Anjaneyulu Kowluru

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.

Evidence for Differential Roles of the Rho Subfamily of GTP-Binding Proteins in Glucose- and Calcium-Induced Insulin Secretion from Pancreatic β 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).

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 β 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 β cells [Sjöholm, FEBS Lett. 367 (1995) 283–286]. Therefore, we investigated the regulation of PP2A‐like activity by ceramides in isolated β (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 μM), with maximal stimulation (+50–100%) at <12.5 μM. 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 μM) 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 β cells leading to altered insulin secretion, and decreased cell viability followed by apoptotic cell demise.

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.

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

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.

Calcium–Calmodulin-Dependent Myosin Phosphorylation by Pancreatic Islets

SUMMARY 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 μM) and calmodulin (Ka = 2 μM) and inhibited by trifluoperazine (Ki = 10 μM), 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.

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.

Secretagogue-responsive and -unresponsive pools of phosphatidylinositol in pancreatic islets

The effect of glucose on phosphatidylinositol turnover was studied. Phosphatidylinositol of rat pancreatic islets was labeled with myo[2-3H]inositol in the presence of various secretagogues (16.7 mM D-glucose, 22 mM D-mannose, 20 mM D-glyceraldehyde) and nonsecretagogues (3.3 mM D-glucose, 20 mM pyruvate, 16.7 mM D-galactose, 16.7 mM L-glucose). Upon subsequent stimulation with 16.7 mM D-glucose, only the islets that were labeled in the presence of secretagogues showed a loss of radioactivity from phosphatidylinositol. No loss of radioactivity from phosphatidylinositol occurred in the presence of 3.3 mM D-glucose even after labeling in the presence of secretagogues. A comparison of the subcellular distribution of labeled phosphatidylinositol in islets before and after stimulation with insulinotropic glucose revealed a loss of radioactivity from the plasma membrane fraction as judged by subcellular fractionation with a sucrose gradient. These results support a hypothesis advanced previously that pancreatic islets contain a unique pool of phosphatidylinositol that undergoes rapid turnover only in the presence of insulinotropic concentrations of D-glucose or other secretagogues [R. S. Rana, R. J. Mertz, A. Kowlura, J. F. Dixon, L. E. Hokin, and M. J. MacDonald (1985) J. Biol. Chem. 260, 7861-7867]. On the basis of the subcellular fractionation studies reported here, the secretagogue-responsive phosphatidylinositol pool appears to be located primarily in the plasma membrane of pancreatic islets.

GTP and Its Binding Proteins in the Regulation of Insulin Exocytosis

Despite considerable evidence for the involvement of the purine nucleotides GTP* and ATP in exocytosis, there is currently no consensus about their exact role(s). With regard to GTP (the focus of this chapter), our recent studies (1) have suggested that there is at least a permissive role for GTP in insulin release. Normal rat pancreatic islets were pretreated overnight with each of four structurally-dissimilar inhibitors of a rate-limiting step in the cytosolic production of GTP (inosine monophosphate dehydrogenase; IMPDH). When islet concentrations of GTP were inhibited by more than about 80% (to levels down to less than 0.6–0.7 pmol/islet), insulin release subsequently stimulated by nutrients (glucose or α-ketoisocaproate) or by aphorbol ester was reduced 40–60%. In contrast, insulin secretion induced by direct depolarization of β-cells (by 50 m M K+) was refractory to inhibition. These results were attributed to a depletion of islet GTP stores, since repletion of GTP by coprovision of guanine (but not the normalization of both ATP and UTP content by the provision of adenine) reversed the inhibitory effects of the drugs both on GTP content and on insulin release. In subsequent studies, we observed that lesser degrees of inhibition of GTP, or, conversely, a stimulation of islet GTP content (by provision of guanine alone) to levels about 25% above basal, did not have any discernible negative or positive modulatory effects on secretion. These findings suggested that islet GTP content is normally saturating, and therefore mild to moderate changes in these levels do not alter secretion. Since the average islet content of GTP approximates 750–1000 μM, it appears that a reduction in total islet GTP levels to less than 150–200 μM (or, possibly, a comparable relative reduction of free GTP levels, which may be one-fifth to one-twentieth of total levels; ref. 2) is needed to vitiate the effects of GTP on secretion. Such drastic changes are not likely to be induced by physiologic agonists (and in fact were not induced by glucose, which had relatively modest effects on total islet GTP content); therefore, we defined the effects of GTP as “permissive.”