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Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within b-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in b-cells selectively activated protein kinase R–like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion. Fil: Ghiasi, Seyed Mojtaba. Universidad de Copenhagen; Dinamarca Fil: Dahlby, Tina. Universidad de Copenhagen; Dinamarca Fil: Andersen, Caroline Hede. Universidad de Copenhagen; Dinamarca Fil: Haataja, Leena. University of Michigan; Estados Unidos Fil: Petersen, Sólrun. Universidad de Copenhagen; Dinamarca Fil: Omar-Hmeadi, Muhmmad. Uppsala Universitet; Suecia Fil: Yang, Mingyu. Uppsala Universitet; Suecia Fil: Pihl, Celina. Universidad de Copenhagen; Dinamarca Fil: Bresson, Sophie Emilie. Universidad de Copenhagen; Dinamarca Fil: Khilji, Muhammad Saad. Universidad de Copenhagen; Dinamarca Fil: Klindt, Kristian. Universidad de Copenhagen; Dinamarca Fil: Cheta, Oana. Universidad de Copenhagen; Dinamarca Fil: Perone, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Copenhagen; Dinamarca Fil: Tyrberg, Björn. Astrazeneca. IMED Biotech Unit; Suecia Fil: Prats, Clara. Universidad de Copenhagen; Dinamarca Fil: Barg, Sebastian. Uppsala Universitet; Suecia Fil: Tengholm, Anders. Uppsala Universitet; Suecia Fil: Arvan, Peter. University of Michigan; Estados Unidos Fil: Mandrup-Poulsen, Thomas. Universidad de Copenhagen; Dinamarca Fil: Marzec, Michal Tomasz. Universidad de Copenhagen; Dinamarca

Epac is a cAMP-activated guanine nucleotide exchange factor that mediates cAMP signaling in various types of cells, including β-cells, where it is involved in the control of insulin secretion. Upon activation, the protein redistributes to the plasma membrane, but the underlying molecular mechanisms and functional consequences are unclear. Using quantitative high-resolution microscopy, we found that cAMP elevation caused rapid binding of Epac2A to the β-cell plasma membrane, where it accumulated specifically at secretory granules and rendered them more prone to undergo exocytosis. cAMP-dependent membrane binding required the high-affinity cyclic nucleotide-binding (CNB) and Ras association domains, but not the disheveled–Egl-10–pleckstrin domain. Although the N-terminal low-affinity CNB domain (CNB-A) was dispensable for the translocation to the membrane, it was critical for directing Epac2A to the granule sites. Epac1, which lacks the CNB-A domain, was recruited to the plasma membrane but did not accumulate at granules. We conclude that Epac2A controls secretory granule release by binding to the exocytosis machinery, an effect that is enhanced by prior cAMP-dependent accumulation of the protein at the plasma membrane.

Chronic palmitate exposure impairs glucose-stimulated insulin secretion and other aspects of β-cell function, but the underlying mechanisms are not known. Using various live-cell fluorescence imaging approaches, we show here that long-term palmitate treatment influences cAMP signaling in pancreatic β-cells. Glucose stimulation of mouse and human β-cells induced oscillations of the subplasma-membrane cAMP concentration, but after 48 h exposure to palmitate, most β-cells failed to increase cAMP in response to glucose. In contrast, GLP-1–triggered cAMP formation and glucose- and depolarization-induced increases in cytoplasmic Ca2+ concentration were unaffected by the fatty acid treatment. Insulin secretion from control β-cells was pulsatile, but the response deteriorated after long-term palmitate exposure. Palmitate-treated mouse islets showed reduced expression of adenylyl cyclase 9, and knockdown of this protein in insulinoma cells reduced the glucose-stimulated cAMP response and insulin secretion. We conclude that impaired glucose-induced generation of cAMP is an important determinant of defective insulin secretion after chronic palmitate exposure.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is important for a variety of cellular processes as a precursor for second messengers and by regulating ion channels, the cytoskeleton, and vesicle traffic in many types of cells, including insulin-secreting beta-cells. Here, we applied evanescent wave microscopy and the PIP(2)-binding pleckstrin homology domain from phospholipase C (PLC)-delta fused to the green fluorescent protein to characterize the regulation of plasma membrane PIP(2) in individual insulin-secreting MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mmol/l evoked antisynchronous oscillations of [PIP(2)] and cytoplasmic Ca(2+)concentration, consistent with PLC being periodically activated by the voltage-dependent Ca(2+) influx. The effect of adenine nucleotides on [PIP(2)] was studied in cells permeabilized with alpha-toxin. ATP dose- dependently stimulated PIP(2) synthesis with half-maximal effect at 300 mumol/l. Omission of the nucleotide resulted in rapid loss of PIP(2) with t(1/2) < 40 s. ADP also stimulated PIP(2) formation, but this effect reflected local ATP formation and was prevented by the adenylate kinase inhibitor diadenosine-pentaphosphate. The ATP-induced PIP(2) synthesis was counteracted by the ADP analog adenosine-5'-O-2-thiodiphosphate. We conclude that plasma membrane PIP(2) is dynamically regulated by intracellular Ca(2+) and the ATP-to-ADP ratio in insulin-secreting cells. The rapid turnover allows maintenance of PIP(2) levels while generating second messengers of critical importance for insulin secretion.

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.

OBJECTIVE cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown. RESEARCH DESIGN AND METHODS The subplasma-membrane cAMP concentration ([cAMP]pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells. RESULTS In islets exposed to 3 mmol/L glucose, [cAMP]pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]pm oscillations in most α-cells, CONCLUSIONS Oscillatory [cAMP]pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion.