Cholecystokinin, neuropeptide Y and galanin modulate the release of pancreatic somatostatin-25 and somatostatin-14 in vitro.

The direct effects of cholecystokinin (CCK), neuropeptide Y (NPY) and galanin on somatostatin-14 (SS-14) and somatostatin-25 (SS-25), containing [Tyr7, Gly10]-SS-14 at its carboxy terminus and presumably derived from a different gene than that giving rise to SS-14, were evaluated on principal islets (Brockmann bodies) removed from rainbow trout, Oncorhynchus mykiss. The potential for peptidergic action was deduced from the extensive neural network innervating the Brockmann body and confirmed by immunohistochemistry. Immunopositive CCK-like staining was located around the periphery of principal islets as well as in centrally-located nerve tracts. Mammalian CCK-33 stimulated SS-25 release at a dose of 10(-11) M in the presence of 1, 3 and 10 mM glucose. CCK-33 inhibited the release of SS-14 at concentrations of 10(-11) and 10(-7) M only in the presence of high (10 mM) glucose. Mammalian NPY stimulated SS-25 release in a dose-related manner in the presence of low (1 mM) glucose; the efficacy of this effect decreased at higher glucose concentrations. SS-14 release was inhibited by NPY in the presence of 10 mM glucose. Mammalian galanin generally inhibited the secretion of SS-25, but stimulated the release of SS-14 at 10(-7) M in the presence of 3 mM glucose. These results suggest that CCK, NPY and galanin modulate the release of the somatostatins present within the trout pancreas. Furthermore, glucose may mediate the overall responsiveness of somatostatin-secreting cells to these peptides.

Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: an antisense study.

To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, alpha TC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 +/- 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 +/- 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

Lipid-stimulated somatostatin secretion in rainbow trout,Oncorhynchus mykiss.

Previous work has shown that somatostatins (SS) affect teleost lipid metabolism indirectly by inhibition of insulin (INS) and directly by stimulation of hepatic lipolysis. In the present study, rainbow trout (Oncorhynchus mykiss) were used to characterize further the lipid-SS relationship by evaluating how lipid, contributes to SS secretion bothin vivo andin vitro. In vivo hyperlipidemia was induced for up to 3 h by short-term (2 min) infusion of a triacylglycerol (TG)-rich lipid emulsion (20% Intralipid(®)). Plasma total lipid concentration increased 118 and 155% over control levels 1 h and 3 h, respectively, after infusion; much of this increase was due to elevated plasma fatty acids (FA), which increased 39 and 520%, respectively, over the same time-frame. The hyperlipidemic pattern was attended by a significant increase in the plasma concentration of SS. The specific effects of fatty acids were evaluated on isolated Brockmann bodies. Palmitic acid and oleic acid stimulated SS release 378 and 82%, respectively, over baseline levels. These results indicate that lipids, and in particular fatty acids, modulate SS secretion in rainbow trout.

Pancreatic somatostatin-14 and somatostatin-25 release in rainbow trout is stimulated by glucose and arginine.

Previous work suggested that teleost fish possess two somatostatin (SS) genes. Regulation of differential secretion of SS gene I (SS-25) and SS gene II products (SS-14) by nutrients was studied using rainbow trout. Hemi-islets were cultured in medium containing different concentrations of glucose and arginine. SS-14 and insulin were determined by radioimmunoassay; SS-25 was determined by a novel enzyme-linked immunosorbent assay. Glucose stimulated SS-25 release in a concentration-related manner, with 10 mM being the most effective concentration; at higher (25 mM) glucose, release decreased. Insulin levels in the medium also were found to be more elevated from islets incubated in 10 mM glucose than those incubated in 25 mM glucose. Maximum SS-14 release was observed at lower (1-5 mM) glucose concentrations. Arginine stimulated SS release so that maximum release of both SS forms occurred at the lowest glucose concentrations (1 mM). A concentration-related effect of arginine was observed on SS-25 release. The results of this study indicate that glucose and arginine regulate secretion of both pancreatic SS gene I and gene II products from rainbow trout islets, suggesting that two different pancreatic SS interact in nutrient homeostasis in this species.

Effects of insulin, glucagon, and somatostatin on the release of somatostatin-25 and somatostatin-14 from rainbow trout, Oncorhynchus mykiss, pancreatic islets in vitro.

Somatostatins are a diverse family of peptides known to modulate insulin and glucagon secretion as well as to stimulate glycogenolysis and lipolysis in salmonid fish. In this study, Brockmann bodies (bisected to yield hemi-islets) isolated from rainbow trout, Oncorhynchus mykiss, were used to study the effects of insulin, glucagon, and somatostatin at various concentrations of glucose (1, 5, and 10 mM) on pancreatic somatostatin release. The release of somatostatin-25, the most predominate form of somatostatin in salmonid pancreas, was stimulated by insulin in the presence of 1 and 5 mM glucose but not in the presence of 10 mM glucose, whereas glucagon stimulated somatostatin-25 release only in the presence of high (10 mM) glucose. Somatostatin-25 release also was stimulated by somatostatin-14. The secretion of somatostatin-14 was suppressed by insulin in the presence of 5 and 10 mM glucose and was stimulated by glucagon in the presence of high (10 mM) glucose. These results indicate that insulin, glucagon, and somatostatin-14 are regulators of somatostatin-14 and somatostatin-25 pancreatic release in rainbow trout and that these effects are modulated by glucose.

Processing of mouse proglucagon by recombinant prohormone convertase 1 and immunopurified prohormone convertase 2 in vitro.

The mouse tumor cell line alpha TC1-6 was used as a model system to examine the post-translational processing of proglucagon. Determination of the mouse preproglucagon cDNA sequence and comparison with the published sequences of rat and human preproglucagons revealed nucleic acid homologies of 89.1 and 84%, respectively, and amino acid homologies of 94 and 89.4%, respectively. Immunohistochemical analyses with antibodies directed against PC2 and glucagon colocalized both the enzyme and substrate within the same secretory granules. PC1 was also immunolocalized in secretory granules. Cells were metabolically labeled with [3H]tryptophan, and extracts were analyzed by reverse-phase high pressure liquid chromatography. Radioactive peptides with retention times identical to those of synthetic peptide standards were recovered and subjected to peptide mapping to verify their identities. To determine the potential role of PC1 and PC2 in proglucagon processing, 3H-labeled proglucagon was incubated in vitro with recombinant PC1 and/or immunopurified PC2. Both enzymes cleaved proglucagon to yield the major proglucagon fragment, glicentin, and oxyntomodulin, whereas only PC1 released glucagon-like peptide-I from the major proglucagon fragment. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. These results suggest a potential role for PC1 and/or PC2 in cleaving several of the normal products, excluding glucagon, from the mouse proglucagon precursor.

Differential effects of somatostatin-14 and somatostatin-25 on carbohydrate and lipid metabolism in rainbow trout Oncorhynchus mykiss.

Previous studies have indicated that teleost fish appear to have two somatostatin genes. In salmonid fish, it is purported that gene I encodes for somatostatin-14 (SS-14), while gene II encodes for somatostatin-25 (sSS-25). In the present study, the physiological effects of SS-14 and sSS-25 on carbohydrate and lipid metabolism in rainbow trout, Oncorhynchus mykiss, were evaluated by in vivo administration of hormone and measuring resulting levels of specific metabolites and hormones present within tissues and plasma. Somatostatin-14 administration caused hyperglycemia without affecting liver glycogen content and increased plasma fatty acid (FA) levels in association with enhanced activity of the lipid mobilizing enzyme, triacylglycerol lipase (TG lipase). Somatostatin-14 injection also resulted in reduced hepatic glucose-6-phosphate dehydrogenase activity, which may indicate a decrease in glucose channeling through the pentose phosphate shunt. In addition, SS-14 reduced plasma glucagon concentration, while having no effect on plasma insulin levels. Salmon SS-25 elevated plasma glucose levels in association with reduced glycogen content and resulted in increased plasma FA levels accompanied by increased hepatic TG lipase activity. Salmon SS-25 injection also resulted in a reduction in plasma glucagon and insulin levels. These results indicate that SS-14 and sSS-25 are important regulators of carbohydrate and lipid metabolism in rainbow trout and that modulation of metabolic activity by these peptides may be accomplished, in part, by alterations in insulin and glucagon levels circulating in the plasma.

Insulin suppression is associated with hypersomatostatinemia and hyperglucagonemia in glucose-injected rainbow trout.

Rainbow trout, Oncorhynchus mykiss, were used to evaluate the effects of carbohydrate loading on plasma levels of pancreatic hormones and associated changes in metabolic indexes in a carnivorous fish. Glucose (3,000 mg/dl, 10 microliters/g body wt) was injected intraperitoneally into fish (mean wt 54 +/- 5 g) that were killed 0.5-24 h after administration. Glucose injection resulted in hyperglycemia with maximum glucose levels of 306 +/- 13 mg/dl observed 60 min after injection. Glucose administration also resulted in hyperlipidemia. Plasma fatty acids increased twofold in glucose-injected animals. Alterations in plasma metabolites reflected changes in energy stores. Although total lipid concentration was unaffected by glucose injection, lipolytic enzyme activity in the liver was enhanced. Biosynthetic capacity, as indicated by NADPH production from glucose-6-phosphate dehydrogenase, was decreased by glucose injection. Liver glycogen content was reduced in glucose-injected animals 1 h after injection. Glucose injection was attended by increases in the plasma levels of gene II somatostatin-25 (predominant form of pancreatic somatostatin in salmonids) and of glucagon. Insulin levels were initially suppressed after glucose injection. These results indicate that metabolic adjustments caused by glucose administration can be related to the regulatory action of pancreatic hormones. Furthermore, these results suggest that the somatostatin-secreting cells of the trout are sensitive to glucose and that somatostatin-suppressed insulin secretion contributes to the glucose intolerance of trout.

Somatostatin-14 and somatostatin-25 stimulate glycogenolysis in rainbow trout, Oncorhynchus mykiss, liver incubated in vitro: a systemic role for somatostatins.

The effects of somatostatin-14 (SS-14) and salmon somatostatin-25 (sSS-25) on hepatic glycogenolysis were studied by incubating rainbow trout liver pieces in vitro. Glycogen content in untreated liver pieces cultured for 3 and 5 hr was 28.6 +/- 7.6 mg/g fresh wt. and 21.5 +/- 6.6 U, respectively. Treatment of liver pieces with either SS-14 or sSS-25 resulted in significant glycogen depletion; sSS-25 appeared more potent in this regard. Equimolar concentrations (10(-8) M) of SS-14 or sSS-25 reduced glycogen content to 10.6 +/- 1.6 and 2.6 +/- 2.2 U, respectively, in liver pieces incubated for 3 hr. Alterations in liver glycogen content were reflected in glucose release into medium. Basal release of glucose into culture medium over the course of a 3-hr incubation was 20 +/- 5.6 mumol/g dry wt. Both SS-14 and sSS-25 stimulated a rapid increase (500 and 600%, respectively) in glucose release during the first 0.5 hr of incubation. After 3 hr, SS-14 and sSS-25 stimulated glucose release over basal levels to 116 +/- 9.3 and 153 +/- 16 U, respectively. Both SS-14 and sSS-25 stimulated glucose release in a dose-dependent manner; ED50 for both peptides was ca. 5 X 10(-8) M. These results indicate that both SS-14 and sSS-25 directly mediate hepatic glycogenolysis in rainbow trout.

Radioimmunoassay for salmon pancreatic somatostatin-25.

A specific and sensitive radioimmunoassay (RIA) for the measurement of plasma levels of somatostatin-25 (SS-25) in salmon was developed using antisera raised against coho salmon (Oncorhynchus kisutch) SS-25. Somatostatin-25 was iodinated by the chloramine-T method and repurified on Sephadex G-25. The RIA was performed using a double antibody (goat anti-rabbit gammaglobulin as second antibody) method under disequilibrium conditions. Plasma from several salmonids (coho, chinook, rainbow trout, brook trout, arctic char, lake trout, and whitefish) as well as plasma from some nonsalmonids (sucker, bluegill) cross-reacted with the antisera; serial dilutions of plasma from rainbow trout, brook trout, chinook salmon, and coho salmon were parallel to the SS-25 standard curve. Plasma from catfish showed negligible cross-reactivity. None of the mammalian somatostatins (somatostatin-14, somatostatin-28). U II, or other pancreatic hormones (insulin, glucagon) tested showed significant cross-reactivity with the antibody in the assay system. The lowest detectable level of SS-25 was 5 pg/tube; especially reproducible results were obtained in the range of 0.15-1.20 ng/ml, which appears to be the normal range of SS-25 circulating in the plasma of salmonids. Intra- and interassay coefficients of variation were 5.7 and 12.6%, respectively. Injection of glucose into chinook salmon resulted in an elevation of plasma SS-25 titers within 30 min and was coincident with hyperglycemia.