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Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
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RELATION BETWEEN
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GROWTH HORMONE AND SOMATOMEDIN! Judson J. Van Wyk, MD.2 and Louis E Underwood, MD.3 Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill,
North Carolina 27514
DEFINITION OF SOMATOMEDIN In 1957, Salmon & Daughaday showed that growth hormone stimulates the incor poration of radiolabeled sulfate into proteoglycans of cartilage by inducing a second ary "sulfation factor" (1). Subsequent studies have indicated that normal plasma contains an unknown number of growth hormone dependent substances which stimulate cellular events concerned with the growth of both extraskeletal and skele tal tissues. Although immunologically distinct from insulin, these substances display many insulin-like metabolic actions in vitro. As a result of these and other studies, the original designation "sulfation factor" became entirely too constrictive. The more open-ended term, "somatomedin," was therefore introduced to encompass all growth hormone dependent substances in plasma which stimulate growth in respon sive tissues (2). Although whole plasma stimulates the growth of many tissues in vitro, very little is known concerning the number of specific factors involved,their chemical natures, or their hormonal dependencies. Because of rapid acquisition of new knowledge concerning the somatomedins, it seems wisest at present to avoid overly strict definitions of what substances should or should not be included under this designa tion. Thus, from our present perspective,it would seem irrational,simply on histori cal grounds, to exclude from this family of compounds growth hormone dependent peptides which might be inactive in cartilage assays but which nevertheless stimulate 'Supported by USPHS Research Grants AMOI022 and HD08299, Training Grant AM05330,and by grants from the Human Growth Foundation, Elsa U. Pardee Foundation, and University of North Carolina Research Council. 2JJVW is a recipient of Research Career Award #4 K06 AM14115 from the National Institutes of Health. "LEU is a Jefferson-Pilot Fellow
in Academic
Medicine, University of North Carolina. 427
.
Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
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VAN WYK & UNDERWOOD
the growth of extraskeletal tissues. Likewise, it now seems wise to drop an earlier suggestion that all members of this family must exhibit insulin-like properties (3, 4). Recent studies now suggest that the growth-promoting effects of somatomedin may be mediated by a different portion of the molecule than are the insulin-like effects. Thus, somatomedins and insulin seem to be related through some' ancestral homology in structure which gives rise to a partial overlap of biological action; the growth-promoting actions of the somatomedins are therefore not necessarily depen dent on the insulin-like effects. The purpose of this review is to survey the assay methods which have been used to measure somatomedin levels in plasma and to monitor purification; to review present knowledge of the purification and chemical nature of these substances; to describe their in vitro biological effects; and to review currently available informa tion on the relationship between linear growth, plasma growth hormone, and plasma somatomedin levels. Since most of our present knowledge of the somatomedins has been garnered from studies which have employed some modification of the original sulfation factor assay, the term "somatomedin" will be used in this review to refer to those substances which are detected by these assays. Where the term is used generically, its broader meaning should be obvious from the context.
THE ORIGINAL SOMATOMEDIN PHENOMENON Salmon & Daughaday (1) reported in 1957 that the incorporation of radioactive sulfate into proteoglycans of cartilage was stimulated when normal serum was incubated with rat costal cartilage and 35S04• When serum from a hypopituitary individual or a hypophysectomized animal was substituted for the normal serum, sulfate incorporation was diminished. Enrichment of the hypopituitary serum by the addition of large quantities of growth hormone caused no change, but the capacity of serum from hypopituitary patients or animals to stimulate sulfate incorporation returned after parenteral administration of growth hormone. From these fundamen tal observations arose the concept that growth hormone does not stimulate skeletal growth directly, but rather acts by stimulating the formation of a secondary growth promoting substance. It was subsequently shown that this "sui fation factor" exerted multiple biologic effects on cartilage including the incorporation of thymidine into DNA, uridine into RNA, amino acids into protein, and the conversion of proline to hydroxyproline (5-7). Because of the possibility that different factors might stimulate the synthesis of cartilage matrix and cell replication, several workers have used the operational term "thymidine factor" when measuring the incorporation of thymidine into DNA (8, 9).
ASSAYS FOR SOMATOMEDIN
Cartilage Bioassays Most bioassay methods for somatomedin are based on the incorporation of 3SS04 into cartilage. Cartilage possesses unique advantages for in vitro studies since the predominant cell type is relatively resistant to low oxygen tension and nearly all of
Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
GROWTH HORMONE AND SOMATOMEDIN
429
the labeled sulfate which is taken up can be identified as sulfated glucosaminogly cans. After a lag phase of 18-20 hr,the incorporation of 3H-methyl thymidine into DNA parallels the incorpor.ation of sulfate (10, II). Using a dual label assay, Van Wyk has concluded that the plasma factor which stimulates sulfate incorporation is the same as that responsible for the synthesis of DNA (11, 12). The original sulfation factor assay utilized costal cartilage segments from hypo physectomized rats. This assay has great sensitivity but is exceedingly cumbersome and costly to perform and has large variances. Yde found satisfactory dose response curves in costal cartilages from fasted, immature rats, but this method sacrifices sensitivity due to high basal uptakes of labeled sulfate (13). The disadvantages of the rat cartitage assay led Hall to introduce a simpler and less time-consuming bioassay which quantitated the incorporation of 35S04 into pelvic leaflets of I I-day chick embryos (14). Although slightly less sensitive than rat cartilage assays, numerous laboratories have reported satisfactory precision with this technique or one of its variations. Van den Brande et al have recently developed an assay of very high precision using identical pieces of costal cartilage from a single pig (15). These results have been confirmed by Phillips et aI,who stressed the importance of using pigs less than 9 months of age (16). The porcine assay is the least sensitive of the cartilage assay systems, but also seems to be the least influenced by inhibitors in plasma. It must be stressed that all somatomedin bioassays are greatly influenced by inhibitory substances and therefore reflect only net somatomedin activity. Salmon has investigated the nature of these inhibitory substances in rat plasma and found that at least parCof the inhibitory material was heat labile, nondialyzable, and destroyed by tryptic digestion (17,18). This inhibitory material is increased in the serum of fasted and hypophysectomized rats,in hypopituitary patients, and in the plasma of patients with protein-calorie malnutrition (19). In addition to the variable quantity of macromolecular inhibitors present in sera, the in vitro uptake of 3SS04 may be inhibited by fatty acids (20) and high cortisol levels (6, 21,22). Likewise,failure to correct for variations in the plasma content of inorganic sulfate may,under certain circumstances,give rise to spurious conclu sions based on the incorporation of 35S04•
Assay of Somatomedin by Cell Culture Techniques Most mammalian cells fail to divide in vitro unless their culture medium is enriched with serum or some similar complex of growth factors (23,24). Gey & Thalhimer (25) demonstrated that insulin at very high dosage would at least partially replace the requirement for serum of cells grown in vitro. Salmon & Hosse (26) first showed that bovine plasma extracts rich in somatome din also stimulated the growth of HeLa cells. Uthne has shown that extracts of human plasma which are rich in somatomedin-Iike activity also stimulate thymidine incorporation by human glial-like cells in culture. With further purification, two separate substances were identified; one stimulated the growth of glial-like cells, whereas the other stimulated sulfate incorporation by embryonic chick pelvic lea flets (27). Both factors qualified as somatomedins,since plasma from hypophysecto-
Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
430
VAN WYK & UNDERWOOD
mized rats was deficient in both activities, and both activities increased following the administration of growth hormone. To differentiate between these substances, he designated the cartilage factor somatomedin-A and the factor which stimulates glial-like cells somatomedin-B. Somatomedin-rich extracts of human plasma prepared in our laboratory have likewise been found to stimulate thymidine incorporation in human fibroblast cul tures, fetal rat liver cell cultures, and in a rat ovarian tumor cell line.4 Since these effects were obtained with only partially purified preparations of somatomedin, it was not clear which component of this material might be responsible for the mito genic activity. More recent studies by Dr. Joel Baseman have demonstrated that an essentially pure preparation of somatomedin-C isolated in our laboratory (see be low) stimulates dramatic increases in thymidine incorporation and mitotic index in primary cultures of chick fibroblasts. These effects were far greater than the maxi mal effect which could be achieved with insulin (J. Baseman, unpublished observa tions). There are as many variations of cell culture assay techniques as there are laborato ries performing them. In general, monolayer cultures of various cell lines are estab lished and grown to confiuency. They are then deprived of serum, and the response of test substances is measured in terms of 3H-thymidine incorporation into DNA, increase in mitotic index, or the incorporation' of 35S04 into proteoglycans (28). Although the use of cell culture assays to define specific growth factors will undoubt edly be of critical importance in future somatomedin research, it is beyond the scope of this review to summarize the diverse literature on this subject. Much of the research on growth factors has originated in laboratories of cell biology and has been carried out without specific consideration to possible hormonal dependencies.
Specific Radioreceptor Assays for Somatomedin Specific radioimmunoassays and cell receptor assays will unquestionably replace bioassay techniques when sufficient quantities of the pure somatomedins become available. Marshall et al have developed a radioreceptor assay for somatomedin-C based on the competition between 125I-somatomedin-C and unlabeled somatomedin for binding to cell-free membrane preparations prepared from human placentas (29, 30). The agreement between results obtained with this assay and the rat cartilage bioassay has been excellent. In addition to its immense value in monitoring the purification of somatomedin from partially purified plasma fractions, the placental receptor assay has been found capable of accurately measuring somatomedin levels in unextracted human plasma. Satisfactory dose response curves are obtained at dosages between J and 20 f.t-I of plasma per milliliter of incubation medium (Figure J). This assay has provided good discrimination between the plasma somatomedin levels of hypopituitary, normal, and acromegalic subjects (Figure 2). It also appears 'Studies of the effects of somatomedin on cultured human fibroblasts were carried out by Michael Swift, M.D., at the University of North Carolina. The fetal liver cell and the rat ovarian tumor cell studies were done by Hyam Leffert, M.D., and Denis Gospodarowicz, Ph.D., respectively, at the Salk Institute, La Jolla, California.
431
GROWTH HORMONE AND SOMATOMEDIN
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Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
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Figure 1
Competition of partially purified somatomedin, acromegalic plasma,and a normal
plasma pool for the human placental somatomedin-C receptor. Approximately 150
J.Lg
of
placental cell membrane protein and tracer quantities of highly purified, radioiodinated soma tomedin-C were incubated overnight at 4° C either alone or with graded dosages of the somatomedin standard or the plasmas. Membrane-bound somatomedin was determined by counting the centrifuged membrane pellet. The competitive effects of the three preparations were expressed as the percentage of controls containing only membrane and radioiodinated somatomedin. Each point is the mean ± SEM of data derived in seven separate assays. Reprinted with permission from Marshall, R. N., Underwood, L. E., Voina, S. J., Foushee, D. 8., Van Wyk, J. J. 1974. J. Clin. Endocrinol. Metab. 39:283-92.
that the factors in serum of starved rats which inhibit sulfate incorporation in rat cartilage assays have no influence on the radioreceptor assay. Yalow et al have developed a highly sensitive radioimmunoassay for somato medin-B which can detect levels in plasma diluted 1 :5000 or greater (30a). They have found that in some patients with acromegaly, definitive therapy leading to a decrease in growth hormone is accompanied by a fall in somatomedin-B. Prelimi nary findings suggest that the levels of somatomedin-B in plasma are in the micro gram/milliliter range rather than in the nanogram range, which is common for most peptide hormones. Of even greater interest is that the species specificity of the assay is the same as that for growth hormone; i.e. the antibody cross-reacts with human and monkey plasma, but not with plasma from subprimate species.
432
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Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
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HYPOPITUITARY
NORMAL
ACROMEGALIC
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ADULTS
Plasma somatomedin levels determined by the competitive placental membrane
binding assay in 35 hypopituitary children, 19 normal children, and 12 adults with acromegaly. Determinations were run in duplicate at plasma concentrations ranging between 1 JLlIml (in acromegalies) and 20 JLlIml (in hypopituitary children). The somatomedin activity in each sample was determined by comparison with the normal plasma pool standard. The mean somatomedin values ± I SD are indicated for each group.
INSULIN-LIKE EFFECTS OF SOMATOMEDIN
Biologic Effects Somatomedin mimics the actions of insulin in all in vitro bioassay systems which measure insulin-like activity. In adipose tissue,it stimulates glucose oxidation and lipid synthesis and inhibits epinephrine-stimulated lipolysis (31-33). At subsaturat ing concentrations, the actions of somatomedin and insulin are additive, thus sug gesting that the insulin-like effects of somatomedin and of insulin itself might operate through a common mechanism (32). I n muscle, somatomedin stimulates membrane transport of sugar and amino acids and promotes the synthesis of protein and RNA (27, 34). In this system, the time course of the somatomedin effect is identical to that of insulin and different from that of growth hormone which is effective only after a period of preincubation (27, 35). Insulin and somatomedin also affect adenylate cyclase in a similar manner. Under certain circumstances, insulin inhibits the epinephrine-induced rise of adenylate
GROWTH HORMONE AND SOMATOMEDIN
433
cyclase in adipose tissue and liver (36, 37). Tell et al found that somatomedin inhibited the rise in adenylate cyclase produced in lymphocytes and fat cells by epinephrine, in chondrocytes by parathyroid hormone, and in liver by prostaglandin (PGE2) (38).
Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
Interactions with Insulin Receptor A rational explanation for these insulin-like effects of somatomedin was provided by the demonstration that in a wide variety of tissues, somatomedin is able to compete with 125I-insulin for binding to the primary insulin receptor on the cell membrane (29, 32, 39). The potency ratio between somatomedin and insulin in insulin receptor assays is remarkably constant from tissue to tissue with one unit of somatomedin having tfle potency of approximately 100-350 /-LU of insulin. This potency ratio agrees very well with the biological potency ratios of the two hormones in the standard fat pad and rat diaphragm bioassays for insulin. Competitive binding assays for insulin-like activity are sensitive to immunoreactive insulin and other insulin-like substances as well as somatomedin. The insulin radioreceptor assay is therefore less specific than the radioreceptor assay for somatomedin-C (29). Using the radioreceptor assay for total insulin-like activity, we have been unable to detect differences in unextracted serum between hypopituitary, normal, and acromegalic patients.
Significance of Insulin-Like Effects of Somatomedin It is now clear that the effects of somatomedin on carbohydrate and fat metabolism in assays for insulin-like activity are attainable only at relatively high somatomedin dosages. These effects probably have little physiologic significance in vivo. Con versely, the growth-stimulating effects of insulin, as measured in cartilage and tissue culture assays, are obtainable only with dosages of insulin far above the concentra tion of insulin in plasma. The marked discrepancy between insulin: somatomedin potency ratios in growth stimulating assays, as contrasted with assays based on insulin-like activity, may be explained by the existence of separate receptors for insulin and somatomedin on the same cell membranes. Membrane receptor assays utilizing 125I-somatomedin-C are 100 times more sensitive to competition by unlabeled somatomedin than are similar assays using 125I-insulin as the trace (29). Similarly, specific somatomedin receptor assays are markedly insensitive to insulin, since insulin concentrations several thou sand fold higher than those found in plasma are required to observe a threshold efef ct ably well with the very large dosages of insulin required to mimic the effect of somatomedin in cell growth assays. The fact that somatomedin and insulin share overlapping biological activities and cross-react with their respective receptors, however, suggests that there may be some structural homology between the two hormones. It also suggests that the active sites on the molecule which bind to the insulin receptor probably differ from the active sites which interact with specific somatomedin receptor.
434
VAN WYK & UNDERWOOD
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RELATIONSHIP BETWEEN SOMATOMEDIN AND NONSUPPRESSIBLE INSULIN-LIKE ACTIVITY
Less than 10% of the total plasma insulin-like activity, as measured in biological assays, can be neutralized by antibodies directed against pancreatic insulin (40). The remainder, which is immunologically distinct from insulin, is called nonsuppresible insulin-like activity (NSILA). Pancreatectomy does not diminish the content of NSILA, and NSILA levels are normal in diabetic patients (41, 42). Froesch and his group in Zurich have pioneered in characterizing the chemical nature and biologic properties of NSILA. They have shown that the portion of NSILA which is soluble in cold acid ethanol is attributable to a peptide with a molecular weight of about 7500. The Zurich group has reported that this peptide, designated NSILA-S, is growth hormone dependent, that it stimulates sulfate incor poration in the rat cartilage assay, and that it competes for the insulin receptor in chick fibroblasts (43,44). Furthermore, NSILA-S inhibits adenylate cyclase in fat cells (45) and is a mitogen for chick fibroblasts (46). Recently, Megyesi et aI, using a preparation of NSILA-S furnished by Froesch, demonstrated that liver cell mem branes possess a specific receptor for NSILA-S which is distinct from the insulin receptor. The potency ratios between insulin and NSILA-S in competing for the separate receptors are similar to the potency ratios between insulin and somatome din-C in competing for the insulin and somatomedin-C receptors (47). Thus, evi dence is rapidly accumulating that NSILA-S is either identical or at least only slightly different from somatomedin-C. It is unlikely that the issue of identity can be further resolved short of complete structural analysis of the two peptides. RELATIONSHIP BETWEEN SOMATOMEDIN AND OTHER GROWTH FACTORS
A number of growth factors sharing some features in common with somatomedin have been described. Pierson & Temin have purified a small peptide from calf serum which stimulates the multiplication of fibroblasts in tissue culture (48). This peptide, which was given the designation "multiplication stimulating activity" (MSA), has activity in rat cartilage assays and in biological and competitive binding assays for insulin. Dulak & Temin have shown that a specific clonal line of rat liver cells synthesizes sufficient quantities of MSA to permit exuberant proliferation in the absence of serum (49, 50). Several closely related peptides of approximately 7000 daltons have been isolated from the media in which these cells are grown. Samples of these peptides have proven highly active in our somatomedin-C receptor assay. Although growth hormone dependency has not been studied, MSA appears to be closely related to the somatomedins identified in human plasma. Nerve growth factor (NGF), a peptide of about 12,000 daltons isolated from submandibular gland of male mice, triggers differentiative processes in the develop ing nervous system and maintains the mature nerve cell. NGF has a high degree of structural homology with proinsulin, and the two peptides have overlapping
Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
GROWTH HORMONE AND SOMATOMEDIN
435
biological activities (51,52). Whether NGF is influenced by growth hormone is not known. Epidermal growth factor (EGF), a peptide of 6000 daltons, was also isolated from submandibular glands of male mice (53). This substance accelerates the eruption of the incisors and opening of the eyelids in young mice and stimulates epithelial cell proliferation in organ cultures. Furthermore,EGF can replace insulin in the growth of mammary gland explants (54) and stimulate DNA synthesis and cell replication in human fibroblasts (55). EGF is highly responsive to stimulation by androgenic hormones (56), but whether growth hormone plays any part in its regulation is not known. Erythropoietin is a peptide growth factor specific for erythrocyte precursors and, like EGF and NGF, is responsive to androgens (57). It is also partially responsive to growth hormone since levels are high in acromegaly,low in hypopituitarism, and rise in response to growth hormone administration (58). Gospodarowicz has recently isolated fibroblast growth factors from bovine brain and pituitary tissue (59). These substances, which are of about 13,000 daltons, appear to stimulate the same train of metabolic responses in fibroblast cultures as do somatomedins B and C, MSA, EGF, NSILA-S,and other less well characterized mitogenic peptides. The brain growth factor has the remarkable property of stimu lating limb regeneration in the frog (59a). No information is yet available on whether this substance is subject to hormonal regulation. The substances described above provide only a sampling of a lengthening list of mitogenic peptides which have in common the capacity to stimulate in their respec tive target tissues the set of biochemical reactions described by Hershko et al as a positive pleiotypic response (60). A rational scheme of classification and nomencla ture will be possible only when the molecular structures of these substances are known and their hormonal control mechanisms delineated. PURIFICATION AND CHEMICAL PROPERTIES OF THE SOMATOMEDINS
In contrast to the situation with other peptide hormones, no organ has been identi fied which contains a higher concentration of somatomedin than that present in native plasma. Progress in isolating and determining the chemical structure of these trace peptides has therefore required the fractionation of enormous quantities of outdated human plasma as starting material. Moreover, until very recently, the rate of progress has been limited by the slow and cumbersome bioassay methods needed to monitor purification. In whole plasma, somatomedin activity is associated with proteins the size of albumin or larger (II, 61). After extraction of plasma with cold acid ethonal, 20-40% of the original biological activity is recovered in the soluble fraction and thus separated from the bulk of plasma proteins. Following gel chromatography of the acid ethanol extract in acid medium, somatomedin is recovered in fractions corresponding to peptides having a molecular weight between 5000 and 8000 (8, II, 32, 62). Somatomedin is stable in acid solution and is not destroyed by boiling. It
Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
436
VAN WYK
& UNDERWOOD
is inactivated by highly alkaline solutions and by treatment with l3-mercaptoe thanol. Thus, at least one disulfide linkage seems to be required for its biological activity. Three separate somatomedins, provisionally designated by the suffixes A, B, and C, have thus far been isolated from acid ethanol extracts of Cohn fraction IV of outdated human plasma.5 The Swedish group of Uthne, Hall, Sievertsson, and Fryklund have monitored their plasma fractionation by two entirely different bioas say systems, the embryonic chick cartilage sulfation factor assay (14) and a cell culture assay which depends on the stimulation of thymidine uptake by human glial-like cells (27). Somatomedin-A is their designation for a neutral peptide of about 7000 daltons which is most active in chick cartilage. Somatomedin-B is an acidic peptide of about 4500 daltons which is most active in glial cells. Using somewhat different methods, we have identified two peptide fractions with activity in the rat cartilage assay. After isoelectric focusing in 6-M urea, a majority of the biological activity focused in the basic region (pH 8.4-9.4), whereas less than one third of this activity could be accounted for in the neutral region. Conversely, the neutral fractions were more active than the basic fractions in competitive binding assays for insulin-like activity. The basic peptide, designated somatomedin-C, was subsequently isolated in essentially pure form by preparative acrylamide gel electro phoresis and a series of additional chromatographic steps (4). In addition to its activity in skeletal tissues, somatomedin-C is highly active in stimulating DNA synthesis and the rate of mitosis in chick fibroblast cultures. The insulin-like activity of our basic fraction and somatomedin activity of our neutral fraction proved to be intrinsic properties of discrete peptides rather than contaminants, since the respective somatomedin:insulin ratios remained constant through subsequent purification procedures. Since, as discussed in previous sections, it seems likely that different portions of the molecule are responsible for these activities, it is possible that the neutral and basic peptides represent chemical vari ants of a single naturally occurring peptide. Since these forms are similar in size N 7000 daltons), it is conceivable that alteration of charge properties during the course of purification might selectively alter that portion of the molecule which interacts with the insulin or somatomedin receptors. Until complete structural comparisons are available between the several peptides isolated in different laborato ries, it is our tentative conclusion that somatomedins A and C and NSILA-S are derived from a single naturally occurring peptide, and that this peptide accounts for most of the "sulfation factor" activity of native plasma. Somatomedin-B, however, appears to represent a quite different substance. PRODUCTION AND DISPOSAL OF SOMATOMEDIN
Since somatomedin does not appear to be stored in any organ, it may be inferred that this substance is not manufactured in a classic endocrine tissue possessing
·We are indebted to Professor Bertil Aberg, M.D., Vice-President and Director of Research, AB Kabi Laboratories, Stockholm, and Knut Uthne, Ph.D., of the Kabi Laboratories for supplying us with the acid ethanol extracts from which purification of somatomedin was accomplished.
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GROWTH HORMONE AND SOMATOMEDIN
437
secretory granules. There is now growing evidence that the liver is a major site of somatomedin generation. Following the intravenous administration of 125I-growth hormone, a high percentage of the radioactive label is taken up by hepatic cells (63). Somatomedin activity in serum declines with partial hepatectomy and rises again as the liver regenerates (64). Lastly, perfusion of isolated rat liver with growth hormone results in a prompt increase of somatomedin activity in the effluent (65). Fragmentary evidence also suggests that muscle and kidney may also be sites of somatomedin generation (66, 67). Other sites of somatomedin generation have not been excluded, and studies so far have left unsettled the question of whether soma tomedin is synthesized de novo or is a cleavage product of growth hormone it self. In comparison to most peptide hormon
Further
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Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
Copyright 1975. All rights reserved
RELATION BETWEEN
+7163
GROWTH HORMONE AND SOMATOMEDIN! Judson J. Van Wyk, MD.2 and Louis E Underwood, MD.3 Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill,
North Carolina 27514
DEFINITION OF SOMATOMEDIN In 1957, Salmon & Daughaday showed that growth hormone stimulates the incor poration of radiolabeled sulfate into proteoglycans of cartilage by inducing a second ary "sulfation factor" (1). Subsequent studies have indicated that normal plasma contains an unknown number of growth hormone dependent substances which stimulate cellular events concerned with the growth of both extraskeletal and skele tal tissues. Although immunologically distinct from insulin, these substances display many insulin-like metabolic actions in vitro. As a result of these and other studies, the original designation "sulfation factor" became entirely too constrictive. The more open-ended term, "somatomedin," was therefore introduced to encompass all growth hormone dependent substances in plasma which stimulate growth in respon sive tissues (2). Although whole plasma stimulates the growth of many tissues in vitro, very little is known concerning the number of specific factors involved,their chemical natures, or their hormonal dependencies. Because of rapid acquisition of new knowledge concerning the somatomedins, it seems wisest at present to avoid overly strict definitions of what substances should or should not be included under this designa tion. Thus, from our present perspective,it would seem irrational,simply on histori cal grounds, to exclude from this family of compounds growth hormone dependent peptides which might be inactive in cartilage assays but which nevertheless stimulate 'Supported by USPHS Research Grants AMOI022 and HD08299, Training Grant AM05330,and by grants from the Human Growth Foundation, Elsa U. Pardee Foundation, and University of North Carolina Research Council. 2JJVW is a recipient of Research Career Award #4 K06 AM14115 from the National Institutes of Health. "LEU is a Jefferson-Pilot Fellow
in Academic
Medicine, University of North Carolina. 427
.
Annu. Rev. Med. 1975.26:427-441. Downloaded from www.annualreviews.org Access provided by University of Wisconsin - Milwaukee on 02/02/15. For personal use only.
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the growth of extraskeletal tissues. Likewise, it now seems wise to drop an earlier suggestion that all members of this family must exhibit insulin-like properties (3, 4). Recent studies now suggest that the growth-promoting effects of somatomedin may be mediated by a different portion of the molecule than are the insulin-like effects. Thus, somatomedins and insulin seem to be related through some' ancestral homology in structure which gives rise to a partial overlap of biological action; the growth-promoting actions of the somatomedins are therefore not necessarily depen dent on the insulin-like effects. The purpose of this review is to survey the assay methods which have been used to measure somatomedin levels in plasma and to monitor purification; to review present knowledge of the purification and chemical nature of these substances; to describe their in vitro biological effects; and to review currently available informa tion on the relationship between linear growth, plasma growth hormone, and plasma somatomedin levels. Since most of our present knowledge of the somatomedins has been garnered from studies which have employed some modification of the original sulfation factor assay, the term "somatomedin" will be used in this review to refer to those substances which are detected by these assays. Where the term is used generically, its broader meaning should be obvious from the context.
THE ORIGINAL SOMATOMEDIN PHENOMENON Salmon & Daughaday (1) reported in 1957 that the incorporation of radioactive sulfate into proteoglycans of cartilage was stimulated when normal serum was incubated with rat costal cartilage and 35S04• When serum from a hypopituitary individual or a hypophysectomized animal was substituted for the normal serum, sulfate incorporation was diminished. Enrichment of the hypopituitary serum by the addition of large quantities of growth hormone caused no change, but the capacity of serum from hypopituitary patients or animals to stimulate sulfate incorporation returned after parenteral administration of growth hormone. From these fundamen tal observations arose the concept that growth hormone does not stimulate skeletal growth directly, but rather acts by stimulating the formation of a secondary growth promoting substance. It was subsequently shown that this "sui fation factor" exerted multiple biologic effects on cartilage including the incorporation of thymidine into DNA, uridine into RNA, amino acids into protein, and the conversion of proline to hydroxyproline (5-7). Because of the possibility that different factors might stimulate the synthesis of cartilage matrix and cell replication, several workers have used the operational term "thymidine factor" when measuring the incorporation of thymidine into DNA (8, 9).
ASSAYS FOR SOMATOMEDIN
Cartilage Bioassays Most bioassay methods for somatomedin are based on the incorporation of 3SS04 into cartilage. Cartilage possesses unique advantages for in vitro studies since the predominant cell type is relatively resistant to low oxygen tension and nearly all of
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the labeled sulfate which is taken up can be identified as sulfated glucosaminogly cans. After a lag phase of 18-20 hr,the incorporation of 3H-methyl thymidine into DNA parallels the incorpor.ation of sulfate (10, II). Using a dual label assay, Van Wyk has concluded that the plasma factor which stimulates sulfate incorporation is the same as that responsible for the synthesis of DNA (11, 12). The original sulfation factor assay utilized costal cartilage segments from hypo physectomized rats. This assay has great sensitivity but is exceedingly cumbersome and costly to perform and has large variances. Yde found satisfactory dose response curves in costal cartilages from fasted, immature rats, but this method sacrifices sensitivity due to high basal uptakes of labeled sulfate (13). The disadvantages of the rat cartitage assay led Hall to introduce a simpler and less time-consuming bioassay which quantitated the incorporation of 35S04 into pelvic leaflets of I I-day chick embryos (14). Although slightly less sensitive than rat cartilage assays, numerous laboratories have reported satisfactory precision with this technique or one of its variations. Van den Brande et al have recently developed an assay of very high precision using identical pieces of costal cartilage from a single pig (15). These results have been confirmed by Phillips et aI,who stressed the importance of using pigs less than 9 months of age (16). The porcine assay is the least sensitive of the cartilage assay systems, but also seems to be the least influenced by inhibitors in plasma. It must be stressed that all somatomedin bioassays are greatly influenced by inhibitory substances and therefore reflect only net somatomedin activity. Salmon has investigated the nature of these inhibitory substances in rat plasma and found that at least parCof the inhibitory material was heat labile, nondialyzable, and destroyed by tryptic digestion (17,18). This inhibitory material is increased in the serum of fasted and hypophysectomized rats,in hypopituitary patients, and in the plasma of patients with protein-calorie malnutrition (19). In addition to the variable quantity of macromolecular inhibitors present in sera, the in vitro uptake of 3SS04 may be inhibited by fatty acids (20) and high cortisol levels (6, 21,22). Likewise,failure to correct for variations in the plasma content of inorganic sulfate may,under certain circumstances,give rise to spurious conclu sions based on the incorporation of 35S04•
Assay of Somatomedin by Cell Culture Techniques Most mammalian cells fail to divide in vitro unless their culture medium is enriched with serum or some similar complex of growth factors (23,24). Gey & Thalhimer (25) demonstrated that insulin at very high dosage would at least partially replace the requirement for serum of cells grown in vitro. Salmon & Hosse (26) first showed that bovine plasma extracts rich in somatome din also stimulated the growth of HeLa cells. Uthne has shown that extracts of human plasma which are rich in somatomedin-Iike activity also stimulate thymidine incorporation by human glial-like cells in culture. With further purification, two separate substances were identified; one stimulated the growth of glial-like cells, whereas the other stimulated sulfate incorporation by embryonic chick pelvic lea flets (27). Both factors qualified as somatomedins,since plasma from hypophysecto-
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mized rats was deficient in both activities, and both activities increased following the administration of growth hormone. To differentiate between these substances, he designated the cartilage factor somatomedin-A and the factor which stimulates glial-like cells somatomedin-B. Somatomedin-rich extracts of human plasma prepared in our laboratory have likewise been found to stimulate thymidine incorporation in human fibroblast cul tures, fetal rat liver cell cultures, and in a rat ovarian tumor cell line.4 Since these effects were obtained with only partially purified preparations of somatomedin, it was not clear which component of this material might be responsible for the mito genic activity. More recent studies by Dr. Joel Baseman have demonstrated that an essentially pure preparation of somatomedin-C isolated in our laboratory (see be low) stimulates dramatic increases in thymidine incorporation and mitotic index in primary cultures of chick fibroblasts. These effects were far greater than the maxi mal effect which could be achieved with insulin (J. Baseman, unpublished observa tions). There are as many variations of cell culture assay techniques as there are laborato ries performing them. In general, monolayer cultures of various cell lines are estab lished and grown to confiuency. They are then deprived of serum, and the response of test substances is measured in terms of 3H-thymidine incorporation into DNA, increase in mitotic index, or the incorporation' of 35S04 into proteoglycans (28). Although the use of cell culture assays to define specific growth factors will undoubt edly be of critical importance in future somatomedin research, it is beyond the scope of this review to summarize the diverse literature on this subject. Much of the research on growth factors has originated in laboratories of cell biology and has been carried out without specific consideration to possible hormonal dependencies.
Specific Radioreceptor Assays for Somatomedin Specific radioimmunoassays and cell receptor assays will unquestionably replace bioassay techniques when sufficient quantities of the pure somatomedins become available. Marshall et al have developed a radioreceptor assay for somatomedin-C based on the competition between 125I-somatomedin-C and unlabeled somatomedin for binding to cell-free membrane preparations prepared from human placentas (29, 30). The agreement between results obtained with this assay and the rat cartilage bioassay has been excellent. In addition to its immense value in monitoring the purification of somatomedin from partially purified plasma fractions, the placental receptor assay has been found capable of accurately measuring somatomedin levels in unextracted human plasma. Satisfactory dose response curves are obtained at dosages between J and 20 f.t-I of plasma per milliliter of incubation medium (Figure J). This assay has provided good discrimination between the plasma somatomedin levels of hypopituitary, normal, and acromegalic subjects (Figure 2). It also appears 'Studies of the effects of somatomedin on cultured human fibroblasts were carried out by Michael Swift, M.D., at the University of North Carolina. The fetal liver cell and the rat ovarian tumor cell studies were done by Hyam Leffert, M.D., and Denis Gospodarowicz, Ph.D., respectively, at the Salk Institute, La Jolla, California.
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placental cell membrane protein and tracer quantities of highly purified, radioiodinated soma tomedin-C were incubated overnight at 4° C either alone or with graded dosages of the somatomedin standard or the plasmas. Membrane-bound somatomedin was determined by counting the centrifuged membrane pellet. The competitive effects of the three preparations were expressed as the percentage of controls containing only membrane and radioiodinated somatomedin. Each point is the mean ± SEM of data derived in seven separate assays. Reprinted with permission from Marshall, R. N., Underwood, L. E., Voina, S. J., Foushee, D. 8., Van Wyk, J. J. 1974. J. Clin. Endocrinol. Metab. 39:283-92.
that the factors in serum of starved rats which inhibit sulfate incorporation in rat cartilage assays have no influence on the radioreceptor assay. Yalow et al have developed a highly sensitive radioimmunoassay for somato medin-B which can detect levels in plasma diluted 1 :5000 or greater (30a). They have found that in some patients with acromegaly, definitive therapy leading to a decrease in growth hormone is accompanied by a fall in somatomedin-B. Prelimi nary findings suggest that the levels of somatomedin-B in plasma are in the micro gram/milliliter range rather than in the nanogram range, which is common for most peptide hormones. Of even greater interest is that the species specificity of the assay is the same as that for growth hormone; i.e. the antibody cross-reacts with human and monkey plasma, but not with plasma from subprimate species.
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Plasma somatomedin levels determined by the competitive placental membrane
binding assay in 35 hypopituitary children, 19 normal children, and 12 adults with acromegaly. Determinations were run in duplicate at plasma concentrations ranging between 1 JLlIml (in acromegalies) and 20 JLlIml (in hypopituitary children). The somatomedin activity in each sample was determined by comparison with the normal plasma pool standard. The mean somatomedin values ± I SD are indicated for each group.
INSULIN-LIKE EFFECTS OF SOMATOMEDIN
Biologic Effects Somatomedin mimics the actions of insulin in all in vitro bioassay systems which measure insulin-like activity. In adipose tissue,it stimulates glucose oxidation and lipid synthesis and inhibits epinephrine-stimulated lipolysis (31-33). At subsaturat ing concentrations, the actions of somatomedin and insulin are additive, thus sug gesting that the insulin-like effects of somatomedin and of insulin itself might operate through a common mechanism (32). I n muscle, somatomedin stimulates membrane transport of sugar and amino acids and promotes the synthesis of protein and RNA (27, 34). In this system, the time course of the somatomedin effect is identical to that of insulin and different from that of growth hormone which is effective only after a period of preincubation (27, 35). Insulin and somatomedin also affect adenylate cyclase in a similar manner. Under certain circumstances, insulin inhibits the epinephrine-induced rise of adenylate
GROWTH HORMONE AND SOMATOMEDIN
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cyclase in adipose tissue and liver (36, 37). Tell et al found that somatomedin inhibited the rise in adenylate cyclase produced in lymphocytes and fat cells by epinephrine, in chondrocytes by parathyroid hormone, and in liver by prostaglandin (PGE2) (38).
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Interactions with Insulin Receptor A rational explanation for these insulin-like effects of somatomedin was provided by the demonstration that in a wide variety of tissues, somatomedin is able to compete with 125I-insulin for binding to the primary insulin receptor on the cell membrane (29, 32, 39). The potency ratio between somatomedin and insulin in insulin receptor assays is remarkably constant from tissue to tissue with one unit of somatomedin having tfle potency of approximately 100-350 /-LU of insulin. This potency ratio agrees very well with the biological potency ratios of the two hormones in the standard fat pad and rat diaphragm bioassays for insulin. Competitive binding assays for insulin-like activity are sensitive to immunoreactive insulin and other insulin-like substances as well as somatomedin. The insulin radioreceptor assay is therefore less specific than the radioreceptor assay for somatomedin-C (29). Using the radioreceptor assay for total insulin-like activity, we have been unable to detect differences in unextracted serum between hypopituitary, normal, and acromegalic patients.
Significance of Insulin-Like Effects of Somatomedin It is now clear that the effects of somatomedin on carbohydrate and fat metabolism in assays for insulin-like activity are attainable only at relatively high somatomedin dosages. These effects probably have little physiologic significance in vivo. Con versely, the growth-stimulating effects of insulin, as measured in cartilage and tissue culture assays, are obtainable only with dosages of insulin far above the concentra tion of insulin in plasma. The marked discrepancy between insulin: somatomedin potency ratios in growth stimulating assays, as contrasted with assays based on insulin-like activity, may be explained by the existence of separate receptors for insulin and somatomedin on the same cell membranes. Membrane receptor assays utilizing 125I-somatomedin-C are 100 times more sensitive to competition by unlabeled somatomedin than are similar assays using 125I-insulin as the trace (29). Similarly, specific somatomedin receptor assays are markedly insensitive to insulin, since insulin concentrations several thou sand fold higher than those found in plasma are required to observe a threshold efef ct ably well with the very large dosages of insulin required to mimic the effect of somatomedin in cell growth assays. The fact that somatomedin and insulin share overlapping biological activities and cross-react with their respective receptors, however, suggests that there may be some structural homology between the two hormones. It also suggests that the active sites on the molecule which bind to the insulin receptor probably differ from the active sites which interact with specific somatomedin receptor.
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RELATIONSHIP BETWEEN SOMATOMEDIN AND NONSUPPRESSIBLE INSULIN-LIKE ACTIVITY
Less than 10% of the total plasma insulin-like activity, as measured in biological assays, can be neutralized by antibodies directed against pancreatic insulin (40). The remainder, which is immunologically distinct from insulin, is called nonsuppresible insulin-like activity (NSILA). Pancreatectomy does not diminish the content of NSILA, and NSILA levels are normal in diabetic patients (41, 42). Froesch and his group in Zurich have pioneered in characterizing the chemical nature and biologic properties of NSILA. They have shown that the portion of NSILA which is soluble in cold acid ethanol is attributable to a peptide with a molecular weight of about 7500. The Zurich group has reported that this peptide, designated NSILA-S, is growth hormone dependent, that it stimulates sulfate incor poration in the rat cartilage assay, and that it competes for the insulin receptor in chick fibroblasts (43,44). Furthermore, NSILA-S inhibits adenylate cyclase in fat cells (45) and is a mitogen for chick fibroblasts (46). Recently, Megyesi et aI, using a preparation of NSILA-S furnished by Froesch, demonstrated that liver cell mem branes possess a specific receptor for NSILA-S which is distinct from the insulin receptor. The potency ratios between insulin and NSILA-S in competing for the separate receptors are similar to the potency ratios between insulin and somatome din-C in competing for the insulin and somatomedin-C receptors (47). Thus, evi dence is rapidly accumulating that NSILA-S is either identical or at least only slightly different from somatomedin-C. It is unlikely that the issue of identity can be further resolved short of complete structural analysis of the two peptides. RELATIONSHIP BETWEEN SOMATOMEDIN AND OTHER GROWTH FACTORS
A number of growth factors sharing some features in common with somatomedin have been described. Pierson & Temin have purified a small peptide from calf serum which stimulates the multiplication of fibroblasts in tissue culture (48). This peptide, which was given the designation "multiplication stimulating activity" (MSA), has activity in rat cartilage assays and in biological and competitive binding assays for insulin. Dulak & Temin have shown that a specific clonal line of rat liver cells synthesizes sufficient quantities of MSA to permit exuberant proliferation in the absence of serum (49, 50). Several closely related peptides of approximately 7000 daltons have been isolated from the media in which these cells are grown. Samples of these peptides have proven highly active in our somatomedin-C receptor assay. Although growth hormone dependency has not been studied, MSA appears to be closely related to the somatomedins identified in human plasma. Nerve growth factor (NGF), a peptide of about 12,000 daltons isolated from submandibular gland of male mice, triggers differentiative processes in the develop ing nervous system and maintains the mature nerve cell. NGF has a high degree of structural homology with proinsulin, and the two peptides have overlapping
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biological activities (51,52). Whether NGF is influenced by growth hormone is not known. Epidermal growth factor (EGF), a peptide of 6000 daltons, was also isolated from submandibular glands of male mice (53). This substance accelerates the eruption of the incisors and opening of the eyelids in young mice and stimulates epithelial cell proliferation in organ cultures. Furthermore,EGF can replace insulin in the growth of mammary gland explants (54) and stimulate DNA synthesis and cell replication in human fibroblasts (55). EGF is highly responsive to stimulation by androgenic hormones (56), but whether growth hormone plays any part in its regulation is not known. Erythropoietin is a peptide growth factor specific for erythrocyte precursors and, like EGF and NGF, is responsive to androgens (57). It is also partially responsive to growth hormone since levels are high in acromegaly,low in hypopituitarism, and rise in response to growth hormone administration (58). Gospodarowicz has recently isolated fibroblast growth factors from bovine brain and pituitary tissue (59). These substances, which are of about 13,000 daltons, appear to stimulate the same train of metabolic responses in fibroblast cultures as do somatomedins B and C, MSA, EGF, NSILA-S,and other less well characterized mitogenic peptides. The brain growth factor has the remarkable property of stimu lating limb regeneration in the frog (59a). No information is yet available on whether this substance is subject to hormonal regulation. The substances described above provide only a sampling of a lengthening list of mitogenic peptides which have in common the capacity to stimulate in their respec tive target tissues the set of biochemical reactions described by Hershko et al as a positive pleiotypic response (60). A rational scheme of classification and nomencla ture will be possible only when the molecular structures of these substances are known and their hormonal control mechanisms delineated. PURIFICATION AND CHEMICAL PROPERTIES OF THE SOMATOMEDINS
In contrast to the situation with other peptide hormones, no organ has been identi fied which contains a higher concentration of somatomedin than that present in native plasma. Progress in isolating and determining the chemical structure of these trace peptides has therefore required the fractionation of enormous quantities of outdated human plasma as starting material. Moreover, until very recently, the rate of progress has been limited by the slow and cumbersome bioassay methods needed to monitor purification. In whole plasma, somatomedin activity is associated with proteins the size of albumin or larger (II, 61). After extraction of plasma with cold acid ethonal, 20-40% of the original biological activity is recovered in the soluble fraction and thus separated from the bulk of plasma proteins. Following gel chromatography of the acid ethanol extract in acid medium, somatomedin is recovered in fractions corresponding to peptides having a molecular weight between 5000 and 8000 (8, II, 32, 62). Somatomedin is stable in acid solution and is not destroyed by boiling. It
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is inactivated by highly alkaline solutions and by treatment with l3-mercaptoe thanol. Thus, at least one disulfide linkage seems to be required for its biological activity. Three separate somatomedins, provisionally designated by the suffixes A, B, and C, have thus far been isolated from acid ethanol extracts of Cohn fraction IV of outdated human plasma.5 The Swedish group of Uthne, Hall, Sievertsson, and Fryklund have monitored their plasma fractionation by two entirely different bioas say systems, the embryonic chick cartilage sulfation factor assay (14) and a cell culture assay which depends on the stimulation of thymidine uptake by human glial-like cells (27). Somatomedin-A is their designation for a neutral peptide of about 7000 daltons which is most active in chick cartilage. Somatomedin-B is an acidic peptide of about 4500 daltons which is most active in glial cells. Using somewhat different methods, we have identified two peptide fractions with activity in the rat cartilage assay. After isoelectric focusing in 6-M urea, a majority of the biological activity focused in the basic region (pH 8.4-9.4), whereas less than one third of this activity could be accounted for in the neutral region. Conversely, the neutral fractions were more active than the basic fractions in competitive binding assays for insulin-like activity. The basic peptide, designated somatomedin-C, was subsequently isolated in essentially pure form by preparative acrylamide gel electro phoresis and a series of additional chromatographic steps (4). In addition to its activity in skeletal tissues, somatomedin-C is highly active in stimulating DNA synthesis and the rate of mitosis in chick fibroblast cultures. The insulin-like activity of our basic fraction and somatomedin activity of our neutral fraction proved to be intrinsic properties of discrete peptides rather than contaminants, since the respective somatomedin:insulin ratios remained constant through subsequent purification procedures. Since, as discussed in previous sections, it seems likely that different portions of the molecule are responsible for these activities, it is possible that the neutral and basic peptides represent chemical vari ants of a single naturally occurring peptide. Since these forms are similar in size N 7000 daltons), it is conceivable that alteration of charge properties during the course of purification might selectively alter that portion of the molecule which interacts with the insulin or somatomedin receptors. Until complete structural comparisons are available between the several peptides isolated in different laborato ries, it is our tentative conclusion that somatomedins A and C and NSILA-S are derived from a single naturally occurring peptide, and that this peptide accounts for most of the "sulfation factor" activity of native plasma. Somatomedin-B, however, appears to represent a quite different substance. PRODUCTION AND DISPOSAL OF SOMATOMEDIN
Since somatomedin does not appear to be stored in any organ, it may be inferred that this substance is not manufactured in a classic endocrine tissue possessing
·We are indebted to Professor Bertil Aberg, M.D., Vice-President and Director of Research, AB Kabi Laboratories, Stockholm, and Knut Uthne, Ph.D., of the Kabi Laboratories for supplying us with the acid ethanol extracts from which purification of somatomedin was accomplished.
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secretory granules. There is now growing evidence that the liver is a major site of somatomedin generation. Following the intravenous administration of 125I-growth hormone, a high percentage of the radioactive label is taken up by hepatic cells (63). Somatomedin activity in serum declines with partial hepatectomy and rises again as the liver regenerates (64). Lastly, perfusion of isolated rat liver with growth hormone results in a prompt increase of somatomedin activity in the effluent (65). Fragmentary evidence also suggests that muscle and kidney may also be sites of somatomedin generation (66, 67). Other sites of somatomedin generation have not been excluded, and studies so far have left unsettled the question of whether soma tomedin is synthesized de novo or is a cleavage product of growth hormone it self. In comparison to most peptide hormon
