Fish Physiology and Biochemistry vol. 11 no. 1-6 pp 421-428 (1993) Kugler Publications, Amsterdam/New York
Insulin stimulates hepatic lipogenesis in rainbow trout, Oncorhynchus mykiss Darrin J. Cowley and Mark A. Sheridan Department of Zoology, North Dakota State University, Fargo, ND 58105, U.S.A.
Keywords: lipogenesis, insulin, glucagon, rainbow trout
Abstract The effects of the pancreatic hormones, insulin and glucagon, on rates of lipid biosynthesis in liver removed from rainbow trout, Oncorhynchus mykiss, were evaluated in vitro. Livers were removed from animals fasted for 30-36h, cut into ca. 1 mm3 pieces, and incubated in the presence of various concentrations of salmon insulin (sINS), bovine insulin (bINS), or a combination of bINS and bovine/porcine glucagon (GLU). Lipid synthesis was evaluated by total lipid concentration, 3 H20 incorporation into total lipid, and by fatty acid synthetase activity. Both mammalian and sINS tended to increase tissue total lipid concentration in hepatic tissue incubated for 5h. Insulin also stimulated 3H 2 0 incorporation into total lipid in a dose-dependent manner. Bovine INS (2 x 10-6 M) stimulated de novo synthesis nearly 6-fold over control rates; sINS (2 x 10-6 M) stimulated label incorporation more than 7-fold over control rates. Glucagon inhibited INS-stimulated 3 H20 incorporation; whereas, GLU alone had no effect on lipid synthesis in liver pieces incubated 5h. Lipid class analysis indicated that bINS significantly stimulated 3H 20 incorporation into phospholipids, fatty acids, and triacylglycerols. The greatest accumulation of label was in the triacylglycerol fraction, where incorporation was stimulated 17-fold over control levels. Hepatic enzymatic analysis indicated that bINS also significantly stimulated lipogenic enzyme activity 9-fold above control levels. These results indicate that INS is an important regulator of lipid synthesis in the liver of trout.
ResumC Les effets des hormones pancr6atiques, insuline et glucagon, sur le taux de biosynthese des lipides dans le foie de truite arc-en-ciel (Oncorhynchus mykiss), ont et6 6valu6s in vitro. Les foies ont 6t6 pr6lev6s sur des animaux mis Ajeun depuis 30-36 heures, puis coups en fragment d'un mm3 et incub6s en presence de concentrations variables d'insuline de saumon (sINS), d'insuline bovine (bINS), ou d'une combinaison de bINS et de glucagon bovin/porcin (GLU). La synthese lipidique est estim6e a partir de la concentration totale de lipides, par l'incorporation de 3 H20 dans les lipides totaux, et par l'activit6 acides gras synth6tase. Les insulines mammaliennes et de saumon ont tendance A augmenter la concentration totale de lipides du tissu h6patique incub6 durant 5 heures. L'insuline stimule aussi l'incorporation de 3 H2 0 dans les lipides totaux de maniere dose-d6pendante. La bINS (2 x 10-8 M) stimule pres de six fois la synthese de novo compare A Correspondence to: Dr. Mark A. Sheridan, Department of Zoology, North Dakota State University, Fargo, ND 58105, U.S.A., Tel: 701-237-8110; Fax: 701-237-7149
422 celle observe dans le contr6le, et la sINS (2 x 10-8 M) plus de 7 fois. Le glucagon inhibe la stimulation de l'incorporation de 1'3 H2 0 par l'insuline, alors que seul, il n'a aucun effet sur la synthese lipidique des explants de foie incub6s durant 5 heures. L'analyse des classes de lipides montre que la bINS stimule significativement l'incorporation de 3H 20 dans les phospholipides, les acides gras et les triglycerides. La plus grande accumulation a lieu dans la fraction des triglycerides, avec une stimulation augment6e de 17 fois. L'analyse des enzymes h6patiques indique que la bINS stimule 9 fois l'activit6 de l'enzyme lipog6nique. Ces r6sultats montrent que l'insuline est un important rgulateur de la synthese des lipides dans le foie.
Introduction Lipid biosynthesis in fish species utilizes the same biochemical mechanisms as those employed by mammals (reviewed by Christiansen and Klungsoyr 1988). Fatty acid synthesis proceeds, first, by the synthesis of long-chain fatty acids from C-2 precursors and, second, by the desaturation and chain elongation of saturated fatty acids. This process involves two steps: 1) ATP-dependent carboxylation of acetyl CoA forming malonyl CoA and 2) decarboxylation of malonyl CoA in a condensation reaction yielding palmitate. These reactions are catalyzed by acetyl CoA carboxylase and by fatty acid synthetase complexes, respectively. In mammals, control of fatty acid biosynthesis has been thoroughly studied (Mabrouk et al. 1990). Insulin (INS) and glucagon (GLU) have been shown to regulate hepatic lipogenesis, as measured by acetyl CoA carboxylase activity (Mabrouk et al. 1990), and adipose tissue lipogenesis, as measured by increased acecyl CoA carboxylase activity (Zammit and Corstorphine 1982) and by incorporation of tritium into total lipids (Jungas 1970). In fish, lipid biosynthesis also has been reported to occur in a number of different depot organs, including adipose tissue, and liver (Christiansen and Klungsoyr 1988). The liver, however, is the major site of fatty acid synthesis in fish (Lin et al. 1977). In contrast to mammals, fish have very little lipid synthesis taking place in adipose tissue. Lipid synthesis in fish appears to be influenced by nutritional, environmental, and hormonal factors. Lin et al. (1977) have reported in coho salmon (Oncorhynchus kisutch) that nutritional status effects lipid metabolism, such that fish which have been fed a high carbohydrate load show increased rates of
hepatic lipid synthesis; whereas, fish fasted or refed a high fat diet show a reduction in lipogenesis. Fish acclimated to cold temperatures show rates of hepatic lipid biosynthesis higher than fish acclimated to warm temperatures (Hazel 1990). Tashima and Cahill (1968) have reported in toadfish (Opsanus tau) that hormonal treatments of INS or GLU had no effect of plasma free fatty acid concentration. In rainbow trout, Oncorhynchusmykiss (Harmon and Sheridan 1992), golden shinner, Notemigunus crysoleucas (de Vlaming and Pardo 1975), and northern pike, Esox lucius (Ince and Thorpe 1975), however, INS stimulates removal of fatty acids from the plasma. Codfish INS administration in vivo also has been shown to increase lipid biosynthesis in liver as well as in muscle of northern pike, as measured by 14C incorporation (Ince and Thorpe 1976). Glucagon, on the other hand, elevates plasma fatty acid concentrations in rainbow trout (Ince and Thorpe 1975). Whether or not INS and GLU have a direct effect on hepatic lipogenesis in fish has yet to be shown. The overall objective of this study was to assess the effects of pancreatic hormones, INS and GLU, on rates of hepatic lipid biosynthesis. The specific questions we asked were: 1) What are the effects of INS and GLU on hepatic total lipid content? 2) What are the effects of INS and GLU on rates of 3 H2 0 incorporation into total lipids? 3) What are the effects of INS and GLU on rates of 3H2 0 incorporation into specific lipid classes? and 4) What are the effects of INS on lipid fatty acid synthetase activity?
423 Materials and methods Experimentalanimals Yearling rainbow trout of both sexes were obtained from the Garrison Dam National Fish Hatchery near Riverdale, ND and maintained indoors under 12L:12D photoperiod. The water (14°C) was continuously recirculated; new dechlorinated municipal water was replaced at a rate of ca. 10% makeup volume per day. Animals were fed ad libitum twice daily with Glencoe Mills trout chow (Glencoe, MN). Prior to experimentation, fish were fasted for 30-36 h and anesthetized with buffered 0.01% (w/v) tricaine methanesulfonate (MS222). Blood was collected from the severed caudal vessels, and the liver exposed for perfusion.
In situ perfusion and tissue preparation The liver was perfused in situ with Hanks buffer (Buffer A: 137 mM NaCl, 5.4 mM KCI, 1.7 mM CaC1 2, 0.8 mM MgSO 4 , 0.5 mM KH 2PO 4, 0.3 mM Na2PO4, 4 mM NaHCO 3, 10 mM HEPES and 5.5 mM glucose, pH 7.6, gassed continuously with 100% 02), at a flow rate of 2 ml/min for a period of at least 15 min. Following perfusion, the liver was removed, placed on an iced petri dish, and diced into ca. 1 mm cubes. Liver pieces were preincubated in Buffer A for 15 min in darkness at 14°C in a gyratory shaker (150 RPM). Pieces were collected by centrifugation (500 x g at 140 C) and washed three times by resuspension-centrifugation. Liver pieces (ca. 160 mg) were then placed into each well of a multiwell culture plate containing Buffer B (135 mM NaCl, 5.4 mM KC1, 1.7 mM CaC12, 0.8 mM MgSO 4, 0.7 mM MnC12, 0.5 mM KH 2PO 4 , 0.3 mM Na 2HPO4 , 4 mM NaHCO 3, 10 mM HEPES, and 0.24%7o (w/v), bovine serum albumin pH 7.6).
To each culture well containing liver pieces and incubation medium (Buffer B, 1.65 ml) was added 0.1 ml substrate mixture (containing 200 mM sodium acetate, 200 mM glucose and 200 mM lactate), 0.05 ml hormone (bovine/porcine glucagon, bovine insulin [Sigma, St Louis, MO]; salmon insulin), and 0.2 ml 3 H2 0 (New England Nuclear, Boston, MA, specific activity 1.0 mCi/g); bringing the total volume of the reaction mixture to 2.0 ml. Control cultures received additional Buffer B in place of hormone. In combination experiments, GLU was added 10 min prior to INS addition. Just after the addition of 3 H 2 0, a 50 zl aliquot of the incubation medium was removed for 3 H2 0 specific activity determination. The incubation proceeded in darkness for 5h at 14°C in a gyratory shaker (150 RPM). After various times, the medium was removed and the tissue pieces rinsed three times with Buffer C (135 mM NaCI, 5.4 mM KCI, 1.7 mM CaC12, 0.8 mM MgSO 4, 0.7 mM MnCI 2, 0.5 mM KH 2PO 4, 0.3 mM Na2 HPO 4, 4 mM NaHCO 3, 10 mM HEPES, 5.5 mM glucose and 0.24% (w/v), bovine serum albumin, pH 7.6) and weighed. Lipids were extracted according to Folch et al. (1957). A 50 t1 aliquot of the redissolved (in chloroform) extract was added to 4.9 ml of liquid scintillation cocktail (Scintisol; Isolab, Akron, OH) and the activity determined by liquid scintillation counting (LSC). In another series of experiments, the lipid extract was saponified (Sheridan et al. 1985), and label incorporation into saponifiable and nonsaponifiable components determined. The possibility of INS causing increased water uptake or retention was evaluated in a third series of experiments, where the liver pieces were incubated in Buffer B (1.95 ml) in the presence (1 x 10-8 M bINS; added in 0.05 ml) and absence of hormone (control, 0.05 ml Buffer B) for 5h. After incubation, the liver pieces were dried (90°C) to constant weight.
Total lipid and lipid class analyses 3
H 2 0 incorporationinto lipids
De novo synthesis of lipid was evaluated by 3 H2 0 incorporation into liver pieces incubated in vitro.
The analysis of lipids in fish has been described previously (Sheridan et al. 1985). The lipid classes containing 3H20 were visualized on thin layer chromatography (TLC) plates, under ultraviolet
424 TB
z 0 o
z
oX
zo X
0~ V)
0b
o=
o v
a-
z U,
='k (n)n E INS
INS
MAI1A LN GLU
GLU
I
Fig. 1. Effects of pancreatic hormones, bovine insulin, salmon insulin and bovine/porcine glucagon, on tissue total lipid concentration in liver removed from rainbow trout and incubated for 5h. Data are presented as means + SEM (n = 6); *p < 0.05 compared to control group. GLU, glucagon; INS, insulin.
light, after spraying with 0.05% (w/v) 2,7-dichlorofluorescein in methanol. The silica matrix containing the individual lipid classes was scraped into conical glass tubes and the lipids re-extracted using a total of 1.2 ml of 2:1 (v/v) chloroform/methanol for the nonpolar lipids and 1:2 (v/v) chloroform/methanol for polar lipids. The re-extracted lipids, collected in glass scintillation vials, were concentrated to dryness in a vacuum oven ( - 10 psi; 60°C). Scintillation cocktail (4.9 ml) was added to each vial and total 3H 20 incorporation estimated by LSC.
Fatty acid synthetase activity Hepatic fatty acid synthetase was determined in liver pieces incubated (150 RPM) in Buffer B (1.95 ml) with (2 x 10-6 M bINS; added in 0.05 ml) and without (control; 0.05 ml additional Buffer B) hormone for 5h at 14°C. After incubation, pieces were washed three times with Buffer A then dispersed by sonication (10 watts; 20 kHz). The resulting suspension was centrifuged (16,000 x g) for 10 min and the supernatant was collected and saved on ice. Synthetase activity was determined as described previously (Sheridan et al. 1985).
GLU
Fig. 2. Effects of pancreatic hormones, bovine insulin, salmon insulin and bovine/porcine glucagon, on tritium incorporation into tissue total lipids of liver removed from rainbow trout and incubated for 5h. Data presented as means + SEM (n = 6); *p < 0.05 compared to control group. GLU, glucagon; INS, insulin.
Statistics Results are expressed as means + SEM. Statistical differences were estimated by analysis of variance; multiple comparisons among means were made by the Student-Newman-Keuls test.
Results Total lipid The effects of INS and GLU on total lipid content are shown in Fig. 1. The control-treated and glucagon-treated liver pieces showed no change in hepatic total lipid content. Both bINS and sINS tended to increase tissue total lipid concentration in trout liver; only the high dose (2 x 10-6 M) of sINS resulted in a significant increase in total hepatic lipid content (Fig. 1). In combination with INS, GLU inhibited INS-stimulated hepatic lipid content. The potential of INS to cause increased water uptake or retention (affecting measures of fresh weight) also was examined. Liver pieces incubated in the presence of 1 x 10-8 M bINS showed a 79.5% + 0.02070 weight loss upon drying liver and pieces incubated in the absence of hormone showed
425 CONTROL
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10
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.
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MAMMALIAN GLU
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.
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Insulin stimulates hepatic lipogenesis in rainbow trout, Oncorhynchus mykiss Darrin J. Cowley and Mark A. Sheridan Department of Zoology, North Dakota State University, Fargo, ND 58105, U.S.A.
Keywords: lipogenesis, insulin, glucagon, rainbow trout
Abstract The effects of the pancreatic hormones, insulin and glucagon, on rates of lipid biosynthesis in liver removed from rainbow trout, Oncorhynchus mykiss, were evaluated in vitro. Livers were removed from animals fasted for 30-36h, cut into ca. 1 mm3 pieces, and incubated in the presence of various concentrations of salmon insulin (sINS), bovine insulin (bINS), or a combination of bINS and bovine/porcine glucagon (GLU). Lipid synthesis was evaluated by total lipid concentration, 3 H20 incorporation into total lipid, and by fatty acid synthetase activity. Both mammalian and sINS tended to increase tissue total lipid concentration in hepatic tissue incubated for 5h. Insulin also stimulated 3H 2 0 incorporation into total lipid in a dose-dependent manner. Bovine INS (2 x 10-6 M) stimulated de novo synthesis nearly 6-fold over control rates; sINS (2 x 10-6 M) stimulated label incorporation more than 7-fold over control rates. Glucagon inhibited INS-stimulated 3 H20 incorporation; whereas, GLU alone had no effect on lipid synthesis in liver pieces incubated 5h. Lipid class analysis indicated that bINS significantly stimulated 3H 20 incorporation into phospholipids, fatty acids, and triacylglycerols. The greatest accumulation of label was in the triacylglycerol fraction, where incorporation was stimulated 17-fold over control levels. Hepatic enzymatic analysis indicated that bINS also significantly stimulated lipogenic enzyme activity 9-fold above control levels. These results indicate that INS is an important regulator of lipid synthesis in the liver of trout.
ResumC Les effets des hormones pancr6atiques, insuline et glucagon, sur le taux de biosynthese des lipides dans le foie de truite arc-en-ciel (Oncorhynchus mykiss), ont et6 6valu6s in vitro. Les foies ont 6t6 pr6lev6s sur des animaux mis Ajeun depuis 30-36 heures, puis coups en fragment d'un mm3 et incub6s en presence de concentrations variables d'insuline de saumon (sINS), d'insuline bovine (bINS), ou d'une combinaison de bINS et de glucagon bovin/porcin (GLU). La synthese lipidique est estim6e a partir de la concentration totale de lipides, par l'incorporation de 3 H20 dans les lipides totaux, et par l'activit6 acides gras synth6tase. Les insulines mammaliennes et de saumon ont tendance A augmenter la concentration totale de lipides du tissu h6patique incub6 durant 5 heures. L'insuline stimule aussi l'incorporation de 3 H2 0 dans les lipides totaux de maniere dose-d6pendante. La bINS (2 x 10-8 M) stimule pres de six fois la synthese de novo compare A Correspondence to: Dr. Mark A. Sheridan, Department of Zoology, North Dakota State University, Fargo, ND 58105, U.S.A., Tel: 701-237-8110; Fax: 701-237-7149
422 celle observe dans le contr6le, et la sINS (2 x 10-8 M) plus de 7 fois. Le glucagon inhibe la stimulation de l'incorporation de 1'3 H2 0 par l'insuline, alors que seul, il n'a aucun effet sur la synthese lipidique des explants de foie incub6s durant 5 heures. L'analyse des classes de lipides montre que la bINS stimule significativement l'incorporation de 3H 20 dans les phospholipides, les acides gras et les triglycerides. La plus grande accumulation a lieu dans la fraction des triglycerides, avec une stimulation augment6e de 17 fois. L'analyse des enzymes h6patiques indique que la bINS stimule 9 fois l'activit6 de l'enzyme lipog6nique. Ces r6sultats montrent que l'insuline est un important rgulateur de la synthese des lipides dans le foie.
Introduction Lipid biosynthesis in fish species utilizes the same biochemical mechanisms as those employed by mammals (reviewed by Christiansen and Klungsoyr 1988). Fatty acid synthesis proceeds, first, by the synthesis of long-chain fatty acids from C-2 precursors and, second, by the desaturation and chain elongation of saturated fatty acids. This process involves two steps: 1) ATP-dependent carboxylation of acetyl CoA forming malonyl CoA and 2) decarboxylation of malonyl CoA in a condensation reaction yielding palmitate. These reactions are catalyzed by acetyl CoA carboxylase and by fatty acid synthetase complexes, respectively. In mammals, control of fatty acid biosynthesis has been thoroughly studied (Mabrouk et al. 1990). Insulin (INS) and glucagon (GLU) have been shown to regulate hepatic lipogenesis, as measured by acetyl CoA carboxylase activity (Mabrouk et al. 1990), and adipose tissue lipogenesis, as measured by increased acecyl CoA carboxylase activity (Zammit and Corstorphine 1982) and by incorporation of tritium into total lipids (Jungas 1970). In fish, lipid biosynthesis also has been reported to occur in a number of different depot organs, including adipose tissue, and liver (Christiansen and Klungsoyr 1988). The liver, however, is the major site of fatty acid synthesis in fish (Lin et al. 1977). In contrast to mammals, fish have very little lipid synthesis taking place in adipose tissue. Lipid synthesis in fish appears to be influenced by nutritional, environmental, and hormonal factors. Lin et al. (1977) have reported in coho salmon (Oncorhynchus kisutch) that nutritional status effects lipid metabolism, such that fish which have been fed a high carbohydrate load show increased rates of
hepatic lipid synthesis; whereas, fish fasted or refed a high fat diet show a reduction in lipogenesis. Fish acclimated to cold temperatures show rates of hepatic lipid biosynthesis higher than fish acclimated to warm temperatures (Hazel 1990). Tashima and Cahill (1968) have reported in toadfish (Opsanus tau) that hormonal treatments of INS or GLU had no effect of plasma free fatty acid concentration. In rainbow trout, Oncorhynchusmykiss (Harmon and Sheridan 1992), golden shinner, Notemigunus crysoleucas (de Vlaming and Pardo 1975), and northern pike, Esox lucius (Ince and Thorpe 1975), however, INS stimulates removal of fatty acids from the plasma. Codfish INS administration in vivo also has been shown to increase lipid biosynthesis in liver as well as in muscle of northern pike, as measured by 14C incorporation (Ince and Thorpe 1976). Glucagon, on the other hand, elevates plasma fatty acid concentrations in rainbow trout (Ince and Thorpe 1975). Whether or not INS and GLU have a direct effect on hepatic lipogenesis in fish has yet to be shown. The overall objective of this study was to assess the effects of pancreatic hormones, INS and GLU, on rates of hepatic lipid biosynthesis. The specific questions we asked were: 1) What are the effects of INS and GLU on hepatic total lipid content? 2) What are the effects of INS and GLU on rates of 3 H2 0 incorporation into total lipids? 3) What are the effects of INS and GLU on rates of 3H2 0 incorporation into specific lipid classes? and 4) What are the effects of INS on lipid fatty acid synthetase activity?
423 Materials and methods Experimentalanimals Yearling rainbow trout of both sexes were obtained from the Garrison Dam National Fish Hatchery near Riverdale, ND and maintained indoors under 12L:12D photoperiod. The water (14°C) was continuously recirculated; new dechlorinated municipal water was replaced at a rate of ca. 10% makeup volume per day. Animals were fed ad libitum twice daily with Glencoe Mills trout chow (Glencoe, MN). Prior to experimentation, fish were fasted for 30-36 h and anesthetized with buffered 0.01% (w/v) tricaine methanesulfonate (MS222). Blood was collected from the severed caudal vessels, and the liver exposed for perfusion.
In situ perfusion and tissue preparation The liver was perfused in situ with Hanks buffer (Buffer A: 137 mM NaCl, 5.4 mM KCI, 1.7 mM CaC1 2, 0.8 mM MgSO 4 , 0.5 mM KH 2PO 4, 0.3 mM Na2PO4, 4 mM NaHCO 3, 10 mM HEPES and 5.5 mM glucose, pH 7.6, gassed continuously with 100% 02), at a flow rate of 2 ml/min for a period of at least 15 min. Following perfusion, the liver was removed, placed on an iced petri dish, and diced into ca. 1 mm cubes. Liver pieces were preincubated in Buffer A for 15 min in darkness at 14°C in a gyratory shaker (150 RPM). Pieces were collected by centrifugation (500 x g at 140 C) and washed three times by resuspension-centrifugation. Liver pieces (ca. 160 mg) were then placed into each well of a multiwell culture plate containing Buffer B (135 mM NaCl, 5.4 mM KC1, 1.7 mM CaC12, 0.8 mM MgSO 4, 0.7 mM MnC12, 0.5 mM KH 2PO 4 , 0.3 mM Na 2HPO4 , 4 mM NaHCO 3, 10 mM HEPES, and 0.24%7o (w/v), bovine serum albumin pH 7.6).
To each culture well containing liver pieces and incubation medium (Buffer B, 1.65 ml) was added 0.1 ml substrate mixture (containing 200 mM sodium acetate, 200 mM glucose and 200 mM lactate), 0.05 ml hormone (bovine/porcine glucagon, bovine insulin [Sigma, St Louis, MO]; salmon insulin), and 0.2 ml 3 H2 0 (New England Nuclear, Boston, MA, specific activity 1.0 mCi/g); bringing the total volume of the reaction mixture to 2.0 ml. Control cultures received additional Buffer B in place of hormone. In combination experiments, GLU was added 10 min prior to INS addition. Just after the addition of 3 H 2 0, a 50 zl aliquot of the incubation medium was removed for 3 H2 0 specific activity determination. The incubation proceeded in darkness for 5h at 14°C in a gyratory shaker (150 RPM). After various times, the medium was removed and the tissue pieces rinsed three times with Buffer C (135 mM NaCI, 5.4 mM KCI, 1.7 mM CaC12, 0.8 mM MgSO 4, 0.7 mM MnCI 2, 0.5 mM KH 2PO 4, 0.3 mM Na2 HPO 4, 4 mM NaHCO 3, 10 mM HEPES, 5.5 mM glucose and 0.24% (w/v), bovine serum albumin, pH 7.6) and weighed. Lipids were extracted according to Folch et al. (1957). A 50 t1 aliquot of the redissolved (in chloroform) extract was added to 4.9 ml of liquid scintillation cocktail (Scintisol; Isolab, Akron, OH) and the activity determined by liquid scintillation counting (LSC). In another series of experiments, the lipid extract was saponified (Sheridan et al. 1985), and label incorporation into saponifiable and nonsaponifiable components determined. The possibility of INS causing increased water uptake or retention was evaluated in a third series of experiments, where the liver pieces were incubated in Buffer B (1.95 ml) in the presence (1 x 10-8 M bINS; added in 0.05 ml) and absence of hormone (control, 0.05 ml Buffer B) for 5h. After incubation, the liver pieces were dried (90°C) to constant weight.
Total lipid and lipid class analyses 3
H 2 0 incorporationinto lipids
De novo synthesis of lipid was evaluated by 3 H2 0 incorporation into liver pieces incubated in vitro.
The analysis of lipids in fish has been described previously (Sheridan et al. 1985). The lipid classes containing 3H20 were visualized on thin layer chromatography (TLC) plates, under ultraviolet
424 TB
z 0 o
z
oX
zo X
0~ V)
0b
o=
o v
a-
z U,
='k (n)n E INS
INS
MAI1A LN GLU
GLU
I
Fig. 1. Effects of pancreatic hormones, bovine insulin, salmon insulin and bovine/porcine glucagon, on tissue total lipid concentration in liver removed from rainbow trout and incubated for 5h. Data are presented as means + SEM (n = 6); *p < 0.05 compared to control group. GLU, glucagon; INS, insulin.
light, after spraying with 0.05% (w/v) 2,7-dichlorofluorescein in methanol. The silica matrix containing the individual lipid classes was scraped into conical glass tubes and the lipids re-extracted using a total of 1.2 ml of 2:1 (v/v) chloroform/methanol for the nonpolar lipids and 1:2 (v/v) chloroform/methanol for polar lipids. The re-extracted lipids, collected in glass scintillation vials, were concentrated to dryness in a vacuum oven ( - 10 psi; 60°C). Scintillation cocktail (4.9 ml) was added to each vial and total 3H 20 incorporation estimated by LSC.
Fatty acid synthetase activity Hepatic fatty acid synthetase was determined in liver pieces incubated (150 RPM) in Buffer B (1.95 ml) with (2 x 10-6 M bINS; added in 0.05 ml) and without (control; 0.05 ml additional Buffer B) hormone for 5h at 14°C. After incubation, pieces were washed three times with Buffer A then dispersed by sonication (10 watts; 20 kHz). The resulting suspension was centrifuged (16,000 x g) for 10 min and the supernatant was collected and saved on ice. Synthetase activity was determined as described previously (Sheridan et al. 1985).
GLU
Fig. 2. Effects of pancreatic hormones, bovine insulin, salmon insulin and bovine/porcine glucagon, on tritium incorporation into tissue total lipids of liver removed from rainbow trout and incubated for 5h. Data presented as means + SEM (n = 6); *p < 0.05 compared to control group. GLU, glucagon; INS, insulin.
Statistics Results are expressed as means + SEM. Statistical differences were estimated by analysis of variance; multiple comparisons among means were made by the Student-Newman-Keuls test.
Results Total lipid The effects of INS and GLU on total lipid content are shown in Fig. 1. The control-treated and glucagon-treated liver pieces showed no change in hepatic total lipid content. Both bINS and sINS tended to increase tissue total lipid concentration in trout liver; only the high dose (2 x 10-6 M) of sINS resulted in a significant increase in total hepatic lipid content (Fig. 1). In combination with INS, GLU inhibited INS-stimulated hepatic lipid content. The potential of INS to cause increased water uptake or retention (affecting measures of fresh weight) also was examined. Liver pieces incubated in the presence of 1 x 10-8 M bINS showed a 79.5% + 0.02070 weight loss upon drying liver and pieces incubated in the absence of hormone showed
425 CONTROL
[ n
CL MAMMALIAN INS
10
I
.
w
MAMMALIAN GLU
3
'C
z"
0 V)
.
2
., 'o 2 o
1
