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U J Lewis

Publications and source records attributed to U J Lewis.

At least 19 recordsLinked to original sources
PubMed2000

Structure and properties of members of the hGH family: a review.

This review will summarize the properties of five variant forms of human growth hormone: a disulfide dimer, a glycosylated form, 20 kD-hGH, and two peptides made up of portions of 22 kD-hGH. The two pituitary peptides (hGH(1-43) and hGH(44-191)) have, respectively, insulin-potentiating and anti-insulin properties. Both have been detected in serum. The shorter peptide may prove to be useful in decreasing the amount of exogenous insulin required by diabetics. The larger, strongly anti-insulin peptide, may be involved in diabetic retinopathy. We believe that this peptide is the long sought after diabetogenic substance of the pituitary gland.

Diabetes Mellitus↗
PubMed1996

Glycosylated human growth hormone (hGH): a novel 24 kDa hGH-N variant.

We have identified a human pituitary protein as a novel glycosylated variant of hGH. Isolation of the denatured protein included separation of human pituitary extract by Sephadex G-100 chromatography in ammonium bicarbonate, followed by Sephadex G-100 chromatography in 10% acetic acid, with subsequent DEAE Sephacryl chromatography in ammonium bicarbonate, and finally by preparative SDS PAGE. The pituitary protein has a molecular weight of 24 kDa as determined by SDS PAGE analysis and is thus larger than the normal 22 kDa hGH. N-Terminal amino acid sequence analysis of the first twenty-six residues reveals that this protein is not derived from the hGH-V gene but is rather a hGH-N gene product. Assays for the detection of glycoconjugates (periodate oxidation, sialidase treatment, trifluoromethanesulfonic acid treatment) indicate that the hGH variant has carbohydrate moieties. The discovery of new hGH raises questions about the role of glycosylation in the structure/function relationships of this hormone.

Amino Acid Sequence↗
PubMed1996

Human growth hormone deletion mutant (hGH44-191) binds with high affinity to lactogenic receptors but not to somatogenic receptors.

The carboxyl-terminal hGH fragment, hGH44-191, displays diabetogenic activity. To understand whether this biological activity is mediated through somatogenic or lactogenic receptors, we investigated the ability of hGH44-191 to bind both receptor classes. We found that hGH44-191 could not compete with [125I]hGH or [125I]bGH for bovine liver somatogenic binding sites. Additionally, hGH44-191 could not displace [125I]hGH from the somatogenic receptor sites when human liver microsomes were used as the receptor source. In contrast, hGH44-191 effectively competed with [125I]hGH for lactogenic receptor sites of lactating mammary gland microsomes and of bovine liver microsomes. In summary, hGH44-191 does not bind to somatogenic receptors but does not bind to lactogenic receptors. These data suggest that the biological actions of hGH44-191 could be mediated through lactogenic receptors.

Animals↗
PubMed1996

Human growth hormone fragments 1-43 and 44-191: in vitro somatogenic activity and receptor binding characteristics in human and nonprimate systems.

Human GH (hGH) fragments 1-43 and 44-191 have potent in vivo effects on glucose homeostasis in rodents but cannot stimulate body growth. To assess the in vitro bioactivity of these hGH fragments we tested their activity against GH-responsive FDC-P1 cell lines expressing full-length human (h), mouse (m), or rabbit (r) GH receptors (GHR). Binding specificity and affinity was tested using GHR-containing membrane preparations from three species and recombinant hGH binding protein (hGHBP). Recombinant hGH 1-43 and recombinant 44-191 stimulated proliferation of FDC-P1-hGHR cells with half-maximal effect at approximately 2000 and 100 nM, respectively, whereas intact hGH stimulates proliferation of FDC-P1-hGHR cells with ED50 of 0.02-0.03 nM. However, these fragments had minimal effect on cells expressing mGHR or rGHR. Although 44-191 did not stimulate proliferation of FDC-P1-rGHR cells, when added to these cells in the presence of 0.23 nM hGH, it antagonized hGH action in a dose-dependent manner (ED50 at 230 nM). Binding of these GH fragments was compared using membrane preparations from rabbit liver, rabbit and mouse adipose tissue, and recombinant hGHBP. Binding competition curves were consistent, with 44-191 having at least a 10-fold lower affinity for rabbit liver GHR and rabbit adipose GHR than bovine GH and a 61-fold lower affinity for hGHBP than hGH. Binding of hGH 1-43 could not be demonstrated to GHRs in rabbit liver microsomes, adipose microsomes, or to hGHBP. HGH 1-43 did not compete for insulin binding sites in adipose microsomes. In conclusion, hGH 44-191 binds with low affinity to the GHR and at supraphysiologic levels stimulates proliferation of FDC-P1-hGHR cells. At high doses, 44-191 can also antagonize GH action in FDC-P1-rGHR cells, presumably by blocking receptor dimerization. Binding of 1-43 to GHR could not be detected, and the basis for its weak in vitro mitogenic effect remains to be elucidated. The low affinity of the fragments for cloned GHRs and low biopotency in these systems suggests that the metabolic actions of these fragments are unlikely to be mediated by the cloned GHR. This raises the possibility of a separate receptor mediating metabolic effects of these fragments.

Adipose Tissue↗
PubMed1991

Isolation and biochemical properties of four forms of glycosylated porcine prolactin.

Four isoforms of glycosylated prolactin (G-pPRL) were isolated from porcine pituitary glands by affinity chromatography and concanavalin A-Sepharose, based upon differences in their affinity for the lectin. Structural analysis indicated differences in the carbohydrate units of the four G-pPRLs. N-glycanase treatment cleaved the oligosaccharide from the G-pPRLs, establishing N-linked glycosylation. The binding of G-pPRLs to receptors from lactating rabbit mammary glands was only 3-8% that of nonglycosylated pPRL (NG-pPRL). The immunological crossreactivity of the G-pPRLs varied from 36 to 65% that of NG-pPRL. When tested in the pigeon crop sac bioassay, G-pPRLs were only 11-40% as active as NG-pPRL. The metabolic clearance rate of one of the G-pPRLs was slower and another faster than that of NG-pPRL. We conclude that there are several forms of G-PRL of variable immuno- and bio-potencies in the porcine pituitary, and that the current radioimmunoassay for the hormone does not measure the actual bioactivity.

Animals↗
PubMed1991

A recombinant-DNA-derived modification of human growth hormone (hGH44-191) with enhanced diabetogenic activity.

A modified human growth hormone (hGH) that lacks the first 43 residues of the intact hormone was prepared by recombinant-DNA technology. For preparative purposes an additional alanine was made the amino terminal residue. Sequence analysis and tryptic peptide mapping combined with amino acid analyses confirmed the structure of the polypeptide. Less than 2% N-terminal methionine was detected. The hGH44-191 was estimated to be at least 10 times more active than hGH in producing glucose intolerance in obese yellow mice (Avy/A) and was equipotent to hGH in increasing serum free fatty acids in fasted, hypophysectomized rats. The peptide did not promote growth in hypophysectomized rats nor did it exhibit early (1h) insulin-like activity in fasted, hypophysectomized rats, as indicated by its failure to lower blood glucose and fatty acids. The modified hGH was inactive in the Nb-2 cell assay but was about one-third as active as hGH in stimulating the pigeon crop sac. In radioimmunoassays using 125I-labeled hGH and polyclonal antibodies to intact hGH, cross-reactivity of hGH44-191 was less than 1%. We conclude that removal of the amino terminal portion of hGH enhances its diabetogenic properties, and that this activity does not depend upon the ability to promote growth. Furthermore, the insulin-like activity can be separated from its diabetogenic action by deletion of the first 43 amino terminal residues. This is the first report of a modified hGH that has anti-insulin effects greater than hGH itself.

Amino Acid Sequence↗
PubMed1991

Comparison of lipolytic and antilipolytic activities of lower vertebrate growth hormones on chicken adipose tissue in vitro.

Mammalian and avian growth hormones (GH) (pituitary derived or biosynthetic) exert two effects on chicken adipose tissue explants in vitro. They (i) increase the basal rate of glycerol release a lipolytic effect) and (ii) inhibit glucagon-stimulated glycerol release (an antilipolytic effect). The ability of lower vertebrate GH preparations to exert lipolytic and antilipolytic effects was examined and biological activity was compared to differences in amino-acid residue sequences and to predicted structure. Irrespective of species origin (blue shark, sturgeon, bonito, yellow tail, salmon, bullfrog, sea turtle), all lower vertebrate GH preparations showed very weak (less than 5% the potency of bovine GH), if any, lipolytic activity, but retained strong antilipolytic activity. The present data indicate that the structural requirements for lipolytic and antilipolytic activities of GH differ in chicken adipose tissue. Despite the high sequence homology (88%) between chicken and sea turtle GH, the latter preparation did not stimulate lipolysis. It is suggested that Pro132, conserved only in lipolytically active GH species (human, bovine, and chicken), represents a major determinant of lipolytic activity in chicken adipose tissue. The structural determinants for antilipolytic activity may comprise any or all of residues 3, 17, 64, 108, 109, and 152.

Adipose Tissue↗
PubMed1990

Effects of prolactin and glycosylated prolactin on (pro)insulin synthesis and insulin release from cultured rat pancreatic islets.

In order to study the possible differential effects of the nonglycosylated and glycosylated forms of prolactin on insulin content and secretion in pancreatic islets, neonatal rat pancreatic islets were exposed for 6 days in vitro to 2 micrograms/ml of nonglycosylated ovine prolactin (oPRL), or to 2 micrograms/ml of glycosylated oPRL (G-oPRL). oPRL stimulated a significant increase (p less than 0.01) in the total amount of insulin released into the medium over the 6 day culture period; however, G-oPRL had no effect. Islets cultured for 6 days in the presence of oPRL showed no increase in the amount of DNA per islet. However, there was a significant (p less than 0.007) increase in the amount of total protein synthesized by the islets exposed to oPRL. These findings suggest that the effect of oPRL on neonatal rat pancreatic islet cells is a nonspecific effect. The nonglycosylated form of PRL may play a role in B-cell function by promoting protein synthesis, which results in augmented insulin synthesis.

Animals↗
PubMed1990

Glycosylated human prolactin: alterations in glycosylation pattern modify affinity for lactogen receptor and values in prolactin radioimmunoassay.

The receptor-binding properties of monomeric nonglycosylated human PRL (hPRL), glycosylated hPRL that does not bind to Concanavalin-A-Sepharose (G1-hPRL) and glycosylated hPRL that binds to Concanavalin-A-Sepharose (G2-hPRL) were tested in the lactating rabbit mammary gland RRA for lactogenic hormones. Variations in the glycosylation pattern of G-hPRL altered its receptor-binding properties, suggesting that the site of glycosylation may be proximal to the receptor-binding region. Relative potencies for the displacement of [125I]hPRL by hPRL, G1-hPRL, and G2-hPRL were 100%, 40%, and 26%, respectively. Relative potencies for displacement of [125I]G1-hPRL by hPRL, G1-hPRL, and G2-hPRL were 100%, 44%, and 69%, respectively; however, the displacement curve for G2-hPRL was not parallel to the others. When G2-hPRL was radiolabeled, there was no specific binding to lactogenic receptors. The presence of PRL receptor subtypes and/or kinetic cooperativity was suggested by the complexity of the binding isotherms. The immunoreactivities of the PRLs were tested in a homologous RIA, using polyclonal antiserum. The modification of the glycosylation pattern of hPRL significantly altered the RIA values for PRL. When hPRL was used as the radiotracer, the percent cross-reactivities of hPRL, G1-hPRL, and G2-hPRL were approximately 100%, 23%, and 17%, respectively. When G1-hPRL was used as the radiotracer, the percent cross-reactivities were approximately 100%, 135%, and 54% for hPRL, G1-hPRL, and G2-hPRL; however, the displacement curve for hPRL was not parallel to those of the glycosylated hPRLs. When G2-hPRL was used as the radiotracer, the percent cross-reactivities were approximately 100%, 32%, and 37% for hPRL, G1-hPRL, and G2-hPRL. These data point out that the glycosylation heterogeneity of hPRL is a factor that affects the diagnostic accuracy of hPRL determinations. Specific RIAs for each PRL are needed so that we can have valid and reliable measurements of each PRL isoform and consequently gain a better understanding of PRL's complex biological role.

Animals↗
PubMed1989

Antibodies to newly recognized murine 13-18 KDa pituitary peptides crossreact with growth hormone and prolactin from several species, including man.

Recently we identified five novel peptides of Mr 13,000 to 18,000 (designated P13, P14, P16, P17, and P18 according to approximate Mr) in the anterior pituitary gland of rat and man that appeared related to GH and PRL in regulation and structure. We have now raised polyclonal antibodies in the rabbit to four of these peptides--P13, P14, P17 and P18--isolated from rat anterior pituitary; the rabbit injected with P16 did not produce antibodies. Besides reacting with their respective immunogens, antisera to all four peptides crossreacted, quite unexpectedly, with human GH and with human, porcine, and ovine PRL. Antiserum to P17, in addition, crossreacted very strongly with rat PRL, while P18 antiserum crossreacted not only with human GH but also with its 20K and cleaved variants. These results provide strong evidence for the structural relatedness of these peptides to GH and PRL, and raise the possibility that they may be related functionally as well.

Animals↗
PubMed1989

The complete amino acid sequence of growth hormone of an elasmobranch, the blue shark (Prionace glauca).

The complete amino acid sequence of growth hormone (GH) from a phylogenetically ancient fish, the blue shark (Prionace glauca), was determined. The shark GH isolated from pituitary glands by U. J. Lewis, R. N. P. Singh, B. K. Seavey, R. Lasker, and G. E. Pickford (1972, Fish. Bull. 70, 933-939) was purified by reversed-phase high-performance liquid chromatography. The hormone was reduced, carboxymethylated, and subsequently cleaved in turn with cyanogen bromide and Staphylococcus aureus protease. The intact protein was also cleaved with lysyl endopeptidase and o-iodosobenzoic acid. The resulting peptide fragments were separated by rpHPLC and submitted to sequence analysis by automated and manual Edman methods. The shark GH consists of 183 amino acid residues with a calculated molecular weight of 21,081. Sequence comparisons revealed that the elasmobranch GH is considerably more similar to tetrapod GHs (e.g., 68% identity with sea turtle GH, 63% with chicken GH, and 58% with ovine GH) than teleostean GHs (e.g., 38% identities with salmon GH and 42% with bonito GH) except for eel GH (61% identity), and substantiates the earlier finding derived from the immunochemical and biological studies (Hayashida and Lewis, 1978) that the primitive fish are less diverged from the main line of vertebrate evolution leading to the tetrapod than are the modern bony fish.

Amino Acid Sequence↗
PubMed1989

Two forms of glycosylated human prolactin have different pigeon crop sac-stimulating activities.

Two forms of glycosylated PRL (G-PRL) which differed in their binding properties to Concanavalin-A (Con-A) were isolated from human pituitary glands. One form, G1-hPRL, was only slightly retarded by Con-A; the other, G2-hPRL, was adsorbed by Con-A and could be eluted with methyl-D-manno-pyranoside, an indication of differing carbohydrate units in the two G-PRLs. Differences in type of glycosylation were also indicated by HPLC peptide mapping of tryptic digests of the two forms. The elution time for the tryptic peptide carrying the asparagine-linked carbohydrate unit varied for the two G-PRLs. The results point to the asparagine at position 31 as being the site of attachment of the carbohydrate. The carbohydrate structure influenced the crop sac-stimulating activity of the G-hPRLs. G1-hPRL had only about one fourth the activity of the reference standard (nonglycosylated ovine PRL, 35 IU/mg). The form that bound to Con-A, G2-hPRL, was equipotent to the reference standard. Because glycosylated forms have varying biological activities and are major components of circulating PRL, the physiological significance of serum concentrations of PRL measured by RIA will have to be reevaluated.

Amino Acid Sequence↗
PubMed1989

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.

Adipose Tissue↗
PubMed1988

Increased sensitivity of adipose tissue to insulin after in vivo treatment of yellow Avy/A obese mice with amino-terminal peptides of human growth hormone.

Treatment of obese yellow Avy/A mice with the human GH (hGH) peptide hGH-(1-43) enhanced the in vitro sensitivity of their adipose tissue to insulin. Insulin-stimulated glucose oxidation, as determined by measurement of 14CO2 production, was enhanced 106% after administration of hGH-(1-43) at a dosage of 1 microgram/day for 3 days. A significant increase in CO2 production was detected with as little as 100 ng peptide/day for 3 days. A single injection of 10 micrograms increased sensitivity to insulin 2-5 times. This enhancing effect of insulin action could not be seen in lean agouti A/a animals nor could it be demonstrated by in vitro addition of hGH-(1-43) to adipose tissue. Synthetic hGH-(1-43) was used for these studies, but initial physiological work was done with peptide isolated from pituitary glands. At equimolar doses, intact hGH, a trypsin digest of either hGH or BSA, carbidomethyl cysteine-hGH-(146-191), and hGH-(32-46) were inactive. Carbidomethyl cysteine-hGH-(1-139) and hGH-(1-15) showed the enhancing property, but were only about 10% as active as hGH-(1-43). HGH-(1-43) did not increase serum insulin concentrations in the obese mice. We conclude that when administered in vivo to obese mice, hGH-(1-43) enhances the sensitivity of adipose tissue to the action of insulin, an indication that the peptide may play a role in carbohydrate metabolism.

Adipose Tissue↗
PubMed1988

Amino-terminal peptide of growth hormone enhances insulin action in normal rats.

Earlier studies demonstrated that the 20K-dalton variant of human GH (hGH), which differs from hGH by deletion of the amino acid residues 32-46, has decreased insulin-like activity. The current study assessed whether a peptide representing this deleted region could enhance insulin-stimulated glucose uptake in the intact rat and if this effect was localized in liver and/or muscle. Peptides hGH-(32-46) and rat GH-(32-45) were used in these studies. Assessment of the action of a peptide was done by determining its effect on the steady state serum glucose (SSSG) concentration during an insulin suppression test. Glucose was infused alone and with two rates of infusion of insulin. The data indicated that neither of the GH peptides affected SSSG in the absence of exogenously administered insulin or at low (40 microU/ml) serum levels of insulin, but when serum insulin was increased to 77 microU/ml, a significant (P less than 0.05) decrease in SSSG was produced by the peptides. In subsequent studies isolated liver and hind limb skeletal muscle were perfused with a solution of hGH-(32-46). Basal glucose release from the liver was suppressed by both hGH-(32-46) and insulin alone, and this decrease was not enhanced by combining insulin with hGH-(32-46). Glucose uptake by skeletal muscle, expressed as a metabolic clearance constant (k), was enhanced by infusion of insulin and further increased with added peptide. Basal uptake was 6.67 microliter/min.g muscle; uptake was 8.17 microliter/min.g (P less than 0.01) after addition of 128 microU/ml insulin. This increased still farther to 9.24 microliter/min.g (P less than 0.05) when peptide was administered with insulin. These findings suggest that GH peptides independently suppress glucose outflow from the liver and potentiate insulin action by facilitating glucose uptake by peripheral tissues.

Animals↗
PubMed1988

Glycosylation selectively alters the biological activity of prolactin.

We have undertaken studies to determine the effect of glycosylation on the lactogenic activity of ovine PRL (oPRL). Measuring casein production in the in vitro mouse mammary gland explant assay, we found that glycosylated oPRL had 80% of the activity of oPRL. In competitive binding studies using lactogen receptors from mammary glands of lactating rabbits, glycosylated oPRL had only 20% the potency of oPRL. In the Nb2 assay also, glycosylated oPRL was approximately 24% as potent as oPRL in stimulating mitogenic activity. Thus, these studies show that the glycosylated variant of PRL has less biological activity than the major PRL form and that the alteration of an activity by glycosylation is selective.

Animals↗
PubMed1988

Glycosylated prolactin in porcine plasma: immunoblotic measurement from birth to one year of age.

As much as 40% of PRL in the pituitary gland of the pig is glycosylated. To help determine the physiological significance of this structural variant of PRL, we have measured glycosylated PRL (G-PRL) in the plasma of growing pigs from birth to 1 yr of age. An immunoblotting method developed originally for human plasma was used. With some modifications, the method could detect G-PRL in as little as 0.2 ml porcine plasma. Concanavalin-A affinity chromatography confirmed the glycoprotein nature of the plasma G-PRL band. Quantitative estimates of the immunoblotting results revealed marked differences with age in the secretion of the two monomeric forms of PRL. G-PRL concentrations averaged 138% higher than those of non-G-PRL between birth and 2 months of age, but lower thereafter. Chronologically, both forms displayed similar patterns between birth and 2 months, the concentration remaining unchanged or decreasing slightly. After 2 months, however, concentrations of non-G-PRL increased markedly; the increase was characterized by great fluctuations. G-PRL concentrations, on the other hand, increased only moderately, but the increase was consistent until the end of the study period. The results demonstrate the circulating nature of G-PRL in the pig and suggest that the variant may play a physiological role in the growth and development of the animal.

Aging↗