Bombesin stimulates insulin secretion by a pancreatic islet cell line

Transcription

1 Pro. Natl. Aad. Si. USA Vol. 81, pp , Marh 1984 Medial Sienes Bombesin stimulates insulin seretion by a panreati islet ell line (gastrin-releasing peptide/somatostatin/gluagon/hit-t15 ells) SHERIDAN L. SWOPE AND AGNES SHONBRUNN* Department of Pharmaology, Harvard Medial Shool, and Laboratory of Toxiology, Harvard Shool of Publi Health, Boston, MA 2115 ommuniated by Konrad Bloh, November 28, 1983 ABSTRAT The amphibian tetradeapeptide, bombesin (BBS) has been shown to stimulate insulin seretion both in vivo and by panreati islet ells in vitro. To determine whether BBS an at diretly on panreati (3 ells, we examined its effets on insulin seretion by HIT-T15 ells (HIT ells), a lonal islet ell line. Addition of 1 nm BBS to HIT ells stimulated insulin release 25-fold within 3 se. The rapid stimulatory effet of BBS on insulin release was short-lived: the seretory rate returned to basal levels after 9 min of BBS treatment. The derease in the rate of insulin release in the ontinued presene of BBS was due not to depletion of intraellular insulin stores but to speifi desensitization to this peptide. Stimulation of insulin seretion by BBS was dose dependent with an ED.o value (.51 ±.15 nm) similar to the onentration of BBS-like immunoreative material in rat plasma. Five BBS analogs, inluding porine gastrin-releasing peptide, were as powerful as BBS in stimulating insulin release. The relative potenies of the analogs tested indiated that the OOH-terminal otapeptide sequene in BBS was suffiient for stimulation of release. In ontrast, 14 peptides struturally unrelated to BBS did not alter insulin seretion. BBS ation was synergisti with that of gluagon; insulin seretion in the presene of maximal onentrations of both peptides was greater than the additive effets of the two peptides added individually. Somatostatin inhibited BBS-stimulated release by 69 ± 1% with an IDso value of 3.2 ±.3 nm. These results show that BBS stimulation of insulin seretion by a lonal panreati ell line losely parallels its effets in vivo and support the hypothesis that BBS stimulates insulin seretion by a diret effet on the panreati j ell. The lonal HIT ell line provides a homogeneous ell preparation amenable for studies on the biohemial mehanisms of BBS ation in the endorine panreas. Bombesin (BBS), a tetradeapeptide originally isolated from the skin of the frog Bombina bombina (1), is one of a group of amphibian peptides with homologous ounterparts in mammalian brain and gut (2-8). BBS modulates many behavioral, metaboli, and endorine proesses in mammals when introdued into the entral nervous system or the blood stream (4, 6, 9). These proesses inlude regulation of body temperature, food and water intake, blood gluose levels, and the release of pituitary, panreati, and gut hormones. Therefore, endogenous BBS-like peptides have been proposed to play a role in the maintenane of metaboli homeostasis (4). In this study, we have foused on the effet of BBS on hormone seretion by the endorine panreas. Intravenous infusion of BBS in humans and dogs auses an inrease in the plasma onentrations of both insulin and gluagon and lowers blood gluose levels (1-14). In vitro, BBS stimulates the seretion of insulin and gluagon by perfused dog panreas (15, 16), whereas it has been shown to either stimulate The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S solely to indiate this fat. (17) or inhibit insulin release by isolated rat panreas (18). Beause panreati islet ells onsist of a mixture of hormone-sereting ell types, these studies annot distinguish between a diret or indiret effet of BBS on panreati 8 ells. To eluidate the nature of any diret effet of BBS on panreati P ells, we examined its ations in a lonal insulin-sereting ell line. HIT-T15 (HIT) ells were derived from simian virus 4-transformed hamster panreati islets and retain many harateristis of normal differentiated (3 ells (19). HIT ells synthesize and proess proinsulin, ontain seretory granules, and respond appropriately to a variety of ompounds known to modulate insulin release in normal islet ells (19). Our results indiate that HIT ells provide a useful in vitro model system for investigating the ation of BBS in the p ell of the panreas. A preliminary report of some of these experiments has been presented.t MATERIALS AND METHODS Materials. Syntheti BBS, [Tyr4]BBS, ranatensin, litorin, porine gastrin-releasing peptide (GRP), GRP(14-27), holeystokinin otapeptide, oxytoin, vasopressin, growth-hormone releasing fator(1-44), seretin, and vasoative intestinal peptide were purhased from Peninsula Laboratories (San arlos, A). Somatostatin, alitonin, substane P, and neurotensin were purhased from Bekman. [Leu]Enkephalin was obtained from Bahem Fine hemials (Torrane, A). Thyrotropin-releasing hormone was provided by Abbott (North hiago, IL). ulture medium, horse serum, and fetal alf serum were purhased from GIBO, and plasti ulture dishes were from Falon. IgGsorb (Staphyloous aureus protein A) was purhased from Enzyme enter (Boston). Gluagon, melatonin, phenylmethylsulfonyl fluoride, and iodoaetamide were purhased from Sigma. Monoomponent porine insulin (lot no J-256) and HIT ells were generous gifts from Eli Lilly. ell ulture. The establishment and properties of the HIT ell line have been desribed (19). After several months of propagation in our laboratory, the ells beame more flattened and more tightly adherent to ulture dishes. The flattened ells ontinued to serete insulin and their responses to gluagon and somatostatin were similar to that desribed by Santerre et al. (19). The new morphology has been retained during ontinuous passage of the ells for 2 years. Beause inreased adherene to the substratum was experimentally advantageous, HIT ells with the flattened morphology were used in all experiments. Experiments were arried out using repliate 35-mm dishes inoulated with 1.5 ml from a single donor suspension. ultures were grown in Ham's F12 medium supplemented with 15% horse serum/2.5% fetal alf serum at 37 in humidified 5% 2/95% air. The ulture medium was hanged Abbreviations: BBS, bombesin; GRP, gastrin-releasing peptide. *To whom reprint requests should be addressed. tswope, S. L. & Shonbrunn, A., Proeedings of the Thirteenth Annual Meeting of the Soiety for Neurosienes, November 6-11, 1983, Boston, MA, abstrat

2 every 3-4 days and experiments were arried out 1 day after the medium was hanged. ells were maintained in ulture for 3-5 hanges of medium before use, and the number of ells per plate was determined at the time of eah experiment using a hemoytometer. Insulin Radioimmunoassay. Radiolabeled insulin (speifi ativity, i/g; 1 i = 37 GBq) was prepared by hloramine T iodination of monoomponent porine insulin (2). Antiserum against monoomponent porine insulin was produed in guinea pigs (Hartley strain) aording to published proedures (21, 22) An antiserum designated 792 and shown to be speifi for insulin was used at a final onentration of 1:16,. Free antigen was separated from antibody-antigen omplex by preipitation with either 2%o polyethylene glyol (Mr, 8)/2% horse serum (23) or IgGsorb (Staphyloous aureus protein A) (24). The limit of assay sensitivity was 15 pg of insulin per.1 ml of sample buffer. HIT ell (hamster) insulin and porine insulin gave parallel displaement urves for inhibition of iodinated antigen binding. The intra-assay variability was ±5% and inter-assay variation was ±27%. Measurement of Insulin Seretion. To determine the effet of BBS and other neuropeptides on insulin seretion, repliate dishes were washed twie with a Hepes-buffered salt solution ontaining 118 mm Nal/4.6 mm Kl/.5 mm al2/1 mm Mgl2/1 mm gluose/5 mm Hepes/.1% NaHO3/.1% bovine serum albumin, ph 7.2. Subsequently, 1 ml of Hepes-buffered salt solution, preequilibrated at E 1- a (n a a) (n H- Medial Sienes: Swope and Shonbrunn Minutes Pro. Natl. Aad. Si. USA 81 (1984) in 5% 2/95% air and ontaining the appropriate onentration of peptide, was added. The ultures were then inubated for the desired time at 37 in humidified 5% 2/95% air. For the short time ourse experiment shown in Fig. la, ells were washed with buffer then inubated in Hepes-buffered salt solution without NaHO3 (ph 7.2) at 37 in ambient atmosphere. At the end of eah inubation, the buffers were olleted, floating ells were removed by entrifugation, and the supernatants were stored at -2' for the subsequent determination of insulin onentration by RIA. To determine the extent of insulin degradation by HIT ells, we measured the reovery of exogenous [1251]insulin and nonradiolabeled insulin from the inubation buffers by preipitation with 1% trihloroaeti aid and by RIA, respetively. After inubation with HIT ells for 6 min at 37, [1251]insulin (.15-6 ng/ml) was degraded only 21 ± 2% and nonradiolabeled insulin (1 ng/ml) was degraded only 25 ± 12%. Furthermore, the addition of 1 nm BBS did not alter the stability of either radiolabeled or nonradioative insulin (data not shown). Therefore, the 5- to 25-fold inrease in insulin aumulation indued by BBS annot be due to inhibition of insulin degradation in the medium. Measurement of Intraellular Insulin. For determination of intraellular insulin levels, HIT ells were extrated into 5 M aeti aid ontaining bovine serum albumin (5 mg/ml)/ phenylmethylsulfonyl fluoride (.3 mg/ml)/iodoaetamide (.3 mg/ml). Samples were snap-frozen and thawed 3 times and then extrated at 4 for 16-2 hr. Extrats were entrifuged and the supernatants were lyophilized. The lyophilized samples were dissolved in a buffer of 1 mm NaH2PO4/1 mm Na2EDTA/.1% Triton X-1/phenylmethylsulfonyl fluoride (.3 mg/ml)/iodoaetamide (.3 mg/ml), ph 7.6. Insulin onentrations in the extrats were determined by RIA. RESULTS The Effet of BBS on Insulin Seretion. The data in Fig. 1 show the effet of 1 nm BBS on insulin seretion by HIT ells. The stimulatory effet of BBS was very rapid; within 3 se BBS aused a 25-fold inrease in insulin release (Fig. LA). Thereafter, the seretory rate dereased and returned to basal levels by 9 min (Fig. 1B). The lak of a prolonged E G) -o 1 I-- { Ha N._ a) 5 (It '-I Hours FIG. 1. Time ourse for BBS stimulation of insulin seretion. HIT ells were inubated at either 37 in the ambient atmosphere in Hepes-buffered salt solution without biarbonate (A) or at 37 in 5% 2/95% air in Hepes-buffered salt solution with biarbonate (B). Inubations were arried out in the absene (o) or presene () of 1 nm BBS for the times shown. The onentration of insulin released into the buffer was measured by RIA. Eah point represents the mean value for tripliate (A) or quadrupliate (B) dishes, and the brakets show the SEM. ell number was 3.7 ±.1 x 16 ells per dish (A) and 5.1 ±.4 x 16 ells per dish (B). L lo11 lo lo-8 io-7 [BBS] (M) FIG. 2. onentration dependene for BBS stimulation of insulin seretion. HIT ells (3.3 ±.2 x 16 ells per dish) were inubated at 37 in 1. ml of Hepes-buffered salt solution ontaining the indiated onentration of BBS. After 6 min, the buffers were olleted and the released insulin was measured by RIA. Eah point represents the mean value of quadrupliate dishes and the bars show the SEM.

3 1824 Medial Sienes: Swope and Shonbrunn Table 1. The effet of different neuropeptides on insulin release by HIT ells Insulin sereted, ng per dish Peptide added ontrol With BBS None.5 ± ±.5 Seretin.42 ± ±.3 Vasoative intestinal peptide.5 ± ±.32 Thyrotropin-releasing hormone.51 ± ±.63 Substane P.44 ± ±.53 Neurotensin.45 ± ±.51 HIT ells (3.9 ±.2 x 16 ells per dish) were inubated with the peptides shown. The final onentration of all peptides was 1 nm. After 6 min, the buffers were removed and the onentration of insulin was determined by RIA. Values represent mean ± SEM of tripliate dishes. stimulatory effet of BBS on seretion ould have been due to depletion of intraellular insulin stores. However, during a 6-min inubation at 37, 1 nm BBS aused the release of only 1.4 ±.7% (mean ± range; n = 2) of intraellular insulin. Furthermore, there was no detetable differene in the insulin ontent of untreated and BBS-treated ells (data not shown). Therefore, stimulation of insulin release by BBS does not deplete intraellular stores. The explanation for the transient nature of the response to BBS is disussed below. The onentration dependene for the stimulation of insulin release by BBS is shown in Fig. 2. In this experiment, a half-maximal response was eliited by.3 nm BBS. In four experiments, the ED5 value for BBS was.51 ±.15 nm (mean ± SEM). The onentration of extratable BBS-like immunoreativity in rat plasma has been reported to be about.2 nm (3). Therefore, BBS stimulates insulin seretion in a dose-dependent manner with an ED5 in the physiologial onentration range. The Effet of Other Neuropeptides on Insulin Seretion. To determine the speifiity of BBS ation, we tested the ability of other peptides to regulate insulin seretion in HIT ells. The results in Table 1 show that five neuropeptides struturally unrelated to BBS did not alter basal insulin release nor did they modulate the stimulatory ation of BBS. In independent experiments, oxytoin, pentagastrin, epidermal growth fator, [Leulenkephalin, vasopressin, growth hormone-releasing fator, holeystokinin otapeptide, melatonin, and alitonin at onentrations of 1 nm had no effet on insulin seretion. Therefore, the ability of BBS to stimulate insulin release by HIT ells is not shared by all brain-gut peptides. To analyze the strutural requirements for biologial ativity in BBS-like peptides, we tested five BBS analogs shown in Table 2. [Tyr4]BBS, whih has a tyrosine instead of a leuine at position four, is the syntheti analog used for radiolabeling in reeptor-binding studies (25, 26). Litorin and ranatensin, peptides originally isolated from amphibian skin (6), both ontain the last eight OOH-terminal amino aids of BBS with a penultimate leuine for phenylalanine substitution. GRP, a mammalian BBS analog isolated from porine gut, has the same OOH-terminal deapeptide sequene as Pro. Natl. Aad Si. USA 81 (1984) Table 3. Poteny of BBS analogs to stimulate insulin seretion Peptide ED5, nm BBS.51 ±.15 [Tyr4]BBS.46 ±.17 Ranatensin 1.11 ±.8 Litorin.63 ±.5 GRP 2.6 ±.61 GRP(14-27).78 ±.3 The onentration of peptide required to produe half-maximal stimulation of hormone release (ED5) was determined in experiments suh as that shown in Fig. 2. Statistial analysis using Dunan's multiple range test showed that the ED5 value for GRP was signifiantly different from that for the other analogs [P <.1 ompared to BBS, litorin, and [Tyr4]BBS; P <.5 ompared to ranatensin and GRP(14-27)]. The ED5 values for the analogs other than GRP did not differ signifiantly from that for BBS or eah other (P >.99). Values represent mean ± SEM of four experiments for BBS or mean ± range of two experiments for BBS analogs. BBS exept for a histidine for glutamine substitution (2). GRP differs from BBS in its NH2-terminal 17 amino aids. GRP(14-27) is the OOH-terminal tetradeapeptide fragment of GRP. Maximal onentrations of every BBS analog tested stimulated insulin release to the same extent as did 1 nm BBS (data not shown). The results in Table 3 show that the four short peptides were equipotent with BBS. However, BBS was 4-fold more potent than GRP, the NH2-terminal extended analog (Table 3). The relative potenies of these peptides indiate that the OOH-terminal otapeptide sequene in BBS is suffiient for stimulation of insulin seretion. The Effet of Somatostatin and Gluagon on BBS-Stimulated Seretion. Both in vivo and in panreati islets in vitro, gluagon stimulates insulin seretion (27), whereas somatostatin inhibits it (28). Beause HIT ells respond in the expeted manner to these two peptides (19), we investigated the effet of gluagon and somatostatin on the stimulatory ation of BBS. A maximal onentration of BBS (1 nm) inreased insulin release 1-fold, whereas, in the same experiment, gluagon (1,uM) aused a 6-fold inrease (Table 4). The simultaneous addition of gluagon and BBS at maximal onentrations resulted in a 4-fold stimulation of insulin seretion (Table 4). In three independent experiments, the effet of gluagon plus BBS was fold greater than the additive effet of the two peptides alone. Therefore, BBS and gluagon at synergistially to stimulate insulin release. Although 1 nm somatostatin had no effet on basal insulin seretion, it inhibited the stimulatory effet of BBS by 71% (Table 4). In four experiments, this onentration of somatostatin inhibited BBS-stimulted insulin release by 69 ± 1% (mean ± SEM). Fig. 3 shows that the inhibitory effet of somatostatin was dose dependent: the ID5 for somatostatin in two experiments was 3.2 ±.3 nm (mean ± range). We have haraterized speifi, high-affinity reeptors for BBS in HIT ells and have shown that the binding of [125I1 Tyr4]BBS is not affeted by either somatostatin or gluagon (unpublished observations). Therefore, the modulatory ef- Table 2. Amino aid sequenes of BBS analogs* BBS pglu-gln-arg-leu-gly-asn-gln-trp-ala-val-gly-his-leu-metnh2 [Tyr4]BBS pglu-gln-arg-tye-gly-asn-gln-trp-ala-val-gly-his-leu-metnh2 Ranatensin pglu Val-Pro -Gln-Trp-Ala-Val-Gly-His -Phe-MetNH2 Litorin pglu Gln-Trp-Ala-Val-Gly-His -Phe-MetNH2 GRP Ala... Met -Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-MetNH2 GRP(14-27) Met -Tyr-Pro -Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-MetNH2 *Summarized from refs. 2 and 6. pglu denotes pyroglutami aid. The NH2-terminal amino aids 2-13 in GRP are symbolized by... Amino aids differing from BBS sequene are underlined. The hyphens indiate amino aid deletions.

4 Table 4. The effet of gluagon and somatostatin on the stimulatory ation of BBS Insulin sereted, ng per dish Peptide added ontrol With BBS None.5 ± ±.5 Gluagon (1,uM) 2.92 ± ± 1.3 Somatostatin (1 nm).48 ± ±.1 HIT ells (3.9 ±.2 x 16 ells per dish) were inubated with the peptides shown in the absene or presene of 1 nm BBS. After 6 min, the buffers were removed and the onentration of insulin was determined by RIA. Values represent mean ± SEM of tripliate dishes. fets of somatostatin and gluagon on BBS ation must be mediated by independent reeptors. BBS-Indued Desensitization. The observation that BBS had a transient effet on insulin release (see Fig. 1) without depleting intraellular insulin stores suggested that HIT ells desensitize to the stimulatory effet of BBS. To test this hypothesis, ells were inubated for 2 hr in the presene or absene of 1 nm BBS and then hallenged in fresh buffer with three different seretagogues. During the first inubation, 1 nm BBS aused a 1-fold inrease in insulin seretion (Fig. 4A). However, ells exposed to BBS in the first inubation did not respond to subsequent treatment with BBS (Fig. 4B). In ontrast, ells preinubated with BBS did respond to gluagon and a high onentration of K+ with inreased insulin release (Fig. 4B). These data show that BBS indues speifi desensitization in HIT ells. DISUSSION BBS is a potent stimulator of insulin seretion when infused intravenously into either humans or experimental animals (1-14). In addition, BBS-like peptides are present in peptidergi neurons of the mammalian panreas, and eletrial stimulation of the splanhni nerve has been shown to inrease BBS levels in peripheral plasma to onentrations suffiient to stimulate insulin release (29, 3). These results strongly suggest that BBS-like peptides play an important role in the physiologial ontrol of insulin seretion. The data presented here show that BBS diretly stimulates - 4 I- 3 I. 'o 2 r._s Medial Sienes: l H 1 ' - ). I- F Swope and Shonbrunn o lo-1i 1-9 lo [SRIF] FIG. 3. onentration dependene for somatostatin inhibition of BBS-stimulated insulin release. HIT ells (3.4 ±.4 x 16 ells per dish) were inubated at 37 in 1. ml of Hepes-buffered salt solution ontaining 1 nm BBS and the indiated onentration of somatostatin (SRIF). After 6 min, the buffers were olleted and the released insulin was measured by RIA. Insulin seretion in the absene of any peptide was 5.1 ±.7 ng/ml. Eah point gives the mean ± SEM of tripliate dishes. (M) E 2 P 1 15 Pro. NatL. Aad Si. USA 81 (1984) 1825 K BBS BBS GLU FIG. 4. BBS-indued desensitization of HIT ells. HIT ells ( X 16 ells per dish) were inubated in 1. ml of Hepes-buffered salt solution at 37 for 2 hr in the absene () or presene of 1 nm BBS. The amount of insulin released during this inubation was measured by RIA and is shown in A. Repliate dishes that had been inubated with 1 nm BBS for 2 hr were subsequently inubated again in fresh buffer ontaining either no seretagogue (), 1 nm BBS, 5 mm KB (K+), or 1M gluagon (GLU). After 9 ± 1in, the buffers were removed and sereted insulin was measured by RIA (B). Eah bar represents the mean A SEM of tripliate dishes. insulin release by a lonal line of hamster panreati ells. The harateristis of the insulin response to BBS in HIT ells are remarkably similar to what is observed both in vivo and in panreati islets in vitro. BBS is effetive in HIT ells at the same low onentrations that stimulate insulin seretion in perfused dog panreas (15, 16) and inrease plasma insulin levels in dogs and humans (1, 14). In all systems, the response to BBS is rapid and transient. Infusion of BBS in dogs and humans produed a maximal stimulation of insulin seretion within 5 to 15 min followed by a derease in insulin levels during ontinued infusion of the peptide (11-14). Similarly, in perfused isolated anine panreas, stimulation of insulin release was maximal after 2 min and dereased thereafter (15, 16). In HIT ells, a 25-fold stimulation of insulin release ourred 3 se after the addition of BBS and the seretory rate subsequently dereased to the basal rate. As in vivo (13), somatostatin inhibited the stimulatory effet of BBS in HIT ells. Finally, a mammalian BBS-like peptide, GRP, inreased insulin release both when infused intravenously into dogs (14) and when added to HIT ells. These observations suggest that the effets of BBS-like peptides on insulin seretion in vivo are due to diret ation on 3 ells. To determine the strutural requirements for BBS ation, we ompared the potenies of several BBS analogs to stimulate insulin seretion by HIT ells. Our data indiate that the OOH-terminal otapeptide sequene in BBS is suffiient for potent stimulation. Of additional interest is the observation that porine GRP was less potent than the amphibian peptide BBS. Similar results have been observed in other systems. For example, when injeted intraisternally into rats, BBS is more potent than GRP in produing hypothermia and hyperglyemia (31) and in ausing inhibition of gastri aid seretion and stimulation of gastrin release (32). Furthermore, BBS has been shown to bind to reeptors in a rat pituitary ell line with a 3-fold higher apparent affinity than does GRP (26). The greater poteny of the amphibian peptide BBS than its mammalian analog probably reflets

5 1826 Medial Sienes: Swope and Shonbrunn differential sensitivity of ertain target ells to the various moleular forms of endogenous BBS-like peptides. The presene of several forms of BBS-like substanes in mammals was reognized in early studies of the distribution of immunoreative BBS-like peptides (3, 6-8). Reently, three biologially ative BBS-like peptides have been purified from anine intestine (5). These peptides share a ommon OOHterminal sequene that has substantial homology with both porine GRP and amphibian BBS. The observation that BBS and GRP(14-27) are more potent than GRP in some target ells (refs. 31 and 32; Table 3) indiates that the OOH-terminal fragments of endogenous BBS-like peptides are physiologially more potent than their NH2-terminal extended ounterparts. However, diverse target tissues do show differential sensitivity to various BBS-like peptides (4, 6, 7). Moleular heterogeneity and differential ellular responsiveness to ative forms have also been reported for the neuropeptides somatostatin, holeystokinin, and gastrin (28, 33, 34). Further studies are learly warranted to determine the relative importane of the different forms of mammalian BBS in the various biologial ations of this peptide. Both in vivo and in perfused panreas the stimulatory effet of BBS on insulin seretion is transient (11-16). Similarly, in HIT ells the time ourse for BBS-stimulated insulin seretion shows a return to the basal seretory rate by 9 min in the ontinued presene of the peptide. This derease in seretory rate is not due to a signifiant derement in intraellular insulin, but rather to speifi desensitization of the ellular response to BBS. Desensitization to BBS has also been observed in dispersed panreati ainar ells (35, 36). Sine BBS-indued desensitization and resensitization in ainar ells our in the absene of protein synthesis, BBS reeptors must be able to yle between ative and inative states. However, the mehanism by whih BBS desensitization ours remains to be determined. An interesting result obtained in our study was the synergisti effet of BBS and gluagon on insulin seretion. Gluagon eliits its biologial effets by inreasing intraellular AMP levels in target ells (37). The intraellular mehanism of BBS ation has been most thoroughly investigated in the exorine panreas where its effets appear not to be mediated by AMP (38-4). In panreati exorine ells BBS has been shown to modulate membrane potential, alium efflux, GMP aumulation, and phosphatidylinositol turnover (38-4). The relative importane of these biohemial hanges in initiating the biologial ations of BBS remains to be larified. Our results indiate distint and synergisti mehanisms for BBS and gluagon ation. HIT ells provide a homogeneous well-defined system for determining the mehanism by whih BBS stimulates insulin seretion both alone and in onjuntion with other regulatory agents. This investigation was supported in part by a researh grant from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (AM 32234) and from the William F. Milton Fund. S.L.S. was supported by a National Researh Servie Award (5 T32 GM736). 1. Anastasi, A., Erspamer, V. & Bui, M. (1971) Experientia 27, MDonald, T. J., Jornvall, H., Nilsson, G., Vagne, M., Ghatei, M., Bloom, S. R. & Mutt, V. (1979) Biohem. Biophys. Res. ommun. 9, Brown, M., Allen, R., Villarreal, J., Rivier, J. & Vale, W. (1978) Life Si. 23, Tahe, Y. & Brown, M. (1982) Trends Neurol. Si. 5, Reeve, J. R., Jr., Walsh, J. H., hew, P., lark, B., Hawke, D. & Shively, J. E. (1983) J. Biol. hem. 258, Pro. NatL Aad Si. USA 81 (1984) 6. Erspamer, V. (198) in Gastrointestinal Hormones, ed. Glass, G. B. J. (Raven, New York), pp Walsh, J. H., Reeve, J. R., Jr., & Vigna, S. R. (1981) in Gut Hormones, eds. Bloom, S. R. & Polak, J. M. (hurhill Livingstone, New York), 2nd Ed., pp Melhiorri, P. (198) in Gastrointestinal Hormones, ed. Glass, G. B. J. (Raven, New York), pp Lezohe, E., Basso, N. & Speranza, V. (1981) in Gut Hormones, eds. Bloom, S. R. & Polak, J. M. (hurhill Livingstone, New York), 2nd Ed., pp Ghatei, M. A., Jung, R. T., Stevenson, J.., Hillyard,. J., Adrian, T. E., Lee, Y.., hristofides, N. D., Sarson, D. L., Mashiter, K., MaIntyre, I. & Bloom, S. R. (1982) J. lin. Endo. Metab. 54, Bruzzone, G., Tamburranao, G., Lala, A., Maueri, M., Annibale, B., Severi,., De Magistris, L., Leonetti, G. & Delle Fave, G. (1983) J. lin. Endo. Metab. 56, Shusdziarra, V., Rouiller, D., Harris, V., Pfeiffer, E. F. & Unger, R. H. (198) Regul. Peptides 1, Vaysse, N., Pradayrol, J. A., hayvialle, F., Pignal, J. P., Esteve,., Susini, F., Desos, R. & Ribet, A. (1981) Endorinology 18, MDonald, T. J., Ghatei, M. A., Bloom, S. R., Trak, N. S., Radziuk, J., Dupre, J. & Mutt, V. (1981) Regul. Peptides 2, Ipp, E. & Unger, R. H. (1979) Endor. Res. ommun. 6, Hermansen, K. (198) Endorinology 17, Martindale, R., Levin, S. & Alfin-Slater, R. (1982) Regul. Peptides 3, Taminato, T., Seino, Y., Goto, Y., Matsukura, S., Imura, H., Sakura, N. & Yanaihara, N. (1978) Endorinol. Japonia 25, Santerre, R. F., ook, R. A., risel, R. M. D., Sharp, J. D., Shmidt, R. J., Williams, D.. & Wilson,. P. (1981) Pro. Natl. Aad. Si. USA 78, Wilson, M. A. & Miles, L. E. M. (1977) in Handbook ofradioimmunoassay, ed. Abraham, G. E. (Dekker, New York), pp Makulu, D. R. & Wright, P. (1971) Metabolism 2, Wright, P. H., Makulu, D. R. & Posey, I. J. (1968) Diabetes 17, Desbuquois, B. & Aurbah, G. D. (1971) J. lin. Endorinol. 33, Jonsson, S. & Kronvall, G. (1974) Eur. J. Immunol. 4, Jensen, R. T., Moody, T., Pert,., Rivier, J. E. & Gardner, J. D. (1978) Pro. Natl. Aad. Si. USA 75, Westendorf, J. M. & Shonbrunn, A. (1983) J. Biol. hem. 258, Marks, V. & Samols, E. (197) Adv. Metab. Disord. 4, Arimura, A. (1981) Biomed. Res. 2, Holst, J. J., Knuhtsen, S., Knigge, U., Jensen, S. L., Nielsen,. V. & Larsson, L. I. (1982) Digestion 25, Bloom, S. R., Edwards, A. V. & Ghatei, M. A. (1982) Diabetologia 23, Brown, M., Marki, W. & Rivier, J. (198) Life Si. 27, Tahe, Y., Marki, W., Rivier, J., Vale, W. & Brown, M. (1981) Gastroenterology 81, Dokray, G. J. (1981) in Gut Hormones, eds. Bloom, S. R. & Polak, J. M. (hurhill Livingstone, New York), 2nd Ed., pp Walsh, J. H. (1981) in Gut Hormones, eds. Bloom, S. R. & Polak, J. M. (hurhill Livingstone, New York), 2nd Ed., pp Lee, P.., Jensen, R. T. & Gardner, J. D. (198) Am. J. Physiol. 238, G213-G Pandol, S. J., Jensen, R. T. & Gardner, J. D. (1982) J. Biol. hem. 257, Sutherland, E. W. & Rall, T. W. (196) Pharmaol. Rev. 12, Peterson,. H. (1982) Br. Med. Bull. 38, Gardner, J. D. (1979) Annu. Rev. Physiol. 41, Shulz, I. & Stolze, H. H. (198) Annu. Rev. Physiol. 42,