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S Harvey and L A Cogburn

Abstract

Complete processing of the TRH precursor in the rat hypothalamus generates TRH and a number of other 'cryptic' peptides that flank the TRH progenitor sequences. Two of these peptides, P4 (Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr; present between amino acids 160 and 169 of rat prepro-TRH) and P5 (Phe-Ile-Asp-Pro-Gly-Leu-Gln-Arg-Ser-Trp-Glu-Glu-Lys-Glu-Gly-Glu-Gly-Val-Leu-Met-Pro-Glu; present between amino acids 178 and 199 of rat prepro-TRH), have recently been shown to modulate TRH-induced GH and thyrotrophin release from rat pituitary glands. The possibility that these peptides might modulate GH secretion in chickens was examined, since TRH is a physiological GH-releasing factor in birds. The administration of P4 and P5 (at doses of 10 and 100 μg/kg) consistently lowered basal plasma GH concentrations 30 and 60 min after a bolus i.v. injection. Pretreatment with P4 and P5 similarly suppressed the GH response to systemic TRH challenge. The GH-releasing activity of maximally stimulatory doses of TRH was also reduced by concomitant injections of either P4 (100 μg/kg) or P5 (100 μg/kg), which blocked the GH-releasing activity of submaximally effective doses of TRH. In marked contrast, neither P4 nor P5 significantly affected basal or TRH-induced GH release from chicken pituitary glands incubated in vitro. These results demonstrate novel actions of P4 and P5 on hypothalamic–pituitary function and, for the first time, indicate extrapituitary sites of action for these cryptic peptides in modulating anterior pituitary function.

Journal of Endocrinology (1996) 151, 359–364

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C L Render, K L Hull and S Harvey

Abstract

It is well established that GH-like proteins and mRNA are present in extrapituitary tissues, but it is not known whether this reflects ectopic transcription of the pituitary GH gene or the expression of a closely related gene. This possibility was, therefore, further investigated.

Immunoreactive (IR) GH-like proteins were readily measured by RIA and immunoblotting in hypothalamic and extrahypothalamic brain tissues of the domestic fowl, in which GH-IR was similar in size and antigenicity to pituitary GH. RT-PCR of mRNA from these brain tissues, with oligonucleotide primers spanning the coding region of pituitary GH cDNA, also generated cDNA fragments identical in size (689 bp) to pituitary GH cDNA. The amplified brain cDNA sequences contained BamH1 and Rsa1 cleavage sites similar to those located in pituitary GH cDNA. These cDNA sequences also hybridized with a cDNA probe for chicken GH cDNA, producing moieties of expected size that were identical to the hybridizing moieties in pituitary tissue. The nucleotide sequences of the PCR products generated from hypothalamic and extrahypothalamic brain tissues, determined by a modified cycle dideoxy chain termination method, were also identical to pituitary GH cDNA. This homology extended over 594 bp of the hypothalamic cDNA fragment (spanning nucleotides 65 to 659 of the pituitary GH cDNA and its coding region for amino acids 4 to 201) and 550 bp of the extrahypothalamic cDNA fragment (spanning nucleotides 76 to 626 of pituitary GH cDNA and its coding region for amino acids 8 to 190).

These results clearly establish that pituitary GH mRNA sequences are transcribed in hypothalamic and extrahypothalamic tissues of the chicken brain, in which GH-IR proteins are abundantly located. However, as GH mRNA could not be detected in the chicken brain by Northern blotting, its turnover may be more rapid than in pituitary tissue. The local production of GH within the brain nevertheless suggests that it has paracrine roles in modulating neural or neuroendocrine function.

Journal of Endocrinology (1995) 147, 413–422

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K. L. Hull, R. A. Fraser and S. Harvey

ABSTRACT

Although GH has no direct effect on GH release from chicken pituitary glands, GH receptor mRNA similar to that in the rabbit liver was identified by Northern blot analysis in extracts of adult chicken pituitaries. Complementary (c) DNA, reverse transcribed from chicken pituitary RNA, was amplified by the polymerase chain reaction (PCR) in the presence of 3′- and 5′-oligonucleotide primers coding for the extracellular domain of the chicken liver GH receptor and was found to contain an electrophoretically separable fragment of 500 bp, identical in size to that in chicken liver. Digestion of this pituitary cDNA with NcoI produced expected moities of 350 and 150 bp. Amplification of chicken pituitary cDNA in the presence of oligonucleotide primers for the intracellular sequence of the chicken liver GH receptor produced an electrophoretically separable fragment of approximately 800 bp, similar to that in chicken liver. This fragment was cut into expected moieties of 530 and 275 bp after digestion with EcoRI. These PCR fragments were identified in extracts of the pituitary caudal lobe, in which somatotrophs are confined and account for the majority of endocrine cell types, and in the cephalic lobe, in which somatotrophs are lacking. Translation of the GH receptor mRNA in the pituitary gland was indicated by the qualitative demonstration of radio-labelled GH-binding sites in plasma membrane preparations, in pituitary cytosol and in nuclear membranes. These results provide evidence for the expression and translation of the GH receptor gene in pituitary tissue, in which GH receptors appear to be widely distributed within cells and in different cell types. GH may therefore have paracrine, autocrine or intracrine effects on pituitary function.

Journal of Endocrinology (1992) 135, 459–468

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S. Harvey, V. L. Trudeau, R. J. Ashworth and S. M. Cockle

ABSTRACT

Pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2) is a tripeptide with structural and immunological similarities to thyrotrophin-releasing hormone (TRH; pGlu-His-ProNH2). Since TRH stimulates GH secretion in domestic fowl, the possibility that pGlu-Glu-ProNH2 may also provoke GH release was investigated. Unlike TRH, pGlu-Glu-ProNH2 alone had no effect on GH release from incubated chicken pituitary glands and did not down-regulate pituitary TRH receptors. However, pGlu-Glu-ProNH2 suppressed TRH-induced GH release from pituitary glands incubated in vitro and competitively displaced [3H]methyl3-histidine2-TRH from pituitary membranes. Systemic injections of pGlu-Glu-ProNH2 had no significant effect on basal GH concentrations in conscious birds, but promptly lowered circulating GH levels in sodiumpentobarbitone anaesthetized fowl. Submaximal GH responses of conscious and anaesthetized birds to systemic TRH challenge were, however, potentiated by prior or concomitant administration of pGlu-Glu-ProNH2. These results demonstrate, for the first time, that pGlu-Glu-ProNH2 has biological activity, with inhibitory and stimulatory actions within the avian hypothalamo-pituitary axis. These results indicate that pGlu-Glu-ProNH2 may act as a TRH receptor antagonist within this axis.

Journal of Endocrinology (1993) 138, 137–147

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K L Hull, W C J Janssens, W R Baumbach and S Harvey

Abstract

Thyroid hormones inhibit the synthesis and release of GH in avian species. This may represent a feedback mechanism, since GH enhances the peripheral production of tri-iodothyronine (T3). The possibility that GH may also have direct effects on thyroidal function was therefore investigated.

The basal and thyrotrophin-induced release of thyroxine (T4) from incubated chicken thyroid glands was not enhanced, however, in the presence of chicken GH. Contrarily, GH impaired T4 release in a dose-related way. These actions were probably mediated by specific receptors, since binding sites for radiolabelled GH were demonstrated on the plasma membranes of chicken thyroid glands. Expression of the GH receptor gene in these tissues was also demonstrated using a cRNA probe for the rabbit liver GH receptor, which specifically hybridized with RNA moieties of 4·4 kb, 2·7 kb and 1·0 kb. Moreover, reverse transcription of thyroidal RNA and its amplification in the presence of 3′- and 5′-oligonucleotide primers coding for the extracellular or intracellular domains of the GH receptor generated electrophoretically separable fragments of 500 bp and 800 bp respectively, as would be expected from analysis of the hepatic GH receptor cDNA sequence. Digestion of the 500 bp fragment with NcoI or EcoRI also produced moieties of expected size (350 bp and 150 bp or 325 bp and 175 bp respectively), as did BamHI or HaeIII digestion of the 800 bp fragment (yielding fragments of 550 bp and 275 bp or 469 bp and 337 bp respectively). Translation of the GH receptor mRNA was also indicated by the immunocytochemical demonstration of GH receptors in thyroid follicular and parafollicular cells, using a specific polyclonal antibody raised against the chicken GH-binding protein.

These results therefore provide evidence, for the first time, of GH receptor gene expression in thyroid tissue and the translation of functional GH receptors in thyroid glands. These results also demonstrate differential effects of GH on the extracellular concentrations of T3 and T4, which may permit subtle regulation within the somatotroph-thyroid axis.

Journal of Endocrinology (1995) 146, 449–458

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K. L. Hull, R. A. Fraser, J. A. Marsh and S. Harvey

ABSTRACT

GH receptor (GHR) mRNA has been identified in peripheral (liver and muscle) and central (brain and hypothalamus) tissues of sex-linked dwarf (SLD) Leghorn chickens. Total RNA was extracted from the tissues of immature (1 week, 4 week), pubertal (16 week) and adult (> 24 weeks) SLD and K (the normally growing strain) Leghorn chickens. In both groups and all tissues, an mRNA moiety of 4·4 kb hybridized with cRNA probes derived from the rabbit hepatic GHR sequence. An additional low-abundance transcript of 2·8 kb was also identified in some tissues. An age-related increase in expression was observed in K and SLD hepatic GHR mRNA, suggesting normal regulation of SLD GHR gene transcription. Amplification of cDNA from K and SLD tissues in the presence of oligonucleotide primers coding for the intracellular or extracellular domains of the chicken GHR generated electrophoretically separable fragments of expected size. Restriction enzyme digestion of the products with EcoRI, BstNI, HaeIII, NcoI or BamHI produced smaller moieties of expected sizes in both strains. These results demonstrate that, in contrast to broiler SLDs, a GHR gene deletion is not responsible for the GHR dysfunction in Leghorn SLDs. Although the actual defect in GHR gene expression in SLD Leghorns remains to be identified, this study demonstrates that sex-linked dwarfism, like Laron dwarfism, is due to a heterogeneity of lesions.

Journal of Endocrinology (1993) 137, 91–98

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H Y Li, Y X Liu, L Harvey, S Shafaeizadeh, E M van der Beek and W Han

The prevalence of gestational diabetes mellitus (GDM) is estimated at 14% globally, and in some countries, such as Singapore, exceeds 20%. Both women and children exposed to GDM have an increased risk of later metabolic diseases, cardiovascular disease and other health issues. Beyond lifestyle changes and pharmaceutical intervention using existing type 2 diabetes medications for expecting women, there are limited treatment options for women with GDM; targeting better outcomes of potentially affected infants is unexplored. Numerous animal models have been generated for understanding of pathological processes of GDM development and for development of treatment strategies. These models, however, suffer from limited windows of opportunity to examine risk factors and potential intervention options. By combining short-term high-fat diet (HFD) feeding and low-dose streptozotocin (STZ) treatments before pregnancy, we have established a mouse model with marked transient gestation-specific hyperglycemia, which allows testing of nutritional and pharmacological interventions before, during and beyond pregnancy.