Nutritional state modulates hormone-mediated hepatic glycogenolysis in chinook salmon (Oncorhynchus tshawytscha)
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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 254~202-206 (1990)
Nutritional State Modulates Hormone-Mediated Hepatic Glycogenolysis in Chinook Salmon (Oncorhynchus tshawytscha)
MICHAEL KLEE, CARMEN EILERTSON, AND MARK A. SHERIDAN Department of Zoology, North Dakota State University, Fargo, North Dakota 581 05
ABSTRACT Juvenile chinook salmon were used to investigate the interaction of nutritional state and hormones (glucagon, epinephrine) on hepatic glycogenolysis. Experiments were con- ducted in vitro on liver removed from animals of varying nutritional states: fed 1 week, fasted 1 week, fed 3 weeks, fasted 3 weeks, and fasted 1 weeklrefed 2 weeks. Basal glycogen breakdown in liver isolated from continuously fed (1 week and 3 weeks) fish proceeded at insignificant levels, whereas glycogen breakdown in liver isolated from fasted animals progressed rapidly during the course of incubation. However, no difference in the pattern of glycogen breakdown between liver isolated from animals fasted for 1 week and those fasted for 3 weeks was observed. The presence of glucose in the incubation medium retarded glycogen breakdown in the livers from fasted fish. Both epinephrine and glucagon stimulated glucose release from liver isolated from fish fed continuously and from fish fasted 1 week; epinephrine appeared more potent in this regard than glucagon. Epinephrine also stimulated glucose release from liver isolated from fish fasted 3 weeks, whereas glucagon was without significant glycogenolytic effect on this tissue. Insulin inhibited epinephrine- and glucagon-stimuluated glucose release. These results suggest that glucagon plays a role in modulating nutrition-associated adjustments in metabolism during the early stages of fasting and that, with extended fasting, the liver loses its sensitivity to glucagon with regard to glycogenolysis.
Nutritional state has been found to have pro- found effects on the metabolism of salmonids. During short periods of food deprivation (1 week), coho salmon (Oncorhynchus kisutch) display a marked hyperglycemia supported by glycogenoly- sis (Sheridan and Plisetskaya, '88). After longer periods of fasting (3 weeks), glycogen mobiliza- tion ceases, and glycemic levels are normal, sup- ported in part by increased gluconeogenesis (Sheridan and Plisetskaya, '88; Sheridan, Pliset- skaya, and Mommsen, unpublished). During pe- riods of food deprivation, plasma concentrations of insulin as well as of glucagon and of glucagon- like peptide (GLP) are reduced in coho salmon (Sheridan and Plisetskaya, '88) and in trout (0. mykiss, formerly Salmo gairdneri) (Moon et al., '89). During such periods, it is notable, however, that glucagon and GLP are depressed to a lesser extent than insulin such that they are relatively more abundant in the plasma of fasting fish (Sheridan and Plisetskaya, '88; Moon et al., '89).
In this study we used chinook salmon (0. tshawytscha) to investigate further the role of glucagon in regulating hepatic glycogenolysis during food deprivation. We asked two questions:
0 1990 WILEY-LISS, INC.
1) what is the effect of nutritional state on basal glycogenolysis in liver cultured in vitro? 2) What is the effect of glucagon on glycogenolysis in liver cultured in vitro isolated from animals of varying nutritional states?
MATERIALS AND METHODS Experimental animals
Juvenile chinook salmon (0. tshawytscha) of both sexes, age 1 + year, were obtained from the Garrison National Hatchery near Riverdale, North Dakota. Fish were maintained in well- aerated fresh water (14C) under 12L: 12D photo- period and fed ad libitum with Glenco Mills (Glenco, MN) trout chow (ca. 41% protein, lo%, fat, 4% fiber, 35% moisture).
For experiments, animals were divided into five groups: fed 1 week, fasted 1 week, fed 3 weeks, fasted 3 weeks, and fasted 1 weeklrefed 2 weeks. During the course of experiments, fish in the fed
Received July 17, 1989; revision accepted November 7, 1989. A part of this work was presented a t the Annual Meeting of the
American Society of Zoologists, Dec. 26-30, 1989, Boston, MA (Am. 2001. 28:47A).
NUTRITION-HORMONE INTERACTIONS IN SALMON LIVER 203
groups received the same diet and ration on which they were maintained; food was withdrawn 24 hr prior to samplings. Animals were killed by a sharp blow to the head, and the liver was removed aseptically and prepared for organ culture.
Organ culture and incubation conditions Livers (generally from 12 fish) were rinsed thor-
oughly with cold (4C) glucose-free Hanks-MEM solution (containing 4 mM NaHC03 and 10 mM HEPES, pH 7.6; Moon et al., '85), placed in an iced sterile petri dish and diced into approximately 1 mm cubes and randomized. Liver pieces were preincubated (150 rpm) in gassed (99.5% 02/0.5% C02) glucose-free Hanks-MEM for 15 min at 14C. Liver pieces were collected by low-speed centrifugation (270g for 5 min at 14C) and washed twice by resuspension-centrifugation.
For experiments, liver pieces were transferred aseptically to 24-well culture plates (four to six pieces per week, ca. 15 mg fresh weight), each well containing 1.0 ml of glucose-free Hanks- MEM or an equivalent volume of Hanks-MEM containing 5.6 mM glucose. In hormone experi- ments, test solutions of epinephrine bitartrate, mammalian (bovineiporcine) glucagon and bo- vine insulin (Sigma, St. Louis, MO), made up in glucose-free Hanks-MEM, were administered to culture wells. In combination experiments, insu- lin was added 5 min prior to other hormones. Con- trol cultures received an equivalent volume of in- cubation medium. Cultures were incubated (100 rpm) in darkness at 14C. In glucose-release stud- ies a single aliquot of the incubation medium was removed at a specified time for glucose analysis. After incubation, liver pieces were either dried to constant weight (glucose-release studies) or fro- zen on dry ice and stored at -90C until subse- quent glycogen assay. Experiments were repeated two or three times; results from replicate groups were combined (after appropriate statistical evaluation).
Biochemical analyses Glucose was determined by o-toluidine col-
orimetric assay (Huvarinen and Nikkila, '62). Liver glycogen was determined by the method of Murat and Serfaty ('74), and results were pre- sented in glucosyl units after subtraction of free glucose.
Statistics Statistical differences were estimated by one-
way analysis of variance. Multiple comparisons
among means were made by the Student- Newman-Keuls test; a was set at 0.05.
RESULTS Effects of nutritional state on hepatic
glycogenolgsis Basal glycogenolysis was examined in liver ob-
tained from animals in each of the various nutri- tional groups; data are presented as percent change from initial since starting glycogen con- tent varied among groups (Fig. 1). Initial glyco- gen levels in fed groups averaged 5.3 mg glucose/g fresh weight and 1.8 mg/g in fasted groups; there was no significant difference in the glycogen con- tent of the two fasted groups. Initial glycogen con- tent in the fastedirefed groups averaged 2.8 mg glucose/g fresh weight.
Basal glycogen breakdown proceeded at only slight insignificant levels in liver isolated from fed animals cultured in glucose-free medium (Fig. 1). There is a tendency of medium glucose to re- tard basal glycogenolysis. Liver isolated from either of the fasted groups displayed significant glycogen depletion when cultured in glucose-free medium. When glucose was added to the medium, however, basal glycogen depletion was significantly retarded (Fig. 1). Liver isolated from fish that were fasted then refed, although recover- ing from the fast with regard to initial glycogen content, behaved in culture in much the same manner as liver isolated from fasted fish.
Enects of hormones on hepatic glycogenolysis
Both epinephrine and glucagon significantly stimulated glucose release from livers isolated from continuously fed fish. Epinephrine appeared more potent in stimulating glucose release than glucagon (Fig. 2). Insulin inhibited both epineph- rine- and glucagon-stimulated glucose release from liver isolated from continuously fed animals.
Basal glucose release from liver isolated from fasted fish (either 1 or 3 weeks) was higher than that from liver isolated from fed animals, an ob- servation that is consistent with our foregoing data on basal glycogenolysis. In liver isolated from animals fasted for 1 week, epinephrine and glucagon exerted glucose mobilizing effects simi- lar to those noted in liver from continuously fed fish; insulin inhibited these glycogenolytic ac- tions. In liver isolated from fish fasted for 3 weeks, epinephrine stimulated glucose release and glucagon was without significant effect (Fig.
204 KLEE ET AL.
FAST 3Wks 4 150 r a, u
0 ' I T \ T FED 3Wks FAST,/REFED
t=Ohrs t=3hrs t=5hrs
FAST 1Wk Y
t=Ohrs t=3hrs t=5hrs
t=Ohrs t=3hrs t=5hrs t
Fig. 1. Relative glycogen content in chinook salmon liver cultured in vitro from fish of varying nutritional states. Liver pieces were cultured in glucose-free medium (0) or in me-
2). Insulin inhibited epinephrine-stimulated glu- cose release from liver isolated from fish fasted for three weeks.
DISCUSSION The results of the present study indicate that
nutritional state influences both basal and hor- mone-mediated hepatic glycogenolysis in chinook salmon. Glycogenolysis in chinook salmon pro- ceeds from the activation of glycogen phosphoryl- ase and results in glucose release (Sheridan and Muir, '88). During a short-term (1 week) fast, coho salmon have been observed to undergo hepatic glycogen depletion; during longer term food depri- vation, however, glycogen mobilization ceases (Sheridan and Plisetskaya, '88)