ANNALS OF CLIN ICAL AND LABORATORY SCIENCE, Vol. 5, No. 6 Copyright © 1975, Institute for Clinical Science

Somatomedin and the Regulation of Skeletal Growth *t

M. JOYCELYN ELDERS, M.D., B. S. WINGFIELD, M.S., M. LORETTA McNATT, M.S., J. A. LEE, Ph.D., and EDWIN R. HUGHES, M.D.

Department of Pediatrics, University of Arkansas School of Medicine, Little Rock, AR 72201 and University of South Alabama School of Medicine, Mobile, AL 36607

ABSTRACT Somatomedin is a polypeptide(s) which “ mediates” the actions of growth hor­ mone. This pituitary dependent hormone was previously called “ sulfation factor,” a term derived from the bioassay technique which measures the incorporation of radioactive sulfate into cartilage glycosaminoglycans. Somatomedin has a more general effect upon cartilage; it is necessary for the cell multiplication and car­ tilage maturation which results in the growth of long bones. Somatomedin is not found in the plasma in growth hormone deficiency and ap­ pears following growth hormone administration. A genetic defect in somatomedin synthesis has been identified in the Laron’s dwarf. Growth hormone is present in excess in the plasma and growth hormone administration does not stimulate so­ matomedin synthesis in this syndrome. Insufficient data are available to delineate the role for somatomedin in other growth disturbances. It has been demonstrated that glucocorticoid hormones in­ terfere with both somatomedin synthesis and its biological activity. Purification of somatomedins in plasma has been achieved and radioreceptor or radioimmunoassays will be available in the future for study of growth problems in children.

Introduction endocrine system. Early in life, thyroid hor­ Disorders of growth are the most common mone is perhaps the most important hor­ .26 problems relating to the skeletal system en­ mone regulating long bone growth After countered in children. Many of these prob­ the second year, growth hormone is lems can be shown to be either nutritional, preeminent in regulating growth while the constitutional, or genetic in origin.73 After sex hormones begin to play a major role at or .60 having ruled out these factors, the problems about the time of puberty The growth of which are left are those relating to the long bones involves an ordered sequence of events which include multiplication of car­ tilage cells, glycosaminoglycan (GAGS) bio­ "Studies at the University of Arkansas were supported synthesis, collagen biosynthesis and cal­ by USPHS Grant AM 15901 and CA 13907. tStudies at the University of South Alabama were cification.73 supported by USPHS Grant AM 15923. The major determinants in this system, 440 SOMATOMEDIN AND THE REGULATION OF SKELETAL GROWTH 4 4 1

DETERMINANTS OF LONG BONE GROWTH nucleic acid (DNA) synthesis, DNA depen­ dent ribonucleic acid (RNA) synthesis, pro­ Gr o w t h Ho r m o n e Re l e a s i n g Ho r m o n e tein synthesis, the biosynthesis of GAGS, y Growth Hormone collagen synthesis and increased thickness of y the epiphyseal growth plate with resultant Somatomedin long bone growth.44 The somatomedin bioassay in plasma is based on sulfate incor­

DNA poration into cartilage glycosaminoglycans PROTEIN (GAGS).54 Although sulfate incorporation is )| Sags the final step in GAGS biosynthesis, an alteration at any step in the biosynthesis

growth could result in decreased sulfate uptake. At present it is not known which of these steps Figure 1. Hormonal factors involved in the longi­ tudinal growth of long bones. somatomedin specifically stimulates or which may be altered by other factors. GAGS is known to be synthesized by the assuming adequate nutritional and normal stepwise addition of sugar residues onto a thyroid function, are depicted in figure 1. In­ protein acceptor.58 cluded is a functioning hypothalamus ca­ Xylose is transferred from UDP-xylose by pable of releasing growth hormone releasing the enzyme xylosyltransferase to serine hormone in response to an appropriate residues of the protein acceptor, thereby stimulus.41 Growth hormone releasing hor­ initiating the biosynthesis of the polysac­ mone is secreted into the portal venous charide chains.21 Synthesis of GAGS con­ system which connects directly to the ante­ tinues by sequential addition of two rior pituitary gland and stimulates the secre­ galactose residues and one glucuronic acid tion of growth hormone.52 Growth hormone residue. These additions occur in sequence then acts on the appropriate target organ to and require specific glycosyltransferase stimulate the secretion of a small enzymes for the transfer of the sugar from polypeptide which mediates the action of the respective uridine diphosphate(UDP)- growth hormone on cartilage.8 This peptide derivative.32 Sulfation of the growing chain was formerly called “ sulfation factor” to occurs simultaneously with polymerization describe its action of stimulating sulfate of the polysaccharide eham.40 Polymeriza­ uptake by the cartilage cell.5 As more in­ tion occurs as the repeating units of formation became available on the action of hexuronic acid and amino sugar are added. somatomedin, it was apparent that this term In figure 2 is depicted, graphically, the was too restricted. The presently accepted process of GAGS biosynthesis in cartilage. term for this factor is somatomedin.7 GAGS are long polysaccharide residues of “Somato” designates a hormonal relation­ approximately 50 dimer units attached to a ship to somatotrophin (growth hormone) protein core. This protein core is 3700 Ang­ and “ soma” indicates the target tissue. The stroms in length.13 All GAGS have a com- suffix “medin” was chosen to indicate that

this hormone mediates the action of growth G A G S S Y N T H E T I C U N IT

hormone. UDP-xyl UDP-Gol UDP-GA Somatomedin is transported via the ACCEPTOR} ^ ^ ^ ^ plasma, from its site of synthesis, to the car­ PROTEIN > Ser-xyl-G ol-Gal- GA-(N-Ac-Gal-GA) x tilage cell where a number of anabolic S04 events are initiated.6 These include the Figure 2. Schematic representation of the glyco- stimulation of mitotic activity, deoxyribo­ saminoglycan (GAGS) biosynthetic unit. 4 4 2 ELD ERS ET AL. mon linkage region of xylose-galactose- may vary as to the type of cartilate used in galactose-glucuronic acid between serine the in vitro assay system, i.e., hypophysec­ and the dimer units.3 The individual tomized rat rib cartilage,48 embryonic chick proteoglycan molecules have a molecular pelvic rudiments,23 porcine rib cartilage,66 or weight of approximately 4 million. In ad­ the rib cartilage of normal rats following dition to the protein core (acceptor protein), prolonged fasting.75 The incubation medium a covalently linked protein, molecular also varies, but all include an enriched nu­ weight approximately 200,000 provides trient mixture suitable to maintain cell secondary structure to the GAGS.43 growth in culture. The basic procedure is de­ The biosynthesis of these compounds picted in figures 3 and 4. takes place in the endoplasmic reticulum and Golgi apparatus of the cartilage cell.55 BASIC SOMATOMEDIN ASSAY PROCEDURE Enzymes responsible for their biosynthesis are bound to the membrane in a sequential fashion.56 The acceptor protein is synthesized in the rough endoplasmic reticulum and trans­ ported to the Golgi where the UDP-sugar transferases are found. The biosynthesis of GAGS in cartilage both in vivo and in vitro has been shown to be growth hormone de­ pendent.113,34 Sulfate incorporation into car­ CHICK PELVIC tilage is reduced by hypophysectomy and is RUDIMENT restored by the administration of growth hormone.34 This action of growth hormone, + however, is indirect since the stimulation of PREINCUBATE sulfate uptake in cartilage cannot be IN TISSUE CULTURE MEDIUM achieved by the addition of growth hormone 4-24 hrs; 37°C in vitro.13 In the initial studies by Salmon and Daughaday,48 it was found that the ADDITION OF PLASMA serum of normal rats contained a factor which stimulated sulfate uptake into hypophysectomized rat rib cartilage in vitro. INCUBATE 16-20 hrs; 37°C Serum from hypophysectomized rats had decreased ability to stimulate sulfate uptake. ADDITION OF 35S04 Treatment of the hypophysectomized rats with growth hormone restored the capacity INCUBATE of the serum to stimulate sulfate uptake, but 24 hrs; 37°C the addition of growth hormone in vitro failed to restore activity. These studies form the basis of the ♦ bioassays of plasma somatomedin which is TERMINATE BY 2 MIN. BOILING, THEN; presently used by many investigators. 1. Wash repeatedly 2. Digest cartilage 3. Take aliquots for: Bioassay for Somatomedin a. radioactivity b. protein Several modifications of the original c. uronic acid hypophysectomized rat rib cartilage bioassay Figure 3. Schematic representation of the basic so­ for somatomedin are presently in use. These matomedin assay procedure. SOMATOMEDIN AND THE REGULATION OF SKELETAL GROWTH 4 4 3

^ S 04 Uptake By Embryonic Chick Cartilage assay has the greatest sensitivity but the least precision. The embryonic chick cartilage assay and the porcine rib cartilage assay have the greatest simplicity and precision, but are less sensitive.45 Van Wyk et al33 have recently developed a competitive placental membrane binding assay for somatomedin which has excellent precision and is extremely sensitive. The activity has been found to be low in patients with growth hormone deficiency, high in patients with acromegaly and also to vary with stature in patients with normal growth hormone secretion. Insulin and proinsulin compete with somatomedin for binding, but at high concentrations. Binding is not 1.25 2.5 5.0 10.0 affected by other growth factors studied. At the present time, purified somatomedin is Figure 4. Representative bioassay using a human very difficult to prepare. However, this assay reference serum in concentrations from 1.25 to 10 per­ or a radioimmunoassay appears to be the cent. assay of choice for future studies with this hormone. The cartilage is removed from its source Chemical Properties of Somatomedin and preincubated in an enriched tissue cul­ ture medium for 4 to 24 hours prior to the Somatomedin circulates in the plasma addition of serum or plasma containing so­ associated with a high molecular weight pro­ matomedin. Serum or plasma is then added tein of 50,000 daltons or greater.5 Cold acid- to the incubation medium. Incubation is ethanol will extract 20 to 40 percent of the allowed to proceed for 24 hours following somatomedin from plasma while precipi­ which 35S0 4 is added and the incubation tating 99 percent of the other plasma pro­ continued for an additional 24 hours. The teins.67 After acid-ethanol extraction, further concentration of serum or plasma used may purification is achieved using gel filtration vary from 1 percent to 20 percent of the in­ and carboxymethyl cellulose ion-exchange cubation medium depending upon the chromatography.69 The greatest resolution amount of somatomedin in the serum or has been obtained using isoelectric focusing. plasma. The cartilage is boiled for two When this procedure is utilized, soma­ minutes to stop the reaction, then washed tomedin activity is recovered in an acid, neu­ repeatedly before digesting and weighing. tral, and/or basic pH range.69 All have Following digestion, aliquots are taken for molecular weights of 6,000 to 11,000 counting of radioactivity, protein, uronic daltons.69 The neutral peptide has been acid or sulfate determinations. For each termed somatomedin A and has been found assay, a symmetrical 4 or 6 point design is to be most active in the chick embryo assay used with equal number of pelvic rudiments system.22 The acidic peptide, termed so­ for each concentration of the reference and matomedin B, has a molecular weight of ap­ test serum. Statistical analysis of validity, proximately 6,000 daltons, slightly less than regression and precision are carried out on somatomedin A. It stimulates thymidine in­ each assay to insure parallelism.19 corporation by human fibroblasts and glial- The hypophysectomized rat rib cartilage like cells.62 The basic peptide has been 4 4 4 ELD ERS ET AL. termed somatomedin C. 68 It is an arginine on the growth hormone sensitive tissues, rich peptide of about 50 amino acid residues such as cartilage, muscle, adipose tissue and and is very active in stimulating 35S0 4 and liver, are shown in table I. Perhaps the best 3H-thymidine uptake in hypophysectomized known biological effects of somatomedin are rat cartilage.70 The molecular weight is very the effects of somatomedin on the cartilage similar to somatomedin A. It has a mobility cell. It is now well accepted that all of the slightly greater than insulin by preparative known actions of growth hormone on car­ gel electrophoresis at pH 2.3 in a 15 percent tilage are attributable to somatomedin. So­ polyacrylamide gel.69 matomedin stimulates the incorporation of At present it is not known if these are 3H-thymidine into DNA11 and 3H-uridine different peptides or if they appear unique into RNA,50 increases the incorporation of because of different methods of isolation 14C-leucine into protein-polysaccharides and/or assay systems. They all, however, complexes,49 increases the incorporation of meet the criteria established for a somato­ 35S0 4 into chondromucoprotein,51 and pro­ medin, that is: (1) they are to some extent vides the conversion of 14C-proline under the control of growth hormone; (2) into hydroxyproline of collagen.9 These they have been shown to be insulin-like in biological effects were previously ascribed to their activity; (3) they stimulate cell growth growth hormone; however, it has now been in one or more tissues; and (4) they stimulate amply demonstrated that the growth hor­ 35S0 4 uptake in cartilage in at least one mone effects are mediated by somatomedin. species.68 Other growth factors have also Growth hormone in vitro is without effect on been isolated from various tissues. These in­ cartilage.48 clude insulin from the pancreas,40 non-sup- In the rat epididymal fat pad, soma­ pressible insulin like activity (NSILA) from tomedin stimulates glucose oxidation.24 In liver,27 multiplication stimulating activity isolated fat cells, somatomedin stimulates (MSA) from calf serum,37 nerve growth lipid synthesis and opposes the effect of factor and epidermal growth factor from epinephrine on stimulating lipolysis.61 It in­ mouse submandibular gland,40,57 and hibits the rise in adenylcyclase production erythropoietin from plasma and urine.28 by epinephrine in lymphocytes and fat cells. Somatomedins have only been partially It also inhibits the rise in adenylcyclase in purified from plasma and as yet the specific chondrocytes produced by parathormone.71 site of synthesis has not been determined. Somatomedin competes with insulin for Several tissues may be able to synthesize so­ binding to the plasma membrane receptors, matomedin but current evidence can only cell-free liver membranes and chondro­ suggest that the liver may be the primary site cytes.25 Only insulin and pro-insulin of synthesis.35 Although somatomedin has have previously been shown to compete for not been purified, it is stable in plasma. It the insulin receptors. Furthermore, so­ can be stored for long periods of time at 4° C matomedin can compete effectively with in­ and is unaffected by repeated freezing, sulin for binding to a common receptor on thawing or lyophilization.5 Plasma can be the cell membrane of adipose tissue acidified and heated to 100° C for one hour lymphocytes and placenta. However, in car­ without destroying the activity. However, tilage membranes, the two hormones do not somatomedin activity is destroyed by compete for the same primary receptor site. proteolytic enzymes. The cartilage cell is much more sensitive to somatomedin than to insulin. Biological Actions of Somatomedin In the rat diaphragm, somatomedin has A comparison of the in vitro effects of so­ been shown to have a direct effect on matomedin with those of growth hormone membrane transport.63 It stimulates amino SOMATOMEDIN AND THE REGULATION OF SKELETAL GROWTH 4 4 5

TABLE I

Biological Actions of Somatomedins and Growth Hormone on Selective Tissues In Vitro

Somatomedin Effect Growth Hormone Effect

Cartilage Stimulates growth Minimal to no effect on cartilage Increases 35S uptake into glycosaminoglycan Increases [JH]3« thymidine uptake into desoxyribonucleic acid Increases [^H] uridine uptake into ribonucleic acid Increases [*4C] proline incorporation into hydroxyproline of collagen Increases [lt+C] leucine uptake into protein Increases amino acid transport

Adipose tissue Stimulates glucose oxidation Stimulates all effects seen with Opposes effect of epinephrine on somatomedin but there is long lag stimulated lipolysis or is phase and action can be blocked by anti-lipolytic puromycin and theophylline Stimulates lipogenesis Lipolytic Competes with insulin for binding

Stimulates protein synthesis Increased somatomedin production Increased protein synthesis but weak and delayed

Rat diaphragm Direct effect on membrane transport Stimulates all effects seen with Stimulates amino acid transport, sugar somatomedin but there is long lag transport and protein systhesis phase and action can be blocked by Action is immediate and not blocked by puromycin and theophylline puromycin and theophylline

HeLa cells Stimulate growth in tissue culture Minimal effect

acid transport, sugar transport and protein Factors Influencing Somatomedin Activity systhesis. These effects are not blocked by either puromycin, an inhibitor of protein Plasma somatomedin activity has been synthesis, or theophylline, a phosphodi­ shown to be influenced by multiple factors, esterase inhibitor. It would appear, including age, nutritional status, constitu­ therefore, that the effect of somatomedin is tional factors and some as yet unidentified not mediated through either protein syn­ factors. These are shown in table II. Van den thesis or cyclic nucleotides. Significantly, Brande et al65 have shown that somatomedin both theophylline and puromycin block the activity is lowest in the very young and in­ action of growth hormone on the rat creases with age. The largest increase occurs diaphragm.63 In addition, the growth hor­ before the age of six years and reaches adult mone effect is delayed and requires rela­ levels about the time of puberty. No sex tively large doses of the hormone. difference was found either before or after In muscle, somatomedin stimulates pro­ puberty. Nutritional status, however, does tein synthesis, the action is immediate and appear to alter somatomedin activity. Severe requires only minimal amounts of the hor­ malnutrition or marasmus results in low so­ mone, whereas the action of growth hor­ matomedin levels.65 Salmon et al47 showed mone on muscle in vitro is very weak and de­ that prolonged fasting in the rat caused low layed. The same situation is true for the levels of somatomedin. The low levels ap­ effects of somatomedin on liver.24,64 pear to be due to the development of 4 4 6 ELDERS ET AL.

TABLE I I

Factors Which Influence Plasma Somatomedin Activity

Effect on Plasma Factors Studied Somatomedin Activity Growth Hormone Concentrations

Endocrine Growth hormone Excess High High Tall Deficiency Low Low Short Limited responders Low-normal Low-normal Short Thyroid hormone Excess Normal Normal Normal-tall Deficiency Normal-low Normal-low Normal Insulin Deficiency Normal -low Normal Normal Excess Normal Normal-low Normal-tall Sex hormones Androgens Normal Normal Tall Estrogens Decreased Normal Normal Environmental Nutritional status Fasting Low High Malnutrition Low Low Short Obesity Normal Normal-low Tall Emotional deprivation syndrome Low High Short Constitutional Stature Short Low Normal Short Tall High Normal Tall Dysmorphic dwarfism Low Normal Short Sex Normal Normal Age Increases Normal Genetic Laron's dwarfism Low High Very short Turner's syndrome High Normal Short Noonan's syndrome High Normal Short Cerebral gigantism Low Normal Tall Achondroplasia High Normal Short Drugs Estrogens Decreased ?Normal Glucocorticoids Decreased ?Normal Short Testosterone No effect Normal Other Free fatty acids Decreased Normal Uremia Decreased Normal

somatomedin inhibitors in the plasma. have low somatomedin levels. Children with Furthermore, children who are markedly emotional deprivation usually have very low underwright have very low levels of so­ levels of somatomedin but have high levels matomedin which may also be related to the of growth hormone.65 Conversely, children presence of inhibitors. In children with with constitutional dwarfism often have documented hypopituitarism, somatomedin normal to low levels of somatomedin but levels are decreased. In response to growth have normal levels of growth hormone.10 hormone administration, there is a rise in Normal plasma somatomedian levels are plasma somatomedin levels which precedes often seen in patients who have undergone any detectable increase in growth.65 surgery for a craniopharyngioma, in spite of Children who have a limited or poor low plasma growth hormone levels. These response to growth hormone stimulating patients also have very high insulin levels tests have been shown to be short and to and are usually obese.18 Likewise, somato­ SOMATOMEDIN AND THE REGULATION OF SKELETAL GROWTH 4 4 7 medin activity is usually normal in obesity recently demonstrated that patients with but growth hormone levels are frequently achondroplasia also have very high so­ low.53 Thus, the increased somatomedin matomedin activity. It is thought that this levels, relative to that growth hormone high somatomedin activity may be related to in both obese and post-operative cranio­ the cartilage hypertrophy seen in these pharyngioma patients, may be due to the patients. same, although unknown, mechanism. Patients with chronic renal disease and However, in both cases, insulin is markedly elevated blood urea nitrogen or nephrec- increased. Quite possibly these paradoxical tomized patients also have decreased so­ somatomedin levels may be related to the matomedin levels, suggesting the kidney high insujin levels. Laron’s dwarfism is may have a role in the synthesis of so­ associated with very low levels of so­ matomedin or, alternatively, elevations of matomedin.30 These patients are extremely the blood urea nitrogen may inhibit the dwarfed but have very high levels of growth effect of somatomedin at the cartilage hormone. Significantly, they do not respond level.45 to administered growth hormone by in­ Elevations of plasma free fatty acids have creasing their somatomedin activity.29 This been shown to be inhibitory to plasma so­ could be due to the absence of growth hor­ matomedin in vitro, suggesting a role for mone receptors in the target tissue, or al­ free fatty acids in the control of cartilage ternatively the inability of the target tissue metabolism.14 to synthesize somatomedin.17 Presumably, Thyroid hormone has been shown to have these patients could successfully be treated an influence on growth hormone. Patients with purified somatomedin. who are severely thyroid hormone deficient In contrast to the patient with Laron’s usually do not release growth hormone in dwarfism, patients with acromegaly have response to stimulation test. However, when very high levels of growth hormones and treated with thyroid hormone, they fre­ also have high levels of somatomedin.31 This quently respond. It is somewhat puzzling is due to overproduction of growth hor­ that the somatomedin activity is normal in mone, which in turn stimulates increased patients with thyroid hormone deficiency.65 production of somatomedin. Androgens have little effect on so­ Constitutional factors have also been matomedin activities. Estrogens in large shown to be associated with alterations in doses, however, have been shown to plasma somatomedin activity. Patients with decrease plasma somatomedin activity74 short stature usually have normal or minimal and, thereby, provide a rational basis for growth hormone as well as low somato­ therapy in some patients with acromegaly. medin activity. In contrast, tall stature is The chronic administration of adrenal glu­ associated with normal growth hormone and cocorticoids universally causes inhibition of a high somatomedin activity.65 growth. Possible mechanisms for this growth Genetic factors also influence somato­ retardation have included inhibition of pro­ medin activity. Patients with Turner’s syn­ tein synthesis, suppression of growth hor­ drome, XO chromosomal abnormality, mone release, inhibition of growth hormone usually have normal growth hormone levels action at the peripheral level and the sup­ and high somatomedin activity, yet they pression of somatomedin generation.4,38 have very short stature.2 This is also true of In figure 5 is shown a summary of the the patients with Noonan’s syndrome,15 sug­ proposed possible sites of cortisol action gesting a possible peripheral defect or a de­ which may inhibit long bone growth. The fect in responsiveness of the peripheral tissue hypothalamus which is responsible for re­ to somatomedin. Van den Brande et al65 leasing growth hormone releasing hormone 4 4 8 ELD ER S ET AL.

Possible Sites o f C ortisol Inhibition of this protein or because the acceptor pro­ tein is uniquely sensitive to glucocorticoids as compared to proteins in general. In­ hibition of synthesis of this acceptor protein would inhibit GAGS biosynthesis at the first step unique to the synthetic pathway causing a decrease in the extracellular matrix with resultant inhibition of long bone growth. In­ hibition of sulfation by cortisol, although un­ likely, is shown at Site 5.

References

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