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W. H. SAWYER
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M. MANNING
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SUMMARY

The 4-threonine analogues of oxytocin and of mesotocin and isotocin were prepared by solid-phase synthesis. [4-Threonine]-oxytocin is about twice as active as oxytocin in rat uterus assays in vitro and in vivo and about three times as active in fowl vasodepressor assays. It is slightly more active than oxytocin in rat or rabbit milk-ejection assays. When infused intravenously into water-loaded rats it causes much less depression of diuresis than does an equal dose of oxytocin. [4-Threonine]-oxytocin has much less vasopressor activity than oxytocin. [4-Threonine]-mesotocin also shows enhanced oxytocin-like properties. Its oxytocic activity is equal to or greater than that of oxytocin and its fowl vasodepressor potency is about the same as that of [4-threonine]-oxytocin, 1500 u./mg. It also has less antidiuretic and vasopressor activities than mesotocin. Thus 4-threonine analogues, containing nothing but common l-amino acids, appear to have more of the specific oxytocin-like properties and less of the vasopressin-like properties than do oxytocin or mesotocin. Thus they may be considered improvements on the natural hormones. In this respect they are unique among the reported synthetic analogues of natural peptide hormones.

Substitution of 4-threonine in the weakly-active analogue [3-leucine]-oxytocin also increases its oxytocic and fowl vasodepressor activities. Thus a threonine in the 4-position appears to endow oxytocin-like peptides with greater specific activities than do the amino acids that occur naturally in this position, glutamine and serine. These observations may be of interest when considered (a) from an evolutionary viewpoint, (b) in attempting to interpret relations between molecular structures and biological activities, and (c) as describing peptides with more of the desired properties of oxytocin and less of the undesired properties which might have therapeutic advantages over the natural hormone.

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Y Arsenijevic
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M Dubois-Dauphin
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E Tribollet
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M Manning
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W H Sawyer
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J J Dreifuss
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Abstract

Arginine vasopressin (AVP) acts in the pituitary gland, in synergy with corticotrophin-releasing factor, to induce ACTH release in response to stressful stimuli. Pituitary AVP receptors in the rat are coupled to phospholipase C, as are the so-called V1-type AVP receptors. The present study examined [3H]AVP binding in membranes prepared from the anterior lobe of the pituitary gland of the pig. [3H]AVP, alone or in competition with analogues, bound to sites in the pig anterior lobe which are pharmacologically similar to those described previously by others in the rat pituitary gland. For comparison, the same competition studies were performed on membrane preparations from the rat liver which contain the classic V1-type AVP receptor. Pituitary and liver AVP-binding sites were dissimilar; both cyclic and linear V1 antagonists had, in general, a much lower affinity for pituitary AVP-binding sites than for those in the liver. Thus, Phaa-d-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 (Phaa=phenylacetyl) has a 2500-fold greater affinity for the latter (negative logarithm of inhibition constant (pK i)=9·64) than for the former (pK i=6·22). One linear antagonist, Pa-d-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 (Pa=propionyl) had about equal affinities for liver and pituitary membranes (pK i=6·39 and 6·53 respectively). Another compound, Phaa-d-Tyr-Phe-Val-Asn-Arg-Pro-Arg-Arg-NH2 had the highest affinity found to date for binding to AVP sites in the pituitary (pK i=7·43). These findings suggest some ideas for the design of more potent and/or selective AVP analogues acting in the pituitary gland.

Journal of Endocrinology (1994) 141, 383–391

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R. M. MANNING
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G. N. HENDY
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S. E. PAPAPOULOS
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J. L. H. O'RIORDAN
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SUMMARY

Antisera to a trichloroacetic-acid precipitate of human parathyroid hormone (PTH) were produced in goats. Two of these antisera (G36 and G31) were of high affinity, and the bovine and porcine hormones were less reactive. Synthetic peptides containing the amino-terminal region of human PTH reacted with both antisera; the 1–34 peptide (PTH-(1–34)), with the sequence proposed by Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts in 1974, was more reactive than that having the sequence proposed by Brewer, Fairwell, Ronan, Sizemore & Arnaud in 1972. The antisera were further characterized with a number of other native and synthetic fragments of human PTH and reacted poorly with fragments from the carboxy-terminal region of the molecule. Since the amino-terminal fragments did not account for all the immunoreactivity, it is assumed that the antisera had some recognition sites for the central part of the molecule.

Highly purified human PTH-(1–84) was labelled with 125I and radioimmunoassays were developed using this tracer and antiserum G36. To avoid the problems associated with labelling human PTH with 125I, a labelled antibody assay was developed with G36 and an immunoadsorbent consisting of human PTH-(1–34) (sequence of Niall et al.) coupled to cellulose. A sensitive homologous amino-terminal specific assay was developed in this way.

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G. N. HENDY
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R. M. MANNING
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M. ROSENBLATT
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G. W. TREGEAR
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H. T. KEUTMANN
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J. L. H. O'RIORDAN
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The immunological properties of a synthetic peptide comprising the carboxyl-terminal 53–84 region of human parathyroid hormone (PTH) have been studied. The immunoreactivity of the synthetic human PTH-(53–84) peptide paralleled that of a 53–84 fragment of the native human hormone prepared by enzymic digestion, in both a standard radioimmunoassay, which was not region-specific, and also a radioimmunoassay specific for the carboxyl-terminal region of PTH. However, in both types of radioimmunoassay the synthetic human PTH-(53–84) peptide was four to five times more reactive than the native human PTH-(53–84) fragment.

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Anthony J Manning Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1 Canada

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Harry M Murray Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1 Canada

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Jeffrey W Gallant Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1 Canada

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Makoto P Matsuoka Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1 Canada

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Emily Radford Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1 Canada

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Susan E Douglas Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1 Canada

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Ghrelin is a conserved vertebrate hormone that affects both GH release and appetite. We have cloned and characterized Atlantic halibut preproghrelin cDNA and examined for the first time preproghrelin expression during fish larval development using quantitative real-time PCR. In addition, cellular sites of expression in larvae and tissue-specific expression in 3-year-old halibut were studied. A full-length cDNA for preproghrelin was isolated from halibut stomach tissue. The 899 bp cDNA encodes an open reading frame of 105 amino acids that is comprised of a signal peptide and two peptides with high similarity to ghrelin and obestatin. The deduced amino acid sequence of halibut ghrelin peptide (GSSFLSPSHKPPKGKPPRA) shows significant conservation relative to other teleostean sequences and is identical to human ghrelin for the first seven amino acids of the sequence. The putative obestatin peptide is well-conserved among fishes but shares limited similarity with its human counterpart. Expression of ghrelin was localized to two different cell types in the stomach of larval halibut by in situ hybridization. However, sensitive PCR assays on tissues collected from 3-year-old fish additionally identified ghrelin transcripts in pyloric caecae, intestine, and in immature ovary and testis. Ontogenetic studies detected ghrelin expression prior to exogenous feeding during larval development (hatching and mouth-opening stages) with increased expression occurring through metamorphosis. This increase was pronounced during climax metamorphosis and coincided with stomach differentiation. Patterns of preproghrelin expression suggest that ghrelin has important roles during and after larval development in halibut, and that ghrelin is associated with digestive and gonadal tissues in this teleost.

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Zhenguang Zhang University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

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Agnes E Coutinho University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

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Tak Yung Man University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

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Tiina M J Kipari Centre for Genomic and Experimental Medicine, MRC Institute of Genetic and Molecular Medicine, The University of Edinburgh, Western General Hospital, Edinburgh, UK

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Patrick W F Hadoke University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

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Donald M Salter Centre for Genomic and Experimental Medicine, MRC Institute of Genetic and Molecular Medicine, The University of Edinburgh, Western General Hospital, Edinburgh, UK

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Jonathan R Seckl University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

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Karen E Chapman University/BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK

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11β-Hydroxysteroid dehydrogenase-1 (11β-HSD1) predominantly converts inert glucocorticoids into active forms, thereby contributing to intracellular glucocorticoid levels. 11β-HSD1 is dynamically regulated during inflammation, including in macrophages where it regulates phagocytic capacity. The resolution of inflammation in some disease models including inflammatory arthritis is impaired by 11β-HSD1 deficiency or inhibition. However, 11β-HSD1 deficiency/inhibition also promotes angiogenesis, which is beneficial in mouse models of surgical wound healing, myocardial infarction or obesity. The cell types responsible for the anti-inflammatory and anti-angiogenic roles of 11β-HSD1 have not been characterised. Here, we generated Hsd11b1 MKO mice with LysM-Cre mediated deletion of Hsd11b1 to investigate whether 11β-HSD1 deficiency in myeloid phagocytes is pro-angiogenic and/or affects the resolution of inflammation. Resolution of inflammatory K/BxN-induced arthritis was impaired in Hsd11b1 MKO mice to a similar extent as in mice globally deficient in 11β-HSD1. This was associated with >2-fold elevation in levels of the endothelial marker Cdh5 mRNA, suggesting increased angiogenesis in joints of Hsd11b1 MKO mice following arthritis. A pro-angiogenic phenotype was confirmed by measuring angiogenesis in subcutaneously implanted polyurethane sponges, in which Hsd11b1 MKO mice showed 20% greater vessel density than their littermate controls, associated with higher expression of Cdh5. Thus, 11β-HSD1 deficiency in myeloid phagocytes promotes angiogenesis. Targeting 11β-HSD1 in macrophages may be beneficial in tissue repair.

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T S McQuaid Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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M C Saleh Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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J W Joseph Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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A Gyulkhandanyan Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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J E Manning-Fox Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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J D MacLellan Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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M B Wheeler Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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C B Chan Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward, C1A 4P3 Canada
Departments of Medicine and Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, M5F 1A8 Canada

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We investigated whether an increase in cAMP could normalize glucose-stimulated insulin secretion (GSIS) in uncoupling protein-2 (UCP2) overexpressing (ucp2-OE) β-cells. Indices of β-cell (β-TC-6f7 cells and rodent islets) function were measured after induction of ucp2, in the presence or absence of cAMP-stimulating agents, analogs, or inhibitors. Islets of ob/ob mice had improved glucose-responsiveness in the presence of forskolin. Rat islets overexpressing ucp2 had significantly lower GSIS than controls. Acutely, the protein kinase A (PKA) and epac pathway stimulant forskolin normalized insulin secretion in ucp2-OE rat islets and β-TC-6f7 β-cells, an effect blocked by specific PKA inhibitors but not mimicked by epac agonists. However, there was no effect of ucp2-OE on cAMP concentrations or PKA activity. In ucp2-OE islets, forskolin inhibited ATP-dependent potassium (KATP) channel currents and 86Rb+ efflux, indicative of KATP block. Likewise, forskolin application increased intracellular Ca2+, which could account for its stimulatory effects on insulin secretion. Chronic exposure to forskolin increased ucp2 mRNA and exaggerated basal secretion but not GSIS. In mice deficient in UCP2, there was no augmentation of either cAMP content or cAMP-dependent insulin secretion. Thus, elevating cellular cAMP can reverse the deficiency in GSIS invoked by ucp2-OE, at least partly through PKA-mediated effects on the KATP channel.

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S Khan Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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D E W Livingstone Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK
Centre for Discovery Brain Science, University of Edinburgh, Hugh Robson Building, Edinburgh, UK

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A Zielinska College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

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C L Doig Department of Biosciences, School of Science & Technology, Nottingham Trent University, Nottingham, UK

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D F Cobice Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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C L Esteves Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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J T Y Man Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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N Z M Homer Mass Spectrometry Core Laboratory, Edinburgh Clinical Research Facility, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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J R Seckl Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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C L MacKay SIRCAMS, School of Chemistry, University of Edinburgh, Joseph Black Building, King's Buildings, Edinburgh, UK

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S P Webster Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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G G Lavery Department of Biosciences, School of Science & Technology, Nottingham Trent University, Nottingham, UK

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K E Chapman Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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B R Walker Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK
Clinical & Translational Research Institute, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne, UK

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R Andrew Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK
Mass Spectrometry Core Laboratory, Edinburgh Clinical Research Facility, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK

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11β-Hydroxysteroid dehydrogenase 1 (11βHSD1) is a drug target to attenuate adverse effects of chronic glucocorticoid excess. It catalyses intracellular regeneration of active glucocorticoids in tissues including brain, liver and adipose tissue (coupled to hexose-6-phosphate dehydrogenase, H6PDH). 11βHSD1 activity in individual tissues is thought to contribute significantly to glucocorticoid levels at those sites, but its local contribution vs glucocorticoid delivery via the circulation is unknown. Here, we hypothesised that hepatic 11βHSD1 would contribute significantly to the circulating pool. This was studied in mice with Cre-mediated disruption of Hsd11b1 in liver (Alac-Cre) vs adipose tissue (aP2-Cre) or whole-body disruption of H6pdh. Regeneration of [9,12,12-2H3]-cortisol (d3F) from [9,12,12-2H3]-cortisone (d3E), measuring 11βHSD1 reductase activity was assessed at steady state following infusion of [9,11,12,12-2H4]-cortisol (d4F) in male mice. Concentrations of steroids in plasma and amounts in liver, adipose tissue and brain were measured using mass spectrometry interfaced with matrix-assisted laser desorption ionisation or liquid chromatography. Amounts of d3F were higher in liver, compared with brain and adipose tissue. Rates of appearance of d3F were ~6-fold slower in H6pdh−/− mice, showing the importance for whole-body 11βHSD1 reductase activity. Disruption of liver 11βHSD1 reduced the amounts of d3F in liver (by ~36%), without changes elsewhere. In contrast disruption of 11βHSD1 in adipose tissue reduced rates of appearance of circulating d3F (by ~67%) and also reduced regenerated of d3F in liver and brain (both by ~30%). Thus, the contribution of hepatic 11βHSD1 to circulating glucocorticoid levels and amounts in other tissues is less than that of adipose tissue.

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