Bok regulates mitochondrial fusion and morphology.

Bok (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, but the cellular role that Bok plays is controversial. Remarkably, endogenous Bok is constitutively bound to inositol 1,4,5-trisphosphate receptors (IP3Rs) and is stabilized by this interaction. Here we report that despite the strong association with IP3Rs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IP3Rs or calcium influx into the mitochondria. Rather, Bok deletion significantly reduces mitochondrial fusion rate, resulting in mitochondrial fragmentation. This phenotype is reversed by exogenous wild-type Bok and by an IP3R binding-deficient Bok mutant, and may result from a decrease in mitochondrial motility. Bok deletion also enhances mitochondrial spare respiratory capacity and membrane potential. Finally, Bok does not play a major role in apoptotic signaling, since Bok deletion does not alter responsiveness to various apoptotic stimuli. Overall, despite binding to IP3Rs, Bok does not alter IP3R-mediated Ca2+ signaling, but is required to maintain normal mitochondrial fusion, morphology, and bioenergetics.

Impaired Store-Operated Calcium Entry and STIM1 Loss Lead to Reduced Insulin Secretion and Increased Endoplasmic Reticulum Stress in the Diabetic ß-Cell.

Store-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in ß-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by ß-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued ß-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve ß-cell health and function.

Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry.

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca2+ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca2+-binding protein, with Ca2+-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca2+ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca2+ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.

Molecular physiology and pathophysiology of stromal interaction molecules.

Ca2+ release from the endoplasmic reticulum is an important component of Ca2+ signal transduction that controls numerous physiological processes in eukaryotic cells. Release of Ca2+ from the endoplasmic reticulum is coupled to the activation of store-operated Ca2+ entry into cells. Store-operated Ca2+ entry provides Ca2+ for replenishing depleted endoplasmic reticulum Ca2+ stores and a Ca2+ signal that regulates Ca2+-dependent intracellular biochemical events. Central to connecting discharge of endoplasmic reticulum Ca2+ stores following G protein-coupled receptor activation with the induction of store-operated Ca2+ entry are stromal interaction molecules (STIM1 and STIM2). These highly homologous endoplasmic reticulum transmembrane proteins function as sensors of the Ca2+ concentration within the endoplasmic reticulum lumen and activators of Ca2+ release-activated Ca2+ channels. Emerging evidence indicates that in addition to their role in Ca2+ release-activated Ca2+ channel gating and store-operated Ca2+ entry, STIM1 and STIM2 regulate other cellular signaling events. Recent studies have shown that disruption of STIM expression and function is associated with the pathogenesis of several diseases including autoimmune disorders, cancer, cardiovascular disease, and myopathies. Here, we provide an overview of the latest developments in the molecular physiology and pathophysiology of STIM1 and STIM2. Impact statement Intracellular Ca2+ signaling is a fundamentally important regulator of cell physiology. Recent studies have revealed that Ca2+-binding stromal interaction molecules (Stim1 and Stim2) expressed in the membrane of the endoplasmic reticulum (ER) are essential components of eukaryote Ca2+ signal transduction that control the activity of ion channels and other signaling effectors present in the plasma membrane. This review summarizes the most recent information on the molecular physiology and pathophysiology of stromal interaction molecules. We anticipate that the work presented in our review will provide new insights into molecular interactions that participate in interorganelle signaling crosstalk, cell function, and the pathogenesis of human diseases.

Stromal Interaction Molecule 1 (STIM1) Regulates ATP-sensitive Potassium (KATP) and Store-operated Ca2+ Channels in MIN6 ß-Cells.

Stromal interaction molecule 1 (STIM1) regulates store-operated Ca2+ entry (SOCE) and other ion channels either as an endoplasmic reticulum Ca2+-sensing protein or when present in the plasma membrane. However, the role of STIM1 in insulin-secreting ß-cells is unresolved. We report that lowering expression of STIM1, the gene that encodes STIM1, in insulin-secreting MIN6 ß-cells with RNA interference inhibits SOCE and ATP-sensitive K+ (KATP) channel activation. The effects of STIM1 knockdown were reversed by transduction of MIN6 cells with an adenovirus gene shuttle vector that expressed human STIM1 Immunoprecipitation studies revealed that STIM1 binds to nucleotide binding fold-1 (NBF1) of the sulfonylurea receptor 1 (SUR1) subunit of the KATP channel. Binding of STIM1 to SUR1 was enhanced by poly-lysine. Our data indicate that SOCE and KATP channel activity are regulated by STIM1. This suggests that STIM1 is a multifunctional signaling effector that participates in the control of membrane excitability and Ca2+ signaling events in ß-cells.

IRS1 deficiency protects ß-cells against ER stress-induced apoptosis by modulating sXBP-1 stability and protein translation.

Endoplasmic reticulum (ER) stress is among several pathological features that underlie ß-cell failure in the development of type 1 and type 2 diabetes. Adaptor proteins in the insulin/insulin-like-growth factor-1 signaling pathways, such as insulin receptor substrate-1 (IRS1) and IRS2, differentially impact ß-cell survival but the underlying mechanisms remain unclear. Here we report that ß-cells deficient in IRS1 (IRS1KO) are resistant, while IRS2 deficiency (IRS2KO) makes them susceptible to ER stress-mediated apoptosis. IRS1KOs exhibited low nuclear accumulation of spliced XBP-1 due to its poor stability, in contrast to elevated accumulation in IRS2KO. The reduced nuclear accumulation in IRS1KO was due to protein instability of Xbp1 secondary to proteasomal degradation. IRS1KO also demonstrated an attenuation in their general translation status in response to ER stress revealed by polyribosomal profiling. Phosphorylation of eEF2 was dramatically increased in IRS1KO enabling the ß-cells to adapt to ER stress by blocking translation. Furthermore, significantly high ER calcium (Ca(2+)) was detected in IRS1KO ß-cells even upon induction of ER stress. These observations suggest that IRS1 could be a therapeutic target for ß-cell protection against ER stress-mediated cell death by modulating XBP-1 stability, protein synthesis, and Ca(2+) storage in the ER.

GPR119 Agonist AS1269574 Activates TRPA1 Cation Channels to Stimulate GLP-1 Secretion.

GPR119 is a G protein-coupled receptor expressed on intestinal L cells that synthesize and secrete the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). GPR119 agonists stimulate the release of GLP-1 from L cells, and for this reason there is interest in their potential use as a new treatment for type 2 diabetes mellitus. AS1269574 is one such GPR119 agonist, and it is the prototype of a series of 2,4,6 trisubstituted pyrimidines that exert positive glucoregulatory actions in mice. Here we report the unexpected finding that AS1269574 stimulates GLP-1 release from the STC-1 intestinal cell line by directly promoting Ca(2+) influx through transient receptor potential ankyrin 1 (TRPA1) cation channels. These GPR119-independent actions of AS1269574 are inhibited by TRPA1 channel blockers (AP-18, A967079, HC030031) and are not secondary to intracellular Ca(2+) release or cAMP production. Patch clamp studies reveal that AS1269574 activates an outwardly rectifying membrane current with properties expected of TRPA1 channels. However, the TRPA1 channel-mediated action of AS1269574 to increase intracellular free calcium concentration is not replicated by GPR119 agonists (AR231453, oleoylethanolamide) unrelated in structure to AS1269574. Using human embryonic kidney-293 cells expressing recombinant rat TRPA1 channels but not GPR119, direct TRPA1 channel activating properties of AS1269574 are validated. Because we find that AS1269574 also acts in a conventional GPR119-mediated manner to stimulate proglucagon gene promoter activity in the GLUTag intestinal L cell line, new findings reported here reveal the surprising capacity of AS1269574 to act as a dual agonist at two molecular targets (GPR119/TRPA1) important to the control of L-cell function and type 2 diabetes mellitus drug discovery research.

Functional role of serotonin in insulin secretion in a diet-induced insulin-resistant state.

The physiological role of serotonin, or 5-hydroxytryptamine (5-HT), in pancreatic ß-cell function was previously elucidated using a pregnant mouse model. During pregnancy, 5-HT increases ß-cell proliferation and glucose-stimulated insulin secretion (GSIS) through the Gαq-coupled 5-HT2b receptor (Htr2b) and the 5-HT3 receptor (Htr3), a ligand-gated cation channel, respectively. However, the role of 5-HT in ß-cell function in an insulin-resistant state has yet to be elucidated. Here, we characterized the metabolic phenotypes of ß-cell-specific Htr2b(-/-) (Htr2b ßKO), Htr3a(-/-) (Htr3a knock-out [KO]), and ß-cell-specific tryptophan hydroxylase 1 (Tph1)(-/-) (Tph1 ßKO) mice on a high-fat diet (HFD). Htr2b ßKO, Htr3a KO, and Tph1 ßKO mice exhibited normal glucose tolerance on a standard chow diet. After 6 weeks on an HFD, beginning at 4 weeks of age, both Htr3a KO and Tph1 ßKO mice developed glucose intolerance, but Htr2b ßKO mice remained normoglycemic. Pancreas perfusion assays revealed defective first-phase insulin secretion in Htr3a KO mice. GSIS was impaired in islets isolated from HFD-fed Htr3a KO and Tph1 ßKO mice, and 5-HT treatment improved insulin secretion from Tph1 ßKO islets but not from Htr3a KO islets. Tph1 and Htr3a gene expression in pancreatic islets was not affected by an HFD, and immunostaining could not detect 5-HT in pancreatic islets from mice fed an HFD. Taken together, these results demonstrate that basal 5-HT levels in ß-cells play a role in GSIS through Htr3, which becomes more evident in a diet-induced insulin-resistant state.

Characterization of mice expressing Ins1 gene promoter driven CreERT recombinase for conditional gene deletion in pancreatic ß-cells.

Gene manipulation using Cre-loxP recombination has proven to be an important approach for studying the impact of gene expression on pancreatic ß-cell biology. We report the generation of a transgenic mouse line that enables a highly specific system for conditional gene manipulation within ß-cells and achieve tissue specific and temporally regulated deletion of the Ctnnb1 (ß-catenin) gene in pancreatic ß-cells. cDNA encoding Cre recombinase fused to modified estrogen receptor (CreERT) under control of mouse insulin 1 gene promoter (Ins1) was used to construct the mouse line Tg(Ins1-Cre/ERT)1Lphi, also termed MIP1-CreERT. In a cross of MIP1-CreERT with a ROSA26/LacZ reporter strain, tamoxifen [Tmx] - dependent ß-galactosidase expression occurred within pancreatic ß-cells but not in other organ systems. Intraperitoneal glucose tolerance tests and glucose-stimulated changes in ß-cell cytoplasmic calcium concentration were not adversely affected in adult MIP1-CreERT. A mouse line with floxed Ctnnb1 gene (Ctnnb1f/f) was crossed with the MIP1-CreERT line to generate a mouse model for inducible ß-cell specific deletion of ß-catenin gene (Ctnnb1f/f:MIP1-CreERT). Ctnnb1f/f:MIP1-CreERT mice and Ctnnb1f/f littermate controls, were injected with Tmx as adults to knock down ß-catenin production in the majority of pancreatic ß-cells. These mice showed normal glucose tolerance, islet cyto-architecture and insulin secretion. A novel protein fraction of 50Kd, immunoreactive with anti-ß-catenin was observed in islet extracts from Ctnnb1f/f:MIP1-CreERT[Tmx] mice but not MIP1-CreERT-negative Ctnnb1f/f[Tmx] controls, indicating possible presence of a cryptic protein product of recombined Ctnnb1 gene. The MIP1-CreERT mouse line is a powerful tool for conditional manipulation of gene expression in ß-cells.

Resveratrol Interferes with Fura-2 Intracellular Calcium Measurements.

Resveratrol, a naturally occurring polyphenol found in some fruits and especially in grapes, has been reported to provide diverse health benefits. Resveratrol's mechanism of action is the subject of many investigations, and some studies using the ratiometric calcium indicator Fura-2 suggest that it modulates cellular calcium responses. In the current study, contradictory cellular calcium responses to resveratrol applied at concentrations exceeding 10 µM were observed during in vitro imaging studies depending on the calcium indicator used, with Fura-2 indicating an increase in intracellular calcium while Fluo-4 and the calcium biosensor YC3.60 indicated no response. When cells loaded with Fura-2 were treated with 100 µM resveratrol, excitation at 340 nm resulted in a large intensity increase at 510 nm, but the expected concurrent decline with 380 nm excitation was not observed. Pre-treatment of cells with the calcium chelator BAPTA-AM did not prevent a rise in the 340/380 ratio when resveratrol was present, but it did prevent an increase in 340/380 when ATP was applied, suggesting that the resveratrol response was an artifact. Cautious data interpretation is recommended from imaging experiments using Fura-2 concurrently with resveratrol in calcium imaging experiments.

Insulin secretion and Ca2+ dynamics in ß-cells are regulated by PERK (EIF2AK3) in concert with calcineurin.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting ß-cell. Although genetic ablation of PERK in ß-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent ß-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca(2+) signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca(2+) entry and sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates ß-cell Ca(2+) signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.

Serum- and glucocorticoid-induced protein kinase 1 (SGK1) is regulated by store-operated Ca2+ entry and mediates cytoprotection against necrotic cell death.

Serum and glucocorticoid-regulated kinase 1 (SGK1) encodes a phosphatidylinositol 3-kinase-dependent serine/threonine kinase that is rapidly induced in response to cellular stressors and is an important cell survival signal. Previous studies have suggested that an increase in cytoplasmic Ca(2+) concentration ([Ca(2+)]c) is required for increased SGK1 expression, but the subcellular source of Ca(2+) regulating SGK1 transcription remains uncertain. Activation of endoplasmic reticulum stress (ERS) with thapsigargin (TG) increased SGK1 mRNA and protein expression in MDA-MB-231 cells. Intracellular Ca(2+) imaging revealed that store-operated Ca(2+) entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca(2+) from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca(2+) levels, however, triggered cell death with a much greater proportion of the cells undergoing necrosis rather than apoptosis. A relative increase in the percentage of cells undergoing necrosis was observed in cells expressing a short hairpin RNA targeted to the SGK1 gene. Necrotic cell death evoked by cytoplasmic Ca(2+) overloading was associated with persistent hyperpolarization of the inner mitochondrial membrane and a modest increase in calpain activation, but did not involve detectable caspase 3 or caspase 7 activation. The effects of cytoplasmic Ca(2+) overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca(2+) entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

ß-Cell-specific protein kinase A activation enhances the efficiency of glucose control by increasing acute-phase insulin secretion.

Acute insulin secretion determines the efficiency of glucose clearance. Moreover, impaired acute insulin release is characteristic of reduced glucose control in the prediabetic state. Incretin hormones, which increase ß-cell cAMP, restore acute-phase insulin secretion and improve glucose control. To determine the physiological role of the cAMP-dependent protein kinase (PKA), a mouse model was developed to increase PKA activity specifically in the pancreatic ß-cells. In response to sustained hyperglycemia, PKA activity potentiated both acute and sustained insulin release. In contrast, a glucose bolus enhanced acute-phase insulin secretion alone. Acute-phase insulin secretion was increased 3.5-fold, reducing circulating glucose to 58% of levels in controls. Exendin-4 increased acute-phase insulin release to a similar degree as PKA activation. However, incretins did not augment the effects of PKA on acute-phase insulin secretion, consistent with incretins acting primarily via PKA to potentiate acute-phase insulin secretion. Intracellular calcium signaling was unaffected by PKA activation, suggesting that the effects of PKA on acute-phase insulin secretion are mediated by the phosphorylation of proteins involved in ß-cell exocytosis. Thus, ß-cell PKA activity transduces the cAMP signal to dramatically increase acute-phase insulin secretion, thereby enhancing the efficiency of insulin to control circulating glucose.

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain.

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.

Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans.

Glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of ß-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.

Epac2-dependent mobilization of intracellular Ca²+ by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in ß-cells of phospholipase C-ε knockout mice.

Calcium can be mobilized in pancreatic ß-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in ß-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the ß-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse ß-cells were loaded with the photolabile Ca(2+) chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca(2+) generated CICR in only 9% of the ß-cells tested, whereas CICR was generated in 82% of the ß-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in ß-cells of PLC- KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC- KO ß-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT ß-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC- exists in ß-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type.

Conditional gene targeting in mouse pancreatic ß-Cells: analysis of ectopic Cre transgene expression in the brain.

OBJECTIVE: Conditional gene targeting has been extensively used for in vivo analysis of gene function in ß-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate ß-cell- or pancreas-specific recombination, also drive Cre expression in the brain. RESEARCH DESIGN AND METHODS: Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by ß-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1(tm1Cvw) lacZ knock-in mice. Cre expression in ß-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. RESULTS: All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)(89.1Dam) mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)(1Lphi) mice were the only line that lacked Cre activity in the brain. CONCLUSIONS: Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet ß-cells.

PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM in human islets of Langerhans.

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca(2+)](i) that reflected both Ca(2+) influx and intracellular Ca(2+) mobilization in islet beta-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca(2+)-dependent exocytosis.

Glucose-dependent potentiation of mouse islet insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM.

Epac2 is a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) that is proposed to mediate stimulatory actions of the second messenger cAMP on mouse islet insulin secretion. Here we have used methods of islet perifusion to demonstrate that the acetoxymethyl ester (AM-ester) of an Epac-selective cAMP analog (ESCA) penetrates into mouse islets and is capable of potentiating both first and second phases of glucose-stimulated insulin secretion (GSIS). When used at low concentrations (1-10 µM), 8-pCPT-2'-O-Me-cAMP-AM activates Rap1 GTPase but exhibits little or no ability to activate protein kinase A (PKA), as validated in assays of in vitro PKA activity (phosphorylation of Kemptide), Ser (133) CREB phosphorylation status, RIP1-CRE-Luc reporter gene activity, and PKA-dependent AKAR3 biosensor activation. Since quantitative PCR demonstrates Epac2 mRNA to be expressed at levels ca. 5.3-fold greater than that of Epac1, available evidence indicates that Epac2 does in fact mediate stimulatory actions of cAMP on mouse islet GSIS.