Pharmac. Ther.B, Vol. 2, pp. 591-604, 1976. PergamonPress. Printed in Great Britain Specialist Subject Editor: C. H. L1

THE

BIOLOGY JACK L .

OF GROWTH

KOSTYO a n d

HORMONE

CHARLES R . REAGAN

Department of Physiology, Emory University, Atlanta, GA 30322, U.S.A.

INTRODUCTION Pituitary growth hormone is generally regarded as one of the principal factors regulating somatic growth in vertebrates. It has been detected in the pituitary glands of representatives of all vertebrate classes from fishes to mammals and shown to be an essential requisite for growth in selected species either by the demonstration of the cessation of growth after hypophysectomy or by the stimulation of growth following the administration of pituitary extracts or purified growth hormone preparations. A notable exception to this i s t h e guinea pig, which produces growth hormone but does not appear to require it in order to grow (Knobil and Hotchkiss, 1964). Growth hormone is not a single chemical entity but exists in the vertebrates as a family of rather closely related peptides, each species apparently producing a growth hormone molecule with a somewhat different primary structure. The amino acid sequences of the growth hormones of only three species have been definitely established to date. These include the human (Li and Dixon, 1971), the ox (Fellows, 1973) and the sheep (Li et al., 1973). Each of these hormones consists of a single peptide chain of 191 amino acid residues with a molecular weight of approximately 22,000 daltons. There are two intrachain disulfide bridges in the molecule. The amino acid sequences of the growth hormones of the ox and sheep are more similar to one another than they are to the human growth hormone sequence, although considerable homology exists in the primary structures of all three species of the hormone. Much less is known about the structural features of the growth hormones of the lower vertebrates. Studies of the immunochemical relatedness of the growth hormones of various species suggest that the mammalian growth hormones are more similar to one another structurally than they are to the growth hormones of the birds, reptiles, amphibians and fishes (for review see Wilhelmi, 1974). These differences in structure may account for the so-called species specificity of the growth hormones. Humans, and other primates that have been studied, do not respond to growth hormones of infraprimate origin, while common laboratory mammals, like the rat, dog and cat, respond to growth hormones of general mammalian origin. The rat, however, does not grow in response to pituitary extracts or purified growth hormones of fishes and gives only minimal responses to amphibian, reptile or bird hormones (see Wilhelmi, 1974). The molecular basis for this species specificity is not understood. It has been proposed (Wilhelmi, 1955; Li et al., 1955) that there is a common amino acid sequence or core in the growth hormones of various species and that it is this core that interacts with receptors on target cells. Differences in the structure of other parts of the molecule would presumably determine whether or not a given growth hormone could interact with receptors on a cell, thereby imparting species specificity. Alternatively, the active core might be released from the molecule as a fragment by the action of proteolytic enzymes in the blood or in the tissues, and it could be this fragment that interacts with receptors. Species specificity, in this case, would be conferred by the ability of a given growth hormone molecule to serve as substrate for the 'fragmenting enzyme'. At the moment, it is difficult to evaluate this latter idea, since the exact nature of the biologically active form of circulating growth hormone is not known. In any event, it is worth noting that various large fragments of the N-terminal two-thirds of the growth hormones of man, ox, sheep and pig have been found to possess substantial biological activity (for review see Kostyo, 1974). Thus, the entire native structure of the growth hormone molecule, i.e. all 591

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191 amino acid residues, does not seem to be necessary for the hormone to exert its biological effects. The major physiological action of growth hormone is to stimulate the synthesis of proteins and nucleic acids, particularly the structural macromolecules of many tissues, thereby promoting somatic growth of the organism. Besides having this effect on the formation of macromolecules, growth hormone, under certain circumstances, also exerts marked effects on other aspects of metabolism. For example, the growth hormone-deficient or hypophysectomized animal responds to the acute administration of growth hormone with a transient hypoglycemia. This insulin-like effect of growth hormone can also be demonstrated on isolated tissues of growth hormone-deficient animals, the effect being a transient stimulation of glucose uptake and utilization by the cells. After giving this brief response, the organism then becomes refractory and will not give another hypoglycemic response to growth hormone for 24-48 hr, i.e. until it becomes growth hormone-deficient again. The development of this refractoriness of long duration probably explains why the insulin-like effect of growth hormone is not readily demonstrated in normal animals. Growth hormone has other effects on carbohydrate metabolism, including stimulation of glucose production by the liver and, particularly when present in excess, causing the development of insulin resistance in the peripheral tissues. Growth hormone also increases the extent to which the/3-cells secrete insulin in response to a glucose stimulus. In certain organisms having a limited pancreatic /3-cell reserve, such as the dog, cat and man, chronic exposure to excessive amounts of growth hormone can result in the development of diabetes. Hence these actions of growth hormone on carbohydrate metabolism have been referred to as the diabetogenic effects of growth hormone. Growth hormone also affects the rate at which fatty acids are utilized by certain tissues, and it influences the process of lipid mobilization. In the latter case, it would appear that the hormone has a permissive effect on the ability of agents to stimulate the process of lipolysis. The nature of each of these biological properties of growth hormone will be discussed in detail in subsequent sections of this article. Much of what is known of these actions of growth hormone has been learned from experiments on common laboratory animals such as the rat, mouse, dog and cat. The species specificity of growth hormone has limited the supply of growth hormone for human investigation to that which can be prepared from human pituitaries. This, coupled with the difficulties and the restrictions placed on human investigation, has limited the extent of our understanding of the biological actions of growth hormone in man. From what is known, however, there is no reason to believe that the human differs, in any great respect, from the other mammals in its responses to growth hormone. Unfortunately, there has been very little investigation of the metabolic effects of growth hormone in the lower vertebrates. Consequently, the discussion to follow will deal mainly with a summary of the biological properties and effects of growth hormone in laboratory mammals and, when possible, to effects established in man. EFFECTS OF GROWTH HORMONE ON PROTEIN METABOLISM In an effort to understand the growth-promoting action of growth hormone, early investigators focused on the effects of pituitary extracts on the chemical composition of the body and body fluids (for review see Engel and Kostyo, 1964; Kostyo and Nutting, 1974). Their findings were later confirmed, in the most part, with purified growth hormone preparations. Further, when the species specificity of growth hormone was recognized in the mid-1950s ~ind human growth hormone became available, the changes seen in the composition of blood and urine of experimental animals after growth hormone treatment were also found in man. The most commonly observed changes in blood composition after growth hormone administration were decreases in non-protein nitrogen, a-amino nitrogen and urea, particularly in fasting animals. When more sophisticated methods for quantitative amino acid analysis became available, it was

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found that growth hormone decreased the levels of most of the amino acids in the plasma. In the human, growth hormone lowered the amounts of all of the amino acids in the blood, and particularly that of glutamine. Many investigators also reported that the excretion of nitrogen, phosphorous, sodium and potassium in the urine declined during growth hormone administration. That growth hormone actually caused the retention of these basic elements of protoplasm was established by elemental balance studies in both animals and man. These basic observations, coupled with the finding that growth hormone treatment increased the amount of protein in the body relative to the other major constituents, such as fat, forced the conclusion (Russell, 1955a, b, 1957) that the hormone acts primarily to stimulate the formation of protein. On the other hand, it does not appear to retard the rate of protein breakdown (Goldberg, 1969). During the last 15 years, considerable effort has been devoted to the study of the action of growth hormone on protein formation. Three tissues have been used primarily for this work, namely skeletal muscle, liver and cartilage. Hence the remainder of this section will deal exclusively with findings on these tissues. The reader is referred to several comprehensive reviews (Kostyo, 1973; Kostyo and Nutting, 1974; Daughaday, 1971; Daughaday et al., 1975) for information on other tissues and for many of the experimental details omitted from the following discussion.

SKELETAL MUSCLE Growth hormone produces a rapid stimulation of protein synthesis in skeletal muscles, such as those of the thigh and diaphragm. This has been demonstrated in young hypophysectomized rats by injecting growth hormone intravenously and then pulse-labeling the muscle proteins with radioactive amino acids (Kostyo and Nutting, 1973). A period of approximately 30 min elapses after injection of the hormone before protein synthesis is stimulated. Once stimulated, however, the rate of protein formation remains elevated for many hours, finally returning to the basal level about 24 hr after hormone administration. Growth hormone also stimulates the incorporation of radioactive amino acids into the protein of the isolated diaphragm of young hypophysectomized rats, when added directly to the bathing medium (for review see Engel and Kostyo, 1964; Kostyo and Nutting, 1974). As in vivo, the stimulation of protein synthesis is not immediate but occurs after a lag-period of approximately 30 min (Rillema and Kostyo, 1971). In such in vitro experiments, the hormone first stimulates the synthesis of proteins of the contractile complex and, somewhat later, the formation of many other proteins (Kostyo, 1968). During the early phase, growth hormone may also stimulate the synthesis of proteins involved in the transport of amino acids across the muscle cell membrane. Some years ago, it was established that growth hormone injected in vivo or added directly to isolated muscle preparations stimulates the transport of some, but not all, amino acids into muscle cells (for review see Kostyo and Nutting, 1974). This effect of the hormone has a lag-phase that is similar to that of the stimulatory effect on protein synthesis, namely the effect appears approximately 30 min after exposure of the cells to the hormone (Rillema and Kostyo, 1971). The rate of amino acid transport remains elevated for about 1-2 hr and then returns to the basal or unstimulated rate (Hjalmarson, 1968). The transport mechanism is then refractory to further stimulation by growth hormone for many hours. Kostyo (1968) and Hjalmarson (1968), using the isolated rat diaphragm, found that when the tissue is incubated with inhibitors of protein synthesis prior to exposure to growth hormone, the effect of the hormone on amino acid transport is inhibited or blocked entirely. An obvious interpretation of this finding is that the hormone stimulates the synthesis of some protein involved in the activation of the transport mechanism. Alternatively, a protein with a high rate of turnover may be involved in mediating the effect of growth hormone on amino acid transport. This protein may be depleted during the period that the tissue is preincubated with inhibitors of protein synthesis, thus preventing the hormone from expressing its stimulatory effect

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on the transport mechanism. Which of these possibilities is correct still remains to be determined. Available evidence indicates that growth hormone exerts its prompt effect on protein synthesis in muscle by influencing the rate at which ribosomes translate messenger RNA. In contrast, the transcription of new messenger RNA or the synthesis of ribosomes does not seem to be involved, judging from the many unsuccessful attempts to show rapid effects of growth hormone on RNA synthesis in muscle (Martin and Young, 1965; Kostyo, 1966; Dawson et al., 1966). Ribosomal RNA does increase in skeletal muscles of hypophysectomized rats 18-24 hr after growth hormone administration (Florini and Breuer, 1966), but this is obviously not involved in mediating the rapid effect of the hormone on protein synthesis. Other evidence suggesting that the formation of new RNA is not necessary for growth hormone to stimulate protein synthesis in muscle has come from experiments in which growth hormone added in vitro still stimulated amino acid incorporation in the isolated rat diaphragm in which precursor incorporation into RNA was abolished with actinomycin D (Martin and Young, 1965; Knobil, 1966). More direct evidence indicating that the initial effect of growth hormone on protein synthesis involves the translation process itself was provided by studies (Kostyo and Rillema, 1971) in which the effects of growth hormone on the catalytic activity of muscle ribosomes was measured using puromycin. In these experiments, the isolated rat diaphragm was incubated with radioactive puromycin to label nascent peptide chains. Incubations were carried out either in the presence of emetine or at low temperature to prevent the formation of new peptide chains. The rate and extent of the reaction of puromycin with nascent peptides was then measured, the rate being an index of the catalytic capability of the ribosomes and the extent of the reaction being an indicator of the number of ribosomes engaged in the translation process. Exposure of the diaphragm to growth hormone only increased the rate of the puromycin reaction, i.e. increased the ability of the ribosomes to carry out the translation process. The mechanism by which growth hormone improves the ability of muscle ribosomes to translate messenger RNA is still unclear. There is some evidence suggesting that changes in cyclic nucleotide metabolism may be involved. Theophylline and other inhibitors of cyclic nucleotide phosphodiesterase block the in vitro effect of growth hormone on protein synthesis and amino acid transport in the isolated rat diaphragm (Payne and Kostyo, 1970; Rillema et al., 1973). The ability of a particular inhibitor to block the action of growth hormone appears to be correlated directly with the degree of glycogenolysis caused by the inhibitor. These observations suggest that a reduction in the concentration of cyclic AMP (cAMP) is an essential step in the series of reactions leading to ribosome activation. While efforts to demonstrate a reduction in the total cAMP content of the isolated rat diaphragm following exposure to growth hormone in vitro have been unsuccessful (lsaksson et al., 1974), a recent attempt (Kostyo et al., 1975) to detect effects of growth hormone on the small free or metabolically vulnerable pool of cAMP in the diaphragm have yielded positive results. In this latter study, the nucleotides in the diaphragm were labeled with radioactive adenine. The tissue was then exposed to growth hormone and then to the polyene, filipin, to render the cell membranes permeable to cAMP. The release of radioactive cAMP from the tissue was then measured, the assumption being made that the amount of labeled cAMP released would reflect the amount present in the free cellular pool. Within 15-30 min after the diaphragm was exposed to growth hormone, the amount of cAMP released from the tissue declined, thus suggesting a reduction in the amount of this nucleotide in the free pool of the cells. Hence, this observation is consistent with the idea that one of the steps leading to ribosome activation by growth hormone is a reduction in the amount of free cAMP in the cells. How growth hormone lowers the amount of free cyclic AMP remains to be established. One recent report (Thompson et al., 1973) indicating that growth hormone injection into rats can stimulate hepatic cyclic nucleotide phosphodiesterase, suggests that the hormone may affect the metabolic degradation of cAMP. In any event, this phase of growth hormone's action on the muscle cell lasts

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only a short time. Kostyo and co-workers (1975) found that the release of labeled cAMP from the diaphragm in the presence of growth hormone was reduced for only a brief period. Within an hour after exposure of the cells to the hormone the amount of cAMP released was markedly increased, and enhanced release persisted for at least another hour. The significance of this secondary rise in free cyclic AMP in the muscle cell is presently unclear. As noted earlier in this section, a single injection of growth hormone into a hypophysectomized rat stimulates the rate of protein synthesis for many hours. If injections of the hormone are repeated at daily intervals, the rate of protein synthesis in the muscles remains elevated for many days (Kostyo and Knobil, 1959; Nutting et al., 1972) and the musculature grows, as does the entire body. The events that follow the initial stimulation of ribosome activity and that eventually lead to growth of the muscles are not well characterized. Within 12-18 hr after the first injection of growth hormone, there is an increase in ribosomal RNA synthesis resulting in an increase in the number of ribosomes in each muscle cell (Florini and Breuer, 1966). This increase in ribosomes in the cells is accompanied by a gradual increase in the capacity of the ribosomes to carry out the translation process. Within 24 hr of the start of hormone treatment, the ability of muscles to form proteins doubles and persists at this rate if treatment is continued for some days. After 7 days of treatment with the hormone, the DNA content of many skeletal muscles in the rat increases (Beach and Kostyo, 1968), perhaps reflecting the proliferation of nuclei, as the cells undergo the hypertrophy characteristic of muscle growth. LIVER The liver, like the musculature, responds quite rapidly to growth hormone by increasing its rate of protein formation. Very large increases in protein synthesis have been observed in the livers of young hypophysectomized rats only 30 min after the intravenous administration of growth hormone (Kostyo and Nutting, 1973). The liver will continue to produce proteins at an enhanced rate for a number of days provided that hormone administration is continued on a daily basis (Korner, 1960). Growth hormone has also been shown to stimulate hepatic protein synthesis under in vitro conditions. Jefferson and Korner (1967) found that the addition of growth hormone to fluid perfusing the isolated livers of either normal or hypophysectomized rats stimulated the incorporation of radioactive amino acids into protein. Again, the rate of protein synthesis increased within 30min of addition of growth hormone to the perfusion fluid. To obtain this effect, however, it was necessary to suppliment the perfusion fluid with amino acids at concentrations several times higher than those normally found in the plasma. Slices of rat liver incubated in vitro also respond with an increase in protein synthesis to the direct addition of growth hormone to the incubation medium, but once again, the medium must be enriched with super-physiological levels of amino acids (Clemens and Korner, 1970). The basis for this high amino acid requirement is presently unclear. As in the case of muscle, little is known of the molecular events involved in mediating the effect of growth hormone on protein synthesis in the liver. Although there were assertions in the earlier literature (for review see Kostyo and Nutting, 1974) that the primary effect of growth hormone on the liver is to stimulate the transcription of new messenger RNA, this does not appear to be the case. Quite rapid effects of growth hormone on hepatic RNA synthesis have been observed, both in vivo (Sells and Takahashi, 1967) and in vitro (Jefferson and Korner, 1967), the earliest effects coinciding with stimulatory effects on protein synthesis. However, the RNA produced within the first hours after growth hormone administration appears to be mainly ribosomal RNA (Jackson and Sells, 1967; Korner, 1965; Talwar et al., 1964; Widnell and Tata, 1966). Unfortunately, most experiments attempting to elucidate the mechanism of action of growth hormone on protein synthesis in the liver have been conducted on animals treated for several hours or many days with the hormone. Thus, little can be

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said concerning the nature of the initial effect of the hormone on the protein synthetic mechanism. It is quite clear, however, from the wealth of literature dealing with later effects of the hormone on the liver (for review see Kostyo and Nutting, 1974) that some hours after growth hormone treatment, the activity and number of protein synthetic units increases (Garren et al., 1967; Korner, 1968; Staehelin, 1965). As in muscle, growth hormone appears to influence some aspect of the translation process. The hormone also appears to increase the synthesis of the cellular membranes to which polyribosomes are bound (Tata, 1970). CARTILAGE

The potent anabolic action of growth hormone is perhaps most vividly displayed by the skeletal system, which grows in a striking and obvious way in response to the hormone. When animals or humans are exposed to excessive or supra-physiological amounts of the hormone for extended periods, the effects of the skeleton are unmistakeable. In the young organism, there is excessive growth of the long bones resulting in time in gigantism. In older organisms, in which the growth centers of the long bones have fused, so-called acromegalic features (abnormal thickening of certain bones) develop. Growth hormone exerts these effects on the skeleton by stimulating the processes of chondrogenesis and osteogenesis. Most investigations of the anabolic action of growth hormone on the skeletal system have focused on the process of chondrogenesis. It was demonstrated a number of years ago (for review see Engel and Kostyo, 1964), that removal of the pituitary gland of the rat reduced the in vivo incorporation of radioactive sulfate into various cartilagenous tissues. Similar results were seen when tissue, such as costal cartilage, was removed from hypophysectomized rats and incubated with radioactive ~ulfate in vitro. These results presumably reflected a reduction in the rate of chondroitin sulfate synthesis by the cartilage. Treatment of the hypophysectomized animals with growth hormone returned the rate of sulfate incorporation toward normal. Subsequent studies have shown that growth hormone administration also affects other metabolic processes in cartilage. For example, amino acid incorporation into mucopolysaccharide complexes and uridine incorporation into RNA are stimulated (Salmon and DuVall, 1970a), and the rate of thymidine incorporation into DNA is enhanced (Daughaday and Reeder, 1966; Murakawa and Raben, 1968). Also, the conversion of proline to hydroxyproline is stimulated (Daughaday and Mariz, 1962), probably reflecting an increase in the rate of collagen formation. It is now generally accepted that these actions of growth hormone are not the result of the direct interaction of growth hormone with the cartilage itself. As Salmon and Daughaday (1957) first showed, growth hormone added directly to medium bathing isolated pieces of cartilage of hypophysectomized rats has no effect on sulfation or on other aspects of metabolism. On the other hand, serum of normal or growth hormonetreated rats stimulates the metabolism of isolated hypophysectomized rat cartilage. The serum of hypophysectomized rats also has some ability to stimulate the sulfation process, but its activity is only 30--40 per cent that of normal rat serum (Philips et al., 1974). The growth hormone-dependent principal in the serum, which presumably mediates the action of growth hormone on cartilage metabolism, was first called sulfation factor (Salmon and Daughaday, 1957), but it is now generally referred to as somatomedin (Daughaday et al., 1972). Somatomedin has now been detected and quantitated in the sera of both experimental animals and man with a variety of in vitro assay systems employing isolated rat costal cartilage, as in the original experiments described above, isolated embryonic chick cartilage and slices of pig cartilage (see Daughaday et al. (1975) and Van Wyk et al. (1974) for current reviews of assay techniques). It exists in the blood as a component of a large molecular weight complex (Daughaday and Kipnis, 1966), i.e. it is bound to plasma proteins. It can be dissociated from its binding protein by employing the acid-ethanol extraction conditions used for the isolation of insulin

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from biological materials (Van Wyk et al., 1971). Studies with preparations of somatomedin purified from human blood in this manner suggest that the substance may actually be a family of small peptides having molecular weights in the range of 6000-9000 daltons (Hall, 1972; Uthne, 1973; Van Wyk et al., 1974). The biological properties of these partially purified somatomedin preparations have proven to be quite interesting. They not only stimulate cartilage metabolism, but they also exert insulinlike effects on other tissues of the body. For instance, somatomedin preparations have been found to stimulate glucose oxidation (Hall and Uthne, 1971) and inhibit catecholamine-induced lipolysis in isolated rat adipose tissue (Underwood et al., 1972). Further, rat (Salmon and DuVall, 1970b) and human (Uthne et al., 1974) somatomedin preparations were shown to stimulate protein synthesis, amino acid transport and sugar uptake by the isolated diaphragm of the hypophysectomized rat, the responses being characteristic of those produced by insulin. These and other observations have led to the suggestion that somatomedin may be identical to the acid-ethanol soluble fraction of the non-suppressible insulin-like activity (NISLA-S) of the blood described by Jakob et al. (1968). Little is known of the mechanism by which growth hormone affects the level of somatomedin in the blood. Following the injection of growth hormone, 6 or more hours usually elapse before somatomedin activity increases in the blood (Salmon and Daughaday, 1957; Phillips et al., 1973). In one clinical study (Hall, 1971), an increase in somatomedin was found 3 hr after the intravenous injection of growth hormone into hypopituitary subjects. The source of the somatomedin appearing in the plasma after growth hormone administration may be the liver. Somatomedin activity has been detected in the fluid perfusing the isolated liver of the rat, and the addition of growth hormone to the perfusate has been found to increase the amount of activity present (McConaghey and Sledge, 1970; McConaghey, 1972). Traces of somatomedin-like activity have been found in a variety of tissues (Salmon, 1972), but no organ, including the liver, appears to contain a high concentration of the material. There have been extensive studies made of the activity of somatomedin in the blood of human subjects (for current review see Daughaday et al., 1975). As might be expected, the somatomedin activity in serum of hypopituitary subjects is sub-normal and can be restored toward normal by the administration of human growth hormone. There appears to be some correlation, also, between the rate of growth of human subjects and the activity of somatomedin in the blood (Hall and Olin, 1972). On the other hand, there is no correlation between the concentration of hGH in the blood and the level of somatomedin, e.g. not all acromegalic subjects have an elevated somatomedin level (Hall, 1972). Whether somatomedin is exclusively responsible for the effects of growth hormone on cartilage metabolism is not altogether clear. The only evidence suggesting that growth hormone does not act on cartilage directly is the finding that the metabolism of isolated cartilage is not affected by the addition of growth hormone to the bathing medium. It is certainly possible that, in vivo, growth hormone or some active fragment of the hormone may act directly to stimulate cartilage metabolism. In any event, one recent observation suggests that somatomedin alone cannot account for the effects of growth hormone on cartilage metabolism. Phillips et al. (1973) have found that when hypophysectomized rats are treated with low doses of growth hormone, sufficient to promote growth, cartilage metabolism is stimulated but the level of somatomedin in the blood is not altered. Only very large doses of growth hormone, greatly in excess of those required to stimulate growth, increase somatomedin activity in the blood of these animals. Thus this observation raises some question regarding the role played by somatomedin in the physiological action of growth hormone on cartilage metabolism and growth.

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A rather prompt hypoglycemia is produced when previously untreated hypophysectomized animals or humans are given an intravenous injection of growth hormone (for review see Altszuler, 1974). The fall in blood glucose is caused by a direct stimulation by growth hormone of glucose uptake and utilization by the peripheral tissues, coupled to the inability of the livers of these individuals to produce adequate glucose to combat the hypoglycemia. Hypoglycemia does not result from an action of the hormone on insulin secretion. It is important to realize that this hypoglycemic or insulin-like effect usually only occurs as an acute response to a single injection of growth hormone. Growth hormone-deficient animals or humans that have received the hormone recently do not respond to another injection of growth hormone with hypoglycemia. Similarly, the intravenous injection of growth hormone into normal animals or humans has little or no effect on the concentration of glucose in the blood. The insulin-like effect of growth hormone can also be demonstrated readily on isolated tissues of hypophysectomized animals. As Park et al. (1952) first showed, the addition of growth hormone to medium bathing the isolated diaphragm of the hypophysectomized rat stimulates the uptake and utilization of glucose by the tissue. The uptake of various non-utilizable sugars by the diaphragm is also stimulated by growth hormone (Ahr6n and Hjalmarson, 1968) suggesting that the hormone affects the process regulating the membrane transport of glucose. Growth hormone added in vitro has also been shown to stimulate the uptake of glucose by the isolated perfused heart of the hypophysectomized rat (Henderson et al., 1961) and the uptake and utilization of glucose by isolated adipose tissue of hypophysectomized rats (Goodman, 1965). Similar effects have been obtained with diaphragm muscle and adipose tissue removed from rats treated acutely with growth hormone. The time-course of the insulin-like effect has been studied most extensively using isolated muscle (Ahr6n and Hjalmarson, 1968) and adipose tissue (Goodman, 1968a). The addition of growth hormone to medium bathing these tissues has no immediate effect on glucose metabolism; stimulation of glucose uptake and utilization occurs about 15-30 min later (Rillema and Kostyo, 1971). The effect persists for 2-3 hr and then subsides. If inhibitors of RNA or protein synthesis are added along with growth hormone to the incubation medium, the stimulatory phase persists for several additional hours (Ahr6n and Hjalmarson, 1968), suggesting that protein synthesis is involved, in some manner, in limiting the duration of the insulin-like effect. Of particular interest is the fact that once the tissues have responded to the insulin-like effect, they become refractory for an extended period (up to 48 hr) and cannot be stimulated further by growth hormone. For example, growth hormone does not stimulate sugar uptake or utilization in diaphragm muscle that has been removed from growth hormone-treated rats or that has been preincubated for several hours with growth hormone (Park et al., 1952; Hjalmarson and Ahr6n, 1967). If either muscle or adipose tissue is exposed to actinomycin D during the initial period of stimulation with growth hormone, refractoriness does not develop (for review see Goodman, 1973). This observation suggests that the development of refractoriness, like the termination of the insulin-like effect, requires protein synthesis. Quite obviously, the refractoriness observed in these in vitro experiments explains the failure of normal animals to give a hypoglycemic response to growth hormone. Since the normal animal is continually under the influence of growth hormone, its peripheral tissues are probably permanently refractory to the insulin-like effect of the hormone. This may be of significant protective value to the organism, considering that growth hormone secretion is rather mercurial and not tied in any particular way to the feeding-fasting cycle. Viewed in this light, the insulin-like action of growth hormone probably has little if any role in the normal regulation of carbohydrate metabolism.

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THE DIABETOGENIC E F F E C T OF GROWTH H O R M O N E Although there are differences in sensitivity among various species of animals, the chronic administration of growth hormone to normal organisms usually results in an elevation in the fasting blood glucose concentration (for review see Engel and Kostyo, 1964; Goodman, 1973; Altszuler, 1974). In the dog, the marked hyperglycemia produced by chronic growth hormone treatment is accompanied by increased glucose output from the liver (Campbell and Rastogi, 1966; Altszuler et al., 1968a, b). The concentration of glycogen in the liver also rises in the fed animal, and increased breakdown of this glycogen may contribute to the enhanced rate of hepatic glucose production observed in these animals (Altszuler, 1974). Whether increased gluconeogenesis contributes to the increased hepatic production of glucose is presently unclear. Chronic treatment of animals with growth hormone also results in a rise in the concentration of insulin in the blood, perhaps due to an increased sensitivity of the /3-cells of the pancreas to circulating glucose and other insulinogenic stimuli (Campbell and Rastogi, 1966; Altszuler, 1974). There is also some evidence suggesting a direct stimulatory effect of growth hormone on insulin secretion by the/3-cells in the chronically-treated animal (Rathgeb et al., 1970). The animal receiving chronic growth hormone treatment also exhibits a marked decrease in the sensitivity of the peripheral tissues to the stimulatory effect of insulin on glucose uptake and utilization. While the mechanism responsible for this decreased insulin sensitivity has been the subject of considerable speculation, very little direct evidence is available bearing on the processes involved. That it may be due in part to a reduction in the rate of glucose phosphorylation in the tissues has been suggested by the finding of reduced phosphorylation of glucose in the heart (Randle et al., 1966) and diaphragm muscle (Ridick et al., 1962) of animals receiving growth hormone treatment. Certain other aspects of glucose metabolism may be involved. The rate of glucose metabolism appears to be controlled in part by the rate of glycolysis and in part by the rate of conversion of pyruvate to acetyl coenzyme A. The activities of key regulatory enzymes in these processes, namely phosphofructokinase and pyruvate dehydrogenase, are inhibited indirectly and directly, respectively, by an increase in the ratio of acetyl coenzyme A to coenzyme A in the cell. It has been proposed (Randle et al., 1966) that the reduction in glucose utilization in the animal treated with growth hormone may result from the hormone's ability to mobilize lipid, the oxidation of which would increase the ratio of acetyl coenzyme A to coenzyme A. This, in turn, would impair the rate of glycolysis, resulting in the accumulation of glucose-6-phosphate and, consequently, the inhibition of glucose phosphorylation by hexokinase. In this situation, the ability of insulin to stimulate the uptake of glucose would necessarily be impaired. Whether this mechanism is indeed responsible for the decrease in insulin sensitivity of the peripheral tissues remains to be established. If some animals, like the dog or cat, or humans are treated for long periods with large doses of growth hormone, frank diabetes mellitus may be produced. For example, treatment of normal dogs with 3 mg of growth hormone per kg body weight per day for 14-21 days results in marked hyperglycemia, glucosuria, elevated free fatty acids and occasionally ketonuria (Campbell, 1955). Plasma insulin is also elevated. If treatment with growth hormone is continued, permanent diabetes and degenerative changes in the /3-cells result (Young, 1953; Campbell, 1955). Animals with a large pancreatic reserve, such as the rat, must be partially pancreatectomized before they will exhibit the manifestations of diabetes in response to growth hormone. Thus it is clear that growth hormone, particularly when given in large doses to appropriate animals, can be a diabetogenic agent. However, this property would appear to be a pharmacological exaggeration of the role played by growth hormone in the physiological regulation of carbohydrate metabolism. The importance of growth hormone for the normal regulation of carbohydrate metabolism is reflected in the metabolic abnormalities of the hypophysectomized animal (DeBodo and Sinkoff, 1953).

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These include a reduced rate of hepatic glucose production (Steele et al., 1956) and a marked increase in the sensitivity of the peripheral tissues to insulin. THE EFFECT OF GROWTH HORMONE ON LIPID METABOLISM The first indication that growth hormone influences lipid metabolism came from the studies of Lee and Schaffer (1934) in which extracts rich in growth-promoting activity were found to decrease the amount of fat in the carcasses of experimental animals. Subsequent studies, with purified preparations of growth hormone, demonstrated that the hormone is ketogenic and that it can cause fat mobilization and reduce the respiratory quotient, the latter being suggestive of an effect on fatty acid oxidation (Astwood, 1955). Early studies dealing with the action of growth hormone on lipid metabolism have been extensively reviewed (Smith et al., 1955; Weil, 1955; DeBodo and Altszuler, 1957; Ketterer et al., 1957). The following discussion will be devoted to more recent findings concerning the effects of growth hormone on lipogenesis, fatty acid mobilization and fatty acid oxidation. Chronic administration of growth hormone to hypophysectomized rats maintained on a high carbohydrate diet results in a dramatic decrease in lipid content of adipose tissue and a decreased ability of adipocytes to incorporate carbon from glucose or amino acids into fatty acids (Goodman, 1963). Normal rats maintained on such a diet also respond to the daily injection of growth hormone with a reduction in the synthesis of both carcass and liver fatty acids (Fain and Wilhelmi, 1962). Similar results have been obtained after a single injection of growth hormone into hypophysectomized and normal animals (Fain and Wilhelmi, 1962). It should be noted that fatty acid synthesis in the liver and adipose tissue of the hypophysectomized animal is lower than normal (Bates et al., 1955; Fain and Wilhelmi, 1962; Goodman, 1964), but this appears to be related to the decreased secretion of insulin by the hypophysectomized animal (Mayhew et al., 1969). While it is still uncertain how growth hormone decreases lipogenesis in adipose tissue, it is clear that the reduction in fat formation is accompanied by a decrease in glucose oxidation and its metabolism to fatty acids (Goodman, 1963). Whether the inhibition of fat synthesis by growth hormone is an indirect consequence of the inhibition of glucose metabolism remains to be established. Early work on the action of growth hormone on lipid mobilization showed that the hormone increases the amount of fat in the liver and the concentration of triglycerides in the blood. Adipose tissue is the source of this lipid. Subsequently, it was established that growth hormone increases the concentration of free fatty acids in the blood of animals and man (for review see Goodman and Schwartz, 1974). After a single injection of growth hormone, approximately 2 hr elapse before the level of free fatty acids rises above control. The increased level then persists for 12-24hr (Engel et al., 1958; Goodman and Knobil, 1959; Raben and Hollenberg, 1959). Very large amounts of growth hormone are usually required to elict this response in most species, however. This action of growth hormone on free fatty acid release appears to be due both to a depression of the re-esterification process (Goodman, 1969) and to accelerated lipolysis, as indicated by parallel increases in both fatty acid and glycerol release (Winkler et al., 1969). Attempts to study the lipolytic effect of growth hormone in vitro have been complicated by the high degree of in vitro lipolytic activity of other pituitary hormones, such as ACTH and TSH, that often contaminate growth hormone preparations. The conclusion that can be drawn from many studies using isolated adipose tissue is that the hormone either has no in vitro effect on lipolysis or that it is effective only when present at inordinately high concentrations. However, in vitro lipolytic effects can be obtained with reasonable concentrations of growth hormone if glucocorticoids are added to the incubation medium and incubation is carried out for several hours (Fain et al., 1965). This combined action of growth hormone and glucocorticoids can be blocked with inhibitors of RNA and protein synthesis, suggesting that their action on lipolysis is

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dependent upon the synthesis of protein. Growth hormone and glucocorticoids also a u g m e n t t h e l i p o l y t i c a c t i o n o f e p i n e p h r i n e ( G o o d m a n , 1969), n o r e p i n e p h r i n e a n d t h e o p h y l l i n e ( F a i n , 1968) o n i s o l a t e d a d i p o s e t i s s u e . T h i s finding, c o u p l e d w i t h t h e f a c t t h a t a n i n t a c t p i t u i t a r y g l a n d is r e q u i r e d f o r a n o r m a l l i p o l y t i c r e s p o n s e to a l m o s t a n y a g e n t ( G o o d m a n , 1968b), l e d G o o d m a n (1968c) to s u g g e s t t h a t g r o w t h h o r m o n e m a y n o t i n i t i a t e t h e l i p o l y t i c p r o c e s s b u t t h a t it c o n d i t i o n s t h e l i p o l y t i c m a c h i n e r y o f a d i p o c y t e s to o t h e r p h y s i o l o g i c a l s t i m u l i f o r l i p i d m o b i l i z a t i o n . Some evidence suggests that the action of growth hormone on the lipolytic process m a y i n v o l v e c y c l i c A M P (for r e v i e w s e e G o o d m a n a n d S c h w a r t z , 1974). G r o w t h hormone together with glucocorticoids has been shown to raise the concentration of c A M P in i s o l a t e d a d i p o c y t e s ( M o s k o w i t z a n d F a i n , 1970). T h e t i m e - c o u r s e f o r t h e effect of these agents on the level of cAMP coincides with that for their effect on lipolysis. Furthermore, theophylline, which inhibits the breakdown of cAMP, potentiates the lipolytic action of growth hormone and glucocorticoids. The exact r e l a t i o n s h i p b e t w e e n g r o w t h h o r m o n e a n d c A M P , w i t h r e s p e c t to t h e l i p o l y t i c p r o c e s s , r e m a i n s to b e e s t a b l i s h e d . Studies of the action of growth hormone on fatty acid oxidation have yielded c o n f l i c t i n g i n f o r m a t i o n ( f o r r e v i e w s e e G o o d m a n a n d S c h w a r t z , 1974). A c o m p l i c a t i o n in this w o r k is t h a t t h e o x i d a t i o n o f f r e e f a t t y a c i d s is d e p e n d e n t u p o n t h e a m o u n t o f f r e e f a t t y a c i d t a k e n u p b y t h e t i s s u e s , w h i c h in t u r n , is r e l a t e d to t h e c o n c e n t r a t i o n in t h e b l o o d . I n t h e in v i v o s i t u a t i o n , g r o w t h h o r m o n e m a y r a i s e t h e l e v e l o f f r e e f a t t y a c i d s in t h e b l o o d a n d t h u s i n d i r e c t l y a f f e c t t h e r a t e o f f a t t y a c i d o x i d a t i o n . A t t h e moment, the bulk of evidence favors the conclusion that growth hormone has no direct effect on fatty acid uptake or oxidation.

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