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Background: Heterozygous loss of function mutations in GH1 are a cause of autosomal dominant isolated growth hormone deficiency (GHD) or IGHD type II. However, this condition is rare and a genotype/phenotype association is often based on single case or pedigree reports. Clinical Case: The male proband was born at 38 weeks following an uncomplicated pregnancy, birthweight 2585 gm (˗1.3SD), length 43 cm (˗2.7SD). At 20 months old: length ˗3.2SD, weight/length 16%. Peak growth hormone (GH) levels 4.42 ng/mL and 3.2 ng/mL following clonidine and arginine stimulation, respectively; cortrosyn-stimulated cortisol was 41.4 mcg/dL. Prolactin was 61.2 ng/mL (reference range 2.6 to 13.1 ng/mL); T4 and TSH levels were normal. Magnetic resonance imaging showed a flattened anterior pituitary gland, consistent with empty sella, with normal bright spot. He has had an intermittent and variable response to GH treatment due to noncompliance; recent recorded height was ˗3.6SD at 13.9 and he was early pubertal. Father had a history of childhood growth hormone treatment and is 151 cm tall, 2 paternal aunts did not receive treatment and are 145 cm tall, 3 children of these aunts are diagnosed with GHD. There is no maternal family history of GHD; mother is 155 cm. Proband testing revealed heterozygous GH1 c.291 + 34G>C (EGL Genetics), classified as a variant of unknown significance; father was also found to have this variant, mother was negative. Genetic testing for of additional family members is under consideration. Conclusion: Nine of 96 heterozygous GH1 variants are located in intron 3. Two have been classified as pathogenic and are predicted to cause exon skipping. Our proband’s variant cannot be considered a canonical splice site or in a mutational hot spot but has extremely low frequency in population databases. Functional studies clan clarify if our proband’s variant results in a 17.5 kDa GH isoform and dominant negative effect on full length GH production with potential detrimental effects on other anterior pituitary hormones. We present the second GHD pedigree with GH1 c.291 + 34G>C mutation. As in the previous report (1), no other hormone deficiencies are identified, despite the appearance of an empty sella for our proband. Reference: (1) Cho SY, Ki, CS, Park, HD, et al. Genetic investigation of patients with undetectable peaks of growth hormone after two provocation tests. Clin Endocrinol 2013; 78: 317-20.

Children with chronic renal failure (CRF) are often growth recarded despite normal serum levels of GH and insulin-like growth factors (IGFs). Recent studies suggest that excess IGF-binding proteins (IGFBPs) in the 35-kDa fractions of CRF serum contribute to CRF growth failure. This report characterizes the relationship between IGFBP-3 and IGF peptides in the serum of growth-retarded CRF children. Size-exclusion chromatography at pH 7.4 found IGFBP-3 and IGFs almost exclusively in the 150-kDa fractions of normal serum, where their molar stoichiometry was approximately 1:1. However, similar chromatography of CRF serum found a molar excess of IGFBP-3 over total IGFs in the 150-kDa fractions and large amounts of IGFs in the 35-kDa fractions. In the 150-kDa fractions of CRF serum, IGFBP-3 was present in normal amounts, but a greater than normal amount was in the form of a 29-kDa IGFBP-3 fragment. Treatment of these CRF children with recombinant human GH increased the molar excess of IGFBP-3 over total IGFs in the 150-kDa fractions, the amount of IGFBP-3 and total IGFs in the 150-kDa fractions, and the amount of IGFs, but not IGFBPs, in the 35-kDa fractions. These data suggest that in untreated CRF children, proteolysis of IGFBP-3 in the 150-kDa fractions releases IGFs to the excess IGFBPs in the 35-kDa fractions, but insufficient IGF is released to overcome the growth-inhibiting effects of these excess IGFBPs. Treatment with recombinant human GH increases levels of IGFs and IGFBP-3 in the 150-kDa fractions, and subsequent IGFBP-3 proteolysis releases sufficient IGF to overcome the growth inhibitory effects of excess IGFBPs in the 35-kDa fractions of CRF serum.

The activity of the hypothalamic-GH-insulin-like growth factor (IGF) network declines with age. It has recently been shown that increased cardiovascular mortality occurs in adults with GH deficiency. As hypercholesterolemia is common in GH-deficient adults, and because there is experimental evidence that GH may play a role in regulating plasma cholesterol, we decided to investigate the activity of the GH-IGF axis in an elderly population by measuring serum IGF-I, IGF-II, and IGF-binding protein-3 (IGFBP-3) levels and to study their relationship with blood lipid levels. One hundred and thirty-two elderly subjects, 52 men and 80 women, were studied (age range, 60–91 yr). Men had significantly lower levels of IGFBP-3, high density lipoprotein cholesterol (HDL-C) and apoprotein A1 (ApoA1) compared to the women, whereas IGF-I and IGF-II were only slightly lower. Using linear regression analysis, we observed an inverse relationship of age with IGF-I (r = −0.35; P < 0.001), IGF-II (r = 0.40; P < 0.001), IGFBP-3 (r = 0.52; P < 0.001), body mass index, and lipid levels. Univariate regression analysis showed a strong and positive correlation of both IGF-I and IGFBP-3 with HDL-C and ApoA1. Partial correlation analysis, after adjustment for age and body mass index, showed that IGFBP-3 and IGF-II were still significantly and positively related to HDL-C and ApoA1. Furthermore, a strong association was documented among IGF-I, IGF-II, and IGFBP-3. These data demonstrate that even in an elderly population, further aging is accompanied by a progressive decline in circulating IGF-I, IGF-II, and IGFBP-3, suggesting a continuing diminution of the GH-IGF axis throughout aging. Moreover, the strong correlation between HDL-C and an index of GH secretion, such as IGFBP-3, suggests that GH might play an important role in lipid metabolism in healthy elderly subjects.

Previous studies suggest that growth retardation in children with chronic renal failure (CRF) results in part from inhibition of insulin-like growth factor (IGF) action by excess serum IGF-binding proteins (IGFBPs). Excess IGFBPs in CRF serum include IGFBP-1, -2, and -3 and a diffuse approximately 24- to 28-kDa IGFBP band identified by [125I]IGF ligand blot. The present studies characterized this diffuse approximately 24- to 28-kDa band. Initial studies identified this band as IGFBP-6, because it was immunoprecipitated by antiserum raised against a synthetic peptide of human IGFBP-6 (hIGFBP-6). Additional [125I]IGF ligand blots found that the immunoprecipitated band was 1) recognized by [125I]IGF-II but not [125I]IGF-1, 2) more abundant in CRF than in normal serum, and 3) more abundant in serum from dialyzed than nondialyzed prepubertal CRF children. Using the hIGFBP-6 antiserum in a specific and sensitive RIA, we found that serum IGFBP-6 levels were 4.7 +/- 1.7 nmol/L in 10 normal prepubertal children, 21.4 +/- 6.1 nmol/L in 44 nondialyzed prepubertal CRF children, 73.5 +/- 14.4 nmol/L in 7 dialyzed prepubertal CRF children, and 94.6 +/- 26.2 nmol/L in 14 dialyzed pubertal CRF children. IGFBP-6 levels were also elevated in 71 nondialyzed European children with CRF. In nondialyzed CRF children, serum IGFBP-6 levels 1) correlated inversely with the glomerular filtration rate, 2) did not correlate with height SD score, and 3) were not altered by 12 months of daily recombinant hGH treatment. In summary, a specific antiserum and RIA were used to demonstrate elevated levels of intact IGF-II-binding IGFBP-6 in serum of CRF children. We postulate that the excess IGFBP-6 may modulate the action of IGF-II on target tissues.

The in vivo physiological relationships among GH, the insulin-like growth factors (IGFs), and the IGF-binding proteins (IGFBPs) are not completely defined, and single random measurements of these serum proteins do not completely reveal their dynamic relationships. We report the kinetic responses of the IGFs and IGF-binding proteins to exogenous GH in 23 subjects with untreated GH deficiency [5 women and 18 men; age, 15.0 +/- 6.2 yr (+/- s.d.), height z-score = -4.4 +/- 2.2 (+/- s.d.); body mass index = 19.3 +/- 2.4 kg/m2]. After an overnight fast, subjects were given a sc dose of recombinant human GH (2.85 i.u./m2), and blood was sampled from an indwelling peripheral venous catheter 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, and 24 h after the injection. Subjects were then treated with recombinant human GH (2.85 i.u./m2.day); fasting samples were obtained at 3 months (n = 22), and timed sampling was repeated at 6 months (n = 21). Fasting levels of IGF-I, free IGF-I, IGF-II, IGFBP-3, and insulin increased significantly within 3 months of GH treatment, whereas IGFBP-1, IGFBP-2, and IGFBP-6 showed no change. In the timed sampling studies at 0 and 6 months, GH levels peaked 3 h after treatment; the degree of rise and the rate of decline were both greater at 6 months. IGF-I levels increased beginning at 4 h, continuing throughout the 24-h period at month 0, whereas a plateau was observed after 6-8 h during the 6-month study. Free IGF-I paralleled total IGF-I except during fasting, when it varied inversely with IGFBP-1. IGFBP-3 and IGF-II both showed late (> 20 h) responses to a dose of GH, whereas IGFBP-2 and IGFBP-6 showed minimal changes. IGFBP-1 varied inversely with insulin, which, in turn, varied with meal intake. Comparative studies in 2 subjects with GH receptor deficiency showed no response to exogenous GH. However, both IGFBP-1 and IGFBP-2 were several-fold elevated, and IGFBP-1 varied inversely with the low insulin levels. Our data are the first to examine multiple elements of the serum IGF system in response to GH in both GH-deficient and replete states. The relationships of the different response patterns provide insight into the physiology of this system and may guide future studies.

Weight loss is a common, persistent characteristic of long term human immunodeficiency virus (HIV-1) infection; its full etiology remains unknown. Because treatment with GH has induced nitrogen retention in various catabolic conditions, we designed this study to determine whether a moderate dose of insulin-like growth factor I (IGF-I) combined with a low GH dose could impede the catabolic response seen in HIV-1 infection. A double blind, placebo-controlled study design was used. Subjects in the GH/IGF-I treatment group (n = 44) and control group (n = 22) continued to receive their routine stable antiretroviral therapy. No patient had a recent history of opportunistic infection, malignancy, or Kaposi's sarcoma and had dietary intakes of at least 25 Cal/kg weight.day at study entry. During the 12-week study period, dietary instruction was given, and subjects were encouraged to maintain an intake of 35 Cal/kg and 1 g protein/kg. All subjects had a body mass index of 19.8 kg/m2 or less at the time of study entry or a weight loss of 10% or more of their premorbid weight and a body mass index below 26.1 kg/m2. The treatment group received 0.34 mg (0.68 mg/day) GH, twice daily, and 5.0 mg (10 mg/day) IGF-I, twice daily. Changes in body composition of total body potassium (TBK), total body nitrogen (TBN), fat-free mass (FFM), and body fat (Fat) were examined at 6 and 12 weeks during the treatment period. TBK, TBN, FFM, and Fat for the treatment and placebo groups were, on the average, below normal at study entry. At 6 weeks, the GH/IGF-I group showed a significant increase in FFM (P0.0001), a minimal increase in TBK (P0.05), and a substantial decrease in Fat (P0.01) compared with baseline values. The loss of body fat continued to be significant (P0.01) in the GH/IGF-I group treatment at 12 weeks, whereas the increase in FFM was minimal (P0.05). No significant changes in the mean body composition occurred at 6 or 12 weeks in the placebo group. By 12 weeks, neither TBK (body cell mass) nor TBN (total protein mass) had significantly increased relative to the values at baseline, although the FFM remained elevated. Thus, the combined GH and IGF-I doses used in this study in adult males with HIV-associated weight loss were ineffective in producing a sustained anabolic response and, in fact, resulted primarily in a significant loss of body fat.

We report the development of a unique radioimmunoassay (RIA) for rat insulin-like growth factor-I (rIGF-I) which does not recognize human IGF-I (hIGF-I). The rIGF RIA uses a specific anti-rIGF-I antiserum with rIGF-I standards and radioligand. Rat sera were extracted by either an abbreviated acid/ethanol procedure or by acid-chromatography with virtually identical results. Assay sensitivity is congruent to 0.5 ng/tube and inter- and intra-assay coefficients of variation were 3.4-7.6% and 4.8-9.2%, respectively. Comparisons of the rIGF-I RIA with a typical heterologous RIA shows that the latter underestimates rIGF-I levels congruent to of 3-fold. Sera from rats treated with hIGF-I were also studied using the rIGF-I RIA and a hIGF-I- specific immunoradiometric assay (IRMA), and results indicate poor correlation between the actual total IGF-I levels (rIGF-I RIA + hIGF-IRMA) and the heterologous RIA estimates. The availability of both rIGF-I and hIGF-I specific immunoassays facilitates more precise studies of hIGF-I in the rat model.

Insulin-like growth factors I and II (IGF-I and II) are present in serum primarily within a ternary complex consisting of IGF, IGF-binding protein-3 (IGF-3) and acid-labile subunit (ALS). Relatively little is known about ALS as compared to the other components of the complex. We report immunoblot studies of ALS using a new rabbit antiserum to human ALS1-34. The antiserum shows high specificity for ALS, labelling only the intact 82-88 kDa doublet in whole serum. Treatment with endoglycosidase-F leads to only a partial deglycosylation of ALS in whole serum, while purified ALS is reduced to M(r) approximately 58 kDa. Acidification of both whole serum and purified ALS leads to a complete loss of ALS ability to bind to cross-linked IGFBP-3:[125I]IGF-II tracer; however, immunoblot studies show no change in the apparent M(r) of the major ALS band. Immunoblot studies of human serum shows that intact ALS is decreased in growth-hormone (GH) deficiency, increases with GH treatment, is elevated in GH excess and is unchanged with IGF-I treatment. These data provide new information regarding the characteristics of ALS and demonstrate the research utility of a highly-specific antiserum for this protein.

Insulin-like growth factor binding protein-1 (IGFBP-1) is a liver-derived protein that modulates the mitogenic actions of the insulin-like growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in vivo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of protein kinase-C (PKC) on HepG2 cell IGFBP-1 production. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 production in a time- and dose-dependent manner, with maximal stimulation occurring at 10-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than 10-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin did not inhibit PMA stimulation. The transient PKC activator diC8 had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein.

Insulin is the principal regulator of hepatic insulin-like growth factor binding protein-1 (IGFBP-1) production, mediating the rapid decrease in plasma IGFBP-1 in response to nutritional intake. In this study, we defined IGFBP-1 regulation by insulin in upper and lower body obesity, conditions associated with insulin resistance and chronic hyperinsulinemia. Overnight postabsorptive IGFBP-1 levels in obese and nonobese women showed an inverse, nonlinear relationship with plasma insulin concentrations. Maximum suppression of IGFBP-1 was seen at 70-90 pmol/L plasma insulin. Both groups of obese women had mean fasting plasma insulin concentrations above this threshold level and, consequently, markedly suppressed IGFBP-1 levels. To assess the dynamics of insulin regulated IGFBP-1, 10 obese and 8 nonobese women were studied during sequential saline infusion (0-90 min), hyperinsulinemia (insulin infusion; 90-210 min) and hypoinsulinemia (somatostatin + GH infusion; 210-330 min). Insulin infusion rapidly decreased plasma IGFBP-1 levels in nonobese subjects (60% decrease in 2 h), but had little or no further suppressive effect in obese subjects. Complete insulin withdrawal resulted in a significant rise in plasma IGFBP-1 concentrations in all subjects, but the response was blunted in obese compared to nonobese groups. In contrast to plasma IGFBP-1, IGF-I concentrations did not vary during hyper- and hypoinsulinemic infusion periods and were not significantly different between groups. Basal GH levels were significantly higher in nonobese when compared to obese women, but did not change with infusions. In conclusion, low IGFBP-1 levels in obesity are related to elevated insulin levels which are, in turn, related to body fat distribution and insulin resistance. The chronically depressed levels of IGFBP-1 may promote IGF bioactivity as well as its feedback regulation of GH secretion, thus contributing to the metabolic and mitogenic consequences of obesity. In addition, our findings imply that hepatic insulin sensitivity in terms of IGFBP-1 production is preserved despite peripheral insulin resistance in obesity.

A human hepatoma cell line, HepG2, secretes a discrete insulin-like growth factor-binding protein (IGFBP-1) into serum-free medium, which is identical to the 25K mol wt BP in amniotic fluid and plasma. IGFBP-1 levels in vivo have been shown to be inversely correlated with circulating insulin concentrations. This study investigated the direct effects of insulin on IGFBP-1 production in vitro. Addition of insulin to HepG2 cultures induced a rapid dose-dependent decrease in IGFBP-1 synthesis and secretion independent of the glucose concentration in the medium. As assessed by ligand binding and specific RIA, levels of IGFBP-1 were 20-50% of control levels in 18-h conditioned medium from insulin-treated cells. Monoclonal antibody studies indicated that the suppressive effect of insulin on IGFBP-1 synthesis was mediated through specific interaction with the insulin receptor. Therefore, HepG2 cells respond to insulin by altering the synthesis and secretion of IGFBP-1 in a manner that mimics many of the changes in plasma IGFBP-1 levels observed in vivo and provide an in vitro model for studies of IGFBP-1 biosynthesis.

Corticosterone methyl oxidase deficiency type II (CMO type II deficiency) is an autosomal recessive disorder characterized by a defect in the terminal step of aldosterone biosynthesis. Previous reports emphasized the diagnostic utility of elevated urinary ratios of 18-hydroxytetrahydro-compound A to tetrahydro-aldosterone, which are primary metabolites of 18-hydroxycorticosterone (18-OHB) and aldosterone, respectively. Limited data suggest that plasma ratios of 18-OHB to aldosterone are also abnormal in affected individuals. We report serum steroid profiles in two siblings with CMO type II deficiency. Serum levels of aldosterone precursors were elevated in both patients before treatment. In particular, the serum ratios of 18-OHB to aldosterone were greatly elevated and declined to normal levels during mineralocorticoid replacement. The possibility of heterozygote detection using this ratio is suggested. We also confirm previous reports of a detrimental effect on linear growth rate after cessation of mineralocorticoid therapy despite maintenance of normal serum electrolytes. This effect is associated with biochemical evidence of chronic salt depletion. Based on our observations, we recommend that serum 18-OHB to aldosterone ratios be routinely measured for the diagnosis and management of patients with CMO type II deficiency.

Nucleotide sequencing of the two known cDNAs encoding human insulin-like growth factor I (IGF-I) predicts two different prohormone forms of IGF-I. The predicted prohormone amino acid sequences (E peptide regions) extend the carboxy-terminus of IGF-I by either an additional 35 (IGF-IA) or 77 (IGF-IB) amino acids. We developed an antiserum directed against a synthetic peptide which is unique to the E peptide region of IGF-IA prohormone. In a RIA using this antiserum, synthetic E peptide immunoreactivity was found in the serum of patients with chronic renal failure. The protein recognized by this antiserum has a mol wt of about 13,000 by neutral gel filtration and about 19,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. These data suggest that the E peptide region of the IGF-IA prohormone is expressed and circulates as part of the prohormone.