Recombinant human insulin-like growth factor: testing the somatomedin hypothesis in hypophysectomized rats

The in-vivo biological activity of recombinant methionyl insulin-like growth factor I (met-IGF-I) was demonstrated in hypophysectomized rats by following blood glucose after an i.v. bolus injection of met-IGF-I; a dose-dependent decrease in blood sugar was seen. Membrane transport was studied using the non-metabolizable amino acid alpha-aminoisobutyric acid; stimulation was obtained with the highest dose used (90 micrograms/rat). To test the original somatomedin hypothesis, growth studies were performed in hypophysectomized rats. Two or three doses of met-IGF-I were given with three different administration regimes (i.v. or s.c. infusion, or s.c. injections twice daily) for 6 or 8 days. Little growth-promoting activity was observed, with a significant effect on body weight gain obtained only when met-IGF-I was given continuously at the highest dose used (180 micrograms/day). No effect was seen on the in-vivo uptake of radioactive sulphate into cartilage. Epiphyseal cartilage width increased slightly at the highest dose of met-IGF-I, but only when the hormone was given by infusion. When 180 micrograms met-IGF-I/day were given by injections, a significant effect on longitudinal bone growth was obtained (90 micron above control). The levels of IGF in the serum were not measurably increased after s.c. administration of met-IGF-I, whereas after i.v. infusion, significantly raised levels were obtained at the higher dose rates (3.0 +/- 0.3 and 2.8 +/- 0.1 units/ml). Growth hormone was much more effective than met-IGF-I even at 50-fold lower doses. Priming the animals with 10 mu. bovine GH/day followed by combined infusions of GH and met-IGF-I did not reveal any potentiating effects of met-IGF-I in the presence of GH. We conclude that met-IGF-I is a relatively poor growth-promoting agent when given systemically, and that somatomedins are more likely to act as local growth factors rather than as circulating mediators of the growth-promoting effects of GH.     

Recombinant human insulin-like growth factor I stimulates growth and has distinct effects on organ size in hypophysectomized rats.

Recombinant human insulin-like growth factor I (rhIGF-I) was infused subcutaneously into hypophysectomized rats for as long as 18 days. Three hundred micrograms (39 nmol) of rhIGF-I per day and 200 milliunits (4.5 nmol) of human growth hormone (hGH) per day increased body weight, tibial epiphyseal width, longitudinal bone growth, and trabecular bone formation similarly. Weight gains of the kidneys and spleen, however, were greater with rhIGF-I than with hGH, whereas the weight of the epididymal fat pads was reduced with rhIGF-I. The weight of the thymus was increased by rhIGF-I treatment. Thus, IGF-I administered over a prolonged period of time mimics GH effects in hypophysectomized rats. Quantitative differences between rhIGF-I and hGH treatment with respect to organ weights may be related to different forms of circulating IGF-I or may be due to independent effects of GH and IGF-I. The results support the somatomedin hypothesis, but they also stress the role of GH as a modulator of IGF-I action.     

Evidence suggesting that the direct growth-promoting effect of growth hormone on cartilage in vivo is mediated by local production of somatomedin.

We have used a catheterization system that permits chronic infusion into the arterial supply of one hindlimb of rats to study the direct effects of rat growth hormone and human somatomedin C on growth of the tibial epiphyseal cartilage plate in hypophysectomized rats. Rat growth hormone (0.4 microgram per day) or human somatomedin C (0.25, 1, or 4 micrograms per day) stimulated growth of the epiphyseal plate of the infused limb but not of that of the contralateral noninfused limb. The somatomedin C had a dose-related effect. Rabbit antiserum to human somatomedin C, but not normal rabbit serum, completely abolished the direct growth effect of the rat growth hormone when it was co-infused with the hormone. These results support the concept that growth hormone stimulates long bone growth by inducing local production of somatomedin, which in turn stimulates cell proliferation in an autocrine or paracrine fashion. However, they do not exclude the possibility that serum somatomedin may also play a role.     

A re-assessment of the effect of thyrotrophin (TSH) on the tibial plate bioassay for growth hormone (GH)

The claim has been made that thyrotrophin (TSH) can augment the action of growth hormone (GH) to stimulate growth of the epiphysial cartilage plate of the hypophysectomized rat's tibia. The TSH induces its effect via secretion of thyroid hormones which in turn enhance the stimulatory action of GH. If this is true then the employment of the tibia test, whose endpoint is the increase in thickness of the epiphysial cartilage plate in response to GH present either in crude pituitary extracts or relatively purified preparations, which also are likely to contain modest or appreciable quantities of TSH, requires further examination. The present study utilized various fractions of crude pituitary extracts from intact and thyroidectomized rats that respectively contained appreciable quantities of GH or essentially no GH. Fractional aliquots of pituitary extracts from thyroidectomized rats were administered concomitantly with graded doses of exogenous GH to hypophysectomized rats to determine the point at which TSH in the extracts was sufficiently able to stimulate significant tibial plate growth when compared to recipients given GH alone. Purified GH and TSH were also administered in various doses to hypophysectomized recipients in a further attempt to delineate the dose range at which TSH augments the action of GH to promote significant chondrogenesis of the epiphysial plate. The results indicate that the enhancement of the GH effect on the cartilage plate by TSH was evident only when quantities above 100 microng bovine GH were co-administered with 100 mU bovine TSH. As little as 40 mU TSH augmented the growth effect of 400 microng GH on the cartilage plate, demonstrating that smaller quantities of TSH could potentiate larger quantities of GH. These data, therefore, suggest that extracts equivalent to not more than one-half of a normal adult rat's anterior pituitary gland should be administered to hypophysectomized rats for bioassay of GH. Fractions of glands greater than this may contain sufficient amounts of TSH to augment the appreciable quantities of GH already present.     

Hypophysectomy and growth hormone receptors in liver membranes of male rats

The effects of hypophysectomy on GH binding were studied in liver membranes of male rats. Ten days after surgery, the specific binding of [125I]iodobovine GH and of [125I] iodohuman GH was 2- to 3-fold higher in microsomal membranes of hypophysectomized rats than in membranes of control male animals. The number of receptors rather than the affinity of the binding was affected. A nonspecific membrane effect due to hypophysectomy is unlikely since membrane markers such as 5' nucleotidase, galactosyl transferase, and insulin binding were not different in liver membranes of hypophysectomized and control rats. The somatogenic specificity and the subcellular distribution of the binding sites were not altered by hypophysectomy; the number of the GH binding sites were increased in plasma membranes as well as in Golgi fractions. Hypophysectomy in male rats creates a situation where growth failure, absence of circulating GH, and lack of plasma somatomedin activity are associated with increased concentration of liver somatogenic receptors. The latter finding could explain why livers of hypophysectomized rats are more sensitive to GH than those of normal rats.     

Effect of somatomedin and growth hormone on bone collagen synthesis in vitro

Although pituitary hormones, particularly growth hormone (GH), are known to influence skeletal growth, there is no evidence for a direct effect of GH or GH-dependent factors (somatomedins) on bone as opposed to cartilage. We have examined the effects of GH, a somatomedin (Sm) preparation, and serum from intact and hypophysectomized rats on bone collagen synthesis in cultures of 21-day fetal rat calvaria. Collagen synthesis and non-collagen protein synthesis were measured by the incorporation of ³H-proline into collagenase-digestible (CDP) and noncollagen protein (NCP). Bovine and rat GH caused a small inhibition in the incorporation of labeled proline into CDP which was not dose related. Sm in doses of 18–540 mU/ml increased the incorporation of proline into CDP up to three-fold after 24 hr in culture. Sm also had a smaller and more variable stimulatory effect on the labeling of NCP. The effects of Sm were maximal after 3 hr of treatment and were maintained for 96 hr. Sm (60 mU/ml) and insulin (10^?8 M) had effects of similar magnitude and were not additive. The addition of 10% serum from hypophysectomized rats stimulated the labeling of both CDP and NCP, but serum from rats with intact pituitaries had a greater effect. Treatment of hypophysectomized rats with thyroxine, corticosterone, and GH. did not increase the bone collagen synthesis stimulating activity of the serum, although GH treatment did increase serum Sm activity by a pig cartilage assay. The results indicate that GH dose not stimulate bone collagen synthesis directly. However, they suggest that the pituitary gland either releases or stimulates the production of factors which stimulate bone collagen synthesis. Sm may be such a factor, but sulfation activity and bone collagen synthesis stimulating activity may be dissociable.     

Regulation of cartilage acid hydrolases by growth hormone

The in vivo regulation of three acid hydrolases, namely cathepsin D, cathepsin B, and acid phosphatase, by GH was investigated. The costal cartilage cathepsin D and acid phosphatase activities of hypophysectomized rats were reduced relative to those found in normal controls. Treatment of hypophysectomized animals with GH enhanced rat growth rate and increased these two enzyme activities toward normal levels. Results of pepstatin experiments suggested that the elevated cartilage cathepsin D activity corresponded to an increase in enzyme concentration. A degree of specificity in this regulation was apparent because cartilage cathepsin B, unlike cathepsin D and acid phosphatase, was refractory to hypophysectomy and GH treatment. In contradistinction to cartilage, none of these hepatic enzymes responded to GH, and only cathepsin B activity was diminished by hypophysectomy. Centrifugational and detergent studies indicated that changes in enzyme activities induced by GH treatment were not due to the differential release of acid hydrolases from subcellular compartments. Overall, our results suggest that costal cartilage cathepsin D and acid phosphatase activities are GH dependent and may be related to cartilage growth. These observations may provide insight into the mechanism of GH action and, derivatively, skeletal growth.     

Local injections of human or rat growth hormone or of purified human somatomedin-C stimulate unilateral tibial epiphyseal growth in hypophysectomized rats

The growth-promoting actions of GH are thought to be mediated by somatomedin(s) (Sms), but unilateral bone growth in hypophysectomized (HX) rats given local injections of human (h) GH has been reported. Using slightly different methods, we have confirmed these results with purified hGH and have extended them to show that four daily injections directly into the tibial epiphyseal plate of 1 or 5 micrograms/day purified rat GH or recombinant DNA-derived hGH, but not of hPRL (6 micrograms/day) caused significant cartilage growth compared to that of the vehicle-injected contralateral tibia in rats that had been HX 14 days before the first injection. Thus, it is unlikely that the effects of GH are due to contaminating growth factors in the GH preparations, because the bacteria-derived preparation of hGH, which is unlikely to have such contaminants, was also active. Furthermore, we have shown that similar injections of 100 or 500 ng/day purified hSm-C caused unilateral tibial growth in rats HX 8 days, but not 14 days, before the first injection. These results demonstrate that both GH and Sm-C have direct growth-promoting effects on cartilage in vivo and are compatible with the theory that GH may act by stimulating local SM synthesis.     

The importance of growth hormone and liothyronine in induced hypocalcaemia in hypophysectomized animals.

The resistance to hypocalcaemia induced by synthetic salmon calcitonin (SCT) was studied in intact and hypophysectomized rats. The recovery from hypocalcaemia was greatly impaired in the latter group, but this was normalised by small doses of purified human growth hormone (GH). The initial hypocalcaemic response to SCT was delayed in the hypophysectomized rats. This was not affected by treatment with GH, but normalised by substitution with liothyronine. We conclude that GH accelerates the recovery from hypocalcaemia in hypophysectomized animals, perhaps by a stimulation of parathyroid activity, while thyroid hormone accelerates the initial hypocalcaemic response to SCT, probably by restoring the reduced rate of bone resorption.     

Circulating growth factor studies in growth plate versus resting cartilage in vitro. II. Recognition of growth factors in rat serum

The growth-promoting effects of GH can be explained in part by the mediation of somatomedins/insulin-like growth factors (IGFs). However, large quantities of the IGFs are required to stimulate growth in vivo, and in some conditions, IGF levels may correlate poorly with GH levels and growth status. These observations suggest that other circulating factors may also be important for growth. We have examined the growth-promoting activity in rat serum, as assessed by stimulation of sulfate and/or thymidine uptake by resting and growth plate cartilage (osteochondral junction) from hypophysectomized rats in vitro. Although stimulation by a low molecular weight somatomedin fraction (approximately 5,000-12,000) accounted for about 90% of serum stimulation of sulfate uptake by resting cartilage, it explained only about 60% of stimulation of the growth plate. Growth plate and resting cartilage appeared equally insensitive to insulin, but the growth plate exhibited reduced sensitivity to inhibitor(s) in diabetic rat serum. Fractionation of normal rat serum by gel filtration at neutral pH revealed comparable stimulation of growth plate and resting cartilage by high molecular weight factors, presumably somatomedins bound to carrier proteins. After gel filtration at acid pH, both growth plate and resting cartilage responded to somatomedins with molecular weights from 5,000-12,000. However, the growth plate also responded to a 12,000-22,000 mol wt factor [Sephadex G-75; 5 X 120 cm; sulfate uptake, 68 +/- 16% above buffer (mean +/- SEM); P less than 0.01] which did not affect resting cartilage (sulfate uptake, 27 +/- 21% above buffer; P = NS). Levels of both the low and higher molecular weight factors were reduced in the serum of hypophysectomized rats. We conclude that circulating growth-promoting activity includes both the low molecular weight somatomedins and a higher molecular weight growth plate growth factor which is not recognized by resting cartilage. Use of the osteochondral junction assay system may permit elucidation of the regulation and nature of this growth factor.     

Somatomedin and analogues of cyclic AMP increase the number of cells synthesizing DNA in cartilage from hypophysectomized rats

The effects of somatomedin and certain nucleotides on nuclear labelling of cartilage cells with [3H]thymidine were determined by autoradiography. Segments of costal cartilage from hypophysectomized rats were incubated for 24 h in a basal medium with or without additions and then pulsed for 2 h with [3H]thymidine in the basal medium. Both somatomedin (0.1 U/ml) and Bt2cAMP (10(-4)M) increased the number of labelled nuclei, and the combined effects were more than additive. A parallelism between the effects of these agents on nuclear labelling and their effects on total thymidine incorporation into DNA was demonstrated. The 8-bromated derivative of cAMP (10(-4)M) also enhanced chondrocyte nuclear labelling, but neither 8-Br-5'-AMP (10(-4)7) nor 8-Br-cGMP (10(-4)M) exhibited actions of the cAMP analogues. It is concluded that in cartilage obtained from hypophysectomized rats and incubated under the specified conditions (1) both somatomedin and cAMP analogues increase the number of cells synthesizing DNA as well as total thymidine incorporation into DNA, (2) the effects of the hormone and cyclic nucleotide in combination are synergistic, and (3) the increased incorporation of labelled thymidine into DNA reflects increased DNA synthesis and not merely an alteration of the specific activity of the intracellular thymidine nucleotide pool.     

Discordance of serum and tissue somatomedin levels in growth hormone-stimulated growth in the rat

Although the somatomedins are believed to mediate GH-induced somatic growth, circulating levels of insulin-like growth factor I (IGF-I) do not always correlate with growth rate. To evaluate a paracrine or autocrine effect that might explain this discordance, the concentrations of plasma and tissue IGF-I were compared with indices of growth in hypophysectomized rats treated with rat GH (rGH). The rats received a total of 250 micrograms rGH by either continuous pump infusion (P) or twice daily injections (I). After 4 days of treatment, the amounts of IGF-I present in acetic acid extracts of liver and kidney and in native serum were determined by RIA and related to proximal tibial epiphyseal plate width. Tibial epiphyseal plate width increased from 198 +/- 6 microns (mean +/- SE) in untreated controls to 339 +/- 13 microns in group P and 347 +/- 9 micron in group I; weight gain was 11.6 +/- 1.0 g in group P and 9.6 +/- 0.8 g in group I, while control animals lost 1.0 g. Serum concentrations of IGF-I were no different between control animals and those in the injection group (0.91 +/- 0.06 vs. 0.90 +/- 0.06 U/ml), whereas levels in the infusion group increased slightly (1.1 +/- 0.05 U/ml). In contrast, rGH administration caused tissue IGF-I to double in liver (control, 0.24 +/- 0.04 U/g; P, 0.51 +/- 0.03 U/g; I, 0.46 +/- 0.05 U/g) and nearly triple in kidney (control, 0.52 +/- 0.05 U/g; P, 1.51 +/- 0.09 U/g; I, 1.36 +/- 0.08 U/g). There was no detectable change in somatomedin-binding protein by gel exclusion chromatography. Since the rGH administered was sufficient to stimulate growth and increase tissue somatomedin levels without corresponding increases in circulating IGF-I, an autocrine or paracrine action of IGF-I appears to mediate GH's initial somatogenic actions in the young rat.     

Human somatomedin A and longitudinal bone growth in the hypophysectomized rat

A somatomedin A preparation, when given at total doses of 14 and 70 U did not increase the longitudinal bone growth in hypophysectomized rats. Growth hormone (WHO) significantly increased the longitudinal bone growth.     

Nutrition and somatomedin. XIV. Altered levels of somatomedins and somatomedin inhibitors in rats with streptozotocin-induced diabetes

Diabetes is associated with poor growth despite elevated levels of growth hormone (GH). Skeletal GH effects are mediated by somatomedins; in diabetes, somatomedins measured by radioassay are normal, yet somatomedin activity measured by bioassay is low. Since bioassay measurements reflect the presence of both somatomedins and somatomedin inhibitors, we asked if diabetes might be associated with discordant regulation of these circulating factors. Graded severity of diabetes was induced in rats by injection of streptozotocin at 37, 73, 146, and 293 mg/kg. After two days, metabolic derangement varied from normal serum beta-hydroxybutyrate with slight increase in glucose and minimal weight loss at 37 mg/kg streptozotocin to beta-hydroxybutyrate 10.6 mmol/L, glucose 447 mg/dL, and 33 g weight loss at 293 mg/kg streptozotocin. After fractionation of serum on Sephacryl S-300 pH 7.0, somatomedins and somatomedin inhibitors were measured by rat cartilage bioassay. Somatomedins (Kav 0.25 to 0.50) were comparable to control levels despite beta-hydroxybutyrate 2 mmol/L, glucose 534 mg/dL, and weight loss 11 g at 73 mg/kg streptozotocin and fell only at higher streptozotocin dosage. In contrast, somatomedin inhibitors (Kav 0.62 to 0.88) began to rise at 37 mg/kg streptozotocin and increased with higher dosage. Levels of somatomedins were correlated weakly only with beta-hydroxybutyrate (r = 0.48, P less than 0.05), while somatomedin inhibitors were correlated significantly with all indices, particularly beta-hydroxybutyrate (r = 0.78, P less than 0.0001). The early rise in somatomedin inhibitors but late fall in somatomedins could explain low somatomedin activity (and poor growth) despite normal levels of somatomedins measured by radioassay; measurement of somatomedin inhibitors may provide an index of growth potential in diabetes mellitus.     

The effects of insulin and growth hormone on the release of somatomedin by the isolated rat liver.

Livers from hypophysectomized (hypox) rats were perfused with oxygenated Waymouth's medium in a system which permitted continuous recirculation for separate 30 minute periods after which fresh medium was supplied. In most experiments 6 changes of medium were carried out over a 3 hour period. The somatomedin activity of each perfusate was determined by measuring its ability to stimulate sulfate uptake in hypox rat cartilage in vitro. For comparison between experiments the results are expressed as the per cent stimulation of sulfate uptake by the perfusate compared with the unperfused buffer. Without hormonal additions there was a progressive fall in the release of somatomedin activity during the 6 periods of study. When compared with the results without hormone, the addition of 1000 muU/ml of insulin per ml of medium during the 2nd to 6th period led to a significant increase in perfusate somatomedin activity at all periods. The addition of 100 muU/ml of insulin was without significant effect. The possible inter-relationship between insulin and growth hormone in the regulation of somatomedin release was studied with a dose of bGH of 250 ng/ml which had previously been shown to be insufficient by itsel to stimulate somatomedin release. When added to a medium containing 1000 muU/ml of insulin, this dose of bGH did not significantly stimulate somatomedin release beyond that obtained with insulin alone. However, when 250 ng/ml was added to a medium containing 100 muU/ml insulin, a significant stimulation of somatomedin release was observed while the addition of each hormone separately was without significant effect. These results support the hypothesis that insulin shares with GH the regulation of somatomedin release by the liver. Differences in insulin concentration may explain some clinical situations in which somatomedin concentrations cannot be correlated with GH levels.     

Expression of GH receptor mRNA in regenerating skeletal muscle of normal and hypophysectomized rats. An in situ hybridization study.

Expression of growth hormone receptor mRNA was investigated by in situ hybridization in skeletal muscle from normal and hypophysectomized rats during the first seven days of regeneration after ischemic injury. A digoxigenin-labelled RNA probe directed against the extracellular part of the rat GH receptor was used. In both normal and hypophysectomized rats distinct expression of GH receptor mRNA could be demonstrated in the regenerating muscle cells at the myoblast/myotube stage. The GH receptor expression appeared to decline with increasing maturation of the regenerated muscle fibres. In hypophysectomized rats, the regeneration process and the expression of GH receptor mRNA was delayed compared with that in normal animals. It is concluded that growth hormone may affect also the early phase of muscle regeneration in normal animals. To what extent lack of growth hormone contributes to the delayed regeneration observed in the hypophysectomized rats remains to be elucidated.     

Nutrition and somatomedin. XV. Growth plate, growth factor and biologically active somatomedins in rats with streptozotocin-induced diabetes

Diabetic children may exhibit poor growth, yet levels of growth hormone and somatomedins measured by specific radioligand assays usually are normal. In the present studies, biological assays based on costal cartilage from hypophysectomized rats were used to test the possibility that diabetes is associated with decreases in circulating growth-related factors. 'Growth plate growth factor' activity was evaluated with tissue from the osteochondral junction (which resembles epiphyseal cartilage), and 'somatomedin' activity was measured with resting cartilage distant from the growth plate. Diabetes was induced in rats by administration of streptozotocin (STZ) at 40, 80, 160 and 310 mg/kg. Two days later, animals receiving STZ 40 mg/kg exhibited slight hyperglycemia but normal beta-hydroxybutyrate and weight gain; glucose and beta-hydroxybutyrate rose, and weight fell progressively with higher dosage. Gel filtration on Sephadex G-75 at pH 2.4 was used to separate rat serum into somatomedins (KAv 0.50-0.75) and growth plate growth factor (KAv 0.38-0.50). Somatomedins were 102, 92, 83 and 68% of control in STZ-treated animals, i.e. unchanged from control at 40 mg/kg STZ and falling only with higher dosage. In contrast, the growth plate growth factor declined at all doses of STZ to 93, 84, 69 and 57% of control. Thus, the growth plate growth factor began to fall with mild hyperglycemia alone (glucose 190 mg/dl), while a comparable fall in somatomedins was not seen until glucose was greater than 400 mg/dl and beta-hydroxybutyrate was three times normal. Only the growth plate growth factor was correlated with changes in body weight (r = 0.44, p less than 0.025). We conclude that decreases in levels of a circulating growth plate growth factor may contribute to growth impairment in diabetes. Measurements of this factor may be useful in examining underlying mechanisms.     

Recombinant DNA-derived human insulin-like growth factor II (hIGF-II) stimulates growth in hypophysectomized rats

Sustained subcutaneous administration of recombinant DNA-derived insulin-like growth factor II to immature female hypophysectomized rats stimulated significant increases in body weight gain, tibial epiphyseal cartilage width, femur hydroxyproline concentrations and a significant decrease in serum urea nitrogen concentrations. Recombinant DNA-derived human growth hormone (Humatrope), administered in the same manner produced similar biological effects. The data support the contention that hIGF-II has anabolic effects when administered to hypophysectomized rats and may be a locally acting mediator of pituitary hormone actions.     

Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats

We have studied potential mechanisms by which zinc deficiency (ZD) may result in growth impairment in young animals. Dietary-induced ZD in young rats resulted in diminished skeletal growth as measured by tibial epiphyseal width. Treatment with bovine GH (bGH) did not increase skeletal growth suggesting GH resistance rather than GH deficiency in zinc-deficient rats. Serum levels of basic somatomedin (SM) were lower in zinc-deficient rats than in control rats receiving a zinc adequate diet, either ad libitum or in pair matched amounts, and were restored to normal by zinc repletion but not by bGH treatment, suggesting that SM production is impaired by ZD. There was a high correlation between tibial epiphyseal widths and serum or femur zinc concentrations. These findings, along with observations that despite similar levels of serum basic SM the bGH-treated zinc-deficient rats had smaller tibial epiphyseal widths than pair fed control rats, additionally suggest that the action of SM on skeletal growth is impaired by ZD.     

Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action.

We have validated a method for extracting and measuring the tissue content of somatomedin C (Sm-C)/insulin-like growth factor I (IGF-I), a growth-hormone-dependent, growth-promoting peptide. The Sm-C content of tissue extracts was strongly growth-hormone dependent because most of the tissues studied from hypophysectomized rats contained significantly less Sm-C than normal tissues. The intraperitoneal administration of ovine growth hormone (oGH) to hypophysectomized rats caused tissue extractable Sm-C to increase in kidney, liver, lung, heart, and testes. Tissue Sm-C responses to oGH were maximal after 12 hr, 6 hr before the maximal increment in serum. In liver and lung, the tissue Sm-C response to various doses of oGH fit linear regression models, and the doses of oGH needed to increase the Sm-C are in the range of those required to increase protein synthesis. Although these results do not exclude the possibility that the somatomedins act by hormone-like endocrine mechanisms, they add support to the concept that these peptides act through autocrine or paracrine mechanisms, being produced at multiple sites and acting at or near their sites of production.