Biology subjects

Ghislain, J.

Publications and source records attributed to Ghislain, J..

9 recordsLinked to original sources

bioRxiv2026

The regulator of G protein signaling RGS9 supports insulin secretion in male mouse islets

Objectives Regulators of G protein signaling (RGS) proteins terminate G protein-coupled receptor signaling and regulate multiple aspects of pancreatic islet function. Although RGS9 is highly enriched in human and rodent islets, its cellular localization and physiological role remain largely unknown. Here, we investigated the expression of Rgs9 in mouse islets and its contribution to insulin secretion. Methods Rgs9 expression was characterized by RNA in situ hybridization and fluorescence-activated cell sorting coupled with quantitative PCR. Rgs9 function was investigated by shRNA-mediated knockdown in wild-type and Dishevelled, Egl-10 and Pleckstrin (DEP) domain-deficient Rgs9 mutant (Rgs9{Delta}DEP/{Delta}DEP) mouse pseudo-islets. Results Rgs9 was detected throughout the endocrine pancreas but was enriched in {beta} and {delta}cells, with the Rgs9-1 isoform representing the predominant splice variant in mouse islets. Rgs9 knockdown in mouse pseudo-islets impaired glucose- and KCl-stimulated insulin secretion without affecting insulin content. In contrast, Rgs9{Delta}DEP/{Delta}DEP mouse islets failed to reproduce this phenotype. Further characterization of the mutant mice revealed persistent expression of Rgs9 transcripts, which when knocked down impaired both glucose- and KCl-stimulated insulin secretion. Conclusions These data indicate that RGS9 supports glucose-stimulated insulin secretion. Given that RGS9 acts exclusively on Gi/o, our results further suggest that RGS9 in {beta} cells dampens inhibitory signaling to promote insulin secretion. Finally, the Rgs9{Delta}DEP/{Delta}DEP mutant does not represent a complete loss-of-function in islets likely due to the retention of the GTPase-activating protein (GAP) activity in the absence of the DEP domain.

cell biology↗

bioRxiv2025

Free fatty acid receptor 4 agonists stimulate insulin secretion via different mechanisms in mouse versus human islets

The free fatty acid receptor FFAR4 is expressed in pancreatic islets, and its activation potentiates insulin and inhibits somatostatin (SST) secretion. We investigated the mechanisms of action of FFAR4 on hormone secretion in mouse and human islets. The effects of the FFAR4 agonist Compound A (Cpd A) on insulin and SST secretion were investigated in islets from mice following ablation of {delta} cells, deletion of SST and deletion of the G protein Gz (Gnaz-/-), in purified mouse {beta} and {delta} cells, in human EndoC-{beta}H5 cells, and in human islets. Ca++ dynamics in response to Cpd A were measured in {delta} cells from Gnaz-/- mouse islets and in human islets. The insulinotropic effect of Cpd A was lost in {delta} cell-ablated and SST-deficient mouse islets and was absent in purified mouse {beta} cells. Gz deletion prevented Cpd A inhibition of SST secretion but not the potentiation of insulin release. Cpd A diminished Ca++ transients in mouse {delta} cells, an effect that was lost in Gz deficient islets. In human islets, FFAR4 activation increased insulin secretion and intracellular Ca++ transient independent of SST secretion. Consistent with a direct effect on {beta} cells, Cpd A potentiated insulin secretion in human EndoC-{beta}H5 cells. We conclude that FFAR4 activation stimulates insulin secretion from mouse islets indirectly via Gz-coupled inhibition of SST secretion from {delta} cells, while in human islets, it stimulates insulin release via a direct effect on {beta} cells. These key species-related differences are to be taken into account as FFAR4 is considered a potential therapeutic target for metabolic diseases. STRUCTURED ABSTRACTO_ST_ABSObjectivesC_ST_ABSThe free fatty acid receptor FFAR4 (GPR120) is expressed in the murine islet where its activation promotes insulin and glucagon and inhibits somatostatin (SST) secretion. However, its precise mechanism of action in different islet cells is still unknown, and potential species-related differences have not been explored. This study was aimed to address three questions: 1-What is the relative importance of {delta} cells and SST in the insulinotropic effect of FFAR4 in mouse islets? 2-Which G protein does FFAR4 couple to in mouse {delta} cells? 3-Does FFAR4 stimulate insulin secretion by similar mechanisms in mouse and human islets? MethodsThe effects of the FFAR4 agonist Compound A (Cpd A) on insulin and SST secretion were investigated in islets from mice following ablation of {delta} cells, deletion of SST and deletion of the G protein Gz (Gnaz-/-), in purified mouse {beta} and {delta} cells, in human EndoC-{beta}H5 cells, and in human islets. Ca++ dynamics in response to Cpd A were measured in {delta} cells from Gnaz-/- mouse islets and in human islets using adenovirally-transduced Ca++ reporters. ResultsThe insulinotropic effect of Cpd A was lost in {delta} cell-ablated and SST-deficient mouse islets and was absent in purified mouse {beta} cells. Gz deletion diminished Ca++ transients in mouse {delta} cells, prevented Cpd A inhibition of SST secretion but did not inhibit the potentiation of insulin release. In human islets, FFAR4 activation increased insulin secretion and intracellular Ca++ transient without affecting SST secretion. Consistent with a direct effect on {beta} cells, Cpd A potentiated insulin secretion in human EndoC-{beta}H5 cells. ConclusionFFAR4 activation stimulates insulin secretion from mouse islets indirectly via Gz-coupled inhibition of SST secretion from {delta} cells, while in human islets, it stimulates insulin release via a direct effect on {beta} cells. These key species-related differences are to be taken into account as FFAR4 is considered a potential therapeutic target for metabolic diseases.

cell biology↗

bioRxiv2022

Beta-cell mass expansion during puberty involves serotonin signaling and determines glucose homeostasis in adulthood

Puberty is associated with transient insulin resistance that normally recedes at the end of puberty; however, in overweight children insulin resistance persists leading to an increased risk of type 2 diabetes. The mechanisms whereby pancreatic {beta} cells adapt to pubertal insulin resistance, and how they are affected by the metabolic status, have not been investigated. Here we show that puberty is associated with a transient increase in {beta}-cell proliferation in rats and humans of both sexes. In rats, {beta}-cell proliferation correlated with a rise in growth hormone (GH) levels. Serum from pubertal rats and humans promoted {beta}-cell proliferation, suggesting the implication of a circulating factor. In pubertal rat islets, expression of genes of the GH/serotonin (5-HT) pathway underwent changes consistent with proliferative effect. Inhibition of the pro-proliferative 5-HT receptor isoform HTR2b blocked the increase in {beta}-cell proliferation in pubertal islets ex vivo and in vivo. Peri-pubertal metabolic stress blunted {beta}-cell proliferation during puberty and led to altered glucose homeostasis later in life. This study identifies a role of GH/GHR/5-HT/HTR2b signaling in the control of {beta}-cell mass expansion during puberty and a mechanistic link between pubertal obesity and the risk of developing type 2 diabetes.

physiology↗

bioRxiv2021

Single-cell RNA sequencing reveals a role for reactive oxygen species and peroxiredoxins in fatty-acid-induced rat β-cell proliferation

The functional mass of insulin-secreting pancreatic {beta} cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing {beta} cells. To decipher the underlying molecular mechanisms, we assessed {beta}-cell proliferation in isolated rat islets exposed to glucose and oleate or palmitate for 48 h and analyzed the transcriptional response by single-cell RNA sequencing. Unsupervised clustering of pooled {beta} cells identified subpopulations, including proliferating {beta} cells. {beta}-cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved energy metabolism and mitochondrial activity. Comparison of proliferating vs. non-proliferating {beta} cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, the antioxidant N-acetyl cysteine and the peroxiredoxin inhibitor Conoidin A both blocked oleate-induced {beta}-cell proliferation. Our data reveal a key role for ROS signaling in {beta}-cell proliferation in response to nutrients.

cell biology↗

bioRxiv2021

Very Long-Chain Unsaturated Sphingolipids Mediate Oleate-Induced Rat β-Cell Proliferation

Fatty-acid (FA) signaling contributes to {beta}-cell mass expansion in the face of nutrient excess, but the underlying mechanisms are poorly understood. Here we tested the hypothesis that sphingolipids, generated by the intracellular metabolism of FA, are implicated in the {beta}-cell proliferative response to FA. Isolated rat islets were exposed to individual FA in the presence of 16.7 mM glucose for 48 h and the contribution of the de novo sphingolipid synthesis pathway was tested using the serine palmitoyltransferase inhibitor myriocin, the sphingosine kinase (SphK) inhibitor SKI II, or adenovirus-mediated knockdown of SphK, fatty-acid-elongase-1 (ELOVL1) and acyl-CoA-binding protein (ACBP). Wistar rat were infused with glucose and the lipid emulsion ClinOleic and received SKI II by gavage. B-cell proliferation was assessed by immunochemistry or flow cytometry. Sphingolipidomic analyses were performed by LC-MS/MS. Amongst the various FA tested, only oleate increased {beta}-cell proliferation. Myriocin, SKI II, and SphK knockdown all decreased oleate-induced {beta}-cell proliferation. Oleate exposure did not increase the total amount of sphingolipids but led to a specific rise in 24:1 species. Knockdown of ACBP or ELOVL1 inhibited oleate-induced {beta}-cell proliferation. We conclude that unsaturated very long-chain sphingolipids produced from the available pool of C24:1 acyl-CoA mediate oleate-induced {beta}-cell proliferation in rats.

cell biology↗

bioRxiv2020

Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labelling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

cell biology↗

bioRxiv2020

FFAR1 and FFAR4 additively cooperate to control glucose tolerance and insulin secretion in male mice

The free fatty-acid receptors FFAR1 (GPR40) and FFAR4 (GPR120) are implicated in the regulation of insulin secretion and insulin sensitivity, respectively. Although GPR120 and GPR40 share similar ligands, few studies have addressed possible interactions between these two receptors in the control of glucose homeostasis. Here we generated mice deficient in gpr120 (Gpr120KO) or gpr40 (Gpr40KO), alone or in combination (Gpr120/40KO), and metabolically phenotyped male and female mice fed a normal chow or high-fat diet. We assessed insulin secretion in isolated mouse islets exposed to selective GPR120 and GPR40 agonists singly or in combination. Following normal chow feeding, body weight and energy intake were unaffected by deletion of either receptor, although fat mass increased in Gpr120KO females. Fasting blood glucose levels were mildly increased in Gpr120/40KO mice, and in a sex-dependent manner in Gpr120KO and Gpr40KO animals. Oral glucose tolerance was slightly reduced in male Gpr120/40KO mice and in Gpr120KO females, whereas insulin secretion and insulin sensitivity were unaffected. In hyperglycemic clamps, the glucose infusion rate was lower in male Gpr120/40KO mice but insulin and c-peptide levels were unaffected. No changes in glucose tolerance were observed in either single or double KO animals under high-fat feeding. In isolated islets from wild-type mice, the combination of selective GPR120 and GPR40 agonists additively increased insulin secretion. We conclude that while simultaneous activation of GPR120 and GPR40 enhances insulin secretion ex vivo, combined deletion of these two receptors only minimally affects glucose homeostasis in vivo in mice.

physiology↗

bioRxiv2020

Free-fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice

ObjectiveMaintenance of glucose homeostasis requires the precise regulation of hormone secretion from the endocrine pancreas. Free fatty-acid receptor 4 (FFAR4/GPR120) is a G protein-coupled receptor whose activation in islets of Langerhans promotes insulin and glucagon secretion and inhibits somatostatin secretion. However, the contribution of individual islet cell types (, {beta}, and {delta} cells) to the insulinotropic and glucagonotropic effects of GPR120 remains unclear. As gpr120 mRNA is enriched in somatostatin-secreting {delta} cells, we hypothesized that GPR120 activation stimulates insulin and glucagon secretion via inhibition of somatostatin release. MethodsGlucose tolerance tests were performed in mice after administration of the selective GPR120 agonist Compound A. Insulin, glucagon and somatostatin secretion were measured in static incubations of isolated mouse islets in response to endogenous ({omega}-3 polyunsaturated fatty acids) and/or pharmacological (Compound A and AZ-13581837) GPR120 agonists. The effect of Compound A on hormone secretion was tested further in islets isolated from mice with global or somatostatin cell-specific knockout of gpr120. Gpr120 expression was assessed in pancreatic sections by RNA in situ hybridization. Cyclic AMP (cAMP) and calcium dynamics in response to pharmacological GPR120 agonists were measured specifically in , {beta} and {delta} cells in intact islets using cAMPER and GCaMP6 reporter mice, respectively. ResultsAcute exposure to Compound A increased glucose tolerance and circulating insulin and glucagon levels in vivo. Endogenous and/or pharmacological and GPR120 agonists reduced somatostatin secretion in isolated islets and concomitantly demonstrated dose-dependent potentiation of glucose-stimulated insulin secretion and arginine-stimulated glucagon secretion. Gpr120 was enriched in {delta} cells. Pharmacological GPR120 agonists reduced cAMP and calcium levels in {delta} cells but increased these signals in and {beta} cells. Compound A-mediated inhibition of somatostatin secretion was insensitive to pertussis toxin. The effect of Compound A on hormone secretion was completely absent in islets from mice with either global or somatostatin cell-specific deletion of gpr120 and was partially reduced upon blockade of somatostatin receptor signaling by cyclosomatostatin. ConclusionsInhibitory GPR120 signaling in {delta} cells contributes to both insulin and glucagon secretion in part via mitigating somatostatin release.

physiology↗

bioRxiv2019

HB-EGF Signaling is Required for Glucose-Induced Pancreatic β-Cell Proliferation in Rats

The molecular mechanisms of {beta}-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced {beta}-cell proliferation in rats is dependent on Epidermal Growth Factor Receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand Heparin-Binding EGF-like Growth Factor (HB-EGF) in the {beta}-cell proliferative response to glucose, a {beta}-cell mitogen and key regulator of {beta}-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates {beta}-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks {beta}-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in {beta} cells in response to glucose in a Carbohydrate Response Element Binding Protein (ChREBP)-dependent manner. In addition, chromatin-immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced {beta}-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in {beta}-cell compensation to increased metabolic demand.

cell biology↗