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U J Lewis

Publications and source records attributed to U J Lewis.

At least 37 records · Page 2Linked to original sources
PubMed1987

In vitro insulin-like actions of human growth hormone: a study with an insulin antibody.

When polyclonal insulin antibodies were preincubated with either adipose tissue from hypophysectomized rats or adipocytes from normal rats, human growth hormone failed to stimulate glucose oxidation. Removal of insulin from adipocytes through incubation with pyruvate at pH 7.0, followed by washing three times, also abolished subsequent in vitro insulin-like action of hGH. Administration of the same insulin antibody to hypophysectomized rats 30 minutes prior to injection of hGH did not inhibit the insulin-like activity of the hGH as measured by its ability to decrease serum glucose and non-esterified fatty acid levels. It is concluded that the in vitro promotion of glucose oxidation by hGH requires insulin. Because of the uncertainty of complete removal of insulin in intact animals, such a conclusion cannot be made regarding in vivo insulin-like action of hGH.

Adipose Tissue↗
PubMed1987

Induction of prolactin receptors in the liver is more closely related to the growth-promoting than to the lactogenic potency of peptides.

The sex differentiated binding 125I-human prolactin (PRL) to rat liver membranes was studied and the present results extend our previous studies on induction of hepatic PRL receptors by growth hormone (GH). In prepubertal female rats, PRL receptor levels are low compared with those in mature female rat livers. Infusion of hGH during one week to 17-day-old female rats resulted in a receptor level typical of adult female rats. The time course of receptor disappearance in male rats treated with hGH was also studied. When the receptor-inducing hormone was removed, receptor levels in hGH-treated male rats returned to the normal level characteristic of male rats after approximately 96 h. The specificity of various GH-like and PRL-like hormones in PRL receptor induction was studied in hypophysectomized rats. The PRL-like hormones were identified by measuring their potency to displace 125I-hPRL from a receptor preparation obtained from female rat livers, and the GH-like hormones were identified by their potency to increase body weight in hypophysectomized rats. Using similar doses of hormones it was found that in vivo administration of growth-promoting peptides (rGH, hGH, bGH) induced PRL receptors, whereas lactogenic hormones (rPRL, hPL) had a very small or no effect on PRL receptor induction. This suggests that binding to a type of GH receptor is the first step in PRL receptor induction.

Animals↗
PubMed1986

A lectin-binding immunoassay indicates a possible glycosylated growth hormone in the human pituitary gland.

By means of a lectin binding immunoassay we detected a glycosylated human growth hormone-immunoreactive substance in the human pituitary gland. The assay involves reacting the test substance with concanavalin A immobilized on a solid support followed by treatment with human growth hormone antibodies. Quantitation is achieved by reaction of the concanavalin A-glycoprotein-antibody complex with an 125I-labeled second antibody. Specificity of the assay for a glycosylated human growth hormone was indicated by inhibition of binding by N-acetyl-D-glucosamine. Furthermore, use of antiserum that had been absorbed with human growth hormone reduced the amount of radioactivity bound to the tubes. Western blotting of pituitary immunoprecipitates after gel electrophoresis in sodium dodecyl sulfate revealed human growth hormone-immunoreactive components that also reacted with concanavalin A.

Binding, Competitive↗
PubMed1986

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.

Adipose Tissue↗
PubMed1986

The growth of cultured rabbit articular chondrocytes is stimulated by pituitary growth factors but not by purified human growth hormone or ovine prolactin.

Recent reports describing a direct stimulation by GH of bone and cartilage growth led us to compare the in vitro mitogenic effects of highly purified human GH and PRL and two pituitary-derived growth factors in rabbit articular chondrocytes. These preparations were tested for their ability to promote [3H]thymidine incorporation into growth-arrested monolayer chondrocyte cultures and were also assayed in cell growth experiments. The factors tested included 22,000-dalton and 20,000-dalton human GH ovine PRL, glycosylated ovine PRL, bovine pituitary fibroblast growth factor (bpFGF), and a partially purified pituitary growth factor distinct from bpFGF. We found that no significant mitogenic effect was produced by either of the human GH or PRL preparations. Both of the pituitary-derived growth factors were potent mitogens, with bpFGF active at a final medium concentration of 10 pg/ml. These studies support the large body of evidence that GH has no significant direct in vitro effect on chondrocyte growth. The very potent effects of the pituitary-derived growth factors raise the possibility that their presence in GH preparations may be responsible for the in vitro mitogenic effects attributed to these preparations.

Animals↗
PubMed1986

Release of the 22,000- and the 20,000-dalton variants of growth hormone in vivo and in vitro by human anterior pituitary cells.

Using a combination of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunoblotting with antihuman GH (anti-hGH) serum, we quantitatively and independently measured the major 22,000-dalton form of hGH (hGH22K) and the 20,000-dalton form (hGH20K). This technique was equally effective in assaying cell culture media or human sera. We studied isolated human pituitary cell cultures during a 16-day incubation period with and without stimulation by GH-releasing hormone (GHRH). Under basal conditions, the cells released 2.83 +/- 0.24 (+/- SEM) microgram hGH22K/ml . day and 0.67 +/- 0.17 microgram hGH20K/ml . day. GHRH (10(-8) M) treatment resulted in stimulation of both hGH22K and hGH20K by 24 h. We also measured both hGH22K and hGH20K in the sera of normal subjects before and after an iv bolus injection of 100 micrograms GHRH. hGH20K increased as did hGH22K. Peak concentrations of both variants occurred 45 min after GHRH administration. The results of this study indicate that the combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting is an accurate and effective means of separately assaying hGH22K and hGH20K. We also demonstrated that primary monolayer cultures of human pituitary cells are an excellent model system for study of the secretion of these two hGH variants.

Adult↗
PubMed1985

Highly purified human growth hormone fails to stimulate lipolysis in rabbit adipocytes in vitro or in rabbits in vivo.

To determine whether the rapid lipolytic effect observed with human growth hormone (hGH) preparations in rabbits and rabbit adipose tissue is an intrinsic property of the hormone, we examined the lipolytic effects in vivo and in vitro of clinical grade preparations, hGH purified by DEAE cellulose chromatography, and hGH prepared by recombinant DNA techniques. Using isolated rabbit perirenal adipocytes, ACTH and clinical-grade hGH preparations both stimulated glycerol release to the same maximal rate with half-maximal hGH effects observed between 8 and 50 micrograms/mL. Purification of the most potent lipolytic preparation of clinical grade hGH by DEAE cellulose chromatography yielded a preparation (2 IU/mg of growth activity that retained insulinlike effects on rat fat pad [U-14C] glucose metabolism) that, at concentrations up to 0.2 mg/mL, failed to stimulate lipolysis by adipocytes incubated for one or four hours in the presence or absence of dexamethasone or trypsin inhibitor or when preincubated for three hours prior to addition of hGH. Recombinant DNA-derived hGH did not stimulate glycerol release at 0.1 mg/mL. While antiserum to purified hGH blocked the increase in glucose oxidation in rat fat pads produced by clinical grade hGH, it did not inhibit its lipolytic effect using rabbit adipocytes. Purified hGH (0.1 mg/kg IV) was also unable to elicit a rise in serum free fatty acid (FFA) levels of conscious rabbits while, at the same dose, clinical grade hGH increased FFA levels to 900 microEq/L over basal. Rapid lipolytic stimulation in rabbit adipocytes by hGH preparations could not be attributed to the 20,000 molecular weight variant of hGH (hGH20K) or the peptide corresponding to positions 32 to 46 of hGH (deletion peptide).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1985

Glycosylated human prolactin.

A glycosylated form of human PRL (G-hPRL) was isolated from pituitary glands. The glycoprotein was separated from the major form of PRL on columns of lentil lectin-Sepharose 4B. The major form of PRL did not bind to the lentil lectin, whereas the glycosylated modification did and could be eluted with methyl-alpha-D-mannopyranoside. By gel electrophoresis in sodium dodecyl sulfate, a mol wt of 25,000 was estimated for the glycosylated PRL. The mol wt of hPRL is 23,000. In a RIA for hPRL, the glycosylated hormone was about one third as reactive as the principal form. Since there is only one Asn-X-Ser(Thr) sequence in hPRL, the asparagine at position 31 is the likely point of N-linked glycosylation.

Chromatography↗
PubMed1984

Glycosylated ovine prolactin.

A modification of prolactin, in which the asparagine at position 31 carries a carbohydrate unit, was isolated from ovine pituitary glands. Sequence and amino acid analyses identified the point of linkage of the carbohydrate. Glucosamine was found in acid hydrolysates, an indication that the carbohydrate is attached through N-acetylglucosamine. The glycosylated prolactin binds to concanavalin A and lentil lectin and is eluted with methyl alpha-D-mannopyranoside. During gel electrophoresis in sodium dodecyl sulfate, the glycosylated hormone migrates as a Mr 25,000 protein; prolactin has a Mr of 23,000. The modified prolactin had a potency of 20 international units/mg, approximately equal to 60% the potency of a reference prolactin preparation when measured by the pigeon crop sac assay. In a radioimmunoassay, the glycosylated form had only 34% the immunoreactivity of ovine prolactin.

Amino Acid Sequence↗
PubMed1981

Altered proteolytic cleavage of human growth hormone as a result of deamidation.

The major desamido form of human growth hormone (hGH) results from deamidation of asparagine 152. Peptide mapping and amino acid sequencing were used in the identification. This desamido form (hGHAsp152) could be produced by incubation of the undeamidated hormone in an alkaline medium. Another minor deamidated form which contained glutamic acid at 137 (hGHGlu137) also was identified in preparations of hGH. This form was not produced by alkaline treatment of hGH. Limited hydrolysis of hGH with subtilisin produced two cleaved forms, one with cleavages at positions 139 and 149 and another with cleavages at 139 and 146. hGHAsp152 underwent only one type of modification, cleavage at positions 139 and 146. Hydrolysis of hGHGlu137 resulted in cleavages in the region of 139 to 149 identical with those noted with hGH, but in addition, proteolysis had occurred in the region of 95 to 127, an area where hGH was not attacked by subtilisin. That Glu at 137 modified cleavage points was also indicated by the greater resistance of hGHGlu137 to hydrolysis by subtilisin as compared to hGH. The results demonstrate that deamidation can alter points of proteolytic cleavage. If proteolytic processing of hGH is found to be of physiologic significance, deamidation may be a way of directing specific cleavages.

Amino Acid Sequence↗
PubMed1981

Procedure for isolation of the 20,000-dalton variant of human growth hormone.

An isolation procedure for the 20,000-dalton variant of human growth hormone has been devised to improve the yield of the final product. The improvement involved elimination of cumbersome steps that decreased yield, and modification of chromatography on DEAE-cellulose to provide better separation of the variant from the major form of growth hormone.

Chromatography, DEAE-Cellulose↗
PubMed1981

Binding characteristics of a biologically active variant of human growth hormone (20K) to growth hormone and lactogen receptors.

The dose-dependent displacement characteristics of a biologically active 20,000 molecular weight human pituitary growth hormone variant (20K) and of human growth hormone (hGH) were compared using hGH liver plasma membrane receptors both from 20-30 day pregnant rabbits and from normal female rats and also using mammary gland plasma membrane receptors from 5-6 day lactating rabbits. Different preparations of 20K were found to be only 3-20% as potent as hGH when compared at the dose necessary to cause 50% displacement of (125I)iodo-hGH from liver receptor and was 22-53% as effective as hGH in the mammary receptor assay system. These findings suggest that 20K and hGH may have separate receptors or that the binding characteristics of the two hormones may be quite different.

Animals↗
PubMed1981

Somatomedin generating activity of the 20,000-dalton variant of human growth hormone.

The 20,000-dalton variant of human growth hormone stimulated production of somatomedins in the hypophysectomized rat. Somatomedin activity was measured by a radioreceptor assay. The results indicate that the insulin--like activities of human growth hormone that are greatly diminished in the variant are not prerequisites for somatomedin production.

Animals↗