p53-independent induction of p21(waf1/cip1) contributes to the activation of caspases in GTP-depletion-induced apoptosis of insulin-secreting cells.

We investigated the role of some key regulators of cell cycle in the activation of caspases during apoptosis of insulin-secreting cells after sustained depletion of GTP by a specific inosine 5'-monophosphate dehydrogenase inhibitor, mycophenolic acid (MPA). p21(Waf1/Cip1) was significantly increased following MPA treatment, an event closely correlated with the time course of caspase activation under the same conditions. MPA-induced p21(Waf1/Cip1) was not mediated by p53, since p53 mass was gradually reduced over time of MPA treatment. The increment of p21(Waf1/Cip1) by MPA was further enhanced in the presence of a pan-caspase inhibitor, indicating that the increased p21(Waf1/Cip1) may occur prior to caspase activation. This notion of association of p21(Waf1/Cip1) accumulation with caspase activation and apoptosis was substantiated by using mimosine, a selective p21(Waf1/Cip1) inducer independent of p53. Mimosine, like MPA, also increased p21(Waf1/Cip1), promoted apoptosis and simultaneously increased the activity of caspases. Furthermore, knocking down of p21(Waf1/Cip1) transfection of siRNA duplex inhibited caspase activation and apoptosis due to GTP depletion. In contrast to p21(Waf1/Cip1), a reduction in p27(Kip1) occurred in MPA-treated cells. These results indicate that p21(Waf1/Cip1) may act as an upstream signal to block mitogenesis and activate caspases which in turn contribute to induction of apoptosis.

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.

Evaluation of nurses and genetic counselors as providers of education about breast cancer susceptibility testing.

PURPOSE/OBJECTIVES: To compare outcomes of pretest education about breast cancer susceptibility testing provided by nurses and genetic counselors. DESIGN: Two-group, post-test only evaluation of an educational intervention. SETTING: A tertiary care hospital. SAMPLE: 87 women who had a first-degree relative with premenopausal breast cancer; six specially-trained providers (four genetic counselors and two nurses). METHODS: Self-administered questionnaire completed immediately following education sessions. MAIN RESEARCH VARIABLES: Subjects' understanding of the limitations of testing, perceived autonomy in decision making, and satisfaction; partnership as perceived by subjects and providers. FINDINGS: After the sessions, 62% of subjects understood the limitations of testing, 98% reported a high degree of perceived autonomy in decision making, 81% were highly satisfied with the session, and 91% reported forming a partnership with their providers. Lower perceived partnership reported by genetic counselors was the only significant difference by provider type. CONCLUSIONS: With training and supervision, nurses and genetic counselors can be equally effective in providing education about genetic testing for breast cancer susceptibility in research settings. Additional research is needed to determine the outcomes of education provided in clinical settings. IMPLICATIONS FOR NURSING PRACTICE: As the demand for education about genetic testing for cancer susceptibility increases, nurses need to be educated and trained to provide this service.

Effects of inhibitors of guanine nucleotide synthesis on membrane potential and cytosolic free Ca2+ levels in insulin-secreting cells.

Adenine nucleotides play an important role in the control of membrane potential by acting on ATP-sensitive K+ (K(ATP)) channels and, in turn, modulating the open probability of voltage-gated Ca2+ channels in pancreatic islet beta-cells. Here, we provide evidence that guanine nucleotides (GNs) also may be involved in the modulation of these events in vivo. GNs were depleted by treatment of HIT-T15 cells with mycophenolic acid (MPA). Resting membrane potential was more depolarized in cells treated for 3 and 6 hr with MPA than in control cells, and this effect was inhibited by diazoxide. After 6 hr of exposure to MPA, basal cytosolic free Ca2+ concentrations ([Ca2+]i) were elevated by 20%. Increments in [Ca2+]i induced by submaximal concentrations of K+ (10-15 mM) or bombesin were enhanced by > 50%. Opening K(ATP) channels with diazoxide lowered basal [Ca2+]i in MPA-treated cells to normal and abrogated the enhanced [Ca2+]i responses. However, an L-type Ca2+ channel blocker only abolished the enhanced [Ca2+]i response to stimuli and had no effect on the elevated basal [Ca2+]i, in contrast to EGTA, which obliterated both, implying that the latter was due to Ca2+ influx via non-L-type Ca2+ channels. These effects on ion fluxes were attributable specifically to GN depletion, since guanosine, which restores GTP content and the GTP/GDP ratio, but not adenosine, prevented all MPA-induced ion changes; furthermore, the latter were mimicked by mizoribine (a structurally dissimilar GTP synthesis inhibitor). It is concluded that, in addition to adenine nucleotides, GNs might contribute to the modulation of K(ATP) channels in intact beta-cells. In addition, GN depletion appeared to be able to reduce stimulated insulin secretion by a mechanism largely independent of the changes of ion fluxes observed above.

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.

Inosine monophosphate dehydrogenase: A molecular switch integrating pleiotropic GTP-dependent beta-cell functions.

Studies of pancreatic islet function in the pathogenesis of type 2 diabetes mellitus have tended to focus on the short-term control of insulin secretion. However, the long-term control of beta-cell mass is also relevant to diabetes, since this parameter is reduced substantially even in non-insulin-dependent diabetes in humans. In animal models of type 2 diabetes, the normal balance between beta-cell proliferation and programmed cell death is perturbed. We take the perspective in this overview that inosine monophosphate dehydrogenase (IMPDH; EC 1.1.1. 205) may represent a previously neglected molecular integrator or sensor that exerts both functional (secretory) and anatomical (proliferative) effects within beta-cells. These properties reflect the fact that IMPDH is a rate-limiting enzyme in the new synthesis of the purine guanosine triphosphate (GTP), which modulates both exocytotic insulin secretion and DNA synthesis, as well as a number of other critical cellular functions within the beta-cell. Alterations in the expression or activity of IMPDH may be central to beta-cell replication, cell cycle progression, differentiation, and maintenance of adequate islet mass, effects that are probably mediated both by GTP directly, and indirectly via low molecular mass GTPases. If GTP becomes depleted, a hierarchy of beta-cell functions becomes progressively paralyzed, until eventually the effete cell is removed via apoptosis.

Participation in breast cancer susceptibility testing protocols: influence of recruitment source, altruism, and family involvement on women's decisions.

OBJECTIVES: We offered education, counseling, and family-based BRCA1/2 testing to women at increased risk of breast cancer and assessed (a) their reasons for participating and (b) whether source of recruitment, desire to help research (altruism), and the need to communicate with their affected relative about testing distinguish those who did and those who did not complete each phase of our protocol. MATERIALS AND METHODS: We sent invitations to 403 women who had completed a questionnaire on BRCA1/2 testing, 178 of whom were considered high risk because they had more than one relative on the same side of the family with early-onset breast cancer. RESULTS: Among the 132 high-risk respondents from the mid-Atlantic states (where testing was offered), 36% (n = 47) were interested in counseling. Those who actually attended counseling were more likely to have some college education, a higher perceived risk of breast cancer, and a greater fear of stigma and were less likely to have a daughter than those who did not attend. The reasons for attending that were rated "very important" were to learn about the test (80%), to have the test (43%), and to help research (38%). High-risk women were eligible for testing only if their affected relative was willing to be tested and tested positive. After the session, 83% intended to ask their affected relative to be tested, but only half of the affected relatives actually came for pretest counseling. The proportion of participants who ultimately involved an affected relative was 2.5 times higher among women from a clinical population (25%) than among those from a registry population (10%); in this latter population, an altruistic desire to help research was a greater motivator for participation than interest in being tested. CONCLUSIONS: Source of recruitment influences both motivations to attend education and counseling and actual testing behavior. These results have implications for interpretation of findings from studies in research settings as well as for informed consent and decision-making in the context of family-based testing.

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.

Use of a soluble tetrazolium compound to assay metabolic activation of intact beta cells.

Although assessments of metabolic activation are central to studies of beta-cell function, available techniques are tedious, insensitive, and/or require cell disruption. We have investigated the use of a new water-soluble tetrazolium salt, MTS (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4- sulfophenyl]-2H-tetrazolium, inner salt), in the presence of phenazine methosulfate (PMS), an intermediate electron acceptor that amplifies its signal (fluorescence at 490 nm). During static incubations of glucose-responsive (HIT-T15 or INS-1) dispersed beta cells with increasing glucose concentrations, there was a progressive increase in MTS reduction, with a maximum signal-to-noise (S/N) ratio of 24 with HIT-T15 cells and 10 with INS-1 cells. This was associated with, but not attributable to, parallel increases in insulin secretion. Pure mitochondrial fuels (alpha-ketoisocaproate [KIC], methyl pyruvate [MP], or L-glutamine [GLN] + L-leucine [LEU]) also increased the reduction of MTS in INS-1 cells (6.5-, 4.8-, and 14.4-fold, respectively), but generally less than glucose, suggesting a major role of glycolysis in the signal induced by glucose. Inhibitors of glucose metabolism (mannoheptulose [MH], lodoacetate [IA], or 2-deoxyglucose [2-DG]) markedly reduced the glucose-stimulated MTS signal. In comparison to another tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), MTS assay provided a better S/N ratio with glucose or other nutrient secretagogues. Extant theory holds that activation of mitochondrial dehydrogenases by increments in Ca2+ influx couples glycolysis to mitochondrial oxidation of glucose-derived fuels. However, reduction of fuel-induced calcium influx (by Ca2+-free medium or diazoxide [DZX]) or direct stimulation of calcium influx (by 40 mmol/L K+) failed to significantly modulate the signal, arguing against this theory. We conclude that the MTS assay is a facile test that reflects the global metabolic function of insulin-secreting beta cells. Furthermore, since this assay does not require disruption of cells to solubilize the formazan product, and therefore also allows concomitant measurement of insulin secretion, it offers considerable advantages over earlier methods.

Purine nucleotide- and sugar phosphate-induced inhibition of the carboxyl methylation and catalysis of protein phosphatase-2A in insulin-secreting cells: protection by divalent cations.

Recently, we demonstrated that the 36 kDa catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes methylation at its C-terminal leucine in normal rat islets, human islets and isolated beta cells; this modification increases the catalytic activity of PP2A [Kowluru et al. Endocrinology. 137:2315-2323, 1996]. Previous studies have suggested that adenine and guanine nucleotides or glycolytic intermediates [which are critical mediators in beta cell function] also modulate phosphatase activity in the pancreatic beta cell. Therefore, we examined whether these phosphorylated molecules specifically regulate the carboxyl methylation and the catalytic activity of PP2A in beta cells. Micromolar concentrations of ATP, ADP, GTP or GDP each inhibited the carboxyl methylation of PP2Ac and, to a lesser degree, the catalytic activity of PP2A. Likewise, the carboxyl methylation of PP2Ac and its catalytic activity were inhibited by [mono- or di-] phosphates of glucose or fructose. Additionally, however, the carboxyl methylation of PP2Ac was significantly stimulated by divalent metal ions (Mn2+ > Mg2+ > Ca2+ > control). The nucleotide or sugar phosphate-mediated inhibition of carboxyl methylation of PP2Ac and the catalytic activity of PP2A were completely prevented by Mn2+ or Mg2+. These data indicate that divalent metal ions protect against the inhibition by purine nucleotides or sugar phosphates of the carboxyl methylation of PP2Ac perhaps permitting PP2A to function under physiologic conditions. Therefore, these data warrant caution in interpretation of extant data on the regulation of phosphatase function by purine nucleotides.

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.

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.

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.

Inhibition of calcium-induced insulin secretion from intact HIT-T15 or INS-1 beta cells by GTP depletion.

Using intact rat islets, we previously observed that GTP depletion (achieved through the use of mycophenolic acid or other synthesis inhibitors) impedes nutrient- but not K+-induced insulin secretion. It was concluded that a proximal nutrient-dependent step in stimulus-secretion coupling (but not the process of Ca2+-induced exocytosis itself) is modulated by ambient GTP levels. To examine Ca2+-dependent steps further in intact beta cells, INS-1 cells (which synthesize GTP and ATP similarly to rat islets) and HIT-T15 cells (whose synthesis of purine nucleotides is different) were studied following cell culture for 1-18 hr in various concentrations of mycophenolic acid (MPA) or mizoribine (MZ). Both agents profoundly reduced GTP content (mean: -78%) and lowered the GTP/GDP ratio by an average of -73%; concomitantly, MPA or MZ reduced insulin secretion induced by 10 mM glucose, 30 or 40 mM KCl, or 100 microM tolbutamide, independent of any changes in cell viability, insulin content, ATP content, the ATP/ADP ratio, or cytosolic free Ca2+ concentrations. In INS-1 cells (which appear to have normal nucleobase transport and "salvage" pathway activities), guanine (but not adenine) restored GTP content, the GTP/GDP ratio, and Ca2+-induced secretion. In HIT cells, the phosphoribosylation of exogenous guanine or hypoxanthine is defective; however, provision of 500 microM guanosine (but not adenosine) reversed the effects of MPA. We conclude that, at least in certain situations, a requisite role for GTP in the distal step(s) of exocytosis can be demonstrated.

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.

Immunolocalization of eicosanoid enzymes and growth factors in human myometrium and fetoplacental tissues in failed labor inductions.

OBJECTIVE: To investigate the correlation between myometrial-derived eicosanoids and growth factors during the onset of parturition. METHODS: Myometrial samples were obtained from patients who were delivered by cesarean for failed induction or abnormal fetal heart rate tracings but who experienced normal labor progression until the occurrence of the abnormal tracing. Placentas and fetal membranes were obtained from patients with normal labor, no labor, and failed labor progression. The tissues were processed and sections were immunostained for cyclooxygenases, prostacyclin synthetase (PGI2-S), thromboxane A2 synthetase (TXA2-S), 5-lipoxygenase, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and EGF receptor, using specific antibodies directed against these molecules. RESULTS: Myometrial and fetoplacental tissues from women with normal labor, no labor, and failed labor contain immunoreactive cyclooxygenases, 5-lipoxygenase, TXA2-S, PGI2-S, EGF, TGF-alpha, and EGF receptor. However, their immunostaining intensity, with the exception of EGF receptor, decreased substantially in myometrium from women with failed labor induction compared with those having normal labor progression. No difference was noted in the immunostaining intensity of growth factors and eicosanoid enzymes in the fetoplacental membranes from these patients, except for cyclooxygenases, which were prominent in fetal membranes from normal labor compared with failed labor and no labor. CONCLUSION: Myometrial-derived eicosanoids and growth factors may be important in processes of parturition because reduction in their production in the myometrium is correlated with failed labor induction. Because of the regulatory action of growth factors in eicosanoid biosynthesis in uterine and fetoplacental tissues, EGF/TGF-alpha may indirectly influence the process of parturition by regulating eicosanoid production in the myometrium.

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.