Action of glucagon and glucagon-like peptide on glycogen metabolism of trout isolated hepatocytes

Action of glucagon and glucagon-like peptide on glycogen metabolism of trout isolated hepatocytes

Comp. Biochem. PhysioL Vol,96B, No. 2, pp. 387-391, 1990 Printed in Great Britain 0305-0491/90$3.00+ 0.00 PergamonPress plc ACTION OF GLUCAGON A N D...

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Comp. Biochem. PhysioL Vol,96B, No. 2, pp. 387-391, 1990 Printed in Great Britain

0305-0491/90$3.00+ 0.00 PergamonPress plc


Abstract--1. The incubation of isolated trout hepatocytes in the presence of salmon glucagon, mammalian glucagon, salmon glucagon-like peptide (GLP) induced an increase in the content of cyclic adenosine 3',5'-monophosphate (cAMP), in the glycogen phosphorylase a activity, in the glucose release, and a decrease in the glycogen level in these cells. 2. GLP showed a greater potency than either glucagon, and the same activity as equimolar doses of epinephrine. 3. Results differ from those obtained in catfish hepatocytes, in which GLP had a very low activity compared to glucagon and epinephrine.

INTRODUCTION The physiological pattern of carbohydrate mobilization in some poikilothermic animals, such as fish, is different from that of mammals. In fish, glycogen is present in all tissues, and its level can vary among fish species, even in the same environmental conditions (Dean and Goodnight, 1964; Plisetskaya, 1968; Valtonen, 1974). In some fish, liver glycogen levels are 3-, 4-fold or higher than in mammals, and can also remain high after prolonged periods of starvation (Dave et al., 1975; French et al., 1981; Ottolenghi et al., 1981; Moon, 1983). Liver glycogenolysis is stimulated by a range of hormones, mainly glucagon and epinephrine. In teleost fish, as in mammals, glucagon elicits hyperglycemia, but its effect on liver glycogen varies according to fish species, as well as to physiological and environmental conditions (Plisetskaya, 1975; Moon, 1988; Ottolenghi et al., 1988). Glucagon-like peptide (GLP), a new member of the glucagon family, has been recently isolated from both mammals and fish (Andrews and Ronner, 1985; Plisetskaya et al., 1986; Pollock et al., 1988). In mammals, GLP stimulates insulin secretion from perfused pancreas (Holst et aL, 1987; Mojsov et al., 1987), and possibly also acts as neurotransmitter (Shimizu et al., 1987). In fish (salmonids and anguillids), GLP participates in the regulation of glucose production by stimulating either gluconeogenesis or glycogenolysis (Mommsen et al., 1987; Plisetskaya et al., 1988; Ottolenghi et al., 1988; Mommsen and Moon, 1989). We have studied the effects of peptides of the glucagon family on glycogen metabolism in catfish *A part of this work has been presented at the 14th Conference of the European Comparative Endocrinologists, Salzburg, 1988. ,Author to whom correspondence should be addressed.

hepatocytes (Ottolenghi et al., 1989), and are now extending this research to hepatocytes isolated from trout since trout has a lower liver glycogen level (French et aL, 1981) than catfish (Ottolenghi et al., 1981), therefore its metabolic pattern may possibly be different. The effects of epinephrine were also tested, since we previously observed (Ottolenghi et al., 1989) that in catfish, contrary to mammals, epinephrine was more effective than glucagon in stimulating glycogenolysis. MATERIALS AND METHODS Materials Reagents for teleost-Ringer solution and buffers, theophylline and 2-mercapto-ethanol were purchased from Merck (Darmstadt, FRG). All other reagents for cell isolation and assays, epinephrine and mammalian glucagon were from Sigma Chemical Company (St Louis, MO). According to Sigma, the purity of mammalian glucagon, extracted from a mixture of bovine and porcine pancreas, was 95% or higher. Salmon giucagon and GLP isolated from coho salmon, Oncorhyncus kisutch (Plisetskaya et al., 1986) were a gift from Dr Plisetskaya. Binding protein for cAMP analysis was prepared from beef adrenals according to Brown et al. (1971). 3H-cAMP was a product of Amersham International (Amersham, UK). Liquid scintillation solution, Atomlight, was purchased from New England Nuclear (Boston, MA). A kit from Boehringer (Mannheim, FRG) was used for glucose determination. Fish were fed a standard diet from Veronesi (Verona, Italy). Animals Experiments were carried out from December to March. Rainbow trout (Salmo gairdneri), weighing approximately 300--400g, were purchased from a local dealer, who bred fish in basins at environmental temperatures (2-10°C). Fish were fed ad libitum with a standard trout diet until the experiment, or fasted 4-6 days. Some trout were killed at the fish farm, and the liver was cut out immediately and freeze-clamped in liquid nitrogen. The transportation of fish to the laboratory took about 30 min. Fish were placed in




groups of three to five in tanks containing 2001 of well aerated, dechlorinated and continuously depurated tap water, at a storehouse temperature of about 15°C. Fish were anaesthetized by immersion in water containing chloretone (1,1,1-trichlor-2-methyl-2-propanol) (60ml of a saturated soln/1). Hepatocyte preparation and incubation

The hepatocyte preparation was performed as reported elsewhere (Ottolenghi et al., 1984). Hepatocytes (about 100 mg of wet weight, corresponding to about 2 × 106 cells) were suspended in 2.5 ml of teleost-Ringer solution, pH 7.4, 10 pl of an appropriate concentration of glucagon (mammalian or salmon), salmon GLP, or epinephrine were added to the incubation medium to give a final concentration varying from 3 × I0 -I° to 3 × 10-7 M. The hormone addition practically did not change the ionic content of the final medium. The incubation was carried out for 1 hr at 25°C in Erlenmeyer flasks, gassed with a O2/CO 2, 99/1% (v/v) mixture, in a shaking bath. At the end of the incubation the samples were centrifuged (10 min, 50 g, 4°C). Aliquots were taken from supernatants for the glucose assay, then the hepatocyte pellets were homogenized in the assay buffer, and used for the determination of glycogen phosphorylase a activity and glycogen level. For testing cAMP content, hepatocytes were incubated as reported by Brighenti et al. (1987a). Analyses

cAMP concentration in cells was determined by the competitive protein-binding assay according to Brown et al. (1971), with the only modification that the standards were treated with exactly the same procedure applied to all other samples. Glycogen phosphorylase a activity was measured as described by Umminger and Benziger (1975). Glycogen content in cells was assayed as glucose after enzymatic hydrolysis with amylo-l,6-glucosidase (Carr and Neff, 1984). Glucose was determined by the glucose oxidase and peroxidase method (Bergmeyer and Bernt, 1963). Protein content in cells and homogenates was measured according to Lowry et aL (195l). Analysis o f data

Values are expressed as the mean _.+SEM. Statistical analysis was carried out using the paired and unpaired Student's t-tests. RESULTS

The glycogen content in total liver and in isolated hepatocytes varied according to feeding and stress conditions, such as the handling and transportation of fish from farm to the laboratory, and the change of environmental temperature (see Methods) (Table 1). In fed trout there was a significant difference between the glycogen level present in liver drawn from fish in the farm just after being caught and that in liver drawn from fish in the laboratory immediately after their arrival, but this did not occur in starved

trout. The preparation of hepatocytes from both starved and fed fish resulted in a drastic lowering of the glycogen level. The effects of glucagon-family peptides on cAMP content, on glycogen phosphorylase a activity in hepatocytes, and on the glucose released from cells, are shown in Table 2. Salmon GLP increased cAMP content in trout liver cells, starting at the hormone concentration of 3 × 10 -8 M; mammalian and salmon glucagons had no significant effects. Mammalian and salmon glucagons also had no effect on glycogen phosphorylase a activity at low concentrations (3 x 10 10, 3 × 10 -gM). Low effect was shown by mammalian and salmon glucagon at 3 × 10 -8 M, while at this concentration the enzyme was very significantly increased by salmon GLP. At 3 × 10-7M concentration all hormones showed strong effects, G L P being significantly the most potent. At 3 x 10-7M concentration all peptides induced a significant glucose release, but GLP was only effective at a concentration of 3 x 10 -8 M. The effects of glucagons and G L P on the glycogen level of isolated hepatocytes from starved fish is presented in Table 3. In spite of a very low starting glycogen level, G L P was again the most effective in enhancing glycogen breakdown. The comparative effect of equimolar concentrations (3 x 10 -s M) of salmon glucagon, salmon G L P and epinephrine on phopshorylase a activity and on glucose release is reported in Fig, 1, Like salmon GLP, epinephrine was significantly more effective than salmon glucagon in increasing phosphorylase activity and glucose release. The magnitude of the increases induced by epinephrine and G L P were not significantly different. DISCUSSION The metabolic responses of fish to different nutritional conditions and to starvation have been extensively studied (Larsson and Lewander, 1973; Nagai and Ikeda, 1973; Ince and Thorpe, 1976; Cowey et al., 1977; Renaud and Moon, 1980; French et al., 1981). Sometimes, fish in the same family present a different pattern in the utilization of glycogen stores (Inui and Oshima, 1966; Butler, 1968; Mayerle and Butler, 1971; Valtonen, 1974; Dave et a1.,1975). As regards trout, decreases of 50--60% in liver glycogen level of rainbow trout were observed after prolonged periods of starvation (2-5 months) and exercise (French et at., 1981; Morata et al., 1982a). In our experiments a faster and greater mobilization of liver glycogen took place, since a decrease of about 85% in the polysaccharide level already occurred

Table 1. Glycogenlevel in rainbow trout liver and isolated hepatocytes Liver Drawn in Farm Laboratory Hepatocytes Glycogen (mg/g) Fed 28.61 +3.67 7.41 +2.25* 3.17+ 1.23 (4) (8) (5) Starved 1.52 ± 0.45§ 1.14± 0.25t 0.20 ± 0.04~ (4)



Mean values+ SEM of (n) experiments. Levels of significance(unpaired Student's t-test): *P < 0.01 as compared to values of livers drawn at the farm; tP <0.1, :~P <0.05, §P <0.01 as compared to values of fed trouts.

Glucagons on trout hepatocyte glycogen metabolism


Table 2. Effects of mammalian and salmon glucagon, and salmon glucagon-like peptid¢ (GLP), on cAMP level, glycogen phoshorylase a activity, and glucose release in isolated trout bepatocytes HolTnon¢

Concentration (M) 0 3 x 10 10 3 x 10-9 3 X l0 -8 3 × 10-7 0 3x 3× 3x 3x

l0 -m 10-9 l0 8 l0 7

0 3x 3x 3x 3x

10-,0 l0 -9 l0 -s 10-7

Mammalian* glucagon

Salmon glucagon

Salmon GLP

cAMP level (pmol/mg prot) 12.2 -+ 0.6 14.9 ± 0.5 13.6±0.9 13.2 ± 0.7 15.4-+ 1.0

14.0 ± 0.6 14.3 ± 0.5 14.4± 1.0 13.4_+ I.l 13.6 ± 0.7 15.0-+ 0.8t§ 14.6± 1.1 19.7_+ 0.8~§¶[ Glycogen phosphorylase a activity (#mol Pl liberated min -1 g cells-I ) 1.70 ± 0.21 1.95 _+0.29 1.95 ± 0.25 2.25 + 0.19 1.95 ± 0.39 1.82 ± 0.25 1.95 ± 0.20 2.55 _+0.48 2.34 _+0.33"t 5.45 -+ 0.55~;I]** 4.30_+ 1.37"[" 4.65_+ 0.95:~ 6.45_+0.94511** Glucose released (mg/g cells) 0.44+0.11 0.52 _-4-0.24 0.70 + 0.36 0.75 + 0.33 0.60 + 0.24 0.78 ___0.31 0.85 _+0.35 0.84 ± 0.34 0.70 ± 0.80 1.42 ± 0.40:~§¶] 2.58 _+0.792 3.42 ± 1.54:~ 3.48 ± 1.70:[:

*Porcine/bovine. Mean values ± SEM of five experiments. Levels of significance (paired Students's t-test): t P < 0.05 and ~P < 0.01 as compared to control values; §P < 0.05 and [IP < 0.01 as compared to samples treated with salmon glucagon; ¶lP < 0.05 and **P < 0.01 as compared to samples treated with mammalian glucagon. after 4--6 days o f fasting. T h e difference m a y be a s c r i b e d to t h e s e a s o n o r to different e x p e r i m e n t a l c o n d i t i o n s . T h e stress o f h a n d l i n g a n d t r a n s p o r t a t i o n r e d u c e d g l y c o g e n c o n t e n t in fed t r o u t to a b o u t a q u a r t e r o f its original level, w h e r e a s it d i d n o t influence t h e a l r e a d y low g l y c o g e n level in s t a r v e d fish. This result agrees with t h o s e o f M o r a t a et al. (1982b), w h o r e p o r t e d t h a t u n d e r stress c o n d i t i o n s t h e d e c r e a s e o f g l y c o g e n o c c u r s d u r i n g t h e first stages o f a n e m e r g e n c y . R e c e n t l y M o o n et al. (1988) o b s e r v e d t h a t a significant h a n d l i n g - i n d u c e d g l y c o g e n d e p l e t i o n in t r o u t was p r e v e n t e d by a p r i o r fla d r e n e r g i c b l o c k a d e with p r o p r a n o l o l . T h e s e findings i n d i c a t e that h a n d l i n g stress c a u s e d a release o f catecholamines, which induced the activation of glycogen phosphorylase, and the breakdown of g l y c o g e n as also f o u n d by M o r a t a et aL (1982b). A similar effect o f p r o p r a n o i o l in b l o c k i n g catec h o l a m i n e a c t i o n o n glycogenolysis was also f o u n d by us in isolated catfish liver cells (Brighenti et al., 1987b). I n the p r e s e n t e x p e r i m e n t s the d r a m a t i c effect o f e p i n e p h r i n e p o s s i b l y indicates a high sensitivity o f t r o u t liver cells to stress c o n d i t i o n s . T h e p r o c e d u r e for h e p a t o c y t e p r e p a r a t i o n f u r t h e r d e c r e a s e d glycogen c o n t e n t , as also o b s e r v e d in t r o u t b y M o o n et al. (1988), a n d in catfish by us ( c o m p a r e basal g l y c o g e n levels in O t t o l e n g h i et al., 1981, 1989). Table 3. Effects of mammalian and salmon glucagon, and glucagonlike peptide (GLP), on the glycogen content in isolated hepatocytes from starved rainbow trout Glycogen Control 0' Control 60' Mammalian glucagon 3 x l0 -s M Salmon glucagon 3 x l0 -~ M Salmon GLP 3 x l0 -s M Mean values of two experiments.

(mg/g cells) 0.17 0.1 l 0.033 0.036 0.018

T h e glucose released by h e p a t o c y t e s into t h e m e d i u m m a y c o m e f r o m t w o different sources: glycogenolysis f r o m e n d o g e n o u s glycogen, a n d de novo synthesis f r o m g l u c o n e o g e n i c p r e c u r s o r s . T h e shift f r o m the g l u c o n e o g e n i c to t h e glycogenolytic process, o r vice versa, m a y d e p e n d o n different

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Fig. 1. Comparative effects of salmon glucagon, salmon glucagon-like peptide (GLP) and epinephrine on (A) glycogen phosphorylase a activity and on (B) glucose release in isolated trout hepatocytes. Hormones ( G L U = s a l m o n glucagon, GLP = salmon glucagon-like peptide, EPIN -- epinephrine) were added to a final concentration o f 3 x 10 -s M. Values are the mean 4- SEM of the per cent variation with respect to control values: 1.71 :i: 0.28 #moles Pi liberated min -j g cells -~, and 0.33 _ 0.15 mg/g cells for glycogen phosphorylase a activity (A) and glucose release (B), respectively. Levels of significance (paired Student's t-test): O, P < 0.01 as compared to control; X, P < 0.01 as compared to values of samples treated with salmon glucagon.



conditions: species and strain of fish, sex and reproductive cycle, season and environmental temperature, nutritional and stress state. It has been suggested (Mommsen, 1986) that the level of glycogenolysis is influenced by the initial glycogen content, which is actually very low in our conditions. However, since no gluconeogenetic precursors were added into the incubation medium, glucose had to originate either from endogenous precursors or from glycogen, even if this polysaccharide was present in very small amounts. The high stimulation of phosphorylase activity indicates that it is mainly the glycogenolytic process that is involved. The amount of glucose released from trout liver cells was low compared to that released in the same conditions from catfish hepatocytes (Ottolenghi et al., 1989), in which high levels of glycogen are present. Concentrations of glucagons and G L P lower than 3 x 10 a M were shown to have no effect on c A M P level, phosphorylase a activity, glycogen content and glucose release in isolated trout liver cells. By increasing the concentrations of the hormones their effects become evident. The higher hormone concentrations in the media (3 x 10-8-3 x 10-TM) used in our tests differed greatly from the physiological ones found in plasma by Gutierrez et al. (1986) (0.2-1.1 x 10 l0 M), and M o o n et al. (1989) (approximately 2.2 x 10 -~° M). However, it should be taken into account that liver cells (mainly the periportal cells) are exposed to much higher levels of pancreatic hormones than all other target cells (Plisetskaya and Sullivan, 1989). The same high concentrations were used by Plisetskaya et al. (1989) in experiments on salmon liver slices. In catfish (Ottolenghi et al., 1989) and in salmon (Plisetskaya et al., 1989) mammalian glucagon was the least active peptide. In the present experiments on trout cells, salmon and mammalian glucagons had similar effects, and G L P showed a greater effect than either glucagon. As in catfish (Ottolenghi et al., 1989), in trout epinephrine had a greater effect than salmon glucagon. This fact could be ascribed to a lower sensitivity of liver cells to the glucagon hormone family in winter, but we have seen that G L P had the same effect as epinephrine. To explain this point further studies are necessary especially at the receptor level. At present, we are not able to explain why catfish and trout hepatocytes respond to a different extent to glucagons and GLP, even if these hormones have many structural similarities. In trout hepatocytes, G L P could possibly bind to specific G L P receptors, and, given the simultaneous increase in c A M P levels, the metabolic pathway would seem to occur through the activation of the adenylate cyclase system via the transducer G protein (Gilman, 1987). Acknowledgements--Dr E. M. Plisetskaya is thanked for her generous supply of salmon glucagon and GLP. This work was supported by a grant for Scientific Research from Ministero della Pubblica lstruzione, Rome, and by Grant No. 0926/87 from NATO, REFERENCES

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