JrnlID 424_ArtID 2262_Proof# 1 - 12/02/2019 Pflügers Archiv - European Journal of Physiology https://doi.org/10.1007/s00424-019-02262-7 1 3 SIGNALING AND CELL PHYSIOLOGY 2 4 5 The functional impact of G protein-coupled receptor 142 (Gpr142) 6 on pancreatic β-cell in rodent 7 Israa Mohammad Al-Amily1 & Pontus Dunér2 & Leif Groop1,3 & Albert Salehi1,3 8 9 Received: 3 October 2018 /Revised: 31 January 2019 /Accepted: 5 February 2019 10 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 11 Abstract 12 We have recently shown that the G protein-coupled receptor 142 (GPR142) is expressed in both rodent and human pancreatic β- 13 cells. Herein, we investigated the cellular distribution of GPR142 within islets and the effects ofOF selective agonists of GPR142 on 14 glucose-stimulated insulin secretion (GSIS) in the mouse islets and INS-1832/13 cells. Double-immunostaining revealed that 15 GPR142 immunoreactivity in islets mainly occurs in insulin-positive cells. Potentiation of GSIS by GPR142 activation was 16 accompanied by increased cAMP content in INS-1832/13 cells. PKA/Epac inhibition markedly suppressed the effect of 17 GPR142 activation on insulin release. Gpr142 knockdown (Gpr142-KD) in islets was accompanied by elevated release of 18 MCP-1, IFNγ, and TNFα during culture period and abolished the modulatoryPRO effect of GPR142 activation on the GSIS. 19 Gpr142-KD had no effect on Ffar1, Ffar2,orFfar3 mRNA while reducing Gpr56 and increasing Tlr5 and Tlr7 mRNA expres- 20 sion. Gpr142-KD was associated with an increased expression of ChrebpD, Txnip, RhoA, and mitochondrial Vdac1 concomitant 21 with a reduced Pdx1, Pax6, and mitochondrial Vdac2 mRNA levels. Long-termE exposure of INS-1832/13 cells to hyperglycemia 22 reduced Gpr142 and Vdac2 while increased Chrebp, Txnip,andVdac1 mRNA expression. GPR142 agonists or Bt2-cAMP 23 counteracted this effect. Glucotoxicity-induced decrease of cell viability in Gpr142-KD INS-1 cells was not affected by 24 GPR142-agonists while Bt2-cAMP prevented it. The results show the importance of Gpr142 in the maintenance of pancreatic 25 β-cell function in rodents and that GPR142 agonists potentiate GSIS by an action, which most likely is due to increased cellular 26 generation of second messenger molecule cAMP. 27 Keywords Type 2 diabetes . β-Cell dysfunction . Apoptosis . Cell viability . Confocal image 28 ORRECT 29 Introduction C body [36]. Although there are several hormones that increase 32 blood glucose, insulin secreted by pancreatic β-cells is the 33 30 A tightly regulated blood glucose level with very narrow fluc- only hormone capable of decreasing blood glucose by increas- 34 31 tuations is paramount to achieveUN optimal cell function in the ing glucose uptake by peripheral tissues [20]. Insulin secretion 35 by the pancreatic β-cell is regulated through various metabol- 36 37 This article is part of the topical collection on Signaling and cell ic, hormonal and neural factors [26, 28]. The secreted insulin physiology in turn, regulates many metabolic aspects of importance to the 38 β 39 Electronic supplementary material The online version of this article body. Thus, dysregulated -cell function would contribute to (https://doi.org/10.1007/s00424-019-02262-7) contains supplementary development of the metabolic syndrome. Disturbed metabolic 40 material, which is available to authorized users. conditions with accelerated and sustained hyperglycemia fur- 41 ther negatively modulate pancreatic β-cell function, ultimate- 42 * Albert Salehi ly resulting in the development of Type 2 diabetes (T2D) [26, 43 [email protected] 28, 39]. The incident of obesity and T2D are increasing rap- 44 idly around the world, independent of ethnicity and conven- 45 1 Department of Clinical Science, SUS, Division of Islet Cell Q1 46 Physiology, University of Lund, Jan Waldenströmsgata 35, Building tional antidiabetic agents and treatment strategies are failing to 91, Floor 11, SE-205 02 Malmö, Sweden preserve insulin-producing β-cells. Since functional β-cells 47 48 2 Experimental cardiovascular research, University of Lund, are very essential in the prevention of T2D new approaches Lund, Sweden aim to preserve β-cell functionality, by targeting specific 49 β 50 3 Department of Neuroscience and Physiology, Metabolic Research GPCRs with positive impacts on the -cell as subsequent Unit, University of Gothenburg, Gothenburg, Sweden diabetes therapies, have gathered tremendous interest. 51 JrnlID 424_ArtID 2262_Proof# 1 - 12/02/2019 Pflugers Arch - Eur J Physiol 52 GPCRs regulate a myriad of physiological and pathophys- Chemicals 100 53 iological signaling processes in the body and constitute a rich 54 source of targets for the pharmacological prevention of vari- Collagenase (IV) was bought from Sigma St. Louis, MO, 101 55 ous human diseases including T2D [5, 37]. The majorities of USA, and fatty acid free bovine serum albumin (BSA) from 102 56 GPCRs expressed in the pancreatic islets is still “orphan” with Boehringer Mannheim, Germany. Polyclonal rabbit anti- 103 57 poorly characterized function and/or still with no identified GPR142, was from LSBio (LS-C383759) (Seattle, WA, 104 58 ligands and have not been utilized as possible antidiabetic USA), guinea pig-raised anti-insulin antibody (Cat# 16049) 105 59 targets [2, 4]. Obviously, a low number of the functionally (Progen), mouse monoclonal anti-glucagon antibody (Cat# 106 60 characterized GPCRs in the body are the targets of more than ab10988) (Abcam), mouse monoclonal anti-somatostatin an- 107 61 30% of all modern drugs, which emphasizes the importance of tibody (ab140665)(Abcam), and monoclonal mouse anti- 108 62 remaining GPCRs currently not functionally mapped in this VDAC1 (AB_2564843) (Calbiochem). Cy2-conjugated anti- 109 63 regard. This could offer novel approaches to treat a number of mouse IgG (715-165-151) and Cy5-conjugated anti-guinea 110 64 human diseases including the metabolic syndrome and pig (Cat# 706-225-148) were from Jackson Immunoresearch 111 65 diabetes. Laboratories Inc. (West Grove, PA, USA). The qPCR primers 112 66 We have recently identified all GPCRs expressed by pan- used were from Applied Biosystems (see below for the ID- 113 67 creatic islet cells [2, 4]. Based on the published results during numbers). Compound 33 and compound A were kindly do- 114 68 recent years, it has become apparent that these GPCRs have a nated by Lilly. The insulinOF ELISA kit was from Mercodia 115 69 great impact on islet cell function and might be suitable drug (Sweden). Myr-PKI (Cat# 2546) was from Tocris, ESI-05 116 70 targets to influence insulin secretory events and islet cell sur- (Cat# M092-05) was from Biolog Life Science and all other 117 71 vival in a positive manner. chemicals were from Merck AG, (Darmstadt, Germany) or 118 72 The aim of the present study was to investigate the cellular Sigma-AldrichPRO (USA). 119 73 localization and the functional impact of the previously The content of Krebs-Ringer bicarbonate buffer (KRB) 120 74 deorphanized Gpr142 (L-tryptophan/L-phenylalanine recep- used forD incubation of islets was (in mM): NaCl (120), KCl 121 75 β 122 tor) on cAMP generation in mouse pancreatic islets -cell. (4.7),E CaCl2 (2.5), KH2PO4 (1.2), MgSO4 (1.2), HEPES (10), 76 The effect of cAMP on secretory process is mediated by two NaHCO3 (25), and 0.1% fatty acid free bovine serum albumin 123 77 ubiquitously important signaling proteins, i.e., protein kinase and the pH was adjusted to 7.4. 124 78 A (PKA) and exchange protein activated by cAMP (Epac) The secretion assay buffer used for INS-1 cell experiments 125 79 [12]. Since PKA and Epac may act independently or converge was also a HEPES balanced salt solution (HBSS) containing 126 80 synergistically in regulating a specific part of insulin secretory (in mM): NaCl (114), KCl (4.7), KH2PO4 (1.2), MgSO4 127 81 processes, we used two specific blockers of these two proteins (1.16), HEPES (20), CaCl2 (2.5), NaHCO3 (25.5), pH was 128 82 to investigate the impact of Gpr142 on the insulin secretion. adjusted to 7.2 and thereafter supplemented with 0.2% fatty 129 83 For comparisons, the effect of phospholipase C (PLC) inhib- acid free bovine serum albumin. 130 84 itor on Gpr142 evoked insulin releaseORRECT was also studied. 85 Attempts were also implementedC to study the impact of 86 Gpr142 on the expression of a number of down-stream sig- Isolation of mouse pancreatic islets 131 87 naling molecules of importance for the β-cell dysfunction. For 88 this purpose, we used twoUN newly developed small molecule All mice used for preparation of pancreatic islets were 132 89 agonists for GPR142 with higher magnitude of selectivity and sacrificed by cervical dislocation. After a midline incision, 133 90 potency compared to naturally occurring endogenous ligands the major duodenal papilla was identified. A hemostatic 134 91 L-tryptophan and L-phenylalanine (Supplementary Fig. 1) clamp was placed on either side of the duodenal papilla, 135 92 [19, 34]. located precisely where the bile duct drains allowing free 136 passage for the solution to enter the pancreas via duodenal 137 papilla. Preparation of pancreatic islets was carried out by 138 retrograde injection of 1.5 ml of a collagenase solution 139 93 Material and methods (1 mg/ml) via the bile-pancreatic duct. The pancreas was, 140 then carefully dissected from surrounding tissues, placed 141 94 Animals in a 50 ml vial, and incubated at 37 °C for 16 min. 142 Thereafter 40 ml ice-cold Hank’s buffer solution was 143 95 Female mice of the c57Bl/6j strain (Janvier Labs, Paris) added and the vial was vigorously shaken to separate the 144 96 weighing 25–30 g were used in our study. A standard pellet islets from acinar tissues.