A missense mutation in zinc finger homeobox-3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice.

A protein altering variant in the gene encoding zinc finger homeobox-3 (ZFHX3) has recently been associated with lower BMI in a human genome-wide association study. We investigated metabolic parameters in mice harboring a missense mutation in Zfhx3 (Zfhx3Sci/+ ) and looked for altered in situ expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence growth and metabolic effects. One-year-old male and female Zfhx3Sci/+ mice weighed less, had shorter body length, lower fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin, and insulin-like growth factor-1 (IGF1) concentrations than Zfhx3+/+ littermates. In a second cohort of 9-20-week-old males and females, Zfhx3Sci/+ mice ate less than wildtype controls, in proportion to body weight. In a third cohort of female-only Zfhx3Sci/+ and Zfhx3+/+ mice that underwent metabolic phenotyping from 6 to 14 weeks old, Zfhx3Sci/+ mice weighed less and had lower lean mass and energy expenditure, but fat mass did not differ. We detected increased expression of somatostatin and decreased expression of growth hormone-releasing hormone and growth hormone-receptor mRNAs in the arcuate nucleus (ARC). Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal expression of orphan G protein-coupled receptor Gpr50 was decreased. We demonstrate for the first time an energy balance effect of the Zfhx3Sci mutation, likely by altering expression of key ARC neuropeptides to alter growth, food intake, and energy expenditure.

CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and ethanol intake.

Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n = 115,865; p = 0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found the evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans from our UK Biobank studies. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co-expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men.

Genome sequencing and transcriptome analyses of the Siberian hamster hypothalamus identify mechanisms for seasonal energy balance.

Synthesis of triiodothyronine (T3) in the hypothalamus induces marked seasonal neuromorphology changes across taxa. How species-specific responses to T3 signaling in the CNS drive annual changes in body weight and energy balance remains uncharacterized. These experiments sequenced and annotated the Siberian hamster (Phodopus sungorus) genome, a model organism for seasonal physiology research, to facilitate the dissection of T3-dependent molecular mechanisms that govern predictable, robust, and long-term changes in body weight. Examination of the Phodopus genome, in combination with transcriptome sequencing of the hamster diencephalon under winter and summer conditions, and in vivo-targeted expression analyses confirmed that proopiomelanocortin (pomc) is a primary genomic target for the long-term T3-dependent regulation of body weight. Further in silico analyses of pomc promoter sequences revealed that thyroid hormone receptor 1ß-binding motif insertions have evolved in several genera of the Cricetidae family of rodents. Finally, experimental manipulation of food availability confirmed that hypothalamic pomc mRNA expression is dependent on longer-term photoperiod cues and is unresponsive to acute, short-term food availability. These observations suggest that species-specific responses to hypothalamic T3, driven in part by the receptor-binding motif insertions in some cricetid genomes, contribute critically to the long-term regulation of energy balance and the underlying physiological and behavioral adaptations associated with the seasonal organization of behavior.

Steady-state energy balance in animal models of obesity and weight loss.

OBJECTIVE: We wanted to exam the steady-state energy balance by using high-fat diet-induced obese (DIO) rats and mice as models for positive energy balance, and gastric bypassed (GB) rats and gene knockout of muscarinic acetylcholine M3 receptor (M3KO) mice as models for negative energy balance. METHODS: One hundred and thirty-two rats and mice were used. Energy balance was measured by a comprehensive laboratory animal monitoring system. Gene expression was analysed by in situ hybridisation in M3KO mice. RESULTS: DIO rats reached the plateau of body weight 28 weeks after starting high-fat diet (25% heavier than controls), whereas DIO mice reached the plateau after 6 weeks (23% heavier than controls). At the plateau, DIO rats had higher calorie intake during the light phase but not during the dark phase, while mice had the same calorie intake per day as controls. DIO rats and mice had lower energy expenditure (EE) and respiratory exchange ratio (RER) than controls. GB-rats reached the plateau (15% weight loss) 2 weeks after surgery and had the same calorie intake as sham-operated controls. EE, but not RER, was higher in GB rats than controls during the dark phase. The lean M3KO mice (25% lighter than wild-type (WT) mice at the plateau between 6 and 15 months of age) had the same calorie intake but higher EE, RER and hypothalamic mRNA expression of NPY, AgRP and leptin receptor than WT mice. CONCLUSION: When body weight gain or loss reached a plateau, the steady-state energy balance was mainly maintained by EE and/or RER rather than calorie intake.

Body weight loss, effective satiation and absence of homeostatic neuropeptide compensation in male Sprague Dawley rats schedule fed a protein crosslinked diet.

Food structure contributes to the induction of satiation and the maintenance of satiety following intake of a meal. There is evidence from human studies that protein-crosslinking of a milk-protein based meal may enhance satiety, but the mechanism underpinning this effect is unknown. We investigated whether a rat model would respond in a similar manner and might provide mechanistic insight into enhanced satiety by structural modification of a food source. Rats were schedule fed a modified AIN-93M based diet in a liquid form or protein-crosslinked to produce a soft-solid form. This was compared to a modified AIN-93M solid diet. Average daily caloric intake was in the order solid > liquid > crosslinked. Body composition was unaltered in the solid group, but there was a loss of fat in the liquid group and a loss of lean and fat tissue in the crosslinked group. Compared to rats fed a solid diet, acute responses in circulating GLP-1, leptin and insulin were eliminated or attenuated in rats fed a liquid or crosslinked diet. Quantification of homeostatic neuropeptide expression in the hypothalamus showed elevated levels of Npy and Agrp in rats fed the liquid diet. Measurement of food intake after a scheduled meal indicated that reduced energy intake of liquid and crosslinked diets is not due to enhancement of satiety. When continuously available ad-libitum, rats fed a liquid diet showed reduced weight gain despite greater 24 h caloric intake. During the dark phase, caloric intake was reduced, but compensated for during the light phase. We conclude that structural modification from a liquid to a solidified state is beneficial for satiation, with less of a detrimental effect on metabolic parameters and homeostatic neuropeptides.

Somatostatin receptor activation is involved in the control of daily torpor in a seasonal mammal.

Siberian hamsters (Phodopus sungorus) show spontaneous daily torpor only after ∼2 mo in winter-like short photoperiods (SP). Although some SP-induced hormonal changes have been demonstrated to be necessary for the occurrence of seasonal torpor, the whole set of preconditions is still unknown. Recent findings provide evidence that the hypothalamic pituitary growth axis is involved in endocrine responses to SP exposure in the photoperiodic hamsters. To examine whether suppression of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) secretion affects the incidence of daily torpor, we used two somatostatin receptor agonists, pasireotide (SOM230) and octreotide, with different affinity profiles for receptor subtypes. Pasireotide strikingly increased the torpor frequency in male hamsters compared with sham-treated controls, and torpor duration was often increased, which in some cases exceeded 12 h. In contrast, administration of octreotide reduced the body weight of SP hamsters but had only a marginal effect on torpor frequency in males and no effect in females. Together with measured concentrations of circulating IGF-1, the present results strongly suggest that reduced activity of the GH/IGF-1 axis is not critical for stimulation of torpor expression but activation of specific somatostatin receptors is critical. This putative role for certain somatostatin receptor subtypes in torpor induction provides a promising new approach to unravel the endocrine mechanisms of torpor regulation.

Photoperiodic expression of two RALDH enzymes and the regulation of cell proliferation by retinoic acid in the rat hypothalamus.

Retinoic acid (RA) has been found to regulate hypothalamic function, but precisely where it acts is unknown. This study shows expression of retinaldehyde dehydrogenase (RALDH) enzymes in tanycytes that line the third ventricle in an area overlapping with the site of hypothalamic neural stem cells. The influence of RA was examined on the proliferation of progenitors lining the third ventricle using organotypic slice cultures. As has been shown in other regions of neurogenesis, RA was found to inhibit proliferation. Investigations of the dynamics of RALDH1 expression in the rat hypothalamus have shown that this enzyme is in tanycytes under photoperiodic control with highest levels during long versus short days. In parallel to this shift in RA synthesis, cell proliferation in the third ventricle was found to be lowest during long days when RA was highest, implying that RALDH1 synthesized RA may regulate neural stem cell proliferation. A second RA synthesizing enzyme, RALDH2 was also present in tanycytes lining the third ventricle. In contrast to RALDH1, RALDH2 showed little change with photoperiodicity, but surprisingly the protein was present in the apparent absence of mRNA transcript and it is hypothesized that the endocytic tanycytes may take this enzyme up from the cerebrospinal fluid (CSF).

Molecular pathways involved in seasonal body weight and reproductive responses governed by melatonin.

Seasonal mammals typically of temperate or boreal habitats use the predictable annual cycle of daylength to initiate a suite of physiological and behavioural changes in anticipation of adverse environmental winter conditions, unfavourable for survival and reproduction. Daylength is encoded as the duration of production of the pineal hormone melatonin, but how the melatonin signal is decoded has been elusive. From the studies carried out in birds and mammals together with the advent of technologies such as microarray analysis of gene expression, progress has been achieved to demystify how seasonal physiology is regulated in response to the duration of melatonin signalling. The critical tissue for the action of melatonin is the pars tuberalis (PT) where melatonin receptors are located. At the molecular level, regulation of cyclic adenosine monophosphate (cAMP) signalling in this tissue is likely to be a key event for melatonin action, either an acute inhibitory action or sensitization of this pathway by prolonged stimulation of melatonin receptors reflecting durational melatonin presence. Melatonin action at the PT has been shown to have both positive and negative effects on gene transcription, incorporating components of the circadian clock as part of the mechanism of decoding the melatonin signal and regulating thyrotrophin-stimulating hormone (TSH) expression, a key output hormone of the PT. Microarray analysis of gene expression of PT tissue exposed to long and short photoperiods has identified important new genes that may be regulated by melatonin and contributing to the seasonal regulation of TSH production by this tissue. In the brain, tanycytes lining the third ventricle of the hypothalamus and regulation of thyroid hormone synthesis by PT-derived TSH in these cells are now established as an important component of the pathway leading to seasonal changes in physiology. Beyond the tanycyte, identified changes in gene expression for neuropeptides, receptors and other signalling molecules pinpoint some of the areas of the brain, the hypothalamus in particular, that are likely to be involved in the regulation of seasonal physiology.

Photoperiodic regulation of glycogen metabolism, glycolysis, and glutamine synthesis in tanycytes of the Siberian hamster suggests novel roles of tanycytes in hypothalamic function.

The objective of this study is to investigate the impact of photoperiod on the temporal and spatial expression of genes involved in glucose metabolism in the brain of the seasonal mammal Phodopus sungorus (Siberian hamster). In situ hybridization was performed on brain sections obtained from male hamsters held in long photoperiod (high body weight and developed testes) or short photoperiod (reduced body weight with testicular regression). This analysis revealed upregulation in expression of genes involved in glycogen and glucose metabolism in short photoperiod and localized to the tanycyte layer of the third ventricle. On the basis of these data and a previously identified photoperiod-dependent increase in activity of neighboring hypothalamic neurons, we hypothesized that the observed expression changes may reflect alteration in either metabolic fuel or precursor neurotransmitter supply to surrounding neurons. Gene expression analysis was performed for genes involved in lactate and glutamate transport. This analysis showed that the gene for the lactate transporter MCT2 and glutamate transporter GLAST was decreased in the tanycyte layer in short photoperiod. Expression of mRNA for glutamine synthetase, the final enzyme in the synthesis of the neuronal neurotransmitter precursor, glutamine, was also decreased in short photoperiod. These data suggest a role for tanycytes in modulating glutamate concentrations and neurotransmitter supply in the hypothalamic environment.

Ovarian hormones induce de novo DNA methyltransferase expression in the Siberian hamster suprachiasmatic nucleus.

The present study investigated neuroanatomically localised changes in de novo DNA methyltransferase expression in the female Siberian hamster (Phodopus sungorus). The objectives were to identify the neuroendocrine substrates that exhibit rhythmic Dnmt3a and Dnmt3b expression across the oestrous cycle and also examine the role of ovarian steroids. Hypothalamic Dnmt3a expression was observed to significantly increase during the transition from pro-oestrous to oestrous. A single bolus injection of diethylstilbestrol and progesterone was sufficient to increase Dnmt3a cell numbers and Dnmt3b immunoreactive intensity in the suprachiasmatic nucleus. In vitro analyses using an embryonic rodent cell line revealed that diethylstilbestrol was sufficient to induce Dnmt3b expression. Up-regulating DNA methylation in vitro reduced the expression of vasoactive intestinal polypeptide, Vip, and the circadian clock gene, Bmal1. Together, these data indicate that ovarian steroids drive de novo DNA methyltransferase expression in the mammalian suprachiasmatic nucleus and increased methylation may regulate genes involved in the circadian timing of oestrous: Vip and Bmal1. Overall, epigenetically mediated neuroendocrine reproductive events may reflect an evolutionarily ancient process involved in the timing of female fertility.

The role of histamine 3 receptors in the control of food intake in a seasonal model of obesity: the Siberian hamster.

Siberian hamsters develop hypophagia and increase catabolism of fat reserves in response to short photoperiods resulting in a natural loss of body weight in winter. We previously found that histamine 3 receptor (H3R) mRNA in the posterior hypothalamus is significantly decreased in short photoperiods. We hypothesized that this lower expression of H3R might contribute to the winter hypophagic state, therefore we examined the effects of the H3R agonist imetit and inverse agonists clobenpropit and thioperamide on food intake. We expressed the Siberian hamster H3R receptor in vitro and confirmed that imetit, clobenpropit and thioperamide are bound specifically, thus validating them as tools to investigate the role of H3R in vivo. Intracerebroventricular administration of histamine decreased food intake in hamsters in the fat summer state. Administration of imetit to hamsters in the lean state increased food intake, whereas administration of inverse agonists decreased food intake, though this was associated with decreased locomotor activity. Both H3R inverse agonists prevented the nocturnal rise in body temperature indicating additional effects on energy expenditure. In summary, our results suggest that increased availability of central histamine or the reduction of H3R activity decrease food intake. These effects are similar to those observed in hamsters in short photoperiods.

A unifying hypothesis for control of body weight and reproduction in seasonally breeding mammals.

Animals have evolved diverse seasonal variations in physiology and reproduction to accommodate yearly changes in environmental and climatic conditions. These changes in physiology are initiated by changes in photoperiod (daylength) and are mediated through melatonin, which relays photoperiodic information to the pars tuberalis of the pituitary gland. Melatonin drives thyroid-stimulating hormone transcription and synthesis in the pars tuberalis, which, in turn, regulates thyroid hormone and retinoic acid synthesis in the tanycytes lining the third ventricle of the hypothalamus. Seasonal variation in central thyroid hormone signalling is conserved among photoperiodic animals. Despite this, different species adopt divergent phenotypes to cope with the same seasonal changes. A common response amongst different species is increased hypothalamic cell proliferation/neurogenesis in short photoperiod. That cell proliferation/neurogenesis may be important for seasonal timing is based on (i) the neurogenic potential of tanycytes; (ii) the fact that they are the locus of striking seasonal morphological changes; and (iii) the similarities to mechanisms involved in de novo neurogenesis of energy balance neurones. We propose that a decrease in hypothalamic thyroid hormone and retinoic acid signalling initiates localised neurodegeneration and apoptosis, which leads to a reduction in appetite and body weight. Neurodegeneration induces compensatory cell proliferation from the neurogenic niche in tanycytes and new cells are born under short photoperiod. Because these cells have the potential to differentiate into a number of different neuronal phenotypes, this could provide a mechanistic basis to explain the seasonal regulation of energy balance, as well as reproduction. This cycle can be achieved without changes in thyroid hormone/retinoic acid and explains recent data obtained from seasonal animals held in natural conditions. However, thyroid/retinoic acid signalling is required to synchronise the cycles of apoptosis, proliferation and differentiation. Thus, hypothalamic neurogenesis provides a framework to explain diverse photoperiodic responses.

Fat, carbohydrate and protein by oral gavage in the rat can be equally effective for satiation.

This study aimed to determine the relative efficacy of the macronutrients, protein, fat and carbohydrate to induce satiation and satiety in rats in relation to macronutrient activation of neurons in the nucleus of the solitary tract (NTS). Male Sprague Dawley rats were schedule-fed twice a day for 2 h, receiving 100% of daily ad-libitum energy intake. On test day 1, 30 min before the first scheduled meal of the day, rats were gavaged with an 8 kcal isocaloric, isovolumetric solution of a glucose, lipid or peptone macronutrient solution or a non-caloric saline solution. To assess satiation, thirty minutes later rats were given access to food for 2 h and food intake determined. A second 2 h food access period 3 h later was used for assessment of satiety. On the second test day, rats were gavaged as before and killed 90 min after food presentation. Blood was collected for measurement of circulating metabolic markers. Brains were removed for analysis of c-Fos expression by in situ hybridization in the NTS. Rats which received saline consumed a similar amount of food compared to pre-gavage intakes. However, rats gavaged with a caloric macronutrient solution all reduced food intake by 18-20 kcal. Interestingly, the reduction in caloric intake was greater than the caloric value of the macronutrient solution gavaged and was sustained following the second scheduled meal. Quantification by in situ hybridization of c-Fos mRNA expression in the NTS 90 min post-gavage, showed a significant increase with each macronutrient, but was 24-29% higher with a lipid or peptone gavage compared to a glucose gavage. In conclusion, when delivered directly to the stomach, all macronutrients can be equally effective in inducing satiation with significant neuronal activation in the NTS of the hindbrain.

Disruption of an enhancer associated with addictive behaviour within the cannabinoid receptor-1 gene suggests a possible role in alcohol intake, cannabinoid response and anxiety-related behaviour.

The cannabinoid-1 receptor (CB1) plays a critical role in a number of biological processes including nutrient intake, addiction and anxiety-related behaviour. Numerous studies have shown that expression of the gene encoding CB1 (CNR1) is highly dynamic with changes in the tissue specific expression of CNR1 associated with brain homeostasis and disease progression. However, little is known of the mechanisms regulating this dynamic expression. To gain a better understanding of the genomic mechanisms modulating the expression of CNR1 in health and disease we characterised the role of a highly conserved regulatory sequence (ECR1) in CNR1 intron 2 that contained a polymorphism in linkage disequilibrium with disease associated SNPs. We used CRISPR/CAS9 technology to disrupt ECR1 within the mouse genome. Disruption of ECR1 significantly reduced CNR1 expression in the hippocampus but not in the hypothalamus. These mice also displayed an altered sex-specific anxiety-related behavioural profile (open field test), reduced ethanol intake and a reduced hypothermic response following CB1 agonism. However, no significant changes in feeding patterns were detected. These data suggest that, whilst not all of the expression of CNR1 is modulated by ECR1, this highly conserved enhancer is required for appropriate physiological responses to a number of stimuli. The combination of comparative genomics and CRISPR/CAS9 disruption used in our study to determine the functional effects of genetic and epigenetic changes on the activity of tissue-specific regulatory elements at the CNR1 locus represent an important first step in gaining a mechanistic understanding of cannabinoid regulatory pharmacogenetics.

G protein-coupled receptor 101 mRNA expression in supraoptic and paraventricular nuclei in rat hypothalamus is altered by pregnancy and lactation.

In a previous study performed in mouse models of energetic challenge, there was evidence to suggest that the orphan G protein-coupled receptor GPCR101 may have a role in the regulation of energy balance. To further investigate this possibility, we utilised in situ hybridisation to determine the effect of energetic challenges experienced by pregnant and lactating rats on GPCR101 mRNA expression. In the rat hypothalamus, GPCR101 mRNA expression was detected in a number of hypothalamic nuclei. During pregnancy and lactation, GPCR101 mRNA level remained unchanged in most nuclei, but had increased in the supraoptic nucleus by the end of pregnancy and remained elevated during lactation. GPCR101 mRNA expression showed a similar pattern of expression in the rostral ventromedial parvocellular subdivision of the paraventricular nucleus. A common feature of these two nuclei is the production of the peptide oxytocin. Dual in situ hybridisation revealed GPCR101 and oxytocin mRNA co-expression in neurons of these two nuclei. In the supraoptic nucleus, in situ hybridisation revealed that the temporal regulation of oxytocin and GPCR101 mRNA expression were similar. In the paraventricular nucleus, although temporal changes in oxytocin mRNA expression were similar to GPCR101, the spatial expression of the two mRNA species was different; in contrast to GPCR101, oxytocin mRNA expression changed in both parvo- and magnocellular neurons during lactation. In conclusion, increased GPCR101 mRNA expression in supraoptic and paraventricular nuclei from late pregnancy to late lactation may reflect the functional importance of this receptor in the regulation of neurons of these nuclei during this period.

Diet induced obesity is independent of metabolic endotoxemia and TLR4 signalling, but markedly increases hypothalamic expression of the acute phase protein, SerpinA3N.

Hypothalamic inflammation is thought to contribute to obesity. One potential mechanism is via gut microbiota derived bacterial lipopolysaccharide (LPS) entering into the circulation and activation of Toll-like receptor-4. This is called metabolic endotoxemia. Another potential mechanism is systemic inflammation arising from sustained exposure to high-fat diet (HFD) over more than 12 weeks. In this study we show that mice fed HFD over 8 weeks become obese and show elevated plasma LPS binding protein, yet body weight gain and adiposity is not attenuated in mice lacking Tlr4 or its co-receptor Cd14. In addition, caecal microbiota composition remained unchanged by diet. Exposure of mice to HFD over a more prolonged period (20 weeks) to drive systemic inflammation also caused obesity. RNAseq used to assess hypothalamic inflammation in these mice showed increased hypothalamic expression of Serpina3n and Socs3 in response to HFD, with few other genes altered. In situ hybridisation confirmed increased Serpina3n and Socs3 expression in the ARC and DMH at 20-weeks, but also at 8-weeks and increased SerpinA3N protein could be detected as early as 1 week on HFD. Overall these data show lack of hypothalamic inflammation in response to HFD and that metabolic endotoxemia does not link HFD to obesity.

VGF-derived peptide, TLQP-21, regulates food intake and body weight in Siberian hamsters.

The Siberian hamster survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. VGF gene expression is photoperiodically regulated in the hypothalamus with significantly higher expression in lean Siberian hamsters. The aim of this study was to investigate the role of VGF in regulating these seasonal cycles by determining the effects of a VGF-derived peptide (TLQP-21) on food intake and body weight. Acute intracerebroventricular administration of TLQP-21 decreased food intake, and chronic treatment caused a sustained reduction in food intake and body weight and decreased abdominal fat depots. Behavioral analysis revealed that TLQP-21 reduced meal size but not the frequency of feeding bouts, suggesting a primary action on satiety. Hamsters treated with TLQP-21 lost a similar amount of weight as a pair-fed group in which food intake was matched to that of the TLQP-21-treated group. Central or peripheral treatment with TLQP-21 did not produce a significant effect on resting metabolic rate. We conclude that the primary action of TLQP-21 is to decrease food intake rather than increase energy expenditure. TLQP-21 treatment caused a decrease in UCP-1 mRNA in brown adipose tissue, but hypothalamic expression of orexigenic and anorexigenic neuropeptide genes remained unchanged after TLQP-21 treatment, although compensatory increases in NPY and AgRP mRNA were observed in the pair-fed hamsters. The effects of TLQP-21 administration are similar to those in hamsters in short days, suggesting that increased VGF activity may contribute to the hypophagia that underlies the seasonal catabolic state.

Hypothalamic thyroid hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction.

Seasonal adaptations in physiology exhibited by many animals involve an interface between biological timing and specific neuroendocrine systems, but the molecular basis of this interface is unknown. In this study of Siberian hamsters, we show that the availability of thyroid hormone within the hypothalamus is a key determinant of seasonal transitions. The expression of the gene encoding type III deiodinase (Dio3) and Dio3 activity in vivo (catabolism of T(4) and T(3)) is dynamically and temporally regulated by photoperiod, consistent with the loss of hypothalamic T(3) concentrations under short photoperiods. Chronic replacement of T(3) in the hypothalamus of male hamsters exposed to short photoperiods, thus bypassing synthetic or catabolic deiodinase enzymes located in cells of the ependyma of the third ventricle, prevented the onset of short-day physiology: hamsters maintained a long-day body weight phenotype and failed to undergo testicular and epididymal regression. However, pelage moult to a winter coat was not affected. Type II deiodinase gene expression was not regulated by photoperiod in these hamsters. Collectively, these data point to a pivotal role for hypothalamic DIO3 and T(3) catabolism in seasonal cycles of body weight and reproduction in mammals.

Regulation of the ovine MT1 melatonin receptor promoter: interaction between multiple pituitary transcription factors at different phases of development.

Pineal secretion of melatonin provides a neuroendocrine representation of the light-dark cycle, which is used to synchronise daily and annual rhythms of physiology and behaviour. In mammals, melatonin primarily acts through MT(1) melatonin receptors that exhibit a highly restricted tissue distribution. Expression of MT(1) receptors is subject to developmental and circadian control, which likely modulates the physiological actions of melatonin. To investigate the mechanisms controlling MT(1) expression we cloned the proximal 1.5kb region of the ovine MT(1) promoter. Sequence analysis revealed putative cis-elements for transcription factors involved in pituitary development, namely Pitx-1 and Egr-1, and multiple putative E-boxes, which are involved in both circadian and developmental gene regulation. Nuclear protein from ovine pars tuberalis (PT) cells, a site of high endogenous MT(1) expression, stimulated gene expression from a MT(1) expression construct, indicating the presence of a functional promoter. Pitx-1 was strongly expressed in the ovine PT and stimulated MT(1) promoter activity in transfection assays. Co-transfection with Egr-1 induced promoter-specific effects: Pitx-1-stimulated MT(1) activity was inhibited, whereas betaLH promoter activity was enhanced. In addition to Pitx-1 the circadian clock genes Clock and Bmal1 were also expressed in the PT. However, despite multiple putative E-boxes in the MT(1) promoter, transfected Clock and Bmal1 were unable to regulate either basal or Pitx-1-stimulated MT(1) promoter activity. The current data, in conjunction with our previous study of the rat MT(1) promoter, suggests a general model in which melatonin receptor expression in the mammalian pituitary is determined by the developmentally changing balance between stimulatory and inhibitory transcription factors. Furthermore, our data suggest that circadian variation in MT(1) gene expression does not depend upon the direct action of circadian clock genes on E-box cis-elements.

Gene expression analysis and microdialysis suggest hypothalamic triiodothyronine (T3) gates daily torpor in Djungarian hamsters (Phodopus sungorus).

Thyroid hormones play an important role in regulating seasonal adaptations of mammals. Several studies suggested that reduced availability of 3,3',5-triiodothyronine (T3) in the hypothalamus is required for the physiological adaptation to winter in Djungarian hamsters. We have previously shown that T3 is involved in the regulation of daily torpor, but it remains unclear, whether T3 affects torpor by central or peripheral mechanisms. To determine the effect of T3 concentrations within the hypothalamus in regulating daily torpor, we tested the hypothesis that low hypothalamic T3 metabolism would favour torpor and high T3 concentrations would not. In experiment 1 gene expression in torpid hamsters was assessed for transporters carrying thyroid hormones between cerebrospinal fluid and hypothalamic cells and for deiodinases enzymes, activating or inactivating T3 within hypothalamic cells. Gene expression analysis suggests reduced T3 in hypothalamic cells during torpor. In experiment 2, hypothalamic T3 concentrations were altered via microdialysis and torpor behaviour was continuously monitored by implanted body temperature transmitters. Increased T3 concentrations in the hypothalamus reduced expression of torpor as well as torpor bout duration and depth. Subsequent analysis of gene expression in the ependymal layer of the third ventricle showed clear up-regulation of T3 inactivating deiodinase 3 but no changes in several other genes related to photoperiodic adaptations in hamsters. Finally, serum analysis revealed that increased total T3 serum concentrations were not necessary to inhibit torpor expression. Taken together, our results are consistent with the hypothesis that T3 availability within the hypothalamus significantly contributes to the regulation of daily torpor via a central pathway.