GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
36, 33-39 (1978)
Hepatic Mediation of Hormonal and Nutritional Factors Influencing the in Vitro Sulfur Uptake by Rainbow Trout Bone1 M. P. KOMOURDJIAN~ AND D. R. IDLER Marine Sciences Research Laboratory,
Memorial University of Newfoundland, Canada AIC 5S7 Accepted May 16, 1978
St. John’s, Newfoundland,
Previous studies have shown that the mammalian liver mediates somatotropin (STH) actions via the release of peptides termed somatomedins. Our study indicated mat the in vitro %S uptake by gill arch bone of hypophysectomized (hypexed) rainbow trout, salvo guirdneri, was significantly (P < 0.05) stimulated by liver slices from fed intact trout. Slices from trout starved for 2 or 5 weeks or from fish hypexed and starved for that period inhibited uptake. However, liver from feeding hypexed trout had no effect on uptake. A combinaticm of thyroxine and STH stimulated uptake in the presence but not in the absence of liver tissue from long-term starved or hypexed animals. Neither hormone was active by itself. This study suggests a nutritional-hormonal control over skeletal sulfur metabolism and presents evidence for the existence of a “somatomedin-like” activity in livers of S. gairdneri.
In mammals, somatotropins (STH) elicit their metabolic actions, which include the promotion of 35S uptake, via the somatomedins (Salmon and Daughaday, 1957; Van Wyk et al., 1974). These peptide mediators are found in plasma and are presumed to originate from various organs including the liver (McConaghey, 1972). More recently, thyroxine and other pituitary principles (Holder and Wallis, 1977) have also been shown to stimulate an increase in somatomedin levels. Furthermore, these mediators are also modulated by nutritional factors (Phillips and Young, 1976a). The elucidation of the modes of action of STH is essential for growth manipulation, the development of a sensitive STH assay, and the identification of growth-promoting substances. Yet, decisive information is lacking in this area for lower vertebrates. Ash ( 1977) suggested that mammalian STH r Contribution No. 299 from the Research Laboratory, supported by 6732 to D. R. I. Z Present address: Department of Natural Sciences, College of Cape Nova Scotia, Canada B IP 6J I.
enhances 35Suptake by teleostean cartiIage and found no evidence for a somatomedinlike mediator in plasma from rainbow trout, Salvo gairdneri, and the cichhd, Tilapia andersoni. However, Shapiro and Pimstone (197’7) found that plasma samples from 26 vertebrate species surveyed9 including S. gairdneri, were all able to promote 35S uptake by porcine cartilagen Essentially no work concerns the role of somatomedins in bone formation save mammalian studies by Thorngren et al. (1977) and Canalis et al. (1977). Sulfur actively enters bone, as it does cartilage, to integrate as chondroitin sulfate in the tissue structure (Belanger, 1954; Dziewiatkowski, 1962), and in S. gairdrwi this process in either tissue is mfluenced by thyroxine in vivo (Barrington and Rawdon 1947). This study seeks to identify same of the factors potentially influencing sulfur uptake in S. gairdneri gill arch bone particularly STH, thyroxine, and nutrition.
Marine Sciences NRC Grant No.
MATERIALS
Mathematics and Yreton, Sydney,
AND METt-iODS
Animals. Rainbow trout, S, guirdneris 14.5 to i7.5 cm in length, were obtained from Goossens trout 33 0016-6480/78/036L0033~01~00/0 Cowright @ 1978 by Academic Press, Inc. All rights of reproduction in any form resewed.
34
KOMOURDJIAN
farm, Otterville, Ontario. They were kept in 11% salinity water -under a l2L:l2D photoperiod. Ewos 5P salmon pellets were presented several times a day. Four groups of animals were used, two intact and two hypophysectomized. One intact and one hypophysectomized group were fed three times a day; the other two groups were starved. Fish were hypophysectomized by the buccal approach of Komourdjian and Idler (1977). To eliminate a possible bias produced by anesthetics the intact fish were exposed to the same propoxate/MS 222 anesthesia used during hypophysectomy (Komourdjian and Idler, 1977). Incubation medium. Tissues were incubated in Cortland medium (Phillips et al., 1957) containing 0.7 g of glucose/liter and the amino acid supplement previously used for cartilage (Daughaday and Reeder, 1966): 0.05 r& cystine, 0.03 ~LW methionine, 0.05 m&i’ phenylalanine, 0.06 W tyrosine, 0.1 n&Z arginine, 0.2 nnV lysine, 0.08 rml4 histidine, 0.05 m&f tryptophan, 0.07 m isoleucine, 0.13 m leucine, 0.12 m&f threonine, 0.25 mJ4 valine, 0.57 ti glutamine, 0. l I m.V serine, and 0.21 ti glycine. The final pH of the medium was adjusted to 7.8 with Trizma (Sigma Chemical). Imubations. Fish were stunned by a swift blow to the cranium. The heart was immediately exposed, and a cannula was inserted into the bulbus arteriosus. The ventricle was then slit open, and 12-15 ml of Cortland medium containing 0.7 g of glucose/liter were flushed through the animal. Most of the blood was thus removed, and the gill filaments appeared blanched. The liver was then quickly removed, sliced, and placed in aerated chilled (O-4’) incubation medium. No significant differences were found between basal (no exogenous hormone added) W incorporation rates by gill bone from hypexed and intact animals in preliminary studies. However, only bone from hypexed fish was used to eliminate the possibility of a contamination of the tissue by endogenous hormones. Gill arches were removed, and the bony sections were microscopically dissected free of covering epithelium. Segments of approximately 1 mm in length (ca. 700 ,.~g dry weight) were thus obtained and placed in chilled and aerated incubation medium. Bone segments and liver slices were preincubated separately in medium for 4 hr under oxygen in a 10 shaking water bath. Segments (three to four per flask) were then incubated for 8 hr in (1) medium containing 66 or 6.6 p g of thyroxine (Sodium L-thyroxine pentahydrate, Sigma Chemical) per ml, (2) medium containing 3.4 X 10mzor 3.4 X IO-* IU of somatotropin (porcine STH, Sigma Chemical) per ml. or (3) a combination of both hormones in a ratio of 66 pg of thyroxine and 3.4 x lO-z IU of STH per ml. Each set of segments was compared to a control group, with
AND
IDLER
similar sample number drawn from the same animals and incubated in medium alone (Table 1). Further segments were incubated in the presence of 8 mg of liver slices in 1 ml of medium containing 3.4 x 10mz IU of STH, 66 pg of thyroxine or the aforementioned combination of thyroxine and STH with 1: 10 and 1: 100 dilutions for samples from fed fish (Table 3). These incubations were repeated several times with liver slices from intact and hypexed trout fed regularly or starved for periods of I to over 5 weeks. A control group for each treatment consisted of segments from the same fish incubated with liver slices but without hormone supplement (Table 2). Additional incubations included bone from the same animals in the presence and absence of liver from the above fish (Table 3). Two sets of four segments were boiled at lOW for 10 min before incubation in the medium. In all cases 1 &i of NaZ?S04 (New England Nuclear) was added to the incubate. After 8 hr the segments were washed in several changes of distilled water and placed in boiling water for 4 min. They were then rinsed in distilled water, blotted on filter paper, and dried overnight. The dried segments were weighed, to the nearest microgram, on a Cahn 4700 electrobalance and individualIy digested in 0.6 ml of formic acid in scintillation vials at 100 for 40 min. To each digest were added 10 ml of Aquasol (New England Nuclear), and the samples were counted in a Packard Tri-Carb scintillation counter. Results were pooled where possible or presented as separate runs. Means of the groups were tested against respective controls by Student’s “t” test. Other comparisons were performed using Duncan’s multiple-range test. Several of the incubated slices were dissolved in Protosol (New England Nuclear) to determine their importance in Yl removal from the medium. Liquifluor (New England Nuclear) was used as the scintillator. Color quenching, which attained 20%, was determined after addition of a known quantity of label to each vial.
RESULTS
The mean uptake of 35S by gill arch bone in medium devoid of liver tissue or hormone supplement (basal rate), determined from 18 simultaneous incubations, was 159 cpm/mg of cartilage/ 8 hr (Table 1). No significant difference was apparent between bone from intact and hypexed fish in preliminary studies, and the bone segments boiled after removal from the animal did not incorporate significant amounts of radiosulfur.
SULFUR
UPTAKE
IN
TABLE THE
EFFECT
OF THYROXINE
AND
TROUT
BQNE
3.5
I
SOMATOTROPIN (STH) ON ?5 UPTAKE TROUT, S. gairdneri
BY GILL
ARCH
BONE
FROM RAINBOW
Medium
Na
Uptake (cpm/mg/8 hr)*
Control (no hormone) Thyroxine (6.6 pg) Thyroxine (66 pg)
10 6 8
165.6 5 19.3 149.0 * 2s.5
-IO
n.s.
135.8
-18
AND
COMPARATIVE
ENDOCRINOLOGY
36, 33-39 (1978)
Hepatic Mediation of Hormonal and Nutritional Factors Influencing the in Vitro Sulfur Uptake by Rainbow Trout Bone1 M. P. KOMOURDJIAN~ AND D. R. IDLER Marine Sciences Research Laboratory,
Memorial University of Newfoundland, Canada AIC 5S7 Accepted May 16, 1978
St. John’s, Newfoundland,
Previous studies have shown that the mammalian liver mediates somatotropin (STH) actions via the release of peptides termed somatomedins. Our study indicated mat the in vitro %S uptake by gill arch bone of hypophysectomized (hypexed) rainbow trout, salvo guirdneri, was significantly (P < 0.05) stimulated by liver slices from fed intact trout. Slices from trout starved for 2 or 5 weeks or from fish hypexed and starved for that period inhibited uptake. However, liver from feeding hypexed trout had no effect on uptake. A combinaticm of thyroxine and STH stimulated uptake in the presence but not in the absence of liver tissue from long-term starved or hypexed animals. Neither hormone was active by itself. This study suggests a nutritional-hormonal control over skeletal sulfur metabolism and presents evidence for the existence of a “somatomedin-like” activity in livers of S. gairdneri.
In mammals, somatotropins (STH) elicit their metabolic actions, which include the promotion of 35S uptake, via the somatomedins (Salmon and Daughaday, 1957; Van Wyk et al., 1974). These peptide mediators are found in plasma and are presumed to originate from various organs including the liver (McConaghey, 1972). More recently, thyroxine and other pituitary principles (Holder and Wallis, 1977) have also been shown to stimulate an increase in somatomedin levels. Furthermore, these mediators are also modulated by nutritional factors (Phillips and Young, 1976a). The elucidation of the modes of action of STH is essential for growth manipulation, the development of a sensitive STH assay, and the identification of growth-promoting substances. Yet, decisive information is lacking in this area for lower vertebrates. Ash ( 1977) suggested that mammalian STH r Contribution No. 299 from the Research Laboratory, supported by 6732 to D. R. I. Z Present address: Department of Natural Sciences, College of Cape Nova Scotia, Canada B IP 6J I.
enhances 35Suptake by teleostean cartiIage and found no evidence for a somatomedinlike mediator in plasma from rainbow trout, Salvo gairdneri, and the cichhd, Tilapia andersoni. However, Shapiro and Pimstone (197’7) found that plasma samples from 26 vertebrate species surveyed9 including S. gairdneri, were all able to promote 35S uptake by porcine cartilagen Essentially no work concerns the role of somatomedins in bone formation save mammalian studies by Thorngren et al. (1977) and Canalis et al. (1977). Sulfur actively enters bone, as it does cartilage, to integrate as chondroitin sulfate in the tissue structure (Belanger, 1954; Dziewiatkowski, 1962), and in S. gairdrwi this process in either tissue is mfluenced by thyroxine in vivo (Barrington and Rawdon 1947). This study seeks to identify same of the factors potentially influencing sulfur uptake in S. gairdneri gill arch bone particularly STH, thyroxine, and nutrition.
Marine Sciences NRC Grant No.
MATERIALS
Mathematics and Yreton, Sydney,
AND METt-iODS
Animals. Rainbow trout, S, guirdneris 14.5 to i7.5 cm in length, were obtained from Goossens trout 33 0016-6480/78/036L0033~01~00/0 Cowright @ 1978 by Academic Press, Inc. All rights of reproduction in any form resewed.
34
KOMOURDJIAN
farm, Otterville, Ontario. They were kept in 11% salinity water -under a l2L:l2D photoperiod. Ewos 5P salmon pellets were presented several times a day. Four groups of animals were used, two intact and two hypophysectomized. One intact and one hypophysectomized group were fed three times a day; the other two groups were starved. Fish were hypophysectomized by the buccal approach of Komourdjian and Idler (1977). To eliminate a possible bias produced by anesthetics the intact fish were exposed to the same propoxate/MS 222 anesthesia used during hypophysectomy (Komourdjian and Idler, 1977). Incubation medium. Tissues were incubated in Cortland medium (Phillips et al., 1957) containing 0.7 g of glucose/liter and the amino acid supplement previously used for cartilage (Daughaday and Reeder, 1966): 0.05 r& cystine, 0.03 ~LW methionine, 0.05 m&i’ phenylalanine, 0.06 W tyrosine, 0.1 n&Z arginine, 0.2 nnV lysine, 0.08 rml4 histidine, 0.05 m&f tryptophan, 0.07 m isoleucine, 0.13 m leucine, 0.12 m&f threonine, 0.25 mJ4 valine, 0.57 ti glutamine, 0. l I m.V serine, and 0.21 ti glycine. The final pH of the medium was adjusted to 7.8 with Trizma (Sigma Chemical). Imubations. Fish were stunned by a swift blow to the cranium. The heart was immediately exposed, and a cannula was inserted into the bulbus arteriosus. The ventricle was then slit open, and 12-15 ml of Cortland medium containing 0.7 g of glucose/liter were flushed through the animal. Most of the blood was thus removed, and the gill filaments appeared blanched. The liver was then quickly removed, sliced, and placed in aerated chilled (O-4’) incubation medium. No significant differences were found between basal (no exogenous hormone added) W incorporation rates by gill bone from hypexed and intact animals in preliminary studies. However, only bone from hypexed fish was used to eliminate the possibility of a contamination of the tissue by endogenous hormones. Gill arches were removed, and the bony sections were microscopically dissected free of covering epithelium. Segments of approximately 1 mm in length (ca. 700 ,.~g dry weight) were thus obtained and placed in chilled and aerated incubation medium. Bone segments and liver slices were preincubated separately in medium for 4 hr under oxygen in a 10 shaking water bath. Segments (three to four per flask) were then incubated for 8 hr in (1) medium containing 66 or 6.6 p g of thyroxine (Sodium L-thyroxine pentahydrate, Sigma Chemical) per ml, (2) medium containing 3.4 X 10mzor 3.4 X IO-* IU of somatotropin (porcine STH, Sigma Chemical) per ml. or (3) a combination of both hormones in a ratio of 66 pg of thyroxine and 3.4 x lO-z IU of STH per ml. Each set of segments was compared to a control group, with
AND
IDLER
similar sample number drawn from the same animals and incubated in medium alone (Table 1). Further segments were incubated in the presence of 8 mg of liver slices in 1 ml of medium containing 3.4 x 10mz IU of STH, 66 pg of thyroxine or the aforementioned combination of thyroxine and STH with 1: 10 and 1: 100 dilutions for samples from fed fish (Table 3). These incubations were repeated several times with liver slices from intact and hypexed trout fed regularly or starved for periods of I to over 5 weeks. A control group for each treatment consisted of segments from the same fish incubated with liver slices but without hormone supplement (Table 2). Additional incubations included bone from the same animals in the presence and absence of liver from the above fish (Table 3). Two sets of four segments were boiled at lOW for 10 min before incubation in the medium. In all cases 1 &i of NaZ?S04 (New England Nuclear) was added to the incubate. After 8 hr the segments were washed in several changes of distilled water and placed in boiling water for 4 min. They were then rinsed in distilled water, blotted on filter paper, and dried overnight. The dried segments were weighed, to the nearest microgram, on a Cahn 4700 electrobalance and individualIy digested in 0.6 ml of formic acid in scintillation vials at 100 for 40 min. To each digest were added 10 ml of Aquasol (New England Nuclear), and the samples were counted in a Packard Tri-Carb scintillation counter. Results were pooled where possible or presented as separate runs. Means of the groups were tested against respective controls by Student’s “t” test. Other comparisons were performed using Duncan’s multiple-range test. Several of the incubated slices were dissolved in Protosol (New England Nuclear) to determine their importance in Yl removal from the medium. Liquifluor (New England Nuclear) was used as the scintillator. Color quenching, which attained 20%, was determined after addition of a known quantity of label to each vial.
RESULTS
The mean uptake of 35S by gill arch bone in medium devoid of liver tissue or hormone supplement (basal rate), determined from 18 simultaneous incubations, was 159 cpm/mg of cartilage/ 8 hr (Table 1). No significant difference was apparent between bone from intact and hypexed fish in preliminary studies, and the bone segments boiled after removal from the animal did not incorporate significant amounts of radiosulfur.
SULFUR
UPTAKE
IN
TABLE THE
EFFECT
OF THYROXINE
AND
TROUT
BQNE
3.5
I
SOMATOTROPIN (STH) ON ?5 UPTAKE TROUT, S. gairdneri
BY GILL
ARCH
BONE
FROM RAINBOW
Medium
Na
Uptake (cpm/mg/8 hr)*
Control (no hormone) Thyroxine (6.6 pg) Thyroxine (66 pg)
10 6 8
165.6 5 19.3 149.0 * 2s.5
-IO
n.s.
135.8
-18
