The influence of physiological doses of thyroxine on the lipid reserves of starved and fed brook trout, salvelinus fontinalis (mitchill)

Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 407 to 412. Pergamon Press. Printed in Great Britain

THE INFLUENCE OF PHYSIOLOGICAL DOSES OF THYROXINE ON THE LIPID RESERVES OF STARVED AND FED BROOK TROUT, SALVELINUS FONTINALIS (MITCHILL) TARA NARAYANSINGHAND J. G. EALES Department of Zoology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2 Canada

(Received 31 July 1974) Abstract--1. The effects of thyroxine (T4) on growth and lipid reserves were followed in brook trout, starved or fed 0'6 or 1.2~o of their body weight per day. 2. Nutritional state influenced growth, hepatosomatic index (HSI), viscerosomatic index (VSI) and the visceral lipid content, but had little effect on liver or muscle lipid. 3. T4 promoted growth in fed fish, decreased HSI of starved fish, tended to increase HSI in fed fish (1"2~0 ration), tended to lower hepatic and visceral lipid reserves in both starved and fed fish, increased the free fatty acid level in visceral adipose tissue, but had little effect on muscle lipid. 4. It is concluded that the growth promoting influence ofT4 is accompanied by a trend towards mobilization rather than deposition of fat.

INTRODUCTION

MATERIALS AND METHODS

THERE are several studies on the relationship between thyroid function and fat metabolism in fish. Rainbow trout treated with thyroid hormones tend to have less abdominal fat (Baraduc, 1954; Barrington et al., 1961) and a lower level of plasma lipid (Takashima et al., 1972). LaRoche et al. (1963, 1966) found that radiothyroidectomized rainbow trout had more abdominal fat than controls. An increase in liver lipid reserves was observed by Baker-Cohen (1961) in radiothyroidectomized platyfish, and by Hopper (1965) in thioureatreated Fundulus. Murat & Serfaty (1970) found that the plasma free fatty acid (FFA) level was doubled in carp by thyroxine (T4) treatment. These findings suggest that thyroid hormones promote mobilization of fat. However, other studies cannot be reconciled with this generalization. Rasquin (1949) claimed no effect of desiccated thyroid or thiouracil on fat deposition of Astyanax. LeRay et al. (1969) found no influence by propylthiouracil or thyroid hormones on the total lipids ofMugil auratus. No clearcut seasonal correlations emerged between thyroid activity and lipid deposits of brown trout (Swift, 1955), while Chambers (1951, 1953) found decreased fat in the livers of Fundulus treated with thiourea. Thyroxine may also entrain rhythms of fattening response to prolactin in fish (Meier, 1970, 1972). These varied relationships suggested between fat metabolism and thyroid function in fish may be the result of a variety of factors, including the nutritional state of the fish and the levels of thyroid hormone administered. In this study we examined the influence of physiological doses o f T 4 on the level of fat in liver, visceral adipose tissue and muscle of brook trout starved or fed different rations. A preliminary study was also made on the effects of T4 on the free fatty acid (FFA) levels and lipase activity in visceral adipose tissue. F r o m these data it was hoped to draw conclusions on the role o f T 4 in regulating fat mobilization or deposition in brook trout.

Stockfish Brook trout from a local hatchery were acclimated for two weeks in the laboratory at 12°C in running dechlorinated Winnipeg City water and fed trout pellets (Victor Fox Foods Ltd., Winnipeg) once per day at a ration of approximately 1% of body weight.

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T4 treatment Control or T4-treated fish were held in plastic tanks containing 32 litres of static aerated Winnipeg City water held at 12°C in a controlled environment room (12L- 12D). Prior to addition of fish, 5 ml of 0.1 N NaOH (control tank) or 5 ml of 0"1 N NaOH containing carrier L-T4 (sodium pentahydrate; Sigma) (T4-treated tank) was mixed with the water to provide ambient T4 (free acid) levels of 0, 5, 10 or 25 #g/ 100ml (#g%). Every 24hr the fish were netted with minimum stress and transferred to a tank set up under identical conditions. This precedure was repeated for 13-28 days depending on the experiment. Plasma and tissue samples Blood was withdrawn from the caudal vessels of anaesthetized (MS 222) fish using a heparinized syringe and the fish killed by a blow to the head. The plasma was separated by centrifugation and stored at -20°C. In Expt. I (total lipid analysis) a sample of flank muscle (white), the entire liver and most of the remaining viscera (alimentary tract from the oesophagus to anus, including gall bladder, spleen and any intraperitoneal fat, but excluding heart, kidney, swim bladder, gonads and associated ducts and any gut contents) were excised, weighed and frozen. Liver and visceral weight (g) were expressed relative to whole body weight (g) as the hepatosomatic index (HSI) and viscerosomatic index (VSI) respectively. In Expt. II, samples (0.1-0.8 g) of adipose tissue were removed from freshly-killed fish. Each tissue sample was pooled with that from 4-7 (usually 4) other similarly-treated fish and homogenized (Sorvall Omnimixer, 50-ml chamber) with cold 0.7% saline (approx. 1:3, w/v) for 2 min. The homogenate was centrifuged at 8000 g for 15 min at 4°C and 0-1 ml or 0.2 ml aliquots (usually 4) used for FFA or lipase analyses.

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TARA NARAYANSINGH AND J. G, EALES

Total lipid analysis Total lipids of liver, visceral and muscle samples were determined after methods described by Folch et al. (1957), Bligh & Dyer (1959) and Overturf & Dryer (1969). The thawed tissue was weighed and homogenized in the cold for 1 min in a mixture of water and fat solvent (chloroform: methanol, 2:1, v/v). The final ratio of water:fat solvent was 1:10. The amount of water included the weight of tissue, which for these purposes was assumed to be all water. The homogenate was filtered through solvent-soaked fatlfree filter paper and collected in either 25 ml of 50 ml graduated cylinders which could be sealed with a groundglass stopper. Fat solvent was used to thoroughly wash the homogenization chamber and pulverized paper and homogenate. This was filtered through fresh solvent-soaked paper into the graduated cylinder. Fat solvent was then added to the graduated cylinder to provide a final water: fat solvent ratio of 1:20, v/v. A 0.2 ml volume of 0.05 N NaC1 was then thoroughly mixed with the contents of the graduated cylinder and allowed to stand overnight. The mixture became biphasic with purified lipids in the lower phase. The volume of the lower phase was recorded and the upper phase removed by aspiration. Aliquots (4~10 ml) of the lower phase were pipetted into tared aluminium dishes and evaporated to dryness exactly 5 rain on a hot-plate set at a constant temperature. The dish was then vacuum dried for 5 min and weighed. The tare for each aluminium dish was obtained by heating and desiccating it exactly as described above. The weight of lipid was expressed as mg lipid/g wet wt of tissue. The lipid weights of the entire liver and viscera were also related to the wet wt of the whole fish.

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Fig. 1. Per cent change in body wt (a) and mean final body wt (b) for control (C) and T4-treated (T) brook trout held at 12°C for 28 days. Fish were starved (0%) or fed either 0'6% or 1.2% of initial mean body wt per day. Per cent change in body wt was calculated from the initial and final weights of each group of fish. Brackets represent 1 S.E. on either side of mean (n = 15).

FFA and lipase measurements FFA levels (#M FFA/mg protein) were measured on supernatant aliquots of visceral adipose tissue by the method of Duncombe (t963) modified by Hollett & Auditore (1967). Lipase activity was measured as the ~tM FFA released/hr/ per mg protein in supernatant from a standard triglyceride substrate mixture during incubation for 2 hr at 20°C in a barbital acetate buffer (pH 8'5). For details of basic procedure see Hollett & Auditore (1967). Preliminary observations showed maximal release of FFA in this system at pH 8'5 for supernatant fractions of brook trout adipose tissue.

was greater (P > 0-05 < 0.1) for T4-treated fish on the higher ration. F o r starved fish HSI was significantly (P < 0-05) reduced by T~ treatment. The percentage of the liver or visceral mass comprised of lipid decreased with increasing food avail0% C

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Experiment I. Influence of starvation,food ration and T4 on body and organ weights and hepatic, visceral and muscle lipids A population of 90 trout selected for uniformity in size were divided into six equal groups. Three groups were held at i2°C for 28 days as control fish a n d three groups were treated with T4 (10/~g% a m b i e n t concentration). One pair of control a n d T4-treated groups was starved throughout. Another pair received Victor Fox pellets (0.6% of initial wet body wt) once per day. The final pair were fed 1-2% of body wt once per day. At the end of the treatment fish were weighed, killed and frozen ( - 2 0 ° C ) for later lipid analyses. Both T~ treatment and food ration influenced the final body wt (Fig. lb), and the m e a n per cent change in weight (Fig. la). T4-treated fish tended to lose less weight during starvation or gain more weight when fed than control fish. The changes in body wt were accompanied by disproportionate changes in hepatic a n d visceral wt (Figs. 2a & 3a). W i t h greater food availability, b o t h the HSI a n d VSI increased. T4-treated fish tended to have lower HSI a n d VSI values than control fish, but HSI

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Fig. 2. Per cent of body wt comprised of liver (a), per cent of liver wt comprised of lipid (b), and per cent of body wt comprised of liver lipid (c) for control (C) and T~-treated (T) brook trout held at 12°C for 28 days. Fish were starved (0%) or fed either 0.6% or 1.2% of initial mean body wt per day. Brackets represent I S.E. on either side of mean (n = 15).

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Fig. 3. Per cent of body wt comprised of viscera (a), per cent of visceral wt comprised of lipid (b), and per cent of body wt comprised of visceral lipid (c) for control (C) and T4-treated (T) brook trout held at 12°C for 28 days. Fish were starved (0%) or fed either 0"6% or 1.2% of initial mean body wt per day. Brackets represent 1 S.E. on either side of mean (n = 15). ability (Figs. 2b & 3b). This was largely due to changes in the non-lipid constituents. When hepatic or visceral fat was related to the body wt of the fish (Figs. 2c & 3c) no such decreases were observed. In fact visceral fat expressed in this way tended to increase slightly with food availability. T4 treatment tended to decrease liver lipid whether calculated relative to liver wt or body wt (Figs. 2b & c), and to decrease the visceral fat as related to body wt (Fig. 3c). Per cent fat in viscera (Fig. 3b) did not appear to be influenced by T4 treatment. Per cent fat in muscle was not influenced by food ration or T4 treatment (Fig. 4).

Six groups of 19-23 brook trout were starved at 12°C for up to 19 days and during this period either treated with T4 (ambient concentration 5, 10 or 25 pg%) or held as controls. Fish were killed at intervals from 8-19 days for FFA and lipase determinations on visceral adipose tissue. Plasma T4 levels were also measured (Higgs & Eales, 1973). Fish were treated with 5 #g% in early July for 15-19 days (average body wt controls 29.1 ___0-98 (S.E.)g, n = 21; experimentals 29.7 + 2"02 g, n = 20). Fish were treated with 10pg% in early August for 8-13 days (average body wt controls 38.5+ 1-19g, n = 2 3 ; experimentals 36"3 + 1.94 g, n = 21. Fish were treated with 25 pg% T4 in late July for 12-18 days (average body wt controls 27"8 + 1.62 g, n = 20; experimentals 36"3 4- 2"29g, n = 19). Prolonged treatment with ambient T4 raised plasma T4 levels significantly (Fig. 5b). For fish treated with 5 and 10 pg% T~ the FFA level in adipose tissue was increased relative to controls (Fig. 5a). The difference was significant (P < 0.05) for fish treated with 10 pg% T4. At 25 #g% T4 the trend was reversed but the difference between controls and T4-treated fish was not significant (P > 0'05). Lipase data (not shown) were extremely variable. There was some tendency for lipase activity to be higher for T4-treated fish, but differences were far from being statistically significant.

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Fig. 4. Per cent of flank muscle (white) comprised of lipid for control (C) and T4-treated (T) brook trout held at 12°C for 28 days. Fish were starved (0%) or fed either 0,6% or 1'2% of initial mean body wt per day. Brackets represent 1 S.E. on either side of mean (n = 15).

Fig. 5. FFA levels in supernatant fraction of homogenates of visceral adipose tissue (a), and plasma T4 levels (b) of starved control brook trout (C) and brook trout treated with ambient T4 (T) at concentrations of 5, 10 Or 25 #g%. Brackets represent 1 S.E. on either side of mean (n = 19-23). For fish exposed to i0 #g% T4, FFA levels were significantly (P < 0.05) greater than control levels. All groups tested with T4 (5, 10, 25/~g%) had plasma T4 levels significantly greater than control levels (P < 0.01 or < if001).

410

TARA NARAYANSINGHAND J. G. EALES DISCUSSION

Effects of starvation and ration The 11~o decrease in body wt due to starvation was accompanied by a decrease in HSI. Reduction in size offish livers due to starvation has been described previously (Tashima & Cahill, 1965; Inui & Oshima, 1966; Kamra, 1966; Bouche et al., 1969; Swallow & Fleming, 1969; Larsson & Lewander, 1973), and may be partly due to glycogen loss. Depletion of liver lipid was not observed in this study. In fact "~o fat in liver" tended to increase during starvation (Fig. 2b), presumably due to mobilization of non-lipid hepatic materials. During starvation the metabolic demands on the liver may be less (Nagayama et al., 1972), and some liver atrophy might be expected. Increased activity of peptidases and cathepsins has been demonstrated in the livers of starved carp (Creach et al., 1969). During prolonged starvation (6-8 months) Bouche et al. (1969) observed a 63~o decrease in hepatic RNA of carp, a 48~o decrease in hepatic protein, but negligible changes in hepatic DNA. The greater growth of the fish fed the higher ration (1-2~o) was accompanied by an increase in HSI. This was not due to fat deposition. Percent fat in liver actually decreased (Fig. 2b). Increased glycogen depoSition may have occurred. However, in more rapidly growing fish receiving a proportionately greater influx of nutrients in each meal, greater metabolic activity and a larger liver might be anticipated. VSI values were also reduced during starvation and in fed fish were greater for those receiving the 1"2~o ration. Contrary to the statement by Tashima & Cahill (1965) that adipose tissue depots are rare in fish, we found abundant visceral fat depots in the brook trout. However, as Figs. 3b & c indicate, the changes in visceral mass due to nutritional state were only partly due to changes in the amount of visceral adipose tissue, and much greater changes occurred in the non-lipid materials. During starvation some atrophy of temporarily redundant gut structures is expected. Conversely, ingestion of a large food ration each day places greater demands on the digestive system than ingestion of a small ration and greater development of the digestive tract under such circumstances is not surprising. Starvation did not alter muscle lipid appreciably. Previous workers found significant increases (Mayerle & Butler, 1971), decreases (Wilkins, 1967; Larsson & Lewander, 1973), or no change (Stimpson, 1965) in fish muscle lipid due to starvation, and it appears that starvation for several months may be required to cause significant changes. Furthermore, white muscle was examined in this study, which in starved rainbow trout is less active in fat metabolism than red lateral line muscle (Bilinski & Gardner, 1968). Effects of T4 T4 tended to reduce loss in body wt during starvation and increase growth of fed fish especially at the higher ration. These findings agree with some of the rather conflicting observations on the relationships between fish thyroid function and growth (early literature reviewed by Pickford & Atz, 1957; Harris, 1959; Hopper, 1961; Barrington et al., 1961; LaRoche et al., 1963, 1966; Gross et al., 1963; Bjorkland, 1965; Sage, 1967; Barrington & Rawdon, 1967; Bonnet, 1970; Barber & Barrington, 1972). The reasons for the con-

troversy concerning the relationship between fish growth and thyroid function are not clear. However, some of the previous investigators might be criticized for the use of non-physiological thyroid hormone treatment or poor control of the amount of food fed to the fish. We feel that these two criticisms do not apply here. The rations of food were carefully controlled, and both data from this study (Fig. 5b) and previously (Eales, 1974) indicate that the hormone treatment was in the physiological range. Liver size was significantly lower in starved trout receiving T4 and greater (almost significant) in fed trout (1.2~o ration) receiving T4. The latter observation agrees with that of Narayansingh & Eales (1974) that physiological doses of T4 increase hepatic protein synthesis in recently fed trout. Takashima et al. (1972) found slightly larger livers in T4-treated rainbow trout, while Hatey (1950) found smaller livers in thioureatreated carp. On the other hand Chambers (1951, 1953) observed an increase in liver size due to thiourea treatment in Fundulus. Most workers have failed to demonstrate any relationship between liver size and thyroid function in fish (Pickford, 1952, 1953, 1954a, b; Swift & Pickford, 1965 Hopper & Yatvin, 1965; Pickford & Grant, 1968). In this study the hepatic response to T4 depended on nutritional state. This could explain some of the previous negative or contradictory findings. Fontaine et al. (1953) observed a decrease in hepatic glycogen due to T 4 treatment. Thus glycogen deposition or mobilization could influence liver weight and would probably be influenced by nutritional state. The present trend for hepatic lipid to be reduced by T4 treatment is consistent with the findings that radiothyroidectomy (Baker-Cohen, 1961) or thiourea treatment (Hopper, 1965) tend to increase, liver lipid reserves. However, Chambers (1951, 1953) reported decreased fat in livers of Fundulus injected with thiourea. Swift (1955) found no correlation between seasonal changes in liver lipid and thyroid activity of brown trout, while Takashima et al. (1972) observed no significant change in liver lipid in rainbow trout treated with T 4. T 4 caused little change in VSI, but visceral lipid relative to body wt was slightly depressed by T4 treatment. This trend agrees with that shown by Baraduc (1954), where T , or iodinated casein lowered the visceral fat reserves of rainbow trout, Barrington et al. (1961) also suggested tentatively that T4 may decrease abdominal fat in rainbow trout. This is further supported by LaRoche et al. (1963, 1966) who found greater reserves of abdominal fat in radiothyroidectomized rainbow trout. However, all workers have not observed the same trend. Rasquin (1949) found no effect of desiccated thyroid or thiouracil on abdominal fat deposition of Astyanax and Swift (1955) found no clearcut correlation between seasonal changes in thyroid activity and abdominal fat stores in brown trout. Mobilization of fat from fish adipose tissue may depend on a variety of control mechanisms (Farkas, 1969). Fat mobilization from trout adipose tissue by physiological doses of T4 was implied by the higher FFA levels in the adipose tissue of trout treated with 5 and 10 #g~o T4 and by the suggestion of increased lipase activity. However, interpretation of the lipase data is difficult as the identity of the lipase (intracellular lipase or extracellular lipoprotein lipase) was not established. The apparent reversal of the trend for T4 to raise the

Physiological doses of thyroxine adipose F F A level at the highest and possibly pharmacological T~ dose (25 #g%) suggests a biphasic response. Biphasic responses to graded T4 doses have been described recently for rainbow trout (Oshima et al., 1972). In our study the treatment with 25 #g% T 4 increased the protein content of the adipose tissue (possibly due to fat mobilization), thereby leading to a lower F F A level when expressed as pM F F A / m g tissue protein. T4 had no effect on muscle lipid, agreeing with previous findings. Swift (1955) observed no correlation between seasonal changes in thyroid activity and muscle lipid in brown trout. Baraduc (1954) found that 2-3 months treatment with T4 or iodinated casein had little effect on muscle lipid in rainbow trout. Takashima et al. (1972) found no effect o f T 4 on muscle lipid of rainbow trout. However, white muscle was probably analyzed in most instances, which in rainbow trout at least metabolizes fat to a far lesser extent than red muscle (Bilinski & Gardner, 1968). Plasma lipid was not measured in this study, but Takashima et al. (1972) found that T 4 caused a significant 50% decrease in plasma lipid in rainbow trout. Murat & Serfaty (1970) observed that T4 treatment stimulated a two-fold increase in plasma F F A which was possibly due to F F A mobilization from storage sites in the fish. Conclusion

The changes in body wt due to 4 weeks of starvation or use of a greater food ration involve little change in the amounts of lipid deposited in liver, viscera or white muscle. However, disproportionate weight changes occur in the liver and viscera in these different nutritional states. Trout presumably adjust their visceral and hepatic masses in accordance with requirements for digestion and assimilation of food. The effects of Ta treatment at physiological doses are not dramatic. However, T4 tends to promote increased growth in fed trout, which is accompanied by mobilization rather than deposition of fat in the liver and viscera. Acknowledgements--We wish to thank Miss Deborah Follett for her willing assistance with the lipid analyses. Trout were kindly supplied by the Manitoba Government, Department of Mines, Resources and Environmental Management. This study was supported by grants-in-aid of research from the National Research Council of Canada and from the Fisheries Research Board to the Aquatic Biology Research Unit, Manitoba.

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