Epac-selective cAMP Analog 8-pCPT-2′-O-Me-cAMP as a Stimulus for Ca2+-induced Ca2+ Release and Exocytosis in Pancreatic β-Cells

The second messenger cAMP exerts powerful stimulatory effects on Ca2+ signaling and insulin secretion in pancreatic β-cells. Previous studies of β-cells focused on protein kinase A (PKA) as a downstream effector of cAMP action. However, it is now apparent that cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac). Although one effector of Epac is the Ras-related G protein Rap1, it is not fully understood what the functional consequences of Epac-mediated signal transduction are at the cellular level. 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′-5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) is a newly described cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2′-O-Me-cAMP acts in human pancreatic β-cells and INS-1 insulin-secreting cells to mobilize Ca2+ from intracellular Ca2+ stores via Epac-mediated Ca2+-induced Ca2+ release (CICR). The cAMP-dependent increase of [Ca2+]i that accompanies CICR is shown to be coupled to exocytosis. We propose that the interaction of cAMP and Epac to trigger CICR explains, at least in part, the blood glucose-lowering properties of an insulinotropic hormone (glucagon-like peptide-1, also known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus. The second messenger cAMP exerts powerful stimulatory effects on Ca2+ signaling and insulin secretion in pancreatic β-cells. Previous studies of β-cells focused on protein kinase A (PKA) as a downstream effector of cAMP action. However, it is now apparent that cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac). Although one effector of Epac is the Ras-related G protein Rap1, it is not fully understood what the functional consequences of Epac-mediated signal transduction are at the cellular level. 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′-5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) is a newly described cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2′-O-Me-cAMP acts in human pancreatic β-cells and INS-1 insulin-secreting cells to mobilize Ca2+ from intracellular Ca2+ stores via Epac-mediated Ca2+-induced Ca2+ release (CICR). The cAMP-dependent increase of [Ca2+]i that accompanies CICR is shown to be coupled to exocytosis. We propose that the interaction of cAMP and Epac to trigger CICR explains, at least in part, the blood glucose-lowering properties of an insulinotropic hormone (glucagon-like peptide-1, also known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus. exchange protein activated by cAMP protein kinase A Ca2+-induced Ca2+ release fetal bovine serum enhanced yellow fluorescent protein cAMP-response elements tetramethylrhodamine isothiocyanate standard extracellular saline cAMP-response element-binding protein inositol trisphosphate ryanodine receptors IP3 receptors inositol phosphate endoplasmic reticulum cAMP-regulated guanine nucleotide exchange factors (referred to here as Epac)1 link cAMP production to the activation of the Ras-related small molecular weight G protein Rap1 (1de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofers A. Bos J.L. Nature. 1998; 396: 474-477Google Scholar, 2Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Google Scholar). Two isoforms of Epac have been described (Epac1, Epac2) (1de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofers A. Bos J.L. Nature. 1998; 396: 474-477Google Scholar, 2Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Google Scholar), and each is proposed to mediate the PKA-independent signal transduction properties of cAMP. Analysis of cAMP-mediated signaling pathways is complicated by the lack of specificity with which cAMP acts. cAMP targets not only PKA and Epac, but also certain cAMP phosphodiesterases and ion channels (3Schwede F. Maronde E. Genieser H.-G. Jastorff B. Pharmacol. Ther. 2000; 87: 199-226Google Scholar). Recent structure-function analyses of cAMP action demonstrate that introduction of a 2′-methoxyl group in place of the 2′-hydroxyl group of cAMP confers Epac specificity to the cyclic nucleotide (4Enserink J.M. Christensen A.E. de Rooij J. Triest M.V. Schwede F. Genieser H.G. Døskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Google Scholar). One such analog is 8-(4-chloro-phenylthio)-2′-O-methyladenosine (8-pCPT-2′-O-Me-cAMP). Rap1 activation assays conductedin vitro demonstrate that 8-pCPT-2′-O-Me-cAMP binds to and activates Epac1 with higher apparent affinity (EC50 2.2 μm) than cAMP itself (EC50 30 μm) (4Enserink J.M. Christensen A.E. de Rooij J. Triest M.V. Schwede F. Genieser H.G. Døskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Google Scholar). Furthermore, 8-pCPT-2′-O-Me-cAMP is a weak activator of PKA (4Enserink J.M. Christensen A.E. de Rooij J. Triest M.V. Schwede F. Genieser H.G. Døskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Google Scholar). To date, the properties of 8-pCPT-2′-O-Me-cAMP in living cells have been evaluated only with respect to its ability to promote Epac-mediated activation of Rap1 (4Enserink J.M. Christensen A.E. de Rooij J. Triest M.V. Schwede F. Genieser H.G. Døskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Google Scholar). We now demonstrate that 8-pCPT-2′-O-Me-cAMP is an effective stimulus for Ca2+-induced Ca2+ release (CICR) and exocytosis in human pancreatic β-cells and an insulin-secreting cell line (INS-1). The action of 8-pCPT-2′-O-Me-cAMP is shown to be independent of PKA but is blocked by overexpression of dominant negative Epac2. Therefore, 8-pCPT-2′-O-Me-cAMP is likely to serve as a specific pharmacological tool for analyses of PKA-independent signaling properties of cAMP in the regulation of intracellular Ca2+ signaling and exocytosis. Human islets of Langerhans were provided under the auspices of the Juvenile Diabetes Research Foundation International Islet Distribution Program. Single cell suspensions of human islet cells were prepared by digestion with trypsin-EDTA, and the cells were plated onto glass coverslips (25CIR-1; Fisher) coated with 1 mg/ml concanavalin A (type V; Sigma). Cell cultures were maintained in a humidified incubator (95% air, 5% CO2) at 37 °C in CMRL-1066 culture medium containing 10% fetal bovine serum (FBS), 100 units/ml penicillin G, 100 μg/ml streptomycin, and 2.0 mm l-glutamine. INS-1 cells (passages 70–90) were maintained in RPMI 1640 culture medium containing 10 mm HEPES, 11.1 mm glucose, 10% FBS, 100 units/ml penicillin G, 100 μg/ml streptomycin, 2.0 mm l-glutamine, 1.0 mm sodium pyruvate, and 50 μm2-mercaptoethanol (5Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Google Scholar). INS-1 cells were passaged by trypsinization and subcultured once a week. All reagents for cell culture were obtained from Invitrogen. A plasmid in which expression of enhanced yellow fluorescent protein (EYFP) was placed under the control of the rat insulin II gene promoter (RIP2) was constructed by fusing a −695-bp BamH1 fragment of RIP2 to the coding sequence of EYFP contained within the pEYFP-N1 expression plasmid (Clontech). LipofectAMINE Plus reagent (Invitrogen) was used to transfect INS-1 cells with this plasmid designated as RIP2-EYFP, and clones of INS-1 cells exhibiting stable transfection were obtained by antibiotic resistance selection using G418. For construction of adenovirus, RIP2-EYFP was PCR-amplified inserting XhoI (5′ end, primer CTC GAG ACC GCG GGC CCG GGA TCC) and KpnI (3′ end, primer GGT ACC CCT CTA CAA ATG TGG TAT GGC TG) digestion sites on either end of the RIP2-EYFP sequence. The PCR product was subcloned into pCR2.1, and RIP2-EYFP was then inserted into a XhoI/KpnI site of AdLox.HTM. The RIP2-EYFP-AdLox.HTM vector was co-transfected with psi5 vector into CRE8 cells expressing CRE recombinase. This resulted in recombination of the RIPYFP-AdLox.HTM vector with the psi5 vector (6Chan C.B. MacDonald P.E. Saleh M.C. Johns D.C. Marban E. Wheeler M.B. Diabetes. 1999; 48: 1482-1486Google Scholar). The psi5 vector acts as a donor virus to supply viral backbone. AdRIPYFP viral particles generated in this manner were passaged three times in CRE8 cells and CsCl gradient-purified. 109 viral particles/ml were used to infect β-cells or islets. Pancreatic islets isolated from male Wistar rats (250 g) were plated on rat tail collagen and infected for 48 h with AdRIP2EYFP. Islets were stained for insulin using guinea pig anti-insulin and a secondary anti-guinea pig IgG conjugated with rhodamine TRITC (Jackson ImmunoResearch Laboratories, West Grove, PA). Islets were stained for glucagon using rabbit anti-glucagon, 08–0064, (Zymed Laboratories Inc., San Francisco, CA) and a secondary anti-rabbit IgG-conjugated with rhodamine TRITC. Laser scanning confocal microscopy was performed (Carl Zeiss, LSM 410), and images were obtained using a 63× oil immersion objective. Expression of EYFP was detected through use of an inverted microscope (Eclipse TE300, Nikon Instruments, Melville, NY) equipped with a 75-W xenon arc lamp serving as a light source. A liquid light guide directed unfiltered excitation light to an EYFP filter set mounted in a filter cube containing an HQ500/20 excitation filter, a Q515LP dichroic beamsplitter, and an HQ535/30 emission filter (HQ filter set 41028, Chroma Tech. Corp., Brattleboro, VT). Once an EYFP-positive cell was identified, the filter cube was switched manually to a second filter cube containing components of the fura-2 filter set. Excitation light provided by the xenon arc lamp was reflected by a rotating chopper mirror through 340/20BP and 380/20BP excitation filters (Chroma) mounted in a motorized filter wheel located at the light source. The filtered light was then directed to the fura-2 filter set by way of the liquid light guide. The fura-2 filter cube contained a 400DCLP dichroic beamsplitter and a 510/80 excitation filter (Chroma). The fura-2 loading solution consisted of a standard extracellular saline (SES) containing (in mm): 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 10 HEPES, and 5.6d-glucose. The SES was supplemented with 1 μm fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes Inc., Eugene, OR), 2% FBS, and 0.02% Pluronic F-127 (w/v; Molecular Probes Inc.). Cells were exposed to fura-2 AM for 20–30 min at 22 °C. The loading solution was removed, and cells were washed and equilibrated in fresh SES for 10 min at 22 °C. Images were acquired using a 100× UVF oil immersion objective (numerical aperature 1.3, Nikon), and dual excitation wavelength microfluorimetry was performed ratiometrically at 0.5-s intervals using a digital video imaging system outfitted with an intensified charge-coupled device camera (IonOptix Corp., Milton, MA). [Ca2+]i was calculated according to methods established (7Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar, 8Holz G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google Scholar). In vitro calibration of raw fluorescence values was performed using fura-2 (K+)5 salt dissolved in calibration buffers from Molecular Probes Inc. (Calcium Calibration Kit 1 with Mg2+). Values of R min andR max were 0.20 and 7.70. Cells were loaded with serotonin (5-HT) by incubation in culture medium containing 0.6 mm 5-HT and 0.6 mm 5-hydroxytryptophan. 5-HT is sequestered in large dense-core secretory granules by active transport, but it is excluded from the small synaptic vesicle-like structures that do not contain insulin (9Ekholm R. Ericson L.E. Lundquist I. Diabetologia. 1971; 7: 339-348Google Scholar). The release of 5-HT serves as a surrogate marker for insulin secretion (10Aspinwall C.A. Huang L. Lakey J.R. Kennedy R.T. Anal. Chem. 1999; 71: 5551-5556Google Scholar). Prolonged exposure of β-cells to high concentrations of 5-HT produces toxic effects (11Zawalich W.S. Tesz G.J. Zawalich K.C. J. Biol. Chem. 2001; 276: 37120-37123Google Scholar). Therefore, exposure to 5-HT was limited to 4–16 h. Carbon fiber electrodes for amperometric detection of secreted 5-HT were prepared as described previously (12Kang G. Holz G.G. J. Scholar). A was to a the of which was placed to the cell of The from the to the cell was was used for detection of the amperometric from of The signal was filtered at at 1 and on a The of was in of INS-1 cells by use of a as described previously G. Holz G.G. Diabetes. 2000; Scholar, Holz G.G. Cell 2002; Scholar, Holz G.G. Endocrinology. 2002; Scholar). a exposure to cells were and for using a and a of and were in analyses were performed using the of with least INS-1 cells were plated in medium in culture was to each and the cells were for 48 h. The medium was then to RPMI 1640 containing 10 mm and the pharmacological to be The incubation was for an min at 37 °C. and inositol were for and as described previously J. Biol. Chem. Scholar). and were obtained from and ryanodine were from and dominant negative for transfection and the expression of were obtained from the of S. N. H. H. S. Nat. Cell Biol. 2000; Scholar). was selection of β-cells in cultures of islets in SES containing A −695-bp fragment of the rat insulin II gene promoter (RIP2) was to the coding sequence of EYFP G. Holz G.G. J. 2001; for expression of EYFP in INS-1 cells A and RIP2-EYFP was then into an and expression of EYFP was to human β-cells by gene using and microscopy in with fluorescence that expression of EYFP was to the β-cells and not the in islets of Langerhans from rat studies of small of human islet cells that the EYFP-positive β-cells an increase of exposed to the insulin expression of EYFP by confocal microscopy of EYFP and in a rat islet infected with of a rat detection of EYFP in this in this of and of insulin and EYFP in β-cells. glucagon but not EYFP was in rat infected with of an detection of EYFP in this G, in this of and G that EYFP was not in of EYFP in human β-cells with of three human islet cells was evaluated for expression of EYFP under of fura-2 The cell 1 an increase of [Ca2+]i in to a of 100 to the of This cell contained EYFP that the the of cells is by a The cells of the not contain EYFP and to to were obtained in a of The cAMP analog 8-pCPT-2′-O-Me-cAMP was a effective stimulus for Ca2+ signaling in human β-cells. Human islet cells infected with were loaded with and EYFP-positive β-cells were using the EYFP filter set. the filter set was manually switched to a fura-2 filter of [Ca2+]i using an intensified charge-coupled device The of an EYFP-positive was by the increase of [Ca2+]i that a of 50 via a and The of this cell was by its to the insulin The increase of in to 8-pCPT-2′-O-Me-cAMP was blocked by of human β-cells with ryanodine The increase of exposure of human β-cells to 8-pCPT-2′-O-Me-cAMP was with as by fiber The increase of [Ca2+]i was to the of amperometric from the of 5-HT on an the of the secretory was secretory were detected in the of an increase of The action of 8-pCPT-2′-O-Me-cAMP was also evaluated in the INS-1 insulin-secreting cell line (5Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Google Scholar). cells Epac-mediated CICR as a increase of [Ca2+]i G. Holz G.G. J. 2001; Scholar). 8-pCPT-2′-O-Me-cAMP was a stimulus for CICR in INS-1 cells and the of the increase of [Ca2+]i that in human β-cells The action of 8-pCPT-2′-O-Me-cAMP was by and by In the increase of [Ca2+]i was by a and increase of [Ca2+]i of 8-pCPT-2′-O-Me-cAMP was blocked by the cAMP and is an of PKA and Epac, but it a affinity for Epac with and J. action of 8-pCPT-2′-O-Me-cAMP was a of μm and was not blocked by 10 μm of the PKA or However, the action of 8-pCPT-2′-O-Me-cAMP was by transfection of INS-1 cells with dominant negative negative not cAMP and have been into the N. H. H. S. Nat. Cell Biol. 2000; properties of a of INS-1 cells was performed and the of cells exhibiting CICR in to 8-pCPT-2′-O-Me-cAMP was The action of 100 μm 8-pCPT-2′-O-Me-cAMP was to 10 μm) and was not blocked by or of INS-1 cells with dominant negative blocked the action of 8-pCPT-2′-O-Me-cAMP transfection with not to cells were by use of RIP2-EYFP as described previously G. Holz G.G. J. 2001; 8-pCPT-2′-O-Me-cAMP to in INS-1 cells with of the PKA activator a of and the action of was by the PKA to or μm in the of This was and the of each of was evaluated in To the specificity with which 8-pCPT-2′-O-Me-cAMP Epac and INS-1 cells were with a plasmid in which expression of was placed under the control of cyclic elements This is activated by cAMP via a G. Holz G.G. Diabetes. 2000; Scholar, Holz G.G. Cell 2002; Scholar, Holz G.G. Endocrinology. 2002; Scholar). The of was by μm at PKA and and was by the PKA However, 8-pCPT-2′-O-Me-cAMP to were obtained the of and 8-pCPT-2′-O-Me-cAMP in cells with not demonstrate that in vitro at a of 8-pCPT-2′-O-Me-cAMP or as a of the and pathways in INS-1 or the Ca2+ from Ca2+ stores in INS-1 cells G. Holz G.G. J. 2001; Scholar). In the we that CICR in to 8-pCPT-2′-O-Me-cAMP was also blocked by ryanodine However, it was that Epac stimulatory of cAMP on inositol trisphosphate Ca2+ stores in cells M. S. P.A. F. H. Nat. Cell Biol. 2001; Scholar, S. J. F. M. P.A. M. J. Biol. Chem. 2002; Scholar). This action of cAMP is proposed to from Epac-mediated activation of with of IP3 production M. S. P.A. F. H. Nat. Cell Biol. 2001; Scholar, S. J. F. M. P.A. M. J. Biol. Chem. 2002; Scholar). Therefore, Ca2+ by 8-pCPT-2′-O-Me-cAMP as a of CICR in INS-1 cells from Ca2+ stores not only by ryanodine receptors but also by inositol trisphosphate receptors INS-1 cells and is a stimulus for inositol phosphate production However, a for in Epac-mediated signal transduction in INS-1 cells of production was in INS-1 cells exposed to or to production of inositol in INS-1 phosphate phosphate production in INS-1 cells was by but not or of inositol into cellular to was not in a phosphate production in INS-1 cells was by but not or of inositol into cellular to was not certain by which to Epac was Here we demonstrate that a newly cAMP analog (8-pCPT-2′-O-Me-cAMP) is a of CICR and exocytosis in human β-cells. 8-pCPT-2′-O-Me-cAMP is also to CICR in an insulin-secreting cell line and this is PKA-independent it is not blocked by or of a dominant negative CICR in to 8-pCPT-2′-O-Me-cAMP as in INS-1 cells an to regulation of Ca2+ signaling G. Holz G.G. J. 2001; Scholar). The specificity with which 8-pCPT-2′-O-Me-cAMP PKA and Epac is by its to the action of in an of that is of signal Therefore, 8-pCPT-2′-O-Me-cAMP is to properties that its use in assays of cellular One the of the and of G. Holz G.G. J. 2001; is that and to the action of effects the action of G. Holz G.G. J. 2001; Scholar). are 8-pCPT-2′-O-Me-cAMP acts via acts not only via but also PKA. is also that in INS-1 cells a increase of [Ca2+]i is in to and it is by CICR and This to 8-pCPT-2′-O-Me-cAMP is of the action of in β-cells G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google Scholar). is an of channels and by it produces activation of Ca2+ and a increase of [Ca2+]i G.G. Habener J.F. 1992; Scholar, G.G. Habener J.F. Nature. Scholar). be of to a of is in to 8-pCPT-2′-O-Me-cAMP as an of In this it is that Epac not only Rap1 within its nucleotide exchange but also interaction within its to and are A of Epac and the of been on the of a N. H. H. S. Nat. Cell Biol. 2000; Scholar), it is to that the targets of Epac action also cell Epac stimulatory of cAMP on in cells M. S. P.A. F. H. Nat. Cell Biol. 2001; Scholar, S. J. F. M. P.A. M. J. Biol. Chem. 2002; Scholar). The increase of serves as a stimulus for CICR by the However, we that the action of 8-pCPT-2′-O-Me-cAMP is to be a of Epac-mediated IP3 8-pCPT-2′-O-Me-cAMP to increase of inositol in INS-1 cells loaded with an for as targets of Epac action G.G. Leech C.A. Heller R.S. Castonguay M. Habener J.F. J. Biol. Chem. 1999; 274: 14147-14156Google Scholar, G. Holz G.G. J. 2001; Scholar, Diabetes. 2002; Scholar). of INS-1 cells with ryanodine CICR in to Furthermore, it was previously that treatment of INS-1 cells with the to CICR in to G. Holz G.G. J. 2001; Scholar). methods of were to use of a dominant negative be what in Ca2+ However, 8-pCPT-2′-O-Me-cAMP is an activator of Epac1 (4Enserink J.M. Christensen A.E. de Rooij J. Triest M.V. Schwede F. Genieser H.G. Døskeland S.O. Blank J.L. Bos J.L. Nat. Cell Biol. 2002; 4: 901-906Google Scholar), and in the it to inositol phosphate Although a for the IP3 as a of Epac1 and action in studies are to a Previous studies of INS-1 cells that mobilize Ca2+ from Ca2+ stores G. Holz G.G. J. 2001; Scholar). Ca2+ stores are likely located in the endoplasmic reticulum the Ca2+ by is One effector that Epac signaling to Ca2+ stores is Rap1, a small molecular weight G protein previously to with E. R. R. J. 1998; Scholar). In an signaling in the Ca2+ signaling by through with the an with of Ca2+ release from the S.O. S. D. N. 2000; Scholar). In this cells the CICR in to and this action with the ability of to cAMP production J. P. S. Scholar). Therefore, the of by or is a signaling not to β-cells. Here we also demonstrate that 8-pCPT-2′-O-Me-cAMP exocytosis in human β-cells. is to be to the increase of [Ca2+]i generated by and exocytosis is in the of that an increase of [Ca2+]i is a and not stimulus for of exocytosis. this not Epac-mediated signaling to For the interaction of Epac with an insulin an in stimulatory effects of cAMP on exocytosis in β-cells N. H. H. S. Nat. Cell Biol. 2000; Scholar, N. M. H. S. J. Biol. Chem. 2001; 276: Scholar, E. L. P. J. Scholar, C.A. Holz G.G. Habener J.F. 2000; Scholar). this it is that the of Ca2+ by cAMP is a of we that CICR serves to exocytosis in INS-1 cells (12Kang G. Holz G.G. J. Scholar). The are we demonstrate a functional of Epac-mediated CICR to exocytosis in human β-cells. This is through the use of a newly cAMP The are of light on the blood glucose-lowering properties of an insulinotropic hormone under investigation for use in the treatment of type-2 diabetes Diabetes. 1998; Scholar, Habener J.F. 1999; Scholar). PKA-independent of have been in and effects of on Ca2+ signaling G. Holz G.G. J. 2001; Scholar, B. R. B. Endocrinology. 1999; Scholar), S. N. M. 2000; Scholar, G. Holz G.G. J. Scholar), N. H. H. S. Nat. Cell Biol. 2000; N. M. H. S. J. Biol. Chem. 2001; 276: Scholar), and insulin gene expression G. Holz G.G. Diabetes. 2000; Scholar, Holz G.G. Endocrinology. 2002; Scholar). here demonstrate that 8-pCPT-2′-O-Me-cAMP is likely to serve as a powerful pharmacological tool for of Epac-mediated signaling that such understood effects of were the of S. G. G. H. also the of human islets from the Juvenile Diabetes Research Foundation islet located at the of B. J. B. J. and the of

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.