G-protein-coupled Receptor 40 Expression Research Articles

Vincamine as an agonist of G protein-coupled receptor 40 effectively ameliorates pulmonary fibrosis in mice

BackgroundPulmonary fibrosis (PF) is an irreversible and fatal lung disease with limited therapeutic options. G protein-coupled receptor 40 (GPR40) has been developed as a promising therapeutic target for metabolic disorders and functions potently in varied pathological and physiological processes. Vincamine (Vin) is a monoterpenoid indole alkaloid originated from Madagascar periwinkle and was reported as a GPR40 agonist in our previous work. PurposeHere, we aimed to clarify the role of GPR40 in PF pathogenesis by using the determined GPR40 agonist Vin as a probe and explore the potential of Vin in ameliorating PF in mice. MethodsPulmonary GPR40 expression alterations were assessed in both PF patients and bleomycin-induced PF mice (PF mice). Vin was used to evaluate the therapeutic potential of GPR40 activation for PF and the underlying mechanism was intensively investigated by assays against GPR40 knockout (Ffar1−/−) mice and the cells transfected with si-GPR40 in vitro. ResultsPulmonary GPR40 expression level was highly downregulated in PF patients and PF mice. Pulmonary GPR40 deletion (Ffar1−/−) exacerbated pulmonary fibrosis as evidenced by the increases in mortality, dysfunctional lung index, activated myofibroblasts and extracellular matrix (ECM) deposition in PF mice. Vin-mediated pulmonary GPR40 activation ameliorated PF-like pathology in mice. Mechanistically, Vin suppressed ECM deposition by GPR40/β-arrestin2/SMAD3 pathway, repressed inflammatory response by GPR40/NF-κB/NLRP3 pathway and inhibited angiogenesis by decreasing GPR40-mediated vascular endothelial growth factor (VEGF) expression in the region of interface to normal parenchyma in pulmonary fibrotic tissues of mice. ConclusionPulmonary GPR40 activation shows promise as a therapeutic strategy for PF and Vin exhibits high potential in treating this disease.

Targeting of G-protein coupled receptor 40 alleviates airway hyperresponsiveness through RhoA/ROCK1 signaling pathway in obese asthmatic mice

Obesity increases the severity of airway hyperresponsiveness (AHR) in individuals with asthma, but the mechanism is not well elucidated. G-protein coupled receptor 40 (GPR40) has been found to induce airway smooth muscle contraction after activated by long-chain fatty acids (LC-FFAs), suggesting a close correlation between GPR40 and AHR in obese. In this study, C57BL/6 mice were fed a high-fat diet (HFD) to induce obesity with or without ovalbumin (OVA) sensitization, the regulatory effects of GPR40 on AHR, inflammatory cells infiltration, and the expression of Th1/Th2 cytokines were evaluated by using a small-molecule antagonist of GPR40, DC260126. We found that the free fatty acids (FFAs) level and GPR40 expression were greatly elevated in the pulmonary tissues of obese asthmatic mice. DC260126 greatly reduced methacholine-induced AHR, ameliorated pulmonary pathological changes and decreased inflammatory cell infiltration in the airways in obese asthma. In addition, DC260126 could down-regulate the levels of Th2 cytokines (IL-4, IL-5, and IL-13) and pro-inflammatory cytokines (IL-1β, TNF-α), but elevated Th1 cytokine (IFN-γ) expression. In vitro, DC260126 could remarkedly reduce oleic acid (OA)-induced cell proliferation and migration in HASM cells. Mechanistically, the effects that DC260126 alleviated obese asthma was correlated with the down-regulation of GTP-RhoA and Rho-associated coiled-coil-forming protein kinase 1 (ROCK1). Herein, we proved that targeting of GPR40 with its antagonist helped to mitigate multiple parameters of obese asthma effectively.

The GPR40 Agonist GW9508 Enhances Neutrophil Function to Aid Bacterial Clearance During E. coli Infections.

G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion, and inflammation. However, the function of this receptor in human neutrophils, one of the first leukocytes to arrive at the site of infection, remains to be fully elucidated. In the present study, we demonstrate that GPR40 is upregulated on activated human neutrophils and investigated the functional effects upon treatment with a selective agonist; GW9508. Interestingly, GPR40 expression was up-regulated after neutrophil stimulation with platelet-activating factor (10 nM) or leukotriene B4 (LTB4, 10 nM) suggesting potential regulatory roles for this receptor during inflammation. Indeed, GW9508 (1 and 10 μM) increased neutrophil chemotaxis in response to the chemokine IL-8 (30 ng/ml) and enhanced phagocytosis of Escherichia coli by approximately 50% when tested at 0.1 and 1 μM. These results were translated in vivo whereby administration of GW9508 (10 mg/kg, i.p.) during E. coli infections resulted in elevated peritoneal leukocyte infiltration with a higher phagocytic capacity. Importantly, GW9508 administration also modulated the lipid mediator profile, with increased levels of the pro-resolving mediators resolvin D3 and lipoxins. In conclusion, GPR40 is expressed by activated neutrophils and plays an important host protective role to aid clearance of bacterial infections.

Trans-11 vaccenic acid improves glucose homeostasis in a model of type 2 diabetes by promoting insulin secretion via GPR40

Abstract It is largely unknown whether and how trans-11 vaccenic acid (VA), a ruminant trans-fatty acid with health benefits, influences glucose homeostasis in type 2 diabetes (T2D). VA was provided in diet to rats and in vitro to human/rat islets with diabetogenic challenges. Compared to rats with T2D induced by high-fat diet/streptozotocin, T2D + VA animals had improved ambient and fasting blood glucose, β-cell area, in vivo insulin secretion, and ex vivo glucose-stimulated insulin secretion (GSIS) in islets from T2D + VA rats, which were accompanied with higher islet mRNA and protein expression of G-protein coupled receptor 40 (GPR40). In vitro, VA enhanced GPR40 protein expression and protein kinase D1 phosphorylation, and promoted GSIS in rat and human islets, an effect blocked by the inhibition of GPR40 and protein kinase D. These results indicate that VA may improve glycemic control by promoting insulin secretion in T2D, at least partly through stimulating GPR40 expression and signaling.

GPR40 modulates epileptic seizure and NMDA receptor function.

Epilepsy is a common neurological disease, and approximately 30% of patients do not respond adequately to antiepileptic drug treatment. Recent studies suggest that G protein-coupled receptor 40 (GPR40) is expressed in the central nervous system and is involved in the regulation of neurological function. However, the impact of GPR40 on epileptic seizures remains unclear. In this study, we first reported that GPR40 expression was increased in epileptic brains. In the kainic acid-induced epilepsy model, GPR40 activation after status epilepticus alleviated epileptic activity, whereas GPR40 inhibition showed the opposite effect. In the pentylenetetrazole-induced kindling model, susceptibility to epilepsy was reduced with GPR40 activation and increased with GPR40 inhibition. Whole-cell patch-clamp recordings demonstrated that GPR40 affected N-methyl-d-aspartate (NMDA) receptor-mediated synaptic transmission. Moreover, GPR40 regulated NR2A and NR2B expression on the surface of neurons. In addition, endocytosis of NMDA receptors and binding of GPR40 with NR2A and NR2B can be regulated by GPR40. Together, our findings indicate that GPR40 modulates epileptic seizures, providing a novel antiepileptic target.

Expression of long-chain fatty acid receptor GPR40 is associated with cancer progression in colorectal cancer: A retrospective study.

An increased risk of colorectal cancer (CRC) is associated with a western style diet, particularly hyperlipidemia. The expression of G-protein coupled receptor 40 (GPR40), a membrane-bound receptor for long-chain fatty acids (LCFAs), was examined in 36 cases of subserosal-invading CRC and compared with clinicopathological parameters as well as triglyceride (TG) and low-density lipoprotein (LDL) levels in the blood. All patients with CRC expressed GPR40, which was positively associated with blood TG levels (P<0.0001) but not with blood LDL levels. GPR40 expression was positively associated with nodal metastasis, distant metastasis (particularly to the liver), stage and poor prognosis. Patients with high GPR40 expression and high TG levels had comparatively worse survival outcomes compared with patients with low GPR40 expression and low TG levels. The results of the present study suggest that activation of GPR40 may be associated with the progression and prognosis of CRCs. High levels of GPR40 and/or concurrent high levels of GPR40 and TG may be a risk for CRC progression.

Trans-11 vaccenic acid improves insulin secretion in models of type 2 diabetes in vivo and in vitro.

Trans-11 vaccenic acid (VA) is a fatty acid produced by ruminants entering the human food supply through meat and dairy products, which appears not to have the health risks associated with industrially produced trans-fatty acids. In this study, we investigated the effect of VA on insulin secretion in vivo in rats and in vitro in human and rat islets after diabetogenic insult. Hyperglycemic clamp showed that VA dietary supplementation for 8 weeks significantly increased glucose turnover in rats with type 2 diabetes (T2D), accompanied by an elevated plasma C-peptide concentration, indicating improved insulin secretion. The β-cell area and proliferation rate were higher in T2D+VA than T2D group. Isolated islets from T2D+VA rats had higher glucose-stimulated insulin secretion (GSIS) than T2D group. In vitro, VA treatment for 24 and 48 h significantly enhanced GSIS in rat and human islets after diabetogenic challenges. The mRNA expression of G-protein-coupled receptor 40 (GPR40) and regenerating islet-derived 1α (REG-1α) were consistently increased by VA in both rat and human islets. These results indicate that VA may improve insulin secretion and growth of islets in T2D, at least partly by altering GPR40 and REG-1α mRNA expression.

Role of GPR40 for Fish Oil PUFA-mediated BDNF Synthesis in the Monkey Hippocampus

Background: Polyunsaturated fatty acids (PUFA) are known to be crucial for learning and memory. However, the detailed mechanism of PUFA effects upon neuronal functions remains almost unknown except for the possible facilitation of membrane fluidity. G-protein coupled receptor 40 (GPR40) was found to induce Ca2+ mobilization in response to diverse PUFA. Thereafter, the authors found GPR40 expression in the newborn neurons of the monkey hippocampus after ischemia. This suggested implications of PUFA-mediated GPR40 signaling for adult neurogenesis underlying learning and memory. Objective: This study aims at evaluating whether PUFA-mediated GPR40 activation can affect synthesis of brain-derived neurotrophic factor (BDNF) with the aid of its proteolytic enzyme furin. Methods: Monkeys underwent 20 min transient whole brain ischemia by clamping both the innominate and left subclavian arteries. On days 7 and 15 after ischemia/reperfusion, when adult neurogenesis was shown to be maximal previously by the authors, the brain samples were resected. By the Western blotting analysis of mature-BDNF (m-BDNF), pro-BDNF and furin, syntheses of BDNF in response to two GPR40 agonists as well as selective GPR40 antagonist GW1100, were studied using normal and post-ischemic monkey dentate gyrus (DG) tissue extracts. Results: Both up-regulation of m-BDNF synthesis in response to two GPR40 agonists; fish oil PUFA and docosahexaenoic acid (DHA) and its down-regulation in response to GW1100, were observed. GPR40 antagonist inhibited m-BDNF synthesis, whereas two GPR40 agonists stimulated m-BDNF synthesis conceivably via furin activation. Cleavage of p-BDNF to m-BDNF by furin as well as syntheses of m-BDNF and furin in the DG tissues, occurred immediately after incubation with fish oil PUFA or DHA. Dynamic changes of GPR40, m-BDNF synthesis, and furin occurred simultaneously. Conclusions: These data, although correlative, suggested that m-BDNF may be synthesized by the cleavage of pre-stocked pro-BDNF and/or released from the cell store in response to PUFA. By activating GPR40 and furin, PUFA may be related to adult neurogenesis and the concomitant synaptic plasticity for learning and memory. To the best of our knowledge, this is the first report suggesting a role of GPR40 in PUFA-mediated m-BDNF synthesis.

Abstract 15968: Rescue of Bupivacaine-induced Cardiac Arrest by Lipid Emulsion is Prevented by Inhibition of Free Fatty Acid Receptor GPR40

Introduction: Bupivacaine overdose leads to lethal arrhythmias and cardiac arrest. Lipid emulsion (LE) is cardioprotective and rescues Bupivacaine toxicity. Although both lipid sink and metabolic theories have been proposed, yet the exact molecular mechanism is not known. G protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor-1 is activated by medium and long chain fatty acids. Among free fatty acid receptors, GPR40 expression is one of the highest in heart. Hypothesis: As GPR40 is activated by medium and long chain fatty acids, we hypothesized that GPR40 may mediate LE’s rescue of Bupivacaine cardiotoxicity. Methods: Male Sprague-Dawley rats were used. Continuous Echocardiography and ECG were done. In protocol-1 (n=6), rats received Bupivacaine bolus (10mg/kg, IV) to induce asystole. Resuscitation with LE 20% (5ml/kg bolus, and 0.5ml/kg/min maintenance) and chest compressions were initiated. In Protocol-2 (n=6) rats were pre-treated with GPR40-antagonist GW1100 (200uM, IV) 30-min before inducing asystole. Heart rate (HR) and ejection-fraction (EF) were measured before and 30-min after GW1100. In both protocols, HR, EF and fractional shortening (FS) were measured before asystole (baseline) and at 1, 5 and 10-min after LE. Means±SEM are reported. Results: In protocol-1, baseline HR and EF were 321±21 beats/min and 72.3±4.6%. Bupivacaine resulted in asystole. LE improved HR gradually within 10min; HR was 86±13 at 1min (27% recovery), 216±10 at 5min (67% recovery), and 228±14 at 10min (71% recovery). LV function fully recovered within 5 min of LE treatment as EF and FS were similar to baseline (EF=72±5%, FS=42±4%). To test our hypothesis, rats were pretreated with GW1100, 30-min before inducing asystole. There were no significant differences between HR and EF before (HR=316.6±3.3, EF=68.0±2.3%) and 30-min after GW1100 (HR=330±5.7, EF=71.6±1.5%) excluding GW1100 effects on hemodynamics. GW1100 pre-treatment however prevented LE rescue, with no recovery of cardiac function even after 10min of LE. Conclusions: This is the first report of involvement of GPR40 in rescue of Bupivacaine-induced cardiac arrest by LE. Discovery of GPR40 as a potential receptor for effects of LE may help us understand LE-induced cardioprotection.

Fetuin A promotes lipotoxicity in β cells through the TLR4 signaling pathway and the role of pioglitazone in anti-lipotoxicity

ObjectiveFetuin A (FetA), a secreted glycoprotein, is known to affect inflammation and insulin resistance (IR) in obese humans and animals. Lipotoxicity from chronic hyperlipidemia damages pancreatic β cells, hastening the onset of diabetes. We sought to determine whether FetA promotes lipotoxicity through modulation of the toll-like receptor 4 (TLR4) inflammatory signaling pathway as well as the protective effect of pioglitazone(PIO) on lipotoxicity. MethodsβTC6, a glucose-sensitive mouse pancreatic β cell line, and Sprague–Dawley rats with diet-induced obesity, were used to investigate FetA-mediated lipotoxicity. Protein expression/activation were measured by Western blotting. Small interfering (si)RNAs for TLR4 were used. Cell apoptosis was quantified by TUNEL analysis or flow cytometry, respectively. Insulin release was assessed with an insulin ELISA. ResultsFetA dose-dependently aggravated palmitic acid (PA)-induced βTC6 cell apoptosis, insulin secretion impairment, and inhibition of the expression of G-protein-coupled receptor 40 (GPR40) and pancreatic duodenal homeobox-1(PDX-1). Combined FetA + PA induced TLR4 expression, and subsequent inhibition of TLR4 signaling or expression was shown to prevent the strengthening effect of FetA on PA-induced lipotoxicity in βTC6 cells. FetA + PA induced p-JNK and nuclear factor-κB (NF-κB) subunit P65 expression, and inhibition of this activity reduced PA+ FetA lipotoxicity in βTC6 cells. PIO could ameliorate PA+ FetA-induced damage to βTC6 cells. Similarly, PIO improved insulin secretion disorder, reduced apoptosis, decreased FetA, TLR4, p-JNK, NF-κB subunit P65 and cleaved caspase 3 expression, and increased GPR40 and PDX-1 expression in islet β cells of diet-induced obese rats. The correlative bivariate analysis showed that increases in Fetuin A were directly proportional to the development of β cell injury. ConclusionsFetA can promote lipotoxicity in β cells through the TLR4-JNK-NF-κB signaling pathway. The protective effects of PIO on lipotoxicity in β cells may involve the inhibition of the activation of the FetA and TLR4 signaling pathway.

Overexpression of G-protein-coupled receptor 40 enhances the mitogenic response to epoxyeicosatrienoic acids.

The cytochrome P450 epoxygenase-dependent arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), are potent survival factors and mitogens for renal epithelial cells, but the molecular identity in the cells that initiates the mitogenic signaling of EETs has remained elusive. We screened kidney cell lines for the expression of G-protein-coupled receptor 40 (GPR40) and found that the porcine renal tubular epithelial cell line LLCPKcl4, which has been previously demonstrated to be sensitive to the mitogenic effect of EETs, expresses higher levels of GPR40 mRNA and protein than the human embryonic kidney cell line HEK293. EETs induced only a weak mitogenic EGFR signaling and mild cell proliferation in HEK293 cells. To determine whether GPR40 expression level is what mediates the mitogenic sensitivity of cells to EETs, we created a human GPR40 (hGPR40) cDNA construct and transfected it into HEK293 cells and picked up a number of stable transfectants. We found that GPR40 overexpression in HEK293 cells indeed significantly enhanced EET-induced cell proliferation and markedly augmented EGFR phosphorylation ERK activation, which were inhibited by the EGFR tyrosine kinase inhibitor, AG1478, or the HB-EGF inhibitor, CRM197. EETs significantly enhanced release of soluble HB-EGF, a natural ligand of EGFR, into the culture medium of hGPR40-transfected HEK293 cells, compared to empty vector-transfected cells. In mouse kidneys, markedly higher level of GPR40 protein was found in the cortex and outer stripe of outer medulla compared to the inner stripe of outer medulla and inner medulla. In situ hybridization confirmed that GPR40 mRNA was localized to a subset of renal tubules in the kidney, including the cortical collecting duct. Thus, this study provides the first demonstration that upregulation of GPR40 expression enhances the mitogenic response to EETs and a relatively high expression level of GPR40 is detected in a subset of tubules including cortical collecting ducts in the mammalian kidney.

Exendin-4 protects pancreatic beta cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway

The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated. The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA. Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitate-induced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitate-induced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis. Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7- and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner.

Long‐term in vitro treatment of INS‐1 rat pancreatic β‐cells by unsaturated free fatty acids protects cells against gluco‐ and lipotoxicities via activation of GPR40 receptors

G-protein coupled receptor 40 (GPR40) is a membrane-bound G-protein-coupled receptor with high binding affinity to medium- and long-chain free fatty acids (FFAs). Acute activation of GPR40 on pancreatic β-cells causes insulin secretion, whereas prolonged activation may contribute to a deterioration of the effect of saturated FFAs on β-cells. It has been documented that different types of FFAs produce various effects on insulin secretion; however, little information is available regarding the expression of GPR40 and its function after long-term exposure of β-cells to unsaturated FFAs. In the present study, GPR40 expression and function were assessed in INS-1 β-cells after 48 h exposure to different types of unsaturated FFAs. The mRNA and protein expression of GPR40 was increased significantly by long-term exposure of cells to polyunsaturated α-linolenic acid, but not to either oleic acid or linoleic acid. Immunocytochemistry revealed a reduction in the number of insulin-containing granules in cells treated with α-linolenic acid, which was correlated with an increase in cellular expression of GPR40. Basal and glucose-stimulated insulin secretion were markedly suppressed by 48 h treatment of cells with saturated palmitic acid, but not unsaturated α-linolenic acid. By testing various FFAs, it was found that FFA-induced suppression of basal and glucose-stimulated insulin secretion was attenuated by an increase in the degree of unsaturation of the FFAs and GPR40 expression in response to FFA treatment in INS-1 cells. The results of the present study indicate that long-term in vitro treatment of INS-1 rat pancreatic β-cells by unsaturated FFAs protects the cells against from gluco- and lipotoxicities and that this coincides with an increase in GPR40 expression.

New insights into “GPR40‐CREB interaction inadult neurogenesis” specific for primates

Polyunsaturated fatty acids (PUFA), such as docosahexaenoic (DHA) and arachidonic acids (ARA) are known to be closely related to the brain development and also have beneficial effects on adult neurogenesis, learning, and mental disorders. Although PUFA were demonstrated as ligands for G protein-coupled receptor 40 (GPR40), their signaling mechanism in the brain, especially in the neurogenic niche, remains unknown. Using a monkey model of ischemia-enhanced hippocampal neurogenesis, we studied the spatial correlation between GPR40 and the phosphorylated cAMP response element-binding protein (pCREB), a transcription factor involved in adult neurogenesis, learning and memory. Furthermore, the brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB), both being downstream gene transcripts of pCREB, were studied. Similar to the dynamic change of GPR40 as the authors reported previously, pCREB was up-regulated significantly after transient global brain ischemia on Western blots, and this was associated with an enhanced hippocampal neurogenesis. Immunofluorescence microscopic analysis showed that GPR40 and pCREB expression patterns were completely identical, and they were coexpressed in both mature and newborn neurons as well as in the astrocytes residing in the subgranular zone (SGZ). GPR40/pCREB double-positive cells significantly increased in the SGZ on day 15 after ischemia. The mature form of BDNF (mBDNF) and TrkB receptor showed no remarkable changes on Western blots, although proBDNF (precursor of mBDNF) was maximal on day 9. Immunofluorescence microscopy showed that the newborn neurons expressed BDNF, but not TrkB. These results altogether suggest that PUFA, GPR40, pCREB, and BDNF may be engaged in the same signaling pathway to promote neurogenesis in the adult primate hippocampus.

The G-Protein−Coupled Receptor GPR40 Directly Mediates Long-Chain Fatty Acid−Induced Secretion of Cholecystokinin

Long-chain fatty acid receptors G-protein-coupled receptor 40 (GPR40) (FFAR1) and GPR120 have been implicated in the chemosensation of dietary fats. I cells in the intestine secrete cholecystokinin (CCK), a peptide hormone that stimulates digestion of fat and protein, but these cells are rare and hard to identify. We sought to determine whether dietary fat-induced secretion of CCK is directly mediated by GPR40 expressed on I cells. We used fluorescence-activated cell sorting to isolate a pure population of I cells from duodenal mucosa in transgenic mice that expressed green fluorescent protein under the control of the CCK promoter (CCK-enhanced green fluorescent protein [eGFP] bacterial artificial chromosome mice). CCK-eGFP cells were evaluated for GPR40 expression by quantitative reverse transcription polymerase chain reaction and immunostaining. GPR40(-/-) mice were bred with CCK-eGFP mice to evaluate functional relevance of GPR40 on long-chain fatty acid-stimulated increases in [Ca(2+)]i and CCK secretion in isolated CCK-eGFP cells. Plasma levels of CCK after olive oil gavage were compared between GPR40(+/+) and GPR40(-/-) mice. Cells that expressed eGFP also expressed GPR40; expression of GPR40 was 100-fold greater than that of cells that did not express eGFP. In vitro, linoleic, oleic, and linolenic acids increased [Ca(2+)]i; linolenic acid increased CCK secretion by 53% in isolated GPR40(+/+) cells that expressed eGFP. In contrast, in GPR40(-/-) that expressed eGFP, [Ca(2+)]i response to linoleic acid was reduced by 50% and there was no significant CCK secretion in response to linolenic acid. In mice, olive oil gavage significantly increased plasma levels of CCK compared with pregavage levels: 5.7-fold in GPR40(+/+) mice and 3.1-fold in GPR40(-/-) mice. Long-chain fatty acid receptor GPR40 induces secretion of CCK by I cells in response to dietary fat.

G-protein-coupled receptor 40 (GPR40) expression and its regulation in human pancreatic islets: The role of type 2 diabetes and fatty acids

Background and aimsGPR40 is a membrane-bound receptor paired with medium and long-chain fatty acids (FFA) as endogenous ligands. Its acute activation potentiates insulin secretion from beta cells, whereas prolonged binding might contribute to the deleterious effects of chronic exposure to FFA. Little information is available on the expression of GPR40 and its regulation in human islets (HI). Material and methodsHI were prepared by enzymatic digestion and gradient separation from the pancreas of 20 non-diabetic (Ctrl) and 13 type 2 diabetic (T2DM) multiorgan donors, and functional and molecular studies were then performed. ResultsBy qualitative and quantitative PCR experiments, mRNA expression was shown in HI. Both in T2DM islets and in Ctrl islets pre-exposed for 24h to 1.0mmol/l FFA (palmitate:oleate, 2:1), GPR40 mRNA expression was significantly reduced (p<0.01) in the T2DM cells as compared to Ctrl cells. A significant positive correlation was found between glucose-stimulated insulin secretion and GPR40 expression. ConclusionsThese results show the expression of GPR40 in human pancreatic islets which are regulated by FFA. The finding that T2DM islets have a lower GPR40 expression, and the correlation of these genes with insulin secretion, raises the possibility of an involvement of GPR40 in human diabetes beta-cell dysfunction.

Fatty acids as regulators of hippocampal neurogenesis: the case of GPR40

The hippocampus of adult mammals including humans continues to generate neurons throughout life. The neural stem/progenitor cells, the cellular phenotype responsible for this regenerative capacity, are a subject of intensive research focused on the mechanisms of their regulation. Intriguing recent data implicate an unexpected molecular player in this regulation: free fatty acids. Polyunsaturated fatty acids (PUFA) such as docosahexaenoic and arachidonic acids were identified to act on the pancreas via a novel receptor, named G-protein coupled receptor 40 (GPR40). However, the pancreas appears not to be the single target organ of PUFA/GPR40 actions. Very recent findings have discovered GPR40 expression in the hippocampal dentate gyrus, including its progenitor cell niche responsible for neuronal renewal throughout life. These data open a possibility that PUFA may regulate adult hippocampal neurogenesis and thus become a therapeutic tool to treat neurological disorders in humans. Biomedical Reviews 2007; 18: 69-73.

Expression of the gene for a membrane-bound fatty acid receptor in the pancreas and islet cell tumours in humans: evidence for GPR40 expression in pancreatic beta cells and implications for insulin secretion

G protein-coupled receptor 40 (GPR40) is abundantly expressed in pancreatic beta cells in rodents, where it facilitates glucose-induced insulin secretion in response to mid- to long-chain fatty acids in vitro. However, GPR40 gene expression in humans has not been fully investigated, and little is known about the physiological and pathophysiological roles of GPR40 in humans. The aim of this study, therefore, was to examine GPR40 expression and its clinical implications in humans. GPR40 mRNA expression in the human pancreas, pancreatic islets and islet cell tumours was analysed using TaqMan PCR. GPR40 mRNA was detected in all human pancreases collected intraoperatively. It was enriched approximately 20-fold in isolated islets freshly prepared from the pancreases of the same individuals. The estimated mRNA copy number for the GPR40 gene in pancreatic islets was comparable to those for genes encoding sulfonylurea receptor 1, glucagon-like peptide 1 receptor and somatostatin receptors, all of which are known to be expressed abundantly in the human pancreatic islet. A large amount of GPR40 mRNA was detected in insulinoma tissues, whereas mRNA expression was undetectable in glucagonoma or gastrinoma. The GPR40 mRNA level in the pancreas correlated with the insulinogenic index, which reflects beta cell function (r=0.82, p=0.044), but not with glucose levels during the OGTT, the insulin area under the OGTT curve or the index for the homeostasis model assessment of insulin resistance (HOMA-IR). The present study provides evidence for GPR40 gene expression in pancreatic beta cells and implicates GPR40 in insulin secretion in humans.