Normal Akt/PKB with reduced PI3K activation in insulin-resistant mice.

Insulin stimulates muscle and adipose tissue to absorb glucose through a signaling cascade that is incompletely understood. Insulin resistance, the inability of insulin to appropriately stimulate glucose uptake, is a hallmark of type 2 diabetes mellitus. The development of experimental systems that model human insulin resistance is important in elucidating the defects responsible for the development of type 2 diabetes. When two strains of mice, BTBR and C57BL/6J (B6), are crossed, the resultant male offspring (BtB6) demonstrate insulin resistance in muscle tissue. Here, we report an insulin resistance phenotype in adipose tissue from lean, nondiabetic BtB6 mice similar to that observed in human muscle. Adipocytes isolated from insulin-resistant male mice display 65% less insulin-stimulated glucose uptake compared with insulin-sensitive female mice. Similarly, adipocytes from insulin-resistant mice have diminished insulin-stimulated IRS-1 phosphorylation and phosphatidylinositol 3-kinase (PI3K) activation. However, normal activation of protein kinase B (Akt/PKB) by insulin is observed. Thus BtB6 mice demonstrate the dissociation of insulin-stimulated PI3K activity and Akt/PKB activation and represent a useful model to investigate the causes of insulin resistance in humans.

Inosine-5'-monophosphate dehydrogenase is required for mitogenic competence of transformed pancreatic beta cells.

The relation of inosine-5'-monophosphate dehydrogenase (IMPDH; the rate-limiting enzyme in GTP synthesis) to mitogenesis was studied by enzymatic assay, immunoblots, and RT-PCR in several dissimilar transformed pancreatic ss-cell lines, using intact cells. Both of the two isoforms of IMPDH (constitutive type 1 and inducible type 2) were identified using RT-PCR in transformed beta cells or in intact islets. IMPDH 2 messenger RNA (mRNA) and IMPDH protein were both regulated reciprocally by changes in levels of their end-products. Flux through IMPDH was greatest in rapidly growing cells, due mostly to increased uptake of precursor. Glucose (but not 3-0-methylglucose, L-glucose, or fructose) further augmented substrate uptake and also increased IMPDH enzymatic activity after either 4 or 21 h of stimulation. Serum or ketoisocaproate also increased IMPDH activity (but not uptake). Two selective IMPDH inhibitors (mycophenolic acid and mizoribine) reduced IMPDH activity in all cell lines, and, with virtually identical concentration-response curves, inhibited DNA synthesis (assessed as bromodeoxyuridine incorporation) in response to glucose, serum, or ketoisocaproate. Inhibition of DNA synthesis was reversible, completely prevented by repletion of cellular guanine (but not adenine) nucleotides, and could not be attributed to toxic effects. Despite the fact that modulation of IMPDH expression by guanine nucleotides was readily detectable, glucose and/or serum failed to alter IMPDH mRNA or protein, indicating that their effects on IMPDH activity were largely at the enzyme level. Precursors of guanine nucleotides failed, by themselves, to induce mitogenesis. Thus, adequate IMPDH activity (and thereby, availability of GTP) is a critical requirement for beta-cell proliferation. Although it is unlikely that further increases in GTP can, by themselves, initiate DNA synthesis, such increments may be needed to sustain mitogenesis.

Genetic obesity unmasks nonlinear interactions between murine type 2 diabetes susceptibility loci.

Nonlinear interactions between obesity and genetic risk factors are thought to determine susceptibility to type 2 diabetes. We used genetic obesity as a tool to uncover latent differences in diabetes susceptibility between two mouse strains, C57BL/6J (B6) and BTBR. Although both BTBR and B6 lean mice are euglycemic and glucose tolerant, lean BTBR x B6 F1 male mice are profoundly insulin resistant. We hypothesized that the genetic determinants of the insulin resistance syndrome might also predispose genetically obese mice to severe diabetes. Introgressing the ob allele into BTBR revealed large differences in diabetes susceptibility between the strain backgrounds. In a population of F2-ob/ob mice segregating for BTBR and B6 alleles, we observed large variation in pancreatic compensation for the underlying insulin resistance. We also detected two loci that substantially modify diabetes severity, and a third locus that strongly links to fasting plasma insulin levels. Amplification of the genetic signal from these latent diabetes susceptibility alleles in F2-ob/ob mice permitted discovery of an interaction between the two loci that substantially increased the risk of severe type 2 diabetes.

A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats.

A widely accepted genetically determined rodent model for human type 2 diabetes is the Goto-Kakizaki (GK) rat; however, the lesion(s) in the pancreatic islets of these rats has not been identified. Herein, intact islets from GK rats (aged 8-14 weeks) were studied, both immediately after isolation and after 18 h in tissue culture. Despite intact contents of insulin and protein, GK islets had markedly deficient insulin release in response to glucose, as well as to pure mitochondrial fuels or a non-nutrient membrane-depolarizing stimulus (40 mmol/l K+). In contrast, mastoparan (which activates GTP-binding proteins [GBPs]) completely circumvented any secretory defect. Basal and stimulated levels of adenine and guanine nucleotides, the activation of phospholipase C by Ca2+ or glucose, the secretory response to pertussis toxin, and the activation of selected low-molecular weight GBPs were not impaired. Defects were found, however, in the autophosphorylation and catalytic activity of cytosolic nucleoside diphosphokinase (NDPK), which may provide compartmentalized GTP pools to activate G-proteins; a deficient content of phosphoinositides was also detected. These studies identify novel, heretofore unappreciated, defects late in signal transduction in the islets of our colony of GK rats, possibly occurring at the site of activation by NDPK of a mastoparan-sensitive G-protein-dependent step in exocytosis.

Prolonged depletion of guanosine triphosphate induces death of insulin-secreting cells by apoptosis.

Inhibitors of IMP dehydrogenase, such as mycophenolic acid (MPA) and mizoribine, which deplete cellular GTP, are used clinically as immunosuppressive drugs. The prolonged effect of such agents on insulin-secreting beta-cells (HIT-T15 and INS-1) was investigated. Both MPA and mizoribine inhibited mitogenesis, as reflected by [3H]thymidine incorporation. Cell number, DNA and protein contents, and cell (metabolic) viability were decreased by about 30%, 60%, and 80% after treatment of HIT cells with clinically relevant concentrations (e.g. 1 microg/ml) of MPA for 1, 2, and 4 days, respectively. Mizoribine (48 h) similarly induced the death of HIT cells. INS-1 cells also were damaged by prolonged MPA treatment. MPA-treated HIT cells displayed a strong and localized staining with a DNA-binding dye (propidium iodide), suggesting condensation and fragmentation of DNA, which were confirmed by detection of DNA laddering in multiples of about 180 bp. DNA fragmentation was observed after 24-h MPA treatment and was dose dependent (29%, 49%, and 70% of cells were affected after 48-h exposure to 1, 3, and 10 microg/ml MPA, respectively). Examination of MPA-treated cells by electron microscopy revealed typical signs of apoptosis: condensed and marginated chromatin, apoptotic bodies, cytosolic vacuolization, and loss of microvilli. MPA-induced cell death was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating a specific effect of GTP depletion. An inhibitor of protein isoprenylation (lovastatin, 10-100 microM for 2-3 days) induced cell death and DNA degradation similar to those induced by sustained GTP depletion, suggesting a mediatory role of posttranslationally modified GTP-binding proteins. Indeed, impeding the function of G proteins of the Rho family (via glucosylation using Clostridium difficile toxin B), although not itself inducing apoptosis, potentiated cell death induced by MPA or lovastatin. These findings indicate that prolonged depletion of GTP induces beta-cell death compatible with apoptosis; this probably involves a direct impairment of GTP-dependent RNA-primed DNA synthesis, but also appears to be modulated by small GTP-binding proteins. Treatment of intact adult rat islets (the beta-cells of which replicate slowly) induced a modest, but definite, death by apoptosis over 1- to 3-day periods. Thus, more prolonged use of the new generation of immunosuppressive agents exemplified by MPA might have deleterious effects on the survival of islet or pancreas grafts.

Interleukin-1 beta inhibits phospholipase C and insulin secretion at sites apart from KATP channel.

Although interleukin-1 beta (IL-1 beta) reduces pancreatic islet content of ATP and GTP, the distal events that mediate its inhibitory effects on insulin secretion remain poorly understood. Herein, the activation of phospholipase C (PLC) was quantified during islet perifusions. An 18-h exposure to IL-1 beta (100 pM) totally vitiated activation of PLC induced by glucose, an effect that requires ATP and GTP and closure of the ATP-dependent K+ (KATP) channel. Surprisingly, however, when islets were depolarized directly using either of two agonists, glyburide (which does not act via generation of purine nucleotides) or 40 mM K+ (which acts distal to KATP channel), PLC and insulin secretion were again obliterated by IL-1 beta. IL-1 beta also reduced the labeling of phosphoinositide substrates; however, this effect was insufficient to explain the inhibition of PLC, since the effects on substrate labeling, but not on PLC, were prevented by coprovision of guanosine or adenosine. Furthermore, when IL-1 beta-treated islets were exposed to 100 microM carbachol (which activates PLC partially independent of extracellular Ca2+), the effects were still obliterated by IL-1 beta. These data (together with the finding that IL-1 beta inhibited Ca(2+)-induced insulin release) suggest that, in addition to its effects on ATP synthesis and thereby on the KATP channel, IL-1 beta has at least two undescribed, distal effects to block both PLC as well as Ca(2+)-induced exocytosis. The latter correlated best with IL-1 beta's effect to impede phosphoinositide synthesis, since it also was reversed by guanosine or adenosine.

Roles of GTP and phospholipase C in the potentiation of Ca(2+)-induced insulin secretion by glucose in rat pancreatic islets.

Glucose can augment insulin secretion independently of K+ channel closure, provided cytoplasmic free Ca2+ concentration is elevated. A role for phospholipase C (PLC) in this phenomenon has been both claimed and refuted. Recently, we have shown a role for GTP in the secretory effect of glucose as well as in glucose-induced PLC activation, using islets pre-treated with GTP synthesis inhibitors such as mycophenolic acid (MPA). Therefore, in the current studies, we examined first, whether glucose augments Ca(2+)-induced PLC activation and second, whether GTP is required for this effect, when K+(ATP) channels are kept open using diazoxide. Isolated rat islets pre-labeled with [3H]myo-inositol were studied with or without first priming with glucose. There was a 98% greater augmentation of insulin secretion by 16.7 mM glucose (in the presence of diazoxide and 40 mM K+) in primed islets; however, the ability of high glucose to augment PLC activity bore no relationship to the secretory response. MPA markedly inhibited PLC in both conditions; however, insulin secretion was only inhibited (by 46%) in primed islets. None of these differences were attributable to alterations in labeling of phosphoinositides or levels of GTP or ATP. These data indicate that an adequate level of GTP is critical for glucose's potentiation of Ca(2+)-induced insulin secretion in primed islets but that PLC activation can clearly be dissociated from insulin secretion and therefore cannot be the major cause of glucose's augmentation of Ca(2+)-induced insulin secretion.

Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells.

We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in [Ca2+]i. Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).

Dual functional effects of interleukin-1beta on purine nucleotides and insulin secretion in rat islets and INS-1 cells.

Interleukin-1beta (IL-1beta) has been shown to inhibit glucose-induced insulin secretion from rat islets and purified beta-cells, primarily through the generation of nitric oxide (NO). However, the mechanisms by which NO exerts its effects remain unclear. To examine the role of purine nucleotides, we cultured intact rat islets or INS-1 (glucose-responsive transformed rat) beta-cells for 18 h in the presence or absence of IL-1beta. In islets, the exposure to IL-1beta (100 pmol/l) inhibited subsequent glucose-induced insulin secretion by 91% with no significant effect on insulin content or basal insulin release. IL-1beta also diminished insulin secretion induced by pure mitochondrial fuels, 40 mmol/l K+, or a phorbol ester. Concomitantly, IL-1beta significantly decreased islet ATP (-45%), GTP (-33%), ATP/ADP (-54%), and GTP/GDP (-46%). These effects were totally reversed by provision of N(omega)-nitro-L-arginine methyl ester (NAME) in arginine-free media that inhibited NO production. In contrast, in INS-1 cells, IL-1beta (10 or 100 pmol/l) reduced both basal and glucose-induced insulin secretion by 50%, but insulin content was also reduced by 35%. Therefore, the INS-1 cells were still able to respond to glucose stimulation with a 1.8-2.0-fold increase in insulin release in either the presence or absence of IL-1beta. Concomitantly, in INS-1 cells, IL-1beta had no effect on ATP/ADP or GTP/GDP ratios, although it modestly decreased ATP (-25%) and GTP (-22%). As in islets, all effects of IL-1beta in INS-1 cells were prevented by NAME. Thus, in rat islets, IL-1beta (via the generation of NO) abolishes insulin exocytosis in association with large decreases in the ATP/ADP (and GTP/GDP) ratio, implying the impairment of mitochondrial function. Furthermore, IL-1beta inhibits cytosolic synthesis of new purine nucleotides (via the salvage pathway), as assessed by a decrease in their specific activity after labeling with [3H]hypoxanthine. In contrast, in INS-1 cells, IL-1beta appears to impair cytosolic synthesis of purine nucleotides and insulin biosynthesis selectively (both possibly reflecting decreased glycolysis) with little direct effect on insulin exocytosis itself.

Role for GTP in glucose-induced phospholipase C activation in pancreatic islets.

We have previously demonstrated a permissive role for GTP in insulin secretion; in the current studies, we examined the effect of GTP on phospholipase C (PLC) activation to explore one possible mechanism for that observation. In rat islets preexposed to the GTP synthesis inhibitors mycophenolic acid (MPA) or mizoribine (MZ), PLC activation induced by 16.7 mM glucose (or by 20 mM alpha-ketoisocaproic acid) was inhibited 63% without altering the labeling of phosphoinositide substrates. Provision of guanine, which normalizes islet GTP content and insulin release, prevented the inhibition of PLC by MPA. Glucose-induced phosphoinositide hydrolysis was blocked by removal of extracellular Ca2+ or by diazoxide. PLC induced directly by Ca2+ influx (i.e., 40 mM K+) was reduced 42% in MPA-pretreated islets but without inhibition of the concomitant insulin release. These data indicate that glucose-induced PLC activation largely reflects Ca2+ entry and demonstrate (for the first time in intact cells) that adequate GTP is necessary for glucose (and Ca(2+)-)-induced PLC activation but not for maximal Ca(2+)-induced exocytosis.

Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

Several GTP-binding proteins (G-proteins) undergo post-translational modifications (isoprenylation and carboxyl methylation) in pancreatic beta cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure beta (HIT or INS-1) cells. GTPgammaS-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or beta cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTPgammaS-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K+) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTPgammaS; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K+-induced) insulin secretion from islets and beta cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S-adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in the stimulus-secretion coupling of nutrient-induced insulin secretion, but not in the exocytotic event itself. Furthermore, AFC blocked glucose-activated phosphoinositide turnover, which may provide a partial biochemical explanation for its effect on secretion, and implies that certain G-proteins must be carboxyl methylated for their interaction with signaling effector molecules, a step which can be regulated by intracellular availability of GTP.

Carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting cells: evidence for functional consequences on enzyme activity and insulin secretion.

We report the carboxylmethylation of a 36-kDa protein in intact normal rat islets and clonal beta (INS-1) cells. This protein was predominantly cytosolic. Its carboxylmethylation, as assessed by vapor phase equilibration assay, was resistant to inhibition by N-acetyl-S-trans, trans-farnesyl-L-cysteine, a competitive substrate for cysteine methyl transferases. These data suggest that the methylated C-terminal amino acid is not cysteine. The methylated protein was identified as the catalytic subunit of protein phosphatase 2A (PP2Ac) by immunoblotting. The carboxylmethylation of the PP2Ac increased its catalytic activity, suggesting a key role in the functional regulation of PP2A. Therefore, we studied okadaic acid, a selective inhibitor of PP2A that acts by an unknown mechanism. Okadaic acid (but not 1-nor-okadaone, its inactive analog) inhibited (Ki = 10 nM) the carboxylmethylation of PP2Ac and phosphatase activity in the cytosolic fraction (from normal rat islets and clonal beta-cells) as well as in intact rat islets. Furthermore, methylated PP2Ac underwent rapid demethylation (t 1/2 = 40 min) catalyzed by a methyl esterase localized in islet homogenates. Ebelactone, a purported inhibitor of methyl esterases, significantly delayed (> 200 min) the demethylation of PP2Ac. Furthermore, ebelactone reversibly inhibited glucose- and ketoisocaproate-induced insulin secretion from normal rat islets. These data identify, for the first time, a methylation-demethylation cycle for PP2Ac in the beta-cell and suggest a key functional relationship between PP2A activity and the carboxylmethylation of its catalytic subunit. These findings thus suggest a negative modulatory role for PP2A in nutrient-induced insulin exocytosis.

Evidence of a role for GTP in the potentiation of Ca(2+)-induced insulin secretion by glucose in intact rat islets.

Glucose initiates insulin secretion by closing K(+)-ATP channels, leading to Ca2+ influx (E1); it also potentiates Ca(2+)-induced secretion (E2) when the K(+)-ATP channel is kept open using diazoxide and depolarizing concentrations of K+ are provided. To examine the roles of purine nucleotides in E2, we compared the effects of glucose to those of the mitochondrial fuel monomethylsuccinate. Either agonist could induce E2 accompanied by significant increases in ATP, ATP/ADP ratio, and GTP/GDP ratio; GTP increased significantly only with glucose. Mycophenolic acid (MPA), an inhibitor of cytosolic GTP synthesis, markedly inhibited glucose-induced E2 (either in perifusions or in static incubations) and decreased GTP and the GTP/GDP ratio, but did not alter the ATP/ADP ratio. Provision of guanine (but not adenine) reversed these changes pari passu. In contrast, MPA had no effect on succinate-induced E2, despite generally similar changes in nucleotides. A similar lack of effect of MPA on E2 was seen with a second mitochondrial fuel, alpha-ketoisocaproic acid (KIC). However, in the absence of diazoxide and K+, MPA blunted the secretory effects of either glucose, succinate, or KIC. These studies suggest that GTP plays a role in both glucose and succinate or KIC-induced insulin secretion at a step dependent on mitochondrial metabolism and the K(+)-ATP channel. In addition to mitochondrial effects, glucose appears to have extramitochondrial effects important to its potentiation of Ca(2+)-induced insulin secretion that are also dependent on GTP.

Cytosolic biosynthesis of GTP and ATP in normal rat pancreatic islets.

GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The present work examines the cytosolic pathways of purine nucleotide synthesis using intact rat islets cultured overnight in RPMI 1640 medium containing either [14C]glycine (to label the de novo pathway) or [3H]hypoxanthine (to mark the salvage pathway), with or without mycophenolic acid or L-alanosine (selective inhibitors of cytosolic GTP and ATP synthesis, respectively). Addition of mycophenolic acid decreased total GTP content (mass) by 73-81%; although the incorporation of labeled hypoxanthine into GTP also fell by 87%, the incorporation of glycine did not change. Similarly, L-alanosine decreased ATP mass by 26-33% in the presence of either label; whereas the incorporation of hypoxanthine into ATP fell 59%, the incorporation of glycine was again not significantly decreased. Thus, both the de novo and salvage purine nucleotide biosynthetic pathways are present in rat islets; however, the salvage pathway appears to be quantitatively the more important source of nucleotides. This conclusion was supported by additional studies of the effects on nucleotide content and insulin secretion of various site-specific inhibitors of purine synthesis. These findings have potential relevance to the processes of mitogenesis, cell proliferation and differentiation of islet cells, as well as for the control of insulin secretion.

Non-specific stimulatory effects of mastoparan on pancreatic islet nucleoside diphosphokinase activity: dissociation from insulin secretion.

We examined whether mastoparan (MAS)-induced insulin secretion might involve the activation of nucleoside diphosphokinase (NDP kinase), which catalyzes the conversion of GDP to GTP, a known permissive factor for insulin secretion. MAS and MAS 7 (which activate GTP-binding proteins), but not MAS 17 (an inactive analog), stimulated insulin secretion from normal rat islets. In contrast to their specific effects on insulin secretion, MAS, MAS 7 and MAS 17 each stimulated formation of the phosphoenzyme-intermediate of NDP kinase, as well as its catalytic activity. These effects were mimicked by several cationic drugs. Thus, caution is indicated in using MAS to study cellular regulation, since some of its effects appear to be non-specific, and may be due, in part, to its amphiphilic, cationic nature.

Subcellular localization and kinetic characterization of guanine nucleotide binding proteins in normal rat and human pancreatic islets and transformed beta cells.

The subcellular localization and the kinetics of the GTPase activities of monomeric and heterotrimeric GTP-binding proteins were investigated in normal rat and human pancreatic islets and were compared to those obtained using a transformed hamster beta cell line (HIT cells). The [alpha-32P]GTP overlay technique revealed the presence of at least four low-molecular-mass proteins (approx. 20-27 kDa) in normal rat islets, which were enriched in the secretory granule fraction compared to the membrane fraction (with little abundance of these proteins in the cytosolic fraction). In contrast, in HIT cells, these proteins (at least six) were predominantly cytosolic. Three of these proteins were immunologically identified as rab3A, rac2, and CDC42Hs in islets as well as in HIT cells. In addition, pertussis toxin augmented the ribosylation of at least one heterotrimeric G-protein of about 39 kDa (probably G(i) and/or G(o)) in the membrane and secretory granule fractions of normal rat and human islets, whereas at least three such Ptx substrates (36-39 kDa) were found in HIT cell membranes. Kinetic activities revealed the presence of at least three such activities (Km for GTP of 372 nM, 2.2 microM, and 724 microM) in islet homogenates which were differentially distributed in various subcellular fractions; similar activities were also demonstrable in HIT cell homogenates. Thus, these studies demonstrate the presence of both monomeric G-proteins intrinsic to the secretory granules of normal rat islets which can be ascribed to beta cells; since these G-proteins are regulated by insulinotropic lipids (as described in the accompanying article), such proteins may couple the activation of phospholipases (endogenous to islets) to the exocytotic secretion of insulin. These findings also suggest that caution is necessary in extrapolating data concerning G-proteins from cultured, transformed beta cell lines to the physiology of normal islets, in view of both qualitative and quantitative differences between the two preparations.

Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins.

Many GTP-binding proteins (GBPs) are modified by mevalonic acid (MVA)-dependent isoprenylation, carboxyl methylation or palmitoylation. The effects of inhibitors of these processes on insulin release were studied. Intact pancreatic islets were shown to synthesize and metabolize MVA and to prenylate several candidate proteins. Culture with lovastatin (to inhibit synthesis of endogenous MVA) caused the accumulation in the cytosol of low-M(r) GBPs (labelled by the [alpha-32P]GTP overlay technique), suggesting a disturbance of membrane association. Concomitantly, lovastatin pretreatment reduced glucose-induced insulin release by about 50%; co-provision of 100-200 microM MVA totally prevented this effect. Perillic acid, a purported inhibitor of the prenylation of small GBPs, also markedly reduced glucose-induced insulin secretion. Furthermore, both N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), which inhibited the base-labile carboxyl methylation of GBPs in islets or in transformed beta-cells, and cerulenic acid, an inhibitor of protein palmitoylation, also reduced nutrient-induced secretion; an inactive analogue of AFC (which did not inhibit carboxyl methylation in islets) had no effect on secretion. In contrast with nutrients, the effects of agonists that induce secretion by directly activating distal components in signal transduction (such as a phorbol ester or mastoparan) were either unaffected or enhanced by lovastatin or AFC. These data are compatible with the hypothesis that post-translational modifications are required for one or more stimulatory GBPs to promote proximal step(s) in fuel-induced insulin secretion, whereas one or more inhibitory GBPs might reduce secretion at a more distal locus.

Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets.

Recent studies suggest a permissive requirement for guanosine 5'-triphosphate (GTP) in insulin release, based on the use of GTP synthesis inhibitors (such as myocophenolic acid) acting at inosine monophosphate (IMP) dehydrogenase; herein, we examine the glucose dependency of GTP synthesis. Mycophenolic acid inhibited insulin secretion equally well after islet culture at 7.8 or 11.1 mM glucose (51% inhibition) but its effect was dramatically attenuated when provided at < or = 6.4 mM glucose (13% inhibition; P < 0.001). These observations were explicable by a stimulation of islet GTP synthesis derived from IMP since, at high glucose: (a) total GTP content was augmented; (b) a greater decrement in GTP (1.75 vs. 1.05 pmol/islet) was induced by mycophenolic acid; and (c) a smaller "pool" of residual GTP persisted after drug treatment. Glucose also accelerated GTP synthesis from exogenous guanine ("salvage" pathway) and increased content of a pyrimidine, uridine 5'-triphosphate (UTP), suggesting that glucose augments production of a common regulatory intermediate (probably 5-phosphoribosyl-1-pyrophosphate). Pathway-specific radiolabeling studies confirmed that glucose tripled both salvage and de novo synthesis of nucleotides. We conclude that steep changes in the biosynthesis of cytosolic pools of GTP occur at modest changes in glucose concentrations, a finding which may have relevance to the adaptive (patho) physiologic responses of islets to changes in ambient glucose levels.

Selective inhibitors of GTP synthesis impede exocytotic insulin release from intact rat islets.

To investigate whether GTP concentrations can be a regulatory step in exocytotic hormone secretion, we treated isolated rat islets with mycophenolic acid (MPA) or mizoribine, two selective inhibitors of de novo GTP synthesis. When islets were cultured overnight in purine-free medium containing the drug, MPA reduced GTP levels by up to 81 +/- 1%; guanine circumvented this block via the nucleotide "salvage" pathway. MPA concomitantly inhibited glucose (16.7 mM)-induced insulin secretion in batch-type incubations (or perifusions), by up to 68% at 50 micrograms/ml. Although the inhibition of secretion occurred over a similar concentration range as the reduction in total GTP content, the two variables were not directly correlated. However, the secretory effects also were prevented by adding guanine, but not hypoxanthine or xanthine, to the culture medium. Similar results for GTP content and insulin release were seen using mizoribine. Insulin content was modestly (-18%) reduced by MPA but indices of fractional release (release/insulin content) were also markedly impaired. Although MPA also reduced ATP levels more modestly (-39%) and increased UTP (+87%), these were not the cause of the secretory defect since adenine restored ATP and UTP nearly to normal, but did not alter the reduction in GTP content or insulin secretion. MPA also inhibited secretion induced by amino acid or by a phorbol ester but had virtually no effect on release induced by a depolarizing concentration of K+, suggesting that GTP depletion does not merely impede Ca+ influx or directly block Ca(2+)-activated exocytosis. However, a severe reduction of GTP content did not prevent the pertussis toxin-sensitive inhibition of insulin release induced by epinephrine, suggesting that the function of heterotrimeric GTP-binding proteins is not limited by ambient GTP concentrations. Although these studies do not elucidate the exact site(s) in the exocytotic cascade which depend on intact GTP stores, they do provide the first direct evidence that GTP is required (and can be rate limiting) for insulin release.

Linoleic acid absorption in children with cystic fibrosis.

When safflower oil (triglyceride) was consumed without pancreatic enzymes by children with cystic fibrosis (CF), there was no rise in mean plasma linoleic acid levels over the next 4 h. When linoleic acid monoglyceride (LAM) was consumed, the increase in plasma linoleic acid levels was significantly greater than for safflower oil at 2 (p less than 0.02), 3 (p less than 0.01), and 4 h (p less than 0.01). When free fatty acid (hydrolyzed safflower oil) was ingested, there was almost no increase in plasma linoleic acid levels in CF or control children. The absorption of linoleic acid from triglyceride, but not from LAM, was greater when the CF children also took pancreatic enzymes. Three children with CF had greater increases in plasma linoleic acid levels following ingestion of safflower oil when they took antacid and cimetidine with their pancreatic capsules, compared to when they only took the pancreatic capsules.