Modification of Ghrelin Receptor Signaling by Somatostatin
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Modification of ghrelin receptor signaling by somatostatin receptor-5 regulates insulin release Seongjoon Park1,2, Hong Jiang1,3, Hongjie Zhang4, and Roy G. Smith5 Department of Metabolism and Aging, Scripps Research Institute Florida, Jupiter FL 33458 Edited by Michael O. Thorner, University of Virginia Health Sciences Center, Charlottesville, VA, and accepted by the Editorial Board October 2, 2012 (received for review June 11, 2012) Both ghrelin and somatostatin (SST) inhibit glucose-stimulated in- conditions of negative energy balance endogenous ghrelin acts by sulin secretion (GSIS) from pancreatic β-cells, but how these inde- establishing inhibitory tone on insulin secretion. pendent actions are regulated has been unclear. The mechanism Endogenous ghrelin is fundamentally important for maintaining must accommodate noncanonical ghrelin receptor (GHS-R1a)–G-pro- euglycemia and for survival under conditions of acute food re- tein coupling to Gαi/o instead of Gαq11 and dependence on energy striction (7, 8). Mice lacking the essential enzyme ghrelin octa- balance. Here we present evidence for an equilibrium model of re- noylacyltransferase (GOAT), which converts inactive ghrelin ceptor heteromerization that fulfills these criteria. We show that peptide into its active octanoylated form, are severely compro- GHS-R1a coupling to Gαi/o rather than Gαq11 requires interactions mised by a 60% reduction in dietary intake (9–11). However, the between GHS-R1a and SST receptor subtype 5 (SST5) and that in the phenotype can be rescued by restoring ghrelin or GH to the con- absence of SST5 ghrelin enhances GSIS. At concentrations of GHS- centrations measured in control WT mice subjected to 60% caloric R1a and SST5 expressed in islets, time-resolved FRET and biolumi- restriction, indicating that ghrelin is critically important for pre- nescence resonance energy transfer assays illustrate constitutive venting neuroglycopenia (11, 12). Ironically, expression cloning of formation of GHS-R1a:SST5 heteromers in which ghrelin, but not GHS-R1a that led to the discovery of ghrelin was achieved using SST, suppresses GSIS and cAMP accumulation. GHS-R1a–G-protein a synthetic molecule that augmented episodic GH release (2, 3). coupling and the formation of GHS-R1a:SST5 heteromers is depen- GHS-R1a is expressed in pancreatic β-cells, and ghrelin inhibits dent on the ratio of ghrelin to SST. A high ratio enhances heteromer GSIS (13, 14). Inhibition of insulin release appears paradoxical, α PHYSIOLOGY formation and G i/o coupling, whereas a low ratio destabilizes het- because generally activation of GHS-R1a ghrelin results in cou- – α eromer conformation, restoring GHS-R1a G q11 coupling. The [ghre- pling to Gαq11, which would enhance insulin secretion. However, lin]/[SST] ratio is dependent on energy balance: Ghrelin levels peak in the islet and β-cells, instead ofGαq11-mediated signal trans- during acute fasting, whereas postprandially ghrelin is at a nadir, duction, ghrelin suppresses GSIS via GHS-R1a coupling to Gαi/o, and islet SST concentrations increase. Hence, under conditions of thereby reducing cAMP accumulation (14). Traditionally; SST low energy balance our model predicts that endogenous ghrelin activation of somatostatin receptor subtype-5 (SST5) is considered rather than SST establishes inhibitory tone on the β-cell. Collectively, the major inhibitor of insulin secretion from β-cells (15–18). SST our data are consistent with physiologically relevant GHS-R1a:SST5 released from islet δ-cells also inhibits glucagon secretion by ac- heteromerization that explains differential regulation of islet func- tivating SST2 on α-cells (19–22). tion by ghrelin and SST. These findings reinforce the concept that How and under what physiological conditions are the in- signaling by the G-protein receptor is dynamic and dependent on hibitory actions of ghrelin and SST on insulin secretion in- protomer interactions and physiological context. dependently controlled? Any model that explains ghrelin action on pancreatic β-cells in vivo must include a mechanism for the GPCR oligomers | glucose homeostasis switch in canonical GHS-R1a–G-protein coupling from Gαq11 to Gαi/o, a role for SST5 and SST, and dependence on reciprocal he growth hormone secretagogue receptor type 1a (GHS- changes in relative concentrations of ghrelin and SST as a con- TR1a), was identified in 1996 as an orphan G-protein–coupled sequence of changes in glucose concentrations. This report receptor (GPCR) that regulates the action of a family of small provides evidence for a model incorporating GHS-R1a:SST5 synthetic molecules designed to rejuvenate the growth hormone heteromers that meet these criteria. (GH) axis in humans (1, 2). Three years later GHS-R1a was deorphanized by discovery of an endogenous agonist made in the Results − − stomach called “ghrelin” (3). Ghsr / mice are refractory to the Ghrelin Inhibition of GSIS Is Dependent on both GHS-R1a and SST5. fi GH-releasing and orexigenic properties of ghrelin, confirming We rst measured expression of ghrelin, GOAT, GHS-R1a, that GHS-R1a is a physiologically relevant ghrelin receptor (4). SST5, and SST2 in rat pancreatic islets (Fig. 1A) and used these The study we describe was prompted by the need for a model that includes endogenous ghrelin as well as somatostatin (SST) as a regulator of pancreatic islet function and that elucidates Author contributions: R.G.S. designed research; S.P., H.J., and H.Z. performed research; the paradox that ghrelin inhibits rather than enhances glucose- S.P., H.J., and R.G.S. analyzed data; and S.P. and R.G.S. wrote the paper. fl stimulated insulin secretion (GSIS). The authors declare no con ict of interest. − − − − Experiments in ghrelin / and ghsr / mice led to the conclusion This article is a PNAS Direct Submission. M.O.T. is a guest editor invited by the Editorial Board. that endogenous ghrelin is a physiologically important regulator of 1 GSIS (5–7). In fed mice endogenous ghrelin concentrations are at S.P. and H.J. contributed equally to this work. 2 a nadir; hence, the majority of GHS-R1a binding sites are un- Present address: Unit of Basic Medical Science, Department of Investigative Pathology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki City 852-8523, occupied. In this context, treating WT mice with exogenous ghrelin Japan. suppresses GSIS. When WT mice are fasted, endogenous ghrelin 3Present address: Texas Therapeutics Institute at Brown Foundation, Institute of Molecu- levels reach a maximum, and the mice are refractory to exogenous lar Medicine, University of Texas, Health Science Center at Houston, Houston, TX 77030. −/− ghrelin. In contrast, fasted ghrelin mice are fully responsive to 4Present address: Internal Medicine Residency Program, York Hospital, York, PA 17403. the suppression of GSIS by exogenous ghrelin. Collectively, these 5To whom correspondence should be addressed. E-mail: [email protected]. results indicate that in fasted WT mice GHS-R1a binding sites This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are fully occupied by endogenous ghrelin, suggesting that under 1073/pnas.1209590109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1209590109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 INS1-SJ cells ghrelin siRNA to knockdown endogenous ghrelin production A Rat B ** (Fig. S1). Under these conditions exogenous ghrelin inhibited Islets INS-1SJ *** insulin release induced by 15 mM glucose (Fig. 2B). When 160 GHSR1a INS1SJ-GHSR-SST5 cells were treated with 3 mM and 15 mM SST2 120 glucose, high glucose increased GSIS, and expression of ghrelin siRNA to knock down production of endogenous ghrelin further SST5 80 enhanced GSIS (Fig. 2C); hence, ghrelin siRNA unmasks the Ghrelin suppression of GSIS caused by endogenous ghrelin. Importantly, 40 enhanced GSIS is accompanied by an increase in cAMP (Fig. GOAT 2D), consistent with ghrelin regulation of GSIS reported in 0 C1 Co15 TC1 TCo15G 15 15 15 15 Glucose (mM) pancreatic islets by noncanonical Gαi/o coupling (14). ACTB (% secretion content) Insulin --++GHSR1a -100- 100Ghrelin (nM) Expression of Equivalent Concentrations of GHS-R1a Prevents SST5 Inhibition of GSIS. We next asked if coexpression of equivalent Fig. 1. (A) Comparison of GHS-R1a, SST2, SST5, ghrelin, and GOAT expression levels of GHS-R1a andSST5 modifies SST5 function. To avoid in rat islets and INS-1SJ cells by PCR. (B) INS1-SJ cells transduced with GHS-R1a– activating SST2 in INS-1SJ cells, we selected the SST5-selective expressing lentivirus causes augmentation of GSIS without suppression by agonist Bim23052. INS-1SJ cells expressing either SST5 alone ghrelin. Data represent means ± SEM (n = 4); **P < 0.01, ***P < 0.001. or with an equal concentration of GHS-R1a were treated with Bim23052. When SST5 was expressed alone, Bim23052 inhibited GSIS, but coexpression of GHS-R1a blocked Bim23052 action results to develop a system to model ghrelin and SST signaling. A Pharmacology studies indicate that SST5 rather than SST2 is (Fig. 3 ). Native SST5 exists as a monomer, but signal trans- primarily involved in inhibiting GSIS from β-cells (15–18). We duction requires agonist-induced formation of homodimers (23); hypothesized that noncanonical GHS-R1a coupling resulting in therefore, antagonism of SST5 signaling could be explained by ghrelin inhibition of GSIS involves molecular interactions be- constitutive formation of GHS-R1a:SST5 heteromers. To test tween GHS-R1a and SST5. To test the relative