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sup- ported in part by increased gluconeogenesis (Sheridan and Plisetskaya, '88; Sheridan, Pliset- skaya, and Mommsen, unpublished).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 (14°C) 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 (4°C) 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 14°C. Liver pieces were collected by low-speed centrifugation (270g for 5 min at 14°C) 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 14°C. 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 -90°C 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

t=Ohrs

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). The present in vitro results, slight glycogenolysis in liver isolated from fed animals and pronounced gly- cogenolysis in fasted animals with no apparent difference in the degree of glycogenolysis between animals fasted 1 week and animals fasted 3 weeks, are consistent with these previous obser- vations. Addition of glucose to the culture me- dium inhibits glycogenolysis in livers isolated from fasted fish and appears to mimic the fed state.

Modulation of glycogenolysis by epinephrine

FED 1Wk

0 Glucose-medium

I Glucose+medium

t=3hrs t=5hrs

dium containing 5.6 mM glucose (W). Liver pieces were ana- lyzed for glycogen content after 0, 3, and 5 hr of culture. Presented as means 4 SEM (n = 18-24). *P < 0.05 vs. to.

(Sheridan and Muir, '88; Janssens and Lowrey, '87; Ottolenghi et al., '86; Vernier and Sire, '78; Birnbaum et al., '76) and by glucagon (Plisets- kaya et al., '89; Ottolenghi et al., '88) has been reported in a number of fish species. In addition, the glycogenetic action of insulin in fish is also well known (Plisetskaya et al., '84). In the present study we present for the first time that the effect of hormones on hepatic glycogenolysis in fish is modulated by nutritional state. Glucagon stimu- lates glycogenolysis in liver isolated from fed and from short-term fasted fish; it fails to stimulate glycogenolysis in liver from longer-term fasted fish. On the contrary, epinephrine maintains its glycogenolytic action regardless of nutritional state. Theses results help to explain several in vivo observations in fasting fish. During early stages of fasting glycogen mobilization occurs and results in hyperglycemia; these effects are cor- related with proportionally more glucagon in the plasma of fasting fish than insulin (Sheridan and Plisetskaya, '88; Moon et al., '89). The present data support a role for glucagon modulating nu- trition-associated adjustments in metabolism of short-term fasting salmon. In longer-term fasting salmon, glycogen mobilization ceases in face of chronically (proportional to insulin) elevated

NUTRITION-HORMONE INTERACTIONS IN SALMON LIVER 205

40

0-0 CONTROL A-A EPINEPHRINE (EP) m-B GLUCAGON (GLU)

FASTED 3 WEEKS

/+

A-A INSULIN + EP 0-0 INSULIN + GLU I

6o t

t FASTED 1 WEEK

Oh' - 60 T

TIME (hr) Fig. 2. Effects of epinephrine (2 x M) and glucagon

(2 x MI, in the presence and absence of insulin (2 x M) on glucose release from chinook salmon liver in-

cubated in vitro isolated from fish of varying nutritional states. Presented as means 2 SEM (n = 18-36).

glucagon levels. The present data suggest that the cessation of hepatic glycogenolysis is due to reduced glucagon sensitivity. A possible explana- tion for the reduced sensitivity is decreased num- bers of glucagon receptors, although we have no evidence to support this contention. As a result of reduced glycogenolysis in longer-term fasted ani- mals, salmon are able to maintain some small pool of glycogen-a strategy that we have re- ferred to as partial conservation (Sheridan and Plisetskaya, '88). The significance of this strategy may be related to life history (Love, '70). The ob- servation that epinephrine maintains its gly- cogenolytic action regardless of nutritional state points out the difference between a chronic and an acute effector and may also have some bearing on the apparent difference in potency. (Differences in potency between epinephrine and glucagon have

also been observed by Ottolenghi et al., '88). Glucagon is a chronic effector in fasting salmon and is accommodated by the animal with pro- longed fasting. Epinephrine is an acute effector and is generally released during periods of stress (Nikano and Tomlinson, '67). In this manner, a fasting animal is still able to draw upon its rap- idly mobilizable, albeit reduced, energy stores-a feature that may have great adaptive significance to the salmon, especially during migratory pe- riods.

ACKNOWLEDGMENTS We are indebted to the North Dakota Depart-

ment of Game and Fish and to Tom Pruitt and his staff at the Garrison National Hatchery for pro- viding experimental animals. We thank Kim Michelsen for her valuable assistance. This work is a result of research supported by the National Science Foundation (RII 8610675, BBS 89704115, and DCB 8901380) and by the U.S. Department of Education, project 2-8-01004.

LITERATURE CITED Birnbaum, M.J., J . Schultz, and J.N. Fain (1976) Hormone-

stimulated glycogenolysis in isolated goldfish hepatocytes. Am. J . Physiol., 231:191-197.

Huvarinen, A,, and E.A. Nikkila (1962) Specific determina- tion of blood glucose with o-toluidine. Clin. Chim. Acta., 7: 140- 143.

Janssens P.A., and P. Lowrey (1987) Hormonal regulation of hepatic glycogenolysis in the carp, Cyprinus carpio. Am. J. Physiol., 252:R653-660.

Love, R.M. (1970) The Chemical Biology of Fishes. Academic Press, New York, p. 547.

Moon, T.W., G.D. Foster, and E.M. Plisetskaya (1989) Changes in peptide hormones and liver enzymes in the rain- bow trout deprived of food for 6 weeks. Can. J . Zool., 679189-2193.

Moon, T.W., P . J . Walsh, and T.P. Mommsen (1985) Fish hepatocytes: A model metabolic system. Can. J . Fish. Aquat. Sci., 42:1772-1782.

Murat, J.C., and A. Serfaty (1974) Simple enzymatic determi- nation of polysaccharide content of animal tissue. Clin. Chem., 20:1576-1577.

Nakano, T., and N. Tomlinson (1967) Catecholamine and car- bohydrate concentrations in rainbow trout (Salmon gaird- nerd in relation to physical disturbance. J. Fish. Res. Bd. Canada, 24:1701- 1715.

Ottolenghi, C., A.C. Puviani, A. Baruffaldi, M.E. Gavioli, and L. Brighenti (1988) Glucagon control of glycogenolysis in catfish tissues. Comp. Biochem. Physiol., 90B:285-290.

Ottolenghi, C., A.C. Puviani, M.E. Gavioli, and L. Brighenti (1986) Epinephrine effect on glycogen phosphorylase activ- ity in catfish liver and muscles. Gen. Comp. Endocrinol., 61 :469-475.

Plisetskaya, E., S. Bhattacharya, W.W. Dickhoff and A. Gorb- man (1984) The effect of insulin on amino acid metabolism

206 KLEE ET AL.

and glycogen content in isolated liver cells of juvenile coho salmon (Oncorhynchus kisutch.) Comp. Biochem. Physiol., 78A:773-778.

Plisetskaya, E.M., C. Ottolenghi, M.A. Sheridan, T. P. Mommsen, and A. Gorbman (1989) Metabolic effects of salmon glucagon and glucagon-like peptide in coho and chinook salmon. Gen. Comp. Endocrinol., 731205-216.

Sheridan, M.A., and N.A. Muir (1988) Effects of epinephrine and norepinephrine on glucose release from chinook salmon

(Oncorhynchus tshawytscha) liver incubated in vitro. J. Exp. Zool., 2481155159.

Sheridan, M.A., and E.M. Plisetskaya (1988) Effects of nutri- tional state on lipid and carbohydrate metabolism of coho salmon. Am. Zool., 28:56A (abstract).

Vernier, J.M., and M.F. Sire (1978) Etude in vitro de la gly- cogene phosphorylase hepatique de la truite arc-en ciel- Son control par le glucose, les corticoides, l’adrenaline et la glucagon. Gen. Comp. Endocrinol., 34:360-369.