Suggested mechanism for the selective excretion of glucosylated albumin. The effects of diabetes mellitus and aging on this process and the origins of diabetic microalbuminuria.

In previous studies in the Sprague-Dawley rat, Williams and coworkers reported the phenomenon of selective urinary excretion of glucosylated albumin (editing, i.e., the percent glucosylation of urinary albumin is more than that of plasma albumin) by the mammalian kidney. Ghiggeri and coworkers subsequently found that the extent of editing is reduced in human diabetics. Moreover, the reduction in editing in diabetes correlates inversely with levels of microalbuminuria. We also find reduction in the extent of editing in diabetic humans. We find a striking inverse correlation not only with the magnitude of microalbuminuria but also with the extent of plasma albumin glucosylation. In contrast, we found little correlation between the reduction in editing and the duration of diabetes in human subjects. Stz induced diabetes in the Sprague-Dawley rat is associated with a striking and rapid reduction in editing which develops virtually with the same kinetics exhibited by the appearance of hyperglycemia. This loss of editing is rapidly reversed by daily administration of insulin but not by aldose reductase inhibitors. Mannitol infusion in anesthetized Wistar rats resulted in an increase in urine volume, GFR, and microalbuminuria, and was also accompanied by a marked reduction in editing. This reduction was rapidly reversed by a cessation of mannitol infusion. We propose here that glucosylated albumin (in contrast to unmodified albumin) is not reabsorbed by the proximal tubule, and thus, is preferentially excreted in the urine. We postulate that the increase in GFR which emerges as a consequence of increased plasma osmolality in diabetes mellitus delivers more albumin to the proximal tubule than can be reabsorbed. This results in a dilution of excreted glucosylated albumin molecules by excreted unmodified albumin, which appears as the early microscopic albuminuria of diabetes. Paradoxically, the fall in apparent editing is accompanied by an absolute increase in the total quantity of glucosylated albumin excreted. In contrast, we found that editing of glucosylated albumin by the normal kidney is found to gradually decline as a function of age without the appearance of microalbuminuria. This suggests that a different mechanism operates to produce the loss of editing seen with aging in man, and as clearly (but in a shorter absolute time intervals) in the Fischer-344 rat.

PP2A contributes to endothelial death in high glucose: inhibition by benfotiamine.

Endothelial death is critical in diabetic vascular diseases, but regulating factors have been only partially elucidated. Phosphatases play important regulatory roles in cell metabolism, but have not previously been implicated in hyperglycemia-induced cell death. We investigated the role of the phosphatase, type 2A protein phosphatase (PP2A), in hyperglycemia-induced changes in signaling and death in bovine aortic endothelial cells (BAEC). We explored also the influence of benfotiamine on this phosphatase. Activation of PP2A was assessed in BAEC by the extent of methylation and measurement of activity, and the enzyme was inhibited using selective pharmacological (okadaic acid, sodium fostriecin) and molecular (small interfering RNA) approaches. BAECs cultured in 30 mM glucose significantly increased PP2A methylation and activity, and PP2A inhibitors blocked these abnormalities. PP2A activity was increased also in aorta and retina from diabetic rats. NF-κB activity and cell death in BAEC were significantly increased in 30 mM glucose and inhibited by PP2A inhibition. NF-κB played a role in the hyperglycemia-induced death of BAEC, since blocking its translocation with SN50 also inhibited cell death. Inhibition of PP2A blocked the hyperglycemia-induced dephosphorylation of NF-κB and Bad, thus favoring cell survival. Incubation of benfotiamine with BAEC inhibited the high glucose-induced activation of PP2A and NF-κB and cell death, as well as several other metabolic defects, which likewise were inhibited by inhibitors of PP2A. Activation of PP2A contributes to endothelial cell death in high glucose, and beneficial actions of benfotiamine are due, at least in part, to inhibition of PP2A activation.

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

The gamma subunits of trimeric G-proteins (gamma1, gamma2, gamma5, and gamma7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure beta [HIT-T15] cells. Of these, GTPgammaS significantly stimulated the carboxyl methylation selectively of gamma2 and gamma5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists--a stimulatory concentration of glucose or a depolarizing concentration of K+--resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of gamma subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from beta cells), also stimulated the carboxyl methylation of gamma subunits in intact HIT cells. Stimulatory effects of glucose or K+ were not demonstrable after removal of extracellular Ca2+ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca2+, implying a mechanism of action that is different from glucose or K+ but also suggesting that dissociation of the alphabetagamma trimer is conducive to gamma subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K+ or mastoparan without altering the rise in intracellular calcium induced by glucose or K+. Glucose-induced carboxyl methylation of gamma2 and gamma5 isoforms was vitiated by coprovision of any of three structurally different cyclooxygenase inhibitors. Conversely, exogenous PGE2, which activates Gi and Go in HIT cells and which thereby would dissociate alpha from beta(gamma), stimulated the carboxyl methylation of gamma2 and gamma5 isoforms and reversed the inhibition of glucose-stimulated carboxyl methylation of gamma subunits elicited by cyclooxygenase inhibitors. These data indicate that gamma subunits of trimeric G-proteins undergo a glucose- and calcium-regulated methylation-demethylation cycle in insulin-secreting cells, findings that may imply an important role in beta cell function. Furthermore, this is the first example of the regulation of the posttranslational modification of G-protein gamma subunits via nonreceptor-mediated activation mechanisms, which are apparently dependent on calcium influx and the consequent activation of phospholipases releasing arachidonic acid.

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.

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.

Regulation of guanine-nucleotide binding proteins in islet subcellular fractions by phospholipase-derived lipid mediators of insulin secretion.

In the accompanying article (Kowluru, A., Rabaglia, M.E., Mose, K.E. and Metz, S.A. (1994) Biochim. Biophys. Acta 1222, 348-359) we identified three specific GTPase activities in islet subcellular fractions; most notably, two of these were enriched in the secretory granules. In the present study, we describe the regulation of GTPase activity in subcellular fractions of normal rat and human islets by insulinotropic lipids with a similar rank order as their insulin-releasing capacity. Arachidonic acid (AA), lysophosphatidylcholine (LPC), or phosphatidic acid (PA) inhibited the GTPase activities significantly (by 60-80%) in islet homogenates; each also selectively inhibited certain GTPases in specific individual fractions. Less insulinotropic fatty acids, such as linoleic acid and oleic acid, inhibited GTPase to a lesser degree, whereas lysophosphatidic acid (LPA), phosphatidylcholine (PC) or palmitic acid, which do not acutely promote secretion, were ineffective. Similar inhibitory effects of these lipids were also demonstrable in fractions of human islets as well as those of transformed beta-cells (HIT cells). The effects of lipids were not attributable to their detergent properties (since several detergents failed to mimic lipid effects) or to inhibition of GTP binding (since they actually increased GTP gamma S binding modestly, and moreover, in reconstituted fractions, they potentiated GDP/GTP exchange activity up to 2-fold). These data indicate that the insulinotropic nature of the lipids might be due, in part, to their ability to maintain G-proteins in their GTP-bound (active) configuration by increasing GTP binding and decreasing its hydrolysis. These studies comprise the first evidence for the regulation by biologically active lipids of endocrine cell G-proteins at a locus distal to plasma membrane events (i.e., on endocrine secretory granules), and provide thereby a possible novel mechanism whereby the activation of islet endogenous phospholipases might culminate in insulin exocytosis.

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.

Calcium-calmodulin-dependent myosin phosphorylation by pancreatic islets.

Pancreatic islets contain enzyme activity which catalyzes the phosphorylation by MgATP of cardiac, skeletal, or smooth muscle myosin light chains. The enzyme is activated by calcium (Ka = 10 microM) and calmodulin (Ka = 2 nM) and inhibited by trifluoperazine (Ki = 10 microM), a known inhibitor of calmodulin and of insulin secretion. The enzyme binds to a calmodulin affinity column when Ca2+ is present and is eluted when Ca2+ is omitted. These are the properties of myosin light chain kinase. Since phosphorylation of smooth muscle myosin is necessary for its activation by actin, the kinase may have a key role in coupling stimuli that increase intracellular calcium to the contractile processes involved in insulin secretion.

Activation of acetyl-CoA carboxylase by a glutamate- and magnesium-sensitive protein phosphatase in the islet beta-cell.

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of long-chain fatty acids, which have been implicated in physiological insulin secretion. The catalytic function of ACC is regulated by phosphorylation (inactive)-dephosphorylation (active). In this study we investigated whether similar regulatory mechanisms exist for ACC in the pancreatic islet beta-cell. ACC was quantitated in normal rat islets, human islets, and clonal beta-cells (HIT-15 or INS-1) using a [(14)C]bicarbonate fixation assay. In the beta-cell lysates, ACC was stimulated by magnesium in a concentration-dependent manner. Of all the dicarboxylic acids tested, only glutamate, albeit ineffective by itself, significantly potentiated magnesium-activated ACC in a concentration-dependent manner. ACC stimulation by glutamate and magnesium was maximally demonstrable in the cytosolic fraction; it was markedly reduced by okadaic acid (OKA) in concentrations (<50 nmol/l) that inhibited protein phosphatase 2A (PP2A). Furthermore, pretreatment of the cytosolic fraction with anti-PP2A serum attenuated the glutamate- and magnesium-mediated activation of ACC, thereby suggesting that ACC may be regulated by an OKA-sensitive PP2A-like enzyme. Streptavidin-agarose chromatography studies have indicated that glutamate- and magnesium-mediated effects on ACC are attributable to activation of ACC's dephosphorylation; this suggests that the stimulatory effects of glutamate and magnesium on ACC might involve activation of an OKA-sensitive PP2A-like enzyme that dephosphorylates and activates ACC. In our study, 5-amino-imidazolecarboxamide (AICA) riboside, a stimulator of AMP kinase, significantly inhibited glucose-mediated activation of ACC and insulin secretion from isolated beta-cells. Together, our data provide evidence for a unique regulatory mechanism for the activation of ACC in the pancreatic beta-cell, leading to the generation of physiological signals that may be relevant for physiological insulin secretion.

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.

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.

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.

Ceramide-activated protein phosphatase-2A activity in insulin-secreting cells.

Okadaic acid (OKA)-sensitive phosphatase (PP2A) activity may modulate nutrient-induced insulin secretion from pancreatic beta cells [Kowluru et al., Endocrinology 137 (1996) 2315-2323]. Ceramides, a new class of lipid second messengers may regulate PP2A [Dobrowsky and Hannun, J. Biol. Chem. (1992) 267, 5048-5051], and might play a role in cytokine-mediated apoptosis in beta cells [Sjöholm, FEBS Lett. 367 (1995) 283-286]. Therefore, we investigated the regulation of PP2A-like activity by ceramides in isolated beta (HIT-T15 or INS-1) cells. Cell-permeable (C2, C6 or C18) ceramides stimulated OKA-sensitive (but not -insensitive) phosphatase activity in a concentration-dependent manner (0-12.5 microM), with maximal stimulation (+50-100%) at < 12.5 microM. C2-dihydroceramide (a biologically inactive analog of C2 ceramide) failed to augment PP2A-like activity. Stimulatory effects of ceramides do not appear to be mediated via activation of the carboxyl methylation of the catalytic subunit of protein phosphatase 2A, since no effects of ceramides (up to 25 microM) were demonstrable on this parameter. These data identify a ceramide-activated protein phosphatase as a possible locus at which ceramides might exert their effects on beta cells leading to altered insulin secretion, and decreased cell viability followed by apoptotic cell demise.

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.

Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells.

In the present study, we have shown that exposure of insulin-secreting clonal beta (HIT-T15) cells to interleukin-1beta (IL-1beta) results in a time- and concentration-dependent increase in nitric oxide (NO) release. These effects by IL-1beta on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells. Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1beta-induced NO release. Pre-exposure of HIT cells to C. sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1beta-mediated NO release. These data indicate that activation of Ras and/or Rac may be necessary for IL-1beta-mediated NO release. Preincubation of HIT cells with C. difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step. Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1beta-mediated NO release from these cells. Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1beta-mediated NO release and, presumably, the subsequent dysfunction of the pancreatic beta cell. Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced beta cell death leading to the onset of insulin-dependent diabetes mellitus.

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).

Inhibitors of post-translational modifications of G-proteins as probes to study the pancreatic beta cell function: potential therapeutic implications.

It is well established that glucose-induced insulin secretion involves generation of intracellular second messengers. Using specific inhibitors of guanosine triphosphate [GTP] biosynthesis [e.g., mycophenolic acid; MPA], we have identified a permissive role for GTP in glucose-stimulated insulin secretion. While the exact site of action for GTP within the islet beta cell remains to be identified and defined, recent evidence from several laboratories, including our own, indicate that it could involve activation of GTP-binding proteins [G-proteins]. These studies have identified both trimeric and monomeric forms of G-proteins within the pancreatic beta cell. Recent data also indicate that these G-proteins, specifically the monomeric G-proteins and the gamma subunits of trimeric G-proteins undergo a series of posttranslational modifications at their C-terminal cysteine. Such modifications include, isoprenylation, carboxyl methylation and palmitoylation. These modification steps appear to be essential for translocation of these proteins to the membrane sites for interaction with their respective effector proteins. This review primarily focuses on recent findings that clearly support the viewpoint that these posttranslational modification steps not only play obligatory roles in fuel-induced insulin secretion, but also in cytokine-mediated apoptotic demise of the beta cell. In this review, we also attempted to describe those findings involving the use of specific inhibitors for each of these pathways, and it is our hope that these aspects of beta cell metabolism and function generate interest in development of therapeutic intervention modalities to states of perturbed insulin release.

Erythrocyte sodium-potassium ATPase activity and thiol metabolism in genetically hyperglycemic mice.

Erythrocyte sodium pump activity, osmotic fragility, and thiol status were measured in genetically hyperglycemic (db/db) mice and compared with their nondiabetic littermates (db/m). The data showed no major differences in these parameters. However, erythrocytes from streptozotocin (Stz)-induced diabetic rats had significantly lower activity of sodium pump and thiols with an almost fourfold increase in osmotic fragility as compared with erythrocytes from nondiabetic rats. Sorbinil (an aldose reductase inhibitor) treatment of Stz-diabetic rats normalized all these lesions, suggesting a key role for polyol pathway. However, sorbitol levels in erythrocytes from db/db and db/m mice were undetectable. The data suggest that in db/db mice, the relative lack of polyol pathway, a potential consumer of NADPH, may provide erythrocytes with optimal NADPH for glutathione reductase system, thus maintaining normal GSH levels even at the height of hyperglycemia. Thus, the genetically hyperglycemic mice may serve as a useful model to study diabetes related complications without involving polyol pathway.

Evidence for phosphorylation of pancreatic islet pyruvate kinase.

Rabbit pancreatic islet cytosol catalyzes the calcium-activated phosphorylation by [gamma 32P]ATP of a protein with a molecular weight of 57,000 that is precipitated with antipyruvate kinase antibodies. We were unable to demonstrate that phosphorylation in the presence of calcium or cAMP had any immediate effect on rat pancreatic islet pyruvate kinase activity. This finding is consistent with our inability to confirm the finding of others that pancreatic islets contain phosphoenolpyruvate carboxykinase activity (Diabetes, 34:246, 1985). Since the carboxykinase catalyzes phosphoenolpyruvate formation and pyruvate kinase catalyzes essentially the opposite reaction, if the carboxykinase were present in the beta cell, pyruvate kinase would need to be inhibited to prevent recycling of phosphoenolpyruvate.

IL-1beta-induced nitric oxide release from insulin-secreting beta-cells: further evidence for the involvement of GTP-binding proteins.

Recently, we have demonstrated regulatory roles for G-proteins (e.g., H-Ras) in IL-1beta induced NO release from HIT-T15 cells. Herein, we report a similar regulatory mechanism for IL-1beta induced NO release from RIN5F and INS-1 cells. Our data indicate that functional inactivation of Ras, either by Clostridial toxins or by specific inhibitors of Ras function, results in a significant inhibition in IL-1beta induced NO release, suggesting that activation of specific G-proteins is essential for IL-1beta induced NO release. In the present study, we report possible loci where IL-1beta treatment might result in functional activation of these G-proteins. For example, IL-1beta treatment resulted in significant reduction in (high-and low-affinity) GTPase activities in lysates derived from normal rat islets; such a scenario might lead to retention of candidate G-proteins in GTP-bound, active conformation. Further, IL-1beta treatment increased the G-protein carboxyl methyl transferase activity as well as carboxyl methylation of endogenous beta-cell proteins; such a modification has been shown to increase the membrane association and interaction of these G-proteins with their respective effector proteins. Also, we report immunologic localization of H-Ras regulatory proteins including its nucleotide exchange factor (GRF-1) and its effector protein (eg., Raf-1) in isolated beta-cells. Together, our data indicate localization, and regulation by IL-1beta, of specific enzymes that are critical to activation of G-proteins. Based on these preliminary findings, we propose a model for the involvement of G-proteins in IL-1beta induced NO release and subsequent demise of the pancreatic beta-cell.