Somatostatin Binding to Hepatocytes Isolated from Rainbow Trout,Oncorhynchus mykiss, Is Modulated by Insulin and Glucagon

Somatostatin Binding to Hepatocytes Isolated from Rainbow Trout,Oncorhynchus mykiss, Is Modulated by Insulin and Glucagon

General and Comparative Endocrinology 112, 183–190 (1998) Article No. GC987154 Somatostatin Binding to Hepatocytes Isolated from Rainbow Trout, Oncor...

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General and Comparative Endocrinology 112, 183–190 (1998) Article No. GC987154

Somatostatin Binding to Hepatocytes Isolated from Rainbow Trout, Oncorhynchus mykiss, Is Modulated by Insulin and Glucagon Marty J. Pesek, Nicole Howe, and Mark A. Sheridan Department of Zoology and Regulatory Biosciences Center, North Dakota State University, Fargo, North Dakota 58105 Accepted July 17, 1998

Previously we reported somatostatin-14 (SS-14) binding in the liver of rainbow trout and that fasting enhanced SS-14-binding capacity. In this study, we used hepatocytes isolated from rainbow trout to study aspects of SS-14 processing and to evaluate the basis of fastingassociated changes in SS-14 binding. Hepatocytes specifically bound 5–8% of the total 125I-SS-14 added. Scatchard analysis revealed the existence of two classes of binding sites. The high-affinity site had a dissociation constant (Kd) of 23.6 6 1.1 nM and a binding capacity (Bmax) of 3459 6 134 receptors/cell. The low-affinity site had a Kd of 764 6 27 nM and a Bmax of 17,432 6 345 receptors/ cell. 125I-SS-14 dissociation was hastened by the presence of 1026 M SS-14 . The internalization of 125I-SS-14 was time dependent; preincubation of hepatocytes with 1026 M SS-14 reduced internalization of 125I-SS-14. The number of high-affinity binding sites was also reduced by 1026 M SS-14. Because plasma levels of insulin (INS) decline relative to those of glucagon (GLU) during fasting of trout, we also investigated the effects of these hormones on SS-14 binding. The number of high-affinity SS-14-binding sites was reduced by 1026 M INS and was increased by 1026 M and 1028 M GLU. These results indicated that SS-14 regulates aspects of SS-14 binding and processing and suggests that INS and GLU play a role in fasting-associated changes in SS-14 binding. r 1998 Academic Press Somatostatin (SS) was first isolated as a 14-aminoacid peptide from the hypothalamus of sheep and was 0016-6480/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

reported to inhibit pituitary growth hormone release by Brazeau et al. in 1973. Since this initial discovery, SSs have been isolated from numerous tissues and have been found to exist in a variety of molecular forms. The molecular heterogeneity of the SS family of peptides among vertebrates reflects tissue-specific differential processing of precursor proteins as well as the presence and expression of multiple SS genes. In mammals, peptides of varying lengths have been isolated (e.g., 14, 25, and 28 amino acids); each possesses SS-14 at its C-terminus, and each is derived from the same precursor, denoted as preprosomatostatin-1 (PPSS-1) (Conlon et al., 1997). Teleost fish express PPSS-1 in addition to at least one other precursor; the alternate precursor of salmonids contains [Tyr7, Gly10]-SS-14 at its C-terminus (Sheridan et al., 1997). Somatostatins also are now known to coordinate a wide array of processes associated with growth, development, and metabolism (Gerich, 1983; Patel, 1992). For example, in vivo administration of SSs results in elevated plasma levels of glucose and fatty acids (FA) in both fish and mammals (cf. Sheridan, 1994). Such effects may be indirect influences mediated through insulin (INS). In rainbow trout, SSs inhibits INS (Eilertson and Sheridan, 1993), and INS deficiency results in enhanced hepatic lipolysis and enhanced hepatic glycogenolysis concomitant with elevated plasma levels of FA and glucose (Plisetskaya et al., 1989). The interrelations of SS and other pancreatic peptides also are

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exemplified by the responses of rainbow trout to food deprivation. Fasted fish show elevated levels of SS-14 and depressed levels of INS and glucagon (GLU; however, GLU is depressed to a lesser extent than INS such that the ratio of GLU:INS in the plasma of a fasting fish is considerably greater than that in fed fish), attended by enhanced lipolysis and glycogenolysis in the liver (Sheridan and Mommsen, 1991; Pesek and Sheridan, 1996). The diversity of SS function can be explained by the structural diversity of the hormone and/or by variations in the signaling system (receptors, transduction pathways) possessed by target cells. In mammals, the existence of five SS receptor subtypes (cf. Reisine and Bell, 1995) helps orchestrate the diverse action of PPSS-1 products; however, knowledge of what controls the differential expression of the various SS receptors appropriate to the developmental and physiological conditions of the animal is lacking. The dynamics of SS ligand–receptor interactions are potentially more complex in fish, where information is just emerging. Recently, we detected SS-14 binding in numerous tissues of rainbow trout and found that the SS-14 binding in liver microsomes was highly specific for SS-14; salmon somatostatin-25, which contains [Tyr7, Gly10]-SS-14 at its C-terminus, was a weak competitor (Pesek and Sheridan, 1996). In addition, hepatic SS-14binding capacity was significantly enhanced in fasted trout. Such fasting-induced alterations in SS-14 binding would contribute to increased sensitivity to SS which, in turn, would facilitate necessary metabolic adjustments (e.g., INS inhibition, increased hepatic lipolysis, increased hepatic glycogenolysis; cf., Sheridan and Mommsen, 1991). The basis of the fastingassociated changes in SS-14 binding is not known; however, INS and GLU are implicated because of the interactions between these factors and SS during fasting (Sheridan and Mommsen, 1991; Pesek and Sheridan, 1996). In this study, we used rainbow trout to study the cellular processing of SS-14 and to evaluate the basis of fasting-associated changes in hepatic SS-14-binding characteristics. Our hypothesis was that INS and GLU modulate SS-14-binding capacity. This is the first study in which a nonmammalian species was used to study aspects of SS-14 processing.

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Pesek, Howe, and Sheridan

MATERIALS AND METHODS Experimental Animals Juvenile rainbow trout (Oncorhynchus mykiss) were obtained from the Garrison National Fish Hatchery near Riverdale, North Dakota, and transported to North Dakota State University. The fish were maintained in 800-liter circular tanks supplied with recirculated (ca. 10% make-up volume per day) dechlorinated municipal water at 14°C under a photoperiod of 12L:12D and were fed twice daily to satiety with Supersweet Feeds (Glenco, MN) trout grower except 24 h before experimentation. The animals were acclimated to laboratory conditions for at least 4 weeks prior to experiments.

Isolation of Hepatocytes Fish were anesthetized in 0.01% tricaine methanesulfonate (MS 222) buffered with 0.2% NaHCO3. Hepatocytes were isolated essentially as described by Moon et al. (1985). The liver was perfused in situ at a rate of 2 ml/min with cold (14°C) medium A (in mM: 176 NaCl, 5.4 KCl, 0.81 MgSO4, 0.44 KH2PO4, 0.35 Na2HPO4, 4 NaHCO3, 10 Hepes, and 5 EGTA, pH 7.6) and gassed continuously with 100% O2. After the liver was cleared of blood, the perfusate was changed to medium B (medium A with 0.6 mg/ml collagenase). After 30–40 min, the liver was removed (free of the gallbladder), gently cut into pieces with fine scissors, and incubated in a sterile culture dish containing fresh medium B for an additional 5–10 min. The medium was then removed with a Pasteur pipette and the pieces were rinsed with cold medium A. The pieces were then transferred to a sterile syringe and gently passed through two successive nylon screens (253 and 73 µm, respectively). The resulting cells were collected by centrifugation (100g for 3 min at 14°C), washed three times in medium A, and then finally resuspended in DMEM containing 5 mM glucose, 10 mM Hepes, 0.1 TIU/ml aprotinin, and 0.5% BSA, pH 7.6 (medium C). The viability of the cells was assessed by trypan blue dye exclusion and ranged between 92 and 96% for all experiments. The isolated cells (ca. 2 3 107 cells/ml) were left in suspension for 2–4 h at 14°C for a ‘‘metabolic recovery period.’’

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Preincubation of Hepatocytes In the experiments which evaluated the effects of hormones on SS-binding characteristics, hepatocytes were diluted to a final concentration of 2.5 3 106 cells/ml and preincubated with saline (control) or various concentrations of SS-14 (Sigma S-9129), mammalian INS (Sigma I-5500), or mammalian GLU (Sigma G-1774) for 3 h at 14°C. The incubation was stopped by centrifugation (1000g for 3 min at 14°C). The cells were rinsed with medium C and surface-bound hormone was removed by a 3-min treatment with acidified (pH 4.5) medium C. After acid treatment, the cells were washed twice and resuspended in medium C. Cell viability and cell count were rechecked prior to analysis of SS binding.

Somatostatin Binding Whole-cell competition binding assays were conducted for 3 h at 14°C by incubating 400 µl of the hepatocyte suspension (ca. 1 3 106 cells) and 50 µl of 125I-SS-14 [approximately 40,000 cpm 3-[125I]iodotyrosyl11-Tyr11-SS-14 (sp act 2000 Ci/mmol; Amersham, Arlington Heights, IL), diluted with 0.25 mM Tris buffer with 0.1 TIU/ml aprotinin, 1 mM PMSF, and 0.5% BSA, pH 7.6] with either 50 µl of medium C (for total binding) or 50 µl of unlabeled SS-14 (diluted in medium C) in concentrations from 10 to 1000 nM in microfuge tubes. The incubation was stopped by centrifugation at 1000g for 5 min at 14°C. Unbound hormone was removed by aspiration, and the cells were washed three times with cold medium C. The tips of the tubes containing the pellets were cut off, placed individually in 12 3 75-mm tubes, and counted in a Beckmann 5500 gamma counter. The percentage of specific binding was calculated as B/T 3 100, where B is the difference between total binding and nonspecific binding (NSB; binding observed in the presence of 1000 nM SS-14) and T is the amount of radioactivity originally added to each tube.

hormone, i.e., centrifugation at 1000g for 5 min at 14°C followed by rinsing the pellet twice with medium C) in medium C with or without SS-14 (final concentration of 1026 M). At various times after resuspension, the cells were again pelleted (1000g for 5 min at 14°C) and washed, and the radioactivity which remained bound to the pellet was determined. In a second series of experiments, SS-14 internalization was evaluated according to Simo´n et al. (1988) with some modifications. Labeled SS-14 was added to hepatocytes (ca. 1 3 106 cells) suspended in medium C. At various times, the suspensions were centrifuged (1000g for 5 min at 14°C); and the pellets were washed twice with medium C. The radioactivity of the pellets was determined and taken to represent the total cell-associated hormone. The pellets were then resuspended in medium C (pH 4.5) and incubated (14°C). After 3 min, the suspensions were centrifuged (1000g for 5 min at 14°C) and the resulting pellets were washed twice with medium C. The washings were combined with the acid-treated supernatant and counted; this combined fraction represented surfacebound hormone. The acid-treated pellet was counted and taken to represent internalized hormone. Degradation of labeled hormone was assessed as described by Pesek and Sheridan (1996).

Statistics Data are presented as means 6 SEM. Statistical differences were estimated by ANOVA; multiple comparisons among means were evaluated by Duncan’s test. Affinity constants and binding capacities were calculated from Scatchard plots employing either a negative cooperativity model or a model of multiple binding sites (LIGAND; Munson, 1992); the latter model fit the data better and was used for interpretation of results.

RESULTS Somatostatin Processing In one set of experiments, 125I-SS-14 dissociation from isolated hepatocytes was evaluated by incubating cells in the presence of labeled SS-14 as in the above-described binding experiments and resuspending the cell pellets (after separation of free from bound

Characterization of Binding Sites Isolated hepatocytes specifically bound 5–8% of the total 125I-SS-14 added. Scatchard analysis (Fig. 1) yielded a curvilinear plot, suggesting the presence of two classes of binding sites: a high-affinity/low-capacity

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site and a low-affinity/high-capacity site. The highaffinity/low-capacity site displayed a Kd of 23.6 6 1.1 nM and was estimated to number 3459 6 134 receptors per cell. The low-affinity/high-capacity site displayed a Kd of 764 6 27 nM and was estimated to number 17,432 6 345 receptors per cell. The physiological significance of the low-affinity receptors is unclear, even in mammals; as a result, most of our subsequent analysis considered the high-affinity class.

Dissociation 125I-SS-14

dissociation from hepatocytes increased with time; dissociation was hastened by the presence of unlabeled SS-14 (Fig. 2).

Internalization and Processing The time course of 125I-SS-14 processing is shown in Fig. 3. Preliminary studies indicated that 1.6 6 0.5% of the labeled hormone was degraded at time 0 compared to 4.8 6 1.2% at the end of the processing experiments (3 h). Internalization of 125I-SS-14 was time dependent;

FIG. 2. Dissociation of 125I-somatostatin-14 (SS-14) from isolated hepatocytes (1 3 106 cells) incubated in the presence and absence of unlabeled SS-14. After separation of free from bound hormone, the pellet was resuspended in DMEM either with or without unlabeled SS-14 (final concentration 1026 M). Data are presented as means 6 SEM (n 5 6). *Significant difference (P , 0.05) from hepatocytes incubated in the absence of SS-14; to samples were significantly different from all other samples.

after 3 h, the internalized pool represented 25% of the cell-associated hormone.

Effects of SS-14 Preincubation of hepatocytes with unlabeled SS-14 reduced cell-associated 125I-SS-14 in a dose-related manner; the most pronounced effect was on surfacebound hormone (Fig. 4). Somatostatin binding to hepatocytes preincubated in the presence and absence of SS-14 is shown in Fig. 5. Somatostatin-14 had no effect on the apparent binding affinity of 125I-SS-14. Binding capacity of hepatocytes for 125I-SS-14 was reduced by preincubation of both 1028 and 1026 M SS-14; however, only the decline associated with 1026 M SS-14 was statistically significant.

Effects of Insulin and Glucagon FIG. 1. Scatchard plot of 125I-somatostatin-14 (SS-14) binding to isolated hepatocytes. Hepatocytes (1 3 106 cells) were incubated for 3 h at 14°C, pH 7.6. Data are presented as means of six determinants.

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The effects of INS and GLU on hepatocyte binding characteristics are shown in Fig. 6. Insulin had no effect

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on the binding affinity of either the high-affinity or the low-affinity binding sites. At 1026 M, insulin significantly reduced the number of high-affinity binding sites; no effect of INS was observed on the number of low-affinity sites. Glucagon also failed to affect the binding affinities of either the high-affinity or lowaffinity class of binding sites. In contrast to INS, GLU at both 1028 and 1026 M significantly increased the number of high-affinity binding sites. Glucagon, like INS, had no effect on the binding capacity of the low-affinity binding sites.

DISCUSSION Previously we demonstrated that liver microsomes prepared from rainbow trout possess specific binding sites for SS-14 and that these sites display features characteristic of hormone receptors (Pesek and Sheridan, 1996). The use of a whole-cell binding approach in the present study enabled us to evaluate for the first time how somatostatin is processed in liver cells or, for that matter, in any nonmammalian cell type. This

FIG. 3. Processing of 125I-SS-14 by isolated hepatocytes. Cells (1 3 106) were incubated with 125I-SS-14 (40,000 cpm) at 14°C for various times and then treated as described under Materials and Methods. Data are presented as means 6 SEM (n 5 6).

FIG. 4. Effects of somatostatin-14 (SS-14) on processing of 125I-SS-14 by isolated hepatocytes. Cells (1 3 106 cells) were preincubated with or without SS-14 for 3 h at 14°C and then evaluated for SS-14 processing as described under Materials and Methods. Data are presented as means 6 SEM (n 5 6). For each fraction, groups with different letters are significantly different (P , 0.05).

approach was particularly useful for the evaluation of hormone internalization and for assessing how SS-14 binding characteristics are modulated by hormones. Our results indicate that SS-14 binding to hepatocytes is followed by internalization of the bound hormone and that SS-14 modulates the binding characteristics of its hepatic binding sites. In addition, our results support our initial hypothesis that INS and GLU modulate SS-14 binding. Isolated hepatocytes specifically bound 5–8% of the total 125I-SS-14 added, which was similar to that reported in rainbow trout liver membrane preparations (Pesek and Sheridan, 1996). Scatchard analysis of the binding data demonstrated a curvilinear plot, suggesting the existence of two classes of binding sites, one with high affinity (Kd 23.6 6 1.1 nM) and low capacity (Bmax 3459 6 134 receptors/cell) and the other with low affinity (Kd 764 6 27 nM) and high capacity (Bmax 17,432 6 345 receptors/cell). The existence of two classes of SS-binding sites in rainbow trout liver is similar to that reported in the lung (Barrios et al., 1987), small intestine (Roca et al., 1990), pituitary (Leitner et al., 1979; Zupanc et al., 1994), pancreas (Mehler et al., 1980), and

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adipocytes (Simo´n et al., 1988) of mammals; affinities of the two classes are similar to those observed in rainbow trout liver membrane preparations (Pesek and Sheridan, 1996). Other authors have reported the existence of a single class of SS receptors using different tissues and/or different types of preparations (e.g., brain, RINm5F insulinoma cells, small intestine, placenta; Raper et al., 1992; Tsalikian et al., 1984; Srikant and Patel, 1981, 1985; Weber et al., 1986; Sullivan and Schonbrunn, 1987). Somatostatin binding to the cell surface of rainbow trout hepatocytes resulted in the internalization of ca. 25% of total bound SS. Internalization of SS also has been reported in rat pituitary cells (Draznin et al., 1985a) and adipocytes (Simo´n et al., 1988). Such internalization probably results from the endocytosis of the SS–receptor complex and represents a mechanism for resensitization of desensitized receptors. Intracellular binding sites were first reported by Ogawa et al. (1977). Since the preponderance of evidence suggests that the SS mechanism of action relies on membrane-mediated

FIG. 6. Effects of insulin and glucagon on SS-14-binding characteristics of isolated hepatocytes. After preincubation with 1026 and 1028 M SS-14 and removal of surface-bound hormone, hepatocytes (1 3 106 cells) were incubated with 125I-SS-14 (40,000 cpm) in the absence or presence of various concentrations of unlabeled SS-14. Binding data were subjected to Scatchard analysis; data are presented as means 6 SEM (n 5 6). For each parameter, groups with different letters are significantly different (P , 0.05).

FIG. 5. Effects of somatostatin-14 (SS-14) on SS-14-binding characteristics of isolated hepatocytes. After preincubation with 1026 and 1028 M SS-14 and removal of surface-bound hormone, hepatocytes (1 3 106 cells) were incubated with 125I-SS-14 (40,000 cpm) in the absence or presence of various concentrations of unlabeled SS-14. Binding data were subjected to Scatchard analysis; data are presented as means 6 SEM (n 5 6). For each parameter, groups with different letters are significantly different (P , 0.05).

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modulation of adenylyl cyclase (Barber et al., 1989), internalization of the SS–receptor complex most likely represents a salvage pathway in which the internalized receptors contribute to an intracellular SS receptor pool (Morel et al., 1986) or are catabolized. The intracellular pool also may contain newly synthesized SS receptors (Draznin et al., 1985b). Ligand-binding studies revealed that SS can bind to intracellular binding sites (Steiner et al., 1986). Modulation of the intracellular pool, in terms of recruitment of receptors to the plasma membrane or internalization of surface receptors, would provide a means of regulating SS action in target cells.

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Down-regulation of hepatic SS-14 receptors by SS-14 is supported by several lines of evidence. First, SS-14 modulated SS processing such that total hormone bound to hepatocytes was reduced; this reduction was primarily from the surface-bound fraction. Second, the pattern of reduced surface-bound receptors also is consistent with the effects of SS-14 on 125I-SS-14 dissociation, where SS-14 not only displaces label but also suggests that there are fewer surfacebinding sites to rebind labeled hormone. Finally, Scatchard analysis revealed that hepatocytes incubated in the presence of SS-14 possess significantly fewer receptors on the cell surface than control-treated cells. Down-regulation of SS receptors by SS-14 also has been observed in mouse pituitary cells (Heisler and Srikant, 1985). Hepatocytes isolated from various species of fish also exhibit auto-down-regulation of insulin (Plisetskaya et al., 1993) and glucagon (Navarro and Moon, 1994) receptors. In each of these studies, only binding capacity of the high-affinity class was affected. The significance of the low-affinity class of binding sites remains to be determined. Whether or not SS-14 modulates receptor characteristics in trout liver cells in vivo is not known; the plasma concentration of SS-14 in trout is somewhat lower (ca. 0.6 ng/ml; Pesek and Sheridan, 1996) than that observed to induce down-regulation in vitro. The regulation of SS-14 receptor characteristics appropriate to the physiological status of the organism most likely involves factors in addition to SS-14. Insulin and GLU modulate rainbow trout hepatic SS-binding characteristics. Insulin (1026 M) significantly reduced the number of high-affinity binding sites while GLU (1026 and 1028 M) significantly increased the number of high-affinity binding sites. As far as we are aware, these are the first such findings of INS and GLU on SS-binding characteristics. The means by which INS and GLU affect SS surface receptor number is not known; however, internalization/recruitment from the intracellular pool and/or altered gene expression are possible mechanisms. In a previous study in mammals, it was shown that glucose rapidly induced INS release from islet cells concomitant with SS receptor recruitment from the intracellular pool to the cell surface; 85% of cellular SS receptors were located intracellularly and glucose treatment caused 10% of these receptors to relocate to the plasma membrane (Draznin et al., 1985b).

Insulin and GLU modulation of rainbow trout hepatic SS binding helps explain many aspects of hormone action on intermediary metabolism in rainbow trout. For example, during feeding when INS levels rise (to levels similar to those used in this study; Plisetskaya, 1990), INS-induced down-regulation of hepatic SS receptors would minimize the catabolic actions of SS (e.g., lipolysis, glycogenolysis; Sheridan, 1994) so that the anabolic actions of INS (e.g., lipogenesis, glycogenesis; Sheridan, 1994) would prevail. During fasting, salmonids display increased plasma levels of SS-14 (Pesek and Sheridan, 1996); elevated SS-14 contributes to the metabolic compensation of fasting fish in two ways: inhibition of INS (Eilertson and Sheridan, 1993) and direct stimulation of hepatic lipid and carbohydrate mobilization (Sheridan, 1994). In addition, the GLU:INS ratio in the plasma of fasting of salmonids increases and the relatively higher GLU concentration (at levels somewhat below those used in the present study; Sheridan and Mommsen, 1991) may further contribute to the metabolic adjustments of the animal (Sheridan, 1994). The up-regulation of SS receptors by GLU would enhance hepatic responsiveness to GLU and facilitate the shift from anabolism to catabolism necessary to compensate to food deprivation.

ACKNOWLEDGMENTS We are grateful to Melissa Ehrman, Yung-Hsi Kao, and Jeff Kittilson for technical assistance. Animals were generously provided by the North Dakota Game and Fish Department and the U.S. Department of the Interior, Fish and Wildlife Service (Garrison National Hatchery). This research project was supported by grants from the National Science Foundation, U.S.A. (IBN 9723058), and the NDEPSCoR Science Bound program to M.A.S.

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