Biosynthetic human growth hormone: effects on growth of Snell dwarf mice

Bacterially synthesized human growth hormone (bhGH) administered to Snell dwarf mice during 4 weeks, induced an increase in body length and weight to a comparable degree as obtained with pituitary-derived human growth hormone (hGH). At a dose of 150 mU/day both bhGH and hGH induced a significant stimulation over saline-treated controls, of the weight of the submandibular salivary glands, the m. quadriceps femoris and gastrocnemius, the heart, liver, kidneys, thymus and spleen. The weight of the brain and the thickness of the skinfold were not influenced by either of the preparations used. When organ weights were expressed as a function of body weight, the contribution of the kidneys to body weight was significantly higher with hGH than with bhGH. The other organs studied did not show differences. As a biochemical parameter of cartilage growth, the sulfate incorporation into costal and epiphyseal cartilage in vitro was measured, and it was found to be stimulated by both hormones after short-term treatment. Thus bacterially synthesized hGH behaves identically to pituitary-derived hGH with respect to body length, sulfate incorporation into costal and epiphyseal cartilage, body weight and organ growth of Snell dwarf mice, with one exception: increase of weight of the kidneys, as a function of body weight, was more pronounced after treatment with hGH than with bhGH.     

Effects of part sequences of human growth hormone on in vivo hepatic glycogen metabolism in the rat.

Acute effects of two part sequences of human growth hormone on the in vivo activity levels of hepatic glycogen synthase and glycogen phosphorylase were examined. The peptide corresponding to residues 6 to 13 of the hormone (hGH 6--13) decreased the percentage of phosphorylase in the active form without affecting synthase activity. This action was indirect and dependent upon insulin. The peptide hGH 177--191 decreased the level of the active form of synthase without affecting phosphorylase activity. This effect was also observed with analogous peptides containing the sequence hGH 178--191 (i.e., hGH 172--191 and hGH 178--191), whereas the peptide hGH 179--191 was inert. The onset of these effects was rapid, and maximum changes in activity were produced in 5 min by both peptides. The effect for hGH 177--191 was short-lived, and synthase activity had returned to normal levels by 15 min, whereas the action of hGH 6--13 was of longer duration and was still quite marked at 60 min. Both peptides showed a linear dependence of response to the log dose of peptide injected over the range 0.1--250 microgram hGH 6--13 per kg body weight and 0.05--25 microgram hGH 177--191 per kg body weight. Hepatic 3',5'-cyclicadenylic acid levels were not affected by either peptide. Incorporation of glycerol carbon into liver glycogen was increased by hGH 6--13 and decreased by hGH carbon into liver glycogen was increased by hGH 6--13 and decreased by hGH 177--191. This is discussed in terms of a futile cycle between glycogen and hexose phosphate in the liver, as the basis for a control mechanism for hepatic glycogen metabolism. The present observations are consistent with other in vivo and in vitro actions of these and related peptides.     

Effects of part sequences of human growth hormone on in vivo hepatic glycogen metabolism in the rat

Acute effects of two part sequences of human growth hormone on the in vivo activity levels of hepatic glycogen synthase and glycogen phosphorylase were examined. The peptide corresponding to residues 6 to 13 of the hormone (hGH 6–13) decreased the percentage of phosphorylase in the active form without affecting synthase activity. This action was indirect and dependent upon insulin. The peptide hGH 177–191 decreased the level of the active form of synthase without affecting phosphorylase activity. This effect was also observed with analogous peptides containing the sequence hGH 178–191 (i.e., hGH 172–191 and hGH 178–191), whereas the peptide hGH 179–191 was inert.The onset of these effects was rapid, and maximum changes in activity were produced in 5 min by both peptides. The effect for hGH 177–191 was short-lived, and synthase activity had returned to normal levels by 15 min, whereas the action of hGH 6–13 was of longer duration and was still quite marked at 60 min. Both peptides showed a linear dependence of response to the log dose of peptide injected over the range 0.1–250 μg hGH 6–13 per kg body weight and 0.05–25 gmg hGH 177–191 per kg body weight. Hepatic 3′,5′-cyclicadenylic acid levels were not affected by either peptide. Incorporation of glycerol carbon liver glycogen was increased by hGH 6–13 and decreased by hGH 177–191. This discussed in terms of a futile cycle between glycogen and hexone phosphate in the liver, as the basis for a control mechanism for hepatic glycogen metabolism. The present observations are consistent with other in vivo and in vitro actions of these and related peptides.     

Normalization of growth in hypophysectomized mice using hydrodynamic transfer of the human growth hormone gene

Nonviral gene transfer was investigated as a potential treatment of growth hormone deficiency (GHD) using hypophysectomized mice as a model. After a single hydrodynamic administration of naked plasmid DNA containing the human growth hormone (hGH) gene controlled by an ubiquitin promoter, sustained elevation of circulating hGH was observed the entire observation period (68 days), with a concomitant normalization of circulating insulin-like growth factor I (IGF-I) and IGF-binding protein-3. Furthermore, longitudinal growth was corrected in terms of normalization of tibia length, tail length, and body weight gain. Liver, spleen, and lung weights were normalized, whereas heart weight was normalized partly. hGH mRNA was expressed exclusively in liver tissue. In conclusion, we showed that nonviral hGH gene transfer normalizes longitudinal growth in hypophysectomized mice, indicating that this method potentially could be relevant as a new therapeutic tool in the clinical handling of GHD.     

Non-conventional use of growth hormone: European experience

Results from several centres in Europe using biosynthetic human growth hormone (hGH) for the promotion of growth in a variety of conditions other than classical hGH deficiency were evaluated. Significant increments in growth rates were achieved by daily administration of hGH in doses appropriate for body size without disproportionate skeletal advances in short normals, Turner syndrome, low birth weight, skeletal dysplasia, central precocious puberty (reared with gonadotrophin-releasing hormone analogue) and renal failure.     

The growth factor from plerocercoid larvae of the tapeworm, Spirometra mansonoides, stimulates growth but is not diabetogenic

A factor produced by plerocercoids of the tapeworm Spirometra mansonoides is similar to human growth hormone (hGH) in that it stimulates body growth, binds to hGH receptors, cross-reacts with anti-hGH antibodies, and has lactogenic and insulin-like activities. The purpose of this study was to determine whether plerocercoid growth factor (PGF) is similar to hGH in expressing diabetogenic activity in the genetically obese (ob/ob) mouse. To determine an effective dose for use in the obese mice, the ability of daily injections of PGF to stimulate growth of phenotypically normal mice of the same strain was assessed in a 10-day weight gain assay. Injections of PGF stimulated a dose-dependent weight gain (r = 0.83) and 25 ng eq/day of PGF stimulated a response not significantly different from that produced by 100 micrograms of bovine growth hormone/day. Diabetogenicity was assessed using fasting blood glucose and glucose tolerance tests in obese mice that had been injected for 3 days with saline, hGH, or PGF. Human growth hormone caused a significant increase (P less than 0.005) in fasting blood glucose and glucose tolerance of the obese mice was impaired (P less than 0.01). All of the doses of PGF used to test diabetogenicity in the obese mice were at least twice that required to stimulate a maximal growth response in normal mice, yet none of the doses of PGF increased fasting blood glucose or decreased glucose tolerance. These results show that PGF was a potent growth stimulant but was not diabetogenic.     

Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine-165 to alanine

We have mutated a synthetic human growth hormone (hGH) gene specifically at the codon for Cys-165 to a codon for Ala by replacement of synthetic deoxyoligonucleotides corresponding to this site. This modification prevented the formation of a disulfide bond between Cys-53 and Cys-165 in the hGH molecule. This mutated protein, [Ala165]hGH was expressed at the same level as the intact hGH, 4 X 10(5) molecules per cell under the control of the tryptophan promoter in Escherichia coli, and retained similar immunological activity to intact hGH. The limited digestion pattern of the mutated protein with human plasmin suggests that the tertiary structure of [Ala165]hGH resembles to that of the intact hGH molecule. [Ala165]hGH revealed full biological activity as examined by the body weight increase of hypophysectomized rats.     

Differential effects of hGH and IGF-I on body proportions

The differential growth effects of hGH and IGF-I on the upper/lower (U/L) body segment in relation to height (Ht) were analyzed in 15 patients with isolated Growth hormone deficiency (IGHD,:7M, 8F) mean age 5.0 +/- 3.2 (SD) years treated with hGH; 21 patients with multiple pituitary hormone deficiency including growth hormone (MPHD: 14M, 7F) aged 10.0 +/- 3.8, treated with hGH; 9 patients with Laron Syndrome (LS) (4M,5F) aged 6.9 +/- 5.6 years treated with IGF-I; 9 boys with intrauterine growth retardation (IUGR) aged 6.3 +/- 1.25 years treated by hGH; and 22 boys with idiopathic short stature (ISS) aged 8.0 +/- 1.55 years treated by hGH. The dose of hGH was 33 microg/kg/day, that of IGF-I 180-200 microg/kg/day. RESULTS: the U/L body segment ratio in IGHD patients decreased from 2.3 +/- 0.7 to 1.1 +/- 0.7 (p <0.001), and the Ht SDS increased from -4.9 +/- 1.3 to 2.3 +/- 1 (p < 0.001) following treatment. In MPHD patients the U/L body segment decreased from 1.1 +/- 1.1 to -0.6 +/- 1.0 (p < 0.001), and the Ht SDS increased from -3.3 +/- 1.4 to -2.5 +/- 1.0 (p < 0.009). In the LS group the U/L body segment ratio did not change with IGF-I treatment but Ht improved from -6.1 +/- 1.3 to -4.6 +/- 1.2 (p < 0.001), The differential growth response of the children with IUGR and with ISS resembled that of the children with LS. CONCLUSIONS: hGH and IGF-I act differentially on the spine and limbs.     

Influence of recombinant bovine growth hormone on growth and feed intake in juvenile Nile crocodiles (Crocodylus niloticus)

• 1.1. Slow-growing juvenile Nile crocodiles were injected with recombinant bovine growth hormone (rbGH) once a week for 6 weeks and then re-treated after 4 weeks.
• 2.2. The feed intake of the treated crocodiles was 26 g/kg/meal during the three periods, while the intakes of the controls were 15, 20 and 2 g/kg.
• 3.3. The treated gained 2.3 and 0.9%/week in weight during the first and second injection period and the feed conversion efficiencies were 28 and 13%. The controls lost weight.
• 4.4. The treated animals grew at rates of 0.98 and 0.43%/week during the first and the second injection period.
• 5.5. Bovine GH enhances growth in juvenile crocodiles and seems to have less adverse effects than human GH.

     

Effects of hypophysectomy and the insulin-like and anti-insulin pituitary peptides on carbohydrate metabolism in yellow Avy/A (BALB/c x VY)F1 hybrid mice

The amino-terminal portion of human growth hormone, residues 1-43 (hGH1-43), has insulin-potentiating action, while a hyperglycemic pituitary peptide (HP), which co-purifies with human growth hormone (hGH), is antagonistic to the action of insulin. The effects of hGH, hGH1-43, and HP on glucose metabolism were assessed in young (4-5 weeks) and adult (6-8 months) hypophysectomized yellow Avy/A mice which lacked any interfering endogenous pituitary hormones, and compared with age-matched intact obese yellow Avy/A and lean agouti A/a mice. Treatment with hGH1-43 or HP did not promote body growth in hypophysectomized yellow mice; but after 2 weeks of treatment with hGH, there was a significant increase in body weight (P less than 0.05). Treatment with HP raised blood glucose and lowered insulin concentrations in obese yellow mice, but not in agouti or hypophysectomized yellow mice. The severely impaired glucose tolerance of the hypophysectomized yellow mice was improved by acute (60 min) and chronic (3 days) treatment with hGH1-43 as well as by 2 weeks of treatment with hGH; in contrast, HP had no effect. Glucose oxidation in adipose tissue from obese yellow mice was low and showed essentially no response to stimulation by insulin at doses lower than 1000 microunits/ml. Basal glucose oxidation rates in adipose tissue taken from agouti and hypophysectomized yellow mice were significantly higher (P less than 0.001) than those in tissue from obese yellow mice, and the rates responded significantly (P less than 0.05) to 100 microunits/ml insulin. The insulin binding affinities in liver membranes from agouti mice were higher than those from either obese or hypophysectomized yellow mice. The insulin receptor densities were similar in both agouti and obese yellow mice, but higher in hypophysectomized yellow mice (P less than 0.05). Treatment with hGH1-43 slightly increased, although not significantly, the insulin receptor density in yellow obese mice while hGH showed essentially no change. Therefore, hypophysectomy appeared to increase tissue response and decrease insulin resistance by increasing receptor numbers and lowering the circulating insulin levels. Furthermore, the insulin-like action of hGH was elicited directly in vivo by hGH1-43 in hypophysectomized yellow mice.     

Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3–0.5 ng/10⁵ cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10⁵ cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.     

Growth-stimulating effects of somatomedin-/insulin-like peptides in Snell dwarf mice

The effect of the somatomedin-/insulin-like growth factors IGF-I, IGF-II and N2, as well as of semi-purified SM fractions separated by isoelectric focusing derived from human Cohn IV on different growth parameters, have been studied in the Snell dwarf mouse. HPLC-pure IGF-II, N2 and IGF-I stimulate to a similar extent the sulphate incorporation into costal cartilage, the osteochondral junction and epiphyseal cartilage. After 4 weeks of treatment, increase in body length and weight as well as the weights of several organs is obtained with SM fractions, focusing at acid and neutral pH, and containing mainly IGF-II- and less than 5% IGF-I-like peptides. Fractions containing mainly IGF-I-like peptides and focusing at basic pH at the dosage used seem to be less stimulatory on most of these parameters. The rump/tail ratio and weight/length ratio is comparable to that obtained after treatment with human growth hormone (hGH). hGH induced a significant stimulation of the weight of the liver, kidneys, heart, thymus and spleen. The acid and neutral SM fractions induced growth of the liver, kidneys and spleen. The basic fractions only produced a significant weight gain in kidneys and spleen. The skinfold thickness is stimulated by the SM preparations and only slightly by hGH.     

人生长激素慢病毒载体的构建及其在鼠骨骼肌成肌细胞中的表达

构建能表达人生长激素(hGH)的pLentivirus6/V5-hGH载体,并实现hGH基因在骨骼肌成肌细胞中大量、长期和稳定的表达。体外培养SD鼠骨骼肌成肌细胞,并通过免疫组织化学方法鉴定所得细胞、用台酚兰染色确定培养细胞的活性并绘制生长曲线。将目的基因hGH亚克隆到真核细胞表达载体pLenti6/V5-D-TOPO载体上,构建重组质粒pLentivirus6/V5-hGH。将pLenti6/V5-hGH及阳性对照质粒pLenti6/V5-EGFP分别用Lipofectamin2000介导转染体外培养的SD乳鼠骨骼肌成肌细胞。在激光共聚焦扫描显微镜下计数,确定阳性对照质粒的转染数,从而估计该基因的转染效率。加入筛选试剂以获得稳定表达异源生长激素(GH)的成肌细胞。收集转染及筛选后的细胞培养基,用放射免疫分析法(RIA)检测重组人生长激素(rhGH)的表达水平。聚合酶链式反应法(PCR)及DNA测序显示hGH基因成功地插入到pLenti6/V5-D-TOPO载体中;阳性对照质粒转染细胞24h后,在激光共聚焦显微镜下观察,其转染效率达40%以上。检测收集的上清,与对照组相比,有极显著差异(P<0.01),观察至第8周,rhGH仍持续稳定表达。通过检测培养的chang-liver肝细胞上清中胰岛素样生长因子-1(IGF-1)的水平,验证了rhGH的生物学活性。实验通过培养高纯度的成肌细胞,构建了能在真核细胞内表达hGH的重组质粒pLenti6/V5-hGH,实现了hGH基因在骨骼肌成肌细胞中大量、长期和稳定的表达,并且获得的rhGH具有较强的促进肝细胞分泌IGF-1的能力。     

Metabolic studies of a synthetic lipolytic domain (AOD9604) of human growth hormone

A synthetic analogue (AOD9604) of the lipolytic domain of human growth hormone (hGH) has been studied for its metabolic actions in obese Zucker rats. Daily treatment with an oral dose of AOD9604 of 500 microg/kg body weight for 19 days reduced over 50% (15.8 +/- 0.6 vs. 35.6 +/- 0.8 g) body weight gain of the animals in comparison with the control. The adipose tissues of the AOD9604--treated animals were found to have an increase in lipolytic activity. In contrast to chronic treatment with intact hGH, chronic treatment with AOD9604 showed no adverse effect on insulin sensitivity of the animals, as demonstrated with euglycemic clamp techniques. The results in the present study suggest that the analogue of the hGH lipolytic domain may have the potential to be developed into an orally usable and safe therapeutic agent for obesity.     

Chemically defined medium for the production of biologically active substances of CHO cells

A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.Abbreviations CHO Chinese hamster ovary - hGH human growth hormone - dFCS dialyzed fetal calf serum - dhfr dihydroforate reductase - MTX methotrexate     

Effects of oral administration of a synthetic fragment of human growth hormone on lipid metabolism

A small synthetic peptide sequence of human growth hormone (hGH), AOD-9401, has lipolytic and antilipogenic activity similar to that of the intact hormone. Here we report its effect on lipid metabolism in rodent models of obesity and in human adipose tissue to assess its potential as a pharmacological agent for the treatment of human obesity. C57BL/6J (ob/ob) mice were orally treated with either saline (n = 8) or AOD-9401 (n = 10) for 30 days. From day 16 onward, body weight gain in AOD-9401-treated animals was significantly lower than that of saline-treated controls. Food consumption did not differ between the two groups. Analyses of adipose tissue ex vivo revealed that AOD-9401 significantly reduced lipogenic activity and increased lipolytic activity in this tissue. Increased catabolism was also reflected in an acute increase in energy expenditure and glucose and fat oxidation in ob/ob mice treated with AOD-9401. In addition, AOD-9401 increased in vitro lipolytic activity and decreased lipogenic activity in isolated adipose tissue from obese rodents and humans. Together, these findings indicate that oral administration of AOD-9401 alters lipid metabolism in adipose tissue, resulting in a reduction of weight gain in obese animals. The marked lipolytic and antilipogenic actions of AOD-9401 in human adipose tissues suggest that this small synthetic hGH peptide has potential in the treatment of human obesity.     

Detection of human growth hormone doping in urine: out of competition tests are necessary

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2–50 ng hGH/1. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 I.U. every day). The u-hGH excretion after treatment showed dramatic increases of 50–100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and β2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.     

Synthesis and purification of a deleted human growth hormone, hGH delta 135-146: sensitivity to plasmin cleavage and in vitro and in vivo bioactivities

Proteolytically cleaved human 22 kDa growth hormone (22K hGH) between the amino acid residues 134 and 150 by plasmin or other proteases in vitro has been reported to be most active in growth promoting activity. In this study a deleted mutant hGH lacking amino acid residues from 135 to 146 and having more sensitivity to plasmin digestion was produced using the inverse polymerase chain reaction method and the Escherichia coli expression system. The mutant, hGH delta 135-146, was folded and purified effectively and found to be more sensitive to plasmin cleavage to form the two-chain form in vitro. The biological activities of this plasmin sensitive hGH delta 135-146 were tested by in vitro cell proliferation assays and in vivo growth promoting assay. In Ba/F3-hGHR cells, which express receptors for hGH, hGH delta 135-146 showed 10-20% less growth promoting activity than 22K hGH, but expressed comparable quantities of IGF-I mRNA to that of 22K hGH. In Nb2 rat lymphoma cells, which proliferate in response to hGH via the lactogenic receptors, hGH delta 135-146 showed equivalent activities to those of 22K hGH at lower concentrations. By the body weight gain test using hypophysectomized rats, a lower dose (2.5 nmol kg-1) of hGH delta 135-146 exhibited an equivalent activity to that of wild type 22K hGH, but a higher dose (25 nmol kg-1) of the mutant showed less growth promoting activity than 22K hGH. These results indicated that the plasmin sensitive recombinant hGH delta 135-146 failed to show higher biological activity than the 22K hGH in vivo, suggesting the unsuccessful formation of the active two-chain form in vivo.