published_or_final_version / Medicine / Master / Master of Philosophy

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Thesis (M. Phil.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 92-112).

In 1971 Schally and co-workers (Schally et al., 1971) isolated gonadotropin-releasing hormone (now called luteinizing hormone-releasing hormone (LH-RH)) from sheep hypothalami and established that the hormone was a decapeptide with the amino acid sequence: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂. The peptide was subsequently synthesised (Matsuo et al., 1971b) and shown to stimulate the release of gonadotropins (luteinizing hormone and follicle-stimulating hormone) in a wide range of mammalian species (Schally et al., 1973, 1976). With the exception of amphibians, nonmammalian vertebrates have a poor gonadotropin response to synthetic mammalian LH-RH (for reviews, see Ball, 1981; Jackson, 1981; King and Millar, 1981a). Since there is considerable molecular heterogeneity in the related neurohypophysial nonapeptide hormones (oxytocin-vasopressin) amongst vertebrates (Acher et al., 1972), we postulated that differences might exist in the structure of hypothalamic LH-RH in different vertebrate classes, Utilising a combination of regionspecific antisera and chromatographic techniques, we established that amphibian hypothalamic LH-RH is identical to the mammalian peptide while avian, reptilian, and piscine hypothalamic LH-RHs differ structurally in the region Gly⁶-Leu⁷-Arg⁸ (King and Millar, 1979a, 1980), We have now conducted further studies on avian hypothalamic LH-RH, which indicate that the arginine residue in position eight of mammalian LH-RH is substituted by glutamine in this vertebrate class. Purification of LH-RH from chicken hypothalami and determination of the amino acid composition have confirmed that the structure of avian LH-RH is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH₂.

Leptin is primarily an adipose-derived hormone that acts in the hypothalamus to regulate body fat levels by suppressing appetite and increasing metabolic rate. Pregnancy is characterised by increased food intake and fat mass to meet the metabolic demands of this physiological state. Leptin concentrations also increase during pregnancy, but this does not prevent the pregnancy-induced hyperphagia, suggesting a state of leptin resistance. The aims of this thesis were to measure hypothalamic leptin responsiveness during pregnancy and to investigate the potential mechanisms underlying pregnancy-induced leptin resistance. The satiety response to intracerebroventricular (i.c.v) leptin was measured in fasted non-pregnant (diestrous), early pregnant (day 7), and mid-pregnant (day 14) rats. Serial blood samples collected from another group of rats demonstrated that despite initial elevated plasma leptin concentrations in pregnant rats, fasting significantly decreased leptin concentrations so that pregnant and non-pregnant groups had similar, low leptin concentrations. Leptin treatment significantly reduced food intake in non-pregnant and early pregnant rats but not in mid-pregnant rats. In addition, there was no post-fasting hyperphagic response in the pregnant rats. These results indicate that pregnant rats become resistant to the satiety action of leptin. To investigate the mechanisms underlying pregnancy-induced leptin resistance, leptin-induced activation of hypothalamic leptin-target neurons was examined. Signal transducer and activator of transcription 3 (STAT3) phosphorylation was measured in non- pregnant and mid-pregnant rats following i.c.v. administration of leptin. Western blot and immunohistochemistry analysis indicated that leptin-induced STAT3 phosphorylation was significantly reduced in the ventromedial nucleus of the hypothalamus (VMH) during pregnancy. A suppression in the amount of leptin-induced STAT3 activation was observed in the arcuate nucleus during pregnancy, yet there was no overall change in the number of leptin responsive neurons compared to non-pregnant rats. This raises the possibility of a decrease in the degree of responsiveness of arcuate nucleus neurons to leptin during pregnancy. Using double-labelled immuno-histochemistry for alpha-melanocyte stimulating hormone (α-MSH) and leptin-induced pSTAT3 it was demonstrated that pro-opiomelanocortin (POMC) neurons remain responsive to leptin during pregnancy. In the VMH, consistent with the reduced pSTAT3, pregnancy also induced a 2-fold reduction in mRNA for the long form of the leptin receptor (Ob-Rb), the only isoform with full signal transduction capabilities. Expression of mRNA for one of the short forms of the leptin receptor (Ob-Ra) in the choroid plexus was decreased in early and late pregnancy, suggesting that reduced leptin transport into the brain may contribute to pregnancy-induced leptin resistance. CSF/plasma leptin concentration ratios did not differ between pregnant and non-pregnant rats however, suggesting unimpaired leptin transport during pregnancy. These results indicate that pregnancy is a state of hypothalamic leptin resistance and is associated with impaired activation of the leptin-induced JAK/STAT3 signalling pathway in the VMH and arcuate nucleus, and reduced expression of Ob-Rb mRNA in the VMH. This state of leptin resistance represents an important adaptation of the maternal brain allowing increased food intake and fat mass so that the maternal body can meet the metabolic demands of pregnancy and prepare for the subsequent demands of lactation.

The hypothalamic-pituitary-thyroid axis operates to maintain the circulating concentration of thyroid hormones. Thyrotropin releasing hormone (TRH) is the major hypothalamic messenger controlling the pituitary-thyroid unit. However, the pituitary-thyroid unit responses to various modalities of TRH exposure are not well characterized. Also, interactions between the thyroid axis and other mammalian organ systems, specifically other hormone axes, to maintain the organism's homeostatic state are not well characterized. This work was designed to clarify the response of the pituitary-thyroid unit to TRH and to assess the effects of physiological levels of the rat's primary adrenal cortical hormone, corticosterone, on the thyroid axis. Adult rats were given equal amounts of TRH by intravenous (I.V.) bolus injection or constant intraperitoneal (I.P.) infusion. Both methods resulted in significant increases in plasma thyroid stimulating hormone (TSH), although the time course and peak plasma value varied with the TRH dosage and administration method. Despite the differences in plasma TSH elicited, the thyroid gland responses were similar. Thus, the pituitary is sensitive to the rate and dose of TRH administration. Also, the thyroid is sensitive to plasma levels of TSH but reaches maximum stimulation at submaximal circulating TSH levels. Adrenalectomized female rats, with I.P. and I.V. catheters, were infused with corticosterone (B) to achieve plasma levels within the rat's physiological range. Plasma samples were drawn before and after submaximal TRH (250 ng/100 g Body Weight) administration for assay of TSH and B concentrations. B in the lower half of its physiological range significantly inhibited the increase in plasma TSH observed 10 and 30 minutes after TRH administration. Also, direct stereotaxic infusion of B (50 ng) followed by TRH (1 ng) into the anterior pituitary inhibited the observed increase in plasma TSH. These studies indicate that homeostatic thyroid axis hormone concentrations are maintained by a feedback loop mechanism which is modulated by adrenal hormones. Specifically, physiological levels of corticosterone decrease pituitary sensitivity to TRH in the rat. In addition, the pituitary and thyroid gland exhibit different response patterns to hormonal stimulation.

Although research has linked central nervous system activity with changes in immunoresponsivity, research on the possible role of the central nervous system in altering a specific class of antibody is lacking. This study was an investigation of the possible relationship between anterior or medial hypothalamic functions on Immunoglobulin G. concentrations in rat serum. Thirty-six male albino rats were randomly assigned to three groups of equal size. Animals within the anterior hypothalamic group received bilateral electrode implants in the anterior hypothalamus while animals in the medial hypothalamic group received electrode implants within the ventromedial area of the hypothalamus. A control group received bilateral electrode implants within the lateral hypothalamus. Electrical brain stimulation was administered to animals in both experimental groups. Control animals spent a comparable time in an operant chamber but did not receive electrical brain stimulation. Following brain stimulation of animals within the experimental groups, Immunoglobulin G. concentrations were determined for all groups 3, 6, 12, and 24 hours post-stimulation sessions.

[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 127-149.