Effect of tamoxifen on plasma insulin-like growth factor I in patients with breast cancer.

Human breast cancer cells secrete and have membrane receptors for insulin-like growth factor I (IGF-I), a growth hormone-dependent peptide that stimulates cell replication. However, little is known about plasma concentrations of IGF-I in breast cancer patients. Plasma IGF-I levels are decreased in malnutrition, decline with advancing age, and are influenced by estrogen. We evaluated the effect of the antiestrogen agent tamoxifen on plasma IGF-I in 32 ambulatory breast cancer patients. Treatment with tamoxifen was associated with lower concentrations of plasma IGF-I (0.48 +/- 0.3 unit/ml in treated versus 1.03 +/- 0.6 units/ml in nontreated patients, P less than 0.01). However, patients treated with tamoxifen did not differ from nontreated patients in age, menopause, duration since diagnosis, metastatic disease, recent weight loss, or measures of nutritional status. We conclude that tamoxifen therapy results in a reduction of plasma IGF-I concentration. We speculate that the antitumor action of tamoxifen in breast cancer is due in part to suppression of IGF-I.

Hepatic insulin gene expression as treatment for type 1 diabetes mellitus in rats.

Type 1 diabetes mellitus is caused by a lack of insulin that results from the autoimmune destruction of the pancreatic beta-cells. Severe diabetes, if not controlled by periodic insulin injections, can lead to ketoacidosis and death. We have previously shown that sustained low level production of insulin in the liver of diabetic rats prevented their death from complications of diabetes. To test the hypothesis that there is a window of serum insulin concentrations that can prevent ketoacidosis without significant risk of hypoglycemia secondary to hyperinsulinemia, rats were infused with various doses of a recombinant retrovirus encoding an engineered rat preproinsulin-1 gene. The gene was engineered to allow processing into mature insulin by the protease furin. At the lower doses tested, fatal ketoacidosis was prevented, but the rats exhibited nonfasting hyperglycemia. At intermediate doses, which resulted in serum insulin concentrations of 1.6 mg/ml, the rats achieved near-normoglycemia and no serum ketones. These rats did not exhibit hypoglycemia even during a 24-h fast. At high virus doses, the animals achieved nonfasting normoglycemia but exhibited hypoglycemia during the fast. In conclusion, we have defined a therapeutic window of hepatic insulin expression that provides protection against ketoacidosis without significant risk of hypoglycemia. This window of sustained hepatic insulin expression might permit its development into a novel treatment modality for the prevention of ketoacidosis in patients with severe insulin-dependent diabetes mellitus.

Vitamin D receptor gene Fok1 polymorphism predicts calcium absorption and bone mineral density in children.

The vitamin D receptor (VDR) gene has been implicated as one of the major genetic components of osteoporosis. We evaluated the relationship between markers of mineral status and restriction fragment length polymorphisms of the VDR gene in 72 healthy children age 7-12 years. Using stable isotope techniques and dual-energy X-ray absorptiometry, we measured dietary calcium absorption, bone calcium deposition rates, and total body bone mineral density (BMD). The Fok1 polymorphism at the VDR translation initiation site was significantly associated with BMD (p = 0.02) and calcium absorption (p = 0.04). Children who were FF homozygotes had a mean calcium absorption that was 41.5% greater than those who were ff homozygotes and 17% greater absorption than Ff heterozygotes. BMD was 8.2% greater in the FF genotype than the ff genotype and 4.8% higher than the Ff genotype. These results suggest a substantial relationship between the VDR gene and bone metabolism at one or more levels, including dietary absorption of calcium and BMD in growing children.

Phenotypic correction of dwarfism by constitutive expression of growth hormone.

GH is normally secreted in a pulsatile fashion. When GH is deficient, dwarfism is the result in both rodents and humans. An adenoviral vector containing the rat GH complementary DNA was used to induce constitutive GH expression in hepatocytes of GH-deficient lit/lit mice. Elevated serum GH increased circulating insulin-like growth factor I concentrations, corrected the growth deficiency, and normalized body composition. The results indicate that correction of the dwarf phenotype can be achieved by constitutive expression of GH at an ectopic site by gene transfer.

Effects of acute high dose and chronic low dose estrogen on plasma somatomedin-C and growth in patients with Turner's syndrome.

Previously reported data with regard to the effects of estrogen on plasma somatomedin-C (Sm-C) concentrations are contradictory. This study was designed to evaluate, in the same subjects with Turner's syndrome, the effects of both acute high dose and chronic low dose estrogen treatment on plasma Sm-C concentrations. Eight girls with Turner's syndrome, aged 10 8/12 to 14 9/12 yr, were admitted to the Clinical Research Center for 3 days. Each received an iv infusion of conjugated estrogens (1.25 or 2.5 mg) in 12 h. Plasma Sm-C and serum GH and estrone levels were measured before, during, and after the infusion. The mean serum concentrations in the girls who received 1.25 mg conjugated estrogen increased from less than 222 pmol/L to 1905 +/- 240 (+/- SE), 825 +/- 166, and 296 +/- 74 pmol/L immediately after, 12 h after, and 24 h after completion of the infusion, respectively. Serum GH concentrations were undetectable (less than 1 microgram/L) before, during, and after the infusion. The mean plasma Sm-C concentrations decreased significantly (P = 0.02) after the infusion was terminated, falling from a mean basal value of 1.3 +/- 0.2 (+/- SE) to a nadir of 0.6 +/- 0.1 U/mL 12 h after completion of the infusion. The same girls then were treated with low dose (5 micrograms ethinyl estradiol) estrogen therapy for 9-14 months. Seven of the eight girls had an increase in growth rate. The mean growth rate increased from 2.0 +/- 0.5 to 5.4 +/- 0.8 and 3.8 +/- 0.4 cm/yr from 3 to 6 (P less than 0.05) and from 9 to 14 months of treatment, respectively. Individual variability was great, however, and two of the eight girls never grew at a rate faster than 4.0 cm/yr during therapy. No consistent alteration in bone age relative to chronological age was found, and the growth response was not predicted by the bone age at the beginning of treatment. Mean plasma Sm-C concentrations did not change. These studies demonstrated a growth spurt in girls with Turner's syndrome during chronic low dose estrogen therapy, which was not associated with any change in plasma Sm-C concentrations. Acute high dose iv estrogen infusion resulted in a significant decline in plasma Sm-C concentrations. These results support the concept that in girls with Turner's syndrome the plasma Sm-C response to estrogen is influenced by multiple, as yet undefined, factors.

Influence of physical activity on insulin-like growth factor-I in healthy younger and older men.

To examine the hypothesis that a lower level of physical activity influences the age-related decline in insulin-like growth factor-I (IGF-I), we measured serum concentrations in healthy nonobese younger and older men, characterized for maximal aerobic capacity (VO2 max) and energy expended in leisure time physical activity. To examine the independent influence of physical activity on IGF-I relative to other lifestyle variables, we also determined fat-free weight, percent body fat, body fat distribution (waist to hip and waist to thigh ratios), and habitual caloric intake in our population. IGF-I was 33% lower (P less than 0.01) in older men than in younger men, inversely related to percent body fat (r = -0.55) and indices of upper body fat distribution (waist to hip ratio, r = -0.45; waist to thigh ratio, r = -0.47), and positively related to VO2 max (r = 0.64) and leisure time physical activity (r = 0.45; P less than 0.01). IGF-I was not related to fat-free weight or daily caloric intake. After controlling for the effects of age by multiple regression analysis, VO2 max (r = 0.29) and leisure time physical activity (r = 0.24) were the sole factors independently related to IGF-I (P less than 0.05). Our results suggest that multiple factors contribute to the age-related decline in IGF-I. Lower levels of IGF-I in aging men are related at least in part to diminished physical activity.

Recombinant human insulin-like growth factor-I increases forearm blood flow.

Several recent reports have described systemic effects associated with insulin-like growth factor-I (IGF-I) infusions in humans, including dizziness, syncope, unconsciousness, bradycardia, and asystole. Eleven healthy volunteers (10 males and 1 female) underwent brachial arterial and deep forearm venous catheterizations to evaluate the effects of an intraarterial infusion of IGF-I on blood flow. Saline was infused into the brachial artery for 1 h, followed by infusions of recombinant human IGF-I (1.25, then 10 micrograms/kg BW.h) over 2 subsequent 90-min periods. Blood flow across the forearm was determined from dye dilution curves after an infusion of indocyanine green dye (20 mg/h) into the proximal arterial port. The rate of saline infusion had no effect on forearm blood flow. Likewise, the low dose arterial IGF-I infusion had no effect on blood flow, even though deep venous IGF-I concentrations increased slightly (127 +/- 37 to 172 +/- 19 ng/mL; P = NS). The 10 micrograms/kg.h infusion rate increased deep venous IGF-I concentrations approximately 4-fold above control values (477 +/- 80 vs. 150 +/- 27 ng/mL, respectively; P < 0.0001), whereas IGF-I concentrations in the peripheral circulation were increased approximately 2-fold (299 +/- 50 ng/mL) above control values (P < 0.0001). Arteriovenous glucose differences, peripheral glucose concentrations, and binding protein-3 concentrations were not influenced by either dose of IGF-I. The 10 micrograms/kg.h dose of IGF-I was associated with a 68% increase in forearm blood flow (71 +/- 8 vs. 42 +/- 6 mL/min, respectively; P < 0.0004). These data suggest that an intravascular infusion of IGF-I results in significant alterations in regional blood flow, which may be related to the observed physiological effects or side-effects associated with its use.

Acute growth hormone effects on amino acid and lipid metabolism.

The anabolic actions of GH are well known, although specific tissue responses and the mechanism of nitrogen conservation are less well understood. This study was designed to examine the acute metabolic effects of GH on whole body and regional protein metabolism, using an experimental protocol which controlled for confounding perturbations in other hormones by a simultaneous infusion of somatostatin. Control subjects received replacement doses of insulin, glucagon, and GH for the entire 7-h study period, whereas GH subjects received an identical protocol, except for an increased dose of GH sufficient to increase serum concentrations into the high-physiological range (12-20 ng/mL) for the final 3.5 h of the study (P < 0.001). Thirteen young, healthy male subjects were studied in the postabsorptive period; five served as control subjects and eight as treatment (GH) subjects. Each received continuous iv infusions of somatostatin, L-[13-C]leucine, and L-[2H5]phenylalanine throughout the study. Femoral arterial and venous sampling allowed for simultaneous measurements across the leg and in the whole body. C-Peptide levels were suppressed throughout the infusion; insulin, glucagon, insulin-like growth factor I, cortisol, epinephrine, norepinephrine, and glucose concentrations were not different between groups. Glycerol concentrations increased 3-fold in GH subjects during the final 3.5-h period (P = 0.04). Concentrations of several amino acids declined through the study, but no differences were observed between treatment groups. Leucine oxidation was reduced in GH compared to control subjects (P = 0.04). No changes in CO2 production or whole body leucine or phenylalanine flux were observed, whereas nonoxidative disposal of leucine was marginally higher in GH compared to control subjects (P = 0.07). By contrast, rates of appearance and disappearance of both leucine and phenylalanine across the leg all were relatively lower in GH compared to control subjects; leucine balance across the leg was reduced by GH (P = 0.03), whereas phenylalanine balance was not influenced by GH. Our data thus demonstrate an acute stimulatory effect of GH on lipolysis, a decrease in leucine oxidation, and no stimulation of muscle protein synthesis in spite of enhanced protein synthesis in nonmuscle tissue.

The efficiency of dietary protein utilization is increased during puberty.

We investigated whether the efficiency of dietary protein utilization for growth increases during the pubertal growth spurt in both nondiabetic and diabetic subjects. We measured leucine oxidation and retention (intake minus oxidation) in orally fed nondiabetic (n = 9) and diabetic (n = 9) human subjects, aged 7-17 yr. Eight subjects were Tanner stage I, and 10 were Tanner stages III-V; groups were not matched for gender. After 3 days of consuming a diet containing approximately 1 g/kg. day protein, subjects drank a commercial liquid nutrition formula, containing L-[1(-13)C]leucine, every 30 min for a total of 6 h to provide 1 g protein/kg. day. Isotopic enrichment of CO2 was used to calculate the fractional leucine oxidation rate and, together with alpha-ketoisocaproate isotopic enrichment, to calculate total leucine oxidation. Leucine oxidation rates decreased with puberty in both nondiabetic subjects (36.0 +/- 10.4 vs. 23.9 +/- 4.2 mumol/kg fat-free mass (FFM).h, prepubertal and pubertal, respectively; P < 0.05) and diabetic (33.6 +/- 4.9% vs. 27.3 +/- 3.4 mumol/kg FFM.h, prepubertal and pubertal, respectively; P < 0.1) subjects. Leucine retention increased with puberty in both nondiabetic (0.27 +/- 3.2 vs. 15.7 +/- 5.3 mumol/kg FFM.h, prepubertal and pubertal, respectively; P < 0.001) and diabetic (1.9 +/- 4.9 vs. 13.2 +/- 4.4 mumol/kg FFM.h, prepubertal and pubertal subjects, respectively; P < 0.05) subjects. The data suggest that the pubertal growth spurt is associated with a marked increase in the efficiency of dietary protein utilization for growth.

Puberty in the chimpanzee: somatomedin-C and its relationship to somatic growth and steroid hormone concentrations.

A relationship between sex steroids and the somatomedins (Sms) is well known, but poorly defined. In some primates, including man, there are pubertal increases in Sms, concurrent with increased growth and sex steroid production. In the current studies, indices of somatic growth [body weight, crown-rump length (CRL), and testis size (testicular volume index)] and circulating concentrations of testosterone (T), estradiol (E2), dehydroepiandrosterone sulfate (DHEA-S), cortisol, and Sm-C were determined (n = 208) in 86 male and female chimpanzees during a 1-yr period. In addition, we have attempted to determine whether plasma Sm-C concentrations correlate with serum levels of estrogen and androgens. In male animals between 6 and 8 yr of age, there was a marked increase in testicular size, concurrent with an increase in serum T and preceding slightly an increase in the rate of body weight gain. There were no detectable increases in serum E2 or the CRL slope. In females between 6 and 8 yr of age, serum T increased, concurrent with an increase in the rate of body weight gain much smaller than that in male animals. Serum E2 increased only after 10 yr of age, and no increased linear growth (CRL) was found. In both sexes, increases in serum DHEA-S were found by 4-6 yr of age, in contrast to cortisol concentrations, which were high and remained unchanged from birth to 12 yr of age, except for lower values in the very youngest and very oldest female animals. An increase in Sm-C occurred in both sexes by 4-6 yr of age, with higher values in female than in male animals 0-2, 4-6, and 6-8 yr of age, and when all ages were considered together. In both sexes, plasma Sm-C concentrations correlated with serum T (r = 0.60 and P less than 0.001; r = 0.68 and P less than 0.001; females and males, respectively), although when both sexes were analyzed together, the correlation was not as good (r = 0.36; P less than 0.001). Sm-C concentrations correlated with serum DHEA-S when the two sexes were analyzed separately (r = 0.44 and P less than 0.001; r = 0.54 and P less than 0.001; females and males, respectively) or together (r = 0.49; P less than 0.001). Sm-C correlated poorly with serum E2 levels in females (r = 0.20; P less than 0.05) and did not correlate with E2 in males.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of a novel arginine vasopressin defect by dideoxy fingerprinting.

Autosomal dominant neurohypophyseal diabetes insipidus is a familial form of diabetes insipidus. This disorder is associated with variable levels of arginine vasopressin (AVP) and diabetes insipidus of varying severity, which responds to exogenous AVP. To determine the molecular basis of autosomal dominant neurohypophyseal diabetes insipidus, the AVP genes of members of a large kindred were analyzed. A new method, called dideoxy fingerprinting, was used to detect an AVP mutation that was characterized by DNA sequencing. The novel defect found changes the last codon of the AVP signal peptide from alanine to threonine, which should perturb cleavage of mature AVP from its precursor protein and inhibit its secretion or action.

Pubertal endocrinology of the baboon: elevated somatomedin-C/insulin-like growth factor I at puberty.

Previous data suggested an increase in the rate of weight gain and linear growth in the baboon between 3 and 4 yr of age, similar to the pubertal growth spurt in man. In this cross-sectional study, radioimmunoassayable concentrations of somatomedin-C/insulin-like growth factor I(SM-C/IGF-I) were compared in prepubertal (less than 3 yr), pubertal (3-4 yr), and adult (greater than 10 yr) animals. SM-C/IGF-I concentrations in prepubertal males (0.97 +/- 0.10 U/ml) were low and were not different from those in prepubertal females (0.98 +/- 0.15 U/ml). Between 3 and 4 yr, SM-C/IGF-I increased significantly in both sexes (8.87 +/- 0.74 and 5.27 +/- 0.52 U/ml, male and female, respectively) and decreased (5.92 +/- 1.2 and 2.75 +/- 0.13 U/ml, respectively) in animals greater than 10 yr of age. Sex differences were significant in the 3- to 4-yr-old animals (male greater than female, P less than 0.001). The pubertal elevation in SM-C/IGF-I concentrations is coincident with increases in indices of somatic growth and sexual maturation in the baboon. These and other data suggest that this animal may be an appropriate model for studies to define hormonal mechanisms of pubertal growth.

Insulin does not stimulate protein synthesis acutely in prepubertal children with insulin-dependent diabetes mellitus.

Insulin treatment in adult type I diabetic patients decreases protein loss primarily by inhibiting protein breakdown without stimulating protein synthesis. In young growing rodents, insulin treatment has been reported to stimulate protein synthesis. We examined whether insulin stimulates protein synthesis in normally growing prepubertal children with insulin-dependent diabetes mellitus. Five prepubertal children with insulin-dependent diabetes mellitus (aged 8.6-11.25 yr) were studied in the postabsorptive state on two occasions: once during insulin deprivation (I-; blood glucose, 325 +/- 67.8 mg/dL; mean +/- SD) and once during insulin administration for 4 h (I+; blood glucose, 96 +/- 23.6 mg/dL). Leucine kinetics were measured using a 4-h primed continuous infusion of L-[1-13C]leucine. Serum insulin concentrations were lower (I- vs. I+, 0.6 +/- 0.3 vs. 7.5 +/- 4.3 microU/mL; mean +/- SD; P = 0.02), whereas serum beta-hydroxy-butyrate (I- vs. I+, 3.4 +/- 0.5 vs. 0.9 +/- 0.5 mg/dL; P < 0.001) and free fatty acid concentrations (I- vs. I+, 2.9 +/- 0.4 vs. 0.9 +/- 0.4 mEq/L; P < 0.001) were higher in the insulin-deprived state than during insulin administration. Leucine Ra, an index of protein breakdown (I- vs. I+, 200.5 +/- 23.4 vs. 167 +/- 17 mumol/kg.h; P = 0.008), and leucine oxidation (I- vs. I+, 56.5 +/- 20.7 vs. 29.6 +/- 9.3 mumol/kg.h; P = 0.03) were reduced by insulin treatment. Nonoxidative leucine disposal, an index of protein synthesis, was not affected by insulin treatment (I- vs. I+, 144 +/- 20.8 vs. 137.5 +/- 13.5 mumol/kg.h; P = 0.4). We conclude that the acute decline in net protein loss during insulin treatment in growing prepubertal children, like that in adults, is due primarily to an inhibition of protein breakdown without stimulation of protein synthesis.

Effect of in vitro action of serum proteases or exposure to acid on measurable immunoreactive somatomedin-C in serum.

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.

Estrogen stimulates growth hormone and somatomedin-C in castrate and intact female baboons.

To evaluate the effects of physiological concentrations of estrogen on plasma concentrations of somatomedin-C (Sm-C) and GH, 16 chronic castrate female baboons were implanted either with blank Silastic capsules (n = 5) or capsules containing crystalline estradiol (E2; n = 11), which remained in place for 7 days. By day 7, serum E2 levels in treated animals rose into the physiological adult female range (mean +/- SEM, 96 +/- 9.2 pg/ml) and were greater (P less than 0.0005) than those in control animals (27.5 +/- 3.4 pg/ml). Sm-C concentrations rose significantly by day 7 in treated animals compared to those in control animals, whether assayed from unprocessed plasma (96% increase; P less than 0.01), plasma pretreated with glycine-HCl (99% increase; P less than 0.01), or plasma extracted with acid-ethanol (72% increase; P less than 0.02). GH concentrations in these animals were low and were not significantly different in E2-treated and control animals. To evaluate the effects of pharmacological doses of E2 on Sm-C and GH concentrations, six intact adult female baboons were treated with six daily injections of 1 mg E2 benzoate in oil. Serum E2 rose to a mean level of 1669 +/- 320 pg/ml on day 7. The plasma Sm-C concentration by day 7 was significantly higher than the pretreatment value whether assayed in untreated plasma (4.3-fold increase; P less than 0.01), plasma pretreated with glycine-HCl (2-fold increase; P less than 0.01), or plasma extracted with acid-ethanol (1.7-fold increase; P less than 0.01). Mean serum GH concentrations rose significantly from 3.3 ng/ml on day 0 to 23.6 ng/ml by day 7 (P less than 0.02). Evaluation of the chromatographic profile of native baboon plasma suggested a marked increase in [125I]Sm-C binding to plasma proteins after in vivo pharmacological E2 treatment; a broad peak of [125I]Sm-C binding over a size range from that of albumin to gamma-globulin was found. These results indicate that estrogen treatment of castrate or intact female baboons with both physiological and pharmacological doses results in an increase in plasma Sm-C concentrations that, at least in pharmacological doses, are mediated through an increase in GH concentrations. Although pharmacological E2 treatment results in a stimulation of plasma proteins that bind Sm-C, the effects on plasma Sm-C concentrations were found after procedures that diminish interference from circulating binding proteins. These data support the concept that estrogen may play a role in the physiological increase in plasma concentrations of Sm-C associated with normal puberty.

Discordant metabolic actions of insulin in extreme lipodystrophy of childhood.

Congenital lipodystrophy includes a group of disorders characterized by total or partial absence of adipose tissue and insulin resistance. In this study we investigated the nature of insulin resistance in an 11-yr-old girl with one form of congenital lipodystrophy. We examined in vivo insulin and glycemic responses to feeding and iv glucose and in vitro amino acid and thymidine incorporation responses of skin fibroblasts to insulin exposure. In addition, we used stable isotope infusions of glucose, glycerol, and amino acids to investigate the in vivo metabolic actions of insulin on carbohydrate, fat, and protein. At 5 yr of age, she first demonstrated clinical glucose intolerance. Her basal insulin levels were normal (129 and 114 pmol/L), but increased markedly (peak values, 1304 and 5045 pmol/L) after iv glucose and a mixed meal. Insulin antibodies were undetectable, and specific [125I]insulin binding to her skin fibroblasts was normal. Both [3H]aminoisobutyric acid transport and [3H]thymidine incorporation by her fibroblasts were similar to responses obtained using control cells. At 11 5/12 yr of age, while receiving an infusion of stable isotopes, infusions of insulin at doses of 0.1 and 0.3 U/kg BW.h were ineffective in reducing her blood glucose despite elevating her serum insulin level to approximately 2500 pmol/L. Resting metabolic rate, respiratory quotient, VCO2, carbohydrate and lipid oxidation rates, glucose production rate, glycerol appearance rate, and plasma glycerol concentrations were unperturbed by the insulin infusions. By contrast, the insulin infusions reduced plasma leucine concentrations (124.2 to 86.1 to 66.7 mumol/L) and 13CO2 production rates (0.034 to 0.017 to 0.011 mumol/kg/min; baseline, 0.1, and 0.3 U insulin/kg.h, respectively). The leucine appearance rate declined (1.96 to 1.72 mumol/kg.min) in response to the 0.1 U/kg.h dose, but did not decline further in response to the 0.3 U/kg.h dose. The leucine oxidation rate also declined (0.87 to 0.39 to 0.25 mumol/kg.min), and there was a dose-related reduction in most plasma amino acid concentrations. Finally, nonoxidative leucine disposal increased progressively (1.09, 1.34, and 1.48 mumol/kg.min), suggestive of an insulin-induced increase in protein synthesis. These data indicate profound metabolic resistance to the carbohydrate and lipid actions of insulin, with preservation of protein anabolism. These observations suggest that in this patient, the biological effects of insulin on carbohydrate, lipid, and protein are distinct metabolic actions, regulated independently.

Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

The gene responsible for X-linked adrenal hypoplasia congenita, DAX1, encodes a member of the nuclear hormone receptor superfamily. We sequenced 8851 bp that contained the DAX1 genomic region. The DAX gene was composed of two exons and one 3.4-kilobase intron. Putative TATA and GC boxes and a putative steroidogenic factor 1 response element were present in the 5'-flanking region. Two potentially polymorphic short tandem repeats were identified. The first exon encoded two putative novel zinc finger motifs within a putative DNA binding domain and part of the ligand binding domain, and the second exon encoded the remainder of the ligand binding domain. Although the putative DNA binding domain of DAX1 does not contain substantial sequence similarity to other nuclear hormone receptor superfamily members, the putative ligand binding domain had remarkable similarity to other family members. Single-strand conformational polymorphism analysis permitted identification of three new mutations in DAX1. In conclusion, single-strand conformational polymorphism analysis facilitates identification of mutations in the DAX1 gene, and the short tandem repeats may permit linkage analysis in families in which mutations are not yet identified. We speculate that DAX1 may be the most primitive member of the nuclear hormone receptor superfamily identified in mammals.

Characterization of two novel homozygous missense mutations involving codon 6 and 259 of type II 3beta-hydroxysteroid dehydrogenase (3betaHSD) gene causing, respectively, nonsalt-wasting and salt-wasting 3betaHSD deficiency disorder.

We identified two homozygous missense mutations in the human type II 3beta-hydroxysteroid dehydrogenase (3/betaHSD) gene, the first in codon 6 of exon II [CTT (Leu) to TTT (Phe)] in a male infant with hyperpigmented scrotum and hypospadias, raised as a male and no apparent salt-wasting since neonatal age, and the second in codon 259 of exon IV [ACG (Thr) to ATG (Met)] in a male pseudohermaphrodite with labial scrotal folds, microphallus, chordee, and fourth degree hypospadias, raised as a female and with salt-wasting disorder since neonatal age. In vitro transient expression of mutant type II 3betaHSD complementary DNAs of L6F, T259M, as well as T259R for comparison was examined by a site-directed mutagenesis and transfection of construct into COS-1 and COS-7 cells. Northern blot analysis revealed expression of similar amounts of type II 3betaHSD messenger ribonucleic acid from the COS-1 cells transfected by L6F, T259M, T259R, and wild-type (WT) complementary DNAs. Western immunoblot analysis revealed a similar amount of L6F mutant protein compared to WT enzyme from COS-1 cells, but neither L6F from COS-7 cells nor T259M or T259R mutant protein in COS-1 or COS-7 cells was detectable. Enzyme activity in intact COS-1 cells using 1 micromol/L pregnenolone as substrate in the medium after 6 h revealed relative conversion rates of pregnenolone to progesterone of 46% by WT enzyme, 22% by L6F enzyme, and 8% by T259M enzyme and less than 4% activity by T259R enzyme. Using 1 micromol/L dehydroepiandrosterone as substrate, the relative conversion rate of dehydroepiandrosterone to androstenedione after 6 was 89% by WT enzyme, 35% by L6F enzyme, 5.1% by T259M enzyme and no activity by T259R enzyme. However, the L6F mutant 3betaHSD activity, despite its demonstration in the intact cells, was not detected in homogenates of COS-1 cells or in immunoblots of COS-7 cells, suggestive of the relatively unstable nature of this protein in vitro, possibly attributable to the decreased 3betaHSD activity. In the case of T259M and T259R mutations, consistently undetectable proteins in both COS cells despite detectable messenger ribonucleic acids indicate severely labile proteins resulting in either no or very little enzyme activity, and these data further substantiate the deleterious effect of a structural change in this predicted putative steroid-binding domain of the gene. In conclusion, the findings of the in vitro study of mutant type II 3betaHSD enzyme activities correlated with a less severe clinical phenotype of nonsalt-wasting and a lesser degree of genital ambiguity in the patient with homozygous L6F mutation compared to a more severe clinical phenotype of salt-wasting and severe degree of genital ambiguity in the patient with homozygous T259M mutation in the gene.

Calcium absorption, bone mass accumulation, and kinetics increase during early pubertal development in girls.

To evaluate the changes in calcium and bone mineral metabolism associated with early pubertal development, we performed longitudinal measurements of calcium absorption, calcium kinetics, bone mineral content, and hormonal markers related to puberty in a multiethnic group of girls beginning when they were 7 or 8 yr old. Girls were Tanner stage 1 (breast) at the start of the study. They were placed on a 1200 mg/day dietary calcium intake and studied at approximately 6-month intervals until they reached Tanner stage 2 (breast). Results at that time point (PUB) were compared to values obtained approximately 1 yr earlier (LatePRE) and those 1 yr before that (EarlyPRE). We found an increase in calcium absorption comparing PUB to LatePRE (n = 34; 36.6 +/- 8.7% vs. 30.7 +/- 9.9%; P = 0.002). Using whole body, dual energy, x-ray absorptiometry scanning, we found an increase in calcium gain during the LatePRE to PUB period compared with that during the EarlyPRE to LatePRE period (135 +/- 53 vs. 110 +/- 45 mg/day; P = 0.04). Calcium kinetic studies showed a significant increase in the bone calcium deposition rate (Vo+) during the PUB compared to the LatePRE period. Hormonal and biochemical markers of bone development were also significantly increased at PUB compared to LatePRE. Hormonal activity, as evidenced by the unstimulated LH level, was significantly correlated with calcium gain between the LatePRE and PUB studies and the bone calcium deposition rate in the PUB study. These data demonstrate, using multiple independent methods, an increase in calcium utilization associated with the earliest physical signs of puberty.

Development of a heterologous radioimmunoassay for somatomedin C in sheep blood.

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.