Glucose-stimulated Cdc42 signaling is essential for the second phase of insulin secretion

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.     

YES,a Src Family Kinase,Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.     

A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis

In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.     

Caveolin-1 functions as a novel Cdc42 guanine nucleotide dissociation inhibitor in pancreatic beta-cells

The cycling of the small Rho family GTPase Cdc42 is required for insulin granule exocytosis, although the regulatory proteins involved in Cdc42 cycling in pancreatic beta-cells are unknown. Here we demonstrate that the caveolar protein caveolin-1 (Cav-1) is a Cdc42-binding protein in beta-cells. Cav-1 associated with Cdc42-VAMP2-bound granules present near the plasma membrane under basal conditions. However, stimulation with glucose induced the dissociation of Cav-1 from Cdc42-VAMP2 complexes, coordinate with the timing of Cdc42 activation. Analyses of the Cav-1 scaffolding domain revealed a motif conserved in guanine nucleotide dissociation inhibitors (GDIs), which suggested a novel role for Cav-1 as a Cdc42 GDI in beta-cells. The novel role was further supported by: 1) in vitro binding analyses that demonstrated a direct interaction between Cav-1 and Cdc42; 2) GST-Cdc42 interaction assays showing preferential Cav-1 binding to GDP-Cdc42 over that of GTP-Cdc42; 3) Cav-1 depletion studies resulting in an inappropriate 40% induction of activated Cdc42 in the absence of stimuli and also a 40% increase in basal insulin release from both MIN6 cells and islets. Expression of wild-type Cav-1 in Cav-1-depleted cells restored basal level secretion to normal, whereas expression of a scaffolding domain mutant of Cav-1 failed to normalize secretion. Taken together, these data suggest that Cav-1 functions as a Cdc42 GDI in beta-cells, maintaining Cdc42 in an inactive state and regulating basal secretion in the absence of stimuli. Through its interaction with the Cdc42-VAMP2-bound insulin granule complex, Cav-1 may contribute to the specific targeting of granules to "active sites" of exocytosis organized by caveolae.     

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.     

Differential Phosphorylation of RhoGDI Mediates the Distinct Cycling of Cdc42 and Rac1 to Regulate Second-phase Insulin Secretion

Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interference-depleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases.     

5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.     

A role for G(z) in pancreatic islet beta-cell biology

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.     

Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.     

Novel regulation by Rac1 of glucose- and forskolin-induced insulin secretion in INS-1 beta-cells

Stimulation of insulin secretion by glucose and other secretagogues from pancreatic islet beta-cells is mediated by multiple signaling pathways. Rac1 is a member of Rho family GTPases regulating cytoskeletal organization, and recent evidence also implicates Rac1 in exocytotic processes. Herein, we report that exposure of insulin-secreting (INS) cells to stimulatory glucose concentrations caused translocation of Rac1 from cytosol to the membrane fraction (including the plasmalemma), an indication of Rac1 activation. Furthermore, glucose stimulation increased Rac1 GTPase activity. Time course study indicates that such an effect is demonstrable only after 15 min stimulation with glucose. Expression of a dominant-negative Rac1 mutant (N17Rac1) abolished glucose-induced translocation of Rac1 and significantly inhibited insulin secretion stimulated by glucose and forskolin. This inhibitory effect on glucose-stimulated insulin secretion was more apparent in the late phase of secretion. However, N17Rac1 expression did not significantly affect insulin secretion induced by high K+. INS-1 cells expressing N17Rac1 also displayed significant morphological changes and disappearance of F-actin structures. Expression of wild-type Rac1 or a constitutively active Rac1 mutant (V12Rac1) did not significantly affect either the stimulated insulin secretion or basal release, suggesting that Rac1 activation is essential, but not sufficient, for evoking secretory process. These data suggest, for the first time, that Rac1 may be involved in glucose- and forskolin-stimulated insulin secretion, possibly at the level of recruitment of secretory granules through actin cytoskeletal network reorganization.     

Oleanolic acid enhances insulin secretion in pancreatic beta-cells

We investigated the effect of oleanolic acid, a plant-derived triterpenoid, on insulin secretion and content in pancreatic beta-cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS-1 832/13 cells and enhanced acute glucose-stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin-4 under glucose-stimulated conditions, yet neither Ca(2+) nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti-diabetic properties of this natural product.     

Resistin induces insulin resistance in pancreatic islets to impair glucose-induced insulin release

An adipokine resistin, a small cysteine-rich protein, is one of the major risk factors of insulin resistance. In the present study, transiently resistin-expressing mice using adenovirus method showed an impaired glucose tolerance due to insulin resistance. We found that resistin-expressing mice exhibited impaired insulin secretory response to glucose. In addition, in vitro treatment with resistin for 1 day induced insulin resistance in pancreatic islets and impaired glucose-stimulated insulin secretion by elevating insulin release at basal glucose (2.8 mM) and suppressing insulin release at stimulatory glucose (8.3 mM). In addition, resistin inhibited insulin-induced phosphorylation of Akt in islets as well as other insulin target organs. Furthermore, resistin induced SOCS-3 expression in beta-cells. In conclusion, resistin induces insulin resistance in islet beta-cells at least partly via induction of SOCS-3 expression and reduction of Akt phosphorylation and impairs glucose-induced insulin secretion.     

13C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells

Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process.     

Calpain facilitates actin reorganization during glucose-stimulated insulin secretion

Calpain-10 (CAPN10) has been identified as a diabetes susceptibility gene. Previous studies have shown that alterations in calpain activity alter both glucose uptake and insulin secretion. In this report, we investigated the role of calpain activity in the actin reorganization required for glucose-stimulated insulin secretion. In pancreatic INS-1 cells, acute exposure to a high glucose environment stimulated CAPN10 gene expression with a concomitant increase in calpain activity. However, high glucose did not significantly alter expression of the two major ubiquitously expressed calpain family members, CAPN1 and CAPN2. Furthermore, glucose stimulation resulted in the reorganization of actin and inhibition of calpain activity impaired this reorganization in INS-1 cells. Finally, we identified a 54 kDa isoform as the major CAPN10 isoform that associates with the actin cytoskeleton. Based on our findings, we propose that calpain plays a role in facilitating the actin reorganization required for glucose-stimulated insulin secretion in INS-1 cells.     

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.     

PACAP is an islet neuropeptide which contributes to glucose-stimulated insulin secretion

Pituitary adenylate cyclase activating peptide (PACAP) is a ubiquitously distributed neuropeptide which also is localized to pancreatic islets and stimulates insulin secretion. We examined whether endogenous PACAP within the islets might contribute to glucose-stimulated insulin secretion by immunoneutralizing endogenous PACAP. Immunocytochemistry showed that PACAP immunoreactivity is expressed in nerve terminals within freshly isolated rat islets, but not in islets that had been cultured for 48 h. In contrast, islet endocrine cells did not display PACAP immunoreactivity. Addition of either of two specific PACAP antisera markedly inhibited glucose (11.1 mmol/l)-stimulated insulin secretion from freshly isolated rat islets, whereas a control rabbit serum did not affect glucose-stimulated insulin secretion. In contrast, the PACAP antisera had no effect on glucose-stimulated insulin secretion in cultured islets. Based on these results we therefore suggest that PACAP is an islet neuropeptide which is required for the normal insulinotropic action of glucose.     

Cool-1/βPIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion

Second-phase insulin release requires the sustained mobilization of insulin granules from internal storage pools to the cell surface for fusion with the plasma membrane. However, the detailed mechanisms underlying this process remain largely unknown. GTP-loading of the small GTPase Cdc42 is the first glucose-specific activation step in the process, although how glucose triggers Cdc42 activation is entirely unknown. In a directed candidate screen for guanine nucleotide exchange factors (GEFs), which directly activate small GTPases, Cool-1/βPix was identified in pancreatic islet beta cells. In support of its role as the beta cell Cdc42 GEF, βPix coimmunoprecipitated with Cdc42 in human islets and MIN6 beta cells in a glucose-dependent manner, peaking just prior to Cdc42 activation. Furthermore, RNAi-mediated βPix reduction by 50% corresponded to full ablation of glucose-induced Cdc42 activation and significant attenuation of basal and glucose-stimulated insulin secretion. Of the two Cdc42 guanine nucleotide dissociation inhibitor (GDI) proteins identified in beta cells, βPix competed selectively with caveolin-1 (Cav-1) but not RhoGDI in coimmunoprecipitation and GST-Cdc42-GDP interaction assays. However, a phospho-deficient Cav-1-Y14F mutant failed to compete with βPix; Cav-1(Tyr14) is an established phosphorylation site for Src kinase. Taken together, these data support a new model, wherein glucose stimulates Cav-1 and induces its dissociation from Cdc42, possibly via Src kinase activation to phosphorylate Cav-1(Tyr14), to promote Cdc42-βPix binding and Cdc42 activation, and to trigger downstream signaling and ultimately sustain insulin release.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.