Recombinant DNA Synthesis of Human Growth HormoneUrban, Jie Jiang, Taylor J. Severe gonadal androgen deficiency can have profound catabolic effects in man. Hypogonadal men develop a loss of lean body mass, increased adiposity, and decreased muscle strength despite normal GH and insulin-like recombinant human growth hormone pdf recombbinant I IGF-I concentrations. We designed recombinant human growth hormone pdf studies to investigate whether GH or IGF-I administration to male subjects with profound hypogonadism can diminish or abolish the catabolic effects of testosterone deficiency. Moreover, we also examined rfcombinant nature of clean bulking nedir interactions among GH, IGF-I, and androgens in specific genes of the im system.
Recombinant DNA Synthesis of Human Growth Hormone | SpringerLink
Urban, Jie Jiang, Taylor J. Severe gonadal androgen deficiency can have profound catabolic effects in man. Hypogonadal men develop a loss of lean body mass, increased adiposity, and decreased muscle strength despite normal GH and insulin-like growth factor I IGF-I concentrations.
We designed these studies to investigate whether GH or IGF-I administration to male subjects with profound hypogonadism can diminish or abolish the catabolic effects of testosterone deficiency. Moreover, we also examined the nature of the interactions among GH, IGF-I, and androgens in specific genes of the im system.
On each study day subjects had infusions of l -[ 13 C]leucine; indirect calorimetry; isokinetic dynamometry of the knee extensors; determination of body composition dual energy x-ray absortiometry and hormone and growth factor concentrations, as well as percutaneous muscle biopsies.
Their data were compared with those of previously studied male subjects who received only GnRHa. Administration of rhIGF-I and rhGH to the hypogonadal men had similar effects on whole body metabolism, with maintenance of protein synthesis rates, fat oxidation rates, and fat-free mass compared with the eugonadal state, preventing the decline observed with hypogonadism alone.
This was further amplified by the molecular assessment of important genes in muscle function. However, rhGH administration was associated with a marked increase in IGF-I and androgen receptor messenger ribonucleic acid concentrations in skeletal muscle with a reciprocal decline in IGF-binding protein-4 expression in the hypogonadal men.
The gene expression for myostatin did not change. These hormones, particularly GH, may play a role in the treatment of hypogonadal men rendered hypogonadal pharmacologically or those unable to take full testosterone replacement. The latter requires further study. GH potently and selectively stimulates nitrogen retention, increases measures of whole body protein synthesis, and stimulates accretion of lean body mass in both GH-deficient GHD and GH-sufficient subjects 1 — 3 , similar to the effects of IGF-I 4 — 9.
Both aromatizable and nonaromatizable androgens enhance measures of protein anabolism similar to those affected by GH and IGF-I. Testosterone and oxandrolone, a nonaromatizable androgen, have been shown to markedly increase measures of skeletal muscle protein synthesis in elderly and young subjects 12 , The effects of these hormones on lipid and carbohydrate metabolism are more divergent however. Whereas GH therapy is associated with a subtle, but significant, increase in insulin concentrations and hence relative insulin resistance 17 , IGF-I has insulin-like effects, lowering glucose concentrations despite suppression of circulating insulins 18 , Androgenic compounds, on the other hand, have been found to have synergistic effects with GH on lipolysis Whether IGF-I has similar synergy of effects with testosterone on lipid metabolism has yet to be studied, however.
To investigate the nature of these hormonal interactions we designed the present studies with the following aims. To accomplish this, a group of young eugonadal men was rendered hypogonadal pharmacologically and studied before and after treatment with recombinant human rh GH or rhIGF-I. A group of 13 healthy young males participated in these studies after informed written consent was obtained. Subjects were instructed to keep the same pattern of weekly exercise during these studies.
The afternoon before the isotope tracer studies, isokinetic dynamometry of the left knee extensors and flexors was performed using a Biodex Dynamometer Biodex Corp. After a min training session, followed by 30 min of rest, maximum torque production and work measures were recorded for isometric and isokinetic tests. Subjects also underwent body composition analysis using sum of skinfolds measurements and dual emission x-ray absorptiometry DEXA; model , Hologic, Inc.
On the morning of the first study D1 , after a h overnight fast, two iv heparin locks were placed, one in the antecubital vein for the infusion of isotopes and another in a contralateral hand vein kept heated for arterialized blood sampling Blood, breath, and urine samples were obtained at frequent intervals as detailed below. Shortly after the isotope infusions were finished, the subjects were fed lunch. Approximately 1 h later, a percutaneous muscle biopsy of the anterior quadriceps was performed under local anesthesia for the measurement of gene expression of different proteins in muscle.
Six weeks from baseline, blood was withdrawn and subjects were started on daily sc injections of either rhIGF-I Genentech, Inc. The subjects who received rhIGF-I were closely monitored for hypoglycemia, and the first three injections were administered at the Clinical Research Center. Glucose monitoring equipment and glucose tablets were provided. Subjects were instructed to take the rhIGF-I within 30 min of a meal to avoid hypoglycemia.
The latter was done because these metabolic studies are performed in the fasted state, and rhIGF-I can cause hypoglycemia when given as a bolus. This strategy maintains IGF-I concentrations constant and avoids hypoglycemia 4.
For both groups, study day 2 D2 was repeated identically to study D1 at 10 weeks Fig. Serum GH concentrations were measured at min intervals during the 4-h studies. A small aliquot of the urine collected during the 4-h morning study was used for determination of urea nitrogen concentration. Plasma enrichment of [ 13 C]KIC was determined at the Nemours metabolic core laboratory by mass chromatography mass spectrometry as previously described 24 , 25 13 CO 2 was measured by isotope ratio mass spectrometer as described previously Plasma glucose concentrations were measured by a glucose oxidase method using a glucose analyzer Beckman Coulter, Inc.
Fat-free mass FFM and percent fat mass were measured using DEXA and the tissue bar as well as by the sum of skin folds as described previously Table 1 summarizes the sense and antisense primers, DNA fragment size, and cycle number for each gene. These cycle numbers were chosen from previous titration assays that showed both GAP and the gene in question to be on the linear phase of the PCR assay.
Actin and myosin were measured by a ribonuclease protection assay as previously described The myostatin assay in the rhGH group was only available in four subjects. We used two different sets of primers for GAP, so that DNA fragment sizes would be sufficiently different to separate on agarose gel from the gene being tested.
Standard isotope dilution methods using the essential amino acid leucine were used in these experiments. Plasma enrichments of [1- 13 C]KIC were used as the index of intracellular enrichment of leucine using the reciprocal pool model 24 , All estimates were made at near steady state, between — min of infusion.
The rate of appearance of leucine, leucine oxidation rates, and nonoxidative leucine disposal were calculated as previously described Combustion equations calculate the oxidation of substrates sugars, lipids, and proteins from the rates of O 2 and CO 2 exchanged and total nitrogen excretion in the urine as previously described DEXA scan data were used to estimate body composition changes. FFM represents the sum of nonfat mass plus bone mineral content as calculated using the tissue composition reference bar of Hologic, Inc.
Data for the hypogonadal and GHD subjects were gathered identically to those in the present study. The statistical analysis used to test for treatment differences was ANOVA with repeated measures on one factor. Significance levels less than 0. Two series of analysis were conducted: Data from men treated with GnRHa only, published previously, are included for comparison This may be due to the great variability in the data and may or not be biologically significant.
A greater number of subjects will need to be studied to better assess this issue. Enrichments of the plasma samples for[ 13 C]KIC were: The 13 CO 2 enrichments were: There were no significant changes in rates of proteolysis leucine rate of appearance and protein synthesis nonoxidative leucine disposal when GnRHa-treated subjects were simultaneously treated with either rhIGF-I or rhGH.
These data are in contrast with the decreased lipid oxidation rates observed after 10 weeks of induced hypogonadism and the increased lipid oxidation rates after rhGH treatment of GHD patients 3 , Systeme International units for insulin and glucose are given in parentheses. Data from men treated with GnRHa only published previously, are included for comparison The expression of the androgen receptor was not significantly different from baseline in the hypogonadal men treated with rhIGF-I, yet it was increased during treatment with rhGH.
The expressions of myostatin, actin, and myosin message were unchanged during systemic rhIGF-I or rhGH treatment in these testosterone-deficient men. The data are expressed as arbitrary units, calculated as the ratio of the band densities of IGF-I over the band densities of GAP, which served as the internal control. This is the same information as that in Fig. There was evidence of mild fluid retention shortly after rhIGF-I initiation, which improved as the treatment progressed.
One subject had a hypoglycemic reaction after a dose of rhIGF-I when he skipped breakfast after the injection. Some subjects had transient tachycardia after the first few doses of rhIGF-I. Overall, there were no significant side-effects after hormone treatment. All subjects completed the week studies.
Under these controlled experimental conditions we were able to assess the effects of both rhIGF-I and rhGH on a multiplicity of metabolic pathways without the confounding effects of androgenic steroids. Our results show that even though neither IGF-I nor GH had the potent protein-anabolic effects observed in normal and GHD subjects 2 , 3 , 6 , 9 , the administration of these hormones nonetheless diminished the protein-wasting effects and helped preserve body composition decreasing the loss of lean tissue in severely hypogonadal men.
In profound testosterone deficiency we previously observed marked changes in body composition, with decreased lean body mass, increased adiposity, decreased lipid oxidation rates, and decreased rates of whole body protein synthesis These negative effects were markedly diminished in the present study in which these men were treated with rhGH or rhIGF-I, with better preservation of lean body mass and protein synthesis rates when they were treated with either of these peptides.
However, we did find a dichotomy of the effects of these hormones when we examined skeletal muscle. Induction of hypogonadism in men with GnRHa results in a decrease in lean body mass and an increase in fat mass These effects were independent of any change in systemic GH production or in IGF-I concentrations, suggesting that androgens, per se have potent anabolic actions in man.
When similar hypogonadal men were concomitantly treated with rhGH, there were no changes in body weight, yet a slight, but significant, increase in weight was observed after rhIGF-I administration. Even though we did not measure changes in total body water after treatment, it is unlikely that the differences observed are related solely to different water contents. Both GH and IGF-I have been observed to cause fluid retention and even mild edema, especially shortly after initiation of treatment 10 , 30 , The data available in humans regarding the accrual of lean body mass after GH treatment do not fully separate how much of the change is due to changes in total body water vs.
However, data in experimental animals treated with GH demonstrate that the net gain in lean body mass after GH treatment is in large part in skeletal muscle tissue. For instance, food-restricted rats treated with GH showed an increase in total muscle protein and an increase in skeletal muscle protein after GH Taken in aggregate, these data suggest that the changes in lean body mass are probably in part associated with an increase in lean soft tissue separate from body water.
Further studies using labeled water and other body composition tools will need to be performed to fully characterize these changes. In addition, rates of protein synthesis as measured by stable isotopes studies were unchanged from baseline rates after treatment with either rhIGF-I or rhGH. This is in sharp contrast to the marked decrease in rates of nonoxidative leucine disposal a measure of whole body protein synthesis observed after 10 weeks of sustained hypogonadism induced by GnRHa, as reported previously The latter suggests that systemic IGF-I cannot fully overcome the protein catabolic effects of hypogonadism as well as GH.
Taken in aggregate, the present data suggest that IGF-I and GH can preserve lean body mass and sustain protein synthesis rates in hypogonadal individuals. The data also suggest that androgens may be needed for the full effects of GH and IGF-I on protein pools to be observed. After 10 weeks of severe hypogonadism, otherwise healthy men showed substantial decreases in the rate of oxidation of lipids, as measured by indirect calorimetry These rates, however, remained invariant in the present study when hypogonadal men were treated with either rhGH or rhIGF-I.