Adipose Tissue Of Hypophysectomized Rats Research Articles

Evidence that the N-terminus of human growth hormone is involved in expression of its growth promoting, diabetogenic, and insulin-like activities.

Recent work with various point and deletion mutants of human GH (hGH) has suggested that the proximal N-terminal end of the hormone molecule is important for its growth promoting action. This study was conducted to examine the growth promoting, diabetogenic, and insulin-like activities of two N-terminal mutants of hGH, the deletion mutant Des-7 hGH (met8, ala11), and a chimeric mutant of bovine GH (bGH) and hGH containing the N-terminal 13 amino acids of bGH (met, ala 1-13/14-191, asp11). The CD spectra of these mutants are similar to that of wild-type hGH and they retain lactogenic activity on Nb2 lymphoma cells, whereas their ability to bind to somatogenic receptors on IM-9 lymphocytes and bovine liver membranes is markedly reduced. In this study, growth promoting activity of the mutants was assessed using the 9-day weight gain test in hypophysectomized rats. Des-7 hGH had a potency of 0.03 IU/mg protein in this assay, whereas the potency of the bGH/hGH chimera was 0.71 IU/mg. Diabetogenic activity was tested in the ob/ob mouse, using the elevation of fasting blood glucose and the worsening of glucose tolerance after a 3-day course of treatment as end-points. Both Des-7 hGH and the bGH/hGH chimera had reduced diabetogenic activity compared to that of biosynthetic wild-type hGH, consistent with their reduced growth activity. Insulin-like activity was assessed by testing the in vitro ability of the mutants to stimulate [14C] glucose oxidation by epididymal adipose tissue of hypophysectomized rats. Des-7 hGH had about 1% the activity of wild-type hGH, whereas the chimera was about 20% as active. When Des-7 hGH was added to the incubation medium along with wild-type hGH in ratios of 5, 12.5, or 25:1 (Des-7 hGH:hGH), the insulin-like action of hGH was significantly inhibited, indicating that the mutant is a modest antagonist of the insulin-like action of hGH. When the ability of Des-7 hGH to compete with [125I] hGH for binding to isolated rat adipocytes was tested, the mutant was about 10% as effective as wild-type hGH. Thus, Des-7 hGH appears to be more effective in binding to adipocyte GH receptors than in triggering an insulin-like response, perhaps accounting for its modest antagonistic activity. The results of this study suggest that the proximal N-terminal end of the hGH molecule is involved in the expression of the growth promoting, diabetogenic and insulin-like activities of GH.

The influence of growth hormone and thyroxine on iodothyronine deiodinase activity in the liver, kidney and brown adipose tissue in hypophysectomized rats

The effects of GH and T4 substitution on peripheral iodothyronine deiodinase activity in the liver, kidney and brown adipose tissue of hypophysectomized rats were investigated. Animals were treated with GH (140 micrograms hGH/day), T4 (3 micrograms/day), GH plus T4 (same doses), or saline. Rats were killed 0, 4, 7 or 11 days after treatment was started. Non-hypophysectomized, age-matched rats were killed after 0 and 11 days and served as controls. GH plus T4 restored body weight gain to normal, whereas GH alone and T4 alone did not. Tissue deiodinase activity and T3 concentrations were severely depressed in the hypophysectomized rats compared with non-hypophysectomized controls (to less than 10%). GH substitution in hypophysectomized rats led to a slight but significant elevation in tissue iodothyronine deiodinase activity in the liver and kidney, without concomitant increases in T3. Deiodinase activity in brown adipose tissue did not differ from that in saline-treated controls. T4 administration normalized deiodinase activity and tissue T3 content in all the evaluated tissues. GH plus T4 resulted in a lesser increase in deiodinase activity than T4 alone in the liver and kidney (p less than 0.01 at day 11), whereas no significant difference was observed in brown adipose tissue. In conclusion, GH stimulates iodothyronine deiodinase activity of the liver and kidney in hypophysectomized rats. Moreover, when GH is administered together with T4, the T4-stimulated enzyme activity in the liver and kidney is downregulated, suggesting that GH attenuates (or modulates) the T4 effect on this specific enzyme activity.

Evidence for a Role of Protein Kinase C in the Stimulation of Lipolysis by Growth Hormone and Isoproterenol*

The mechanism that underlies activation of lipolysis by GH is not yet understood. Although cAMP is thought to be involved, the biochemical linkages between GH and cAMP are unknown, and lipolysis produced by GH differs from that produced by such typical activators of adenylate cyclase as the catecholamines with regard to time course, maximum response, and dependence on other factors such as glucocorticoids or theophylline. The present studies were undertaken to evaluate the possibility that activation of protein kinase C by GH may play an important role in the production of the delayed increase in lipolysis. Phorbol myristate acetate (PMA), a known activator of protein kinase C, increased lipolysis in segments of adipose tissue of hypophysectomized rats, and, as with GH, this effect was potentiated by theophylline. The lipolytic effects of PMA were concentration-dependent, required a shorter lag period than those of human GH, increased as the concentration of PMA was raised from 0.1 to 10 microM, and were additive at all concentrations with lipolysis produced by saturating concentrations of GH. The lipolytic actions of GH, but not PMA, were potentiated by dexamethasone in adipose tissue of normal rats. Sphingosine and staurosporine, which are known to inhibit protein kinase C, blocked the lipolytic effects of PMA and severely reduced lipolysis in response to GH in tissues of both normal and hypophysectomized rats. Although higher concentrations of sphingosine interfered with the specific binding of 125I-labeled human GH to isolated adipocytes, the inhibitory effects of sphingosine cannot be attributed to interference with GH binding, since it decreased lipolysis by at least 50% when used at concentrations that were too low to reduce binding significantly. Staurosporine produced little (approximately 20%) or no decrease in binding. At concentrations that severely reduced lipolysis in response to GH and dexamethasone, sphingosine had little or no effect on lipolysis in response to dibutyryl cAMP or forskolin. Staurosporine and sphingosine, however, severely inhibited lipolysis in response to isoproterenol. We conclude that protein kinase C activity plays an important role in hormone-stimulated lipolysis probably by an action exerted on the transduction pathway proximal to cAMP. The present data are equally consistent with the possibilities that GH and/or isoproterenol activate protein kinase C, or that protein kinase C is constitutively active to some extent. It is likely that protein kinase C activity is permissive for, rather than a mediator of, the lipolytic actions of GH and isoproterenol.

Glycosylation abolishes an in vitro insulin-like action of prolactin

Ovine prolactin stimulated 14C-CO2 production from labeled glucose in adipose tissue of hypophysectomized rats in vitro, an insulin-like activity. Glycosylation of the hormone by attachment of a carbohydrate unit at asparagine31 abolished this in vitro insulin-like action. However, neither nonglycosylated nor glycosylated prolactin exhibited in vivo insulin-like action, as they did not lower serum glucose or non-esterified fatty acids in fasted hypophysectomized rats. Hindrance of receptor binding by the carbohydrate unit may account for the absence of in vitro insulin-like activity in glycosylated prolactin, but the dichotomy between in vivo and in vitro insulin-like actions for prolactin remains obscure.

Biosynthetic 20-kilodalton methionyl-human growth hormone has diabetogenic and insulin-like activities.

The anterior pituitary gland produces a 20-kilodalton (kDa) variant of human growth hormone (hGH) that differs from the predominant 22-kDa form of hGH in that amino acid residues 32-46 are deleted. Previous work has suggested that the 20-kDa variant possesses the full growth-promoting and lactogenic activities of 22-kDa hGH but lacks its intrinsic diabetogenic and insulin-like activities. In the present study, recombinant DNA techniques were used to prepare biosynthetic 20-kDa hGH, and some of the biological properties of the purified hGH variant were examined. The biosynthetic 20-kDa hGH variant was found to share the propensity for aggregation exhibited by its native counterpart. Moreover, like the native variant, biosynthetic 20-kDa hGH possessed full growth-promoting activity in the weight gain test in hypophysectomized rats. However, contrary to previous work suggesting that native 20-kDa hGH lacks diabetogenic and insulin-like activities, biosynthetic 20-kDa hGH was found to have substantial diabetogenic activity when administered chronically to ob/ob mice and to possess approximately 20% the in vitro insulin-like activity of biosynthetic 22-kDa hGH on isolated epididymal adipose tissue of hypophysectomized rats. The diabetogenic and insulin-like activities of biosynthetic 20-kDa hGH cannot be ascribed to contamination of the hormone preparation with the 22-kDa form of hGH or with other diabetogenic or insulin-like pituitary peptides. Therefore, the results strongly suggest that diabetogenic and insulin-like activities are also intrinsic properties of the 20-kDa variant of hGH.

Effect of temperature on lipoprotein lipase and lipogenic enzyme activities in brown adipose tissue of hypophysectomized rats.

It has been shown that the same modifications on the composition of brown adipose tissue (BAT) which are normally induced following cold stimulation are also observed in hypophysectomized rats acclimated either at 28 degrees C or 15 degrees C. To test the possibility of BAT stimulation in hypophysectomized rats, we have determined some enzymatic activities known to modulate the energy supply to that organ. Seven week old Long-Evans rats were hypophysectomized. Three weeks later, they were exposed to either 28 degrees C or 15 degrees C ambient temperature for five or six weeks. Hypophysectomized rats were compared to age matched or weight matched controls. Total lipoprotein lipase activity (LPL) (triglyceride uptake) was enhanced in BAT of 28 degrees C hypophysectomized rats compared to controls. Cold acclimation led to a large increased activity. Total LPL activity was comparable in BAT of hypophysectomized and control rats. Total malic enzyme and glucose-6-phosphate dehydrogenase activities (in situ lipogenesis) were doubled in BAT of 28 degrees C hypophysectomized compared to controls. A large enhancement was observed in BAT of either 15 degrees C control or 15 degrees C hypophysectomized rats. Among the studied organs (liver, white adipose tissue, heart, BAT) hypophysectomy promotes the three enzyme activities only in BAT. These variations were discussed with relation to the effect of hypophysectomy on brown adipose tissue at 15 degrees C and 28 degrees C.

Biological activity of bacterial derived human growth hormone in adipose tissue of hypophysectomized rats.

Segments of epididymal fat from hypophysectomized rats were incubated in Krebs-Ringer bicarbonate buffer for 1-4 h. Responses to three bacterially synthesized human GH (hGH) preparations were compared with responses to two highly purified pituitary hGH preparations. Hormones of both bacterial and pituitary origin increased the rate of oxidation of [U14C] glucose to 14CO2 and antagonized epinephrine-stimulated lipolysis. These insulin-like effects were produced over a range of 30-300 ng/ml hGH, and their concentration dependence was quite similar regardless of the source of hormone. Exposure of tissues to GH in the first hour of incubation makes them refractory to the insulin-like actions of a test dose of GH added in the fourth hour of incubation. hGH of either pituitary or bacterial origin also appeared to be equipotent in producing such refractoriness. The minimal effective concentration for this response fell between 10 and 30 ng/ml. Hormones from both sources were also capable of displacing 125I-labeled hGH from specific binding sites on isolated adipocytes, and the displacement curves were superimposable. Fifty percent displacement was seen at concentrations in the range of 10-20 ng/ml. Cell preparations produced a delayed increase in lipolysis which was evident in the presence of 0.3 mg/ml of theophylline in the fourth hour of incubation. In this regard, two of the bacterial hGH preparations were more potent than pituitary hGH and produced statistically significant increases in lipolysis at concentrations 10-30 times lower than pituitary hGH studied simultaneously in tissues of the same rats. Despite the presence of less than 1% contamination with bacterial protein, the greater potency of the bacterial hGH may be due to material of bacterial origin, since one bacterial hGH preparation, which was further purified to remove 99% of the remaining bacterial contamination was equipotent with pituitary hGH. These data indicate that a protein hormone synthesized in bacteria, and therefore likely to be free of peptides which might contaminate hGH purified from pituitary extracts, has all of the diverse physiological effects in adipose tissue that have been observed with preparations of GH derived from pituitary glands. These findings lend strong support to the conclusion that both insulin-like and lipolytic actions of GH are intrinsic properties of the molecule and cannot be attributed to contaminants present in the pituitary extract.

A Hybrid Noncovalent Complex of Fragments of Porcine and Human Growth Hormones with Diabetogenic Activity*

Although porcine GH (pGH) and human GH (hGH) are structurally related, their structures differ to the extent that, unlike hGH, pGH is not active in primates and does not exhibit lactogenic activity. Therefore, it was of interest to determine whether hybrid noncovalent complexes could be formed by complementation of large fragments of pGH with fragments of hGH and to study the immunological and biological properties of such hybrids. For this purpose, pGH was digested with bovine thrombin conjugated to Sepharose. After reduction and S-carbamidomethylation of the digested hormone, two large fragments consisting of residues 1-133 (p1-133T) and 134-191 (p134-191T) were isolated by gel filtration. The human GH fragments used in this work were S-carbamidomethylated peptides 1-134 (h1-134T) and 135-191 (h135-191T) isolated from thrombin-digested hGH. Noncovalent complementation of the peptide fragments was attempted by dissolving equimolar amounts of the materials in 0.5% (wt/vol) ammonium bicarbonate solution containing 6 M guanidine-HCl and then gradually removing the guanidine-HCl by dialysis. Using this method, a hybrid noncovalent complex of peptides p1-133T and h135-191T was produced in 50% yield by weight of starting material. Attempts to produce the reciprocal complex between h1-134T and p134-191T, as well as attempts to recombine the contiguous pGH fragments, p1-133T, and p134-191T, were unsuccessful. The hybrid complex (p1-133T + h135-191T) had 40% the cross-reactivity of native pGH in a rat GH RIA, but it was inert in an hGH RIA. It had very low growth-promoting activity in the 9-day weight gain test in hypophysectomized rats and no detectable in vitro ability to stimulate [14C]phenylalanine incorporation into the protein of the diaphragm of the hypophysectomized rat. When tested for insulin-like activity (stimulation of glucose oxidation in vitro in adipose tissue of hypophysectomized rats), the hybrid complex had less than 1% the activity of native pGH. In contrast, the hybrid complex had approximately 10-20% the diabetogenic activity of native pGH in the ob/ob mouse. The fact that the hybrid complex has substantial diabetogenic activity, although lacking significant anabolic or insulin-like activities, suggests that the structural requirements differ for the expression of these various activities of GH.

Complementation of human growth hormone (GH) peptide 1-134 with C-terminal fragments of human GH produced by digestion with bromelain.

The digestion of human GH (hGH) with the proteolytic enzyme, bromelain, results in a major product consisting of a mixture of three large fragments, i.e. residues 1-135 + 143-191, 1-135 + 145-191, and 1-135 + 146-191. In the case of each fragment, the N-terminal peptide is joined to the C-terminal fragment by the disulfide bridge between residues 56 and 165. A C-terminal fragment mixture consisting of peptides 143-191, 145-191, and 146-191 was isolated from this major digestion product after reduction and S-carbamidomethylation of its disulfide bonds. In the present study, the noncovalent complementation of the peptides in this mixture with S-carbamidomethylated peptide 1-134 derived from thrombin-digested hGH was investigated. Noncovalent complementation of these peptides was accomplished by dissolving equimolar amounts of the materials in 0.5% ammonium bicarbonate-6 M guanidine-HCl and dialyzing the mixture slowly to remove the guanidine-HCl. The recombinant mixture was recovered in 26% yield by gel filtration of the peptide mixture and was found to contain three noncovalent recombinant species, i.e. peptides 1-134 + 143-191, 1-134 + 145-191, and 1-134 + 146-191. Thus it would appear that residues 135-145 are not required to obtain noncovalent complementation between the N- and C-terminal regions of the hGH molecule. In an RIA for hGH the recombinant mixture was found to possess approximately 40% the cross-reactivity of the native hormone. In contrast, it had only about 10% the activity of native hGH in the weight gain test in hypophysectomized rats, in stimulating phenylalanine incorporation into the protein of the isolated hypophysectomized rat diaphragm, and in stimulating glucose oxidation by isolated adipose tissue of hypophysectomized rats. The limited biological activity of the recombinant mixture is of interest, since the major bromelain digestion product from which the C-terminal peptides were derived consists of a mixture of rather similar molecules (i.e. peptides 1-135 + 143-191, 1-135 + 145-191, 1-135 + 146-191; and with intact disulfide bridges), which exhibits substantial growth-promoting and insulin-like activities.

Fragments of human growth hormone produced by digestion with bromelain: Chemistry and biological properties

In an effort to produce small discrete fragments of human growth hormone (GH), we examine the action of the proteolytic enzyme, bromelain, on this molecule. Purified human GH incubated for 40 min at 22°C with crude bromelain and gel-filtered on Sephadex G-100 resulted in a major digestion product, peak 2. SDS-urea gel electrophoresis in the presence of β-mercaptoethanol suggested that peak 2 was composed of two polypeptide chains. Two polypeptide fractions were isolated by the reduction and S-alkylation of peak 2 in 6 M guanidine-HCl and subsequent chromatography on Sephacryl S-200 in 6 M guanidine-HCl. These two fractions, A and B, had the same mobilities as the two components of peak 2 on SDS-urea gels. Amino-terminal analysis, tryptic peptide mapping, carboxypeptidase digestion, cyanogen bromide cleavage, and amino acid analysis of fraction A indicated that it was peptide 1–135. Amino-terminal analysis and tryptic peptide mapping of fraction B suggested the presence of a mixture of peptides 143–191, 145–191 and 146–191. Thus, peak 2 is heterogeneous and appears to be a mixture consisting of peptide 1–135 + peptide 143–191, peptide 1–135 + peptide 145–191 and peptide 1–135 + peptide 146–191, in each case the N-terminal peptide being joined to the C-terminal peptide by the disulfide bridge between residues 53 and 165. In the weight-gain test in hypophysectomized rats, two preparations of peak 2 appeared to be somewhat less active than the native human GH preparations from which they were derived. Several preparations of peak 2 showed equivalent potency in stimulating [ 14C]glucose oxidation to 14CO 2 by isolated epididymal adipose tissue of hypophysectomized rats. Also, most of the peak 2 preparations were somewhat less active than native human GH in displacing 125I-labeled human GH bound to antibodies to human GH.

Ability of growth hormone fragments to compete with 125I-iodinated human growth hormone for specific binding to isolated adipocytes of hypophysectomized rats

Several noncovalent complexes of large fragments of human GH, which are less active than native human GH in stimulating glucose metabolism in adipose tissue of hypophysectomized rats, were tested for their ability to compete with 125I-iodinated human GH for specific binding to isolated adipocytes of hypophysectomized rats. The complexes tested were A (residues 1–134 + residues 141–191; S-carbamidomethylated), B (residues 1–134 + residues 135–191; S-carbamidomethylated) and C (residues 1–134 + residues 135–191; S-carboxymethylated). When compared to native human GH, the complexes were less active in competing with 125I-iodinated human GH for specific binding to adipocytes, and their order of potency in the binding assay (A > B > C) was similar to that of their respective activities in stimulating glucose metabolism in isolated adipose tissue of hypophysectomized rats.

Fragments of human growth hormone produced by digestion with thrombin: chemistry and biological properties.

Human GH (hGH) was digested with bovine thrombin conjugated to Sepharose, resulting in the cleavage of only one peptide bond between amino acid residues 134 and 135 in the large disulfide loop of the molecule. hGH was quite sensitive to the proteolytic action of thrombin, with digestion going essentially to completion. Thrombin-digested hGH (TDhGH) was equipotent with hGH in promoting growth in hypophysectomized rats, in stimulating the in vitro oxidation of glucose by isolated adipose tissue of hypophysectomized rats, in inducing N-acetyllactosamine synthase activity in mammary tissue explants from pregnant mice, and in competing with [l25I]hGH for binding to hGH antibodies. TD-hGH was reduced and alkylated to produce S-carbamidomethylated (RCAM-TDhGH), S-aminoethylated (RAE-TD-hGH), and S-carboxymethylated (RCM-TD-hGH) derivatives. These materials showed substantial alterations in their profiles of biological activity when compared to intact hGH. RCAM-TD-hGH and RAE-TD-hGH had 50% of the growth-promotin...

Effects of growth hormone on glycogen metabolism in adipose tissue of hypophysectomized rats.

ARTICLESEffects of growth hormone on glycogen metabolism in adipose tissue of hypophysectomized ratsT. W. Honeyman, and H. M. GoodmanT. W. Honeyman, and H. M. GoodmanPublished Online:01 Apr 1980https://doi.org/10.1152/ajpendo.1980.238.4.E389MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByThe Metabolic Actions of Growth Hormone1 January 2011 More from this issue > Volume 238Issue 4April 1980Pages E389-E394 Copyright & PermissionsCopyright © 1980 the American Physiological Societyhttps://doi.org/10.1152/ajpendo.1980.238.4.E389PubMed6990777History Published online 1 April 1980 Published in print 1 April 1980 Metrics

The effects of growth hormone on the utilization of L-leucine in adipose tissue.

Growth hormone (1 μg/ml) increased the oxidation of L-[l-14C]leucine to 14CO2 in adipose tissue of hypophysectomized rats whether or not glucose was also present. Glucose alone also increased the rate of leucine consumption, and the magnitude of the growth hormone effect was greater when glucose was also present. The stimulation of leucine oxidation by growth hormone required a lag period of the order of 30 min, was maximal between 30 and 90 min, and thereafter declined and disappeared by 180 min. Addition of actinomycin D or cycloheximide along with growth hormone caused the accelerated rate of leucine consumption to persist through at least the fourth hour. After the response to growth hormone had disappeared, the tissues were refractory to renewed stimulation with additional growth hormone. Growth hormone produced comparable effects on the consumption of α-ketoisocaproate, the deaminated product of leucine, suggesting that the site of stimulation is distal to the transamination reaction. In the absence...

Biological properties of plasmin digests of S-carbamidomethylated human growth hormone.

Reduction and carbamidomethylation of the intrachain disulfide bridges of human growth hormone did not destroy its ability to stimulate weight gain or cartilage metabolism in hypophysectomized rats. The reduced and alkylated hormone also stimulated glucose oxidation in isolated adipose tissue of hypophysectomized rats when added in vitro. When the S-carbamidomethylated hormone was incubated overnight with human plasmin, approximately 95% of the starting material was completely digested, as judged by polyacrylamide gel electrophoresis. The plasmin digest retained the ability to stimulate weight gain, cartilage metabolism, and glucose oxidation. A fraction consisting of two major electrophoretic components was isolated from the digest by chromatography on tsephadex G-50. This fraction possessed the biological properties of the whole digest.

Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver

The major objectives of this study were to define the roles of adrenal glucocorticoids and glucagon in the long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase of mammalian adipose tissue and liver. Particular emphasis was given to elucidation of the mechanisms whereby these hormones produce their regulatory effects on enzymatic activity. To dissociate the important effects of alterations in food consumption produced by experimental manipulation, nutritional conditions were rigidly controlled in the experiments described. Administration of glucocorticoids to adult rats led to a marked reduction in activities of fatty acid synthetase and carboxylase in adipose tissue but no change occurred in liver. Adrenalectomy produced an increase in activities of these lipogenic enzymes in adipose tissue, but, again, no change was noted in liver. The decrease in enzymatic activities in adipose tissue with glucocorticoid administration correlated well with a decrease in fatty acid synthesis, determined in vivo by the 3H 2O method. The mechanisms whereby glucocorticoids led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the decrease in fatty acid synthetase activity observed in adipose tissue was shown to reflect a decrease in content of enzyme, and not a change in catalytic efficiency. The mechanism underlying the decrease in enzyme content is a decrease in synthesis of the enzyme. The relation of the effects of glucocorticoids to the effects of certain other hormones involved in regulation of lipogenesis was investigated in hypophysectomized and in diabetic animals. Thus, the observation that the glucocorticoid effect on synthetase and carboxylase occurred in adipose tissue of hypophysectomized rats indicated that alterations in levels of other pituitaryregulated hormones were not necessary for the effect. That glucocorticoids play some role in regulation of synthetase and carboxylase in liver, at least in the diabetic state, was shown by the observation that the low activities of these enzymes in diabetic animals could be restored to normal by adrenalectomy. An even more pronounced restorative effect was apparent in adipose tissue of adrenalectomized, diabetic animals. Administration of glucagon during the refeeding of starved rats resulted in a marked reduction in the induction of fatty acid synthetase, acetyl-CoA carboxylase and in the rate of incorporation of 3H from 3H 2O into fatty acids in liver, but no change in these parameters occurred in adipose tissue. Administration of theophylline resulted in intermediate reduction in liver. The mechanisms whereby glucagon led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the changes in fatty acid synthetase activity were shown to reflect reductions in content of enzyme. The mechanism underlying these reductions in content is reduced synthesis of enzyme.

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 hormone's growthpromoting, anabolic, diabetogenic, and adipokinetic actions for rat and man.

Effect of growth hormone in vitro on phosphofructokinase activity of rat epididymal adipose tissue

The effect of pituitary growth hormone in vitro on phosphofructokinase of epididymal adipose tissue from hypophysectomized rats has been investigated. Of the four key glycolytic enzymes, hexokinase, phosphofructokinase, aldolase, and pyruvate kinase, assayed in the epididymal adipose tissue of hypophysectomized rats, phosphofructokinase was observed to have the least enzyme activity. Phosphofructokinase extracted from epididymal adipose tissue of hypophysectomized rats incubated before homogenization at 37 ° for 10 min with growth hormone (5 μg/ml) had a higher activity (100%) as compared to enzyme preparations from tissues incubated without growth hormone in the medium. The hormone added directly to the homogenates or supernatant extracts did not give any activation. The apparent increase in enzyme activity was not due to de novo synthesis of the enzyme as cycloheximide did not block the effect. Data presented in this communication suggest that as a result of the action of the hormone on intact tissue, there is a stabilization of the enzyme and/or a stable transformation of less active to a more active form of phosphofructokinase. A correlation is observed between the hormonal stimulation of lipogenesis and of phosphofructokinase activity in epididymal adipose tissue.

Biologically active cyanogen bromide fragments of bovine growth hormone.

When bovine growth hormone is cleaved with cyanogen bromide, several fragments are produced: an N-terminal pentapeptide (fragment E); a 109-residue peptide (fragment A) which is adjacent to the N-terminal pentapeptide and is connected to a 30-residue peptide (fragment B) by a disulfide bridge; a 25-residue peptide (fragment C) which probably lies between fragments A and B in the intact molecule; and a C-terminal dodecapeptide (fragment D). In the present study, several of these fragments were tested for biological activities typical of native bovine growth hormone. Fragment AB (fragments A and B linked by a disulfide bond) was found to stimulate the incorporation of leucine-14C into the protein of diaphragms isolated from hypophysectomized rats, which had received 2 injections of the fragment over a 25-hr period. In addition, fragment AB added in vitro stimulated the conversion of 14C-glucose into CO2 and fatty acids by isolated adipose tissue of hypophysectomized rats. The ability to stimulate glucose utilization was also possessed by fragment A, which was obtained by reduction and carboxymethylation of fragment AB. In contrast, fragments B, C and D did not have consistent in vitro effects on glucose utilization by isolated adipose tissue at the concentrations tested. (Endocrinology86: 416, 1970)