Fatty acid binding protein 5 suppression attenuates obesity-induced hepatocellular carcinoma by promoting ferroptosis and intratumoral immune rewiring.

Due to the rise in overnutrition, the incidence of obesity-induced hepatocellular carcinoma (HCC) will continue to escalate; however, our understanding of the obesity to HCC developmental axis is limited. We constructed a single-cell atlas to interrogate the dynamic transcriptomic changes during hepatocarcinogenesis in mice. Here we identify fatty acid binding protein 5 (FABP5) as a driver of obesity-induced HCC. Analysis of transformed cells reveals that FABP5 inhibition and silencing predispose cancer cells to lipid peroxidation and ferroptosis-induced cell death. Pharmacological inhibition and genetic ablation of FABP5 ameliorates the HCC burden in male mice, corresponding to enhanced ferroptosis in the tumour. Moreover, FABP5 inhibition induces a pro-inflammatory tumour microenvironment characterized by tumour-associated macrophages with increased expression of the co-stimulatory molecules CD80 and CD86 and increased CD8+ T cell activation. Our work unravels the dual functional role of FABP5 in diet-induced HCC, inducing the transformation of hepatocytes and an immunosuppressive phenotype of tumour-associated macrophages and illustrates FABP5 inhibition as a potential therapeutic approach.

Heterogeneity of hepatocyte dynamics restores liver architecture after chemical, physical or viral damage.

Midlobular hepatocytes are proposed to be the most plastic hepatic cell, providing a reservoir for hepatocyte proliferation during homeostasis and regeneration. However, other mechanisms beyond hyperplasia have been little explored and the contribution of other hepatocyte subpopulations to regeneration has been controversial. Thus, re-examining hepatocyte dynamics during regeneration is critical for cell therapy and treatment of liver diseases. Using a mouse model of hepatocyte- and non-hepatocyte- multicolor lineage tracing, we demonstrate that midlobular hepatocytes also undergo hypertrophy in response to chemical, physical, and viral insults. Our study shows that this subpopulation also combats liver impairment after infection with coronavirus. Furthermore, we demonstrate that pericentral hepatocytes also expand in number and size during the repair process and Galectin-9-CD44 pathway may be critical for driving these processes. Notably, we also identified that transdifferentiation and cell fusion during regeneration after severe injury contribute to recover hepatic function.

Kisspeptin upregulates ß-cell serotonin production during pregnancy.

During pregnancy the maternal pancreatic islets of Langerhans undergo adaptive changes to compensate for gestational insulin resistance. The lactogenic hormones are well established to play a key role in regulating the islet adaptation to pregnancy, and one of the mechanisms through which they act is through upregulating ß-cell serotonin production. During pregnancy islet serotonin levels are significantly elevated, where it is released from the ß-cells to drive the adaptive response through paracrine and autocrine effects. We have previously shown that placental kisspeptin (KP) also plays a role in promoting the elevated insulin secretion and ß-cell proliferation observed during pregnancy, although the precise mechanisms involved are unclear. In the present study we investigated the effects of KP on expression of pro-proliferative genes and serotonin biosynthesis within rodent islets. Whilst KP had limited effect on pro-proliferative gene expression at the time points tested, KP did significantly stimulate expression of the serotonin biosynthesis enzyme Tph-1. Furthermore, the islets of pregnant ß-cell-specific GPR54 knockdown mice were found to contain significantly fewer serotonin-positive ß-cells when compared to pregnant controls. Our previous studies suggested that reduced placental kisspeptin production, with consequent impaired kisspeptin-dependent ß-cell compensation, may be a factor in the development of GDM in humans. These current data suggest that, similar to the lactogenic hormones, KP may also contribute to serotonin biosynthesis and subsequent islet signalling during pregnancy. Furthermore, upregulation of serotonin biosynthesis may represent a common mechanism through which multiple signals might influence the islet adaptation to pregnancy.

Loss of cis-PTase function in the liver promotes a highly penetrant form of fatty liver disease that rapidly transitions to hepatocellular carcinoma.

Obesity-linked fatty liver is a significant risk factor for hepatocellular carcinoma (HCC)1,2; however, the molecular mechanisms underlying the transition from non-alcoholic fatty liver disease (NAFLD) to HCC remains unclear. The present study explores the role of the endoplasmic reticulum (ER)-associated protein NgBR, an essential component of the cis-prenyltransferases (cis-PTase) enzyme3, in chronic liver disease. Here we show that genetic depletion of NgBR in hepatocytes of mice (N-LKO) intensifies triacylglycerol (TAG) accumulation, inflammatory responses, ER/oxidative stress, and liver fibrosis, ultimately resulting in HCC development with 100% penetrance after four months on a high-fat diet. Comprehensive genomic and single cell transcriptomic atlas from affected livers provides a detailed molecular analysis of the transition from liver pathophysiology to HCC development. Importantly, pharmacological inhibition of diacylglycerol acyltransferase-2 (DGAT2), a key enzyme in hepatic TAG synthesis, abrogates diet-induced liver damage and HCC burden in N-LKO mice. Overall, our findings establish NgBR/cis-PTase as a critical suppressor of NAFLD-HCC conversion and suggests that DGAT2 inhibition may serve as a promising therapeutic approach to delay HCC formation in patients with advanced non-alcoholic steatohepatitis (NASH).

The selective serotonin reuptake inhibitor fluoxetine has direct effects on beta cells, promoting insulin secretion and increasing beta-cell mass.

AIM: This study investigated whether therapeutically relevant concentrations of fluoxetine, which have been shown to reduce plasma glucose and glycated haemoglobin independent of changes in food intake and body weight, regulate beta-cell function and improve glucose homeostasis. METHODS: Cell viability, insulin secretion, beta-cell proliferation and apoptosis were assessed after exposure of MIN6 beta cells or isolated mouse and human islets to 0.1, 1 or 10 µmol/L fluoxetine. The effect of fluoxetine (10 mg/kg body weight) administration on glucose homeostasis and islet function was also examined in ob/ob mice. RESULTS: Exposure of MIN6 cells and mouse islets to 0.1 and 1 µmol/L fluoxetine for 72 hours did not compromise cell viability but 10 µmol/L fluoxetine significantly increased Trypan blue uptake. The dose of 1 µmol/L fluoxetine significantly increased beta-cell proliferation and protected islet cells from cytokine-induced apoptosis. In addition, 1 µmol/L fluoxetine induced rapid and reversible potentiation of glucose-stimulated insulin secretion from islets isolated from mice, and from lean and obese human donors. Finally, intraperitoneal administration of fluoxetine to ob/ob mice over 14 days improved glucose tolerance and resulted in significant increases in beta-cell proliferation and enhanced insulin secretory capacity. CONCLUSIONS: These data are consistent with a role for fluoxetine in regulating glucose homeostasis through direct effects on beta cells. Fluoxetine thus demonstrates promise as a preferential antidepressant for patients with concomitant occurrence of depression and diabetes.

MicroRNAs and Circular RNAs in Lipoprotein Metabolism.

PURPOSE OF REVIEW: Non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and circular RNAs (circRNAs) are pivotal regulators of mRNA and protein expression that critically contribute to cardiovascular pathophysiology. Although little is known about the origin and function of such ncRNAs, they have been suggested as promising biomarkers with powerful therapeutic value in cardiovascular disease (CVD). In this review, we summarize the most recent findings on ncRNAs biology and their implication on cholesterol homeostasis and lipoprotein metabolism that highlight novel therapeutic avenues for treating dyslipidemia and atherosclerosis. RECENT FINDINGS: Clinical and experimental studies have elucidated the underlying effects that specific miRNAs impose both directly and indirectly regulating circulating high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) metabolism and cardiovascular risk. Some of these relevant miRNAs include miR-148a, miR-128-1, miR-483, miR-520d, miR-224, miR-30c, miR-122, miR-33, miR-144, and miR-34. circRNAs are known to participate in a variety of physiological and pathological processes due to their abundance in tissues and their stage-specific expression activation. Recent studies have proven that circRNAs may be considered targets of CVD as well. Some of these cirRNAs are circ-0092317, circ_0003546, circ_0028198, and cirFASN that have been suggested to be strongly involved in lipoprotein metabolism; however, their relevance in CVD is still unknown. MicroRNA and cirRNAs have been proposed as powerful therapeutic targets for treating cardiometabolic disorders including atherosclerosis. Here, we discuss the recent findings in the field of lipid and lipoprotein metabolism underscoring the novel mechanisms by which some of these ncRNAs influence lipoprotein metabolism and CVD.

Direct Stimulatory Effects of the CB2 Ligand JTE 907 in Human and Mouse Islets.

AIMS: The endocannabinoid system is a complex cell-signaling network through which endogenous cannabinoid ligands regulate cell function by interaction with CB1 and CB2 cannabinoid receptors, and with the novel cannabinoid receptor GPR55. CB1, CB2, and GPR55 are expressed by islet ß-cells where they modulate insulin secretion. We have previously shown that administration of the putative CB2 antagonist/inverse agonist JTE 907 to human islets did not affect the insulinotropic actions of CB2 agonists and it unexpectedly stimulated insulin secretion on its own. In this study, we evaluated whether the lack of antagonism could be related to the ability of JTE 907 to act as a GPR55 agonist. MATERIALS AND METHODS: We used islets isolated from human donors and from Gpr55+/+ and Gpr55-/- mice and quantified the effects of incubation with 10 µM JTE 907 on dynamic insulin secretion, apoptosis, and ß-cell proliferation by radioimmunoassay, luminescence caspase 3/7 activity, and immunofluorescence, respectively. We also measured islet IP1 and cAMP accumulation using fluorescence assays, and monitored [Ca2+]i elevations by Fura-2 single cell microfluorometry. RESULTS: JTE 907 significantly stimulated insulin secretion from islets isolated from human donors and islets from Gpr55+/+ and Gpr55-/- mice. These stimulatory effects were accompanied by significant elevations of IP1 and [Ca2+]i, but there were no changes in cAMP generation. JTE 907 also significantly reduced cytokine-induced apoptosis in human and mouse islets and promoted human ß-cell proliferation. CONCLUSION: Our observations show for the first time that JTE 907 acts as a Gq-coupled agonist in islets to stimulate insulin secretion and maintain ß-cell mass in a GPR55-independent fashion.

Assessing Mouse Islet Function.

Islets of Langerhans are clusters of endocrine cells embedded within the exocrine pancreas. Islets constitute only approximately 1-2% of the total pancreas mass in all species, so methods have been developed to digest the pancreas and purify islets from the surrounding acinar cells. This chapter provides detailed protocols for isolation of mouse islets and their in vitro functional characterization in terms of assessments of islet viability, hormone content and secretion, second messenger generation and ß-cell proliferation.

The cannabinoid ligands SR141716A and AM251 enhance human and mouse islet function via GPR55-independent signalling.

AIMS: Endocannabinoids are lipid mediators involved in the regulation of glucose homeostasis. They interact with the canonical cannabinoid receptors CB1 and CB2, and it is now apparent that some cannabinoid receptor ligands are also agonists at GPR55. Thus, CB1 antagonists such as SR141716A, also known as rimonabant, and AM251 act as GPR55 agonists in some cell types. The complex pharmacological properties of cannabinoids make it difficult to fully identify the relative importance of CB1 and GPR55 in the functional effects of SR141716A, and AM251. Here, we determine whether SR141716A and AM251 regulation of mouse and human islet function is through their action as GPR55 agonists. METHODS: Islets isolated from Gpr55+/+ and Gpr55-/- mice and human donors were incubated in the absence or presence of 10 µM SR141716A or AM251, concentrations that are known to activate GPR55. Insulin secretion, cAMP, IP1, apoptosis and ß-cell proliferation were quantified by standard techniques. RESULTS: Our results provide the first evidence that SR141716A and AM251 are not GPR55 agonists in islets, as their effects are maintained in islets isolated from Gpr55-/- mice. Their signalling through Gq-coupled cascades to induce insulin secretion and human ß-cell proliferation, and protect against apoptosis in vitro, indicate that they have direct beneficial effects on islet function. CONCLUSION: These observations may be useful in directing development of peripherally restricted novel therapeutics that are structurally related to SR141716A and AM251, and which potentiate glucose-induced insulin secretion and stimulate ß-cell proliferation.

Short chain fatty acids stimulate insulin secretion and reduce apoptosis in mouse and human islets in vitro: Role of free fatty acid receptor 2.

AIMS: To evaluate the role of free fatty acid receptor 2 (FFAR2)/G-protein coupled receptor 43 in mediating the effects of the short chain fatty acids (SCFAs) sodium acetate (SA) and sodium propionate (SP) on islet function in vitro, and to identify the intracellular signalling pathways used in SCFA-induced potentiation of glucose-induced insulin secretion. MATERIALS AND METHODS: Islets of Langerhans were isolated from wild-type and FFAR2-/- mice and from human donors without diabetes. The effects of SA and SP on dynamic insulin secretion from perifused islets were quantified by radioimmunoassay, signalling downstream of SCFAs was profiled by single-cell calcium microfluorimetry, and measurement of cAMP was performed using a fluorescence assay. Islet apoptosis was induced by exposure to cytokines or sodium palmitate, and the effects of SA and SP in regulating islet apoptosis were assessed by quantification of caspase 3/7 activities. RESULTS: Deletion of FFAR2 did not affect islet morphology or insulin content. SA and SP reversibly potentiated insulin secretion from mouse islets in a FFAR2-dependent manner. SCFA-induced potentiation of insulin secretion was coupled to Gq activation of phospholipase C and protein kinase C, with no evidence of Gi-mediated signalling. SA and SP protected human and mouse islets from apoptosis, and these pro-survival properties were dependent on islet expression of FFAR2. CONCLUSION: Our results indicate that FFAR2 directly mediates both the stimulatory effects of SA and SP on insulin secretion and their protection against islet apoptosis. We have also shown that SCFA coupling in islets occurs via Gq-coupled intracellular signalling.

Identifying Signalling Pathways Regulated by GPRC5B in ß-Cells by CRISPR-Cas9-Mediated Genome Editing.

BACKGROUND/AIMS: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 ß-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. METHODS: The optimal transfection reagent for use in MIN6 ß-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 ß-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 ß-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. RESULTS: JetPRIME® was the best candidate for MIN6 ß-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 ß-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-ß and IFNγ. CONCLUSION: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 ß-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to ß-cell proliferation and apoptosis.

Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets.

INTRODUCTION: Islets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets. MATERIALS AND METHODS: GPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay. RESULTS: We have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion. DISCUSSION: The detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.

C3aR and C5aR1 act as key regulators of human and mouse ß-cell function.

AIMS: Complement components 3 and 5 (C3 and C5) play essential roles in the complement system, generating C3a and C5a peptides that are best known as chemotactic and inflammatory factors. In this study we characterised islet expression of C3 and C5 complement components, and the impact of C3aR and C5aR1 activation on islet function and viability. MATERIALS AND METHODS: Human and mouse islet mRNAs encoding key elements of the complement system were quantified by qPCR and distribution of C3 and C5 proteins was determined by immunohistochemistry. Activation of C3aR and C5aR1 was determined using DiscoverX beta-arrestin assays. Insulin secretion from human and mouse islets was measured by radioimmunoassay, and intracellular calcium ([Ca2+]i), ATP generation and apoptosis were assessed by standard techniques. RESULTS: C3 and C5 proteins and C3aR and C5aR1 were expressed by human and mouse islets, and C3 and C5 were mainly localised to ß- and α-cells. Conditioned media from islets exposed for 1 h to 5.5 and 20 mM glucose stimulated C3aR and C5aR1-driven beta-arrestin recruitment. Activation of C3aR and C5aR1 potentiated glucose-induced insulin secretion from human and mouse islets, increased [Ca2+]i and ATP generation, and protected islets against apoptosis induced by a pro-apoptotic cytokine cocktail or palmitate. CONCLUSIONS: Our observations demonstrate a functional link between activation of components of the innate immune system and improved ß-cell function, suggesting that low-level chronic inflammation may improve glucose homeostasis through direct effects on ß-cells.

LH-21 and abnormal cannabidiol improve ß-cell function in isolated human and mouse islets through GPR55-dependent and -independent signalling.

AIMS: To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55-/- mice. MATERIALS AND METHODS: Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca2+ ]i, cAMP, apoptosis, ß-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. RESULTS: Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca2+ ]i in human islets and islets from both GPR55+/+ and GPR55-/- mice. LH-21 also increased insulin secretion and [Ca2+ ]i in human islets and GPR55+/+ mouse islets, but concentrations of LH-21 up to 0.1 µM were ineffective in islets from GPR55-/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55+/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased ß-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55-/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. CONCLUSIONS: This study showed that Abn-CBD and LH-21 improve human and mouse islet ß-cell function and viability. Use of islets from GPR55-/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised.

Dynamic Profiling of Insulin Secretion and ATP Generation in Isolated Human and Mouse Islets Reveals Differential Glucose Sensitivity.

BACKGROUND/AIMS: Rodent islets are often used for basic science research but they do not always recapitulate signalling events in human islets. This study evaluated the glucose-dependent responses of human and mouse islets in terms of dynamic insulin secretion, metabolic coupling and the role of glucose transporters. METHODS: Glucose-induced insulin secretion from isolated mouse and human islets was profiled by perifusion and islet ATP levels were measured by chemoluminescence assay. Glucose transporter expression was determined by qPCR and western blotting. RESULTS: Human islets show a left-shifted glucose concentration-insulin secretion profile compared to mouse islets. These data are consistent with glucose transporter expression, with human islets expressing mainly GLUT1 and GLUT3, and GLUT2 being the predominant transporter in mouse islets. Using the GLUT1 inhibitor STF-31 we unveiled an important role for GLUT1 for differences in glucose-induced insulin secretion profiles observed between the two species. CONCLUSION: The high affinity of GLUT1/3 for glucose reflects the left-shifted glucose-induced insulin secretory response of human islets and the impairment of insulin secretion from human islets after STF-31 treatment indicates an important role for GLUT1 in human islet stimulus-secretion coupling. Our data provide further insight into key differences between insulin secretion regulation in mouse and human islets.

The cannabinoid ligand LH-21 reduces anxiety and improves glucose handling in diet-induced obese pre-diabetic mice.

LH-21 is a triazol derivative that has been described as a low-permeant neutral CB1 antagonist, though its pharmacology is still unclear. It has been associated with anti-obesity actions in obese rats. However, its role in preventing type 2 diabetes (T2D) onset have not been studied yet. Given CB1 receptors remain as potential pharmacological targets to fight against obesity and T2D, we wanted to explore the metabolic impact of this compound in an animal model of obesity and pre-diabetes as well as the lack of relevant actions in related central processes such as anxiety. C57BL/6J mice were rendered obese and pre-diabetic by feeding a high-fat diet for 15 weeks and then treated with LH-21 or vehicle for two weeks. Food intake, body weight and glucose handling were assessed, together with other relevant parameters. Behavioural performance was evaluated by the open field test and the elevated plus maze. LH-21 did not affect food intake nor body weight but it improved glucose handling, displaying tissue-specific beneficial actions. Unexpectedly, LH-21 induced anxiolysis and reverted obesity-induced anxiety, apparently through GPR55 receptor. These results suggest that LH-21 can be a new candidate to fight against diabetes onset. Indeed, this compound shows potential in counteracting obesity-related anxiety.

A comparative analysis of human and mouse islet G-protein coupled receptor expression.

G-protein coupled receptors (GPCRs) are essential for islet function, but most studies use rodent islets due to limited human islet availability. We have systematically compared the GPCR mRNA expression in human and mouse islets to determine to what extent mouse islets can be used as surrogates for human islets to study islet GPCR function, and we have identified species-specific expression of several GPCRs. The A3 receptor (ADORA3) was expressed only in mouse islets and the A3 agonist MRS 5698 inhibited glucose-induced insulin secretion from mouse islets, with no effect on human islets. Similarly, mRNAs encoding the galanin receptors GAL1 (GALR1), GAL2 (GALR2) and GAL3 GALR3) were abundantly expressed in mouse islets but present only at low levels in human islets, so that it reads (GALR3) and galanin inhibited insulin secretion only from mouse islets. Conversely, the sst1 receptor (SSTR1) was abundant only in human islets and its selective activation by CH 275 inhibited insulin secretion from human islets, with no effect on mouse islets. Our comprehensive human and mouse islet GPCR atlas has demonstrated that species differences do exist in islet GPCR expression and function, which are likely to impact on the translatability of mouse studies to the human context.

GPR55-dependent stimulation of insulin secretion from isolated mouse and human islets of Langerhans.

AIMS: The novel cannabinoid receptor GPR55 is expressed by rodent islets and it has been implicated in ß-cell function in response to a range of ligands. This study evaluated the effects of GPR55 ligands on intracellular calcium ([Ca2+ ]i ) levels and insulin secretion from islets isolated from GPR55 knockout (GPR55 -/- ) mice, age-matched wildtype (WT) mice and human pancreas. MATERIALS AND METHODS: GPR55 expression was determined by Western blotting and fluorescent immunohistochemistry. Changes in [Ca2+ ]i were measured by Fura-2 microfluorimetry. Dynamic insulin secretion was quantified by radioimmunoassay following perifusion of isolated islets. RhoA activity was monitored using a Rho binding domain pull down assay. RESULTS: Western blotting indicated that MIN6 ß-cells, mouse and human islets express GPR55 and its localization on human ß-cells was demonstrated by fluorescent immunohistochemistry. The pharmacological GPR55 agonist O-1602 (10 µM) significantly stimulated [Ca2+ ]i and insulin secretion from WT mouse islets and these stimulatory effects were abolished in islets isolated from GPR55 -/- mice. In contrast, while the putative endogenous GPR55 agonist lysophosphatidylinositol (LPI, 5 µM) and the GPR55 antagonist cannabidiol (CBD, 1 µM) also elevated [Ca2+ ]i and insulin secretion, these effects were sustained in islets from GPR55 -/- mice. Stimulatory effects of O-1602 on [Ca2+ ]i and insulin secretion were also observed in experiments using human islets, but O-1602 did not activate RhoA in MIN6 ß-cells. CONCLUSIONS: Our results therefore suggest that GPR55 plays an important role in the regulation of mouse and human islet physiology, but LPI and CBD exert stimulatory effects on islet function by a GPR55-independent pathway(s).

The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice.

The endocannabinoid system (ECS) is an intercellular signalling mechanism that is present in the islets of Langerhans and plays a role in the modulation of insulin secretion and expansion of the ß-cell mass. The downstream signalling pathways mediating these effects are poorly understood. Mammalian target of rapamycin complex 1 (mTORC1) signalling is a key intracellular pathway involved in energy homeostasis and is known to importantly affect the physiology of pancreatic islets. We investigated the possible relationship between cannabinoid type 1 (CB1) receptor signalling and the mTORC1 pathway in the endocrine pancreas of mice by using pharmacological analysis as well as mice genetically lacking the CB1 receptor or the downstream target of mTORC1, the kinase p70S6K1. In vitro static secretion experiments on islets, western blotting, and in vivo glucose and insulin tolerance tests were performed. The CB1 receptor antagonist rimonabant decreased glucose-stimulated insulin secretion (GSIS) at 0.1 µM while increasing phosphorylation of p70S6K1 and ribosomal protein S6 (rpS6) within the islets. Specific pharmacological blockade of mTORC1 by 3 nM rapamycin, as well as genetic deletion of p70S6K1, impaired the CB1-antagonist-mediated decrease in GSIS. In vivo experiments showed that 3 mg/kg body weight rimonabant decreased insulin levels and induced glucose intolerance in lean mice without altering peripheral insulin sensitivity; this effect was prevented by peripheral administration of low doses of rapamycin (0.1 mg/kg body weight), which increased insulin sensitivity. These findings suggest a functional interaction between the ECS and the mTORC1 pathway within the endocrine pancreas and at the whole-organism level, which could have implications for the development of new therapeutic approaches for pancreatic ß-cell diseases.