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Effect of bovine growth hormone on growth, organ weights, tissue composition and adipose tissue metabolism in young castrated male goats
Livestock Production Science 55 (1998) 215–225
Effect of bovine growth hormone on growth, organ weights, tissue composition and adipose tissue metabolism in young castrated male goats 1 ˇ J. Skarda
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 104 00 Prague 10, Czech Republic Received 5 May 1997; accepted 17 March 1998
Abstract Young castrated male goats (n 5 22) were used to investigate the effects of administration of a sustained release recombinant bovine growth hormone (GH; 100 mg) at seven-day intervals on body growth, organ weights and lipid content, and on lipogenic responses of cultured explants of omental adipose tissue. Average body weight gain was improved by 22% and the relative weights of the liver, kidney and adrenal glands were increased 16, 19 and 20%, respectively, by GH treatment. The fat content in m. longissimus dorsi and omentum was less in GH-treated animals than in controls by 29.5 and 44.4%, respectively. The rate of fatty acid synthesis was determined in acute incubations both in freshly prepared and chronically cultured omental adipose explants. Adipose explants remained metabolically active and retained their ability to respond to hormones when maintained in tissue culture medium. Cortisol acted synergistically with insulin to produce a higher rate of lipogenesis than that in cultures with insulin alone, but this was the case only in tissues from GH-treated animals; however, cortisol alone decreased lipogenesis in explants from both groups of animals. GH inhibited insulinstimulated lipogenesis in tissues from control animals and insulin plus cortisol-stimulated lipogenesis in tissues from both control and GH-treated animals. GH treatment in vivo and in vitro did not increase the explant’s responsiveness to noradrenaline in vitro; however, responsiveness (inhibition of lipogenesis) to isoprenaline was greater in GH-treated animals than in controls. 1998 Elsevier Science B.V. Keywords: Goat; Growth; Adipose tissue; Lipogenesis; Growth hormone; Cortisol
1. Introduction Many aspects of metabolism in ruminants and in a variety of animal species are influenced by growth hormone (GH). In the short term, GH acts to preserve body protein and glucose pools during a 1
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period of nutrient deprivation at the expense of fat which is mobilized for the provision of material for energy generation and for the formation of the carbon skeleton of non-essential amino acids (Weil, 1965). In the long term, GH has a vital homeorhetic function to channel nutrients to support such complex processes as growth, pregnancy or lactation. Under GH control, the absorbed and depot nutrients are directed toward or diverted from different tissues
0301-6226 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 98 )00138-9
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in a highly coordinated manner (Bauman and Currie, 1980). The mechanisms involved have not been clearly defined. GH can act in tissues directly on the cells themselves or indirectly through the actions of locally and systemically produced insulin-like growth factor I (IGF-I) and IGF binding proteins (IGFBPs) (Scott et al., 1985) or through the alterations of the cell’s ability to respond to homeostatic signals such as from insulin and catecholamines (Sechen et al., 1989). Most of the effects of GH in ruminants have been reported for sheep and cattle, and very little is known about GH action in goats and other ruminant species. There are, however, considerable differences in food intake and rates of gastrointestinal and tissue metabolism between different ruminant species. For example, overall feed digestibility in goats is higher than in sheep and tends to be higher in low protein diets (Tolkamp and Brouwer, 1993). Molar % of acetate in the rumen is higher in goats but molar % of propionate and butyrate is higher in sheep (Jones et al., 1973). There are considerable differences in the rate of metabolism of cows, sheep and goats. High-producing dairy goats may consume approximately twice as much feed per unit of body weight as dairy cows, with a greater proportion of this total feed consumption being utilized for milk production (Larson, 1978). Ruminant adipose tissue responsiveness to GH and catecholamines may be changed in a species-specific manner and one can even expect the existence of breed variations in domestic ruminants, as was shown for pigs (Bidanel et al., 1991). Thus, it is of interest to compare the responses to GH in cattle and sheep with those in goats. The purpose of the present experiments was to document the effect of long-term GH treatment on growth, carcass composition and lipogenesis in omental adipose tissue of young castrated male goats. Cultures with adipose tissue explants were conducted to determine whether GH treatment in vivo affects lipogenesis directly and how GH treatment in vivo affects lipogenic responses of explants cultured in the presence of insulin, cortisol and GH alone or in different combinations (insulin plus cortisol; insulin plus GH; insulin plus cortisol plus GH). In addition, the abilities of these hormones to modulate the lipogenic responses to catecholamines
in explants from control and GH-treated animals were compared in acute incubations.
2. Materials and methods
2.1. Animals and general procedures The experiments were carried out with young castrated male goats (Czech white breed). Twentytwo animals were castrated at 2–3 weeks of age and used for experiments between two and eight months of age. Animals were arranged in pairs (placebotreated and bGH-treated) based on age (pairs did not differ by more than 1 d in age) and body weight (pairs did not differ by more than 2 kg body weight). Animals had free access to grass hay and fresh grass. A concentrate mixture (oats and barley meal) was given two times daily according to body weight (from 0.2 to 0.4 kg day 21 animal 21 ). Starting between two and three months of age, placebotreated animals were injected subcutaneously (s.c.) with olive oil and bGH-treated animals received 100 mg of recombinant methionyl bGH (sometribove) in a lipid-based sustained release vehicle at seven-day intervals in a 147-day experiment. Body weights were recorded weekly. After 147 days of treatment all animals were exsanquinated following stunning with a captive bolt. The adipose tissue of the greater omentum was aseptically removed and placed immediately into sterile phosphate buffered saline at about 388C. The visible blood vessels were stripped away and adipose explants of about 5–10 mg were cut with scissors. In addition, portions of the longissimus dorsi muscle (5-cm long) just behind the last rib (at the level of the 1st and 2nd lumbar vertebrae) were removed as quickly as possible after slaughter (approx. 10 min postmortem) and kept frozen at 2 208C until analysed. Liver, heart, kidneys, adrenals and pituitary gland were removed, weighed and kept frozen at 2 208C until analysed.
2.2. Source of chemicals Sodium acetate (l- 14 C) was purchased from the Institute for Research, Production and Application of
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Radioisotopes (Prague, Czech Republic). Waymouth’s tissue culture medium 752 / 1 (Waymouth, 1959) was a gift from the Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (Prague, Czech Republic). Two forms of recombinantly derived bGH were used. The first, Somidobove (Eli Lilly / Elanco, Indianapolis, IN, USA), was supplied as a substance containing 81.9% bGH. It was solubilized in 0.5 mmol l 21 NaOH and added to culture medium to obtain a concentration of 4.5 nmol l 21 bGH as was recommended by Watt et al. (1991). The second, Sometribove (Monsanto Agricultural, St.Louis, MO, USA), was supplied as a formulated product (Somatech) in syringes (500-mg Sometribove) in a prolonged release lipid based vehicle and, in our experiments, was s.c. injected to experimental animals. Glucagon-free porcine insulin was a gift from Eli Lilly Research Labs. (Indianapolis, IN, USA). Insulin was freshly dissolved in 3 mmol l 21 HCl and added to culture medium to obtain a concentration of 17 nmol l 21 (Watt et al., 1991). Cortisol was purchased from Sigma (St. Louis, MO, USA) and dissolved in ethanol, the amount of which in culture medium never exceeded 0.05%. Cortisol concentration in culture medium was 138 nmol l 21 . Bovine serum albumin (BSA) fraction V was obtained from Armour (Kankakee, IL, USA). Norepinephrine bitartarate (Ne; Noradrenalin Spofa) and isoprenaline hydrochloride (Iso; Isoprenalin Spofa) were bought from Leciva (Prague, Czech Republic).
2.3. Tissue culture Adipose tissue explants (6–8) intended for culture were transferred to 5-ml plastic Petri dishes containing 3 ml of modified Waymouth’s medium, in which the glucose concentration was 3.5 mmol l 21 , sodium bicarbonate concentration was 10.1 mmol l 21 and medium was supplemented with 4 mmol l 21 of sodium acetate, 25 mmol l 21 of Hepes, 100 mg ml 21 of penicillin, 50 mg ml 21 of streptomycin sulphate, 2.5 mg ml 21 of amphotericin B and 1 mg ml 21 of BSA; pH 7.35. Explants were cultured at 378C under O 2 –CO 2 (95:5) for 24 h. To study the effect of different hormones during culture on the lipogenic activity, adipose tissue explants were cultured in the
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presence of insulin, cortisol and bGH alone or in different combinations, after which the explants were washed in saline (at 388C) and transferred to Krebs– Henseleit bicarbonate buffer (Krebs and Henseleit, 1932) for incubation.
2.4. Tissue incubation Fresh adipose tissue explants or cultured (and washed in saline at 388C) adipose explants were transferred to 30-ml polyethylene flasks containing 3 ml of modified Krebs–Henseleit bicarbonate buffer with half of the original calcium concentration (1.27 mmol l 21 ) and with only 10.1 mmol l 21 of sodium bicarbonate. Buffer was supplemented with 25 mmol l 21 of Hepes, 100 mg ml 21 of penicillin, 50 mg ml 21 of streptomycin sulphate, 2.5 mg ml 21 of amphotericin B, 10 mg ml 21 of BSA, 3.5 mmol l 21 of glucose and 4 mmol l 21 of sodium acetate pH 7.35; 388C. Hormones present during culture were not present during acute incubation as their presence had no effect on lipogenesis (data not shown). The lipogenic activities were assessed also in the presence and absence of either Ne (10 mmol l 21 ) or Iso (10 mmol l 21 ) to measure responsiveness to catecholamines. Tissue explants were incubated for 2 h at 388C in an atmosphere of O 2 –CO 2 (95:5, v / v) with reciprocal shaking at 90 strokes per min. All incubations were run under conditions of linear incorporation of acetate and were terminated by cooling in an ice water bath. Explants were then collected, rinsed in cold saline and frozen at 2 208C until analysed. All tissue incubations were carried out in triplicate.
2.5. Adipose tissue fatty acid synthesis Almost 100% of radioactivity from 14 C-acetate was recovered in fatty acids in incubated goat adipose tissues (Bartos and Skarda, 1970) and, therefore, the incorporation of acetate into total lipids is a good measure of the rate of fatty acid synthesis. Fatty acid synthesis was assessed by measuring incorporation of sodium (1- 14 C)acetate (20 kBq ml 21 ) into total lipids of adipose explants over a 2-h period of incubation. When the labelling period was over, the explants were removed from plastic flasks,
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rinsed with cold saline, extracted in 6 ml of chloroform–methanol (2:1, v / v) and radioactivity determined by liquid scintillation spectrometry. In the present experiments, the synthetic activities of adipose explants were expressed as nmol of acetate incorporated per mg protein per hour and statistical comparisons regarding the effect of different hormones on lipogenesis were done either for the control or bGH-treated groups of animals.
2.6. Protein determination The protein content of adipose tissue explants was determined after lipid extraction according to Lowry et al. (1951) with BSA as the standard. Lowry’s assay was also used for determination of proteins in liver (the caudate lobe), heart (muscle of the left ventricle), kidney (cortical region) and longissimus muscle (a central part of the transverse section, taken from behind the last rib).
2.7. Lipid determination Lipid contents of liver, heart, longissimus muscle and omental adipose tissue (the same portions as those used for protein assay) were determined gravimetrically after extraction according to Folch et al. (1957).
2.8. Statistical methods Results are expressed as means6S.E.M. Body weights were subjected to analysis of covariance with initial body weight as a linear covariate. ANOVA was used for statistical comparison of organ weights and contents of proteins and lipids. Values for rates of lipogenesis were analysed by a repeated measures ANOVA, factors comprising hormones in culture, catecholamines in incubation and their interaction. The least squares means and their standard errors (one for paired values for comparisons within a treatment group and a second for unpaired values for comparing placebo- and bGH-treated animals) were calculated. All calculations were carried out with the GLM Procedure (SAS, 1989).
3. Results
3.1. Body growth and tissue composition Recombinant bGH-treated animals grew faster (P , 0.05) than controls. Live weight gain over the 147-day treatment period for the control group averaged 8.5 kg per animal and, for the bGH-treated group, 12.1 kg per animal (an increase of 42%). Liver, adrenals and kidneys (adjusted for final body weights) were heavier in bGH-treated animals; however, weights of heart and pituitaries were not changed (Table 1). The composition of adipose and muscle tissues was markedly affected by bGH treatment. The concentration of total lipids per unit of omental adipose tissue (mg g 21 ) decreased by 44% due to bGH and, simultaneously, protein content increased by 160%. In the longissimus muscle, lipid content was decreased by 30% whereas protein content was not significantly affected by bGH. Lipid and protein concentrations in the liver, heart and kidneys were not affected by bGH (Table 2).
3.2. Lipogenesis in omental adipose explants The rate of fatty acid synthesis in freshly-prepared adipose explants in 2-h incubations in a modified Krebs–Henseleit buffer was established to provide both information on the effect of long term bGH treatment on fatty acid synthesis in vitro and to provide a measure of the maintenance of lipogenesis in subsequent incubations of explants cultured in the presence of different hormones. The data showed that caprine adipose tissue remained metabolically active for at least 24 h in a modified Waymouth’s tissue culture medium. Comparison of the lipogenesis of freshly-prepared explants with explants cultured in the absence of hormones for 24 h showed that the rate of incorporation of acetate into fatty acids in both control and bGH-treated animals was the same after 24 h of culture. Inclusion of hormones in culture medium changed lipogenesis in tissues from both groups of animals in a similar manner. Mean values of acetate incorporation into fatty acids in explants first cultured in different hormonal
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Table 1 Growth and organ weights of castrated male goats receiving s.c. injections of recombinant methionyl bGH in a sustained release vehicle at seven-day intervals for 147 days Control
Initial body weight, kg Final body weight, kg Gain, kg Liver wt., g Relative liver weight a , % Heart wt., g Relative heart wt., % Adrenal wt., g Relative adrenal wt., % Kidney wt., g Relative kidney wt., % Pituitary gland wt, mg Relative pituitary gland wt., %
bGH
n
x¯ 6S.E.M.
n
x¯ 6S.E.M.
11 11 11 11 11 11 11 11 11 4 4 4 4
14.561.0 23.061.2 8.560.9 377.5618.1 1.6560.06 84.265.4 0.36560.011 1.4860.08 0.006560.0003 68.862.3 0.30860.014 18566 0.0008460.00003
11 11 11 11 11 11 11 11 11 4 4 4 4
14.261.3 NS 26.361.9 * 12.161.0 *** 497.6625.8 *** 1.9260.04 *** 95.867.0 * 0.36560.011 NS 2.0160.12 ** 0.007860.0004 * 88.365.4 * 0.36860.029 * 226625 NS 0.0008960.00007 NS
Czech white male goats were castrated at two to three weeks of age and used for experiments between two and three months of age. Control animals were treated s.c. with olive oil. Values are means6S.E.M. a Relative weights are organ weights expressed as a percentage of final body weight. * P,0.05; ** P,0.01; *** P,0.001; NS 5not significant.
Table 2 Organ composition of castrated male goats receiving s.c. injections of recombinant methionyl bGH in a sustained release vehicle at seven-day interval for 147 days Control
bGH
n
x¯ 6S.E.M.
n
x¯ 6S.E.M.
Liver Fat (mg / g) Protein (mg / g)
11 11
4261 17668
11 11
4562 NS 16466 NS
Adipose tissue Fat (mg / g) Protein (mg / g)
11 11
780620 1561
11 11
512660 ** 3969 *
Skeletal muscle a Fat (mg / g) Protein (mg / g)
11 11
2062 19364
11 11
1461 * 19665 NS
Heart Fat (mg / g) Protein (mg / g)
11 11
2962 14665
11 11
2461 NS 15368 NS
Kidney Fat (mg / g) Protein (mg / g)
4 4
2761 14366
4 4
2861 NS 144610 NS
Animals were castrated and treated as described in Table 1. a Skeletal muscle was m. longissimus dorsi, sample taken just behind the last rib. * P,0.05; ** P,0.01; NS 5not significant.
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combinations and then incubated in the absence of catecholamines were consistently lower (P , 0.0001) in bGH-treated animals than in controls (overall means – control, 57.064.23; bGH, 26.7364.23 nmol mg 21 h 21 ; Table 3). Fatty acid synthesis in explants cultured in the absence of hormones was taken as a basic measure of lipogenic response to different hormones added to the culture medium. Addition of insulin stimulated (44.8%) the rate of fatty acid synthesis in tissues of control animals but a much lower (22.2%) effect of insulin was observed in bGH-treated animals. However, these effects of insulin were not significant. The presence of cortisol alone in culture medium decreased lipogenesis in explants from both control (P , 0.05) and bGH-treated (non-significantly) animals. In tissues from bGH-treated animals, cortisol acted synergistically with insulin to produce a higher rate of lipogenesis than that observed in cultures with insulin alone. In control animals, however, no significant effect of cortisol plus insulin on fatty acid synthesis was noted when compared with insulin alone (Table 3). With the combination of insulin plus bGH, the rates of lipogenesis in tissues from control animals were lower than those cultured with insulin alone. No such inhibitory effect of bGH was noted in tissues from bGH-treated animals. When goat adipose explants were maintained in a modified Waymouth’s medium in the presence of insulin plus cortisol plus bGH the rates of lipogeneses were always lower (P , 0.05) than those in cultures with insulin plus cortisol in both control and bGH-treated animals. In vitro rates of lipogenesis were decreased (P , 0.05) by catecholamines in freshly-prepared adipose tissue explants of both control and bGH-treated animals. Iso decreased (P , 0.05) the rate of fatty acid synthesis (5.2560.76 nmol mg 21 h 21 ) more than Ne (13.3661.70 nmol mg 21 h 21 ) in bGHtreated animals but not in controls. The mean values of lipogenesis in explants cultured in the presence of different hormonal combinations and then incubated in the presence of Ne or Iso are shown in Table 3. The difference between pooled means of acetate incorporation into fatty acids in the presence of Ne in control (9.6261.70 nmol mg 21
h 21 ) and bGH-treated animals (13.3661.70 nmol mg 21 h 21 ) was not significant (P , 0.10); however, the rate of lipogenesis in explants incubated in the presence of Iso was lower (P , 0.05) in bGH-treated (5.2560.76 nmol mg 21 h 21 ) than in control animals (8.4160.76 nmol mg 21 h 21 ). These results suggest that Ne and Iso were equally effective in control goats whereas, in GH-treated goats, Iso was more effective than Ne.
4. Discussion
4.1. Growth performance, organ weights and tissue chemical composition The growth rate of GH-treated lambs, growing steers, heifers and bulls has been very variable between studies: cattle ranging from 3–25% and lambs ranging from 0–45% above controls (Enright, 1989; Zainur et al., 1989a,b). The increase in growth rate in our long term bGH-treated goats (42%) is in agreement with some previous reports in lambs. Likewise, the decrease in the proportion of fat in the muscle of bGH-treated goats by 30% agrees with previous reports in cattle and sheep (32%; Wagner and Veenhuizen, 1978; Wise et al., 1988; Beermann et al., 1990). In goats, the increase in gain was accompanied by a decrease in muscle fat but this was not always demonstrated in lambs. For example, no effect of GH on fat was observed by Pullar et al. (1986) even though GH increased daily gain by 30%. Organ weights were expressed as a fraction of final liveweight and therefore normalized. The significant positive effect of GH on liver weight in goats was similar to previous studies with lambs, heifers and steers (Wagner and Veenhuizen, 1978; Sandles and Peel, 1987; Beermann et al., 1990; Early et al., 1990; Pell et al., 1990; Bass et al., 1991; McLaughlin et al., 1993; Schwarz et al., 1993) and pigs (Klindt et al., 1992; Lefaucheur et al., 1992). Heavier livers in GH-treated animals could reflect the increased rates of direct and indirect (e.g., by IGF-I) GH-mediated changes in liver metabolism (e.g., increased gluconeogenesis, increased release of nutrient to support increased growth, etc.). Kidney weight was increased in bGH-treated
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Table 3 Effects of long-term treatment with bGH in vivo and chronic (24 h) culture in the presence of various hormones on the rate of lipogenesis during acute incubations (2 h) in the presence and absence of noradrenaline and isoprenaline in adipose explants from young castrated male goats Acetate incorporation into fatty acids (nmol mg 21 h 21 ) Culture
Incubation variables
variables
No catecholamines
Ne
Iso
Placebo-treated animals Before culture No hormones
63.20 ABCa
18.71 Ab
After culture No hormones Insulin (I) bGH Cortisol (H) I1H I1bGH I1H1bGH
56.15 ABCa 81.33 ABa 40.65 CDa 19.89 Da 88.86 Aa 58.20 ABCa 47.75 BCDa
5.24 Bb 8.83 ABb 6.01 ABb 10.43 ABac 13.70 ABb 5.97 ABb 8.09 ABb
Overall mean
57.00 a
bGH-treated animals Before culture No hormones
40.22 ABa
19.22 ABb
6.85 ABbc
After culture No hormones Insulin bGH Cortisol I1H I1bGH I1H1bGH
27.05 ABa 33.06 ABa 13.65 Ba 8.44 Bac 51.34 Aa 25.31 ABa 14.79 Ba
12.63 ABb 13.39 ABb 10.03 ABac 12.42 ABab 22.62 Ab 6.62 Bb 9.98 ABac
6.08 ABb 8.48 Ab 3.28 ABbc 1.74 Bc 5.59 ABc 6.70 ABb 3.30 ABbc
Overall mean
26.73 a
13.36 b
5.25 c
Pooled S.E.M. 1 Pooled S.E.M. 2
11.97 4.23
4.81 1.70
2.15 0.76
Effect bGH treatment
P,0.0001
9.62 b
NS
9.25 ABb 9.04 ABb 12.46 Ab 6.04 Bb 4.41 Bbc 10.44 ABb 8.50 ABb 7.17 ABb 8.41 b
P,0.0052
Omental adipose explants of four control (placebo-treated) and four bGH-treated (100 mg of recombinant methionyl bGH in a sustained release vehicle at seven-day intervals for 140 days) animals were exposed before and after culture to (1- 14 C)acetate (20 kBq ml 21 ) for a 2-h period of incubation in the presence or absence of noradrenaline (Ne; 10 mmol l 21 ) or isoprenaline hydrochloride (Iso; 10 mmol l 21 ) in Krebs–Henseleit buffer supplemented with sodium acetate (4 mmol l 21 ) and glucose (3.5 mmol l 21 ). Cultured explants were maintained in tissue culture for 24 h in a modified Waymouth’s medium (containing 3.5 mmol l 21 glucose) in the absence of hormones or in the presence of insulin (17 nmol l 21 ), recombinant bGH (Somidobove; 4.5 nmol l 21 ) and cortisol (138 nmol l 21 ) alone or in different combinations. All values are the means of four experiments, each performed in triplicate, and expressed per mg protein. For each column of results, pooled S.E.M. 1 (standard error LS mean for paired values) is for comparison of mean values from animals within the same treatment (placebo- or bGH-treated); pooled S.E.M. 2 (standard error LS mean for unpaired values) is for comparing mean values of placebo-treated with those for bGH-treated animals. Values within a column which do not have the same upper case superscript (A, B, C, D) differ significantly (P,0.05). Values within a row which do not have the same lower case superscript (a, b, c) differ significantly (P,0.05).
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castrated goats similar to what was observed in bGH-treated heifers (Schwarz et al., 1993), porcine GH-treated pigs (Klindt et al., 1992; Lefaucheur et al., 1992) and acromegalic humans (Binnerts et al., 1988). However, in GH-treated lambs, kidney weight was increased variably; non-significantly increased in experiments by Zainur et al. (1989a); Pell et al. (1990) and Bass et al. (1991), and significantly increased in experiments by McLaughlin et al. (1993). Increased kidney weights possibly reflected increased glomerular filtration. The absolute weights of hearts were increased in bGH-treated goats; however, fractional weights were not affected by treatment as was seen in lambs (Zainur et al., 1989a,b; Pell et al., 1990) and pigs (Klindt et al., 1992). However, in cases of acromegaly in humans, an excess of GH increased the fractional weights of the heart (Steele and EvockClover, 1993). GH-treated castrated young goats had heavier adrenal glands than controls and this could reflect an enhanced secretion of corticosteroids and / or catecholamines. Heavier adrenal glands in porcine GH-treated pigs have been previously reported (Sillence and Etherton, 1989; Lefaucheur et al., 1992).
4.2. Regulation of lipogenesis in adipose explants Goat adipose tissue remained metabolically active when maintained in culture in a modified Waymouth’s tissue culture medium as was seen for ovine or bovine adipose tissue maintained in medium 199 (Vernon, 1979; Etherton et al., 1987). The present study has shown that chronic administration of recombinant bGH in vivo can lead to a loss of lipid from omental adipose tissue (and from longissimus muscle) of castrated male goats. Acute incubation of both fresh and cultured omental adipose tissue explants in the presence of (1- 14 C)acetate demonstrated that loss of lipid in bGH-treated animals could at least in part result from the reduced rate of fatty acid synthesis. The mean values of acetate incorporation into fatty acids in explants cultured in the presence of different hormonal combinations were consistently lower in bGH-treated animals than in controls. The direct comparison of the rate of lipogenesis between control and bGHtreated animals presents a problem. It is not accurate under conditions of expressing data per unit number
of adipocytes or per unit weight of DNA or protein content, because non-adipocytes comprise the majority of cells in the tissue; however, adipocytes account for the majority of metabolic activities (Vernon et al., 1987). Results from our studies with adipose explants of both control and bGH-treated animals in chronic tissue culture (24 h) show that bGH has an inhibitory action on lipogenesis before any detectable change in adipocyte size can be expected. Similarly, Borland et al. (1994) have demonstrated with sheep adipose tissue explants that GH caused a decrease in the rate of lipogenesis after a short lag phase of 6 h. The mechanism by which GH decreases lipogenesis is not clear. There is a close relationship between lipogenesis and glucose utilization. In both non-ruminants and ruminants, GH decreases uptake, oxidation and utilization of glucose, due in part to decreased synthesis of glucose transporters (Goodman, 1993; Vernon et al., 1995; Zhao et al., 1996). GH can also affect activity and insulin activation of key lipogenic enzymes such as acetyl-CoA carboxylase (Vernon et al., 1991). In addition, we observed that bGH antagonized insulin action on lipogenesis. It is probable that bGH antagonizes the effects of insulin via some insulindependent expression of glucose transporter gene or via insulin-dependent enzymes in the pathway of both glucose utilization and fatty acid synthesis. In bovine adipose tissue, bGH also antagonized the ability of insulin to maintain lipogenic capacity (Etherton et al., 1987). This antagonism was, however, dependent upon the presence of cortisol. On the other hand, in pig (Walton et al., 1986), sheep (Vernon and Taylor, 1988) and goat adipose tissue (present study), the inhibitory effect of GH on insulin action was seen both in the presence of insulin alone and in the presence of insulin plus cortisol. The addition of cortisol alone to the culture medium decreased lipogenesis in both control and bGH-treated animals. A similar inhibitory effect of cortisol has been found in cultured bovine (Etherton et al., 1987), ovine (Vernon and Finley, 1988) and pig (Walton et al., 1986) adipose tissue. Glucocorticoids are other hormones which act on lipogenesis as a glucose antagonist and it has been shown that they reduce cellular glucose uptake and utilization (Vernon and Taylor, 1988). Glucocorticoids act by
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inhibiting the glucose transport per se, perhaps by affecting glucose transporter (GLUT4) subcellular trafficking (Weinstein et al., 1995). In explants from our placebo-treated animals, an inhibitory effect of cortisol on lipogenesis was eliminated by the simultaneous addition of insulin. However, in bGH-treated animals, insulin acted synergistically with cortisol to produce a higher rate of lipogenesis than that in the presence of insulin alone. Synergism between insulin and cortisol was also observed in non-lactating sheep (Vernon and Finley, 1988), heifers (Etherton et al., 1987) and pigs (Walton et al., 1986). It was suggested that glucocorticoids can potentiate the effects of insulin by enhancing the level of an intracellular mediator of insulin action (Walton et al., 1986) and by stimulating synthesis of insulin receptors and insulin binding (Salhanick et al., 1983; Wastie et al., 1995). Glucocorticoids can potentiate the responses of glucose-6-phosphate dehydrogenase, L-pyruvate kinase and acetyl-CoA carboxylase to insulin (Hillgartner et al., 1995) and, thus, fatty acid synthesis from acetate could be stimulated by enzymes involved in both glucose utilization and fatty acid synthesis. In control goats, Ne and Iso were equally effective whereas, in bGH-treated goats Iso was more effective than Ne. These results are consistent with an increase in the number of b-adrenergic receptors in sheep adipocytes (Watt et al., 1991) and increased isoprenaline-stimulated lipolysis in bovine adipose tissue cultured in the presence of bGH (Lanna et al., 1995). The higher activity of Iso in bGH-treated animals could also be due to GH increasing the number of a 2 -adrenergic receptors (Vernon et al., 1995), which could limit effects of Ne but not Iso.
Acknowledgements I am grateful to Dr. H. Mader and Dr. P. Kubasa from Eli Lilly and Elanco Ges, Vienna, Austria and to Dr. R.J. Collier and Dr. D.L. Hard from Monsanto, St. Louis, MO, USA for the gifts of recombinant bovine GH. I thank Dr. W.W. Bromer from Eli Lilly for the gift of porcine insulin. The author is grateful to E. Urbanova, Ing. P. Krejci and Ing. J. Slaba for their technical assistance and Dr. J. Wolf
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for the statistical analysis. I would like to acknowledge the Grant Agency of the Academy of Sciences of the Czech Republic for funding (project No. A7-045608).
References Bartos, S., Skarda, J., 1970. Effect of insulin on metabolism of adipose tissue in goats during ontogeny. Biol. Neonate 16, 209–214. Bass, J.J., Oldman, J.M., Hodgkinson, S.C., Fowke, P.J., Sauerwein, H., Molan, P., Breier, B.H., Gluckman, P.D., 1991. Influence of nutrition and bovine growth hormone (GH) on hepatic GH binding, insulin-like growth factor-I and growth of lambs. J. Endocrinol. 128, 181–186. Bauman, D.E., Currie, W.B., 1980. Partitioning in nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis and homeorhesis. J. Dairy Sci. 63, 1514–1529. Beermann, D.H., Houge, D.E., Fishell, V.K., Aronica, S., Dickson, H.W., Schricker, B.R., 1990. Exogenous human growth hormone-releasing factor and ovine somatotropin improve growth performance and composition of gain in lambs. J. Anim. Sci. 68, 4122–4133. Bidanel, J.-P., Bonneau, M., Pointillart, A., Gruand, J., Mourot, J., Demade, I., 1991. Effects of exogenous porcine somatotropin (pST) administration on growth performance, carcass traits and pork meat quality of Meishan, Pietrain and crossbred gilts. J. Anim. Sci. 69, 3511–3522. Binnerts, A., Wilson, J.H., Lamberts, S.W., 1988. The effects of human growth hormone administration in elderly adults with recent weight loss. J. Clin. Endocrinol. Metab. 67, 1312–1316. Borland, C.A., Barber, M.C., Travers, M.T., Vernon, R.G., 1994. Growth hormone inhibition of lipogenesis in sheep adipose tissue: requirement for gene transcription and polyamines. J. Endocrinol. 142, 235–243. Early, R.J., McBride, B.W., Ball, R.O., 1990. Growth and metabolism in somatotropin-treated steers: III. Protein synthesis and tissue energy expenditures. J. Anim. Sci. 68, 4153–4166. Enright, W.J., 1989. Effects of administration of somatotropin on growth, feed efficiency and carcass composition of ruminants: A review. In: Sejrsen, K., Vestergaard, M., Neimann-Sorensen, A. (Eds.), Use of Somatotropin in Livestock Production. Elsevier Applied Science, London, pp. 132–156. Etherton, T.D., Evock, C.M., Kensinger, R.S., 1987. Native and recombinant bovine growth hormone antagonize insulin action in cultured bovine adipose tissue. Endocrinology 121, 699– 703. Folch, G., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Goodman, H.M., 1993. Growth hormone and metabolism. In: Schreibman, M.P., Scanes, C.G., Pang, P.K.T. (Eds.), The Endocrinology of Growth, Development and Metabolism in Vertebrates. Academic Press, San Diego, CA, pp. 93–115.
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Hillgartner, F.B., Salati, L.M., Goodridge, A.G., 1995. Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis. Physiol. Rev. 75, 47–76. Jones, G.M., Larsen, R.E., Javed, A.H., Donefer, E., Gaudreau, J.M., 1973. Voluntary intake and nutrient digestibility of forages by goats and sheep. J. Anim. Sci. 34, 830–838. Klindt, J., Buonomo, F.C., Yen, J.T., 1992. Administration of porcine somatotropin by a sustained-release implant: growth and endocrine reponses in genetically lean and obese barrows and gilts. J. Anim. Sci. 70, 3721–3733. Krebs, H.A., Henseleit, M.K., 1932. Untersuchungen uber die Harnstoffbildung im Tierkorper. (Investigation of urea formation in animals). Hoppe-Seyler’s Zeitsch. Physiol. Chem. 210, 33–66. Lanna, D.P.D., Houseknecht, K.L., Haris, D.M., Bauman, D.E., 1995. Effect of somatotropin treatment on lipogenesis, lipolysis, and related cellular mechanisms in adipose tissue of lactating cows. J. Dairy Sci. 78, 1703–1713. Larson, B.L., 1978. The dairy goat as a model in lactation studies. J. Dairy Sci. 61, 1032–1039. Lefaucheur, L., Missohon, A., Ecolan, P., Monin, G., Bonneau, M., 1992. Performance, plasma hormones, histochemical and biochemical muscle traits and meat quality of pigs administered exogenous somatotropin between 30 or 60 kilograms and 100 kilograms body weight. J. Anim. Sci. 70, 3401–3411. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. McLaughlin, C.L., Hedrick, H.B., Veenhuizen, J.J., Finn, R.F., Hintz, R.L., Hartnell, G.F., Kasser, T.R., Baile, C.A., 1993. Comparison of performance, clinical chemistry, and carcass characteristics of finishing lambs treated with recombinant ovine or bovine somatotropins. J. Anim. Sci. 71, 1453–1463. Pell, J.M., Elcock, C., Harding, R.L., Morrell, D.J., Simonds, A.D., Wallis, M., 1990. Growth, body composition, hormonal and metabolic status in lambs treated long-term with growth hormone. Br. J. Nutr. 63, 431–445. Pullar, R.A., Johnsson, I.D., Chadwick, P.M.C., 1986. Recombinant bovine somatotropin is growth promoting and lipolytic in fattening lambs. Anim. Prod. 42 (Abstr.), 433–434. Salhanick, A.I., Krupp, M.N., Amatruda, J.M., 1983. Dexamethasone stimulates insulin receptor synthesis in cultured rat hepatocytes. J. Biol. Chem. 258, 14130–14135. Sandles, L.D., Peel, C.J., 1987. Growth and carcass composition of pre-pubertal dairy heifers treated with bovine growth hormone. Anim. Prod. 44, 21–27. SAS, 1989: The GLM Procedure. In: SAS / STAT User’s Guide, Version 6, Vol. 2, 4th ed. SAS. Inc., Cary, NC, pp. 891–996. Scott, C.D., Martin, J.L., Baxter, R.C., 1985. Rat hepatocyte insulin-like growth factor I and binding protein: effects of growth hormone in vitro and in vivo. Endocrinology 116, 1102–1107. Sechen, S.J., McCutcheon, S.N., Bauman, D.E., 1989. Response to metabolic challenges in early lactation dairy cows during treatment with bovine somatotropin. Dom. Anim. Endocr. 6, 141–154.
Schwarz, F.J., Schams, D., Ropke, R., Kirchgessner, M., Kogel, J., Matzke, P., 1993. Effects of somatotropin treatment on growth performance, carcass traits, and the endocrine system in finishing beef heifers. J. Anim. Sci. 71, 2721– 2731. Sillence, M.N., Etherton, T.D., 1989. Chronic effects of recombinant porcine growth hormone on adrenal weight and activity in pigs. J. Anim. Sci. 67, 1740–1743. Steele, N.C., Evock-Clover, C.M., 1993. Role of growth hormone in growth of homeotherms. In: Schreibman, M.P., Scanes, C.G., Pang, P.K.T. (Eds.), The Endocrinology of Growth, Development and Metabolism in Vertebrates. Academic Press, New York, pp. 73–91. Tolkamp, B.J., Brouwer, B.O., 1993. Statistical review of digestion in goats compared with other ruminants. Small Rumin. Res. 11, 107–123. Vernon, R.G., 1979. Metabolism of ovine adipose tissue in tissue culture. Int. J. Biochem. 10, 57–60. Vernon, R.G., Faulkner, A., Finley, E., Pollock, H., Taylor, E., 1987. Enzymes of glucose and fatty acid metabolism of liver, kidney, skeletal muscle, adipose tissue and mammary gland of lactating and non-lactating sheep. J. Anim. Sci. 64, 1395– 1411. Vernon, R.G., Finley, E., 1988. Roles of insulin and growth hormone in the adaptations of fatty acid synthesis in white adipose tissue during the lactation cycle in sheep. Biochem. J. 256, 873–878. Vernon, R.G., Taylor, E., 1988. Insulin, dexamethasone and their interactions in the control of glucose metabolism in adipose tissue from lactating and nonlactating sheep. Biochem. J. 256, 509–514. Vernon, R.G., Barber, M.C., Finley, E., 1991. Modulation of the activity of acetyl-CoA carboxylase and other lipogenic enzymes by growth hormone, insulin and dexamethasone in sheep adipose tissue and relationship to adaptations to lactation. Biochem. J. 274, 543–548. Vernon, R.G., Faulkner, A., Finley, E., Watt, P.W., Zammit, V.A., 1995. Effect of prolonged treatment of lactating goats with bovine somatotropin on aspects of adipose tissue and liver metabolism. J. Dairy Sci. 62, 237–248. Wagner, J.F., Veenhuizen, E.L., 1978. Growth performance, carcass composition and plasma hormone levels in wether lambs when treated with growth hormone and thyrotropin. J. Anim. Sci. 45 (Abstr.) (Suppl. 1), 397. Walton, P.E., Etherton, T.D., Evock, C.M., 1986. Antagonism of insulin action in cultured pig adipose tissue by pituitary and recombinant porcine growth hormone: potentiation by hydrocortisone. Endocrinology 118, 2577–2581. Wastie, S., Buttery, P.J., Vernon, R.G., 1995. Glucocorticoids and insulin but not growth hormone modulate insulin binding to adipocyte membranes from sheep. Comp. Biochem. Physiol. 111C, 13–18. Watt, P.W., Finley, E., Cork, S., Clegg, R.A., Vernon, R.G., 1991. Chronic control of the b- and a 2 -adrenergic systems of sheep adipose tissue by growth hormone and insulin. Biochem. J. 273, 39–42.
ˇ / Livestock Production Science 55 (1998) 215 – 225 J. Skarda Waymouth, C., 1959. Rapid proliferation of subline of NCTC colony 929 (strain L) mouse cells in a simple chemically defined medium (MB 752 / 1). J. Natl. Cancer Inst. 22, 1003– 1017. Weil, R., 1965. Pituitary growth hormone and intermediary metabolism. I. The hormonal effect on the metabolism of fat and carbohydrate. Acta Endocrinol. 49 (Suppl. 98), 1–92. Weinstein, S.P., Paquin, T., Pritsker, A., Haber, R.S., 1995. Glucocorticoid-induced insulin resistence: dexamethasone inhibits the activation of glucose transport in rat skeletal muscle by both insulin and non-insulin-related stimuli. Diabetes 44, 441–445. Wise, D.F., Kensinger, R.S., Harpster, H.W., Schricker, B.R., Carbaugh, D.E., 1988. Growth performance and carcass merit of lambs treated with growth hormone-releasing factor (GRF) or somatotropin (ST). J. Anim. Sci. 66 (Abstr.) (Suppl. 1), 275.
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Zainur, A.S., Tassell, R., Kellaway, R.C., Dodemaide, W.R., 1989. Recombinant growth hormone in growing lambs: effect on growth, feed utilization, body and carcass characteristics and on wool growth. Aust. J. Agric. Res. 40, 195–206. Zainur, A.S., Tassell, R., Kellaway, R.C., McDowell, G., Dodemaide, W.R., Kirby, A.C., 1989. Recombinant growth hormone in growing lambs: effects on growth, body and carcass characteristics, hormone and metabolites. Aust. J. Agric. Res. 40, 663–674. Zhao, F.-Q., Kennelly, J.J., Moseley, W.M., Tucker, H.A., 1996. Regulation of the gene expression of glucose transporter in liver and kidney of lactating cows by bovine growth hormone and bovine growth hormone-releasing factor. J. Dairy Sci. 79, 1537–1542.
Effect of bovine growth hormone on growth, organ weights, tissue composition and adipose tissue metabolism in young castrated male goats 1 ˇ J. Skarda
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 104 00 Prague 10, Czech Republic Received 5 May 1997; accepted 17 March 1998
Abstract Young castrated male goats (n 5 22) were used to investigate the effects of administration of a sustained release recombinant bovine growth hormone (GH; 100 mg) at seven-day intervals on body growth, organ weights and lipid content, and on lipogenic responses of cultured explants of omental adipose tissue. Average body weight gain was improved by 22% and the relative weights of the liver, kidney and adrenal glands were increased 16, 19 and 20%, respectively, by GH treatment. The fat content in m. longissimus dorsi and omentum was less in GH-treated animals than in controls by 29.5 and 44.4%, respectively. The rate of fatty acid synthesis was determined in acute incubations both in freshly prepared and chronically cultured omental adipose explants. Adipose explants remained metabolically active and retained their ability to respond to hormones when maintained in tissue culture medium. Cortisol acted synergistically with insulin to produce a higher rate of lipogenesis than that in cultures with insulin alone, but this was the case only in tissues from GH-treated animals; however, cortisol alone decreased lipogenesis in explants from both groups of animals. GH inhibited insulinstimulated lipogenesis in tissues from control animals and insulin plus cortisol-stimulated lipogenesis in tissues from both control and GH-treated animals. GH treatment in vivo and in vitro did not increase the explant’s responsiveness to noradrenaline in vitro; however, responsiveness (inhibition of lipogenesis) to isoprenaline was greater in GH-treated animals than in controls. 1998 Elsevier Science B.V. Keywords: Goat; Growth; Adipose tissue; Lipogenesis; Growth hormone; Cortisol
1. Introduction Many aspects of metabolism in ruminants and in a variety of animal species are influenced by growth hormone (GH). In the short term, GH acts to preserve body protein and glucose pools during a 1
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period of nutrient deprivation at the expense of fat which is mobilized for the provision of material for energy generation and for the formation of the carbon skeleton of non-essential amino acids (Weil, 1965). In the long term, GH has a vital homeorhetic function to channel nutrients to support such complex processes as growth, pregnancy or lactation. Under GH control, the absorbed and depot nutrients are directed toward or diverted from different tissues
0301-6226 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 98 )00138-9
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in a highly coordinated manner (Bauman and Currie, 1980). The mechanisms involved have not been clearly defined. GH can act in tissues directly on the cells themselves or indirectly through the actions of locally and systemically produced insulin-like growth factor I (IGF-I) and IGF binding proteins (IGFBPs) (Scott et al., 1985) or through the alterations of the cell’s ability to respond to homeostatic signals such as from insulin and catecholamines (Sechen et al., 1989). Most of the effects of GH in ruminants have been reported for sheep and cattle, and very little is known about GH action in goats and other ruminant species. There are, however, considerable differences in food intake and rates of gastrointestinal and tissue metabolism between different ruminant species. For example, overall feed digestibility in goats is higher than in sheep and tends to be higher in low protein diets (Tolkamp and Brouwer, 1993). Molar % of acetate in the rumen is higher in goats but molar % of propionate and butyrate is higher in sheep (Jones et al., 1973). There are considerable differences in the rate of metabolism of cows, sheep and goats. High-producing dairy goats may consume approximately twice as much feed per unit of body weight as dairy cows, with a greater proportion of this total feed consumption being utilized for milk production (Larson, 1978). Ruminant adipose tissue responsiveness to GH and catecholamines may be changed in a species-specific manner and one can even expect the existence of breed variations in domestic ruminants, as was shown for pigs (Bidanel et al., 1991). Thus, it is of interest to compare the responses to GH in cattle and sheep with those in goats. The purpose of the present experiments was to document the effect of long-term GH treatment on growth, carcass composition and lipogenesis in omental adipose tissue of young castrated male goats. Cultures with adipose tissue explants were conducted to determine whether GH treatment in vivo affects lipogenesis directly and how GH treatment in vivo affects lipogenic responses of explants cultured in the presence of insulin, cortisol and GH alone or in different combinations (insulin plus cortisol; insulin plus GH; insulin plus cortisol plus GH). In addition, the abilities of these hormones to modulate the lipogenic responses to catecholamines
in explants from control and GH-treated animals were compared in acute incubations.
2. Materials and methods
2.1. Animals and general procedures The experiments were carried out with young castrated male goats (Czech white breed). Twentytwo animals were castrated at 2–3 weeks of age and used for experiments between two and eight months of age. Animals were arranged in pairs (placebotreated and bGH-treated) based on age (pairs did not differ by more than 1 d in age) and body weight (pairs did not differ by more than 2 kg body weight). Animals had free access to grass hay and fresh grass. A concentrate mixture (oats and barley meal) was given two times daily according to body weight (from 0.2 to 0.4 kg day 21 animal 21 ). Starting between two and three months of age, placebotreated animals were injected subcutaneously (s.c.) with olive oil and bGH-treated animals received 100 mg of recombinant methionyl bGH (sometribove) in a lipid-based sustained release vehicle at seven-day intervals in a 147-day experiment. Body weights were recorded weekly. After 147 days of treatment all animals were exsanquinated following stunning with a captive bolt. The adipose tissue of the greater omentum was aseptically removed and placed immediately into sterile phosphate buffered saline at about 388C. The visible blood vessels were stripped away and adipose explants of about 5–10 mg were cut with scissors. In addition, portions of the longissimus dorsi muscle (5-cm long) just behind the last rib (at the level of the 1st and 2nd lumbar vertebrae) were removed as quickly as possible after slaughter (approx. 10 min postmortem) and kept frozen at 2 208C until analysed. Liver, heart, kidneys, adrenals and pituitary gland were removed, weighed and kept frozen at 2 208C until analysed.
2.2. Source of chemicals Sodium acetate (l- 14 C) was purchased from the Institute for Research, Production and Application of
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Radioisotopes (Prague, Czech Republic). Waymouth’s tissue culture medium 752 / 1 (Waymouth, 1959) was a gift from the Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic (Prague, Czech Republic). Two forms of recombinantly derived bGH were used. The first, Somidobove (Eli Lilly / Elanco, Indianapolis, IN, USA), was supplied as a substance containing 81.9% bGH. It was solubilized in 0.5 mmol l 21 NaOH and added to culture medium to obtain a concentration of 4.5 nmol l 21 bGH as was recommended by Watt et al. (1991). The second, Sometribove (Monsanto Agricultural, St.Louis, MO, USA), was supplied as a formulated product (Somatech) in syringes (500-mg Sometribove) in a prolonged release lipid based vehicle and, in our experiments, was s.c. injected to experimental animals. Glucagon-free porcine insulin was a gift from Eli Lilly Research Labs. (Indianapolis, IN, USA). Insulin was freshly dissolved in 3 mmol l 21 HCl and added to culture medium to obtain a concentration of 17 nmol l 21 (Watt et al., 1991). Cortisol was purchased from Sigma (St. Louis, MO, USA) and dissolved in ethanol, the amount of which in culture medium never exceeded 0.05%. Cortisol concentration in culture medium was 138 nmol l 21 . Bovine serum albumin (BSA) fraction V was obtained from Armour (Kankakee, IL, USA). Norepinephrine bitartarate (Ne; Noradrenalin Spofa) and isoprenaline hydrochloride (Iso; Isoprenalin Spofa) were bought from Leciva (Prague, Czech Republic).
2.3. Tissue culture Adipose tissue explants (6–8) intended for culture were transferred to 5-ml plastic Petri dishes containing 3 ml of modified Waymouth’s medium, in which the glucose concentration was 3.5 mmol l 21 , sodium bicarbonate concentration was 10.1 mmol l 21 and medium was supplemented with 4 mmol l 21 of sodium acetate, 25 mmol l 21 of Hepes, 100 mg ml 21 of penicillin, 50 mg ml 21 of streptomycin sulphate, 2.5 mg ml 21 of amphotericin B and 1 mg ml 21 of BSA; pH 7.35. Explants were cultured at 378C under O 2 –CO 2 (95:5) for 24 h. To study the effect of different hormones during culture on the lipogenic activity, adipose tissue explants were cultured in the
217
presence of insulin, cortisol and bGH alone or in different combinations, after which the explants were washed in saline (at 388C) and transferred to Krebs– Henseleit bicarbonate buffer (Krebs and Henseleit, 1932) for incubation.
2.4. Tissue incubation Fresh adipose tissue explants or cultured (and washed in saline at 388C) adipose explants were transferred to 30-ml polyethylene flasks containing 3 ml of modified Krebs–Henseleit bicarbonate buffer with half of the original calcium concentration (1.27 mmol l 21 ) and with only 10.1 mmol l 21 of sodium bicarbonate. Buffer was supplemented with 25 mmol l 21 of Hepes, 100 mg ml 21 of penicillin, 50 mg ml 21 of streptomycin sulphate, 2.5 mg ml 21 of amphotericin B, 10 mg ml 21 of BSA, 3.5 mmol l 21 of glucose and 4 mmol l 21 of sodium acetate pH 7.35; 388C. Hormones present during culture were not present during acute incubation as their presence had no effect on lipogenesis (data not shown). The lipogenic activities were assessed also in the presence and absence of either Ne (10 mmol l 21 ) or Iso (10 mmol l 21 ) to measure responsiveness to catecholamines. Tissue explants were incubated for 2 h at 388C in an atmosphere of O 2 –CO 2 (95:5, v / v) with reciprocal shaking at 90 strokes per min. All incubations were run under conditions of linear incorporation of acetate and were terminated by cooling in an ice water bath. Explants were then collected, rinsed in cold saline and frozen at 2 208C until analysed. All tissue incubations were carried out in triplicate.
2.5. Adipose tissue fatty acid synthesis Almost 100% of radioactivity from 14 C-acetate was recovered in fatty acids in incubated goat adipose tissues (Bartos and Skarda, 1970) and, therefore, the incorporation of acetate into total lipids is a good measure of the rate of fatty acid synthesis. Fatty acid synthesis was assessed by measuring incorporation of sodium (1- 14 C)acetate (20 kBq ml 21 ) into total lipids of adipose explants over a 2-h period of incubation. When the labelling period was over, the explants were removed from plastic flasks,
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rinsed with cold saline, extracted in 6 ml of chloroform–methanol (2:1, v / v) and radioactivity determined by liquid scintillation spectrometry. In the present experiments, the synthetic activities of adipose explants were expressed as nmol of acetate incorporated per mg protein per hour and statistical comparisons regarding the effect of different hormones on lipogenesis were done either for the control or bGH-treated groups of animals.
2.6. Protein determination The protein content of adipose tissue explants was determined after lipid extraction according to Lowry et al. (1951) with BSA as the standard. Lowry’s assay was also used for determination of proteins in liver (the caudate lobe), heart (muscle of the left ventricle), kidney (cortical region) and longissimus muscle (a central part of the transverse section, taken from behind the last rib).
2.7. Lipid determination Lipid contents of liver, heart, longissimus muscle and omental adipose tissue (the same portions as those used for protein assay) were determined gravimetrically after extraction according to Folch et al. (1957).
2.8. Statistical methods Results are expressed as means6S.E.M. Body weights were subjected to analysis of covariance with initial body weight as a linear covariate. ANOVA was used for statistical comparison of organ weights and contents of proteins and lipids. Values for rates of lipogenesis were analysed by a repeated measures ANOVA, factors comprising hormones in culture, catecholamines in incubation and their interaction. The least squares means and their standard errors (one for paired values for comparisons within a treatment group and a second for unpaired values for comparing placebo- and bGH-treated animals) were calculated. All calculations were carried out with the GLM Procedure (SAS, 1989).
3. Results
3.1. Body growth and tissue composition Recombinant bGH-treated animals grew faster (P , 0.05) than controls. Live weight gain over the 147-day treatment period for the control group averaged 8.5 kg per animal and, for the bGH-treated group, 12.1 kg per animal (an increase of 42%). Liver, adrenals and kidneys (adjusted for final body weights) were heavier in bGH-treated animals; however, weights of heart and pituitaries were not changed (Table 1). The composition of adipose and muscle tissues was markedly affected by bGH treatment. The concentration of total lipids per unit of omental adipose tissue (mg g 21 ) decreased by 44% due to bGH and, simultaneously, protein content increased by 160%. In the longissimus muscle, lipid content was decreased by 30% whereas protein content was not significantly affected by bGH. Lipid and protein concentrations in the liver, heart and kidneys were not affected by bGH (Table 2).
3.2. Lipogenesis in omental adipose explants The rate of fatty acid synthesis in freshly-prepared adipose explants in 2-h incubations in a modified Krebs–Henseleit buffer was established to provide both information on the effect of long term bGH treatment on fatty acid synthesis in vitro and to provide a measure of the maintenance of lipogenesis in subsequent incubations of explants cultured in the presence of different hormones. The data showed that caprine adipose tissue remained metabolically active for at least 24 h in a modified Waymouth’s tissue culture medium. Comparison of the lipogenesis of freshly-prepared explants with explants cultured in the absence of hormones for 24 h showed that the rate of incorporation of acetate into fatty acids in both control and bGH-treated animals was the same after 24 h of culture. Inclusion of hormones in culture medium changed lipogenesis in tissues from both groups of animals in a similar manner. Mean values of acetate incorporation into fatty acids in explants first cultured in different hormonal
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Table 1 Growth and organ weights of castrated male goats receiving s.c. injections of recombinant methionyl bGH in a sustained release vehicle at seven-day intervals for 147 days Control
Initial body weight, kg Final body weight, kg Gain, kg Liver wt., g Relative liver weight a , % Heart wt., g Relative heart wt., % Adrenal wt., g Relative adrenal wt., % Kidney wt., g Relative kidney wt., % Pituitary gland wt, mg Relative pituitary gland wt., %
bGH
n
x¯ 6S.E.M.
n
x¯ 6S.E.M.
11 11 11 11 11 11 11 11 11 4 4 4 4
14.561.0 23.061.2 8.560.9 377.5618.1 1.6560.06 84.265.4 0.36560.011 1.4860.08 0.006560.0003 68.862.3 0.30860.014 18566 0.0008460.00003
11 11 11 11 11 11 11 11 11 4 4 4 4
14.261.3 NS 26.361.9 * 12.161.0 *** 497.6625.8 *** 1.9260.04 *** 95.867.0 * 0.36560.011 NS 2.0160.12 ** 0.007860.0004 * 88.365.4 * 0.36860.029 * 226625 NS 0.0008960.00007 NS
Czech white male goats were castrated at two to three weeks of age and used for experiments between two and three months of age. Control animals were treated s.c. with olive oil. Values are means6S.E.M. a Relative weights are organ weights expressed as a percentage of final body weight. * P,0.05; ** P,0.01; *** P,0.001; NS 5not significant.
Table 2 Organ composition of castrated male goats receiving s.c. injections of recombinant methionyl bGH in a sustained release vehicle at seven-day interval for 147 days Control
bGH
n
x¯ 6S.E.M.
n
x¯ 6S.E.M.
Liver Fat (mg / g) Protein (mg / g)
11 11
4261 17668
11 11
4562 NS 16466 NS
Adipose tissue Fat (mg / g) Protein (mg / g)
11 11
780620 1561
11 11
512660 ** 3969 *
Skeletal muscle a Fat (mg / g) Protein (mg / g)
11 11
2062 19364
11 11
1461 * 19665 NS
Heart Fat (mg / g) Protein (mg / g)
11 11
2962 14665
11 11
2461 NS 15368 NS
Kidney Fat (mg / g) Protein (mg / g)
4 4
2761 14366
4 4
2861 NS 144610 NS
Animals were castrated and treated as described in Table 1. a Skeletal muscle was m. longissimus dorsi, sample taken just behind the last rib. * P,0.05; ** P,0.01; NS 5not significant.
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combinations and then incubated in the absence of catecholamines were consistently lower (P , 0.0001) in bGH-treated animals than in controls (overall means – control, 57.064.23; bGH, 26.7364.23 nmol mg 21 h 21 ; Table 3). Fatty acid synthesis in explants cultured in the absence of hormones was taken as a basic measure of lipogenic response to different hormones added to the culture medium. Addition of insulin stimulated (44.8%) the rate of fatty acid synthesis in tissues of control animals but a much lower (22.2%) effect of insulin was observed in bGH-treated animals. However, these effects of insulin were not significant. The presence of cortisol alone in culture medium decreased lipogenesis in explants from both control (P , 0.05) and bGH-treated (non-significantly) animals. In tissues from bGH-treated animals, cortisol acted synergistically with insulin to produce a higher rate of lipogenesis than that observed in cultures with insulin alone. In control animals, however, no significant effect of cortisol plus insulin on fatty acid synthesis was noted when compared with insulin alone (Table 3). With the combination of insulin plus bGH, the rates of lipogenesis in tissues from control animals were lower than those cultured with insulin alone. No such inhibitory effect of bGH was noted in tissues from bGH-treated animals. When goat adipose explants were maintained in a modified Waymouth’s medium in the presence of insulin plus cortisol plus bGH the rates of lipogeneses were always lower (P , 0.05) than those in cultures with insulin plus cortisol in both control and bGH-treated animals. In vitro rates of lipogenesis were decreased (P , 0.05) by catecholamines in freshly-prepared adipose tissue explants of both control and bGH-treated animals. Iso decreased (P , 0.05) the rate of fatty acid synthesis (5.2560.76 nmol mg 21 h 21 ) more than Ne (13.3661.70 nmol mg 21 h 21 ) in bGHtreated animals but not in controls. The mean values of lipogenesis in explants cultured in the presence of different hormonal combinations and then incubated in the presence of Ne or Iso are shown in Table 3. The difference between pooled means of acetate incorporation into fatty acids in the presence of Ne in control (9.6261.70 nmol mg 21
h 21 ) and bGH-treated animals (13.3661.70 nmol mg 21 h 21 ) was not significant (P , 0.10); however, the rate of lipogenesis in explants incubated in the presence of Iso was lower (P , 0.05) in bGH-treated (5.2560.76 nmol mg 21 h 21 ) than in control animals (8.4160.76 nmol mg 21 h 21 ). These results suggest that Ne and Iso were equally effective in control goats whereas, in GH-treated goats, Iso was more effective than Ne.
4. Discussion
4.1. Growth performance, organ weights and tissue chemical composition The growth rate of GH-treated lambs, growing steers, heifers and bulls has been very variable between studies: cattle ranging from 3–25% and lambs ranging from 0–45% above controls (Enright, 1989; Zainur et al., 1989a,b). The increase in growth rate in our long term bGH-treated goats (42%) is in agreement with some previous reports in lambs. Likewise, the decrease in the proportion of fat in the muscle of bGH-treated goats by 30% agrees with previous reports in cattle and sheep (32%; Wagner and Veenhuizen, 1978; Wise et al., 1988; Beermann et al., 1990). In goats, the increase in gain was accompanied by a decrease in muscle fat but this was not always demonstrated in lambs. For example, no effect of GH on fat was observed by Pullar et al. (1986) even though GH increased daily gain by 30%. Organ weights were expressed as a fraction of final liveweight and therefore normalized. The significant positive effect of GH on liver weight in goats was similar to previous studies with lambs, heifers and steers (Wagner and Veenhuizen, 1978; Sandles and Peel, 1987; Beermann et al., 1990; Early et al., 1990; Pell et al., 1990; Bass et al., 1991; McLaughlin et al., 1993; Schwarz et al., 1993) and pigs (Klindt et al., 1992; Lefaucheur et al., 1992). Heavier livers in GH-treated animals could reflect the increased rates of direct and indirect (e.g., by IGF-I) GH-mediated changes in liver metabolism (e.g., increased gluconeogenesis, increased release of nutrient to support increased growth, etc.). Kidney weight was increased in bGH-treated
ˇ / Livestock Production Science 55 (1998) 215 – 225 J. Skarda
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Table 3 Effects of long-term treatment with bGH in vivo and chronic (24 h) culture in the presence of various hormones on the rate of lipogenesis during acute incubations (2 h) in the presence and absence of noradrenaline and isoprenaline in adipose explants from young castrated male goats Acetate incorporation into fatty acids (nmol mg 21 h 21 ) Culture
Incubation variables
variables
No catecholamines
Ne
Iso
Placebo-treated animals Before culture No hormones
63.20 ABCa
18.71 Ab
After culture No hormones Insulin (I) bGH Cortisol (H) I1H I1bGH I1H1bGH
56.15 ABCa 81.33 ABa 40.65 CDa 19.89 Da 88.86 Aa 58.20 ABCa 47.75 BCDa
5.24 Bb 8.83 ABb 6.01 ABb 10.43 ABac 13.70 ABb 5.97 ABb 8.09 ABb
Overall mean
57.00 a
bGH-treated animals Before culture No hormones
40.22 ABa
19.22 ABb
6.85 ABbc
After culture No hormones Insulin bGH Cortisol I1H I1bGH I1H1bGH
27.05 ABa 33.06 ABa 13.65 Ba 8.44 Bac 51.34 Aa 25.31 ABa 14.79 Ba
12.63 ABb 13.39 ABb 10.03 ABac 12.42 ABab 22.62 Ab 6.62 Bb 9.98 ABac
6.08 ABb 8.48 Ab 3.28 ABbc 1.74 Bc 5.59 ABc 6.70 ABb 3.30 ABbc
Overall mean
26.73 a
13.36 b
5.25 c
Pooled S.E.M. 1 Pooled S.E.M. 2
11.97 4.23
4.81 1.70
2.15 0.76
Effect bGH treatment
P,0.0001
9.62 b
NS
9.25 ABb 9.04 ABb 12.46 Ab 6.04 Bb 4.41 Bbc 10.44 ABb 8.50 ABb 7.17 ABb 8.41 b
P,0.0052
Omental adipose explants of four control (placebo-treated) and four bGH-treated (100 mg of recombinant methionyl bGH in a sustained release vehicle at seven-day intervals for 140 days) animals were exposed before and after culture to (1- 14 C)acetate (20 kBq ml 21 ) for a 2-h period of incubation in the presence or absence of noradrenaline (Ne; 10 mmol l 21 ) or isoprenaline hydrochloride (Iso; 10 mmol l 21 ) in Krebs–Henseleit buffer supplemented with sodium acetate (4 mmol l 21 ) and glucose (3.5 mmol l 21 ). Cultured explants were maintained in tissue culture for 24 h in a modified Waymouth’s medium (containing 3.5 mmol l 21 glucose) in the absence of hormones or in the presence of insulin (17 nmol l 21 ), recombinant bGH (Somidobove; 4.5 nmol l 21 ) and cortisol (138 nmol l 21 ) alone or in different combinations. All values are the means of four experiments, each performed in triplicate, and expressed per mg protein. For each column of results, pooled S.E.M. 1 (standard error LS mean for paired values) is for comparison of mean values from animals within the same treatment (placebo- or bGH-treated); pooled S.E.M. 2 (standard error LS mean for unpaired values) is for comparing mean values of placebo-treated with those for bGH-treated animals. Values within a column which do not have the same upper case superscript (A, B, C, D) differ significantly (P,0.05). Values within a row which do not have the same lower case superscript (a, b, c) differ significantly (P,0.05).
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castrated goats similar to what was observed in bGH-treated heifers (Schwarz et al., 1993), porcine GH-treated pigs (Klindt et al., 1992; Lefaucheur et al., 1992) and acromegalic humans (Binnerts et al., 1988). However, in GH-treated lambs, kidney weight was increased variably; non-significantly increased in experiments by Zainur et al. (1989a); Pell et al. (1990) and Bass et al. (1991), and significantly increased in experiments by McLaughlin et al. (1993). Increased kidney weights possibly reflected increased glomerular filtration. The absolute weights of hearts were increased in bGH-treated goats; however, fractional weights were not affected by treatment as was seen in lambs (Zainur et al., 1989a,b; Pell et al., 1990) and pigs (Klindt et al., 1992). However, in cases of acromegaly in humans, an excess of GH increased the fractional weights of the heart (Steele and EvockClover, 1993). GH-treated castrated young goats had heavier adrenal glands than controls and this could reflect an enhanced secretion of corticosteroids and / or catecholamines. Heavier adrenal glands in porcine GH-treated pigs have been previously reported (Sillence and Etherton, 1989; Lefaucheur et al., 1992).
4.2. Regulation of lipogenesis in adipose explants Goat adipose tissue remained metabolically active when maintained in culture in a modified Waymouth’s tissue culture medium as was seen for ovine or bovine adipose tissue maintained in medium 199 (Vernon, 1979; Etherton et al., 1987). The present study has shown that chronic administration of recombinant bGH in vivo can lead to a loss of lipid from omental adipose tissue (and from longissimus muscle) of castrated male goats. Acute incubation of both fresh and cultured omental adipose tissue explants in the presence of (1- 14 C)acetate demonstrated that loss of lipid in bGH-treated animals could at least in part result from the reduced rate of fatty acid synthesis. The mean values of acetate incorporation into fatty acids in explants cultured in the presence of different hormonal combinations were consistently lower in bGH-treated animals than in controls. The direct comparison of the rate of lipogenesis between control and bGHtreated animals presents a problem. It is not accurate under conditions of expressing data per unit number
of adipocytes or per unit weight of DNA or protein content, because non-adipocytes comprise the majority of cells in the tissue; however, adipocytes account for the majority of metabolic activities (Vernon et al., 1987). Results from our studies with adipose explants of both control and bGH-treated animals in chronic tissue culture (24 h) show that bGH has an inhibitory action on lipogenesis before any detectable change in adipocyte size can be expected. Similarly, Borland et al. (1994) have demonstrated with sheep adipose tissue explants that GH caused a decrease in the rate of lipogenesis after a short lag phase of 6 h. The mechanism by which GH decreases lipogenesis is not clear. There is a close relationship between lipogenesis and glucose utilization. In both non-ruminants and ruminants, GH decreases uptake, oxidation and utilization of glucose, due in part to decreased synthesis of glucose transporters (Goodman, 1993; Vernon et al., 1995; Zhao et al., 1996). GH can also affect activity and insulin activation of key lipogenic enzymes such as acetyl-CoA carboxylase (Vernon et al., 1991). In addition, we observed that bGH antagonized insulin action on lipogenesis. It is probable that bGH antagonizes the effects of insulin via some insulindependent expression of glucose transporter gene or via insulin-dependent enzymes in the pathway of both glucose utilization and fatty acid synthesis. In bovine adipose tissue, bGH also antagonized the ability of insulin to maintain lipogenic capacity (Etherton et al., 1987). This antagonism was, however, dependent upon the presence of cortisol. On the other hand, in pig (Walton et al., 1986), sheep (Vernon and Taylor, 1988) and goat adipose tissue (present study), the inhibitory effect of GH on insulin action was seen both in the presence of insulin alone and in the presence of insulin plus cortisol. The addition of cortisol alone to the culture medium decreased lipogenesis in both control and bGH-treated animals. A similar inhibitory effect of cortisol has been found in cultured bovine (Etherton et al., 1987), ovine (Vernon and Finley, 1988) and pig (Walton et al., 1986) adipose tissue. Glucocorticoids are other hormones which act on lipogenesis as a glucose antagonist and it has been shown that they reduce cellular glucose uptake and utilization (Vernon and Taylor, 1988). Glucocorticoids act by
ˇ / Livestock Production Science 55 (1998) 215 – 225 J. Skarda
inhibiting the glucose transport per se, perhaps by affecting glucose transporter (GLUT4) subcellular trafficking (Weinstein et al., 1995). In explants from our placebo-treated animals, an inhibitory effect of cortisol on lipogenesis was eliminated by the simultaneous addition of insulin. However, in bGH-treated animals, insulin acted synergistically with cortisol to produce a higher rate of lipogenesis than that in the presence of insulin alone. Synergism between insulin and cortisol was also observed in non-lactating sheep (Vernon and Finley, 1988), heifers (Etherton et al., 1987) and pigs (Walton et al., 1986). It was suggested that glucocorticoids can potentiate the effects of insulin by enhancing the level of an intracellular mediator of insulin action (Walton et al., 1986) and by stimulating synthesis of insulin receptors and insulin binding (Salhanick et al., 1983; Wastie et al., 1995). Glucocorticoids can potentiate the responses of glucose-6-phosphate dehydrogenase, L-pyruvate kinase and acetyl-CoA carboxylase to insulin (Hillgartner et al., 1995) and, thus, fatty acid synthesis from acetate could be stimulated by enzymes involved in both glucose utilization and fatty acid synthesis. In control goats, Ne and Iso were equally effective whereas, in bGH-treated goats Iso was more effective than Ne. These results are consistent with an increase in the number of b-adrenergic receptors in sheep adipocytes (Watt et al., 1991) and increased isoprenaline-stimulated lipolysis in bovine adipose tissue cultured in the presence of bGH (Lanna et al., 1995). The higher activity of Iso in bGH-treated animals could also be due to GH increasing the number of a 2 -adrenergic receptors (Vernon et al., 1995), which could limit effects of Ne but not Iso.
Acknowledgements I am grateful to Dr. H. Mader and Dr. P. Kubasa from Eli Lilly and Elanco Ges, Vienna, Austria and to Dr. R.J. Collier and Dr. D.L. Hard from Monsanto, St. Louis, MO, USA for the gifts of recombinant bovine GH. I thank Dr. W.W. Bromer from Eli Lilly for the gift of porcine insulin. The author is grateful to E. Urbanova, Ing. P. Krejci and Ing. J. Slaba for their technical assistance and Dr. J. Wolf
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for the statistical analysis. I would like to acknowledge the Grant Agency of the Academy of Sciences of the Czech Republic for funding (project No. A7-045608).
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