Louis E. Underwood

Dietary components that regulate serum somatomedin-C concentrations in humans.

Dietary components responsible for the regulation of somatomedin-C in humans were assessed in five adult volunteers of normal weight who were fasted for 5 d on three occasions, then refed three diets of differing composition. The serum somatomedin-C decreased from a mean prefasting value of 1.85 +/- 0.39 U/ml (+/- 1 SD) to 0.67 +/- 0.16 U/ml at the end of fasting (P less than 0.005). After refeeding for 5 d with a normal diet, the mean serum somatomedin-C increased to 1.26 +/- 0.20 U/ml. A protein-deficient (32% of control), isocaloric diet resulted in a significantly smaller increase, to a mean value of 0.90 +/- 0.24 U/ml (P less than 0.05). A diet deficient in both protein and energy led to a further fall 0.31 +/- 0.06 U/ml. The changes in somatomedin-C during fasting and refeeding correlated significantly with mean daily nitrogen balance (r = 0.90). We conclude that both protein and energy intake are regulators of serum somatomedin-C concentrations in adult humans, and energy intake may be of greater importance. The correlation between changes in somatomedin-C and nitrogen balance suggests that the former are directly related to changes in protein synthesis and may be helpful in assessing the response to nutritional therapy.

Evidence that somatomedin is synthesized by multiple tissues in the fetus.

Abstract Production of somatomedin-C, a growth hormone-dependent peptide believed to mediate the growth-promoting actions of growth hormone, has been assessed using explants of fetal mouse tissues. Quantitation of this peptide in media of explants cultured for 3 days has been accomplished with a membrane receptor assay for somatomedin and a specific radioimmunoassay for somatomedin-C. Somatomedin-C is produced by the 11-day-gestation fetal mouse liver, increases exponentially in parallel with liver growth until the 16th day of gestation, and falls postnatally. Media somatomedin is believed to be derived by de novo synthesis since saline extracts of liver and most other fetal tissues contain only a small fraction of the activity in culture media. The immunoreactive material secreted into media appears to be closely related to human somatomedin-C since it produces dilution curves which are parallel to those of pure hormone, migrates on Sephacryl 200 at a size similar to that of one of the components of human serum somatomedin-C, dissociates into small molecular weight material with acid treatment, and isofocuses in a range comparable with that of somatomedin-C purified from human serum. Eleven-day limb bud mesenchymal micromass cultures and 17-day-gestation intestine, heart, brain, kidney, and lung also synthesize immunoreactive somatomedin-C in serum-free medium. For these tissues, the media activity was far in excess of the tissue extractable activity. Somatomedin activity in excess of the tissue extractable activity, however, was not found in media from 17-day-gestation placenta. The finding that multiple tissues synthesize somatomedin-C raises the possibility that the primary biological actions of this hormone are exerted locally at its sites of origin. Although a function of this type by a peptide has not been widely suspected, it seems plausible that the cells of fetal tissues are capable of producing local mitogens in much the same manner as the postulated inducers of tissue differentiation.

Enhancement of the anabolic effects of growth hormone and insulin-like growth factor I by use of both agents simultaneously.

The use of growth hormone (GH) as an anabolic agent is limited by its tendency to cause hyperglycemia and by its inability to reverse nitrogen wasting in some catabolic conditions. In a previous study comparing the anabolic actions of GH and IGF-I (insulin-like growth factor I), we observed that intravenous infusions of IGF-I (12 micrograms/kg ideal body wt [IBW]/h) attenuated nitrogen wasting to a degree comparable to GH given subcutaneously at a standard dose of 0.05 mg/kg IBW per d. IGF-I, however, had a tendency to cause hypoglycemia. In the present study, we treated seven calorically restricted (20 kcal/kg IBW per d) normal volunteers with a combination of GH and IGF-I (using the same doses as in the previous study) and compared its effects on anabolism and carbohydrate metabolism to treatment with IGF-I alone. The GH/IGF-I combination caused significantly greater nitrogen retention (262 +/- 43 mmol/d, mean +/- SD) compared to IGF-I alone (108 +/- 29 mmol/d; P < 0.001). GH/IGF-I treatment resulted in substantial urinary potassium conservation (34 +/- 3 mmol/d, mean +/- SE; P < 0.001), suggesting that most protein accretion occurred in muscle and connective tissue. GH attenuated the hypoglycemia induced by IGF-I as indicated by fewer hypoglycemic episodes and higher capillary blood glucose concentrations on GH/IGF-I (4.3 +/- 1.0 mmol/liter, mean +/- SD) compared to IGF-I alone (3.8 +/- 0.8 mmol/liter; P < 0.001). IGF-I caused a marked decline in C-peptide (1,165 +/- 341 pmol/liter; mean +/- SD) compared to the GH/IGF-I combination (2,280 +/- 612 pmol/liter; P < 0.001), suggesting maintenance of normal carbohydrate metabolism with the latter regimen. GH/IGF-I produced higher serum IGF-I concentrations (1,854 +/- 708 micrograms/liter; mean +/- SD) compared to IGF-I only treatment (1,092 +/- 503 micrograms/liter; P < 0.001). This observation was associated with increased concentrations of IGF binding protein 3 and acid-labile subunit on GH/IGF-I treatment and decreased concentrations on IGF-I alone. These results suggest that the combination of GH and IGF-I treatment is substantially more anabolic than either IGF-I or GH alone. GH/IGF-I treatment also attenuates the hypoglycemia caused by IGF-I alone. GH/IGF-I treatment could have important applications in diseases associated with catabolism.

Hormonal Control of Immunoreactive Somatomedin Production by Cultured Human Fibroblasts

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.

Nutritional regulation of insulin-like growth factor-I

Several lines of evidence indicate that in the human, insulin-like growth factor-I (IGF-I) is nutritionally regulated. Both energy and protein availability are required for maintenance of IGF-I. Measurements of serum IGF-I constitute a sensitive means for monitoring the response of acutely ill patients to nutritional intervention. Serum IGF-I may also serve as a marker for evaluation of nutritional status. Our findings and those of others in animal models suggest that nutrients influence synthesis and action of IGF-I and its binding proteins (IGFBPs) at multiple levels. In fasting, liver growth hormone (GH) binding is decreased, providing one explanation for decreased IGF-I. In protein restriction, GH receptors are maintained, but there is evidence for a postreceptor defects. The latter results from pretranslational and translational defects. Amino acid availability to the hepatocytes is essential for IGF-I gene expression. Protein malnutrition not only decreases IGF-I production rate, but also enhances its serum clearance and degradation. Finally, there is evidence for selective organ resistance to the growth-promoting effects of IGF-I in protein-restricted rats.

Identification of somatomedin/insulin-like growth factor immunoreactive cells in the human fetus.

ABSTRACT: 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.

3 Paracrine functions of somatomedins

Summary 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.

Clinical Uses of Insulin-like Growth Factor I

Dr. Carolyn Bondy (Developmental Endocrinology Branch, National Institute of Child Health and Human Development): Insulin-like growth factor I (IGF-I; somatomedin-C) is an anabolic polypeptide that is structurally homologous to insulin [1]. Its actions are mediated primarily by the IGF-I receptor, which is structurally and functionally homologous to the insulin receptor. The ligand-binding domains of these receptors are sufficiently different that each binds its cognate hormone with about ten times more affinity than does the related ligand [2]. The signal-transducing, tyrosine kinase domains of the two receptors, however, are very similar [2] and activate common intracellular pathways [3]. Thus, it appears that the difference in physiologic effects of insulin and IGF-I are not due primarily to intrinsic differences in signaling capacities of their receptors [4]. Furthermore, with a few notable exceptions, both receptors are widely expressed, with some tissues apparently expressing hybrid receptors that combine insulin and IGF-I receptor subunits [5, 6]. Because insulin and IGF-I are subject to very different regulatory influences and have markedly different patterns of secretion and circulating profiles, hormone bioavailability is probably an important factor in determining the different roles served by IGF-I and insulin. Recombinant human IGF-I recently became available for clinical studies, allowing, for the first time, direct investigation of the metabolic and anabolic effects of IGF-I and its relations with insulin and growth hormone. Our view of the regulatory relations among IGF-I, growth hormone, and insulin is outlined in Figure 1. Growth hormone and insulin stimulate the constitutive secretion of IGF-I from the liver [7] and IGF-I, in turn, suppresses growth hormone and insulin secretion, even under euglycemic conditions [8-11]. In contrast to the highly regulated secretory patterns and fluctuating serum profiles of growth hormone and insulin, circulating IGF-I levels are relatively stable. This stability is due to its constitutive pattern of secretion and to the fact that most circulating IGF-I is bound to high-affinity IGF-binding proteins, which prolong the half-life and titrate the supply of this hormone to its receptors [12, 13]. Six IGF binding proteins have been identified, but clinical data are most abundant for IGF-binding protein-3. This IGF-binding protein binds IGF-I and another component, the acid-labile subunit, and forms a high molecular weight ternary complex, which constitutes the primary reservoir of circulating IGF-I. Circulating levels of this complex are positively regulated by growth hormone. Insulin-like growth factor-binding protein-1 binds a smaller fraction of the total circulating IGF-I, but this fraction may be disproportionately influential in terms of the effects of IGF-I on intermediary metabolism, because IGF-binding protein-1 levels are potently suppressed by insulin. Figure 1. Relations between insulin-like growth factor I (IGF-I) and IGF-binding proteins, growth hormone (GH), and insulin. Originally, the somatomedin hypothesis [1] suggested that circulating IGF-I mediates most of the effects of growth hormone on linear growth. Recently, however, growth hormone was found to stimulate the local production of IGF-I in several tissues in addition to the liver in rodents [1], and thus local autocrine or paracrine effects of IGF-I appeared to be important for normal growth. There is, however, little evidence for growth hormone-stimulated IGF-I synthesis in human tissues other than the liver, and the apparent success of systemic IGF-I treatment in producing linear growth in growth hormone-resistant children, discussed in the following section by Dr. Underwood, suggests that neither local IGF-I production nor direct anabolic effects of growth hormone are essential for statural growth in children. Local autocrine/paracrine growth processes in humans might be regulated by another member of the insulin family of peptides. Insulin-like growth factor II is structurally closely related to IGF-I [1] and binds the IGF-I receptor with high affinity, but unlike IGF-I it is not regulated by growth hormone. In rodents, IGF-II expression is abundant during embryonic development but is largely suppressed after birth. In humans, however, IGF-II levels are equal to or greater than IGF-I in the circulation and in many tissues during adulthood [1, 14-16]. Growth hormone and IGF-I have continuing roles in fuel metabolism and in the maintenance of musculoskeletal mass in adults. Many of the changes in body composition, such as increasing adiposity and decreasing muscle mass, that occur during aging correlate specifically with decreasing levels of these hormones [17]. Several clinical situations exist in which the anabolic or metabolic effects of growth hormone, IGF-I, or both may prove to have substantial therapeutic benefit. Starvation, cachexia, hyperalimentation, and insulin-dependent diabetes mellitus are all associated with a state of functional growth hormone resistance in which, despite normal or high growth hormone levels, circulating IGF-I levels are low and do not respond to growth hormone treatment. A common factor in these conditions is under-insulinization of the liver, which impairs normal IGF-I and IGF-binding protein synthesis. Recent clinical trials evaluated the short-term metabolic effects of IGF-I administration in calorically deprived adult volunteers, as described by Dr. Clemmons, and in insulin-dependent diabetic patients, as described by Dr. Bach. Another area in which IGF-I may have important therapeutic benefit is the hyperglycemic disorders characterized by insulin resistance. In the short term, recombinant IGF-I reduces blood glucose and triglyceride levels in obese patients with noninsulin-dependent diabetes mellitus [11]. These salutary effects have been attributed to improved insulin sensitivity due to suppression of growth hormone and insulin secretion by IGF-I and to the direct, insulin-like metabolic effects of IGF-I. A few studies have reported that recombinant IGF-I treatment improves the hyperglycemia of patients with extreme insulin resistance caused by genetic defects in the insulin receptor, thus suggesting that IGF-I may act through its own receptor to regulate blood glucose [18-21]. Not all insulin-resistant patients respond well to IGF-I treatment, however, as reported by Drs. Guler and Skarulis in a following section. Insulin-like Growth Factor I in Growth Hormone-Resistant Short Stature Dr. Louis Underwood (Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina): We are treating two kinds of patients with short stature secondary to growth hormone insensitivity: patients with Laron-type dwarfism, now called the Laron syndrome [22], and growth hormone-deficient patients in whom large amounts of growth-attenuating antibodies have developed after treatment with growth hormone. Normally, growth hormone binds to the growth hormone receptor to induce hepatic IGF-I production, which in turn stimulates growth and feeds back at the level of the pituitary and hypothalamus to suppress growth hormone secretion (Figure 2). Patients with the Laron syndrome lack functional growth hormone receptors and thus do not respond to growth hormone; their IGF-I levels are very low, growth is slow, and circulating growth hormone levels are high because of decreased feedback suppression of growth hormone by IGF-I (Figure 2). Patients with a deletion of the growth hormone gene may recognize growth hormone as a foreign protein, and large numbers of antibodies may develop that attenuate or obliterate their response to it. Figure 2. Diagram of the growth hormone (GH) and insulin-like growth factor I (IGF-I) growth axis in healthy persons (left), those with the Laron syndrome (middle), and those with a deletion of the gene-encoding growth hormone (right). We studied a boy with the Laron syndrome [23] who was very short (111 cm at 9 years) and had the physical appearance of a person with growth hormone deficiency. Basal serum levels of growth hormone were elevated (10 to 12 g/L) and increased to 40 to 60 g/L after pharmacologic stimulus. His serum IGF-I level was low (5 to 6 g/L; normal for age, 100 g/L), and he had no increase in serum IGF-I after injections of growth hormone. He received growth hormone therapy for 6 months without an increase in growth rate. We admitted him to our Clinical Research Center for 5 weeks and ensured a constant dietary intake. In the second week, he was given three injections of growth hormone at therapeutic doses, and in weeks 3 and 4 he received continuous infusion of recombinant IGF-I (Genentech, San Francisco, California). This treatment was followed by 1 week of postinfusion observation. He showed no metabolic responses to growth hormone, but he had a marked decrease in urinary excretion of urea and in serum urea nitrogen with IGF-I infusion. His urinary calcium level increased and his urinary phosphate and sodium excretion levels decreased [24]. These all are fairly typical growth hormone-like effects and are similar to those that would occur in patients with growth hormone deficiency who are sensitive to growth hormone. Because of the insulin-like effects of IGF-I, he tended to become hypoglycemic when he was infused overnight in a fasting state. However, in the postprandial state, his glucose increased to high levels and his insulin level was suppressed, the latter because of a direct effect of IGF-I on insulin secretion. He was treated with subcutaneous injections of recombinant IGF-I (120 g/kg every 12 hours). After IGF-I injection, serum IGF-I concentrations were in the normal range for at least 7 hours. In general, however, acute metabolic responses to subcutaneous injections are less pronounced than are those observed with intravenous infusion. He has been treated with IGF-I for nearly 2 years and has grown at

Academic achievement and psychological adjustment in short children

Limited information is available on the educational and behavioral functioning of short children. Through 27 participating medical centers, we administered a battery of psychologic tests to 166 children referred for growth hormone (GH) treatment (5 to 16 years) who were below the third percentile for height (mean height = -2.7 SD). The sample consisted of 86 children with isolated growth-hormone deficiency (GHD) and 80 children with idiopathic short stature (ISS). Despite average intelligence, absence of significant family dysfunction, and advantaged social background, a large number of children had academic underachievement. Both groups showed significant discrepancy (p < .01) between IQ and achievement scores in reading (6%), spelling (10%), and arithmetic (13%) and a higher-than-expected rate of behavior problems (GHD, 12%, p < .0001; ISS, 10%, p < .0001). Behavior problems included elevated rates of internalizing behavior (e.g., anxiety, somatic complaints) and externalizing behavior (e.g., impulsive, distractable, attention-seeking). Social competence was reduced in school-related activities for GHD patients (6%, p < .03). The high frequency of underachievement, behavior problems, and reduced social competency in these children suggests that short stature itself may predispose them to some of their difficulties. Alternately, parents of short, underachieving children may be more likely to seek help. In addition, some problems may be caused by factors related to specific diagnoses.

CLINICAL STUDIES WITH RECOMBINANT-DNA-DERIVED METHIONYL HUMAN GROWTH HORMONE IN GROWTH HORMONE DEFICIENT CHILDREN

Thirty-six children with growth hormone deficiency were treated for up to 48 months with methionyl human growth hormone (hGH) synthesised by DNA recombinant methods. The growth rate for these children increased from 3.2 +/- 1.1 cm/yr to 10.5 +/- 2.2 cm/yr (mean +/- SD). This was similar to the effect of pituitary hGH in ten GH deficient children, 3.8 +/- 1.0 to 10.1 +/- 1.1 cm/yr. Serum somatomedin C rose from 0.26 +/- 0.23 U/ml to 0.79 +/- 0.53 U/ml after 6 months of methionyl-hGH therapy, similar to the effect of pituitary hGH. The incidence of antibody formation to methionyl-hGH was higher than that observed with pituitary hGH (Kabi) but poor growth was observed only in the one patient on methionyl-hGH who acquired high-titre high-binding-capacity antibodies to hGH. No consistent changes in levels of antibodies to Escherichia coli proteins were detected. No other allergic manifestations or systemic side-effects were demonstrable.