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We examined the effects of maternal dietary protein restriction on fetal growth and expression of IGF-I and -II, and IGF-binding proteins (IGFBP). We sought to dissociate the respective effects of maternal protein versus calorie restriction on growth indices and IGF synthesis by the neonates of protein-restricted dams. Pregnant Wistar rats (six to eight per group) fed a low (5%) protein diet throughout gestation had impaired body weight gain compared with controls fed a normal (20%) protein diet (by 45%, p < 0.001). Their serum and liver IGF-I concentrations and liver IGF-I mRNA concentrations were also reduced by 60, 80, and 50%, respectively. Serum IGFBP-3 was reduced by 60% in protein-restricted dams within 1 to 2 h after delivery (p < 0.001 versus controls), although IGFBP-1, -2, and -4 were not significantly affected by the dietary protein intake. In pups of protein-restricted dams, the mean body and liver weight at birth was 15-20% less than that observed in the progeny from normal protein-fed dams (p < 0.01). Their plasma and liver IGF-I concentrations were 30 and 60% lower, respectively, whereas liver IGF-I mRNA abundance was reduced by 50% (p < 0.01). In contrast, neonatal plasma IGF-II and liver IGF-II mRNA concentrations were not significantly affected by the maternal protein malnutrition. Also, the plasma levels of IGFBP were not altered in the growth-retarded pups. Maternal protein restriction did not affect fetal and placental growth, plasma and liver IGF-I levels, and liver IGF-I mRNA abundance in 20-d-old fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutrition is one of the main regulators of circulating IGF-I. In humans, serum IGF-I concentrations are markedly lowered by energy and/or protein deprivation. Both energy and proteins are critical in the regulation of serum IGF-I concentrations. Indeed, after fasting, optimal intake of both energy and protein is necessary for the rapid restoration of circulating IGF-I. We believe, however, that in adult humans energy may be somewhat more important than protein in this regard. While the lowest protein intake is able to increase IGF-I in the presence of adequate energy, there is a threshold energy requirement below which optimal protein intake fails to raise IGF-I after fasting. When energy intake is severely reduced, the carbohydrate content of the diet is a major determinant of responsiveness of IGF-I to GH. The essential amino acid content of the diet is also critical for the optimal restoration of IGF-I after fasting, when protein intake is reduced. The exquisite sensitivity of circulating IGF-I to nutrients, the nycthemeral stability of its concentrations and its relative short half-life constitute the basis for its use as a marker of both nutritional status and adequacy of nutritional rehabilitation. For these indications, IGF-I measurement is more sensitive and more specific than measurement of the other nutrient-related serum proteins (albumin, prealbumin, transferrin, retinol-binding protein). Animal models have been developed to investigate the mechanisms responsible for the nutritional regulation of IGF-I. There is no doubt that many mechanisms are involved (Fig. 12). Decline of serum IGF-I in dietary restriction is independent of the diet-induced alterations in pituitary GH secretion. The role of the liver GH receptors is dependent on the severity of the nutritional insult. In severe dietary restriction (fasting), a marked decrease of the number of somatogenic receptors supports the role of a receptor defect in the decline of circulating IGF-I. In contrast, in less severe forms of dietary restriction (protein restriction), the decline of IGF-I results from a postreceptor defect in the GH action at the hepatic level. Nutritional deprivation decreases hepatic IGF-I production by diminishing IGF-I gene expression. Decline in IGF-I gene expression is mainly caused by nutrient deficiency and less importantly by the nutritionally induced hormonal changes (insulin and T3). Diet restriction also increases the clearance and degradation of serum IGF-I through changes in the levels of circulating IGFBPs.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum concentrations of insulin-like growth factor-I (IGF-I) in rats are reduced dramatically in the latter half of pregnancy, decreasing from 1758 +/- 356 ng/ml at 12 days of pregnancy (mean +/- SD) to 761 +/- 192 ng/ml at 15 days. After parturition, IGF-I increases to nonpregnant values in 4 days. Using ligand blotting, we have demonstrated that most of the serum IGF binding proteins (IGFBPs) are concurrently reduced during pregnancy. IGFBP-3, the predominant IGFBP in nonpregnant serum, is reduced to 1.3% of nonpregnant values by 21 days of pregnancy and begins to rise within 1 h postpartum (PP). The sera of 21-day pregnant (but not nonpregnant) rats degrade IGFBP-3 in vitro, and this degradation is prevented by the protease inhibitor antipain. Decreased serum IGF-I concentrations during pregnancy, therefore, may result from reduced IGFBP-3 concentrations causing increased IGF-I clearance. In addition, steady state IGF-I mRNA and peptide levels in liver are decreased in 21-day pregnant rats (37% and 42% of 4 day PP levels, respectively), suggesting that decreased synthesis of IGF-I may also lead to lower serum IGF-I concentrations. After bolus injection, [125I]IGF-I is cleared from the serum of pregnant rats nearly 5 times faster than that of 4 day PP rats (1.21 vs. 0.25 ml/min/kg, respectively). Urinary clearance is relatively insignificant (less than 4%), and [125I]IGF-I does not cross the placenta. The intermediate distribution phase of IGF-I is slower in pregnant rats than in PP rats (t1/2 alpha, 17.1 vs. 5.4 min), whereas the terminal elimination of IGF-I is twice as fast (t1/2 beta, 228.1 vs. 106.4 min). The prolonged IGF-I distribution phase in the pregnant rats may result from decreased concentrations of 34,000 and 30,000 mol wt IGFBPs, which may transport IGF-I to tissues. The faster serum elimination half-life may result from diminished IGFBP-3, leading to greater IGF-I availability to tissues in pregnancy.

Pregnant rats were fasted or allowed access to ad libitum feeds for the last 3 days of gestation to determine if the fetal growth retardation that results from maternal nutrient deprivation correlates with reductions in serum insulin-like growth factor-I (IGF-I) and IGF-II. In addition, IGF-I and IGF-II mRNA concentrations in liver and lung were measured by specific solution hybridization assays to determine if changes in steady state levels of mRNA correlate with changes in serum values. Fetal serum IGF-I concentrations were 30% lower in fasted than in control fetuses, although fasting did not significantly reduce the abundance of IGF-I mRNA in their livers or lungs. Serum IGF-I concentrations in the fasted dams were 34% lower than those in controls. IGF-I mRNA concentrations in the livers and lungs of the fasted dams were also lower than those in controls and correlated with serum IGF-I values (liver: r = 0.833; P less than 0.001; lung: r = 0.610; P less than 0.05). Therefore, whereas IGF-I appears to be transcriptionally regulated by fasting in dams, regulation of circulating IGF-I in fetuses may occur at a post-transcriptional step. Serum IGF-II and liver IGF-II mRNA concentrations were much higher than IGF-I levels in the fetuses and were not influenced by maternal fasting. Dam serum IGF-II concentrations were low compared to those in fetal serum and also were not reduced by fasting. We conclude that one mechanism by which maternal malnutrition causes intrauterine growth retardation is through decreased expression of IGF-I. On the other hand, short term nutrient restriction does not appear to be a regulator of IGF-II during either fetal or adult life.

Protein deprivation in young rats retards growth and decreases serum insulin-like growth factor-I (IGF-I) concentrations, neither of which is prevented by injections of GH once daily. Since four time daily injections of GH in hypophysectomized rats increase serum IGF-I concentrations more efficiently than single daily injections, we assessed whether this mode of GH delivery could overcome the GH resistance of protein malnutrition. Also, we evaluated whether continuous GH infusion could override this GH resistance. We fed 4-week-old female Wistar rats a low (5%) protein diet (P5) or a normal (15%) protein diet (P15) for 7 days. In a first experiment, rats fed a P5 diet received 40 or 400 micrograms/100 g BW.day rat GH (rGH) in four daily sc injections, while control P5 rats were injected at the same frequency with vehicle. In a second experiment, rats fed a P5 diet received 200 micrograms rGH/100 g BW.day by continuous infusion, while P5 sham-operated rats served as controls. IGF-I was measured by RIA on extracted serum, and free and total liver GH binding were determined by incubation of [125I]bovine GH with water- or MgCl2-treated homogenates, respectively. Neither continuous infusion nor repeated injections of rGH normalized the indices of growth or restored the serum IGF-I level to P15 control values. Injections of 400 micrograms rGH increased serum IGF-I 2-fold (P less than 0.01), but did not promote growth. Continuous GH infusion increased total and free liver GH binding to P15 control values, but had no effect on serum IGF-I. The discordance between liver GH binding and IGF-I confirms that a postreceptor defect is responsible for the GH resistance in protein restriction. These observations demonstrate that the consequences of protein restriction on growth are not overridden by intermittent or continuous administration of GH. The increase in IGF-I in response to 400 micrograms GH given intermittently in the absence of growth-promoting effects suggests that nutritional sufficiency is essential for IGF-I to promote growth.

The resistance to GH and the low serum concentrations of insulin-like growth factor-I (IGF-I) that occur during fasting are accompanied by decreased GH receptors in liver homogenates. In protein restriction, however, serum IGF-I but not GH receptors are decreased, suggesting that a post-receptor defect exists. Because conclusions about the status of GH receptors during dietary manipulation are based on studies using liver homogenates, the present study was undertaken to determine whether changes in GH binding by homogenates are paralleled by changes in receptors on the cell surface considered to mediate the GH signal. Collagenase-dispersed hepatocytes or liver homogenates from 7-week-old female Wistar rats fed various diets were evaluated for changes in somatogenic receptors. Fasting for 24 h reduced significantly (P less than 0.001) the plasma concentrations of IGF-I (-31%). Likewise, GH-binding sites were decreased on hepatocytes (-55%; P less than 0.01) and in liver homogenates (-60%; P less than 0.001) compared with controls, as was the velocity of initial binding (-77%; P less than 0.001). Protein restriction for 1 week decreased plasma concentrations of IGF-I (-42%; P less than 0.001) but GH-binding sites were not significantly reduced on hepatocytes or in homogenates. The velocity of initial binding was also not decreased. We conclude that observations on changes in homogenate binding of bovine GH during dietary manipulation provide a reliable means of assessing changes in cell-surface GH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated whether the low serum levels of somatomedin-C (SM-C) observed in protein malnutrition could be related to changes in liver growth hormone and prolactin binding. Growing female rats were fasted for 3 days and subsequently refed for 14 days with isocaloric diets containing 5% (low) or 25% (normal) protein. Control rats were fed a normal protein diet during the entire study. The numbers and affinity constants of somatogenic (GH) and lactogenic (PRL) binding sites were determined by analysis of saturation curves using liver homogenates incubated with 125I-labelled bovine growth hormone and 125I-labelled ovine prolactin. Serum SM-C and growth hormone concentrations were measured by radioimmunoassay. After fasting for 3 days body weight dropped by 21% (P less than 0.01 vs. controls) and serum SM-C by 53% (P less than 0.01), while GH and PRL binding capacities decreased respectively by 63% (P less than 0.01) and 62% (P less than 0.01). On refeeding with a normal protein diet, body weight, serum SM-C, GH and PRL binding capacities returned to control values. In contrast, with low protein intake, body weight, SM-C, GH and PRL binding capacities remained respectively 16%, 55%, 49% and 81% lower than controls (P less than 0.01). No significant changes in serum growth hormone concentrations occurred with fasting or refeeding.(ABSTRACT TRUNCATED AT 250 WORDS)

A heterologous radioimmunoassay for the measurement of somatomedin C in sheep serum has been developed using purified 125I-labelled human somatomedin C and an antiserum to human somatomedin C. It was observed that the GH dependence of somatomedin C could not be verified when unprocessed sheep sera were assayed, because of interference by somatomedin binding proteins. After incubation of samples at pH 3.8, however, concentrations of somatomedin C mirrored GH status. Assays results from sera after prolonged exposure to acid agreed with results from which had undergone acidification and gel chromatography. Using the methods developed in this study, 12 sera from normal sheep had a mean concentration of somatomedin C of 2.35 +/- 0.27 (S.E.M.) units/ml while nine sera from hypophysectomized sheep contained 0.45 +/- 0.04 units/ml (P less than 0.001), and one serum from a ewe with a prolonged GH excess associated with a pituitary tumour had a value of 6.9 units/ml. The radioimmunoassay resulting from these studies should provide a tool for investigation of the physiological control of somatomedin C in sheep.

Most of the somatomedin in serum exists as a high molecular weight complex (approximately equal to 140,000) composed of binding protein subunits and small molecular weight somatomedin. These binding proteins may interfere with measurements of the somatomedins by various radioligand assays. In a companion paper, we reported that when serum is incubated at neutral pH the detectability of the somatomedin-C (Sm-C) is increased. Using gel chromatography, however, it was not possible to demonstrate release of free Sm-C from the macromolecular complex. In the studies reported here, incubation of heparinized plasma at pH 7.4 followed by gel chromatography in a heparin-containing buffer caused 70-80% of the immunoreactive Sm-C to shift from the gamma-globulin region to a molecular weight which approximates that of free Sm-C. This conversion is a time-dependent process which is inhibited by the proteolytic enzyme inhibitor antipain. Similar changes in the elution profile of Sm-C were observed when heparinized plasma was acidified and chromatographed in heparin. These findings suggest that 1) at neutral pH, serum proteolytic enzymes reduce the affinity between small molecular weight Sm-C and its binding proteins. 2) At acid pH, a similar effect is observed. 3) In the presence of heparin, reassociation of these components does not occur. These results suggest a possible mechanism whereby the somatomedin macromolecular complex could be disrupted so that small molecular weight somatomedin is made available to tissues.

We have reported the presence of insulin-related poly A+ RNA sequences in human placenta by RNA to DNA hybridization. In this study we have used a monoclonal antibody to somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) to identify somatomedin-like proteins whose synthesis is directed by placental mRNA. Poly A+ RNA from first trimester and term placenta was translated in a cell-free system using micrococcal nuclease-treated reticulocyte-lysate and [35S]methionine as a label. From 2.0 X 10(6) cpm of specifically incorporated [35S]methionine labeled protein, an immunoprecipitate with an apparent molecular weight of 14,000 represented about 0.1% of total radioactivity in the translational products of poly A+ RNA of first trimester placenta. A less prominent band (0.006%) of the same apparent molecular weight was also evident from translational products of term placental mRNAs. This protein could be competed with either acromegalic serum or synthetic Sm-C/IGF-I when added prior to immunoprecipitation. Translational products synthesized from mRNA of term placenta showed a second labeled band of 24,000 daltons. This band was less effectively competed by acromegalic serum and not competed with either Sm-C/IGF-I or IGF-II and therefore its identity is uncertain. A protein similar to Sm-C/IGF-I is, therefore synthesized in first trimester placenta and to a lesser extent at term, suggesting developmental changes in Sm-C/IGF-I synthesis. Because Sm-C/IGF-I may act in a paracrine fashion, our findings suggest a role for Sm-C/IGF-I in growth of the placenta during early gestation.

Somatomedin-C/insulin-like growth factor I was purified from human plasma using a monoclonal antibody affinity column. Combining immunoaffinity chromatography with standard protein purification methods resulted in an overall recovery of 18%. The 35 micrograms of somatomedin-C/insulin-like growth factor I purified from 500 ml of plasma appeared as a single band when analyzed by polyacrylamide gel electrophoresis and could be used in radioimmunoassay and receptor binding studies.

Plasma somatomedin-C (Sm-C)/insulin-like growth factor I (IGF-I) concentrations have been shown to reflect changes in nitrogen balance induced by manipulation of nutrient intake. To assess the Sm-C/IGF-I response to refeeding a protein restricted diet in which the nitrogen source was supplemented with essential amino acids, six normal adults were fasted for five days, then refed for nine days diets consisting of 35 kcals/kg and 0.48 g protein/kg body weight. In one diet, 80% of the nitrogen was supplied as essential amino acids, and in the other, 80% was supplied as nonessential amino acids. Following the first fast/refeed cycle, a control diet was eaten for two weeks before the fast was repeated and the other test diet was ingested. The refeeding diets were given a random order. Plasma Sm-C/IGF-I fell from a pre-fast mean of 1.64 +/- 0.24 U/mL (mean +/- 1 SEM) to 0.67 +/- 0.18 (P less than 0.001) following fasting, and rose to 1.41 +/- 0.19 U/mL (P less than 0.001) following ingestion of the diet with supplemental essential amino acids. In contrast, when the same subjects were refed a diet in which 80% of the nitrogen was in the form of nonessential amino acids, the plasma Sm-C/IGF-I concentrations rose from 0.74 +/- 0.17 to 1.15 +/- 0.15 U/mL (P less than 0.01). This increase was significantly less than that observed after ingestion of the essential amino acid supplemented diet (0.74 +/- 0.10 U/mL v 0.40 +/- 0.11 U/mL; P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

We have assessed the effect of fasting for 10 days on plasma concentrations of immunoreactive somatomedin C and urinary urea excretion in seven obese male volunteers. From a mean prefast value of 0.83 U/ml, plasma somatomedin C fell to 0.21 U/ml after 10 days of fasting. A prompt increase was observed with refeeding. The change in somatomedin C during fasting showed a highly significant correlation with the change in urinary urea nitrogen excretion (r = 0.74; P less than 0.001). It also was shown that inhibitors which interfere with quantitation in somatomedin bioassays are not observed in the RIA for somatomedin C. The results of this study suggest that measurement of plasma somatomedin C provides a sensitive indicator of nitrogen loss and may be useful in monitoring the changes in protein metabolism that occur during alterations in nutritional status.

To determine if diet composition influences responses to GH, we fed 11 obese women diets containing 12 Cal/kg ideal BW (IBW) for 2 5-week study intervals. Nonprotein calories were supplied to 6 subjects as 72% carbohydrate (high carbohydrate diet), and 5 subjects received 80% of their nonprotein calories as lipid (high lipid diet). Protein intake was constant (1.0 g/kg IBW) in both groups. During 1 study interval we gave injections of GH (0.1 mg/kg IBW) every other day for 3 weeks (weeks 2-4), and in the other interval injections of vehicle were given. Subjects ingesting the high carbohydrate diet excreted significantly less urinary nitrogen [660.2 +/- 124.1 mmol/day (mean +/- SD)] than those who received high lipid (794.2 +/- 198.5 mmol/day; P less than 0.001), and GH injections reduced nitrogen excretion in the high carbohydrate subjects (532.8 +/- 123.8 mmol/day), but not in the high lipid subjects (743.7 +/- 126.6 mmol/day). The subjects receiving the high carbohydrate diet had a significant increase in serum insulin-like growth factor-I (IGF-I; from 36.2 +/- 9.7 to 55.9 +/- 6.6 nmol/L) and urinary C-peptide excretion (from 43.9 +/- 25.6 to 60.8 +/- 29.4 nmol/day) in response to GH. The IGF-I response attenuated slowly over the 3-week treatment interval. On the other hand, the high lipid group had lesser increases in IGF-I (from 31.0 +/- 6.5 to 41.7 +/- 8.8 nmol/L) and C-peptide excretion (from 24.3 +/- 28.9 to 29.8 +/- 32.8 nmol/day), and IGF-I concentrations declined to control values after only 5 days of GH injection. We believe that this initial IGF-I response was due to an antecedent 35-Cal balanced diet. The mean increment in serum FFA 4 h after GH injection was significantly less in subjects fed the high lipid diet (0.53 +/- 0.40 meq/L) than in those fed the high carbohydrate diet (0.83 +/- 0.43 meq/L). GH injections produced more body fat loss than injections of vehicle whether expressed as percent body fat lost (GH, 3.7 +/- 1.0%; vehicle, 2.8 +/- 0.7%, P less than 0.05) or as the fraction of weight lost as fat (fat loss/weight loss; GH, 0.81 +/- 0.13; vehicle, 0.64 +/- 0.08; P less than 0.005). There was an inverse correlation between the percentage of body fat lost and mean urinary C-peptide excretion during GH injections (r = -0.70; P less than 0.05), suggesting that stimulation of insulin secretion by GH might antagonize the tendency of GH to promote fat loss.(ABSTRACT TRUNCATED AT 400 WORDS)

Human growth hormone (hGH) is known to be a potent stimulator of somatomedin secretion in vivo. The induction of somatomedin by growth hormone has been difficult to study in vitro, however, because no organ containing a high concentration of somatomedin has been identified. Because fetal mouse explants have been shown to produce somatomedin in vitro, we have undertaken studies to determine whether postnatal human fibroblast monolayers also produce somatomedin, and if so, whether its production is regulated by other hormones. Quiescent human fibroblasts were exposed to serum-free minimum essential medium, and the medium was assayed for somatomedin concentration using a specific radioimmunoassay for somatomedin-C. A progressive rise in immunoreactive somatomedin to 0.08 U/ml per 10(5) cells per 24 h was observed over 72 h of incubation. This was an underestimation of the actual concentration of immunoreactive somatomedin since the amount measured following acid treatment was at least fourfold higher than in the untreated medium. Growth hormone stimulated immunoreactive somatomedin production in a dose-dependent manner: 5 ng hGH/ml = 0.1 U/ml per 10(5) cells; 50 ng hGH/ml = 0.25 U/ml per 10(5) cells. Platelet-derived growth factor and fibroblast growth factor were also stimulatory, but epidermal growth factor, thyroxine, or cortisol had no effect. Media that had been exposed to human fibroblasts stimulated DNA synthesis in BALB/c 3T3 fibroblasts (a cell type that does not produce somatomedin). Medium-derived immuno-reactive somatomedin eluted from Sephacryl S-200 in two major peaks (150,000 and 8,000 mol wt). The higher molecular weight peak is similar to the one observed when whole serum was used. These studies provide a model system for studying the humoral and nonhumoral factors that control the biosynthesis of somatomedin by human tissues. Since immunoreactive somatomedin production may be a rate-limiting factor for fibroblast growth, the delineation of the hormonal control of somatomedin production should lead to a better understanding of the mechanisms controlling human fibroblast growth.

In insulin-dependent diabetic rats, plasma somatomedin (Sm) levels are low and are not corrected by GH treatment, suggesting GH resistance. To define the mechanism of this GH-resistant state, the number and affinity constant of bovine liver GH-binding sites and the serum Sm-C responses to injections of bovine GH were determined in control (diluent-injected) and diabetic (streptozotocin-injected; 40 mg/kg BW) hypophysectomized rats. The affinity constants (Ka) of the GH-binding sites of control (0.92 +/- 0.07 X 10(9) M-1) and diabetic animals (0.68 +/- 0.04 X 10(9) M-1) were not significantly different (P less than 0.1). Likewise, there were no significant differences in the liver GH-binding capacities between control and diabetic hypophysectomized rats, whether these capacities were expressed as picomoles per liver (26.99 +/- 3.43 vs. 22.27 +/- 2.55, controls vs. diabetics), picomoles per mg DNA (1.26 +/- 0.15 vs. 1.10 +/- 0.12), or femtomoles per mg protein (30.95 +/- 4.08 vs. 29.98 +/- 2.70). Despite the absence of alterations in liver GH-binding sites, the Sm-C responses 24 h after sc injections of graded doses of bovine GH were severely blunted in the diabetic animals. The maximal serum Sm-C response in the controls was 0.81 +/- 0.12 U/ml, but was only 0.09 +/- 0.01 U/ml in the diabetics (P less than 0.01). The dose of GH required to achieve the half-maximal Sm-C response (ED50) was similar in diabetic and nondiabetic rats (700-900 micrograms). The absence of significant alterations in liver GH binding and the decreased maximal serum Sm-C response without changes in the ED50 suggest that the GH-resistant state in insulin-dependent diabetes is due to a postreceptor defect.

Ovine placental lactogen (oPL) and bovine growth hormone (bGH) increase the circulating concentration of somatomedin in hypophysectomized rats. This finding indicates that some of the somatotropic properties of oPL may be due to the induction of somatomedin and raises the possibility that oPL has a role in the control of fetal growth.

The insulin-like growth factors (IGFs) are small peptides that are present in serum and extracellular fluids and stimulate the growth of many cell types. In extracellular fluids the IGFs are bound to carrier proteins that are believed to modify the biological actions of the IGFs. At least three structurally distinct IGF-binding proteins (IGF-BPs) have been identified, and the serum concentrations of one of these has been shown to be regulated by pituitary GH. We report here that this GH responsive protein [39,000-45,000 mol wt (Mr)] can be induced (7-fold) by infusion of IGF-I in hypophysectomized rats or (3.5-fold) in protein-deprived rats, whereas two other forms of IGF-BP (e.g. 31,000-34,000 and 24,000 Mr) showed no change in the hypophysectomized animals and minimal increases in the protein-deprived animals. Likewise, GH injections in hypophysectomized animals resulted in a 7-fold increase in the 39,000-45,000 Mr form and no change in the 31,000-34,000 and 24,000 Mr forms. The protein-deprived animals showed a 3.2-fold increase in the 39,000-45,000 Mr and 2.4- to 1.8-fold increases in the 31,000-34,000 and 24,000 Mr forms, respectively. Changes in the larger Mr IGF-BP in these experimental models are paralleled by changes in serum IGF-I, suggesting that the GH dependence of the former protein is mediated at least partially via IGF-I. Our findings also suggest that the secretion of IGF-I and at least one IGF-BP may be linked, providing a mechanism by which their extracellular fluid concentrations are coordinated. Because IGF-BPs are present in extracellular fluids and can modulate IGF-I-receptor interaction, induction of this protein may be an important mediator of IGF action.

To determine the role of hypoinsulinemia and liver somatogenic (GH) receptors in growth retardation and decreased serum insulin-like growth factor I (IGF-I) levels during protein restriction, we have used a rat model where the effects of a low protein intake on body weight (BW), serum IGF-I concentration, and liver GH binding could be evaluated in the presence of low or high insulin concentrations. Two days after being made diabetic with streptozotocin (60 mg/kg BW), 6-week-old female rats (nine per group) were begun on a low (5%) or normal (15%) protein diet, without or with insulin supplementation (3 U lente daily). Nondiabetic rats fed both diets were used as controls (nine per group). In the nondiabetic animals, 7 days of protein restriction reduced BW gain by 50% (P less than 0.001), serum insulin by 44% (P less than 0.025), and serum IGF-I concentrations by 28% (P less than 0.001) without significantly changing liver GH binding. By day 9, BW was decreased in the diabetic animals by 12%, serum insulin by 80%, serum IGF-I by 55%, and liver GH binding by 62%; these effects were similar in the 5% and 15% protein-fed rats (P less than 0.001 vs. the corresponding controls). In the diabetes fed the normal diet, insulin treatment restored BW gain, serum IGF-I, and liver GH binding to normal values. In contrast, in the diabetics fed a protein-restricted diet and treated with insulin, BW gain and serum IGF-I concentrations remained low, similar to those in the malnourished controls. This diet-induced growth attenuation was observed despite high circulating insulin (2-3 times normal values), appropriate glucose control (63 +/- 9 mg/dl), and near restoration of liver GH binding. We conclude that while both protein restriction and diabetes attenuate growth and reduce IGF-I concentrations, the effects of protein restriction are independent of the effects of insulin and probably act by alteration of postreceptor mechanisms.

The predominant form of somatomedin-C (Sm-C) in human postnatal serum elutes from gel chromatographic columns at an approximate molecular weight of 150,000 daltons (150 K). Nine of 10 infants of 30 weeks gestation or more had elution profiles similar to postnatal sera, while in 7 of 9 infants of 27 weeks or less, the immunoreactive Sm-C eluted predominantly at an apparent molecular weight of 40,000 daltons (40 K). Five infants between 26-32 weeks exhibited a transitional pattern. One 43 week gestation anencephalic infant exhibited only 40 K Sm-C, suggesting that the 150 K proteins which bind Sm-C are acquired in response to growth hormone or other pituitary hormones. Even though the 40 K form of Sm-C is the only form found in mid-gestation fetal serum and in media from in vitro fetal mouse liver explants, it probably does not represent the primary gene product. This is suggested by the observation that 40 K Sm-C also binds 125-I-Sm-C and can be dissociated by acid and, therefore, probably is a complex of Sm-C non-covalently bound to other proteins.

Amniotic fluid was assayed by a radioreceptor assay utilizing 125I-somatomedin C and placental membranes. Growth hormone and prolactin levels were measured by radioimmunoassay at different gestational ages (8-41 weeks). Somatomedin receptor activity, growth hormone, and prolactin reached high levels during early gestation (8-26 weeks) displaying different patterns of appearance which reflect fetal serum levels of these hormones. After 26 weeks gestation all these hormones decreased in concentration. This decrease showed a strong correlation with fetal renal maturation as measured by amniotic fluid creatinine levels.

To determine if the pattern of GH delivery is important for the regulation of serum somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) and liver somatogenic receptors, we have measured serum Sm-C/IGF-I concentrations and free (H2O-treated homogenates) and total (MgCl2-treated homogenates) liver GH-binding sites in hypophysectomized rats treated for 7 days with rat GH (rGH), given either continuously by osmotic minipumps (50 and 250 micrograms/day) or intermittently (four sc injections of 12.5 micrograms/day). At a daily dose of 50 micrograms, intermittent rGH produced greater weight gain [+29.7 +/- 0.8 g (mean +/- SE)] than continuous GH infusion (23.3 +/- 2.0 g; P less than 0.01). Likewise, the serum Sm-C/IGF-I concentration rose more with intermittent (0.33 +/- 0.1 U/ml) than with continuous delivery (0.17 +/- 0.01 U/ml; P less than 0.01). The serum Sm-C/IGF-I level achieved with repeated GH injections was even greater than that after continuous delivery of a 5-fold higher GH dose (250 micrograms/day; 0.27 +/- 0.02 U/ml; P less than 0.05). Continuous infusions of 50 and 250 micrograms rGH/day increased the number of liver total GH receptors by 2.5-fold over that of controls. In contrast, frequent GH injections did not affect GH binding, and the serum Sm-C/IGF-I concentration did not correlate with liver GH-binding sites in the GH-injected rats (r = 0.189; P = NS). Induction of hepatic PRL receptors was 10-fold higher when GH was given continuously than when it was given intermittently. The close correlation observed between GH- and PRL-binding sites in all GH-treated rats (r = 0.955; P less than 0.001) suggests that their regulation may be linked. These data suggest that the regulatory mechanism controlling Sm-C/IGF-I production and growth might be different from those that regulate GH receptor concentrations, with GH pulses being crucial for the maximal stimulation of Sm-C/IGF and growth, but continuous exposure to GH being required for up-regulation of liver GH receptors.

The proportion of immunoreactive somatomedin-C (IR-Sm-C) in blood that is available for measurement in the RIA is influenced by whether the sample is processed as serum or plasma, by how promptly the sample is chilled and frozen, by whether the reaction is carried out in glass or polystyrene tubes and by whether the incubation mixture contains protamine or heparin. Although protamine buffers and polystyrene tubes increase the availability of purified somatomedin-C (Sm-C), they decrease the detectability of Sm-C in serum. By incubating serum at neutral or acid pH, this IR-Sm-C can be made available for measurement, suggesting that incubation alters the nature of the linkage between Sm-C and its binding proteins or causes a conformational change in the binding protein, resulting in greater exposure of IR-Sm-C. The increment in measurable IR-Sm-C that occurs at neutral pH appears to be due to the action of proteolytic enzymes since it is time, temperature, and pH dependent and is inhibited by a variety of protease inhibitors and chelating agents. The increment which occurs at acid pH is not inhibited by chelating agents or elevated temperature and must be due in part to acid hydrolysis of Sm-C and its binding proteins. However, since the acid-induced increment is optimal within a narrow pH range and falls off sharply below pH 3.5, acid proteases may also be involved. These observations, which provide insight into the nature of the serum proteins with which Sm-C is associated, bear on the interpretation of results of serum somatomedin measurements carried out with different methods. They also may aid in delineating the mechanisms by which the somatomedin contained in the macromolecular complex in serum is made available to tissues.

The somatomedins, a group of serum growth factors, have in common the ability to stimulate proteoglycan synthesis in cartilage. All are growth hormone dependent and have insulinomimetic properties in extraskeletal tissues. All stimulate DNA synthesis and mitosis in a variety of cell lines. Somatomedin-A and multiplication-stimulating activity are neutral peptides of 7,000--10,000 daltons, whereas nonsuppressible insulin-like activity and somatomedin-C are basic peptides of 5,700--7,000 daltons. Whether some of these substances are identical remains to be established. By use of a highly specific radioimmunoassay for somatomedin-C and less specific radioreceptor assays for insulin-like activity and somatomedin-C, evidence has been obtained for the existence of two major classes of somatomedins: neutral somatomedin, which is more insulin-like, and basic somatomedin, which is more rigorously growth hormone dependent, and which, in some systems, is s more powerful stimulant of growth.

We have determined if dietary protein restriction for 1 wk has differential effects on growth, serum IGF-I, and liver growth hormone receptors at various stages of development. Female Wistar rats were fed a low (5%) protein diet for 7 d at 3, 4, 6, 8, and 12 wk of age, whereas controls were maintained on a normal (15%) protein diet. Body wt gain was impaired in the groups fed the low protein diet, despite normal energy intake, and the effect was attenuated with age. Liver cell number (DNA content) was reduced by low protein feeding in the 3-, 4-, and 6-wk age groups (p less than 0.01), but not in the older animals. Protein restriction caused a dramatic decrease in serum IGF-I in the younger animals (90 and 82% reduction versus normal fed age-matched controls, at 3 and 4 wk, respectively; p less than 0.001), and this effect was progressively attenuated with increasing age (49, 40, and 25% reductions of serum IGF-I at 6, 8, and 12 wk, respectively). Changes in serum IGF-I correlated with those of liver cell number (r = 0.80; p less than 0.001). Total and free liver growth hormone receptors were slightly decreased in the low protein diet groups at 4 (p less than 0.05) and 6 wk (total: p less than 0.001; free: p less than 0.01) but not in the other age groups. The occurrence of profound diet induced reductions in IGF-I without proportional reductions in liver GH receptors suggest that the apparent GH resistance occurs at a postreceptor level.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatomedin-C (SM-C) and insulin-like growth factor I (IGF-I) produced identical curves of competition in a RIA for SM-C, and in placental cell membrane receptor assays for SM-C and insulin. Somatomedin-A (SM-A), insulin-like growth factor II (IGF-II) and two forms of multiplication stimulating activity (MSA) were less than 5% as potent as SM-C/IGF-I in the RIA and less than 50% that of SM-C/IGFi in the SM-C receptor assay. In the insulin receptor assay, IGF-II and MSA III-2 were much more potent than SM-C/IGF-I. The present data suggest that SM-C and IGF-I are functionally identical substances.

The somatomedins comprise a family of insulin-like peptide growth factors, whose concentrations in postnatal life are growth hormone-dependent, that stimulate sulfate uptake by cartilage and that stimulate the proliferation of a variety of types of cultured cells. Several lines of evidence now support the concept that the somatomedins stimulate fetal growth: 1) they act as mitogens for a number of embryonic and fetal derived cells grown in culture; 2) specific plasma membrane receptors for somatomedins exist in many fetal tissues; 3) multiple fetal tissues appear to be capable of somatomedin synthesis; 4) in some studies blood somatomedin concentrations correlate with birth size. It is proposed that the somatomedins might act at or near their sites of synthesis, and that under the influence of placental lactogen, as well as other factors, they have a significant stimulatory effect on both organ-specific and generalized somatic fetal growth. A hypothesis that integrates the possible growth-promoting actions of both classic hormones and growth factors in the fetus is presented.

To determine the mechanism(s) for the growth retardation associated with malnutrition early in life, the relationships among plasma somatomedin-C (SM-C), plasma GH, and hepatic bovine GH-binding sites were assessed in rat pups that were milk-deprived from birth until weaning (21 days). After this period of malnutrition, body weight, tail length, plasma SM-C, and liver GH-binding capacity of the malnourished animals were significantly (P less than 0.001) reduced below those of control, well fed rats [SM-C at 21 days, 0.06 +/- 0.01 vs. 0.30 +/- 0.04 U/ml (mean +/- SE); GH binding, 2.96 +/- 0.39 vs. 7.19 +/- 0.80 pmol/liver]. The number of GH-binding sites per mg DNA was also reduced (0.59 +/- 0.06 vs. 1.08 +/- 0.11 pmol/mg DNA; P less than 0.001). After 1 week of ad libitum refeeding (days 21-28), body weight and tail length of malnourished pups increased significantly but showed no signs of catching up with that of control pups. Plasma SM-C and liver GH-binding capacity in the malnourished rats also rose significantly (P less than 0.005) after refeeding, but remained as far below controls as at 21 days [SM-C on day 28, 0.19 +/- 0.02 vs. 0.53 +/- 0.02 U/ml (P less than 0.001); GH binding, 6.59 +/- 0.99 vs. 12.94 +/- 1.60 pmol/liver (P less than 0.005)]. After 7 weeks of refeeding (days 21-70), tail length but not body weight recovered, while plasma SM-C levels were normalized. The mean number of GH binding sites per mg DNA in the malnourished rats was not significantly different from that in controls and reached for the males and the females, respectively, 79% and 86% of control binding. When expressed per liver, GH binding followed a similar pattern; for the males, binding capacity returned to 73% of the control value (20.45 +/- 1.82 vs. 27.93 +/- 3.49 pmol/liver; P = NS), and for the females, it returned to 77% of the control value (36.42 +/- 1.76 vs. 47.42 +/- 4.13 pmol/liver; P less than 0.01). In the young rats up to day 28, liver GH-binding capacities correlated with plasma SM-C concentrations (r = 0.81; P less than 0.01), while in the adult rats no correlation was present. During malnutrition and refeeding, there were no changes in the affinity constants of the hepatic GH-binding sites or in plasma GH concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

We have attempted to determine the relative importance of dietary intake of protein and energy in restoring plasma immunoreactive somatomedin-C (Sm-C) concentrations after fasting. Ten healthy human volunteers were fasted for 5 days, then divided into two refeeding groups. One group (variable energy) was refed 1.0 g protein/kg ideal body weight, and in 9-day sequences, 11, 18 and 25 kcal of energy/kg. The other group (variable protein) was given 35 kcal energy/kg, and in 9-day sequences, 0.2, 0.4, and 1.0 g protein/kg. When subjects were refed the variable energy diets there was no significant increase in Sm-C at 11 kcal/kg (0.47 +/- 0.13 to 0.45 +/- 0.12 U/ml), suggesting that there is a threshold energy requirement below which optimal protein intake is not sufficient to raise the Sm-C. When subjects were refed 18 and 25 kcal/kg, it became apparent that the more energy added to the diet, the greater the absolute concentration of Sm-C attained (0.66 U/ml on 18 kcal/kg and 0.97 U/ml on 25 kcal/kg). Sm-C correlated with nitrogen balance (r = 0.58) during refeeding with the variable energy diets and was a good indicator of acute directional change in nitrogen balance. However, Sm-C was not a reliable indicator of nitrogen repletion, since it rose almost to control levels on the 25 kcal/kg diet while nitrogen balance remained slightly negative.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of a radioimmunoassay for somatomedin-C has for the first time made it possible to discriminate between serum concentrations of a single peptide or closely related group of peptides and the net somatomedin activity measured by less specific bioassay and radioreceptor techniques. Antibodies to human somatomedin-C were raised in rabbits using a somatomedin-C ovalbumin complex as the antigen. A variety of peptide hormones at concentrations up to 1 muM are not recognized by the antibody. Insulin at concentrations >0.1 muM cross reacts in a non-parallel fashion; purified somatomedin-A is only 3% as active as somatomedin-C; and radiolabeled cloned rat liver multiplication stimulating activity does not bind to the antibody. Immunoreactive somatomedin-C can also be quantitated in the sera of a variety of subhuman species. Unusual assay kinetics, which are manifest when reactants are incubated under classic "equilibrium" assay conditions, appear to result from the failure of (125)I-somatomedin-C to readily equilibrate with the somatomedin-C serum binding protein complex. It is, therefore, necessary to use nonequilibrium assay conditions to quantitate somatomedin-C in serum. With this assay it is possible to detect somatomedin-C in normal subjects using as little as 0.25 mul of unextracted serum. Serum somatomedin-C concentrations in normal subjects were lowest in cord blood and rose rapidly during the first 4 yr of life to near adult levels. In 23 normal adult volunteers, the mean serum somatomedin-C concentration was 1.50+/-0.10 U/ml (SEM) when compared to a pooled adult serum standard. 19 children with hypopituitary dwarfism had concentrations below 0.20 U/ml. 17 of these were below 0.1 U/ml, the lower limit of sensitivity of the assay. The mean concentration in 14 adults with active acromegaly was 6.28+/-0.37 U/ml (SEM), five times greater than the normal volunteers. Significant increases in serum somatomedin-C concentrations were observed in 8 of 10 hypopituitary children within 72 h after the parenteral administration of human growth hormone. Three patients with Cushing's disease had elevated serum somatomedin-C concentrations (2.61+/-0.14 U/ml [SEM]). Three patients with hyperprolactinemia had normal concentrations (1.74+/-0.11 U/ml [SEM]).The important new discovery brought to light by quantitation of immunoassayable somatomedin in patient sera is that all previously used assays detect, in addition to somatomedin-C, serum substances that are not under as stringent growth hormone control.

Evidence is growing that the somatomedins act by a paracrine and/or autocrine mechanism. The importance of these mechanisms relative to the traditional endocrine actions is not clear, and it is possible that these growth factors act through all three mechanisms. Supporting the possible paracrine/autocrine mechanisms are reports that production of somatomedins or somatomedin-like peptides is widespread throughout the body. Additionally, the somatomedins have biological actions on remarkably diverse cell types, and these responsive cells are found in close proximity to cells known to produce somatomedin. Finally, factors that alter the growth rate of cultured cells produce parallel changes in somatomedin secretion, suggesting that these phenomena are closely linked.

Recent studies suggest a role for insulin-like growth factor I (IGF-I) in the regulation of hormone release from placental, gonadal, and pituitary tissues. To examine whether IGF-I may also regulate the release of PRL from human decidual tissue, we have investigated the effect of recombinant human IGF-I on PRL release from monolayer cultures of human decidual cells exposed to IGF-I for up to 4 days. IGF-I (10-1000 ng/ml) stimulated a sustained dose-dependent increase in PRL release (half-maximal concentration, 25 ng/ml) beginning 48 h after initial exposure, but had no effect on the intracellular PRL content. The amounts of PRL released from maximally stimulated cultures on days 3 and 4 were 168 +/- 3% (mean +/- SEM) and 258 +/- 8% of control values, respectively. IGF-I-mediated effects were inhibited by cycloheximide (3.6 microM), suggesting that the increase in PRL was the result of newly synthesized hormone. The increase in PRL release was not due to a generalized effect on protein release, since IGF-I had no effect on the release of trichloroacetic acid-precipitable [35S]methionyl proteins. Radioligand competition studies indicate that the biological actions of IGF-I are mediated through interaction with the IGF-I receptor. Binding of radiolabeled IGF-I to decidual cells in suspension was specific, saturable, and displacable by unlabeled IGF-I, with a potency nearly 10 times greater than that of insulin. Furthermore, exposure of decidual cells to a monoclonal antibody to the IGF-I receptor (alpha-IR3) completely inhibited both IGF-I-mediated PRL release and specific binding of [125I]IGF-I to decidual cells. Since the actions of IGF-I occurred at physiological concentrations, these findings strongly support a role for IGF-I in the regulation of PRL secretion by human decidua.

In protein-calorie malnutrition, serum IGF-I concentrations are low despite high GH. This GH resistance might be due to a reduced number of liver GH binding sites as suggested by studies performed in fasted rats that were refed a low protein diet. To determine whether a postreceptor defect in GH action might also contribute to the GH resistance, we measured the number and the affinity constant of the liver GH binding sites and the serum IGF-I responses to injections of recombinant bGH in hypophysectomized female rats, fed a standard (15% protein) diet (N = 25) or a low (5%) protein diet (N = 25) for 8 days. There were no significant differences in the liver GH binding capacities between the 15% and the 5% protein-fed rats, whether expressed as pmol per liver (20.6 +/- 3.5 vs 14.4 +/- 1.3; mean +/- SEM; P less than 0.2; N = 5, respectively), pmol per mg DNA (1.08 +/- 0.16 vs 0.84 +/- 0.07; P less than 0.4) or fmol per mg of protein (28.98 +/- 5.04 vs 30.26 +/- 2.00; P greater than 0.5). Likewise, the affinity constants of the GH binding sites of the 15% and the 5% protein-fed rats were not significantly different (0.78 +/- 0.05 vs 0.78 +/- 0.07 x 10(9) l/mol; P greater than 0.5). Despite these non-significant reductions in liver GH binding sites, the IGF-I responses 24 h after sc injections of increasing doses of bovine GH were blunted in the rats fed the 5% protein diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Because inadequate intake of energy or protein causes reduction in the plasma concentrations of somatomedin-C/insulinlike growth factor I (Sm-C/IGF-I), we have completed studies to determine whether the energy deficit induced by vigorous exercise sustained for 1 week would also be associated with a reduction in this peptide. When six healthy, exercise-conditioned males were fed a diet consisting of 35 kcal/kg ideal body weight (IBW) and 1.0 g protein/kg and exercised so that 14.1 to 16.3 kcal/kg of energy was expended each day, their plasma Sm-C/IGF-I concentrations declined from a mean basal value of 1.15 +/- 0.78 U/mL (1 +/- SD) to 0.62 +/- 0.41 U/mL during the last two days of a seven-day exercise period (P less than .05). This decrease in Sm-C/IFG-I occurred almost entirely in the first four days of the study period. After 3 weeks for reacclimation, the calorie intake of each volunteer was reduced by the same number of calories as had been expended in the form of exercise during each day of the exercise week (mean of 15.1 kcal/kg IBW/d). On this regimen the mean plasma Sm-C/IGF-I declined from 1.12 +/- 0.57 U/mL, to 0.69 +/- 0.37 U/mL on the last two days of dietary restriction. The magnitude of fall with dietary restriction was not significantly different from that observed during exercise. During the last three days of the exercise period the mean daily nitrogen balance was -1.60 +/- 1.70 g/d, while this value was -3.50 +/- 1.73 g/d during the last three days of dietary restriction (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Somatomedins/insulin-like growth factors (Sm/IGFs) are present in blood and in extracts from multiple tissues of the human fetus and induce the proliferation of cultured human fetal cells. To identify the cellular location of immunoreactive Sm/IGF in human fetal tissues, we have performed immunocytochemistry in tissues from prostaglandin-induced human fetal abortuses of 12 to 20 wk in gestation. Every tissue studied except the cerebral cortex contains Sm/IGF immunoreactive cells. Cells staining positively include hepatocytes, hepatic hemopoietic cells, columnar epithelia of the pulmonary airways, intestine and kidney tubules, adrenal cortical cells, dermal cells, skeletal and cardiac muscle fibers, and pancreatic islet and acinar cells. Immunostaining was specific for Sm/IGFs, but because of the cross-reactivity of the antibodies it was not possible to determine whether the immunoreactivity represented Sm-C/IGF I, IGF II, or both. Liver contained the greatest proportion of immunoreactive cells, while the thymus and spleen had only a few immunostained cells. With the possible exception of dermal and some adrenal cortical cells, the immunoreactive cells do not appear to be the primary sites of Sm/IGF synthesis, because parallel in situ hybridization histochemical studies using Sm/IGF oligodeoxyribonucleotide probes show that Sm/IGF mRNAs are localized predominantly to fibroblasts and mesenchymal cells. Therefore the immunoreactive cells identified in this study may define sites of action of Sm/IGFs.

The effect of purified somatomedin-C (Sm-C)/insulin-like growth factor I on the uptake of alpha-aminoisobutyric acid (AIB) by confluent cultures of human fibroblasts was studied. An increase in [3H]AIB uptake was observed within 30 min of Sm-C addition, and a maximal effect was reached at 2.5 h (averaging 200% of control AIB uptake). Under the conditions employed, less Sm-C was required for maximal stimulation of [3H]AIB uptake (10 ng/ml in most cell lines) than for a maximal effect on [3H]thymidine incorporation (greater than 30 ng/ml). In multiple experiments with different lines of foreskin-derived and nongenital fibroblasts, the concentration of Sm-C resulting in half-maximal stimulation of [3H]AIB uptake was reproducible and was between 1.7 and 4.8 ng/ml in all cell lines tested except one. No significant difference was observed in the Sm responsiveness of cells from newborns and that of those from normal older children. Determination of binding of [125I]Sm-C to confluent monolayers of one fibroblast line revealed that the concentration of Sm-C resulting in half-maximal binding was nearly identical to that producing half-maximal [3H]AIB uptake stimulation. Stimulation of AIB uptake by an optimal concentration of epidermal growth factor (5 ng/ml) was also determined in several normal cell lines and was consistently close to 140% of the control value. Since epidermal growth factor and Sm interact with different receptors, this response may be a useful measure of cell integrity which is independent of the Sm receptor. The techniques for measurement of AIB uptake described in this report may prove useful in determining whether some children with growth failure of unknown etiology have target cell resistance to Sms.