The effects of amino acid supply on the sensitivity of IGF-1 secretion to growth hormone stimulation in ovine hepatocytes

1999 ◽  
Vol 1999 ◽  
pp. 160-160
Author(s):  
N.M. Wheelhouse ◽  
D.G. Hazlerigg ◽  
J.C. MacRae ◽  
M.A. Lomax
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Venous Blood ◽  
Tissue Growth ◽  
Portal Venous Blood ◽  
Protein Supply ◽  
Amino Acid Concentrations ◽  
Blood Data

Many of the anabolic effects of growth hormone (GH), including promotion of lean tissue growth, are mediated by the actions of insulin-like growth factor-1 (IGF-1) (Gluckman et al, 1987). It is known that the response of hepatic IGF-1 synthesis to GH may be modulated by alterations in levels of feeding or changes in protein supply (Pell et al, 1993). The aim of the current study was to develop an in vitro model to characterise interactions between amino acid supply and GH on hepatic IGF-1 release in ruminants.Ovine hepatocyes were prepared by a modification of a published method (Luo et al, 1992) from the median lobe of livers removed immediately after slaughter from sheep killed at a local abattoir. Free amino acid concentrations in test media were 5x, 1x and 0.2x physiological concentrations based upon in vivo ovine portal venous blood data (Lobley et al, 1995).

Reduced and S-carboxymethylated human growth hormone: a probe for diabetogenic action

1984 ◽  
Vol 247 (5) ◽  
pp. E639-E644
Author(s):  
C. M. Cameron ◽  
J. L. Kostyo ◽  
J. A. Rillema ◽  
S. E. Gennick
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Human Growth Hormone ◽  
Human Growth ◽  
Mouse Mammary Gland ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Hypophysectomized Rats

The biological activity profile of reduced and S-carboxymethylated human growth hormone (RCM-hGH) was determined to establish its suitability for study of the diabetogenic property of hGH. RCM-hGH was found to have greatly attenuated in vivo growth-promoting activity in the 9-day weight-gain test in hypophysectomized rats (approximately 1%) and to have a similar low order of in vitro activity in stimulating amino acid incorporation into the protein of the isolated rat diaphragm. RCM-hGH also only had approximately 1% of the in vitro insulin-like activity of the native hormone on isolated adipose tissue from hypophysectomized rats. In contrast, RCM-hGH retained substantial in vivo diabetogenic activity in the ob/ob mouse, appearing to have approximately 50% of the activity of the native hormone. RCM-hGH was also found to retain significant, although attenuated (25%), in vitro lactogenic activity when tested for the ability to stimulate amino acid incorporation into a casein-rich protein fraction in mouse mammary gland explants. Because RCM-hGH exhibits a high degree of diabetogenic activity, although lacking significant anabolic or insulin-like activities, it will be useful as a "monovalent" probe for the study of the molecular mechanism of the diabetogenic action of GH.

Rapid changes in hepatic glucose output after a pulse of growth hormone in dogs

1984 ◽  
Vol 246 (1) ◽  
pp. E14-E20
Author(s):  
P. Vaitkus ◽  
A. Sirek ◽  
K. H. Norwich ◽  
O. V. Sirek ◽  
R. H. Unger ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Venous Blood ◽  
Compartment Model ◽  
Glucose Turnover ◽  
Base Line ◽  
Indwelling Catheter ◽  
Two Compartment Model ◽  
Portal Venous Blood ◽  
Glucose Output

In response to a single intravenous injection of bovine growth hormone (GH, 100 micrograms/kg) the non-steady-state turnover of glucose, as well as portal levels of insulin (IRI), glucagon (IRG), somatostatin (SRIF), and glucose were determined in normal conscious dogs. Using the two-compartment model validated to calculate rapid turnover changes and tracer infusion methods, the rate of hepatic output of glucose [Ra(t)] was found to be increased, reaching a maximum of 224 mg/min, 7.4 times the basal rate, 4 min after injection of GH. Ra(t) returned to its basal level 35 min later in a damped oscillatory manner. Hormone determinations were carried out in portal venous blood drawn every 2 min for 2 h from an indwelling catheter. IRG peaked 2 min after GH injection and levels of IRI, SRIF, and glucose peaked between 4 and 8 min. Hormone concentrations returned to normal, i.e., were oscillating around base-line levels, about 30 min after GH. These experiments demonstrate for the first time in vivo that a pulse of GH causes transient changes of glucose turnover and measurable alterations of the hormonal homeostasis in the splanchnic area.

Isolation of Somatostatin (a Somatotropin Release Inhibiting Factor) of Ovine Hypothalamic Origin

1974 ◽  
Vol 52 (11) ◽  
pp. 1067-1072 ◽  
Author(s):  
P. Brazeau ◽  
W. Vale ◽  
R. Burgus ◽  
R. Guillemin
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partition Chromatography ◽  
Inhibiting Factor ◽  
Layer Chromatography

Isolation of somatostatin, a tetradecapeptide of ovine origin inhibiting somatotropin secretion, is reported. About 490 000 hypothalamic fragments were submitted to alcohol–chloroform extraction, countercurrent distribution, ion-exchange chromatography, gel filtration, and partition chromatography. Of the 8.5 mg material thus obtained, 77% was accounted for by a peptide shown homogeneous by electrophoresis, thin-layer chromatography, and amino acid analysis. The peptide inhibits the secretion of radioimmunoassayable growth hormone at doses of ≥ 1.0 nM in vitro and 400 ng per rate in vivo.

scholarly journals Metabolomic Profiling of Portal Blood and Bile Reveals Metabolic Signatures of Primary Sclerosing Cholangitis

2018 ◽  
Vol 19 (10) ◽  
pp. 3188 ◽  
Author(s):  
Pamela Tietz-Bogert ◽  
Minsuk Kim ◽  
Angela Cheung ◽  
James Tabibian ◽  
Julie Heimbach ◽  
...  
Keyword(s):  
Liver Disease ◽  
Primary Sclerosing Cholangitis ◽  
Sclerosing Cholangitis ◽  
Enterohepatic Circulation ◽  
Biological Fluids ◽  
Venous Blood ◽  
Bile Formation ◽  
Portal Venous Blood

Primary sclerosing cholangitis (PSC) is a pathogenically complex, chronic, fibroinflammatory disorder of the bile ducts without known etiology or effective pharmacotherapy. Emerging in vitro and in vivo evidence support fundamental pathophysiologic mechanisms in PSC centered on enterohepatic circulation. To date, no studies have specifically interrogated the chemical footprint of enterohepatic circulation in PSC. Herein, we evaluated the metabolome and lipidome of portal venous blood and bile obtained at the time of liver transplantation in patients with PSC (n = 7) as compared to individuals with noncholestatic, end-stage liver disease (viral, metabolic, etc. (disease control, DC, n = 19)) and to nondisease controls (NC, living donors, n = 12). Global metabolomic and lipidomic profiling was performed on serum derived from portal venous blood (portal serum) and bile using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and differential mobility spectroscopy-mass spectroscopy (DMS-MS; complex lipid platform). The Mann–Whitney U test was used to identify metabolites that significantly differed between groups. Principal-component analysis (PCA) showed significant separation of both PSC and DC from NC for both portal serum and bile. Metabolite set enrichment analysis of portal serum and bile demonstrated that the liver-disease cohorts (PSC and DC) exhibited similar enrichment in several metabolite categories compared to NC. Interestingly, the bile in PSC was uniquely enriched for dipeptide and polyamine metabolites. Finally, analysis of patient-matched portal serum and biliary metabolome revealed that these biological fluids were more homogeneous in PSC than in DC or NC, suggesting aberrant bile formation and enterohepatic circulation. In summary, PSC and DC patients exhibited alterations in several metabolites in portal serum and bile, while PSC patients exhibited a unique bile metabolome. These specific alterations in PSC are amenable to hypothesis testing and, potentially, therapeutic pharmacologic manipulation.

scholarly journals SAT-283 Generation of a Long-Acting Human Growth Hormone Receptor Antagonist by Site-Specific Pegylation

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Yue Wang ◽  
Ries J Langley ◽  
Kyle Tamshen ◽  
Heather D Maynard ◽  
Stephen M F Jamieson ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Acid Site ◽  
Half Life ◽  
In Vitro Bioactivity ◽  
Site Specific ◽  
Sds Page ◽  
Long Acting

Abstract Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR), activating key signalling pathways including the JAK/STAT pathway. Excess GH secretion leads to acromegaly and tumoral expression has been implicated in cancer progression, suggesting that GH is also a potential target for anticancer therapy. Pegvisomant is the only GHR antagonist approved for clinical use. This antagonist is a PEGylated form of a mutated GH (B2036) that binds and blocks the receptor. Conjugation to polyethylene glycol (PEG) at multiple amine residues reduces in vitro bioactivity but extends the serum half-life resulting in improved in vivo bioactivity. We investigated whether we could generate a long-acting PEGylated GHR antagonist through site-specific conjugation of PEG. A codon optimised GHR antagonist, with an introduced free cysteine residue at amino acid site 144 (S144C), was generated by gene synthesis and recombinantly engineered by gene fusion with thioredoxin. Recombinant protein was expressed in E. coli and purified using a series of chromatographic methods. Antagonists were PEGylated using cysteine-specific conjugation chemistry. In vitro activity was determined using a Ba/F3-GHR viability assay, and in vivo pharmacokinetic and bioactivity was determined in mice. Fusion to thioredoxin was found to improve soluble protein expression at 30℃, resulting in dramatically increased yield. After a series of purification steps, including Ni-NTA, 3C protease cleavage and ion-exchange chromatography, a single band with a molecular mass of 22 kDa was observed by SDS-PAGE analysis. The recombinant antagonist was conjugated to 20 kDa or 30 kDa-PEG at amino acid site S144C. After purification, a single band with an effective molecular size of approximately 60 kDa (PEG-20kDa conjugate) or 70 kDa (PEG-30kDa conjugate) was observed by SDS-PAGE analysis. The unconjugated antagonist inhibited the proliferation of Ba/F3-GHR cells in a dose-dependent manner with a half maximal inhibitory concentration (IC50) of 10.1 ± 2.5 nM. Following PEGylation and purification, the PEG-20kDa and PEG-30kDa conjugates retained high in vitro bioactivity with an IC50 of 66.2 ± 3.8 nM and 106.1 ± 7.1 nM, respectively. Pharmacokinetic analysis demonstrated that PEGylation increased the serum half-life to approximately 15 hours in mice. Subcutaneous administration of the PEG-30kDa conjugate (10 mg/kg/day) reduced serum IGF-I levels in mice. In conclusion, we have generated a novel long-acting human GHR antagonist conjugate by introducing a free cysteine at a non-essential site of the antagonist and targeted attachment of PEG.

scholarly journals Identification of a cDNA encoding a novel amphibian growth hormone-releasing peptide and localization of its transcript

2002 ◽  
Vol 174 (3) ◽  
pp. 395-402 ◽  
Author(s):  
K Sawada ◽  
K Ukena ◽  
S Kikuyama ◽  
K Tsutsui
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Cellular Localization ◽  
Hormone Secretion ◽  
Basic Amino Acid ◽  
Pituitary Hormone ◽  
Amino Acid Residues ◽  
Rapid Amplification

Recently, we identified in the bullfrog brain a novel neuropeptide with a C-terminal Leu-Pro-Leu-Arg-Phe-NH(2) sequence. This amphibian neuropeptide was shown to stimulate growth hormone (GH) release in vitro and in vivo and so was designated frog GH-releasing peptide (fGRP). In this study, we cloned a cDNA encoding fGRP from the bullfrog brain by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced fGRP precursor consisted of 221 amino acid residues, encoding one fGRP and three putative fGRP-related peptides that included Leu-Pro-Xaa-Arg-Phe-NH(2) (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Northern blot analysis detected a single band of approximately 1.0 kb, indicating that no alternatively spliced forms were present. Such an apparent migration was in agreement with the estimated length of the cDNA, 902 bp. In situ hybridization further revealed the cellular localization of fGRP mRNA in the suprachiasmatic nucleus in the hypothalamus. In addition to fGRP, its related peptides may be hypothalamic factors involved in pituitary hormone secretion.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

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