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Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic β-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study, we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion. miRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2-fold in INS-1 832/3 cells, which were subsequently reproduced in freshly isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the protein kinase A inhibitor H-89 but not affected by the exchange protein activated by cAMP activator 8-pCPT-2'-O-Me-cAMP-AM. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced glucose-stimulated insulin secretion in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/protein kinase A-dependent pathway in pancreatic β-cells. Overexpression of miR-132 or miR-212 enhances glucose and GLP-1-stimulated insulin secretion.

We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptin(ob) mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis.

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.

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.

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.

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).

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.

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.

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.

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.