Fatty-acid receptors and regulatory proteins in islets

Summary
The deorphanization of the G-protein coupled receptor (GPCR) FFA1/GPR40 in 2003 and the demonstration that it is activated by medium-to-long-chain fatty acids and selectively expressed in pancreatic beta-cells (1-3) sparked tremendous interest in this and other fatty acid receptors as potential therapeutic targets to enhance insulin secretion in a glucose-dependent manner in T2D (4). Over the last 15 years we have conducted a series of studies aimed to understand the role of fatty acid receptors in pancreatic islet function and glucose homeostasis and to identify their mechanisms of action. 
Using GPR40 knock-out mice, we have shown that GPR40 mediates approximately 50% of the stimulatory effect of fatty acids on insulin secretion in vitro and in vivo (5) (in collaboration with Dan Lin, Amgen), but is not implicated in their long-term, deleterious effects on beta-cell function (6). We observed that GPR40 plays a role in the maintenance of glucose homeostasis in vivo via a mechanism of action that does not involve changes in intracellular fuel metabolism in islets (7) (in collaboration with Tom Metz, Pacific Northwest National Laboratories; and Marc Prentki, U Montréeal). We identified the mechanisms by which glucose regulates the expression of GPR40 (8) (in collaboration with Rohit Kulkarni, U Harvard and Michael Walker, Weizmann Institute) and showed that activation of GPR40 triggers a signaling cascade that involves protein kinase D1 (PKD1) and p21-activated kinase 4, and leads to depolymerization of cortical actin and stimulation of second-phase insulin secretion (9; 10) (in collaboration with Patrick MacDonald, U Alberta). In vivo, beta-cell PKD1 controls adaptive insulin secretion in response to high-fat feeding and hence contributes to the maintenance of glucose homeostasis during metabolic stress (11). In collaboration with the group of Michel Bouvier (U Montréal), we have demonstrated that GPR40 is subject to biased agonism (12). 
Downstream of GPCRs, G proteins are inactivated by GTPase-activating proteins (GAP), most of which belong to the Regulators of G-protein Signalling (RGS) protein family (13). RGS proteins are key regulators of the termination of GPCR signaling yet little is known about their role in beta-cell function. We have demonstrated that the RGS protein RGS16 positively regulates insulin secretion and beta-cell proliferation by alleviating the tonic inhibitory action of somatostatin in islets (14).
We have expanded the scope of this project beyond GPR40 to explore the physiological role of the other long-chain fatty acid receptor, FFA4/GPR120, in glucose homeostasis and beta-cell function using a conditional knockout mouse and investigating possible interactions between GPR40 and GPR120 in double knockouts. We have recently completed a study (15) demonstrating that GPR120 is predominantly active in islet delta cells and that its activation lowers cAMP levels, inhibits somatostatin secretion, and thereby alleviates tonic inhibition of insulin and glucagon secretion by beta and alpha cells, respectively (in collaboration with Mark Huising, UC Davis).  We are also examining the additive effects of GPR40 and GPR120 on glucose homeostasis and insulin secretion in male mice. 
Following our previous work on RGS16, we are now interrogating the role of RGS9 in the control of insulin secretion and beta-cell proliferation. 

Funding
These projects are funded by Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC).

References
1. Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka Y, Uejima H, Tanaka H, Maruyama M, Satoh R, Okubo S, Kizawa H, Komatsu H, Matsumura F, Noguchi Y, Shinohara T, Hinuma S, Fujisawa Y, Fujino M: Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature 2003;422:173-176
2. Briscoe CP, Tadayyon M, Andrews JL, Benson WG, Chambers JK, Eilert MM, Ellis C, Elshourbagy NA, Goetz AS, Minnick DT, Murdock PR, Sauls HR, Jr., Shabon U, Spinage LD, Strum JC, Szekeres PG, Tan KB, Way JM, Ignar DM, Wilson S, Muir AI: The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem 2003;278:11303-11311
3. Kotarsky K, Nilsson NE, Flodgren E, Owman C, Olde B: A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs. Biochem Biophys Res Commun 2003;301:406-410
4. Ghislain J, Poitout V: The Role and Future of FFA1 as a Therapeutic Target. Handb Exp Pharmacol 2017;236:159-180
5. Latour MG, Alquier T, Oseid E, Tremblay C, Jetton TL, Luo J, Lin DC, Poitout V: GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo. Diabetes 2007;56:1087-1094
6. Kebede M, Alquier T, Latour MG, Semache M, Tremblay C, Poitout V: The fatty acid receptor GPR40 plays a role in insulin secretion in vivo after high-fat feeding. Diabetes 2008;57:2432-2437
7. Alquier T, Peyot ML, Latour MG, Kebede M, Sorensen CM, Gesta S, Kahn CR, Smith RD, Jetton TL, Metz TO, Prentki M, Poitout V: Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets. Diabetes 2009;58:2607-2615
8. Kebede M, Ferdaoussi M, Mancini A, Alquier T, Kulkarni RN, Walker MD, Poitout V: Glucose activates free fatty acid receptor 1 gene transcription via phosphatidylinositol-3-kinase-dependent O-GlcNAcylation of pancreas-duodenum homeobox-1. Proceedings of the National Academy of Sciences of the United States of America 2012;109:2376-2381
9. Ferdaoussi M, Bergeron V, Zarrouki B, Kolic J, Cantley J, Fielitz J, Olson EN, Prentki M, Biden T, Macdonald PE, Poitout V: G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1. Diabetologia 2012;55:2682-2692
10. Bergeron V, Ghislain J, Poitout V: The P21-activated kinase PAK4 is implicated in fatty-acid potentiation of insulin secretion downstream of free fatty acid receptor 1. Islets 2016;In Press
11. Bergeron V, Ghislain J, Vivot K, Tamarina N, Philipson LH, Fielitz J, Poitout V: Deletion of Protein Kinase D1 in Pancreatic beta-Cells Impairs Insulin Secretion in High-Fat Diet-Fed Mice. Diabetes 2018;67:71-77
12. Mancini AD, Bertrand G, Vivot K, Carpentier E, Tremblay C, Ghislain J, Bouvier M, Poitout V: beta-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1. J Biol Chem 2015;290:21131-21140
13. Sjogren B: Regulator of G protein signaling proteins as drug targets: current state and future possibilities. Advances in pharmacology 2011;62:315-347
14. Vivot K, Moulle VS, Zarrouki B, Tremblay C, Mancini A, Maachi H, Ghislain J, Poitout V: The regulator of G-protein signaling RGS16 promotes insulin secretion and β-cell proliferation in rodent and human islets. Molecular Metabolism 2016;5:988-996
15. Croze ML, Flisher M, Guillaume A, Tremblay C, Granziera S, Noguchi GM, Vivot K, Ghislain J, Huising MO, Poitout V: Free-fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice. bioRxiv 2020:2020.2007.2017.208637

The beta-cell response to metabolic stress

Summary
The pancreatic beta cell has a strong capacity to adjust to a changing metabolic environment. For example, the vast majority of people who gain weight and become resistant to the action of insulin do not develop diabetes because their beta cells are able to compensate for insulin resistance by two mechanisms: 1- a large increase in insulin secretion; 2- cell proliferation which leads to enhanced functional beta-cell mass. In about 20% of individuals, however, these compensatory mechanisms are deficient or absent and T2D develops. Our laboratory seeks to understand the cellular and molecular basis of these compensatory mechanisms and of their failure under conditions of metabolic stress.
Since its inception, our lab has focused on elucidating the mechanisms of glucolipotoxicity, the combination of excessive levels of glucose and fatty-acids which is proposed to mediate, at least in part, the deterioration of beta-cell function in T2D (1). In vitro, we have shown that prolonged exposure of isolated islets of Langerhans to elevated levels of fatty acids and glucose impairs insulin gene expression (2; 3) via transcriptional mechanisms that involve de novo synthesis of ceramide (4; 5). Inhibition of insulin gene transcription involves reduction of MafA expression as well as exclusion of PDX-1 from the nuclear compartment (6). We showed that this transcriptional effect is mediated by the enzyme PAS kinase (7; 8) (in collaboration with Chris Rhodes, then at U Chicago and Jared Rutter, U Utah). We have established an in vivo model to study glucolipotoxicity in rats (9) and showed that prolonged infusion of glucose and Intralipid in this model also leads to a decrease in insulin gene expression and nuclear exclusion of PDX-1 (10) which, in older animals, in turn impairs insulin biosynthesis and secretion (11) (in collaboration with Tom Jetton, U Vermont; Raghu Mirmira, U Indiana; and Marc Prentki, U Montréal). 
During the course of these project we have generated novel research tools, including a transgenic rat expressing a Renilla luciferase (RLuc)-enhanced yellow fluorescent protein (YFP) fusion under the control of a 9-kb genomic fragment from the rat ins2 gene (RIP7-RLuc-YFP) (12). This rat line is available from the Rat Resource and Research Center (RRRC) at http://www.rrrc.us/Strain/?x=757&log=yes.
The observation that infusion of excess nutrients in rats leads to a marked increase in beta-cell mass (11) led us to investigate the underlying mechanisms. We found that the increase in beta-cell mass in infused rats is a compensatory mechanism involving the growth factor HB-EGF, the EGF receptor, the kinase mTOR and the transcription factor FoxM1 which promote beta-cell proliferation (13). Further examination of this phenomenon revealed that excess glucose and lipids synergistically and reversibly promote beta-cell proliferation via direct action on the beta-cell (14) but also indirectly through the brain via the autonomic nervous system (15) (in collaboration with Thierry Alquier, U Montréal). We have recently expanded these findings by showing that the HB-EGF/EGFR signaling pathway is implicated in glucose-induced beta-cell proliferation in rodent and human islets (16) (in collaboration with Don Scott, Mount Sinai School of Medicine). 
We have also examined the beta-cell response to other situations or disease states. We showed that mice carrying the most frequent human mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis do not have an intrinsic beta-cell secretory defect but develop insulin resistance and a beta-cell mass deficit with age (17) (in collaboration with Yves Berthiaume, U Montréal). We have demonstrated that the uremic toxin urea impairs insulin secretion via excessive O-glycosylation of the glycolytic enzyme phosphofructokinase-1, a phenomenon that may explain, at least in part, the prevalence of dysregulations of glucose homeostasis in chronic kidney disease (18). 
To further identify the mechanisms by which fatty acids induce beta-cell proliferation using an unbiased approach, we recently performed single-cell RNA sequencing of rodent islets exposed to palmitate or oleate and are currently analyzing these data in collaboration with Rob Sladek at the McGill Genome Center. In addition, we are exploring the role of the de novo ceramide synthesis pathway in oleate-induced beta-cell proliferation by sphingolipidomic analysis in collaboration with Scott Summers and Will Holland at U Utah. A strong emphasis is placed on the translational value of our findings by systematically testing their applicability to human islets, which we receive on a regular basis from the NIH-funded Integrated Islet Distribution Program and the Islet Core of U Alberta. 
We are currently conducting a study to examine the mechanisms of beta-cell compensation to insulin resistance during puberty. 

Funding
These projects are funded by the US National Institutes of Health (NIH), the Canadian Institutes of Health Research (CIHR) and the Quebec Cardiometabolic Health, Diabetes and Obesity Research Network. 
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References
1. Poitout V, Robertson RP: Glucolipotoxicity: fuel excess and beta-cell dysfunction. Endocr Rev 2008;29:351-366
2. Jacqueminet S, Briaud I, Rouault C, Reach G, Poitout V: Inhibition of insulin gene expression by long-term exposure of pancreatic beta-cells to palmitate is dependent upon the presence of a stimulatory glucose concentration. Metabolism 2000;49:532-536
3. Briaud I, Harmon JS, Kelpe CL, Segu VB, Poitout V: Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes 2001;50:315-321
4. Kelpe CL, Moore PC, Parazzoli SD, Wicksteed B, Rhodes CJ, Poitout V: Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis. J Biol Chem 2003;278:30015-30021
5. Moore PC, Ugas MA, Hagman DK, Parazzoli SD, Poitout V: Evidence against the involvement of oxidative stress in fatty acid inhibition of insulin secretion. Diabetes 2004;53:2610-2616
6. Hagman DK, Hays LB, Parazzoli SD, Poitout V: Palmitate inhibits insulin gene expression by altering PDX-1 nuclear localization and reducing MafA expression in isolated rat islets of Langerhans. J Biol Chem 2005;280:32413-32418
7. Fontes G, Semache M, Hagman DK, Tremblay C, Shah R, Rhodes CJ, Rutter J, Poitout V: Involvement of Per-Arnt-Sim Kinase and extracellular-regulated kinases-1/2 in palmitate inhibition of insulin gene expression in pancreatic beta-cells. Diabetes 2009;58:2048-2058
8. Semache M, Zarrouki B, Fontes G, Fogarty S, Kikani C, Chawki MB, Rutter J, Poitout V: Per-Arnt-Sim kinase regulates pancreatic duodenal homeobox-1 protein stability via phosphorylation of glycogen synthase kinase 3beta in pancreatic beta-cells. J Biol Chem 2013;288:24825-24833
9. Fergusson G, Ethier M, Zarrouki B, Fontes G, Poitout V: A model of chronic nutrient infusion in the rat. Journal of visualized experiments : JoVE 2013;
10. Hagman DK, Latour MG, Chakrabarti SK, Fontes G, Amyot J, Tremblay C, Semache M, Lausier JA, Roskens V, Mirmira RG, Jetton TL, Poitout V: Cyclical and alternating infusions of glucose and intralipid in rats inhibit insulin gene expression and Pdx-1 binding in islets. Diabetes 2008;57:424-431
11. Fontes G, Zarrouki B, Hagman DK, Latour MG, Semache M, Roskens V, Moore PC, Prentki M, Rhodes CJ, Jetton TL, Poitout V: Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass. Diabetologia 2010;53:2369-2379
12. Ghislain J, Fontes G, Tremblay C, Kebede MA, Poitout V: Dual-Reporter beta-Cell-Specific Male Transgenic Rats for the Analysis of beta-Cell Functional Mass and Enrichment by Flow Cytometry. Endocrinology 2016;157:1299-1306
13. Zarrouki B, Benterki I, Fontes G, Peyot ML, Seda O, Prentki M, Poitout V: Epidermal Growth Factor Receptor Signaling Promotes Pancreatic beta-Cell Proliferation in Response to Nutrient Excess in Rats through mTOR and FOXM1. Diabetes 2014;63:982-993
14. Moulle VS, Vivot K, Tremblay C, Zarrouki B, Ghislain J, Poitout V: Glucose and fatty acids synergistically and reversibly promote beta cell proliferation in rats. Diabetologia 2017;60:879-888
15. Moulle VS, Tremblay C, Castell AL, Vivot K, Ethier M, Fergusson G, Alquier T, Ghislain J, Poitout V: The autonomic nervous system regulates pancreatic beta-cell proliferation in adult male rats. Am J Physiol Endocrinol Metab 2019;
16. Maachi H, Fergusson G, Ethier M, Brill GN, Katz LS, Honig LB, Metukuri MR, Scott DK, Ghislain J, Poitout V: HB-EGF Signaling Is Required for Glucose-Induced Pancreatic beta-Cell Proliferation in Rats. Diabetes 2020;69:369-380
17. Fontes G, Ghislain J, Benterki I, Zarrouki B, Trudel D, Berthiaume Y, Poitout V: The DeltaF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Is Associated With Progressive Insulin Resistance and Decreased Functional beta-Cell Mass in Mice. Diabetes 2015;64:4112-4122
18. Koppe L, Nyam E, Vivot K, Manning Fox JE, Dai XQ, Nguyen BN, Trudel D, Attane C, Moulle VS, MacDonald PE, Ghislain J, Poitout V: Urea impairs beta cell glycolysis and insulin secretion in chronic kidney disease. J Clin Invest 2016;126:3598-3612