Modification of Ghrelin Receptor Signaling by Somatostatin

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 contributions of this possibility, we used time-resolved (Tr)-FRET SNAP-tag GHS-R1a and SST5, we sought a subclone of INS-1 cells that technology (24, 25), which has the high sensitivity necessary to would allow independent manipulation of GHS-R1a and SST5 detect the association of GHS-R1a with SST5 at the concen- to match concentrations present in pancreatic islets. A subclone trations found in pancreatic islets. was identified, INS-1SJ, that expresses SST2, ghrelin, and GOAT In isolation, GHS-R1a exists as homodimers. Previously, we at levels similar to those found in rat pancreatic islets with low showed by Tr-FRET that dopamine receptor-2 (DRD2) is a com- expression of GHS-R1a and undetectable SST5 (Fig. 1A). petitive inhibitor of GHS-R1a homodimerization, resulting in the Increasing GHS-R1a levels alone in INS-1SJ cells by trans- formation of GHS-R1a:DRD2 heteromers (26). Using similar ducing the cells with a GHS-R1a–expressing lentivirus enhanced methods employing SNAP-GHS-R1a, Tr-FRET illustrates the insulin secretion in either the presence or absence of ghrelin, formation of GHS-R1a:GHS-R1a homodimers on the plasma consistent with GHS-R1a canonical Gαq11 signaling and pro- membrane of INS-1SJ cells (Fig. 3B). Concentration-dependent duction of endogenous ghrelin (Fig. 1B). Because INS1-SJ cells inhibition of the Tr-FRET signal is observed when SST5 is coex- do not express SST5, this result suggested that SST5 is required pressed with SNAP-GHS-R1a at ratios of 1/1 and 2/1 (Fig. 3B). for ghrelin suppression of GSIS. We then established conditions Control experiments confirmed that attenuation of the Tr-FRET for expressing GHS-R1a and SST5 at levels closely matching signal was not a consequence of coexpressed SST5 inhibiting those in pancreatic islets (Fig. 2A). INS1-SJ cells expressing transport of SNAP-GHS-R1a to the plasma membrane (Fig. S2); GHS-R1a and SST5 (INS1SJ-GHSR-SST5) were treated with therefore, the marked inhibition of GHS-R1a homodimerization

A Rat islet B + Ghrelin siRNA Electroporated 120 ** 1.2 Vehicle INS-1SJ cells 100 Ghrelin 0.8 80 60 0.4 40 (% content) (% expression QPCR QPCR expression 0.0 Insulin secretion 20 SST5/ Ghrelin/ GHSR1a/ 0 Fig. 2. Suppression of insulin secretion by ghrelin ACTB ACTB pcDNA3.1 GHSR1a + SST5 ACTB is dependent on coexpression of GHS-R1a and SST5 in INS-1SJ cells electroporated with GHS-R1a and C *** D Con- SST5 to match levels expressed in rat pancreatic * siRNA islets. (A) Quantitation by real-time PCR of GHS-R1a, *** Con- 1.2 * SST5, and ghrelin expression in rat islets and elec- ** Ghrelin siRNA ** troporated INS-1SJ cells. (B) Ghrelin inhibits insulin 400 siRNA Ghrelin secretion induced by 15 mM glucose in INS-1SJ cells 300 siRNA 0.8 in the presence but not in the absence of GHS-R1a and SST5 following suppression of endogenous 200 ghrelin production with ghrelin siRNA. (C) Knock- 0.4 down of endogenous ghrelin production increases cAMP(nM) (% content) (% 100 GSIS. (D) Knockdown of endogenous ghrelin pro- Insulin secretion Insulin duction increases intracellular cAMP. Data repre- 0 0.0 sent means ± SEM (n = 4); *P < 0.05, **P < 0.01, 3 mM 15 mM Glucose 3 mM 15 mM Glucose ***P < 0.001.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209590109 Park et al. Downloaded by guest on October 1, 2021 A ** B *** 120 *** 120 *** 100 100 80 80 60 60 Fig. 3. Expression of an equivalent concentration 40 * of GHS-R1a blocks SST5 agonist-induced inhibition 40 of GSIS in INS-1SJ cells by a mechanism consistent 20 (%) efficiency Fret with the formation of GHS-R1a:SST5 heteromers. (A) Coexpression of SST5 with an equivalent concen- Insulin secretion (% Insulincontent) (% secretion 0 20 Glucose (mM) 151234 15 15 15 tration of GHS-R1a blocks suppression of GSIS by the pcDNA3.1(+) ++ - - SST5 agonist Bim23052. (B) Tr-FRET using N-terminal 0 SNAP-tagged GHS-R1a illustrates competitive in- SST5 ++++ 1:1 1:1 1:2 hibition of GHS-R1a:GHS-R1a homomer formation GHSR1a --++ SNAP-GHSR1a SNAP-GHSR1a : SST5 by equal (1/1) or excess (1/2) concentrations of SST5. : pcDNA3.1(+) In each case, data represent means ± SEM (n = 4); Bim23052 nM ) -2-2 ( *P < 0.05, **P < 0.01, ***P < 0.001.

by SST5 is a consequence of high-afﬁnity interactions between the inhibition of cAMP production, we selected cAMP accu- GHS-R1a and SST5. mulation as a surrogate for GSIS. We next tested whether interactions between GHS-R1a and SST5-Dependent Modiﬁcation of GHS-R1a–G-protein Coupling Is SST5 affected SST suppression of cAMP accumulation. In Controlled by the [Ghrelin]/[SST] Ratio. Both ghrelin and SST are HEK293 cells expressing SST5, SST dose-dependently suppresses capable of inhibiting insulin secretion by a mechanism requiring forskolin-induced cAMP accumulation with maximum inhibition

– PHYSIOLOGY Gαi/o suppression of cAMP accumulation. Above we showed that at 1 10 nM (Fig. S3A). Remarkably, as in the INS-1SJ cells, ghrelin inhibition of GSIS and suppression of cAMP accumula- coexpressing GHS-R1a with SST5 made the cells refractory to tion, as well as GHS-R1a–dependent antagonism of inhibition of SST (Fig. S3B); however, treatment with ghrelin, but not with des- C GSIS by a selective SST5 agonist, are dependent on both GHS- acyl ghrelin (Fig. S3 ), inhibits forskolin-induced cAMP accumulation by a pertussis toxin (PTX)-sensitive mechanism (Fig. R1a and SST5. To elucidate the mechanism more completely S3D). Hence, the noncanonical GHS-R1a–G-protein coupling to and to deduce the relative contributions of SST2 and SST5, a Gαi/o observed in pancreatic β-cells and INS-1SJ β-cells is re- cell system was needed in which the relative concentrations of capitulated in HEK293 cells coexpressing GHS-R1a and SST5. ghrelin, SST, GHS-R1a, SST5, and SST2 could be controlled. Tr-FRET analysis in INS-1SJ cells used ﬂuorophor tagging at Precise control requires a null background; therefore, we selected the GHS-R1a N terminus. As an additional test of heteromer HEK293 cells. Because inhibition of GSIS by ghrelin and SST in formation, we used bioluminescence resonance energy transfer pancreatic β-cells and in the INS-1SJ β-cell line is dependent on (BRET) with GHS-R1a and SST5 tagged at the C terminus with

A 0.12 C GHSR1a-RLuc/SST5-GFP GHS-R1a + SST5 40 0.08 30

0.04 20 BRET Ratio SST5-RLuc/SST5-GFP 10 Fig. 4. BRET analyses and cAMP assays illustrating that GHS-R1a forms constitutive heteromers with SST5 but not with SST2 and blocks SST/SST5-induced 0.00 0 04 81218cAMP nmol/L/per well 0 0.1 1 10 100 1000 suppression of cAMP accumulation. The BRET ratio RLuc::GFP SST (nM) is expressed as a function of the acceptor/donor DNA ratio and is deﬁned as [(emission at 515 nm) − B 0.12 D (background emission at 515 nm)]/[(emission at GHSR1a-RLuc/SST2-GFP 410 nm) − (background emission at 410 nm)]. (A) 20 GHS-R1a + SST2 The BRET ratio in HEK293 cells cotransfected with μ – 0.08 0.1 g GHS-R1a-Rluc and increasing amounts (0.1 15 1.8 μg) of SST5-GFP (upper curve; BRET50 = 2) or 0.1 μg R² = 0.84 SST5-Rluc DNA and increasing amounts (0.1–1.8 μg) 10 of SST5-GFP DNA (lower curve). (B) The BRET ratio 0.04 measured in HEK293 cells cotransfected with 0.1 μg BRET Ratio – μ 5 GHS-R1a-Rluc and 0.1 1.8 g ST2-GFP. (C) HEK293 cells coexpressing GHS-R1a and SST5 are refractory to SST suppression of forskolin-induced cAMP ac- 0.00 cAMP nmol/L/per well 0 cumulation. (D) SST inhibition of forskolin-induced 0 481218 0 0.1 1 10 100 RLuc::GFP SST (nM) cAMP accumulation in HEK293 cells coexpressing GHS-R1a and SST2.

Park et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 either the energy donor luciferase or GFP as acceptor (27). A A fixed DNA concentration of donor and increasing concentrations - ghrelin of acceptor at ratios of 1/1–1/18 were transfected into HEK293 cells, and the BRET ratio was plotted as a function of the relative 45 100 nM ghrelin concentrations of donor and acceptor. The hyperbolic function obtained with the GHS-R1a-Rluc and SST5-GFP pair and the 35 fi BRET50 ratio indicates high-af nity interactions (upper curve in 25 * ∼ Fig. 4A; BRET50 2). An identical result is obtained when donor- * * and acceptor-tagged receptors are reversed (SST5-Rluc and GHS- 15 * R1a-GFP). In contrast, titration of SST5-Rluc + SST5-GFP (lower curve in Fig. 4A) and GHS-R1a-Rluc + SST2-GFP (Fig. 5 0 4B) produces linear BRET functions indicating random collisions well nmol/L/per cAMP 00.1110100 rather than heteromer formation. We next asked whether heteromer formation correlated with resistance to SST inhibition of SST (nM) cAMP accumulation. Cells coexpressing GHS-R1a with either B SST5 or SST2 were treated with forskolin and SST. With SST5 0.25 coexpression, SST did not inhibit cAMP accumulation (Fig. 4C), * whereas with SST2 coexpression SST inhibited forskolin-induced 0.2 cAMP accumulation (Fig. 4D). Hence, as observed in INS1SJ- GHSR-SST5 cells, formation of GHS-R1a:SST5 heteromers 10 nM ghrelin blocks SST responsiveness in favor of ghrelin responsiveness. 0.15 * [Ghrelin]/[SST] Ratio Modifies GHS-R1a–G Protein Coupling and Is 0.1 Associated with Heteromer Formation. In vivo, the concentration of ghrelin is increased during an acute fast and is suppressed 0.05 after a meal. The postprandial increase in blood glucose suppresses ghrelin and releases SST in pancreatic islets. To de- α α 0 termine if GHS-R1a coupling to G i/o vs. G q11 is dependent on Luminescence Fractional the relative concentrations of ghrelin and SST, we asked if the 0 1 10 100 [ghrelin]/[SST] ratio (1,000/1–1/1) influences signal transduction. SST (nM) High [ghrelin]/[SST] ratios result in ghrelin suppression of cAMP accumulation, whereas lowering the ratio antagonizes ghrelin C 0.5 inhibition of cAMP accumulation (Fig. 5A). To test if SST an- * Control 100 nM ghrelin tagonism of ghrelin inhibition of cAMP accumulation is ac- 0.4 companied by restoration of canonical GHS-R1a–Gαq11 coupling, HEK293 cells expressing GHS-R1a, SST5, and aequorin were 0.3 * used (1, 28). Consistent with an equilibrium between GHS-R1a 0.2 * coupling to Gαi/o and Gαq11, lowering the [ghrelin]/[SST] ratio produces a dose-dependent increase in the internal calcium 2+ Ratio BRET 0.1 * concentration ([Ca ]i) mobilization as measured by aequorin bioluminescence (Fig. 5B). Next we performed BRET assays 0 in the absence or presence of ghrelin with increasing SST con- 0 1 10 100 centrations to test whether the specificity of GHS-R1a–G-protein coupling correlates with heteromer formation. Ghrelin SST (nM) markedly increased the BRET ratio which was dose-dependently attenuated by SST (Fig. 5C). These results are consistent with Fig. 5. In HEK293 cells coexpressing GHS-R1a and SST5, GHS-R1a coupling α α agonist concentration-dependent equilibrium between GHS- to G i/o vs. G q11 is dependent on the [ghrelin]/[SST] ratio, which correlates R1a:SST5 and GHS-R1a:GHS-R1a dimers, which in turn regu- with GHS-R1a:SST5 heteromer formation. (A) Decreasing the [ghrelin]/[SST] ratio antagonizes ghrelin suppression of forskolin-induced cAMP accumu- late noncanonical and canonical GHS-R1a signal transduction, lation. (B) HEK293-AEQ17 cells coexpressing GHS-R1a and SST5. Decreasing respectively. 2+ the [ghrelin]/[SST] ratio increases ghrelin-induced [Ca ]i.(C) HEK293 cells cotransfected with GHS-R1a-Rluc (0.2 μg) and SST5-GFP (1.8 μg). A high Discussion [ghrelin]/[SST] ratio increases the BRET ratio; a low [ghrelin]/[SST] ratio Based on our results, we propose a model of regulation of pan- decreases the BRET ratio. The data represent the means ± SD of three in- creatic islet function incorporating endogenous ghrelin and SST. dependent experiments, each performed in triplicate. *P < 0.05 compared with absence of both agonists. Although GHS-R1a normally couples to Gαq11, both ghrelin and SST inhibit GSIS secretion via GHS-R1a and SST5 coupling to Gαi/o. To test conclusively for GHS-R1a and SST5 molecular interactions and to deduce how combinations of ghrelin and SST secretion. However, when GHS-R1a is coexpressed with SST5 in might regulate signal transduction differentially, we identified INS-1SJ cells at concentrations and relative ratios commensurate a clone of INS-1 cells (INS-1SJ) that formed a basis for our with those measured in native rat islets, ghrelin inhibits insulin studies. By matching concentrations of GHS-R1a and SST5 in secretion, supporting our hypothesis of physiologically relevant INS-1SJ cells to those expressed in pancreatic islets, we conclude molecular interactions between GHS-R1a and SST5. that, contrary to current belief, ghrelin rather than SST suppresses To determine the mechanism by which ghrelin inhibits insulin GSIS and cAMP accumulation through a PTX-sensitive mecha- secretion via noncanonical GHS-R1a–G-protein coupling, we tested nism. This outcome is surprising, because canonical ghrelin sig- for GHS-R1a:SST5 heteromer formation using highly sensitive 2+ naling through GHS-R1a (via Gαq11) would mobilize [Ca ]i and Tr-FRET and BRET assays. Tr-FRET used receptors tagged at stimulate rather than inhibit insulin secretion. Indeed, transducing the N terminus, and for BRET receptors were tagged at the C INS-1SJ cells with lentivirus encoding GHS-R1a enhanced insulin terminus. Each method used relative and absolute expression levels

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209590109 Park et al. Downloaded by guest on October 1, 2021 of GHS-R1a and SST5 closely matching those in rat pancreatic suppression of α-cell activity (29–31). As well as inhibiting GSIS, islets. The results of the two different experimental methods are ghrelin inhibits arginine-induced SST secretion (32). In the con- in complete agreement and illustrate high-afﬁnity interactions text of positive energy balance, high glucose lowers circulating between GHS-R1a and SST5 at equimolar concentrations. In- ghrelin (14, 33), thereby relieving the ghrelin suppression of indeed, these results support our hypothesis that the atypical GHS- sulin release from β-cells and stimulating the release of SST from R1a–G-protein coupling essential for ghrelin suppression of GSIS δ-cells (34); SST, in turn, activates SST2 on α-cells, suppressing is a consequence of the formation of GHS-R1a:SST5 heteromers. glucagon secretion (34). Therefore, only in states of low energy Most importantly, we show that differential GHS-R1a–G-protein balance does ghrelin play a dominant role in regulating glucose coupling and signal transduction are inﬂuenced by the relative homeostasis. concentrations of ghrelin and SST. In cells coexpressing GHS- Recent studies show that glucose inhibits glucagon secretion in − − R1a and SST5, when the relative [ghrelin]/[SST] ratio is high, WT mice but not in sst / mice, as is consistent with a direct role ghrelin suppresses cAMP accumulation; when the ratio is low, for glucose on SST secretion (33). The rapid cessation of insulin − − the cells are refractory to ghrelin inhibition of cAMP production, release upon glucose removal is the same in WT and sst / mouse 2+ and ghrelin increases [Ca ]i mobilization, consistent with res- islets, indicating that termination of the insulin-secretory response – α toration of GHS-R1a G q11 coupling. Furthermore, reducing is not regulated by SST from δ-cells. This result is consistent with the [ghrelin]/[SST] ratio lowers the BRET ratio, consistent with our model, which proposes that when glucose levels fall in vivo, it dissociation of GHS-R1a:SST5 heteromers, thereby implicating is ghrelin rather than SST that establishes an inhibitory set point an agonist concentration-dependent equilibrium model of het- on the β-cell to control insulin secretion (Scheme 1). This effect is eromerization. Of course, we cannot preclude the possibility that not observed in isolated mouse islets after glucose removal because, changes in the BRET ratio also might be explained by a change unlike rat and human islets, mouse islets do not produce ghrelin. in conformation of the GHS-R1a:SST5 heteromer resulting in In summary, we provide evidence for an equilibrium model in altered G-protein coupling. which the formation of GHS-R1a:SST5 heteromers is controlled Constitutive formation of GHS-R1a:SST5 heteromers explains by the [ghrelin]/[SST] ratio; hence, ghrelin and SST differentially why GHS-R1a antagonizes SST5 suppression of GSIS and cAMP establish a set point for β-cell insulin secretion. Of course, im- accumulation. Because SST-induced formation of SST5:SST5 plicit in our equilibrium model is dependence on the relative homomers is required for signal transduction (23), the insensitivity concentrations of GHS-R1a and SST5. In rat islets, we show both to SST argues that formation of SST5:SST5 or GHS-R1a:GHS-

are expressed at approximately equal concentrations. Clearly the PHYSIOLOGY R1a homomers is energetically unfavorable compared with GHS- results obtained from model systems do not necessarily equate to R1a:SST5 formation. Indeed, we speculate that the heteromer primary β-cells; nevertheless, the proposed mechanism is con- acts as a buffer preventing ghrelin-induced insulin secretion by ceptually consistent with observations made in isolated islets. Fi- GHS-R1a–Gα coupling and oversuppression of insulin release by q nally, our results reinforce the notion that regulation of intracellular unopposed SST5 coupling to Gα . i/o signaling by GPCRs is not simply a linear relationship but is We propose a model that deﬁnes a role for ghrelin and GHS- dependent on modulation via receptor signaling networks that R1a for controlling glucose homeostasis according to energy can be modiﬁed according to relative agonist concentrations and balance (Scheme 1). We show that when GHS-R1a and SST5 relative GPCR concentrations. are coexpressed, they are functionally and physically associated, α resulting in coupling to G i/o. The physical association of GHS- Experimental Procedures R1a and SST5 allows signal transduction to be regulated tightly Expression Constructs. GHS-R1a-GFP was constructed as described previously according to the relative concentrations of ghrelin and SST. (27). The GHS-R1a-Rluc:human GHS-R1a cDNA fragment was inserted in- Under conditions of low energy balance, ghrelin concentrations frame into the EcoRI/EcoRV sites of the pRluc-N1 vector (Perkin Elmer). Hu- are elevated, resulting in GHS-R1a:SST5–mediated inhibition man SST5 and SST2 cDNA constructs were purchased from Missouri S&T ofinsulinsecretion.Ghrelinalsostimulatesglucagonsecretion cDNA Resource Center (www.cdna.org). The SST5-GFP:SST5 cDNA fragment by direct effects on α-cells and indirectly by disinhibiting insulin was inserted in-frame into the EcoRI/EcoRV sites of the pGFP-N3 vector

High Energy Balance Low Energy Balance Low [Ghr]/[SST] High [Ghr]/[SST] GHS-R SST5 GHS-R :SST5

Ghr Scheme 1. Equilibrium model of ghrelin regula- Gα tion of islet function mediated by GHS-R1a:SST5 q11 Gαi/o heteromers. Our data support a model of ghrelin High [Ghr] Low Energy Balance signaling dependent on the ratio of ghrelin to SST according to energy balance. Low glucose increases Insulin Ghrelin ghrelin concentrations; ghrelin suppresses insulin β Low [SST] secretion from -cells and increases glucagon re- α Low lease from -cells. High glucose reduces ghrelin but High [SST] increases SST release from islet δ-cells. Hence, when Glucose glucose is low, the [ghrelin]/[SST] ratio is high. In Glucagon High Somatostan this context, GHS-R1a activation by ghrelin results in Insulin noncanonical coupling to Gαi/o that lowers cAMP accumulation and inhibits insulin secretion, which is dependent on GHS-R1a:SST5 heteromer forma- Ghrelin Insulin Low [Ghr] tion. Conversely, by lowering the [ghrelin]/[SST] High Energy Balance ratio, high glucose destabilizes GHS-R1a:SST5 het-

eromers, reducing GHS-R1a coupling to Gαi/o;asa consequence, insulin secretion no longer is regu- β α-cell δ -cell -cell lated by ghrelin.

Park et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 (Perkin Elmer). The SST5-Rluc:SST5 cDNA fragment was inserted in-frame detached using nonenzymatic cell dissociation solution (Sigma), were washed into the EcoRI/EcoRV sites of the pRluc-N1vector (Perkin Elmer). The integrity with HBSS, and were resuspended in stimulation buffer [1× PBS with 0.04% of all tagged GHS-R1a, SST5, and SST2 expression constructs was confirmed BSA (pH 7.4) and 0.5 mmol/L 3-isobutyl-1-methylxanthine]. cAMP accumu- by nucleotide sequencing and tested to check that they displayed functional lation also was determined using the Lance cAMP 384 kit from Perkin Elmer. characteristics identical to those of WT receptors in transfected cells. SNAP- GHS-R1a was prepared as described previously (26). Live-Cell Tr-FRET and BRET. Cells were labeled 48 h after being transfected by electroporation as described previously (26). Briefly, SNAP-GHS-R1a–trans- INS-1SJ Cell Line and GSIS. INS-1 832/13 cells that exhibit robust GSIS derived fected cells were incubated in the presence of 25 nM of donor benzyl guanine- from a rat insulinoma were a kind gift from Chris Newgard (Duke University conjugated terbium cryptate (BG-TbK) (SNAP-Lumi4-Tb; Cisbio) and 250 nM of Medical Center, Winston Salem, NC). A subclone of these cells, which we acceptor benzyl guanine-conjugated d2 fluorophore (BC-647) (SNAP-surface “ ” fi named INS-1SJ, was selected speci cally for our experiments because it 647; New England BioLabs) for 1 h at 37 °C in 0.5% FBS containing RPMI allowed testing of the hypothesis that ghrelin attenuation of GSIS is de- medium. Tr-FRET signals were measured at 665 nm and 620 nm after excita- pendent on GHS-R1a and SST5 by independently matching expression levels tion at 337 nm with 50-μs delay and integration time of 400 μsbyusingan of GHS-R1a and SST5 to match levels in rat pancreatic islets. Eighteen hours EnVision plate reader (Perkin Elmer). The ratio of fluorescence (665 nm/620 before GSIS experiments, the standard tissue culture medium for INS-1 cells nm) was calculated for each sample. BRET experiments were based on previous containing 11.1 mM glucose was changed to fresh medium containing studies (27). Details are given in SI Experimental Procedures. 5 mM glucose (35). Insulin secretion was assayed in HBSS (114 mM NaCl,

4.7 mM KCl, 1.2 mM KH2PO4, 116 mM MgSO4, 20 mM Hepes, 2.5 mM CaCl2, 25.5 mM NaHCO3, and 0.2% BSA, pH 7.2). At confluence, cells were washed Aequorin Bioluminescence Assay. The aequorin bioluminescence assay was with 1 mL HBSS containing 3 mM glucose followed by a 2-h preincubation in carried out as described in ref. 28. Human ghrelin and SST (Phoenix) were fi 2 mL of the same buffer. Then insulin secretion was measured after static diluted with modi ed HBSS (25 mM Hepes at pH 7.3) and distributed into incubation or 2 h in 0.8 mL of HBSS containing the glucose concentrations 96-well plates. Assays were performed with the Luminoskan Luminometer and/or ghrelin, SST, or Bim23052 as indicated in the figures. Insulin was (ThermoFisher Labsystems). The fractional luminescence for each well was measured using the rat/mouse insulin ELISA kit (Linco Research). Acid alcohol calculated by taking the ratio of the integrated response to the initial was added to extract total protein, which was assayed using the Coomassie challenge to the total integrated luminescence, as well as the Triton X-100 protein assay reagent kit (Pierce Biotechnology). Secreted insulin was nor- lysis response. Fractional luminescence data for each point represent the malized to total protein. average of triplicate measurements. Statistical analyses were performed using the Student t test. Measurements of Intracellular cAMP. cAMP was measured in INS-1SJ cells using Additional methods are described in SI Experimental Procedures. the cAMP dynamic 2 kit (Cisbio) using homogeneous time-resolved fluorescence technology. cAMP levels were calculated by fluorescence ratio (665 nm/ ACKNOWLEDGMENTS. This work was supported by National Institutes of 620 nm). Forty-eight hours after transfection, the transfected HEK293 cells were Health/National Institute of Aging Grant R01 AG019230 (to R.G.S.).

1. Howard AD, et al. (1996) A receptor in pituitary and hypothalamus that functions in 19. Rossowski WJ, Coy DH (1994) Specific inhibition of rat pancreatic insulin or glucagon growth hormone release. Science 273(5277):974–977. release by receptor-selective somatostatin analogs. Biochem Biophys Res Commun 2. Smith RG, et al. (1997) Peptidomimetic regulation of growth hormone secretion. 205(1):341–346. Endocr Rev 18(5):621–645. 20. Rohrer SP, et al. (1998) Rapid identification of subtype-selective agonists of the so- 3. Kojima M, et al. (1999) Ghrelin is a growth-hormone-releasing acylated peptide from matostatin receptor through combinatorial chemistry. Science 282(5389):737–740. stomach. Nature 402(6762):656–660. 21. Yang L, et al. (1998) Synthesis and biological activities of potent peptidomimetics 4. Sun Y, Wang P, Zheng H, Smith RG (2004) Ghrelin stimulation of growth hormone selective for somatostatin receptor subtype 2. Proc Natl Acad Sci USA 95(18): release and appetite is mediated through the growth hormone secretagogue re- 10836–10841. ceptor. Proc Natl Acad Sci USA 101(13):4679–4684. 22. Zambre Y, et al. (1999) Inhibition of human pancreatic islet insulin release by re- 5. Sun Y, Asnicar M, Saha PK, Chan L, Smith RG (2006) Ablation of ghrelin improves the ceptor-selective somatostatin analogs directed to somatostatin receptor subtype 5. – diabetic but not obese phenotype of ob/ob mice. Cell Metab 3(5):379–386. Biochem Pharmacol 57(10):1159 1164. 6. Sun Y, Asnicar M, Smith RG (2007) Central and peripheral roles of ghrelin on glucose 23. Rocheville M, et al. (2000) Subtypes of the somatostatin receptor assemble as func- – homeostasis. Neuroendocrinology 86(3):215–228. tional homo- and heterodimers. J Biol Chem 275(11):7862 7869. 7. Sun Y, Butte NF, Garcia JM, Smith RG (2008) Characterization of adult ghrelin and 24. Gautier A, et al. (2008) An engineered protein tag for multiprotein labeling in living – ghrelin receptor knockout mice under positive and negative energy balance. Endo- cells. Chem Biol 15(2):128 136. crinology 149(2):843–850. 25. Maurel D, et al. (2008) Cell-surface protein-protein interaction analysis with time- 8. Szentirmai E, Kapás L, Sun Y, Smith RG, Krueger JM (2009) The preproghrelin gene is resolved FRET and snap-tag technologies: Application to GPCR oligomerization. Nat Methods 5(6):561–567. required for the normal integration of thermoregulation and sleep in mice. Proc Natl 26. Kern A, Albarran-Zeckler R, Walsh HE, Smith RG (2012) Apo-ghrelin receptor forms Acad Sci USA 106(33):14069–14074. heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic 9. Gutierrez JA, et al. (2008) Ghrelin octanoylation mediated by an orphan lipid trans- effects of DRD2 agonism. Neuron 73(2):317–332. ferase. Proc Natl Acad Sci USA 105(17):6320–6325. 27. Jiang H, Betancourt L, Smith RG (2006) Ghrelin amplifies. dopamine signaling by 10. Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL (2008) Identification of the crosstalk involving formation of GHS-R/D1R heterodimers. Mol Endocrinol 20(8): acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. 1772–1785. Cell 132(3):387–396. 28. Feighner SD, et al. (1998) Structural requirements for the activation of the human 11. Zhao TJ, et al. (2010) Ghrelin O-acyltransferase (GOAT) is essential for growth hor- growth hormone secretagogue receptor by peptide and nonpeptide secretagogues. mone-mediated survival of calorie-restricted mice. Proc Natl Acad Sci USA 107(16): Mol Endocrinol 12(1):137–145. – 7467 7472. 29. Qader SS, Lundquist I, Ekelund M, Håkanson R, Salehi A (2005) Ghrelin activates 12. Nass RM, Gaylinn BD, Rogol AD, Thorner MO (2010) Ghrelin and growth hormone: neuronal constitutive nitric oxide synthase in pancreatic islet cells while inhibiting – Story in reverse. Proc Natl Acad Sci USA 107(19):8501 8502. insulin release and stimulating glucagon release. Regul Pept 128(1):51–56. 13. Gromada J, Franklin I, Wollheim CB (2007) Alpha-cells of the endocrine pancreas: 35 30. Chuang JC, et al. (2011) Ghrelin directly stimulates glucagon secretion from pancre- – years of research but the enigma remains. Endocr Rev 28(1):84 116. atic alpha-cells. Mol Endocrinol 25(9):1600–1611. 14. Dezaki K, Kakei M, Yada T (2007) Ghrelin uses Galphai2 and activates voltage-de- 31. Kawamori D, et al. (2009) Insulin signaling in alpha cells modulates glucagon secre- + + pendent K channels to attenuate glucose-induced Ca2 signaling and insulin release tion in vivo. Cell Metab 9(4):350–361. in islet beta-cells: Novel signal transduction of ghrelin. Diabetes 56(9):2319–2327. 32. Hauge-Evans AC, et al. (2009) Somatostatin secreted by islet delta-cells fulfills mul- 15. Brazeau P, et al. (1973) Hypothalamic polypeptide that inhibits the secretion of im- tiple roles as a paracrine regulator of islet function. Diabetes 58(2):403–411. munoreactive pituitary growth hormone. Science 179(4068):77–79. 33. Briatore L, Andraghetti G, Cordera R (2003) Acute plasma glucose increase, but not 16. Patel YC, Greenwood MT, Warszynska A, Panetta R, Srikant CB (1994) All five cloned early insulin response, regulates plasma ghrelin. Eur J Endocrinol 149(5):403–406. human somatostatin receptors (hSSTR1-5) are functionally coupled to adenylyl cyclase. 34. Egido EM, Rodriguez-Gallardo J, Silvestre RA, Marco J (2002) Inhibitory effect of Biochem Biophys Res Commun 198(2):605–612. ghrelin on insulin and pancreatic somatostatin secretion. Eur J Endocrinol 146(2): 17. Mitra SW, et al. (1999) Colocalization of somatostatin receptor sst5 and insulin in rat 241–244. pancreatic beta-cells. Endocrinology 140(8):3790–3796. 35. Hohmeier HE, et al. (2000) Isolation of INS-1-derived cell lines with robust ATP-sen- 18. Hoyer D, et al. (1995) Classification and nomenclature of somatostatin receptors. sitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Trends Pharmacol Sci 16(3):86–88. Diabetes 49(3):424–430.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209590109 Park et al. Downloaded by guest on October 1, 2021