Biomedical subjects

Perry Barrett

Publications and source records attributed to Perry Barrett.

15 recordsLinked to original sources

PubMed2006

Photoperiodic regulation of insulin receptor mRNA and intracellular insulin signaling in the arcuate nucleus of the Siberian hamster, Phodopus sungorus.

During the last 5 years it has been well established that photoperiod-induced changes in body weight in the seasonal hamster, Phodopus sungorus, are accompanied by a marked seasonal cycle in leptin sensitivity. In the present study, we investigated the possible involvement of insulin signaling in seasonal body weight regulation. We analyzed the expression pattern and relative intensity of insulin receptor (IR), phosphatidylinositol 3-kinase (PI3-kinase), and protein tyrosine phosphatase 1B (PTP1B) mRNAs by in situ hybridization in the brains of juvenile female hamsters acclimated to either long- (LD) or short-day length (SD) for 8 wk, with or without superimposed food deprivation for 48 h. Furthermore, the hypothalamic concentration and distribution of phospho-AKT, a marker of PI3-kinase activity was determined by immunoblotting and immunohistochemistry. Eight weeks of acclimation to SD led to a substantial downregulation of IR, PTP1B gene expression, and phospho-AKT concentration in this brain region, whereas PI3-kinase mRNA was unchanged. Food deprivation induced a decrease in PTP1B and a trend toward lowered IR gene expression in LD but not in SD. Additionally, a striking increase in PTP1B gene expression in the thalamus was observed after food deprivation in both photoperiods. The direction of change in neuronal insulin signaling contrasts to the central catabolic nature of this pathway described in other species. SD-induced reduction in insulin signaling may be due to decline in body fat stores mediated by enhanced central leptin sensitivity. Increased anorexigenic tone of leptin may overwrite central insulin signaling to prevent catabolic overdrive.

Animals↗

PubMed2006

What can we learn from seasonal animals about the regulation of energy balance?

Weight loss in humans requires, except during an illness, some form of imposed restriction on food intake or increase in energy expenditure. This necessitates overcoming powerful peripheral and central signals that serve to protect against negative energy balance. The identification of the systems and pathways involved has come from mouse models with genetic and targeted mutations, e.g., ob/ob and MC4 R(-/-) as well as rat models of obesity. Study of seasonal animals has shown that they undergo annual cycles of body fattening and adipose tissue loss as important adaptations to environmental change, yet these changes appear to involve mechanisms distinct from those known already. One animal model, the Siberian hamster, exhibits marked, but reversible, weight loss in response to shortening day length. The body weight is driven by a decrease in food intake with the magnitude of the loss of body weight being directly related to the length of time of exposure to short photoperiod. The most important facet of this response is that the point of energy balance is continuously re-adjusted during the transition in body weight reflecting an apparent 'sliding set point'. Studies have focused on identifying the neural basis of this mechanism. Initial studies of known genes (e.g., NPY, POMC, and AgRP) both through the measurement of gene expression in the arcuate nucleus as well as following intracerebroventricular (i.c.v.) injection indicated that the systems involved are not those involved in restoring energy balance following energy deficits. Instead, a novel mechanism of regulation is implied. Recent studies have begun to explore the neural basis of the seasonal body weight response. A discrete and novel region of the posterior arcuate nucleus, the dorsal medial posterior arcuate nucleus (dmpARC) has been identified, where a battery of gene expression changes for signalling molecules (vgf and histamine H3 receptor) and transcription factors (RXRgamma and RAR) occur in association with seasonal changes in body weight. This work provides the basis of a potentially novel mechanism of energy balance regulation.

Animals↗

PubMed2006

Negative energy balance and leptin regulate neuromedin-U expression in the rat pars tuberalis.

Central neuromedin U (NMU) functions in energy balance, the hypothalamic-pituitary-adrenal axis, LH release and circadian rhythmicity. In rats, high levels of NMU occur in the hypothalamic suprachiasmatic nuclei and the pars tuberalis of the pituitary. NMU expression in the pars tuberalis appears to be downregulated in the Zucker fatty (fa/fa) rat, lacking functional leptin receptors. In contrast, in the dorsomedial (DMH) nuclei of the mouse, NMU expression is higher in the ob/ob mouse, lacking leptin, and is upregulated by fasting. However, leptin appears not to change NMU gene expression in either the mouse DMH or the rat pars tuberalis. Thus, the present study aims to better identify factors influencing central NMU expression in the rat pars tuberalis. Sprague-Dawley rats were fasted and/or challenged with intracerebroventricular leptin or ghrelin and gene expression was measured using real-time reverse transcriptase-PCR and quantitative in situ hybridisation with riboprobes specific for NMU and NMU receptor (NMU-R2). NMU expression in the rat pars tuberalis was elevated by fasting. Ghrelin administration had no effect on the level of NMU expression, but leptin was found to diminish the expression in a concentration- and time-dependent manner. NMU-R2 expression was unchanged in any of the groups measured. These results suggest that NMU expression in rat pars tuberalis is upregulated in states of negative energy balance, and this may be mediated indirectly by changes in leptin levels. These results demonstrate a link between energy balance and NMU expression in the pars tuberalis of the pituitary.

Animals↗

PubMed2006

Photoperiodic regulation of cellular retinol binding protein, CRBP1 [corrected] and nestin in tanycytes of the third ventricle ependymal layer of the Siberian hamster.

Tanycytes in the ependymal layer of the third ventricle act both as a barrier and a communication gateway between the cerebrospinal fluid, brain and portal blood supply to the pituitary gland. However, the range, importance and mechanisms involved in the function of tanycytes remain to be explored. In this study, we have utilized a photoperiodic animal to examine the expression of three unrelated gene sequences in relation to photoperiod-induced changes in seasonal physiology and behaviour. We demonstrate that cellular retinol binding protein [corrected] (CRBP1), a retinoic acid transport protein, GPR50, an orphan G-protein-coupled receptor and nestin, an intermediate filament protein, are down-regulated in short-day photoperiods. The distribution of the three sequences is very similar, with expression located in cells with tanycyte morphology in the region of the ependymal layer where tanycytes are located. Furthermore, CRBP1 expression in the ependymal layer is shown to be independent of a circadian clock and altered testosterone levels associated with testicular regression in short photo-period. Pinealectomy of Siberian hamsters demonstrates CRBP1 expression is likely to be dependent on melatonin output from the pineal gland. This provides evidence that tanycytes are seasonally responsive cells and are likely to be an important part of the mechanism to facilitate seasonal physiology and behaviour in the Siberian hamster.

Animals↗

PubMed2005

Temporal changes in gene expression in the arcuate nucleus precede seasonal responses in adiposity and reproduction.

In anticipation of seasonal climate changes, Siberian hamsters display a strategy for survival that entails profound physiological adaptations driven by photoperiod. These include weight loss, reproductive quiescence, and pelage growth with shortening photoperiod and vice versa with lengthening photoperiod. This study reports gene expression changes in the hypothalamus of Siberian hamsters switched from short days (SD) to long days (LD), and also in photorefractory hamsters. Siberian hamsters were maintained in either LD or SD for 14 wk, conditions that generate physiological states of obesity under LD and leanness under SD. After 14 wk, SD lighting was switched to LD and gene expression investigated after 0, 2, 4, and 6 wk by in situ hybridization. Genes encoding nuclear receptors (RXR/RAR), retinoid binding proteins (CRBP1 and CRABP2), and histamine H3 receptor were photoperiodically regulated with significantly lower expression in SD, whereas VGF mRNA expression was significantly higher in SD, in the dorsomedial posterior arcuate nucleus. After a SD-to-LD switch, gene expression changes of CRABP2, RAR, H3R, and VGF occurred relatively rapidly toward LD control levels, ahead of body weight recovery and testicular recrudescence, whereas CRBP1 responded less robustly and rxrgamma did not respond at the mRNA level. In this brain nucleus in photorefractory animals, the CRABP2, RAR, H3R, and VGF mRNA returned toward LD levels, whereas CRBP1 and rxrgamma remained at the reduced SD level. Thus, genes described here are related to photoperiodic programming of the neuroendocrine hypothalamus through expression responses within a subdivision of the arcuate nucleus.

Adaptation, Physiological↗

PubMed2005

Neuromedin-U is regulated by the circadian clock in the SCN of the mouse.

Neuromedin-U (NMU) has been reported to drive several physiological or behavioural responses following i.c.v. injection of the peptide into the third ventricle of rodent brains. Many of these responses are mediated through a change in corticotrophin-releasing factor (CRF) output from the paraventricular nucleus (PVN). A number of the physiological or behavioural responses are regulated in a circadian manner, e.g. feeding. We have previously reported NMU gene expression in the suprachiasmatic nucleus (SCN) and NMU-2 receptor expression in the PVN, dorsal medial hypothalamus (DMH) and other regions of the mouse brain. We therefore hypothesized that NMU would be regulated by the circadian clock and may consequently drive a circadian rhythm of CRF expression in the PVN. Here we report that NMU is regulated in a circadian manner with peak expression during the light phase of a light-dark cycle. In C3H mice held in constant darkness, the NMU rhythm free runs with a period predicted by the free running period of locomotor activity in this mouse. The NMU mRNA transcript colocalizes with cells expressing AVP in the SCN and shows a coincident rhythm of expression with AVP. On the other hand, CRF did not express a circadian rhythm of expression in a light-dark cycle, although a rhythm was evident in constant darkness with a peak of expression prior to the rise of NMU in the same conditions. This would suggest that the circadian rhythm in NMU expression in the SCN does not drive a circadian rhythm in CRF in the PVN to be translated into physiological and behavioural responses mediated by NMU.

Animals↗

PubMed2004

Photoperiodic regulation of histamine H3 receptor and VGF messenger ribonucleic acid in the arcuate nucleus of the Siberian hamster.

To survive winter the Siberian hamster has evolved profound physiological and behavioral adaptations, including a moult to winter pelage, regression of the reproductive axis, onset of daily torpor and increased capacity for thermogenesis. However, one of the most striking adaptations is the catabolism of intraabdominal and sc fat reserves contributing to the loss of up to 40% of body weight. These physiological and behavioral adaptations are photoperiodically driven, yet neither the site(s) in the brain nor the molecular mechanism(s) involved in the regulation of these profound adaptations is known. Here we report a dynamic regulation of gene expression in a dorsal region of the medial posterior area of the arcuate nucleus (dmpARC) of the Siberian and Syrian hamster brain in response to altered photoperiod. We show mRNA for the histamine H3 receptor is down-regulated and VGF is up-regulated in the dmpARC in hamsters switched from long- to short-day photoperiod. These data provide further evidence to support the view that the dmpARC is a major site to relay photoperiodic changes and as a site for the long-term regulation of seasonal physiology and behavior.

Animals↗

PubMed2004

Molecular pharmacology of the ovine melatonin receptor: comparison with recombinant human MT1 and MT2 receptors.

The variations of the pharmacological properties of melatonin receptors between different mammalian species in transfected cell lines have been poorly investigated. In the present study, melatonin analogues have been used to characterize the pharmacology of the recombinant ovine melatonin receptor (oMT1) expressed in CHO cell lines and the native oMT1 from the pars tuberalis (PT). Studies with selective ligands on native and transfected oMT1 showed similar properties for binding affinities [r2(PT/CHO) = 0.85]. The affinities and the functional activities of these ligands were compared with the human receptors (hMT1 or hMT2) expressed in CHO cells as well. The oMT1 and hMT1 receptors had similar pharmacological profiles (r2=0.82). Nevertheless, some of the selective compounds at the human receptor presented a reduced affinity at the ovine receptor. Furthermore, some compounds showed marked different functional activities at oMT1 vs. hMT1 receptors. Our findings demonstrated differences in the pharmacological properties of melatonin receptors in ovine and human species.

Animals↗

PubMed2003

Photoperiodic regulation of hypothalamic retinoid signaling: association of retinoid X receptor gamma with body weight.

This study reports novel events related to photoperiodic programming of the neuroendocrine hypothalamus. To investigate photoperiod-responsive genes, Siberian hamsters were maintained in long or short photoperiods that generate physiological states of obesity or leanness. Microarray expression analysis first identified CRBP1 as a photoperiod-responsive gene, and then further studies using in situ hybridization and immunocytochemistry revealed that expression levels of several related retinoid-signaling genes were modulated in response to photoperiod changes. Genes of the retinoid-signaling pathway, encoding nuclear receptors (RXR/RAR) and retinoid binding proteins (CRBP1 and CRABP2) are photoperiodically regulated in the dorsal tuberomamillary nucleus (DTM): Their expression is significantly lower in short photoperiods and parallels body weight decreases. Studies in pinealectomized hamsters confirm that the pineal melatonin rhythm is necessary for these seasonal changes, and studies in testosterone-treated hamsters reveal that these changes in gene expression are not the secondary consequence of photoperiod-induced changes in steroid levels. Comparative studies using Syrian hamsters, which show divergent seasonal body weight responses to Siberian hamsters when exposed to short photoperiods, showed a distinct pattern of changes in retinoid gene expression in the DTM in response to a change in photoperiod. We infer that the DTM may be an important integrating center for photoperiodic control of seasonal physiology and suggest that the changes in retinoid X receptor gamma expression may be associated with seasonal changes in body weight and energy metabolism.

Animals↗

PubMed2003

Gonadotrophin-releasing hormone drives melatonin receptor down-regulation in the developing pituitary gland.

Melatonin is produced nocturnally by the pineal gland and is a neurochemical representation of time. It regulates neuroendocrine target tissues through G-protein-coupled receptors, of which MT(1) is the predominant subtype. These receptors are transiently expressed in several fetal and neonatal tissues, suggesting distinct roles for melatonin in development and that specific developmental cues define time windows for melatonin sensitivity. We have investigated MT(1) gene expression in the rat pituitary gland. MT(1) mRNA is confined to the pars tuberalis region of the adult pituitary, but in neonates extends into the ventral pars distalis and colocalizes with luteinizing hormone beta-subunit (LH beta) expression. This accounts for the well documented transient sensitivity of rat gonadotrophs to melatonin in the neonatal period. Analysis of an upstream fragment of the rat MT(1) gene revealed multiple putative response elements for the transcription factor pituitary homeobox-1 (Pitx-1), which is expressed in the anterior pituitary from Rathke's pouch formation. A Pitx-1 expression vector potently stimulated expression of both MT(1)-luciferase and LH beta-luciferase reporter constructs in COS-7 cells. Interestingly, transcription factors that synergize with Pitx-1 to trans-activate gonadotroph-associated genes did not potentiate Pitx-1-induced MT(1)-luciferase activity. Moreover, the transcription factor, early growth response factor-1, which is induced by gonadotrophin-releasing hormone (GnRH) and trans-activates LH beta expression, attenuated Pitx-1-induced MT(1)-luciferase activity. Finally, pituitary MT(1) gene expression was 4-fold higher in hypogonadal (hpg) mice, which do not synthesize GnRH, than in their wild-type littermates. These data suggest that establishment of a mature hypothalamic GnRH input drives the postnatal decline in pituitary MT(1) gene expression.

Animals↗

PubMed2003

Digging deep--structure-function relationships in the melatonin receptor family.

The melatonin receptor family is a small group of receptors within the G protein-coupled receptor (GPCR) superfamily. The group comprises of three subtypes which bind melatonin and one member, the melatonin related receptor (MRR), that shares >40% sequence identity with the other melatonin receptors but does not bind melatonin. Identification of two subtypes expressed in the mouse suprachiasmatic nucleus, one of which (MT1) inhibits neuronal firing and the other (MT2) mediating the phase advancing properties of melatonin has given renewed interest to the development of subtype specific compounds for each of the mammalian melatonin receptors. Towards this goal site-directed and chimaeric receptor mutagenesis studies have been performed which have provided some insight into the structure-function relationships of the melatonin receptors. Furthermore, these studies may lead to the identification of the ligand for the orphan MRR.

Amino Acid Sequence↗

PubMed2002

Differential coupling of the extreme C-terminus of G protein alpha subunits to the G protein-coupled melatonin receptors.

Melatonin receptors interact with pertussis toxin-sensitive G proteins to inhibit adenylate cyclase. However, the G protein coupling profiles of melatonin receptor subtypes have not been fully characterised and alternative G protein coupling is evident. The five C-terminal residues of Galpha subunits confer coupling specificity to G protein-coupled receptors. This report outlines the use of Galphas chimaeras to alter the signal output of human melatonin receptors and investigate their interaction with the C-termini of Galpha subunits. The Galphas portion of the chimaeras confers the ability to activate adenylate cyclase leading to cyclic AMP production. Co-transfection of HEK293 cells expressing MT(1) or MT(2) melatonin receptors with Galphas chimaeras and a cyclic AMP activated luciferase construct provided a convenient and sensitive assay system for identification of receptor recognition of Galpha C-termini. Luciferase assay sensitivity was compared with measurement of cyclic AMP elevations by radioimmunoassay. Differential interactions of the melatonin receptor subtypes with Galpha chimaeras were observed. Temporal and kinetic parameters of cyclic AMP responses measured by cyclic AMP radioimmunoassay varied depending on the Galphas chimaeras coupled. Recognition of the C-terminal five amino acids of the Galpha subunit is a requisite for coupling to a receptor, but it is not the sole determinant.

Cell Line↗

PubMed2002

CART gene promoter transcription is regulated by a cyclic adenosine monophosphate response element.

OBJECTIVE: To define the regulatory DNA sequence controlling the expression of the cocaine-amphetamine-regulated transcript (CART) gene expression. RESEARCH METHODS AND PROCEDURES: A rat genomic library was screened for genomic clones containing the CART gene and upstream DNA sequence. A clone containing exons 1 and 2 of the rat CART gene plus 1.2 kb of upstream sequence was isolated. The 1.2-kb upstream sequence and truncated segments of this sequence were cloned into a luciferase reporter vector for analysis of transcriptional activity in the CART expressing pituitary GH3 cell line. Luciferase reporter assays, gel shift assays, and site-directed mutagenesis were used to analyze potential regulatory regions in the 1.2-kb 5' DNA sequence. RESULTS: Sequence analysis of the 1.2-kb upstream sequence reveals several potential regulatory elements. Luciferase reporter assays demonstrate that within the first 162 bp of the start codon is a functional cyclic adenosine monophosphate (cAMP) response element that can induce cAMP-regulated gene expression of the CART promoter in GH3 cells. Mutation of this cAMP response element abolishes cAMP-stimulated transcription [corrected]. Furthermore, the promoter is active in a neuronal cell line where it also demonstrates cAMP responsiveness. DISCUSSION: cAMP-mediated transcription of the CART promoter may be an important aspect of the regulation of the CART gene. However, the cellular context of expression is also important because the CART promoter is not responsive to cAMP stimulation in all cell types. Other transcription factor binding sequences are likely to play a key role in CART promoter regulation.

Animals↗

PubMed2002

Hypothalamic bHLH transcription factors are novel candidates in the regulation of energy balance.

The basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.

Animals↗

PubMed2002

Pituitary adenylate cyclase-activating polypeptide acts as a paracrine regulator of melatonin-responsive cells of the ovine pars tuberalis.

The pars tuberalis (PT) region of the anterior pituitary plays a physiological role in seasonal animals. The primary signal transduction mechanism of the melatonin receptor in this tissue is an inhibition of cAMP signaling. However, nothing is known about the endocrine signals that activate cAMP synthesis in the cells of the PT, as previous studies relied on the pharmacological tool, forskolin, to stimulate cAMP synthesis. Here we show that pituitary adenylate cyclase-activating polypeptide (PACAP) activates cAMP synthesis in the cells of the PT. The pharmacology of cAMP activation by PACAP peptides suggests that cAMP activation is mediated by the type I PACAP receptor. PACAP treatment of PT cells results in cellular responses that are consistent with cAMP activation in these cells, including activation of MAPK and elevation of melatonin receptor mt1 mRNA expression. These responses can be inhibited by melatonin, demonstrating that activation of cAMP occurs within the melatonin-responsive cells. However, although PACAP activates cAMP in the cells of the PT, the effect of PACAP may not be direct, as colocalization in situ hybridization studies demonstrates that the type I PACAP receptor and the melatonin mt1 receptor do not colocalize on the cells of the PT.

Adenylyl Cyclases↗