Metabolic effects of cortisol in the European eel, Anguilla anguilla (L.)

Camp. Biochvm Physrol.. Vol. 63A. pp 339 to 344 0 Pergamon Press Ltd 1979 Prmted I” Great Br~tam

METABOLIC EUROPEAN ULF LIDMAN.

G~~RAN

Department

OXlO-Y629.79;0701-0339SO2.WO

EFFECTS OF CORTISOL IN THE EEL, ANGUILLA ANGUlLLA (L.)

DAVE, M.AJ-LIS JOHANSSON-SJBRECK.

of Zoophysiology.

University (Receioed

of GGteborg, 29 September

LARSSON and KERSTIN LEWANDER

AKE

FACK.

S-400 33 Gliteborg.

Sweden

1978)

Yellow European eels were intraperitoneally injected with cortisol (5 mg/kg body weight of hydrocortisone-21-phosphate-Na-salt) in 0.9”:, saline or saline only daily during 14 days. 2. Sampling of blood and tissues was performed after 1, 4 and 14 days and parameters of inorganic ion, carbohydrate, protein and lipid metabolism were studied. 3. As expected, the blood plasma cortisol levels were significantly elevated at all sampling events as an effect of cortisol injections. 4. These elevated cortisol levels had a stabilizing effect on osmoregulation and weak hypokalemic and hyperphosphatemic actions. 5. Further. a stimulated liver eluconeogenesis and carbohydrate metabolism together with evidence for an enhanced lipolysis were olbserved. Abstract--l.

INTRODUCTION

and identity of corticosteroids in teleost fish has been known for about 20 years (Bondy ef al., 1957; Phillips & Chester Jones, 1957). Among the different steroids identified, cortisol seems to be the most abundant and biologically active form (reviewed by Idler & Truscott, 1972; Leloup-Hatey, 1976). During the past 20 years, a lot of interest has been focused on the regulation and biological activity of corticosteroids and on their metabolic role in various organs in fish (reviews by Chester Jones ef al., 1969; Chester Jones rt a/., 1972; Butler, 1973; Johnson, 1973; Chester Jones et al., 1974; Bentley, 1976). Cortisol has proven to play an important role in the osmo- and electrolyte regulation by controlling the sodium flux across epithelial membranes, probably by stimulation of sodium-potassium activated adenosinetriphosphatase (Na-K-ATPase) in different organs (Jampol & Epstein, 1970; Epstein et al., 1971; Milne et al., 1971; Butler & Carmichael, 1972; Forrest et al., l973a; Ando, 1974). Also the potassium homeostasis has been shown to be affected by cortisol (Chan et al., 1967; Chan et al., 1968). Another part of the metabolism known to be partially regulated by corticoid hormones is the carbohydrate metabolism. Thus, cortisol is known to produce hyperglycemia and to stimulate liver gluconeogenesis in most fish species (reviewed by Butler. 1973). The substrate for this gluconeogenesis is probably amino acids derived from catabolized parietal muscle (Storer, 1967; Pora & Precup, 1971) or other extrahepatic tissue. Furthermore, cortisol is known to induce liver transaminases, namely glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GTP) (Storer, 1967; Freeman & Idler, 1973; Inui 8t Yokote, 1975a). With the exception of protein from extra-hepatic tissue, liver protein may also be used up in the early phases of liver gluconeogenesis (Bellamy, 1968). Regarding the effects of cortisol on lipid metabolism in fish, existing knowledge is very incomplete. The

presence

TSP. 63;3~-~

339

From studies on mammals, the corticosteroids have been assigned a direct or permissive lipolytic effect and/or a prevention of re-esterification (reviews by Thompson & Lippman, 1974; Fain & Czeck, 1975). Evidence for the same qualitative effects on fish is found in a work by Butler & Mayerle (cf. Butler, 1973), where cortisol injections into starved North American eel (Anguillu rostrata), caused a rapid increase in the plasma free fatty acids (FFA). Further, Phleger (1971) pointed out a pronounced inability of triglyceride synthesis in post-spawned salmon, Onchorhynchus gorhusha, a stage which is supposed to have elevated blood cortisol levels (Robertson rt al., 1961; Idler et ul., 1963; Fagerlund, 1967). The purpose of the present investigation was to study the in uitlo effects of daily cortisol treatment during two weeks on the intermediary metabolism of the European eel, Anguilla anguilla L. The effects of cortisol on the fatty acid composition of the total blood plasma lipids (Dave et al., 1979) and on haematological parameters (Johansson-SjGbeck et al., 1978) have been studied in two parallel investigations.

MATERIALS AND METHODS Animcrls Yellow European eels (Anguilla anyuilln L.) were caught by fyke at SmGgen on the Swedish West Coast. They were transported to the laboratory and kept in basins with filtered, recirculating and aerated sea water with a salinity of 337;” and a temperature of 10°C. Under these conditions the eels (average weight 12Og) were acclimatized for two weeks prior to the experiment which took place at the beginning of November. During the acclimatization period as well as during the experiment the eels were starved. Experimental

treatment

At the start of the experiment,

the eels were at random divided into two groups, both receiving daily intraperitoneal injections of either 5 mg/kg body weight of hydrocortisone-21-phosphate, sodium salt (Sigma Chem. Comp.) dissolved in 0.9% saline or 0.9% saline only.

340

11:

i\-----,

Plasma

_-i

cortlsol

l

l

i ‘O.

1

I

Plasma

170

sodium

I

I60 I

I

P'OSmO

Ch,Or,de

Plosmo

potassium

140 I30

L_+_~_--__1

I

-____

Plosmo

~norgonlc

phosphate

31

I

4 Time;

days

14 oftor

first

Injection

Fig. 1. Effects of dail) intraperitoneal injections of cortisol on plasma cortisol and different plasma electrolyte levels in the eel. Each point represents the mean value for 8- 12 animals and vertical bars indicate the standard error of the mean. Continuous lines correspond to cortisol treated eels. broken lines to saline injected controls. *Indicates significant differences (P -c 0.05) between groups.

Sampling was performed at I4 days after the first injection. I2 eels from each group (cortisol were taken out. The sampling analytical methods used have in detail by Dave c’t al. (1975).

three occasions: I. 4 and On every occasion 8 to injected v&-vis controls) procedure as well as the previously been described

mwf~twnt

Sfutistid

Student’s

r-test was

compare the cortisol treated saline injected groups at the different sampling occasions. Statistically significant differences were established at the 0.05 level. used

to

groups with corresponding

RESULTS AND DISCUSSION Plasn7~

cortisol

As was expected, the cortisol injected eels show. at all sampling occasions. significantly elevated plasma cortisol levels in comparison with corresponding control eels (Fig. 1). Concerning the blood cortisol levels of the control eels, these are somewhat elevated when compared to values obtained in previous invesgigations on the European eel (Ball c’f rrl.. 1971: Lewander et ul., 1974; Dave c’f ul.. 1975; Lewander it al., 1976). but nevertheless of the same magnitude. Plasma inorganic

ions

The values obtained for plasma inorganic ion levels are presented in Fig. I. Concerning the sodium and

chloride levels. no significant differences. as an effect of cortisol treatment, can be observed. Nevertheless. the concomitant increase in sodium and chloride in the controls at the 4th day sampling event ought to be mentioned. This result suggests an osmoregulatory dysfunction in these animals at that time. The cause of this effect in the control animals might have been the handling in connection with the injections (Mayer & Mae@ 1967: Wedemayer. 1972). The absence of a corresponding effect in the cortisol treated eels, on the other hand, might be contributed to a stabilizing effect of cortisol on osmoregulation as previously pointed out by Butler & Carmichael (1972) and by Forrest cut al. (1973b). The persisting lower, although not statistically significant, plasma potassium levels in the cortisol treated eels (Fig. 1) might reflect a weak hypokalemic effect of cortisol during the experimental conditions present. Such an action of cortisol has been reported earlier for both fish (Chan et al.. 1967; Chan et ul., 1968; Pickford c’f ul.. 1970) and mammals (Harper. 1975). The suggestion that cortisol gives rise to a hypokalemia is further supported by the observation that the cortisol treated eels showed a pronounced muscle weakness towards the end of the experiment, a finding in line with observations by Epstein ut ul. (1971).

Metabolic effects of cortisol in the eel Blood

60

341

glucose

-E :: q 40 @

I

Time; Fig.

2. Effects

. . . . . . . . . . . . . . . . . ‘.‘.’ .-..- .-.;

.. ..

doyr

after

fmt

mjsction

of daily intraperitoneal injections of cortisol on plasma and tissue carbohydrate metabolites in the eel. Symbols as in Fig. 1.

The transient significantly higher level of plasma inorganic phosphate in the cortisol treated eels at the day 1 sampling occasion (Fig. 1) is hard to explain, as not very much is known about the inorganic phosphate homeostasis in fish. However, the present finding is in agreement with the significantly increased serum phosphate in hypophysectomized killifish (Fundulus heteroclitus), after chronic administration of cortisol (Pickford et al., 1970). Furthermore, another study on the same fish (Srivastava & Pickford, 1972) showed that hypophysectomy only resulted in a significantly decreased serum phosphate level. Carbohydrates

From a general point of view, cortisol is considered to exert a pronounced glucocorticoid action involving stimulation of liver transaminases and gluconeogenesis accompanied by elevated blood glucose levels. In the present investigation, the stimulatory effects of cortisol on the carbohydrate metabolism can be seen from Fig. 2. The significantly higher levels of blood glucose and liver glycogen in the cortisol treated eels are probably due to an enhanced liver gluconeogenesis. These findings are in line with results obtained from previous investigations on fish (reviewed by Butler, 1973; Inui & Yokote, 1975a). However, in the present study the stimulated liver gluconeogenesis did not produce any increased but rather maintained and stabilized levels of both liver glycogen and blood glucose in comparison with the controls. In the latter group, a depletion of liver gly-

cogen and a subsequent decrease in blood glucose took place, probably a handling effect. Concerning the blood lactate levels in the present study, a statistically higher value for the cortisol treated eels compared to controls was observed at the 14 days sampling event. The explanation to this may be that a stimulated liver gluconeogenesis in the cortisol treated eels keeps pace with the glucose catabolism, while in the controls, liver glycogen reserves are depleted at that stage. On the other hand, the muscle glycogen levels are kept constant in both experimental groups except for at the first sampling event (Fig. 2). This drop might be a result of a cortisol-induced arrest in glucose uptake similar to that noted for mammalian adipose tissue (reviewed by Munck, 1971; Fain & Czeck, 1975). In mammals, however, this process does not involve muscle tissue, but in many fish species, the muscle mass can be considered as in part consisting of adipose tissue intermingled with the muscle fibres (Tashima & Cahill, 1965). The restoration of the muscle glycogen level in the cortisol treated animals after four days may be a compensatory effect of an increased insulin secretion (Fain & Czeck, 1975; Lewander et al., 1976). Protein No effects of cortisol

administration on blood and protein contents could be observed in the present investigation except for a significantly lower liver protein level at the last sampling event (14 days) (Fig. 3). Concerning the effect on liver protein, the

tissue

342 Plosmo

of the decrease may be ;I gluconcogenesis from liver protein, a suggestion supported by Bellamy 01 uI. (1968) and Dave c’t rrl. (1975). The nil effect of cortisol on muscle protein in the present study suggests that cortisol does not induce any catabolism of muscle tissue in the eel. This suggestion is in accordance with the investigations of lnui & Yokote (19752~ b) on the Japanese eel. .Aqui//rt jupor~ic~. where cortisol was shown not to increase the plasma amino acid levels. C~LIS~

P~osma

prole~n

Lipill.\ As ;I whole. the corticosteroid trcatcd eels in the present investigation seemed to show decreased trigI,ccride levels in muscle and liver tissue 21s well as in blood plasma when compared to corresponding controls (Fig. 4). When considering plasma phospholipids and free. estcrifed or total blood plasma cholesterol contents (Fig. 5)%no effects from the cortisol trcatmcnt were observed. The trend of decrease m triglyceride

thJlycsrldes

-~~--~

....-....--” __ ___.,..__,. I .

I ____ ___,_._,__..........

E

..-

i

5

2J

i

P

e

1 ,

LlV.3

trlgtyCer,dss

levels observed

Metabolic

5J

effects of cortisol

343

in the eel

Plasma

pharphalipids

Plasma

ChOleS1Sro1,

free

Plasma

choiss1srol.

esreriflrd

.

1

1

6

I

4 Time; days

Fig. 5. Effects of daily

14 af(er

first

injection

intraperitoneal injections of cortisol on plasma levels in the eel. Symbols as in Fig. 1.

in the cortisol treated eels in the present investigation may be an indication of an impaired re-esterification of triglycerides and/or an increased lipolysis as an effect of cortisol. The basis for these suggestions are the statements about a general lipolytic action of glucocorticoids in mammals (Thompson & Lippman, 1974; Fain & Czeck, 1975). Also in fish, a previous investigation supports these ideas. Thus, in the pink salmon, Onchorhynchus gorhuscha. the triglyceride synthesis is impaired in post-spawned fish (Phleger, 1971), a stage in which high levels of corticosteroids in the blood is strongly suspected (Robertson et al., 1961; Idler rt al., 1963; Fagerlund, 1967). Concerning the plasma free fatty acids (FFA). no differences between groups were observed (Fig. 4). This is contradictory to the observation by Butler & Mayerle (cf. Butler, 1973). showing that cortisol exerts a lipolytic effect, i.e. an increase in plasma FFA, in the North American eel. Anguilla rostrata. Such a lipolytic action of cortisol is also generally accepted for mammals (Fain & Czeck, 1975). On the other hand, in mammals the cortisol-induced increase in plasma FFA is very sensitive to the action of insulin (Fain & Czeck, 1975). Such an anti-lipolytic effect of insulin in the European eel has earlier been pointed out in an investigation by Lewander et al. (1976). Thus, a possible lipolytic effect of cortisol in the present investigation may have been abolished by a compensatory insulin release. In summary, the results from the present investigation show that cortisol affects the osmoregulation and ion homeostasis as well as the intermediary metabolism in the yellow phase of the European eel. Anguilla unguilla L. The effects are manifested as a stabilizing action on osmoregulation and a hypokalemic and hyperphosphatemic trend together with stimulated gluconeogenesis and lipolysis.

phospholipid

and cholesterol

Acknowledyrmenfs-The authors wish to thank Mrs Siv &tling for excellent technical assistance during the course of this study. This work was supported by grants from the National

Board

of Fisheries,

Sweden.

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