7 Somatomedin activity in disorders of nutrition and metabolism

7 Somatomedin Activity in Disorders of Nutrition and Metabolism L. S. P H I L L I P S T. G . U N T E R M A N

The existence of the somatomedin peptides was originally hypothesized to explain questions based on studies of growth hormone (GH) action (Salmon and Daughaday, 1957). The demonstration of serum factors which appeared GH-responsive and stimulated growing cartilage in vitro provided evidence for such mediators of GH effects. Subsequent measurements of somatomedins in disorders of the GH axis (GH deficiencyacromegaly) further supported the hypothesis by showing a general correlation with circulating levels of GH. However, extension of somatomedin measurements to other conditions suggested that somatomedins might be regulated by other mechanisms as well. Situations of 'no growth despite GH' and 'growth without GH' were found in which circulating somatomedins or somatomedin activity appeared to parallel levels of insulin and nutritional status more closely than levels of GH (Table 1). Thus, in kwashiorkor and diabetes, growth may be poor despite normal to elevated levels of GH (Hansen and Johansen, 1970; Grant et al, 1973); in these conditions, low levels of somatomedin activity appear to parallel insulin and nutrition more than GH. Conversely, children who are obese or who have had hypothalamic surgery may grow well despite low levels of GH (Van den Brande and Du Caju, 1974; Costin et al, 1976); in such conditions, normal levels of somatomedin activity again appear to parallel levels of insulin and nutrition more than levels of GH. In combination, such observations indicate that somatomedins - - and growth - - are modulated by insulin and nutrition as well as by GH. In view of the anabolic insulin-like actions of the somatomedin peptides (Phillips and Vassilopoulou-Sellin, 1980), such non-GH regulation suggests that somatomedin mechanisms may play an important role in diverting calorie use toward or away from 'growth' as a reflection of nutritional/ hormonal status. While a number of tissues appear to be able to generate somatomedins de novo under tissue culture conditions, the best evidence to date indicates that non-GH regulation of circulating somatomedin activity occurs in the liver. Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - -

Vol. 13, No. 1, March 1984

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L. S. P H I L L I P S A N D T. G . U N T E R M A N T a b l e 1. Non-GH regulation o f somatomedins a

State Obesity Hypothalamic surgery Kwashiorkor Diabetes

Growth

GH

SM

Nutrition

Insulin

Good Good Poor Poor

+ +

+ + -

+ + -

+ + -

a A l t h o u g h s o m a t o m e d i n s w e r e first c h a r a c t e r i z e d as r e g u l a t e d o n l y b y G H , t h e s e clinical o b s e r v a t i o n s s u g g e s t r e g u l a t i o n b y n u t r i t i o n a n d i n s u l i n as well.

This chapter will review the present status of somatomedin regulation and actions in selected disorders which do not involve the GH axis. Emphasis will be placed on altered somatomedin regulation in disorders of nutrition and insulin status, since these have been best studied. Altered somatomedin regulation in conditions of glucocorticoid excess and chronic renal failure will also be reviewed as representative metabolic disorders in which circulating somatomedin activity does not appear to reflect changes in GH levels per se. Finally, the role of the liver will be discussed as a potential locus of 'integrated' nutritional/hormonal regulation of somatomedins. NUTRITION Nutrient Energy and Growth Growth requires a net positive energy balance. Energy requirements for growth include nutrients to sustain basal needs as well as those of deposition of new tissue (Panemangalore, Clark and Clark, 1978), and energy for physical activity must also be provided (Waterlow, Hill and Spady, 1976). The energy cost for tissue synthesis appears to exceed the amount of energy stored within the peptide bonds that are formed (Waterlow, Hill and Spady, 1976), and is in part reflected in the marked postprandial increase in metabolic rate seen in children gaining weight (Brooke and Ashworth, 1972). Nevertheless, except for the early months of postnatal life when up to 30 per cent of caloric expenditure may be directed towards growth (Graystone and Cheek, 1975), and during times of recovery from malnutrition when weight velocity may be 7 to 15 times normal (Whitehead and Biol, 1977), the percentage of total caloric expenditure directed towards growth is relatively small; by the age of one to two years, the energy costs of growth are exceeded by the energy costs of physical activity (Payne and Waterlow, 1971). Because the energy of growth is a relatively small part of an individual's total caloric expenditure, and because ongoing maintenance needs must first be met if the individual is to survive, one might expect the growth process to be very sensitive to nutritional restriction. In fact, decrease in growth is a useful and sensitive clinical marker of undernutrition, with a decrease in weight velocity preceding a decrease in height velocity, and both of these measurements being more sensitive than a decrease in height for age (Tanner, 1976).

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Nutrition and Growth Patterns

The particular form of nutritional restriction (protein, protein-calorie, carbohydrate, and/or fat) is important for determining how the individual will handle available food. For example, protein restriction is accompanied by increased heat loss at the expense of fat and/or carbohydrate oxidation while the efficiency of food nitrogen use is maintained (Heard et al, 1977; Tulp, Gambert and Horton, 1979). In contrast, in food-restricted dogs, an adequate carbohydrate-to-rat ratio is necessary to maintain the efficiency of nitrogen retention (Rosenthal, 1952). The impact of altered nutrition on growth varies not only with the quality of the deficiency, but also with'the timing of the nutritional insult in relation to the stage of the individual within the pattern of normal growth and maturation. In general, tissues are thought to grow in a triphasic sequence, which begins with an increase in cell number, followed by a period of increase in both cell number and cell size, and is completed with a phase of continued cellular hypertrophy (Enesco and Leblond, 1962; Winick and Noble, 1966). The period of cellular proliferation varies between organs within a given species (Hirsch and Han, 1969), and between species there are even greater differences in the patterns of growth - - for example, at birth rats are less mature physiologically than humans. The limited time during which cell number increases in a particular tissue appears to be a 'critical period' during which cells may proliferate if the environment (including nutrition) is favourable; after this period, cellular hyperplasia is not fully compensatory even though nutrition may be improved (McCance, 1976). For example, if the developmental period for brain hyperplasia occurs in a setting of nutritional deprivation, a permanent deficit in brain cell number may result (Winick, Brasel and Rosso, 1972). Thus, it is thought that early malnutrition may affect the organism permanently, and because of differences in the timing of growth and growth regulation among organs, different tissues may be affected unequally. In general, animals exposed to early malnutrition have smaller organs with fewer cells and, relative to their overall small size, they have disproportionately large brains and bones, normal sized hearts and kidneys, and a decrease in skeletal muscle (Panemangalore, Clark and Clark, 1978). In addition, within single organs there may be variation in the effects of exposure to malnutrition during development. When pregnant rats are protein-restricted, the brains of their offspring show a decrease in D N A content which is most marked in the areas of the lateral ventricle and cerebellum, followed by the third ventricle and subiculum, while the DNA content of the grey and white matter of the cerebrum is least affected (Winick, Brasel, and Rosso, 1972). While states of nutritional restriction clearly decrease the growth of organisms, tissues, and cells, excessive nutrition can increase growth. Rat pups given the opportunity to overfeed (by reduction of litter size) show accelerated early growth with early maturation in some areas (sexual development and bone age), but a normal pattern of development in other areas (tooth eruption) (Dickerson and Widdowson, 1960; Widdowson and

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McCance, 1960). Rats with such early growth tend to remain larger than normal through adulthood (Widdowson and McCance, 1960). In human studies, obese children are tall as well as heavy for age (Wolff, 1955; Forbes, 1977), and have increased lean body mass (Forbes, 1977). When their food intake is restricted, their height velocity is reduced (Forbes, 1977). As in the studies in rats, obese children show advanced skeletal and sexual maturation (Wolff, 1955; Garn and Haskell, 1960). Because of their advanced bone age, such tall obese human children end up with relatively normal stature as adults (Garn and Haskell, 1960). Hormone Changes with Nutritional Alteration

While some of the effects of altered nutrition on growth are direct, nutritional status has an additional impact on endocrine function; cell and tissue growth appear to be controlled both by the supply of nutrients, and by the growth-stimulating and growth-inhibiting factors in the environment. Endocrine adaptations may not only modulate the levels of circulating nutrients, but also direct utilization of nutrients in the periphery for maintenance and growth. Growth hormone

Since growth hormone (GH) plays a critical role in promotion of growth (see Chapter 6), there has been great interest in the effect of nutrition on GH secretion. The well-knowfi phen~/nenon of GH secretion in response to acute hypoglycaemia may well be a response to stress and not to hypoglycaemm per se. Nevertheless, short-term fasting in adults results in G H elevation (Cahill et al, 1966). However, GH status is less clear in chronically malnourished children. While GH levels may be normal in patients with balanced protein-calorie deficiency (Raghuramalu and Jaya Rao, 1974), children with kwashiorkor (protein > calorie deficiency) have high basal G H levels (Beas and Muzzo, 1973; Pimstone, Becker and Hansen, 1973; Robinson et al, 1973; Raghuramalu and Jaya Rao, 1974) which correlate inversely with serum albumin (Lunn et al, 1973) and fall to normal with protein feeding (Beas and Muzzo, 1973) or oral amino acid administration. Growth hormone dynamics are abnormal in kwashiorkor; there is little response to arginine infusion, failure to normalize with carbohydrate feeding, and sometimes a paradoxical increase with carbohydrate loating (Alvarez et al, 1972). Lunn et al (1979) have reported an inverse correlation between GH and height-weight velocity - - suggesting poor growth despite increased GH. Others have shown that GH administration does not benefit children with malnutrition (Hadden and Rustishauser, 1967). Although G H dynamics in rats differ from those in humans, studies in these animals lead to similar conclusions. Starved rats have a decrease in plasma and pituitary GH, as well as a decrease in hypothalamic 'GH-RF' activity (Dickerman, Negro-Vilar and MeRes, 1969; Stephan et al, 1971). However, in protein-restricted weanling rats, G H treatment fails to increase cartilage growth activity as measured by sulphate uptake (Shapiro

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149

and Pimstone, 1977). Thus, in protein-depleted rats (which may be GH deficient), there appears to be GH insensitivity as well, similar to that of malnourished humans. While human protein malnutrition results in growth retardation despite elevated GH, in obesity growth is increased (Wolff, 1955; Forbes, 1977) despite diminished GH responses to provocative testing (Cacciore et al, 1972) (see section 'Growth in hyperinsulinism', below). Insulin

Hypoinsulinaemia may play a role in the growth failure of undernutrition, and insulin appears to be a growth regulator and potential OH-sensitizer in states of overnutrition. Plasma insulin levels are decreased with short-term fasting in adults (Cahill et al, 1966), and with chronic malnutrition in children (Lunn et al, 1973; Pimstone et al, 1973; Robinson et al, 1973), where insulin is correlated with albumin levels (Lunn et al, 1973). In such patients, glucose and amino acid challenge elicit a blunted insulin response which improves with nutritional therapy (Pimstone et al, 1973; Robinson et al, 1973). Part of this secretory defect may be due to potassium depletion (Pimstone et al, 1973). Lunn et al (1979) have reported a positive correlation between insulin and height-velocity in malnourished children, suggesting a role for insulin in growth regulation. Obesity has been associated with high insulin levels. While obesity may induce resistance to some of the effects of insulin (Reaven, 1983), other insulin-mediated effects, including growth, may be increased. For example, ob ob mice exhibit a decrease in peripheral insulin sensitivity yet have an increase in the insulin-dependent liver enzyme, glucose kinase (Hellerstr6m et al, 1970). They also have increased growth (Herbai, Westman and Hellerstr6m, 1970), and increased sensitivity to exogenous G H (Herbai, Westman and Hellerstr6m, 1970), despite low GH levels. In combination, these data suggest that in obesity, despite peripheral insulin resistance, insulin may make important contributions to growth - - perhaps by increasing sensitivity to OH (see later). Cortisol

While reduced anabolism due to decreased insulin secretion may contribute to the lack of growth seen with undernutrition, increased catabolism due to corticosteroids appears important as well. Cortisol levels are increased in patients with marasmus and kwashiorkor (Alleyne and Young, 1966; Waterlow and Alleyne, 1971; Lunn et al, 1979). While it is difficult to separate out the stress-related effects of infections frequently present in these patients (Whitehead, Rowland and Cole, 1976), Lunn has shown an inverse relationship between cortisol levels and gain in height and weight (Lunn et al, 1979). Cortisol facilitates the mobilization of carcass protein, and increases the availability of amino acids for gluconeogenesis by the liver. Cortisone-treated rats receiving a low, protein diet have increased weight loss and increased loss of muscle protein (Lunn et al, 1976), while weight loss and decreased DNA synthesis induced by

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L. S. PHILLIPSAND T. G. UNTERMAN

fasting can be blunted by adrenalectomy (Goldberg and Goldspink, 1975). In addition to such catabolic effects on body nitrogen stores, corticosteroids may also decrease the production of growth factors and/or inhibit skeletal growth directly or indirectly (see below). Thyroid

Hypothyroidism is a recognized cause of growth failure. However, the growth-related effects of the low T3 seen in malnutrition (Wartofsky and Berman, 1982) have not been well-studied. Both protein and carbohydrate intake play a role in the modulation of the peripheral conversion of T 4 t o T3 (Glass et al, 1978). In malnutrition, the fall in T3 may be homeostatic, in that a reduced metabolic rate might help to conserve body protein and lean body mass (Burman et al, 1979). Although low levels of thyroid hormones may contribute to growth failure in primary thyroid disease because GH synthesis and release are reduced (Hervas, Morreale de Escobar and Escobar del Ray, 1975; Mosier et al, 1977), G H deficiency does not appear important in the growth failure of nutritional deprivation (see above). Since T3 may sensitize growing cartilage to somatomedins (Froesch et al, 1976), and may have a permissive role in GH-induced somatomedin generation (Burstein et al, 1979), it is possible that low T3 exacerbates the consequences of altered somatomedin metabolism in malnutrition (see below). Nutrition and Somatomedins

Since in malnutrition - - and particularly with protein deficiency - - normal growth does not occur despite the presence of normal or increased GH, there is interest in the level of the presumed defect in GH action. While some anabolic effects of GH (e.g., on muscle) may be direct (Kostyo and Rillema, 1971), effects on skeletal growth appear to be mediated by the somatomedins (see Chapter 1). These circulating peptides exhibit (1) GH-dependence, (2) broad stimulatory effects on cartilage, (3) insulin-like activity on fat and muscle, (4) mitogenic activity on cultured cells, and (5) proinsulin-like amino acid composition and tertiary structure (Phillips and Vassilopoulou-Sellin, 1980). Somatomedins can be measured by specific radioassays, and also by bioassays - - in which somatomedin activity reflects the presence of both somatomedins and inhibitory factors which can antagonize somatomedin action. Deficient growth in the setting of adequate GH could occur because of decreased somatomedins, increased somatomedin inhibitors, or changes in the target organ (altered sensitivity to somatomedins and/or somatomedin inhibitors). Initial studies in humans

Most studies of humans with gross malnutrition have revealed decreased somatomedin activity as measured by bioassay, and/or decreased somatomedins by radioassays. Children with kwashiorkor or marasmus have been found to have low levels of circulating somatomedin activity (Grant et al, 1973; Van den Brande and Du Caju, 1974, Hintz et al, 1978). Grant et

SOMATOMEDINS AND NUTRITIONAL/METABOLICDISORDERS

151

al (1973) noted an increase in somatomedin activity after nine days of nutritional therapy, while Hintz et al (1978) found a slower response. Undernutrition has been associated with a reduction of somatomedin activity in other disorders, including cystic fibrosis (Lee et al, 1980) and coeliac disease (Lecornu, David and Francois, 1978). In Crohn's disease, where the need for adequate nutrition for growth has been carefully studied (Kirschner et al, 1981; Motil et al, 1982) somatomedin levels have been low when measured by RIA (Kelts et al, 1979). Somatomedin activity is also low in these patients, and increases with nutritional supplementation (Kirschner, 1981). In subjects with anorexia nervosa, somatomedin activity is decreased (Rappaport, Prevot and Czernichow, 1978); this decrease is correlated with weight deficit (Rappaport, Prevot and Czernichow, 1979), occurs despite elevated G H levels, and cannot be reversed by treatment with GH but is normalized with weight gain (Rappaport, Prevot and Czernichow, 1978). Takano et al 1979b and Megyesi et al (1975) have also reported a decrease in somatomedins measured by R R A in such patients. Van den Brande and Du Caju (1974) noted that the somatomedin activity of normal serum was blunted by addition of serum from malnourished patients, suggesting that the decrease in measured somatomedin activity might be due in part to an increase in circulating somatomedin inhibitors. Using a similar technique, Hintz et al (1978) showed the presence of significant inhibitory activity in ten of 27 malnourished patients shortly after hospitalization, but no detectable inhibitors after one month of nutritional therapy. Although it has been suggested that patients with marasmus may have normal levels of somatomedins (after heat-labile inhibitors were inactivated) (Van den Brande et al, 1975), the finding of low somatomedins measured by R R A in other malnourished subjects suggests that both somatomedins and inhibitors are involved. In combination, these early studies suggested that malnutrition in humans is accompanied by a decrease in net serum somatomedin activity, in part because of the presence of somatomedin inhibitors, but also because of a decrease in serum somatomedins. (Further discussion on inhibitors is provided in the section 'Insulin and diabetes.) Animal models: acute studies

Attempts to characterize the relationship between nutrition and somatomedin activity in more detail were then carried out using the rat as a model. Fasting resulted in a decrease in cartilage growth activity (Salmon, 1975; Phillips and Young, 1976a; Price et al, 1979), reaching hypopituitary levels after 72 hours (Figure 1). Low cartilage growth activity did not reflect a change in target organ sensitivity, since in vitro exposure of the cartilage to somatornedins resulted in stimulation of sulphate incorporation (Salmon, 1975; Phillips and Young, 1976a; Price et al, 1979). The fall in cartilage growth activity was preceded by a fall in serum somatomedin activity which occurred within 24 hours (Phillips and Young, 1976a). Decreases in serum sornatomedin activity and cartilage growth activity occurred despite administration of GH (Phillips and Young, 1976a) indicating that they were essentially GH-independent. Refeeding was

152

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DAYS Figure 1. Temporal relationship between changes in somatomedin activity and cartilage growth activity (cartilage incorporation of sulphate in vitro) in fasted and refed rats. From 16 experiments: n = 3 to 16 for each point. Fasting produced a prompt decrease in serum somatomedin activity, which was below control levels at 24 hours and reached hypopituitary levels at 72 hours. These changes were followed by parallel changes in cartilage growth activity. With refeeding, somatomedin activity increased within six hours, without a comparable increase in cartilage growth activity. With continued refeeding, somatomedin activity reached control levels after 24 hours, and was followed by a comparable increase in cartilage growth activity. From Phillips and Young (1976a) with kind permission of the editor of Endocrinology.

followed by an increase in somatomedin activity within six hours, with a return to normal by 24 hours. These changes again preceded rises in cartilage activity (Phillips and Young, 1976a; Phillips, Orawski and Belosky, 1978). Similar fasting-induced decreases in somatomedins were found with R R A or RIA measurements (Takano et al, 1978; Furlanetto et al, 1979), suggesting that the fall in somatomedin activity was not due to an increase in inibitors alone. However, because somatomedins measured by radioassay rise more slowly than somatomedin activity after refeeding (Phillips and Young, 1976a; Furlanetto et al, 1979; Takano et al, 1980), inhibitors may well play a role: conceivably, during refeeding the inhibitors might decrease rapidly while somatomedins are returned more slowly to the circulation. In combination, these animal studies demonstrated a close relation between nutrition, somatomedins, and growth (as measured by 'cartilage growth activity'). Rat models have also been used to examine the role of dietary quantity and quality in the regulation of circulating somatomedin activity and cartilage growth activity. In our laboratory, increases in somatomedin activity and cartilage growth activity were closely related to food intake during refeeding after a 72-hour fast (Figure 2) (Phillips, Orawski and

153

SOMATOMEDINS AND NUTRITIONAL/METABOLIC DISORDERS

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CON- FAST 0 O- 31- 61- 100% TROL 3 DAYS 30% 60% 90% % Ad Lib Food Consumption Figure 2. Responses to refeeding for 24 hours with different quantities of a vitaminsupplemented balanced diet. Mean + s.e.m, for six experiments. Refeeding with increasing quantities of balanced diet produced a progressive increase in weight, serum somatomedin activity, and cartilage growth activity (measured as cartilage sulphate in vitro). Significant increases in serum somatomedin activity and cartilage growth activity were obtained by calorie intakes greater than 60 per cent of ad lib. food consumption. From Phillips, Orawski and Belosky (1978) with kind permission of the editor of Endocrinology.

Belosky, 1978). Unless protein was included in the diet, fasting followed by refeeding produced only a transient increase in somatomedin activity, with no stimulation of cartilage growth activity (Table 2); with continued protein deficiency, somatomedin activity fell again to hypopituitary levels. These short-term studies were consistent with the decrease in somatomedin activity seen in humans with kwashiorkor (Grant et al, 1973; Van den Brande and Du Caju, 1974; Hintz et al, 1978, and indicated that protein intake may also be important in the response of growing cartilage to somatomedins (Phillips, Orawski and Belosky, 1978). More recent studies by Takano et al (1980) have demonstrated an increase of somatomedin A with fasting followed by refeeding with a high protein diet, but not after high carbohydrate-low protein refeeding. Somatomedin A did not increase after G H treatment in the fasted state, consistent with our earlier studies in rats (Phillips and Young, 1976a) and with the GH resistance of human malnutrition (Hadden and Rustishauser, 1967).

154

L.S. PHILLIPS AND T. G. UNTERMAN Table 2. Effect of refeeding on sornatomedin activity and cartilage sulphate uptake ~

Control 3-day fast

Somatomedin activity

Cartilage 80 4 uptake

1.29 +_ 24 U/ml 0.44 +_ 0.11 U/ml

100% 45 ± 4%

1.10 ± 0.24 U/ml 1.09 ± 0.13 U/ml

64 ± 10% 78 + 8%

0.78 _+ 0.10 U/ml 0.50 ± 0.09 U/ml

42 ± 11% 34 ± 3%

Balanced diet (80% ad lib):

Day 1 Day 2 Low-protein diet (80% ad lib):

Day 1 Day 2

aSomatomdin activity and cartilage growth activity (cartilage sulphate uptake in vitro) in rats fasted for three days, then refed with either a balanced laboratory diet (80 per cent of ad libitum intake) or an equicaloric diet containing only fat and carbohydrate (1:1). Mean + s.e.m, for four experiments. Three days of fasting produced a marked decline in serum somatomedin activity and cartilage growth activity. Refeeding with a balanced diet for 24 h produced an increase to initial levels of somatomedin activity, and a rise in cartilage growth activity. This was continued over the next 24 h. In contrast, refeeding with an equicaloric low protein diet produced a blunted but significant (P<0.05) rise in somatomedin activity, without an accompanying rise in cartilage growth activity. Continued consumption of this diet led to a return of somatomedin activity to fasted levels, with a fall in cartilage growth activity. These studies show the importance of dietary protein for maintenance of circulating somatomedin activity, and suggest that protein must be present if somatomedins are to stimulate cartilage growth (since the transient rise in somatomedin activity was not accompanied by rise in cartilage growth activity). From Phillips, Orawski and Belosky (1978) with kind permission of the editor of Endocrinology.

Animal models: chronic studies It has b e e n m o r e difficult to c o n s t r u c t a m o d e l of c h r o n i c m a l n u t r i t i o n in rats b e c a u s e of t h e i r l i m i t e d g r o w t h p e r i o d . I n o u r l a b o r a t o r y , rats w e r e f e d for f o u r d a y s with e q u i c a l o r i c diets d e f i c i e n t in fat, c a r b o h y d r a t e , o r p r o t e i n (Phillips a n d Y o u n g , 1976a); p r o t e i n r e s t r i c t i o n r e s u l t e d in significant d e c r e a s e s in b o t h s e r u m s o m a t o m e d i n activ~ity a n d c a r t i l a g e g r o w t h activity ( T a b l e 3), c o m p a r a b l e to levels in a n i m a l s p r o v i d e d with 70 p e r cent less c a l o r i e s . a s - a b a l a n c e d .diet. I n o t h e r studies, S h a p i r o a n d P i m s t o n e (1977) f o u n d t h a t w e a n l i n g rats e x p o s e d to 20 d a y s of p r o t e i n r e s t r i c t i o n e x h i b i t e d d e c r e a s e d c a r t i l a g e s u l p h a t e u p t a k e , with e p i p h y s i a l t h i n n i n g a n d small, f l a t t e n e d c h o n d r o c y t e s - - similar to findings in h y p o p h y s e c t o m i z e d a n i m a l s . U n d e r such c o n d i t i o n s , t r e a t m e n t w i t h high d o s e s of G H d i d n o t i n c r e a s e e i t h e r s e r u m s o m a t o m e d i n activity o r e p i p h y s e a l w i d t h . L o w circulating s o m a t o m e d i n activity a p p e a r e d to b e d u e in p a r t to t h e p r e s e n c e of s o m a t o m e d i n i n h i b i t o r s ( S h a p i r o a n d P i m s t o n e , 1977). A s d e s c r i b e d b y Price et al (1979), rats with m a r a s m u s a n d m a r a s m i c - k w a s h i o r k o r h a v e d e c r e a s e d c a r t i l a g e g r o w t h activity. O v e r t i m e , s o m a t o m e d i n activity also fell, initially m o r e in m a r a s m i c -

SOMATOMEDINS AND NUTRITIONAL/METABOLIC DISORDERS

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kwashiorkor, later m o r e p r o f o u n d l y in marasmus. A l t h o u g h the marasmick w a s h i o r k o r animals had higher s o m a t o m e d i n activity but lower cartilage g r o w t h activity than the m a r a s m i c animals, suggesting that protein restriction limited cartilage responses to s o m a t o m e d i n s , this could not be shown directly.

Table

Control

3. Chronic dietary depletion in rats a Somatomedin activity

Cartilage S04 uptake

1.10 _4-0.21 U/ml

100%

0.55 ± 0.10 U/ml 1.06 ± 0.07 U/ml

64 _+ 9 114 _+ 14

0.58 _+ 0.08 U/ml 0.96 -4- 0.08 U/ml 0.91 ± 0.06 U/ml

77 + 4 107 + 3 115 + 9

Balanced diet:

35% ad lib 95% ad lib Deficient diet:

Low-protein Low-carbohydrate Low-fat (all 95% ad lib)

aSomatomedin activity and cartilage growth activity (cartilage sulphate uptake in vitro) in rats refed for four days with either a balanced laboratory diet or diet in which the protein content was reduced to 10 per cent of the level in the balance diet, while the ratio of the other caloric constituents was held constant. Mean _+ s.e.m, for four experiments. Consumption of a balanced diet at 35 per cent ad libitum calories led to a decrease in serum somatomedin activity and cartilage growth activity. Consumption of diets deficient in carbohydrate or fat did not change somatomedin activity or cartilage growth activity. However, consumption of a diet deficient in protein reduced serum somatomedin activity and cartilage growth activity to levels comparable to those found in animals consuming a diet with two-thirds fewer calories. From Phillips and Young (1976a) with kind permission of the editor of Endocrinology.

In m o r e recent examinations by R e e v e s et al (1979), groups of y o u n g male rats were fed four levels of dietary protein and three levels o f dietary fat for 30 days. Plasma s o m a t o m e d i n activity fell in the low-protein g r o u p s irrespective of fat intake, and rose with increasing protein intake, again for all levels of fat c o n s u m e d ( R e e v e s et al, 1979). S o m a t o m e d i n activity was correlated with weight gain, as well as with f o o d efficiency (gain/100 kcal). In o t h e r chronic studies, Prewitt et al (1982) fed y o u n g rats diets with three levels of p r o t e i n (5, 10 and 15 per cent lactalbumin) at three levels of e n e r g y (ad lib, 75 per cent or 50 per cent) with dietary fat held constant. In the first week, s o m a t o m e d i n C levels were d e p e n d e n t largely on protein intake; with c o n t i n u e d nutritional restriction, e n e r g y intake also began to affect s o m a t o m e d i n levels. Levels of s o m a t o m e d i n C w e r e strongly correlated with weight gain and tail length after two, five and eight weeks. In c o m b i n a t i o n with the acute studies in animal models, these m o r e chronic examinations emphasize the close relationship b e t w e e n diet (quantity and quality - - particularly protein content), s o m a t o m e d i n s and growth.

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Figure 3. Comparison of the effects of protein (left panel) and of energy (right panel) on serum somatomedin C during refeeding of normal adults following five days of fasting. Each point represents the percentage of control of the mean prefast value. The effect of protein is depicted as the difference between the two equicaloric diets. The effect of energy is the difference between the two protein-deficient diets. From Isley, Underwood, and Clemmons (1983) with kind permission of the authors and the editor of Journal of Clinical Investigation.

R e c e n t s t u d i e s in h u m a n s

Very recent clinical examinations have provided additional information. Seven obese males were monitored by Clemmons et al (1981) during a ten-day fast and were found to have a progressive fall in somatomedin C, reaching hypopituitary levels by day five. A prompt increase toward normal occurred with refeeding. Of particular interest was the relation to overall protein metabolism; nitrogen balance in these patients was correlated with the change in somatomedin C level. These investigators (Isley, Underwood and Clemmons, 1983) have also examined normal subjects during a five-day fast, followed by five-day periods of refeeding with normal, equicaloric low-protein, or low-protein/calorie-deficient diets. Levels of somatomedin C declined with fasting (more rapidly than in the obese subjects), and continued to fall with low protein/calorie deficient refeeding; some increase occurred with equicaloric, low-protein refeeding, but recovery was blunted compared to that with the balanced diet. Significant correlations were again noted between somatomedin C and nitrogen balance. Analysis of the somatomedin C responses during refeeding showed clearly the importance of both total dietary energy and dietary protein (Figure 3). This demonstration in humans confirms the roles of energy and protein as shown previously in studies of nutritional regulation of somatomedin activity in animal models (above).

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Other workers have examined the effects of nutrition on GH-induced somatomedin generation in hypopituitary dwarfs. Merimee, Zapf and Froesch (1982) showed that IGF I and II were stimulated much less by G H in the fasted than in the fed state; the blunted response was more marked for IGF I than IGF II. Although IGFs I and II decreased when normal subjects were fasted, there was no change when hypopituitary subjects were fasted - - perhaps because somatomedins were already at minimal levels.

Potential Applications The studies reviewed above suggest that somatomedin levels in humans are both protein- and calorie-dependent, and that the different somatomedins may be influenced to varying degrees. They also suggest that somatomedin measurements may be of clinical utility in nutritional assessment. When compared to the difficulties involved in accurate assessment of nitrogen balance, somatomedin levels are relatively easy to obtain, and appear to reflect nitrogen balance in a variety of nutritional states. In children who do not have G H deficiency, the presence of a low somatomedin level would suggest that nutritional insufficiency should be considered. Also, the speed with which somatomedin C returns toward normal with refeeding may indicate that such measurements may be more sensitive to metabolic status than older indices such as levels of albumin. Although circulating somatomedin inhibitors have been noted in malnourished animals and humans (see above) - - and their measurement might also be useful as indices of nutritional status - - such potential application must await characterization of their relation to body energy and protein status.

INSULIN AND DIABETES Growth Actions of Insulin If growth is viewed as coupled calorie storage and skeletal elongation (Phillips, 1981), insulin appears to have several different actions which are related to these processes. Insulin promotes calorie storage via direct effects on fat and muscle and is permissive for the protein anabolic effects of GH. Thus, hypophysectomized pancreatectomized animals given G H have little nitrogen retention unless they are treated with insulin as well (Milman, DeMoor and Lukens, 1961). While direct actions on fat and muscle appear to involve insulin binding to specific receptors, it is less clear how insulin facilitates GH-induced anabolism. Studies by Baxter, Brown and Turtle (1980) suggest that hepatic G H receptors are decreased in diabetic animals; if parallel effects occur in muscle, this could be one pathway by which insulin modulates the protein anabolic effects of GH.

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L. S. PHILLIPS AND T. G. U N T E R M A N

Although at physiological concentrations insulin appears to have little direct effect on skeletal growth processes as studied with cartilage incubations in vitro (Salmon and Daughaday, 1957), a variety of observations indicate that insulin must be present to sustain normal levels of circulating somatomedin activity (see below). Thus, insulin appears to promote skeletal growth indirectly via generation of somatomedin activity; since somatomedin activity reflects a balance of somatomedin and somatomedin inhibitors, insulin might promote generation of somatomedins and/or reduction in inhibitors. Such actions may involve direct effects of insulin and/or permissive effects on somatomedin generation by GH. While direct insulin binding to somatomedin receptors has generally been low (Rechler et al, 1980), it is also possible that insulin may potentiate somatomedin action in some tissues by facilitating the binding of somatomedins. Finally, it should be kept in mind that secretion of insulin is partially coupled to secretion of GH. Insulin levels rise with GH treatment in hypopituitarism via mechanisms which could involve either direct effects of GH, or GH-induced changes in insulin sensitivity (Fineberg, Merimee and Rabinowitz, 1970); such increases in insulin presumably potentiate both caloric storage and skeletal elongation. Growth in Hyperinsulinism There are no 'pure' hyperinsulinaemic syndromes, for hypoglycaemia supervenes unless nutritional intake is increased. However, when increased insulin is coupled with increased nutrition, growth is increased. Under such conditions, enhancement of skeletal growth is most marked when GH is present; thus, obese children (with hyperinsulinaemia) are tall as well as heavy for age (Laron et al, 1972) despite GH levels which are relatively low following provocative stimulation (Cacciore et al, 1972). When G H is absent, the combination of insulin and nutrition appears to produce a more disproportionate increase in calorie storage, although some skeletal growth occurs as well. Thus, hypohysectomized rats given graded doses of insulin exhibit substantial increases in body fat, less marked increases in protein, and small but significant increases in tibial epiphysial width (Salter and Best, 1953). Similarly, after hypothalamic surgery for tumours such as craniopharyngioma, children may develop hyperphagia with hyperinsulinaemia, and exhibit marked weight gain and some 'catch-up' in height as well (Holmes et al, 1968; Costin et al, 1976). However, they may be less tall than normal children of comparable weight, perhaps reflecting the presence of low but physiologically meaningful levels of GH in simple obesity. As noted previously, levels of circulating somatomedin activity are frequently normal - - despite undetectable GH - - in children who have had hypothalamic surgery (Holmes et al, 1968; Costin et al, 1976). This supports the notion that insulin, together with increased food intake, can support somatomedin generation (and growth). It might be expected that individuals with simple obesity, where GH is present at low levels, would

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have elevated somatomedins. However, obese children and adults have normal levels of somatomedin activity as measured by bioassay (Van den Brande and Du Caju, 1974; Chaussain et al, 1978), and normal somatomedins by RRA (Takano et al, 1979b); it is conceivable that somatomedins might rise if GH were raised to normal levels in obese individuals, but no such studies have been reported. Growth

in Hypoinsulinism

Hypoinsulinism provides a relatively pure model where altered growth (and changes in somatomedins) can be attributed largely to defective utilization of nutrients. Diabetes mellitus is characterized by elevated levels of circulating metabolic fuels - - glucose, amino acids and free fatty acids - - with limited anabolism due to insulin insufficiency, and perhaps glucagon excess as well. Compared with malnutrition of the whole organism as discussed above, diabetes may thus be considered a form of cellular malnutrition which exists despite extracellular overnutrition. In the pre-insulin era, diabetes in childhood was almost always accompanied by growth failure. According to Joslin, Root and White (1925): 'If a diabetic child gained weight before the discovery of insulin, it was considered an extraordinary event . . . 12 children not yet taking insulin . . . have shown an average gain in weight of 2.2 kg a year . . . obviously mild cases'. Even when insulin became available, diabetic children usually had diminished gain in height and weight (Joslin, Root and White, 1925). There is controversy as to whether or not diabetic children are taller than normal at onset of their disease, possibly reflecting a period of hyperinsulinaemia prior to full B cell decompensation. Although Laron et al (1972), Pond (1970) and Edelsten et al (1981) have reported such a 'prediabetic' increase in height, others have been unable to confirm this observation (Tattersall and Pyke, 1973; Draminsky Peterson et al, 1978); in the studies of identical twins reported by Tattersall and Pyke (1973), differences in height were minimal at the time of diagnosis. However, relative growth failure after onset of diabetes in childhood has persisted despite over 50 years of experience with insulin management. Laron, Volovitz and Karp (1977) used 25 children as their own controls, and reported a uniform fall in growth velocity after onset of disease, most with a change of - 0 . 7 s.d. Similarly, Draminsky Peterson et al (1978) have reported decreases in height velocity of about 0.5 cm per year in children with long-standing diabetes. Most studies have shown that earlier onset and greater duratio of diabetes in childhood tend to lead to a greater deficit in adult stature. Evans, Robinson and Lister (1972) found significant short stature in children with diabetes for over six years, and Pond (1970) reported that final height percentiles averaged 27, 37 and 49 per cent in children with onset of disease at 0-4, 5-9 and 10-14 years, respectively. Finally, Tattersall and Pyke (1973) have reported that height deficit averaged over 5 cm in identical twins where the affected twin developed diabetes before puberty. (Although most studies probably

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represent mainly insulin-dependent diabetics, in many reports it is not possible to determine how many children had non-insulin-dependent diabetes.) There is also a tendency for growth impairment to be associated with poorer diabetic control. Pond (1970) noted that short diabetics tended to have poor control and Birkbeck (1972) reported that children with poor control tend to be short. Although Craig (1970) and Jivani and Rayner (1973) found no relation between growth and 'control' measured by urine test indices of control, more recent evaluations using haemoglobin Ale as an index of control have revealed that high haemoglobin Ale is associated with growth impairment (Williams and Savage, 1979). Baumer et al (1982) have also found that higher mean blood glucose (determined by multisample home glucose monitoring) was associated with lower height velocity. Finally, we (Winter et al, 1980) and others have found that children with the Mauriac syndrome of diabetes, dwarfism, and hepatomegaly have substantial improvement in growth rate if better control can be instituted. Somatomedins

in D i a b e t e s

The role of somatomedins in altered growth due to diabetes mellitus has been examined in animal models and humans, and probed both with radioassays which are sensitive and specific, and with bioassays which are less specific and presumably respond to a broad spectrumof growth-related stimulators and inhibitors. Animal models

Virtually all examinations in diabetic animal models have shown decreased levels of somatomedins measured by radioassay and somatomedin activity determined by bioassay. In our laboratory (Phillips and Young, 1976b; Phillips and Orawski, 1977), [3-cell destruction by streptozotocin administration in rats resulted in a prompt fall of cartilage growth activity and circulating somatomedin activity (cartilage bioassay) to hypopituitary levels (Figure 4). This could be prevented by administration of insulin, indicating that insulin deficiency was the probable cause. Changes in cartilage growth activity and somatomedin activity were refractory to G H - - indicating again that GH-induced somatomedin generation is impaired when insulin is deficient. In rats with a spectrum of disease produced by treatment with varying amounts of insulin, circulating somatomedin activity was correlated significantly with cartilage growth activity, change in weight, and diabetic control as measured by urine glucose or plasma glucose or ~-hydroxybutyrate. Animals given insulin sufficient to restore somatomedin activity to normal levels had weight gain comparable to that of normal animals. In combination, these animal studies suggest a major role for insulin in the regulation of circulating somatomedin activity - and, presumably via somatomedin activity, on skeletal growth. Other workers have confirmed these observations using different bioassays and radioassays. Eigenmann et al (1977) have also reported decreased somatomedin activity (fat pad bioassay) in pancreatectomized

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Figure 4. Serum somatomedin activity (porcine cartilage bioassay) and cartilage growth activity (sulphate uptake in vitro) in rats given streptozotocin and protamine-zinc insulin. Somatomedin activity was expressed relative to a standard serum and cartilage growth activity as per cent of control values. From 14 experiments: n = 3 to 14 for each point. Administration of streptozotocin was followed by a marked decrease in somatomedin activity, reaching hypopituitary levels after one day and falling below hypopituitary levels after two days. These changes were followed by a decrease in cartilage growth activity. With insulin administration, somatomedin increased modestly but significantly (P < 0.05) after 6 and 12 hours with little change in cartilage growth activity. However, after 24 and 48 hours of insulin administration, both somatomedin activity and cartilage growth activity were returning towards control levels. From Phillips (1979) with kind permissionLofAcademic Press.

dogs and s t r e p t o z o t o c i n - t r e a t e d rats. Franklin, R e n n i e and C a m e r o n (1979) also f o u n d low s o m a t o m e d i n s m e a s u r e d by fat cell bioassay in diabetic rats, and Miller, Schalch and D r a z n i n (1981) and Baxter, B r o w n and Turtle (1980) have o b s e r v e d similar decreases in s o m a t o m e d i n s in diabetic rats w h e n m e a s u r e d by competitive protein-binding assay and r a d i o r e c e p t o r assay, respectively. Human

studies

A g r e e m e n t is less u n i f o r m on changes in s o m a t o m e d i n s in diabetic h u m a n s ; in general, radioassay evaluations have revealed s o m a t o m e d i n levels in the n o r m a l range, while bioassays have often indicated r e d u c e d levels of s o m a t o m e d i n activity. S o m a t o m e d i n s have been m e a s u r e d by R 1 A as Sm C / I G F I in five studies of diabetic h u m a n s , with m e a n levels c o m p a r a b l e to normals in each r e p o r t ( Z a p f et al, 1980; B l e t h e n et al, 1981a; H o r n e r , K e m p and Hintz, 1981; T a m b o r l a n e et al, 1981; R u d o l f et al, 1982). Cross-sectional examinations with such a p p r o a c h e s have generally revealed no correlations with diabetic control, except for the r e p o r t of B l e t h e n et al (1981a) w h e r e a relation to control a p p e a r e d after multiple regression analysis. A l t h o u g h

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Blethen et al (1981b) have also reported decreased levels of somatomedin C in a case of transient neonatal diabetes, it is not clear that values were lower than expected for infants. There are two reports of increases in somatomedin C after improvement of control by insulin-infusion-pump therapy. In the study by Tamborlane et al (1981), it is not clear whether somatomedins rose due to insulin or instead to onset of puberty. However, in the study by Rudolf et al (1982) it seems likely that intensive insulin therapy brought on a significant rise in somatomedin C - - although both pre- and post-pump values were generally within the normal range. In contrast, most bioassay studies of human diabetes have found reduced levels of circulating somatomedin activity, with a readily demonstrable relation to diabetic control. In the 1960s, Samaan and Fraser (1963) measured somatomedins as 'atypical' (non-suppressible) insulin-like activity (fat pad bioassay) in untreated diabetics, and found low levels in ketosis-prone subjects and thin non-insulin-dependent diabetics, although obese non-insulin-dependent diabetics had normal values. These early observations were essentially duplicated by Yde (1969), who found significant decreases in somatomedins measured by cartilage bioassay in both non-obese and obese newly-diagnosed adult diabetics; 'sulphation factor' levels exhibited a significant inverse correlation with fasting serum glucose only in the non-obese subjects. Somatomedin activity was also low in insulin-treated subjects with severe diabetic retinopathy. Since several other groups were unable to confirm these observations, our laboratory re-examined the relationship. Forty non-obese diabetic children were selected at random, all with height above the third percentile for age. Somatomedin activity was measured by porcine cartilage bioassay, and mean values averaged 50 per cent below levels in normal children (Winter et al, 1979). Moreover, somatomedin activity was inversely related to diabetic control as measured by haemoglobin Ale (Figure 5). The relation between circulating somatomedin activity and diabetic control was further examined in our studies of two children with the Mauriac syndrome (Winter et al, 1980). Both had brittle diabetes, dwarfism and hepatomegaly, and both had circulating somatomedin activity in the hypopituitary range (Figure 6). In the first, low somatomedin activity was refractory to GH administration - - similar to our observations in animal models (Phillips and Young, 1976b) and consistent with the notion that insulinization is required for GH to induce somatomedin generation. The second subject had low somatomedin activity at age seven, when he presented with a history of poor diabetic control and poor growth. With altered management, diabetic control and growth improved, and somatomedin activity was normal at age nine. Unfortunately, control again became poor with accompanying growth impairment, and somatomedin activity was again low at age 12. He was then hospitalized, and after three days of improved control, somatomedin activity had again risen into the normal range. In combination with our previous cross-sectional studies, these observations support the hypothesis that circulating somatomedin activity is significantly related to diabetic control - - and growth.

SOMATOMEDINS AND NUTRITIONAL/METABOLICDISORDERS •

163

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Figure 5. Serum somatomedin activity and haemoglobin Ale in 40 children with insulindependent diabetes, all of whom had height above the third percentile for age. There was a significant inverse relationship between somatomedin activity and diabetic control as measured by haemoglobin Ale (r = -0,39, P<0.0I). From Winter et al (1979) with kind permission of the editor of Diabetes. Somatomedin inhibitors

The apparent discrepancy between radioassay and bioassay studies in diabetics could be explained if the bioassays were sensitive to circulating factors not detected by the radioassays. Indeed, circulating inhibitory factors have been described in hypoinsulinaemic states: malnutrition (Salmon, 1972; Van den Brande et al, 1975; Hintz et al, 1978), hypophysectomy (Salmon, 1974) and, as studied in our laboratory, diabetes (Phillips and Young, 1976b; Phillips, Belosky and Reichard, 1979; Phillips, Vassilopoulou-Sellin and Reichard, 1979; Phillips et al, 1979; Phillips and Scholz, 1982). Their nature is poorly understood, but they appear to be proteins (Salmon, 1975) with a major species having a molecular weight of about 25000 (Bajaj, Fusco and Phillips, 1982) and minor species with molecular weights of about 250 000 and 1000. Such sizes would exclude these factors from the serum fractions separated by acid gel filtration prior to somatomedin R I A or R R A ; thus, the inhibitors are probably not measured by somatomedin radioassays. The inhibitors have broad antianabolic effects on cartilage, blunting the ability of both somatomedins and insulin to stimulate formation of proteoglycan, R N A and D N A (Phillips, Vassilopoulou-Sdlin and Reichard, 1979; Phillips et al, 1979). Since such interactions appear to be

164

L. S. PHILLIPS AND T. G. UNTERMAN

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Figure 6. Measurements of somatomedin activity (porcine cartilage bioassay) in two children with Mauriac syndrome (poorly controlled diabetes, hepatomegaly and short stature). In Case 1, poor diabetic control was associated with serum somatomedin activity in the hypopituitary range. This low somatomedin activity was refractory to five days of treatment with human growth hormone, consistent with the hypothesis that insulin is needed in order to generate somatomedin activity. In Case 2, initial somatomedin activity was also in the hypopituitary range. Treatment with insulin and proper diet was associated with a rise in somatomedin activity to normal levels, associated with an improvement in growth. The patient was then lost to follow up between ages 9 and 12 and returned with a history of recurring poor diabetic control and growth failure. Somatomedin activity had fallen into the hypopituitary range. Three days of intensive insulin therapy raised somatomedin activity into the normal range, suggesting that serum somatomedin activity can respond promptly to changes in insulin status. Adapted from Winter et al (1980). n o n - c o m p e t i t i v e (Phillips, V a s s i l o p o u l o u - S e l l i n a n d R e i c h a r d , 1979), t h e i n h i b i t o r s m a y act e i t h e r at a r e c e p t o r o r at a p o s t r e c e p t o r level. T h e i n h i b i t o r s also a n t a g o n i z e t h e a n a b o l i c effects o f insulin - - b l u n t i n g s t i m u l a t i o n of glucose o x i d a t i o n in a d i p o s e tissue a n d g l y c o g e n f o r m a t i o n in m u s c l e in vitro, a n d s t i m u l a t i o n o f fat a n d m u s c l e in vivo as well (Phillips a n d Scholz, 1982). T h e i n h i b i t o r s a p p e a r to b e m e t a b o l i c a l l y r e g u l a t e d . L e v e l s a r e h i g h e r in d i a b e t i c t h a n in n o r m a l a n i m a l s (Phillips et al, 1979), a n d studies n o w in p r o g r e s s in o u r l a b o r a t o r y s h o w a significant c o r r e l a t i o n with circulating glucose a n d [3-hydroxybutyrate. Since t h e i n h i b i t o r s a r e e l e v a t e d in p o o r l y c o n t r o l l e d d i a b e t e s a n d affect b i o a s s a y s , b u t a r e p r o b a b l y n o t d e t e c t e d in r a d i o a s s a y s , such factors m a y a c c o u n t for d e c r e a s e d circulating s o m a t o m e d i n activity in d i a b e t e s in t h e face of r e l a t i v e l y n o r m a l levels o f s o m a t o m e d i n s m e a s u r e d b y r a d i o a s s a y . M o r e o v e r , if t h e i n h i b i t o r s b l u n t p u t a t i v e s o m a t o m e d i n f e e d b a c k on b r a i n c e n t r e s ( B e r e l o w i t z et al, 1981; T a n n e n b a u m , G u y d a a n d P o s n e r , 1983), such a c t i o n c o u l d a c c o u n t for t h e e l e v a t i o n in h G H which o c c u r s in p o o r l y c o n t r o l l e d d i a b e t e s ( H a n s e n a n d J o h a n s e n , 1970) d e s p i t e n o r m a l levels o f s o m a t o m e d i n s . F i n a l l y , since i n h i b i t o r s a p p e a r to o r i g i n a t e in t h e liver (see b e l o w ) , t h e c o r r e l a t i o n s o f

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inhibitors with levels of glucose and ketoacids - - indices of insulinization - - suggest that inhibitors may provide a potential mechanism for the liver to modulate utilization of fuels as well as their production. Potential Applications

At present, there is no well-defined basis for measurement of somatomedins as useful determinations in diabetes. However, it seems likely that quantification of three facets of somatomedin metabolism could be of potential benefit to the clinician. (1) Measurement of somatomedin levels (IGF I and/or IGF II) by radioassay could be a useful index of nutritional status (above) in subjects with varying nutrient intake and irregular loss of glucose and nitrogen in the urine. (2) Measurement of somatomedin activity (by bioassay) could be a useful indicator of growth potential as a function of diabetic control; in subjects with widely varying glucose levels, somatomedin activity would appear to be more stable than glucose levels but fluctuate more rapidly than haemoglobin Ale, and might be a sensitive index as to whether or not diabetic control was sufficient to support growth. (3) Finally, measurements of somatomedin inhibitors - - as apparent major contributors to the fall in net somatomedin activity in diabetes - - might prove to be particularly useful indices of growth potential in children with fluctuating diabetic control. ADDITIONAL METABOLIC DISORDERS Glucocorticoid Excess

Glucocorticoid excess is another 'non-GH-axis' disorder associated wtih poor growth despite normal to increased levels of insulin and GH secretion. As in uraemia (see section 'Renal failure', below) somatomedin activity as determined in bioassays appears to correlate better with growth than do somatomedins measured with radioassays. Growth abnormalities Decreased statural growth with retarded skeletal maturation (Blodgett et al, 1956; Falliers et al, 1963) is a common finding in children with Cushing's syndrome. Poor growth was present in 11 of 13 cases seen at the Mayo Clinic, and may be the most prominent physical finding in this disorder (Lee, Weldon and Migeon, 1975). Decrease in height velocity may occur despite an increase in weight velocity. Children receiving exogenous steroids (e.g., for asthma, juvenile rheumatoid arthritis, adrenogenital syndrome, or hypopituitarism) are also at risk, exhibiting decreased height for age (Blodgett et al, 1956; Falliers et al, 1963; Rappaport et al, 1973; Preece, 1976). In children receiving oral hydrocortisone for treatment of the adrenogenital syndrome, this effect appears to be dose-related (Rappaport et al, 1973). Evidence for steroid-induced intrauterine growth retardation has also been found in both animal and human studies (Reinisch et al, 1978).

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As potential mechanisms for steroid-induced growth retardation, investigators have examined the effects of steroids on GH secretion, GH action, somatomedin generation, and somatomedin action. Growth hormone While studies of steroid effects on GH secretion have provided somewhat mixed results, the effects of glucocorticoid excess on GH action are less controversial. In general, many adults with glucocorticoid excess may exhibit blunted GH secretion. Krieger and Glick (1972) found that patients with 'central' Cushing's disease (possibly of hypothalamic origin) had a decreased GH response to provocative stimuli, and that this abnormality persisted even after the state of hypercortisolism had been resolved. This suggests that 'hypothalamic' Cushing's disease may be associated with an intrinsic defect in GH secretion, or that long-term steroid excess could induce such a defect. Some patients with adrenal adenomas have normal GH responsiveness (Cohen and Deller, 1966), although there are conflicting reports (Demura et al, 1972). Adults receiving chronic exogenous steroids appear to have a blunted GH response to hypoglycaemic stress (Frantz and Rabkin, 1964). This effect is dose-related, and is less marked in patients receiving short-term steroids. Adults given daily prednisone have a decrease in mean plasma GH concentration and production rate (as measured by continuous plasma withdrawal over 24 hours), with less effect seen when steroids are given on alternate days (Thompson et al, 1972). However, findings in children are quite different. Children receiving steroids for asthma (Morris, Jorgensen and Jenkins, 1968), Still's disease (Sanders and Norman, 1969) or the adrenogenital syndrome (Sturge et al, 1970) have intact GH responsiveness to insulin-induced hypoglycaemia, as do children receiving ACTH therapy for a variety of disorders. Similarly, children with endogenous Cushing's syndrome may also have normal GH responsiveness in the presence of growth retardation (Strickland et al, 1972). Thus, while GH secretion appears to be diminished in adults exposed to excess corticosteroids, most studies suggest that children with steroid-induced growth failure have normal GH secretion. Corticosteroids appear to blunt the anabolic responses to GH. Although GH can induce transient and limited nitrogen retention in children with hypercortisolism (Root, Bongiovanni and Eberlain, 1969), Morris et al (1968) noted a marked decrease of GH-induced nitrogen, phosphate, calcium and potassium retention. These findings parallel the clinical observation that steroid-induced growth failure is refractory to GH therapy (Solomon and Schoen, 1976) and that hypopituitary children may respond to GH only after steroids have been discontinued, In normal subjects, there is a diurnal variation in the metabolic response to GH which is opposite to serum cortisol levels, i.e., a greater anabolic response when GH is given at night. Based on this observation, Rudman et al (1973) have recommended that GH be given in the evening when cortisol levels are low. Experimentally, when hypophysectomized rats are given GH, their growth response is diminished by corticosteroids in a dose-related fashion.

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In combination, these data suggest that whether or not steroids induce a state of GH deficiency, they do produce a state of GH resistance - including growth failure despite GH administration. Because the growth response to GH is thought to be mediated via somatomedins, the failure to respond to GH administration may be due to (1) a decrease in somatomedin generation (low levels of both somatomedin activity as measured in bioassays and somatomedins determined in specific radioassays); (2) a decrease in somatomedin action (low somatomedin activity, but normal levels of somatomedins); or (3) direct effects of steroids on growing cartilage. Corticosteroids and circulating somatomedins Clinical studies have demonstrated low serum somatomedin activity in patients with Cushing's syndrome with a return to normal following appropriate therapy (Van den Brande and Du Caju, 1974; Gourmelen, Girard and Binoux, 1982). Van den Brande et al (1975) have reported a diurnal variation in somatomedin activity in normal children which appeared inversely related to serum cortisol levels. Oral or intravenous steroid administration has been followed by a decrease in somatomedin activity (Elders et al, 1975; Green et al, 1978), and Mosier et al (1976) have also found that somatomedin activity falls when normal rats are treated with steroids. Such decreases in somatomedin activity have generally not been accompanied by a fall in somatomedins. Levels of somatomedin C (Furlanetto et al, 1977) and somatomedin A (Thoren, Hall and Rahn, 1981) measured by RIA have been normal in humans with Cushing's syndrome. Thoren, Hall and Rahn (1981) have also claimed that GH administration produces a normal rise in somatomedin A in such patients. In the only report to date where both bioassays and radioassays were performed on samples from the same subjects, Gourmelen, Girard and Binoux (1982) showed that patients with exogenous or endogenous Cushing's syndrome had normal somatomedin C levels by RRA, but decreased somatomedin activity as measured by embryonic chick cartilage bioassay (Table 4). However, these findings in humans were not duplicated by Asakawa et al (1982) in animals. After rats were given large doses of cortisone acetate for 18 days, somatomedin A measured by RIA was decreased, and resistant to GH administration. The discrepancy between these results and the human studies may reflect species difference and the high dosage of steroids administered. The weight of the evidence indicates that circulating somatomedins are normal under conditions of glucocorticoid excess where somatomedin activity (and growth) are decreased. These results suggest that somatomedin activity may be decreased due to the presence of circulating inhibitors of somatomedin action - - presumably not detected by the specific somatomedin radioassays. Corticosteroids and somatomedin action The apparent discrepancy between somatomedin levels and somatomedin activity in states of corticosteroid excess suggests that either steroids

168

L. S. PHILLIPS AND T. O. UNTERMAN Table 4. I G F level and somatomedin activity in Cushing's syndrome a Control (N)

IGF (U/ml)

1.05 _+ 0.03 (54) Sm activity (U/ml) 0.91 + 0.04 (16)

Cushing's - no treatment (N)

Cushing's- post-treatment (N)

Idiopathic hypopituitarism (N)

0.97 ± 0.06 (39) 0.65 ± 0.06b (21)

1.11 ± 0.08 (23) 'Normal' (5)

0.20 ± 0.04 (12) 0.27 ± 0.04 (14)

aLevels of insulin-like growth factor measured by a radioreceptor assay, and somatomedin activity measured by chick-cartilage bioassay in control subjects and patients with Cushing's syndrome or hypopituitarism. Somatomedins measured by radioassay in patients with Cushing's syndrome were comparable to control levels. In contrast, somatomedin activity measured by bioassay was significantly lower than control levels. These findings suggest the presence of inhibitory factors in conditions of glucocorticoid excess, which are detected in bioassays but not measured by this somatomedin radioassay. bCompared to hypopituitary or normal, P < 0.001. Adapted from Gourmelen, Girard and Binoux (1982).

themselves or steroid-induced factors may inhibit the action of somatomedins on cartilage. Long-term steroid excess produces persistent histological changes in cartilage which are qualitatively different from those of hypopituitarism, and there is limited catch-up growth following withdrawal from steroid exposure ( G r e e n et al, 1978). These findings indicate a decrease in the potential of cartilage to respond to growth stimuli, presumably a direct effect of glucocorticoids. In addition, several studies have shown that supraphysiological levels of corticosteroids added in vitro can inhibit unstimulated sulphate uptake into cartilage chondroitin sulphate and proline conversion to hydroxyproline (Daughaday and Mariz, 1962). H o w e v e r , changes in cartilage structure and responsiveness following chronic exposure in vivo, and changes in cartilage activity on direct exposure to high levels of steroids in vitro, do not necessarily explain the decrease in serum s o m a t o m e d i n activity observed with short-term bioassays. Therefore, m o r e direct studies have been done to establish whether corticosteroids could be the putative 'inhibitors' which blunt somatomedin action in bioassays. Phillips, Herington and D a u g h a d a y (1975) have reported that serum cortisol increases of 100 pg/dl did not blunt s o m a t o m e d i n action on rat or chick cartilage, and cortisol increases of 50 ~tg/dl did not inhibit s o m a t o m e d i n action on porcine cartilage. More recently, Hill (1981) confirmed the lack of effect of physiological concentrations of cortisol on s o m a t o m e d i n action on bovine growth-plate cartilage. Phillips and Weiss (1982) reported similar findings with rat growth-plate cartilage. Thus, in several bioassay systems, exposure to levels of cortisol attained in human disease has not resulted in inhibition of s o m a t o m e d i n action. This indicates that direct effects of glucocorticoids cannot explain decreased s o m a t o m e d i n activity in the serum of patients with hypercortisolism.

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Glucocorticoid induction of somatomedin inhibitors

Our laboratory has examined the possibility that steroids induce ci~qulating somatomedin inhibitors (Unterman et al, 1983). In renal transplant patients receiving alternate-day methylprednisolone, somatomedin activity decreased 66 per cent six hours after treatment, then returned to initial levels by 24 hours. The day off steroids, somatomedin activity did not change. This time-course was then used to determine if steroids lower somatomedin bioactivity by decreasing somatomedins or by increasing putative somatomedin inhibitors. Six normal subjects provided serum six hours after taking prednisone, and at the same time on another day. Somatomedin C by RIA did not change. Serum was then fractionated, and somatomedins and inhibitors measured by cartilage bioassay. After dissociation from carrier proteins, biologically active somatomedins were comparable on and off prednisone. However, somatomedin inhibitors of MW about 20000 were found off prednisone and levels doubled on prednisone (P<0.005). These observations suggest that glucocorticoids may increase levels of circulating somatomedin inhibitors, which could produce growth failure in states of steroid excess. Potential applications

From the studies reviewed above, it would seem that there is little current justification in measurement of somatomedins (by radioassay) in subjects with glucocorticoid excess, except perhaps for evaluation of nutritional status. However, measurement of circulating somatomedin activity (by bioassay) might be a useful index of growth potential in subjects with endogenous hypercortisolism or patients receiving pharmacological steroids. Circulating levels of the steroid-induced 'inhibitor' - - not generally available at present - - would appear to be a more specific indicator of the adverse growth effects of glucocorticoids; such determinations might be useful therapeutic guides in management of steroid therapy in growing children. Renal Failure

Chronic renal failure is a condition in which growth may be poor despite normal levels of circulating insulin and GH, and somatomedin activity as measured in bioassays may be a better index of growth potential than levels of somatomedins measured by more specific radioassays. Growth in uraemia

Uraemic children characteristically grow poorly (West and Smith, 1956; Broyer, 1982). Potential contributing factors include chronic metabolic acidosis (West and Smith, 1956), superimposed infection (West and Smith, 1956), poor nut~:ition (Simmons et al, 1972), and abnormal bone metabolism (Lewy and New, 1975) as well as the uraemic state itself. In a recent review, Potter and Greifer (1978) noted the prevalence of growth retardation (height less than 3rd percentile) to be 36 to 67 per cent across a broad range of renal function, and despite dialysis or transplantation.

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Children wtih renal failure are retarded in bone age relative to chronological age, and in height age relative to bone age (Ritz et al, 1977). There is little improvement with haemodialysis (Mehls et al, 1978), and the recent introduction of chronic ambulatory peritoneal dialysis, which may provide somewhat better control of the uraemic syndrome, has not resulted in reversal of the growth deficiency (Salusky et al, 1982). Growth may improve after renal transplantation (Saenger et al, 1974), but rejection, infection, complications of immunosuppression, and high glucocorticoid requirements may limit growth even after transplantation. Considerable attention has been devoted to the potential for improved nutrition to enhance the growth of uraemic children (Simmons et al, 1972; Betts and MaGrath, 1974; Holliday and Chandler, 1978). In general, children with renal failure tend to have diminished calorie, protein and vitamin intake (Betts and MaGrath, 1974). When nutritional supplementation is provided, growth may improve (Simmons et al, 1972; Betts, MaGrath and White, 1977) but usually cannot be normalized. Despite such promise, studies in laboratory animal models indicate that uraemia is generally associated with decreased gain of length and weight even when compared with pair-fed controls (Ritz et al, 1977; Schalch et al, 1981). Moreover, poor growth and wasting may persist despite good nutrition, minimal acidosis, and when infections are not a problem (Betts, MaGrath and White, 1977; Hecking et al, 1978). Hormonal changes It is clear that poor growth occurs in uraemia despite levels of insulin and growth hormone which are normal or increased. Although insulin resistance and glucose intolerance are frequent, secondary diabetes is unusual and tends not be severe enough to invoke as a cause of growth failure. Although levels of thyroxine tend to be low-normal, triiodothyronine (T3) appears to be significantly decreased in uraemia (Ramirez et al, 1976). Since levels of TSH are usually normal, it appears that low T3 is a physiological manifestation of chronic non-thyroidal illness rather than a consequence of primary hypothyroidism. There is evidence for 'tissue hypothyroidism' in uraemia (Lim et al, 1980), thyroid hormones are recognized as important for growth, and thyroid hormones may potentiate somatomedin production (Schalch et al, 1979) and action (Froesch et al, 1976)(on chick cartilage); thus, it is possible that low T3 contributes to growth failure in uraemia. However, since somatomedins are usually normal in uraemia (see below) and thyroid hormones have not been shown to enhance somatomedin action on mammalian cartilage (Phillips and Weiss, 1982), there is little reason to expect improved growth from thyroid supplementation. Somatomedins in uraemia Somatomedins in renal failure have been evaluated both by radioassays which are relatively specific, and by bioassays which are less specific but presumably sensitive to growth-inhibiting as well as growth-stimulating factors in the circulation.

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Early evaluations of somatomedins with radioassays revealed uniform elevations in humans with chronic renal failure (Takano et al, 1979a). These observations were consistent whether or not somatomedins were separated from binding proteins prior to assay. Both Takano et al (1979a) and Spencer, Uthne and Arnold (1979) reported that radioreceptor-active somatomedins rose with increasing serum creatinine, but did not change before and after haemodialysis. Schalch et al (1981) also reported elevations in somatomedins in partially nephrectomized rats; even though uraemic animals gained less weight than pair-fed controls, levels of somatomedins were higher in the uraemics. More recently, both Baxter, Brown and Turtle (1982) and Goldberg et al (1982) have reported that Sm C/IGF I measured by RIA is decreased 25 to 50 per cent in uraemia; Goldberg et al (1982) also found a threefold increase in IGF II (Table 5). Since the cartilage-stimulating potency of IGF II is about half that of IGF I (Zapf, Schoenle and Froesch, 1978), these observations in combination suggest that total biologically active somatomedins are not decreased in uraemia. Table 5.1GF 1 a n d I G F H levels in u r a e m i a a

Normal IGF I (U/ml) RIA

1.17 _+ 0.12 b (0.58-1.84) c

IGF II (U/ml) RRA

0.73 + 0.03 (0.39-1.05)

Uraemia 0.41 + 0.05 3.9 _+ 0.29

aLevels of insulin-like growth factor I measured by radioimmunoassay, and insulin-like growth factor II measured by radioreceptor assay in normal and 22 uraemic subjects. IGF I was decreased by 65 per cent in the uraemic subjects (P < 0.001), while levels of IGF I! were increased by 430 per cent ( P < 0.001). bMean + s.e.m. CNormal range (mean + 2 s.d.). Adapted from Goldberg et al, 1982.

In contrast to these findings with somatomedins measured by radioassay, most bioassay studies have revealed decreased circulating somatomedin activity in uraemia (Saenger et al, 1974; Stuart, Lazarus and Hayes, 1974; Takano et al, 1979a). Since most of the bioassays measure stimulation of cartilage 35SO4 uptake, yet circulating inorganic sulphate is elevated in uraemia, our laboratory has emphasized the need for appropriate corrections if determinations are to be accurate (Phillips et al, 1978). After such corrections, bioassayable somatomedin activity was found to be decreased in renal failure (Phillips et al, 1978; Phillips and Kopple, 1981), and correlated inversely with serum urea nitrogen or creatinine (Figure 7). Since somatomedin activity increases with dialysis in vivo or in vitro (Phillips et al, 1978; Phillips and Kopple, 1981) yet there is little change in somatomedins measured by radioassay, these observations suggest that somatomedin activity may be decreased in uraemia due to the presence of circulating inhibitors of low molecular weight.

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L. S. PHILLIPS AND T. G. UNTERMAN

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Figure 7. Serum somatomedin activity versus serum urea nitrogen in 47 men with normal or impaired renal function. With progressive rise in urea nitrogen (indicating renal impairment) somatomedin activity decreased from normal to hypopituitary levels (<0.65 U/ml). From Phillips and Kopple (1981) with kind permission of the editor of Metabolism.

Our laboratory recently investigated such a possibility by examining somatomedins and inhibitors in fractionated normal and uraemic serum (Phillips, Fusco and del Greco, 1982). We found that serum content of biologically active somatomedins (presumably both IGF I and IGF II) was normal in uraemia, as were levels of high molecular weight inhibitory factors (as found in diabetes or malnutrition). In contrast, uraemic sera contained increased levels of inhibitors of molecular weight about 900. These low molecular weight inhibitors could blunt the ability of somatomedins to stimulate synthesis of R N A and DNA as well as chondroitin sulphate in cartilage. They appear to be peptides and were found in normal urine as well as in uraemic serum. Minuto et al (1982) have also reported the presence of a circulating inhibitor in uraemic subjects, but did not characterize it. Based on this work, it seems likely that, under conditions of renal failure, accumulation of such inhibitory factors in the circulation leads to a decrease in somatomedin activity and consequent impairment of growth.

Potential applications From this review, it would appear that in situations of renal failure, measurement of total somatomedins by R R A or IGF II by specific radioassay has no established value at present. Measurement of IGF I by specific R I A may afford some prediction of growth potential. However, the best correlations with growth at this time appear to be provided by measurements of circulating somatomedin activity with bioassays. Such determinations may offer an integrated index of growth potential as

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reflecting both nutritional and renal status. In the future, determination of the low molecular weight somatomedin inhibitors might provide a measure superior to BUN or creatinine as an indicator of the extent to Which 'toxic' uraemic factors are controlled by conservative therapy, dialysis or transplantation. S O M A T O M E D I N / I N H I B I T O R R E G U L A T I O N BY THE LIVER

Accumulating evidence indicates that both GH- and non-GH-mediated changes in circulating somatomedin activity may reflect regulation by the liver; our review will focus on the latter. Sources of Circulating Somatomedins and Inhibitors

As noted above, protein malnutrition, insulin deficiency, and corticosteroid excess are accompanied b y growth failure and a non-GH-mediated decrease in serum somatomedin activity. The site(s) and mechanism(s) by which these changes occur are subjects of active investigation. Somatomedins, like other peptide hormones, now appear to be present in many tissues, including liver, muscle, spleen, lung, brain, peripheral nerve, pituitary, kidney, pancreas, gut and salivary gland (Liske and Reber, 1976; Sara et al, 1982; Vassilopoulou-Sellin and Phillips, 1982); at these sites, somatomedins may serve local functions beyond their role as circulating anabolic growth factors. For example, production of somatomedins in muscle could be involved in the trophic effects of GH on muscle (Kostyo et al, 1975), while neural somatomedins (present in peripheral nerves and released by electrical stimulation (Sara et al, 1982) may account for the trophic effect of nerves on muscle. While GH increases somatomedin release by cultured human fibroblasts (Atkison et al, 1980; Clemmons, Underwood and Van Wyk, 1981), human connective tissue explants (Schimpff, Donnadieu and Gautier, 1981), and perfused rat kidney (McConaghey and Dehnel, 1972), but not rat kidney slices (McConaghey and Dehnel, 1972), non-GH regulation of somatomedin activity has been studied only in the liver (see below). The liver has received greatest attention as a source of somatomedins and potential locus of somatomedin regulation. Patients with chronic liver disease have lowered levels of serum somatomedin activity (Wu et al, 1974), somatomedin A by R R A (Takano et al, 1977), and IGF by RIA and R R A (Zapf et al, 1980). These changes correlate with measures of disease severity, such as serum albumin (Wu et al, 1974; Takano et al, 1977). Similarly, Uthne and Uthne (1972) reported a decrease in serum somatomedin activity in partially hepatectomized rats. Venous catheterization studies in dogs and humans have suggested the presence of a step-up in somatomedin activity at the level of the hepatic vein (Schimpff, Lebrec and Donnadieu, 19"/7) which increased with GH administration, although subsequent studies by the same investigators have failed to reproduce these results. Nevertheless, various in vitro techniques, including studies of tissue slices (McConaghey, 1972), isolated organ perfusion (McConaghey

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and Sledge, 1970), liver explant culture (Rechler et al, 1979; Binoux et al, 1980; D ' E r c o l e , Applewhite and U n d e r w o o d , 1980; Schimpff, Donnadieu and Gautier, 1981), liver cell suspensions (Liberti, 1978), liver cell culture (Spencer, 1979) and cell-free protein synthesis using liver-derived R N A as a template (Acquaviva et al, 1982), have all shown that liver can produce and export somatomedins.

Influence on Hepatic Modulation of Somatomedins and Inhibitors Growth hormone G H regulation of hepatic somatomedins has been demonstrated clearly. Liver extracts from hypophysectomized animals lack somatomedin activity, while administration of G H in vivo restores hepatic content of somatomedin-like material (Vassilopoulou-Sellin and Phillips, 1982). Moreover, exposure to G H in vitro increases s o m a t o m e d i n release by liver explants (Binoux et al, 1980; D ' E r c o l e , Applewhite and U n d e r w o o d , 1980; Schimpff, D o n n a d i e u and Gautier, 1981) (Table 6), and perfused livers (McConaghey, 1972; Daughaday, Phillips and MueUer, 1976; Shapiro, Waligora and Pimstone, 1978; Kogawa et al, 1982).

Table 6. Somatomedin and inhibitor export by liver explants a Control (no added Plus hormone) growth hormone

Plus insulin

Plus cortisol

IGF by RRA (% control)

100

275b 240b 50b (1.0 p~g/mlGH) (10 mU/ml insulin) (1.0 ~tg/mlcortisol)

Inhibitory activity (% control)

100

75b 5 146b (1 mU/ml GH) (1 mU/ml insulin) (0.1 ~g/ml cortisol)

~Liver explants were incubated with or without supplemental hormones for three days, after which conditioned medium content of insulin-like growth factor (IGF) reactivity was measured by radioreceptor assay, and inhibitory activity was measured by chick-cartilage bioassay. Added growth hormone produced a marked increase in IGF reactivity, with a small change in inhibitory activity; insulin produced a comparable increase in IGF reactivity, but also produced a large decrease in medium inhibitory activity; added cortisol decreased medium content of IGF reactivity while increasing medium inhibitory activity. ~P< 0.005 versus hormone-free control medium. Adapted from Binoux et al, 1980; Binoux, Lassarre and Seurin, 1980; Binoux, Lassarre and Hardouin, 1982.

Non-growth hormone-mediated regulation The techniques of liver extraction, explant culture and organ perfusion have also yielded important information about n o n - G H hepatic regulation of s o m a t o m e d i n activity. In particular, the effects of nutrition, insulin, and corticosteroids have been examined.

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Nutrition. Nutritional restriction appears to lower hepatic content and

release of somatomedin activity. Perfused livers from protein-restricted animals exhibit diminished release of somatomedin activity (Shapiro, Waligora and Pimstone, 1978), and perfused livers from fasted animals release decreased quantities of IGF as measured by R R A (Miller, Schalch and Draznin, 1981). Nutritional status appears to affect hepatic regulation of inhibitors as well as somatomedins. Thus, it has been observed that liver extracts (Vassilopoulou-Sellin, 1982) and perfusates (VassilopouluSellin, Phillips and Reichard, 1980) from fasted animals have a net inhibitory effect on cartilage sulphate uptake - - suggesting that, under conditions of nutritional deprivation, the liver not only contains and releases less somatomedins, but also generates and exports inhibitors of somatomedin activity. Moreover, Vassilopoulou-Sellin et al (unpublished data) have recently found that with fasting, the fall in liver perfusate and extract somatomedin activity precedes the decrease seen in serum activity. Taken together, these findings suggest that, with changes in nutritional status, the liver may play an important role in the regulation of serum somatomedin activity. Insulin. The hepatic response to insulin added in vitro has been variable,

perhaps reflecting the difficulty of maintaining tissue metabolic integrity. Early experiments showed that exposure to high levels of insulin in vitro (1000 ~tU/ml) increased liver perfusate somatomedin activity in both well-fed (Daughaday, Phillips and Mueller, 1976) and proteinmalnourished rats (Shapiro, Waligora and Pimstone, 1978). Subsequent efforts did not reproduce such effects on either liver perfusate bioactivity (Vassilopoulou-Sellin, Phillips and Reichard, 1980) or IGF content as measured by R R A (Miller, Schalch and Draznin, 1981). However, more recent studies (Kogawa et al, 1982) have confirmed that high levels of insulin can increase the release of somatomedin A by perfused livers from either normal or hypophysectomized rats. Interestingly, insulin appeared to augment the response to added GH as well (Kogawa et al, 1982). While perfusion studies allow the liver to be studied intact and in situ, explant experiments allow more prolonged exposure to a controlled environment, and thus may reveal more delayed effects of added hormones. It has been shown (Binoux, Lassarre and Seurin, 1980; Binoux et al, 1980; Binoux, Lassarre and Hardouin, 1982) that explants exposed to insulin for three days export increased amounts of IGF measured by R R A (Table 6). In the absence of insulin, the liver explant conditioned medium had a net inhibitory effect on cartilage sulphate uptake, similar to that found with perfusates of diabetic rat livers (see below). With heating, inhibitory effects were lost, and the medium became stimulatory for cartilage, suggesting that somatomedins were present but their effects masked by heat-labile somatomedin inhibitors. When liver explants were exposed to added insulin, medium had net stimulatory activity which did not increase after heating, suggesting that insulin diminished the release of somatomedin inhibitors. (In contrast, while GH exposure increased explant release of IGF, it had less effect than insulin on the release of

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L. S. PHILLIPSAND T. G. UNTERMAN

inhibitors, suggesting that GH acts primarily to increase somatomedin generation, while insulin acts on the liver both to increase somatomedin release, and decrease the release of somatomedin inhibitors.) The effect of insulin deficiency in vivo has been a consistent fall in hepatic content and release of somatomedin activity. Miller, Schalch and Draznin (1981) found that perfused livers from diabetic rats release very little IGF measured by RRA. In our laboratory (Vassilopoulou-Sellin, Phillips and Reichard, 1980), livers from rats with streptozotocin-induced diabetes yielded extracts with little activity and perfusates with net inhibitory bioactivity. Thus, like the fasted liver, the perfused diabetic liver appears to release not only less somatomedins, but somatomedin inhibitors as well. Vassilopoulou-Sellin (1982) and Vassilopoulou-Sellin et al (unpublished data) have found that liver extract and perfusate somatomedin activity fall before a change in serum activity is seen, suggesting that the liver may regulate serum somatomedin activity in states of insulin deficiency as it appears to in states of nutritional restriction.

Glucocorticoids. In contrast to the normal serum levels of IGF reported by Gourmelen, Girard and Binoux (1982) in patients with Cushing's syndrome, liver explants exposed to cortisol export less IGF (Binoux, Lassarre, and Hardouin, 1982). When exposed to high levels of cortisol, liver explant medium exhibits somatomedin inhibitory a c t i v i t y apparently requiring protein synthesis, since it is blocked by cycloheximide (Binoux, Lassarre and Seurin, 1980). Thus, under explant conditions and in the absence of other serum factors, cortisol appears to induce the synthesis and release of hepatic factors which inhibit somatomedin activity. It should be noted that Schalch et al (1979) have reported that perfused livers from hypophysectomized rats released more IGF after pretreatment with a combination of GH, T3, and cortisol than after pretreatment with GH and T3 alone. Thus, while cortisol excess may be accompanied by increased somatomedin inhibitors in vivo (see section 'Glucocorticoid induction of somatomedin inhibitors', above) and in vitro (Binoux, Lassarre and Seurin, 1980), some amount of cortisol may be required for normal somatomedin production. In this system, thyroid hormone may also play a permissive role in hepatic somatomedin generation. Potential Mechanisms

Hepatic hormone receptors have been examined as putative mediators of nutritional/hormonal effects on hepatic generation of somatomedins and somatomedin inhibitors. As noted, in malnutrition there is a decrease in serum somatomedins and hepatic somatomedin release despite an increase in GH. This could be mediated by a decrease in hepatic GH receptors. Postel-Vinay, Cohen-Tough and Charrier (1982) have shown that hepatic GH receptors are decreased in fasted rats and return to normal with refeeding, and that GH receptor number is correlated with circulating somatomedin activity; Baxter, Bryson and Turtle (1981) also found a fall in hepatic GH receptors with fasting (Table 7). Other mechanisms may also

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177

Table 7. Rat hepatic bGH receptors in fasting and diabetes Receptor number Fed Fasting Control Diabetic Treated

3.23 +__1.52" 1.19 +_ 0.82 6.4 + 2.7 1.0 + 0.5b 4.0 + 2.2c

"Compared to fasting, P < 0.05. bCompared to control, P < 0.02. cCompared to diabetics, P < 0.01. Values are for females; males show similar trends. Values are means + s.d., n = 4. Association constants for high-affinity bGH binding sites did not change significantly in these studies. Both fasting and streptozotocin-induced diabetes produced a marked decrease in somatogenic receptor number; treatment of diabetes with insulin restored receptor number toward control levels. Adapted from Baxter, Bryson and Turtle, 1980, 1981.

be important. Our laboratory has found that s o m a t o m e d i n activity may be released by normal rat liver only in the presence of enriched m e d i u m but not with simple buffers (Vassilopoulou-Sellin, Phillips and Reichard, 1980), suggesting that the immediate nutrient supply, independent of hormonal factors, may also affect hepatic regulation of s o m a t o m e d i n activity. The mechanisms by which insulin contributes to hepatic regulation of s o m a t o m e d i n activity a p p e a r multifaceted as well. As noted above, insulin can increase hepatic release of I G F s in the absence of G H (Binoux, Lassarre and I t a r d o u i n , 1982), but also appears to augment the hepatic response of liver to G H (Kogawa et al, 1982). Baxter, Brown and Turtle (1980) and Baxter, Bryson and Turtle (1980) have reported that hepatic G H receptors are reduced in rats with streptozotocin-induced diabetes; circulating somatomedins were correlated with hepatic G H receptors, suggesting that insulin m a y sensitize the liver to G H at the receptor level. Since insulin has a b r o a d impact on hepatic intracellular processes, some of its effects on hepatic regulation of s o m a t o m e d i n activity may be due to events within the cell, as well as changes at the cell surface. CONCLUSIONS O u r overall scheme of regulation of circulating s o m a t o m e d i n activity is shown m Figure 8. G r o w t h h o r m o n e , in concert with insulin and nutritional factors, acts at the level of the liver (and perhaps other body tissues) to p r o m o t e the synthesis and release of s o m a t o m e d i n s into the circulation. Somatomedins can be measured directly by sensitive and specific radioassays. U n d e r conditions of malnutrition, insulin deficiency and/or glucocorticoid excess, the liver appears to release reduced levels of somatomedins, and increased levels of s o m a t o m e d i n inhibitors. The

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ACTIVITY

MITOGENIC ACTIVITY

Figure 8. Schematic representation of factors affecting somatomedin regulation. Growth hormone, together with growth hormone-like peptides (such as prolactin and placental lactogen), insulin and good nutrition act at the level of the liver (and perhaps other sites) to promote the generation of somatomedins; malnutrition, glucocorticoids, oestrogens, and perhaps other systemic illnesses act to promote generation of inhibitors. In renal failure, inhibitors accumulate in the circulation, presumably due to impaired clearance by the kidney. The net biological effect of circulating somatomedins and inhibitors can be determined as somatomedin activity, in bioassays which monitor tissue responses including growth activity, insulin-like activity and/or mitogenic activity. Somatomedins feed back at both hypothalamic and pituitary levels to decrease growth hormone release; in the hypothalamus, somatomedins appear to increase release of somatostatin, whereas in the pituitary, somatomedins appear to blunt growth hormone release directly. c o m b i n a t i o n of s o m a t o m e d i n s and inhibitors in the circulation can be d e t e r m i n e d as s o m a t o m e d i n activity in bioassays. H e p a t i c release of s o m a t o m e d i n s and/or inhibitors has not b e e n e x a m i n e d in chronic renal failure; in this condition, it appears that low molecular weight somat o m e d i n inhibitors accumulate in the circulation as a result of impaired renal function, antagonizing s o m a t o m e d i n action and p r o d u c i n g r e t a r d e d growth. S o m a t o m e d i n s provide negative f e e d b a c k to the h y p o t h a l a m i c pituitary system to decrease release of G H ; since G H levels m a y be elevated in diabetes, despite the presence o f n o r m a l circulating levels of s o m a t o m e d i n s , it w o u l d a p p e a r that circulating s o m a t o m e d i n inhibitors m a y blunt s o m a t o m e d i n action at the hypothalamic-pituitary level, resulting in increased release of G H . As reviewed above, g r o w t h m a y be p o o r despite n o r m a l to elevated levels of G H in conditions o f malnutrition, insulin deficiency, glucocorticold excess and chronic renal failure. In these states, it seems likely that

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poor growth is mediated by a decrease in circulating somatomedins and/or an increase in circulating somatomedin inhibitors. Although measurements of such factors have not yet been validated as useful prognostic indices in these conditions, such validation is currently being tested in a number of different laboratories. Given the information now available, these potential applications are apparent: 1. When malnutrition is suspected - - particularly protein deficiency - determinations of specific somatomedins (such as somatomedin C), net circulating somatomedin activity, and/or measurement of circulating somatomedin inhibitors (currently available only in research laboratories) may be useful iwassessing the nutritional status of the patient, and in monitoring the response to nutritional therapy. 2. In diabetes mellitus, measurement of specific somatomedins may provide a partial index of nutritional status, but does not appear strongly related to diabetic control. Determinations of total circulating somatomedin activity and/or somatomedin inhibitors may be useful indices for assessing diabetic control and providing a prognostic measure as to whether or not diabetic control is sufficient to support good growth. 3. In states of glucocorticoid excess (of exogenous or endogenous origin), measurements of specific somatomedins currently appear to be of little value. However, determination of net circulating somatomedin activity and/or somatomedin inhibitors may be useful prognostic indices of growth and anabolic potential. 4. In chronic renal failure, it appears that measurement of I G F II is of little value, although determination of I G F I/somatomedin C may provide an index of nutritional status. Net circulating somatomedin activity and/or low molecular weight inhibitors may provide indices of growth and anabolic potential and reflect control of the uraemic state. It can be expected that, as our knowledge of the circulating somatomedins and their inhibitory antagonists becomes more refined and assays for the critical factors become more sensitive, specific, and widely available, determinations of somatomedins and somatomedin-related factors will come to play an increasingly important role in the assessment and therapy of malnutrition, insulin deficiency, steroid excess and kidney failure. ACKNOWLEDGEMENTS This work was supported in part by DHHS Training Grant AM-07169 and by Research Grants AM-10699, AM-21483 and MRP HD-11021 from the National I n s t i t n t e s . f Health. We thank Dr Norbert Freinkel for his encouragement and support, and Ms Concepcion Mora for assistance in preparation of the manuscript. REFERENCES

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