0013-7227/78/1031-0121$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 1 Printed in U.S.A.
Somatomedin and Nutrition. IV. Regulation of Somatomedin Activity and Growth Cartilage Activity by Quantity and Composition of Diet in Rats* LAWRENCE S. PHILLIPS, ARTHUR T. ORAWSKI, AND D. C. BELOSKY Center for Endocrinology, Metabolism, and Nutrition and Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611 ABSTRACT. To examine the relation of somatomedin to nutritional status, bioassayable serum somatomedin activity and growth cartilage activity (SO4 uptake in vitro) were determined in rats fasted for 3 days and then either fasted for a fourth day or refed with diets varying in quantity and nutrient composition. Animals refed for 6 or 12 h had modest increases in somatomedin activity and cartilage SO4 uptake which persisted after food was withdrawn and weight loss had begun. Refeeding for 24 h produced greater increases in somatomedin activity and cartilage SO4 uptake; this model was used to study the effects of alterations in diet. Refeeding with different quantities of a balanced diet produced dose-response increases in somatomedin activity and cartilage SO4 uptake. In animals refed isocalorically, diets of fat and carbohydrate or fat alone led to increases in somatomedin activity comparable to those with a balanced diet, yet little increase in cartilage SO4 uptake. In contrast, diets of protein and carbohydrate or protein alone
led to smaller increases in somatomedin activity with greater increases in cartilage SO4 uptake (P < 0.05). Diets of fat and protein or carbohydrate alone produced intermediate responses. Although 24-h refeeding with protein-deficient diets produced an increase in somatomedin activity, continuation of refeeding to 48 h led to a decrease in somatomedin activity (P < 0.05) with cartilage SO4 uptake comparable to fasting levels. A similar decrease in somatomedin activity with low cartilage SO4 uptake was found in animals fed for 4 days with a protein-deficient diet. We conclude that serum somatomedin activity and growth cartilage activity may be determined by both quantity and nutritional composition of the diet. Dietary protein may be of particular importance, as deficiency of this constituent appears to lead acutely to a decrease in growth cartilage activity and chronically to decreases in both serum somatomedin activity and growth cartilage activity. (Endocrinology 103: 121, 1978)
S
We have reported previously that when normal rats were fasted 1) their serum somatomedin activity decreased to hypopituitary levels, 2) this decrease was followed by a similar fall in the activity of their growth cartilage, and 3) these changes could not be prevented by the concurrent administration of pharmacologic amounts of GH (4). When the animals were refed, their somatomedin activity returned promptly to normal levels, followed by a comparable rise in growth cartilage activity (4). In the present studies, we have used the Received July 27, 1977. refeeding of fasted rats as a model in which to Address reprint request to: Dr. Lawrence S. Phillips, Center for Endocrinology, Metabolism, and Nutrition, examine the regulation of serum somatomedin Northwestern University Medical School, 303 East Chi- activity and growth cartilage activity by quancago Avenue, Chicago, Illinois 60611. * This work was presented in part at the 59th Annual tity and nutritional composition of the diet.
OMATOMEDINS were characterized initially as circulating growth factors regulated by GH (1). However, there is now evidence that the cartilage-stimulating activity of somatomedins may be modulated by nutritional status and insulin as well. Thus, somatomedin activity is decreased (despite high levels of GH) in children with kwashiorkortype malnutrition (2), and somatomedin activity is normal (despite low levels of GH) in children who are hyperphagic and hyperinsulinemic after hypothalamic surgery (3).
Meeting of the Endocrine Society, Chicago, June 1977. It was supported in part by Research Grants AM-10699 and AM-21483 and Training Grant AM-07169 from NIAMDD, a research grant and research and development award from the American Diabetes Association, and a research award from the Greater Chicago and Northern Illinois Affiliate of the American Diabetes Association.
Materials and Methods Animals
Male Charles River rats, weighing 90-110 g at the onset of fasting, were housed in a constant 121
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122
PHILLIPS, ORAWSKI, AND BELOSKY
temperature (23 C) room lighted from 0800-1800 h. Fasting and refeeding began between 1000-1200 h. Control animals were maintained four to six per 20 X 33-cm plastic cage containing pine wood shavings (coprophagy not prevented). Groups of animals were given water ad libitum and were deprived of food for approximately 72 h in plastic cages [seven to nine animals per cage (46 X 35-cm)] containing pine wood shavings; weight loss was comparable to that in our previous studies (4). In studies of the time course of the response to refeeding, groups of fasted animals were refed in plastic cages [three to four animals per cage (20 x 33 cm)] with access to both food and water. After a given period of time, they were either sacrificed immediately or placed in similar cages without food and sacrificed as described below. In studies of the effect of diet quantity and composition, animals were refed individually in metabolic cages in which food consumption could be measured. All animals were sacrificed under pentobarbital anesthesia. Serum somatomedin activity Serum somatomedin activity in blood obtained by aortic puncture was measured in a porcine costal cartilage disc bioassay, as described (4, 5).
Endo • 1978 Vol 103 • No 1
added to make all diets comparable in bulk and caloric concentration (approximately 4.6 kcal/g) and minerals and vitamins were supplemented, as above. Saccharine (Sucaryl) was added to all diets at 30 jitl/g, as this provided greater uniformity of food consumption. Experimental design and analysis The effects of diet were studied in 31 separate experiments, each experiment involving 26-29 rats. Within experiments, groups of both control and experimental rats (two to four rats each) were sacrificed at each time period to allow a comparison of serum somatomedin activity and growth cartilage activity. In each experiment, a single value at each time period for control animals and for each group of experimental animals was determined by averaging measurements on individual animals. In the experiments below, n refers to the number of experiments of which each contributed a single pooled value to the pooled data for each time period. Statistical significance was determined by t test.
Results Time course of the response to refeeding
The time course of the response to short term refeeding was first examined in an atGrowth cartilage activity tempt to develop a simple system in which to The activity of the animals' costal cartilage (used measure the effects of diet on somatomedin as an index of stimulation in vivo by somatomedins activity. Figure 1 shows the changes in weight, and other factors) was measured by sulfate incorsomatomedin activity, and cartilage SO4 upporation in vitro, as described (4). take (a measure of growth cartilage activity) observed when fasted rats were allowed free Diets access to a balanced diet for 6 h and then The time course of the response to refeeding was withdrawn from food. With 72 h of fasting, all examined in animals allowed access to pellets of animals lost weight and both serum somatoPurina rat chow. In subsequent studies, animals medin activity and cartilage SO4 uptake fell to were given either balanced or nutrient-depleted powdered diets mixed in our laboratory from con- hypopituitary levels. With 6 h of refeeding, stituents obtained from ICN Pharmaceuticials, Inc. animals initially regained weight, but again The balanced diet contained dextrin, vitamin-free lost weight when withdrawn from food. (Alcasein, corn oil, 4% salts (as USP XVIII), and 1% though these acute changes in weight may vitamins (as "Vitamin Diet Fortification Mixture") represent alterations in lean body mass, they per 100 g. This provided 56%, 28%, and 16% of undoubtedly reflect changes in body water calories as carbohydrate, protein, and fat, respec- and intestinal contents as well.) Serum somatively, proportions similar to those of standard rat tomedin activity rose significantly above fastdiets. With nutrient-depleted diets, calories were provided as 1) fat only (lard), 2) carbohydrate only ing levels with 6 h of refeeding, and rose (dextrin), 3) protein only (vitamin-free casein), 4) further after food was withdrawn even though fat plus carbohydrate (50% corn oil, 50% dextrin), weight loss had already begun. With contin5) fat plus protein (50% corn oil, 50% vitamin-free ued lack of food, somatomedin activity again casein), or 6) protein plus carbohydrate (50% vi- declined. No significant changes in cartilage tamin-free casein, 50% dextrin). Cellulose was SO4 uptake were observed during and after
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 12:55 For personal use only. No other uses without permission. . All rights reserved.
REGULATION OF SOMATOMEDIN BY DIET CARTILAGE S 0 4 UPTAKE (%CONTROL) ^ \
» = p
Vol. 103, No. 1 Printed in U.S.A.
Somatomedin and Nutrition. IV. Regulation of Somatomedin Activity and Growth Cartilage Activity by Quantity and Composition of Diet in Rats* LAWRENCE S. PHILLIPS, ARTHUR T. ORAWSKI, AND D. C. BELOSKY Center for Endocrinology, Metabolism, and Nutrition and Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611 ABSTRACT. To examine the relation of somatomedin to nutritional status, bioassayable serum somatomedin activity and growth cartilage activity (SO4 uptake in vitro) were determined in rats fasted for 3 days and then either fasted for a fourth day or refed with diets varying in quantity and nutrient composition. Animals refed for 6 or 12 h had modest increases in somatomedin activity and cartilage SO4 uptake which persisted after food was withdrawn and weight loss had begun. Refeeding for 24 h produced greater increases in somatomedin activity and cartilage SO4 uptake; this model was used to study the effects of alterations in diet. Refeeding with different quantities of a balanced diet produced dose-response increases in somatomedin activity and cartilage SO4 uptake. In animals refed isocalorically, diets of fat and carbohydrate or fat alone led to increases in somatomedin activity comparable to those with a balanced diet, yet little increase in cartilage SO4 uptake. In contrast, diets of protein and carbohydrate or protein alone
led to smaller increases in somatomedin activity with greater increases in cartilage SO4 uptake (P < 0.05). Diets of fat and protein or carbohydrate alone produced intermediate responses. Although 24-h refeeding with protein-deficient diets produced an increase in somatomedin activity, continuation of refeeding to 48 h led to a decrease in somatomedin activity (P < 0.05) with cartilage SO4 uptake comparable to fasting levels. A similar decrease in somatomedin activity with low cartilage SO4 uptake was found in animals fed for 4 days with a protein-deficient diet. We conclude that serum somatomedin activity and growth cartilage activity may be determined by both quantity and nutritional composition of the diet. Dietary protein may be of particular importance, as deficiency of this constituent appears to lead acutely to a decrease in growth cartilage activity and chronically to decreases in both serum somatomedin activity and growth cartilage activity. (Endocrinology 103: 121, 1978)
S
We have reported previously that when normal rats were fasted 1) their serum somatomedin activity decreased to hypopituitary levels, 2) this decrease was followed by a similar fall in the activity of their growth cartilage, and 3) these changes could not be prevented by the concurrent administration of pharmacologic amounts of GH (4). When the animals were refed, their somatomedin activity returned promptly to normal levels, followed by a comparable rise in growth cartilage activity (4). In the present studies, we have used the Received July 27, 1977. refeeding of fasted rats as a model in which to Address reprint request to: Dr. Lawrence S. Phillips, Center for Endocrinology, Metabolism, and Nutrition, examine the regulation of serum somatomedin Northwestern University Medical School, 303 East Chi- activity and growth cartilage activity by quancago Avenue, Chicago, Illinois 60611. * This work was presented in part at the 59th Annual tity and nutritional composition of the diet.
OMATOMEDINS were characterized initially as circulating growth factors regulated by GH (1). However, there is now evidence that the cartilage-stimulating activity of somatomedins may be modulated by nutritional status and insulin as well. Thus, somatomedin activity is decreased (despite high levels of GH) in children with kwashiorkortype malnutrition (2), and somatomedin activity is normal (despite low levels of GH) in children who are hyperphagic and hyperinsulinemic after hypothalamic surgery (3).
Meeting of the Endocrine Society, Chicago, June 1977. It was supported in part by Research Grants AM-10699 and AM-21483 and Training Grant AM-07169 from NIAMDD, a research grant and research and development award from the American Diabetes Association, and a research award from the Greater Chicago and Northern Illinois Affiliate of the American Diabetes Association.
Materials and Methods Animals
Male Charles River rats, weighing 90-110 g at the onset of fasting, were housed in a constant 121
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 12:55 For personal use only. No other uses without permission. . All rights reserved.
122
PHILLIPS, ORAWSKI, AND BELOSKY
temperature (23 C) room lighted from 0800-1800 h. Fasting and refeeding began between 1000-1200 h. Control animals were maintained four to six per 20 X 33-cm plastic cage containing pine wood shavings (coprophagy not prevented). Groups of animals were given water ad libitum and were deprived of food for approximately 72 h in plastic cages [seven to nine animals per cage (46 X 35-cm)] containing pine wood shavings; weight loss was comparable to that in our previous studies (4). In studies of the time course of the response to refeeding, groups of fasted animals were refed in plastic cages [three to four animals per cage (20 x 33 cm)] with access to both food and water. After a given period of time, they were either sacrificed immediately or placed in similar cages without food and sacrificed as described below. In studies of the effect of diet quantity and composition, animals were refed individually in metabolic cages in which food consumption could be measured. All animals were sacrificed under pentobarbital anesthesia. Serum somatomedin activity Serum somatomedin activity in blood obtained by aortic puncture was measured in a porcine costal cartilage disc bioassay, as described (4, 5).
Endo • 1978 Vol 103 • No 1
added to make all diets comparable in bulk and caloric concentration (approximately 4.6 kcal/g) and minerals and vitamins were supplemented, as above. Saccharine (Sucaryl) was added to all diets at 30 jitl/g, as this provided greater uniformity of food consumption. Experimental design and analysis The effects of diet were studied in 31 separate experiments, each experiment involving 26-29 rats. Within experiments, groups of both control and experimental rats (two to four rats each) were sacrificed at each time period to allow a comparison of serum somatomedin activity and growth cartilage activity. In each experiment, a single value at each time period for control animals and for each group of experimental animals was determined by averaging measurements on individual animals. In the experiments below, n refers to the number of experiments of which each contributed a single pooled value to the pooled data for each time period. Statistical significance was determined by t test.
Results Time course of the response to refeeding
The time course of the response to short term refeeding was first examined in an atGrowth cartilage activity tempt to develop a simple system in which to The activity of the animals' costal cartilage (used measure the effects of diet on somatomedin as an index of stimulation in vivo by somatomedins activity. Figure 1 shows the changes in weight, and other factors) was measured by sulfate incorsomatomedin activity, and cartilage SO4 upporation in vitro, as described (4). take (a measure of growth cartilage activity) observed when fasted rats were allowed free Diets access to a balanced diet for 6 h and then The time course of the response to refeeding was withdrawn from food. With 72 h of fasting, all examined in animals allowed access to pellets of animals lost weight and both serum somatoPurina rat chow. In subsequent studies, animals medin activity and cartilage SO4 uptake fell to were given either balanced or nutrient-depleted powdered diets mixed in our laboratory from con- hypopituitary levels. With 6 h of refeeding, stituents obtained from ICN Pharmaceuticials, Inc. animals initially regained weight, but again The balanced diet contained dextrin, vitamin-free lost weight when withdrawn from food. (Alcasein, corn oil, 4% salts (as USP XVIII), and 1% though these acute changes in weight may vitamins (as "Vitamin Diet Fortification Mixture") represent alterations in lean body mass, they per 100 g. This provided 56%, 28%, and 16% of undoubtedly reflect changes in body water calories as carbohydrate, protein, and fat, respec- and intestinal contents as well.) Serum somatively, proportions similar to those of standard rat tomedin activity rose significantly above fastdiets. With nutrient-depleted diets, calories were provided as 1) fat only (lard), 2) carbohydrate only ing levels with 6 h of refeeding, and rose (dextrin), 3) protein only (vitamin-free casein), 4) further after food was withdrawn even though fat plus carbohydrate (50% corn oil, 50% dextrin), weight loss had already begun. With contin5) fat plus protein (50% corn oil, 50% vitamin-free ued lack of food, somatomedin activity again casein), or 6) protein plus carbohydrate (50% vi- declined. No significant changes in cartilage tamin-free casein, 50% dextrin). Cellulose was SO4 uptake were observed during and after
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REGULATION OF SOMATOMEDIN BY DIET CARTILAGE S 0 4 UPTAKE (%CONTROL) ^ \
» = p
