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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23766/1/0000002.pd

Isolated adipocytes from growth hormone-treated obese (ob/ob) mice exhibit insulin resistance

The genetically obese (ob/ob) mouse is a useful model for the study of the diabetogenic action of growth hormone (GH), because treatment of these animals with GH results in decreased responsiveness of their adipose tissue to insulin in vitro. Studies of the mechanisms involved in GH-induced insulin resistance using isolated adipocytes of ob/ob mice have not been possible, however, because of their extreme fragility and the lack of an adequate system for the maintenance of these cells. This study describes a new method for the isolation of ob/ob mouse adipocytes. The isolated cells are stable, viable and metabolically responsive to insulin. In addition, these adipocytes have been maintained in primary culture, in serum-free medium, for up to 3 days. During culture, the cells exhibit large increases in 125I-hGH binding (10-20-fold) and porcine 125I-insulin binding (5-10-fold). The induction of insulin resistance by GH has also been demonstrated in these freshly isolated ob/ob mouse adipocytes. The studies to date indicate that the ob/ob mouse adipocyte system should provide a useful model for detailed studies of the cellular and molecular mechanisms of GH induced insulin resistance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28578/1/0000381.pd

Trypsin-resistant forms of human growth hormone have diabetogenic and insulin-like activities

Although diabetogenic and insulin-like activities are intrinsic properties of the growth hormone (GH) molecule, it has been frequently suggested that the hormone must be proteolytically processed for these activities to be expressed. If this is correct, then derivatives of GH having resistance to appropriate proteolytic attack might not have diabetogenic and/or insulin-like activity. The purpose of the present study was to prepare derivatives of human GH that are resistant to digestion by trypsin and to determine whether they posses diabetogenic or insulin-like activity. Three derivatives were prepared from purified native human GH in which lysine residues were modified with methyl acetimidate, citraconic anhhydride of S-ethylthioltrifluoroacetate, and one in which arginine residues were modified with camphorquinone-10-sulfonic acid. Comparisons of peptide maps of tryptic digests of these derivatives with that of unmodified human GH indicated that all four were resistant to proteolysis by trypsin. All of these trypsin-resistant forms of human GH were found to posses ssignificant growth-promoting, diabetogenic and insulin-like activities, although all activities were attenuated to some extent in each derivative. The relative potencies of the human GH derivatives in a radioimmunoassay for human GH were somewhat similar to their order of potency in the growth-promoting and diabetogenic assays. These results suggest that if proteolytic processing of the GH molecule is involved in the expression of one or more of its biological activities, such processing probably does not involve a trypsin-like proteinase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25573/1/0000115.pd

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth human (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa Gh was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26576/1/0000115.pd

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[deg]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 [beta]-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 14CO2 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25324/1/0000769.pd