Jack L. Kostyo

Metabolic Effects of Plasmin Digests of Human Growth Hormone in the Rat and Man

As a first step in our study of structure-function relationships among primate and non-primate growth hormones, human growth hormone (hGH) was subjected to the limited digestive activity of human plasmin. The lyophilized whole digest, containing less than 2% of unchanged hormone, had an average of 2.3 new amino-terminal groups per mole. The digest had the same potency as the native hormone (a) in causing weight gain in hypophysectomized rats; (b) in stimulating somatomedin production in hypophysectomized rats; (c) in stimulating upake of [(3)H]leucine into isolated diaphragm of hypophysectomized rats; (d) in accelerating transport of [(14)C]alpha-aminoisobutyric acid into isolated diaphragm of hypophysectomized rats; (e) in stimulating uptake of [3-0-methyl-(14)C]glucose by isolated adipose tissue of hypophysectomized rats; (f) in accelerating conversion of [(14)C]glucose to (14)CO(2) by isolated epididymal adipose tissue of hypophysectomized rats. The digest also caused glucosuria in partially pancreatectomized rats treated with dexamethasone. These metabolic actions of plasmin-digested hGH in the array of animal tests were confirmed by comparable effects elicited in 11 human subjects (nine pituitary-deficient children and adolescents and two nondeficient adults). A single injection of the plasmin digest caused an increase in plasma free fatty acids and a fall in plasma amino acids. Seven daily injections caused positive balances of nitrogen, phosphorous, sodium, and potassium, gain in body weight, and in two of three subjects impairment of glucose tolerance. The potency of the plasmin digest in producing these metabolic effects in man was comparable to that of native hGH.Thus, 2-3 bonds in the hGH molecule can be cleaved by plasmin without impairing the hormones growthpromoting, anabolic, diabetogenic, and adipokinetic actions for rat and man.

Inhibition of metabolic effects of growth hormone by various inhibitors of cyclic nucleotide phosphodiesterase

Abstract When isolated diaphragms of hypophysectomized rats were incubated with bovine growth hormone in the presence of the cyclic nucleotide inhibitors theophylline, quinine and papaverine, the stimulatory effects of the hormone on leucine incorporation into protein, α-aminoisobutyric acid and 3- O -methylglucose transport were suppressed or abolished entirely. The degree of suppression of the hormone effects appeared to correlate with the extent of glycogenolysis caused by the drugs. Thoephylline also rapidly reversed the stimulation of protein synthesis and amino acid and sugar transport produced by growth hormone. When protein synthesis and transport were stimulated by preincubation of the diaphragm with growth hormone, the subsequent addition of theophylline to the medium inhibited the hormonal effects on protein synthesis and sugar transport within 15 min and the effect on amino acid transport within 60 min. These results may mean that the rapid in vitro effects of growth hormone on protein synthesis and membrane transport in rat diaphragm muscle are mediated by a reduction in the cellular level of cyclic AMP or some other nucleotide. Attempts to block the action of growth hormone on 3- O -methylglucose transport by preincubation of the diaphragm with high concentrations (10 mM) of cyclic GMP, cyclic UMP, cyclic TMP and cyclic CMP were unsuccessful. Also an effort was made to mimic the action of growth hormone on sugar transport by incubating the diaphragm with high concentrations of imidazole and histamine, agents known to activate cyclic nucleotide phosphodiesterase. Slight stimulatory effects were obtained, but they could not be correlated with any certainty to the actions of imidazole and histamine on phosphodiesterase. Like growth hormone, insulin also stimulates protein synthesis and amino acid and sugar transport in the isolated rat diaphragm. However, the actions of insulin on these processes were not abolished by theophylline, suggesting some basic difference in the mode of action of these two hormones on protein synthesis and membrane transport in muscle.

The search for the active core of pituitary growth hormone

Abstract The conclusion reached from studies involving the biologic characterization of fragments of pituitary growth hormone produced by chemical or enzymatic cleavage or by synthesis is that the entire native growth hormone molecule is not required to produce most and perhaps all of the metabolic effects of the hormone. Thus, the early speculation that the growth hormone molecule contains an active core or amino acid sequence responsible for its metabolic activities appears to be correct. In all studies with growth hormone fragments of known structure, the biologic activities of the hormone have been found in the N-terminal two thirds of the molecule (up to residue 134). Fragments of the C-terminal portion of the hormone molecule have usually been inactive. However, there is evidence suggesting that the C-terminal third of the native molecule may serve to stabilize or protect the hormone from enzymatic destruction in the circulation. The region of the hormone molecule consisting of residues 95–134 appears to compose part of the active core, since some natural and synthetic peptides spanning this region have biologic activity. However, these peptides have only weak activity compared to native growth hormone, suggesting that additional amino acid residues N-terminal to position 95 are required for full biologic activity. Thus, the active core of growth hormone may consist of a substantial portion of the N-terminal two thirds of the hormone molecule.

In Vitro Effects of Growth Hormone on Cyclic AMP Metabolism in the Isolated Rat Diaphragm

Publisher Summary This chapter discusses the in vitro effects of the growth hormone on cyclic AMP metabolism in the isolated rat diaphragm. To test the hypothesis that growth hormone reduces the amount of cAMP in the small free or metabolically relevant pool of nucleotide in the diaphragm, an indirect method was devised for obtaining a relative estimate of the amount of free cAMP present in the tissue. The method is based upon the experiments of Kuo and De Renzo with isolated rat adipose tissue. These investigators labeled the cAMP of the isolated adipose tissue with 14 C-adenine and then monitored the release of 14 C-cAMP from the tissue after treating it with various hormones and drugs. To facilitate the release of 14 C-cAMP from the cells, the plasma membrane was made more permeable to the nucleotide by treating the tissue with polyenes, such as filipin, which remove membrane sterols. This procedure was utilized to test the in vitro effects of the growth hormone on the release of labeled cAMP from the isolated rat diaphragm, to learn whether the hormone has any influence on the free pool of cAMP in the muscle cell. The results of this study are discussed in detail in the chapter.

Anabolic actions of reduced and S-carbamidomethylated human growth hormone and its plasmin digest in man.

Six children aged 12-15 yr, deficient in endogenous growth hormone, were each treated, after a 7-day control period, for 7 days with 0.0168, 0.052, and 0.168 U/kg body wt3/4 human growth (hGH) (doses A, B, and C, respectively) in separate metabolic balance studies. Doses B and C caused a dose-related retention of N, P, K, Na, and Cl in ratios of 1/0.069/4.5/7.5/5.6. These ratios indicate increments in masses of protoplasm/extracellular fluid (ECF)/bone in ratios of 1/2.0/ less than 0.001. Three of the children were also treated with doses A, B, and C of reduced and carbamidomethylated hGH (RCAM-hGH). Doses B and C caused 1.2-2.8 times as much retention of N, P, and K, and 0.3-0.5 times as much retention of Na and Cl, as did the corresponding doses of hGH. The plasmin digest of RCAM-hGH gave results generally similar to RCAM-hGH. For RCAM-hGH and its plasmin digest, N, P, K, Na, and Cl were retained in ratios of about 1/0.14/5.4/2.2/2.1, indicating increments of protoplasm/ECF/bone of about 1/0.8/0.05. These findings indicate that reduction and carbamidomethylation alter the anabolic actions of hGH in man in both quantitative and qualitative manner. RCAM-hGH is more potent in stimulating enlargement of protoplasm and bone, and less potent in stimulating expansion of ECF, than is the native hormone. The profile of anabolic actions of RCAM-hGH in man does not appear to be further altered by digestion with plasmin.