal effect on glucose-induced Ca2+ mobilization (< 8% of cells nonresponding), but microinjection of anti-G alpha o increased the proportion of nonresponding cells to 37%. The results suggest that, in beta-TC3 cells, distinct signal transduction pathways with specific G-protein involvement may interact with secretagogue-induced Ca2+ mobilization and, ultimately, with insulin secretion.

Glucose-induced insulin receptor tyrosine phosphorylation in insulin-secreting beta-cells.

In the beta TC3 insulin-secreting beta-cell line, glucose rapidly induces the tyrosine phosphorylation of the 97-kDa insulin receptor beta-subunit. Phosphorylation is transient, with fourfold stimulation by 2 min and subsequent dephosphorylation to basal levels by 10-15 min. Elevating the extracellular KCl concentration equipotently initiates receptor phosphorylation. Preventing insulin secretion with 1 mumol/l epinephrine or by removing extracellular Ca2+ blocks the effect. In the absence of glucose-induced secretion, exogenous insulin also stimulated insulin receptor autophosphorylation transiently and with an ED50 of 4 x 10(-9) mol/l. In addition, functional insulin-like growth factor I (IGF-I) receptors are also expressed by these beta-cells, as indicated by IGF-I-induced receptor tyrosine phosphorylation (ED50 = 5 x 10(-9) mol/l) and also by detection of hybrid insulin/IGF-I receptor autophosphorylation at 10(-7) mol/l IGF-I. Both glucose and insulin stimulate the tyrosine phosphorylation of the insulin receptor substrate (IRS) IRS-1 and increase by two- to fivefold the rapid association of IRS-1 with the 85-kDa alpha-subunit of the phosphatidylinositol-3-kinase, as determined by co-immunoprecipitation assays. These results demonstrate that in these beta-cells, glucose-induced insulin secretion activates the beta-cell surface insulin receptor tyrosine kinase and its intracellular signal transduction pathway, suggesting a new autocrine mechanism for the regulation of beta-cell function.

Interleukin 1-induced prostaglandin E2 accumulation by isolated pancreatic islets.

Recombinant human interleukin 1 alpha (IL-1) has been found to induce prostaglandin E2 (PGE2) accumulation by isolated rat islets of Langerhans at concentrations similar to those at which the cytokine inhibits glucose-induced insulin secretion and islet glucose oxidation. Maximal stimulation of PGE2 accumulation (5 times control value) occurred at 200 pM IL-1, and half-maximal stimulation occurred at 25 pM IL-1. Significant augmentation of PGE2 accumulation by IL-1 required 10-18 h of exposure to the cytokine. Islets that had been pretreated with IL-1 for 18 h showed elevated rates of PGE2 production at basal (3-mM) and stimulatory (16.5-mM) glucose concentrations and converted exogenous arachidonic acid to PGE2 at twice the maximal rate of control islets. Exogenous PGE2 did not mimic the inhibitory effects of IL-1 on glucose-induced insulin secretion or glucose oxidation. To rule out the possibility that endogenous PGE2 is involved in the inhibitory effects of IL-1, the effect of a cyclooxygenase inhibitor on IL-1-treated islets was examined. Pharmacological blockade of PGE2 biosynthesis by 10 microM indomethacin did not influence the inhibitory effects of IL-1 on glucose-induced insulin secretion or glucose oxidation. Thus, exogenous PGE2 does not mimic the effects of IL-1 on islets, and inhibition of endogenous PGE2 biosynthesis does not suppress the effects of IL-1 on islets. These results suggest that PGE2 is not a principal mediator of the inhibitory effects of IL-1 on glucose-induced insulin secretion or glucose oxidation.

Interleukin 1 is potent modulator of insulin secretion from isolated rat islets of Langerhans.

The effects of interleukin 1 (IL-1) on glucose-induced insulin secretion from isolated rat islets of Langerhans have been examined. IL-1 both inhibits and stimulates glucose-induced insulin secretion depending on the experimental design. Inhibition of glucose-induced insulin secretion was observed after a 15-h treatment of islets with either purified IL-1, murine recombinant IL-1 (rIL-1), or human rIL-1, rIL-1 inhibition of glucose-induced insulin secretion was dose dependent with half-maximal inhibition observed at 25 pM human rIL-1. Basal insulin secretion was not affected by rIL-1 treatment. Mannose- and leucine-induced insulin secretion was also inhibited by a 15-h treatment with human rIL-1. Islets treated 15 h with inhibitory concentrations of murine IL-1 were morphologically intact, well granulated, and retained normal concentrations of insulin compared with control islets. Furthermore, human rIL-1 treatment did not affect the islet plasma membrane permeability as assessed by the measurement of the islet intracellular volume. Finally, the viability of islets treated 15 h with murine rIL-1 was demonstrated by the observation that the inhibitory effects of murine rIL-1 on glucose-induced insulin secretion were reversible. In addition to the inhibitory effects of IL-1 on glucose-induced insulin secretion, purified IL-1 and human rIL-1 had stimulatory effects on glucose-induced insulin secretion under the following conditions: a 90-min incubation with purified IL-1 (10% vol/vol) or in the presence of human rIL-1 (1400 pM) or a 15-h incubation with relatively low concentrations of human rIL-1 (0.5 or 5 pM).(ABSTRACT TRUNCATED AT 250 WORDS)

Descriptive and mechanistic considerations of interleukin 1 and insulin secretion.

Insulin-dependent diabetes mellitus (IDDM) may be mediated in part by an autoimmune mechanism, as suggested by associated cytologic and serologic phenomena, e.g., insulitis, beta-cell necrosis, and the presence of both islet cell and insulin antibodies. Immunological approaches to the prediction and intervention in the progression of beta-cell destruction in this disease are under evaluation. A recent hypothesis is that cytokines, including interleukin 1 (IL-1), play causative roles in such autoimmune processes. Several studies have convincingly demonstrated that IL-1 is a potent modulator of beta-cell function and can potentiate or inhibit glucose-induced insulin secretion, depending on the concentration and length of exposure to IL-1. IL-1 alone or in concert with other cytokines is cytotoxic to beta-cells. The cellular mechanisms responsible for the potent effects of IL-1 on the beta-cell are unknown and just beginning to emerge. Although speculative at this time, this perspective delineates cellular mechanisms that are likely to represent possible primary sites for the IL-1 action on beta-cells. A mechanistic understanding of the effects of IL-1 on the beta-cell may clarify its role in modulating insulin release in vivo or yield insight into the pathogenesis of IDDM.

Glucose regulation of glutaminolysis and its role in insulin secretion.

Leucine or the nonmetabolized leucine analog +/- 2-amino-2-norbornane-carboxylic acid (BCH) (both at 10 mmol/l) induced biphasic insulin secretion in the presence of 2 mmol/l glutamine (Q2) in cultured mouse islets pretreated for 40 min without glucose but with Q2 present. The beta-cell response consisted of an initial peak of 20- to 25-fold above basal and a less marked secondary phase. However, BCH produced only a delayed response, while leucine was totally ineffective when islets were pretreated with 25 mmol/l glucose plus Q2. With Q2, 10 mmol/l BCH or leucine caused a nearly threefold increase, a twofold increase, or had no effect on cytosolic Ca2+ levels in islets pretreated for 40 min with 0, 5, or 15 mmol/l glucose, respectively. Thus, pretreatment of islets with high glucose inhibited BCH- and leucine-induced cytosolic Ca2+ changes and insulin release. Glucose decreased glutamine oxidation in cultured rat islets when BCH was present at 10 mmol/l, but not in its absence, with a lowest effective level of approximately 0.1 mmol/l, a maximum of 18-30 mmol/l, and an inhibitory concentration, 50%, of approximately 3 mmol/l. The data are consistent with the hypothesis that glucose inhibits glutaminolysis in pancreatic beta-cells in a concentration-dependent manner and hence blocks leucine-stimulated insulin secretion. We postulate that in the basal interprandial state, glutaminolysis of beta-cells is partly turned on because glutamate dehydrogenase (GDH) is activated by a decreased P-potential due to partial fuel depletion and sensitization to endogenous activators such as leucine. Additionally, it may contribute significantly to basal insulin release, which is known to be responsible for about half of the insulin released daily. The data explain "leucine-hypersensitivity" of beta-cells during hypoglycemia and contribute to the elucidation of the GDH-linked syndrome of hyperinsulinism associated with elevated serum ammonia levels. Thus, understanding the precise regulation and role of beta-cell glutaminolysis is probably central to our concept of normal blood glucose control.

Expression of insulin receptor mRNA and insulin receptor substrate 1 in pancreatic islet beta-cells.

The expression of insulin receptor mRNA was examined in rat pancreatic islet cells by single-cell reverse transcriptase (RT)-polymerase chain reaction (PCR). Single cells from disaggregated islets were individually isolated in a microcapillary pipet, and the beta-cells were identified by amplification of the mRNA for insulin. We found that in single beta-cells, the mRNA for the insulin receptor was also expressed. The fraction of single islet cells expressing both insulin receptor and insulin mRNAs corresponds closely to the fraction of beta-cells in the disaggregated islet cell preparation. These results indicate that normal beta-cells have the potential to express authentic insulin receptors. Immunohistochemical analysis was insufficiently sensitive for assaying insulin receptor protein; however, insulin receptor substrate 1 (IRS-1) was readily immunolocalized in islet beta-cells. Since IRS-1 links several cell surface receptors, including those for insulin and IGF-I, to distal signal transduction pathways, our observations indicate that hormonal regulation of islet beta-cells potentially involves the same signal transduction pathway that mediates insulin and growth factor signaling in peripheral insulin target tissue cell types.

Wortmannin inhibits insulin secretion in pancreatic islets and beta-TC3 cells independent of its inhibition of phosphatidylinositol 3-kinase.

Glucose is the primary stimulus for insulin secretion by pancreatic beta-cells, and it triggers membrane depolarization and influx of extracellular Ca2+. Cholinergic agonists amplify insulin release by several pathways, including activation of phospholipase C, which hydrolyzes membrane polyphosphoinositides. A novel phospholipid, phosphatidylinositol 3,4,5- trisphosphate [PtdIns(3,4,5)P3], a product of phosphatidylinositol 3-kinase (PI 3-kinase), has recently been found in various cell types. We demonstrate by immunoblotting that PI 3-kinase is present in both cytosolic and membrane fractions of insulin-secreting beta-TC3 cells and in rat islets. The catalytic activity of PI 3-kinase in immunoprecipitates of islets and beta-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (PtdIns) in the presence of [gamma-32P]ATP. Wortmannin, a fungal metabolite, dose dependently inhibited PI 3-kinase activity of both islets and beta-TC3 cells, with an IC50 of 1 nmol/l and a maximally effective concentration of 100 nmol/l, when it was added directly to the kinase assay. However, if intact islets were incubated with wortmannin and PI 3-kinase subsequently was determined in islet immunoprecipitates, approximately 50% inhibition of PI 3-kinase activity (but no inhibition of glucose- and carbachol-stimulated insulin secretion) from intact islets was obtained at wortmannin concentrations of 100 nmol/l. Wortmannin, at higher concentrations (1 and 10 micromol/l), inhibited glucose- and carbachol-induced insulin secretion of Intact rat islets by 58 and 92%, respectively. Wortmannin had no effect on the basal insulin release from rat islets. A similar dose curve of inhibition of glucose- and carbachol-induced insulin secretion by wortmannin was obtained when beta-TC3 cells were used. Cellular metabolism was, not changed by any wortmannin concentrations tested (0.01-10 micromol/l). Both basal cytosolic [Ca2+]i and carbamyl choline-induced increases of [Ca2]i were unaffected by wortmannin in the presence of 2.5 mmol/l Ca2+, while Ca2+ mobilization from intracellular stores was partially decreased by wortmannin. Together, these data suggest that wortmannin at concentrations that inhibit PI 3-kinase does not affect insulin secretion. PI 3-kinase is unlikely to have a major role in insulin secretion induced by glucose and carbachol.

Arachidonic acid metabolism and insulin secretion by isolated human pancreatic islets.

Isolated human pancreatic islets converted [3H8]arachidonate to compounds with the high-performance liquid-chromatographic mobility of cyclooxygenase products, including prostaglandin E2 (PGE2), PGF2 alpha, and the lipoxygenase product 12-HETE. Human islet synthesis of PGE2, PGF2 alpha, and 12-HETE from endogenous arachidonate was demonstrated with stable isotope dilution-gas chromatographic-negative ion-chemical ionization-mass spectrometric analysis. Pharmacologic inhibition of arachidonate metabolism by both lipoxygenase and cyclooxygenase pathways with BW 755C strongly suppressed glucose-induced insulin secretion from perifused human islets, and the selective cyclooxygenase inhibitor indomethacin enhanced insulin secretion. These findings are similar to those reported for islets isolated from rats and suggest that arachidonate metabolites may modulate glucose-induced insulin secretion in humans.

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets.

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K0.5 of 10 microM) and maximum activation was observed at 50 microM arachidonate. Arachidonic acid did not affect the Ca2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 microM) inhibited (greater than 90%) calmodulin-dependent protein kinase activity (K0.5 = 50-100 microM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K0.5 = 24 microM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50-100 microM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 microM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 microM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.