Khat (Catha edulis) upregulates lipolytic genes in white adipose tissue of male obese mice (C57BL/6J).

ETHNOPHARMACOLOGICAL RELEVANCE: Khat (Catha edulis (Vahl) Forssk.) is a herb from the Celastraceae family (also known as qat, gaad, or mirra) that is widely-consumed in East Africa and in the Arabian peninsula. The green leaves and small stems are consumed primarily at recreational and social gatherings, and medicinally for their antidiabetic and appetite-suppression effects. AIMS: The objectives of this study were to determine the effects of khat and its active alkaloid, cathinone, on food intake and body weight in mice maintained on a high-fat diet, and to investigate its mechanism of action in white adipose tissue and in the hypothalamus. MATERIALS & METHOD: Adult male mice (C57BL/6J) were fed a high fat diet (HFD) for 8 weeks (n = 30), then divided into 5 groups and treated daily for a further 8 weeks with HFD + vehicle [control (HFD)], HFD + 15 mg/kg orlistat (HFDO), HFD + 200 mg/kg khat extract (HFDK200), HFD + 400 mg/kg khat extract (HFDK400) and HFD + 3.2 mg/kg cathinone (HFDCAT). Treatments were carried out once daily by gastric gavage. Blood and tissue samples were collected for biochemical, hormonal and gene expression analyses. RESULTS: Khat extracts and orlistat treatment significantly reduced weight gain as compared to control mice on HFD, and cathinone administration completely prevented weight gain in mice fed on HFD. Khat treatment caused a marked reduction in body fat and in serum triglycerides. A dose-dependent effect of khat was observed in reducing serum leptin concentrations. Analysis of gene expression in adipose tissue revealed a significant upregulation of two lipolysis pathway genes:(adipose triglyceride lipase (PNPLA-2) and hormone-sensitive lipase (LIPE). In the hypothalamic there was a significant (P < 0.05) upregulation of agouti-related peptide (AgRP) and cocaine-amphetamine regulated transcript (CART) genes in the HFDK400 and HFDCAT groups. CONCLUSION: Cathinone treatment blocked body weight gain, while high dose khat extract significantly reduced the weight gain of mice on an obesogenic diet through stimulation of lipolysis in white adipose tissue.

Role of hypothalamic tanycytes in nutrient sensing and energy balance.

Animal models are valuable for the study of complex behaviours and physiology such as the control of appetite because genetic, pharmacological and surgical approaches allow the investigation of underlying mechanisms. However, the majority of such studies are carried out in just two species, laboratory mice and rats. These conventional laboratory species have been intensely selected for high growth rate and fecundity, and have a high metabolic rate and short lifespan. These aspects limit their translational relevance for human appetite control. This review will consider the value of studies carried out in a seasonal species, the Siberian hamster, which shows natural photoperiod-regulated annual cycles in appetite, growth and fattening. Such studies reveal that this long-term control is not simply an adjustment of the known hypothalamic neuronal systems that control hunger and satiety in the short term. Long-term cyclicity is probably driven by hypothalamic tanycytes, glial cells that line the ventricular walls of the hypothalamus. These unique cells sense nutrients and metabolic hormones, integrate seasonal signals and effect plasticity of surrounding neural circuits through their function as a stem cell niche in the adult. Studies of glial cell function in the hypothalamus offer new potential for identifying central targets for appetite and body weight control amenable to dietary or pharmacological manipulation.

Tanycytes and hypothalamic control of energy metabolism.

Studies from a number of areas of neuroendocrinology indicate that hypothalamic tanycytes play a key role in control of energy metabolism. First, profound annual changes in gene expression have been identified in these unusual glial cells in seasonal mammals, for example in genes relating to the transport and metabolism of thyroid hormone into the hypothalamus. The consequent changes in local thyroid hormone availability in the hypothalamus have been shown experimentally to regulate annual cycles in energy intake, storage and expenditure in seasonal species. This is reflected in overt seasonal changes in appetite, body fat composition and torpor. Second, studies in laboratory rodents demonstrate that hypothalamic tanycytes possess transport mechanisms and receptors that indicate they have a cellular function as nutrient sensors. Ex vivo studies with organotypic tanycyte cultures confirm that acute changes in nutrient availability alter calcium and purinergic signalling within and between tanycytes. Finally, tanycytes are components of a stem cell niche in the hypothalamus whose activity can be regulated by the nutritional environment. Experimental depletion of cell division in the hypothalamus alters the homeostatic response to nutrient excess in mice raised in high fat diets. These convergent lines of evidence suggest that tanycytes are nutrient and metabolite sensors that impact upon plasticity and neuronal function in the surrounding hypothalamus, and consequently have an important role in energy intake and expenditure.

Hypothalamic over-expression of VGF in the Siberian hamster increases energy expenditure and reduces body weight gain.

VGF (non-acronymic) was first highlighted to have a role in energy homeostasis through experiments involving dietary manipulation in mice. Fasting increased VGF mRNA in the Arc and levels were subsequently reduced upon refeeding. This anabolic role for VGF was supported by observations in a VGF null (VGF-/-) mouse and in the diet-induced and gold-thioglucose obese mice. However, this anabolic role for VGF has not been supported by a number of subsequent studies investigating the physiological effects of VGF-derived peptides. Intracerebroventricular (ICV) infusion of TLQP-21 increased resting energy expenditure and rectal temperature in mice and protected against diet-induced obesity. Similarly, ICV infusion of TLQP-21 into Siberian hamsters significantly reduced body weight, but this was due to a decrease in food intake, with no effect on energy expenditure. Subsequently NERP-2 was shown to increase food intake in rats via the orexin system, suggesting opposing roles for these VGF-derived peptides. Thus to further elucidate the role of hypothalamic VGF in the regulation of energy homeostasis we utilised a recombinant adeno-associated viral vector to over-express VGF in adult male Siberian hamsters, thus avoiding any developmental effects or associated functional compensation. Initially, hypothalamic over-expression of VGF in adult Siberian hamsters produced no effect on metabolic parameters, but by 12 weeks post-infusion hamsters had increased oxygen consumption and a tendency to increased carbon dioxide production; this attenuated body weight gain, reduced interscapular white adipose tissue and resulted in a compensatory increase in food intake. These observed changes in energy expenditure and food intake were associated with an increase in the hypothalamic contents of the VGF-derived peptides AQEE, TLQP and NERP-2. The complex phenotype of the VGF-/- mice is a likely consequence of global ablation of the gene and its derived peptides during development, as well as in the adult.

Photoperiod Regulates vgf-Derived Peptide Processing in Siberian Hamsters.

VGF mRNA is induced in specific hypothalamic areas of the Siberian hamster upon exposure to short photoperiods, which is associated with a seasonal decrease in appetite and weight loss. Processing of VGF generates multiple bioactive peptides, so the objective of this study was to determine the profile of the VGF-derived peptides in the brain, pituitary and plasma from Siberian hamsters, and to establish whether differential processing might occur in the short day lean state versus long day fat. Antisera against short sequences at the C- or N- termini of proVGF, as well as against NERP-1, TPGH and TLQP peptides, were used for analyses of tissues, and both immunohistochemistry and enzyme linked immunosorbent assay (ELISA) coupled with high-performance liquid (HPLC) or gel chromatography were carried out. VGF peptide immunoreactivity was found within cortex cholinergic perikarya, in multiple hypothalamic nuclei, including those containing vasopressin, and in pituitary gonadotrophs. ELISA revealed that exposure to short day photoperiod led to a down-regulation of VGF immunoreactivity in the cortex, and a less pronounced decrease in the hypothalamus and pituitary, while the plasma VGF levels were not affected by the photoperiod. HPLC and gel chromatography both confirmed the presence of multiple VGF-derived peptides in these tissues, while gel chromatography showed the presence of the VGF precursor in all tissues tested except for the cortex. These observations are consistent with the view that VGF-derived peptides have pleiotropic actions related to changing photoperiod, possibly by regulating cholinergic systems in the cortex, vasopressin hypothalamic pathways, and the reproductive axis.

Antibody-Mediated Inhibition of the FGFR1c Isoform Induces a Catabolic Lean State in Siberian Hamsters.

Hypothalamic tanycytes are considered to function as sensors of peripheral metabolism. To facilitate this role, they express a wide range of receptors, including fibroblast growth factor receptor 1 (FGFR1). Using a monoclonal antibody (IMC-H7) that selectively antagonizes the FGFR1c isoform, we investigated possible actions of FGFR1c in a natural animal model of adiposity, the Siberian hamster. Infusion of IMC-H7 into the third ventricle suppressed appetite and increased energy expenditure. Likewise, peripheral treatment with IMC-H7 decreased appetite and body weight and increased energy expenditure and fat oxidation. A greater reduction in body weight and caloric intake was observed in response to IMC-H7 during the long-day fat state as compared to the short-day lean state. This enhanced response to IMC-H7 was also observed in calorically restricted hamsters maintained in long days, suggesting that it is the central photoperiodic state rather than the peripheral adiposity that determines the response to FGFR1c antagonism. Hypothalamic thyroid hormone availability is controlled by deiodinase enzymes (DIO2 and DIO3) expressed in tanycytes and is the key regulator of seasonal cycles of energy balance. Therefore, we determined the effect of IMC-H7 on hypothalamic expression of these deiodinase enzymes. The reductions in food intake and body weight were always associated with decreased expression of DIO2 in the hypothalamic ependymal cell layer containing tanycytes. These data provide further support for the notion the tanycytes are an important component of the mechanism by which the hypothalamus integrates central and peripheral signals to regulate energy intake and expenditure.

The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo.

INTRODUCTION: The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. NEW METHOD: To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. RESULTS: Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. COMPARISON WITH OLD METHOD: The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. CONCLUSION: The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects.

Hypothalamic control of seasonal changes in food intake and body weight.

Seasonal cycles of fattening and body weight reflecting changes in both food intake and energy expenditure are a core aspect of the biology of mammals that have evolved in temperate and arctic latitudes. Identifying the neuroendocrine mechanisms that underlie these cycles has provided new insights into the hypothalamic control of appetite and fuel oxidation. Surprisingly, seasonal cycles do not result from changes in the leptin-responsive and homeostatic pathways located in the mediobasal and lateral hypothalamus that regulate meal timing and compensatory responses to starvation or caloric restriction. Rather, they result from changes in tanycyte function, which locally regulates transport and metabolism of thyroid hormone and retinoic acid. These signals are crucial for the initial development of the brain, so it is hypothesized that seasonal neuroendocrine cycles reflect developmental mechanisms in the adult hypothalamus, manifest as changes in neurogenesis and plasticity of connections.

On the value of seasonal mammals for identifying mechanisms underlying the control of food intake and body weight.

This article is part of a Special Issue "Energy Balance". Seasonal cycles of adiposity and body weight reflecting changes in both food intake and energy expenditure are the norm in mammals that have evolved in temperate and polar habitats. Innate circannual rhythmicity and direct responses to the annual change in photoperiod combine to ensure that behavior and energy metabolism are regulated in anticipation of altered energetic demands such as the energetically costly processes of hibernation, migration, and lactation. In the last decade, major progress has been made into identifying the central mechanisms that underlie these profound long-term changes in behavior and physiology. Surprisingly they are distinct from the peptidergic and aminergic systems in the hypothalamus that have been identified in studies of the laboratory mouse and rat and implicated in timing meal intervals and in short-term responses to caloric restriction. Comparative studies across rodents, ungulates and birds reveal that tanycytes embedded in the ependymal layer of the third ventricle play a critical role in seasonal changes because they regulate the local availability of thyroid hormone. Understanding how this altered hormonal environment might regulate neurogenesis and plasticity in the hypothalamus should provide new insight into development of strategies to manage appetite and body weight.

Hypothalamic ventricular ependymal thyroid hormone deiodinases are an important element of circannual timing in the Siberian hamster (Phodopus sungorus).

Exposure to short days (SD) induces profound changes in the physiology and behaviour of Siberian hamsters, including gonadal regression and up to 30% loss in body weight. In a continuous SD environment after approximately 20 weeks, Siberian hamsters spontaneously revert to a long day (LD) phenotype, a phenomenon referred to as the photorefractory response. Previously we have identified a number of genes that are regulated by short photoperiod in the neuropil and ventricular ependymal (VE) cells of the hypothalamus, although their importance and contribution to photoperiod induced physiology is unclear. In this refractory model we hypothesised that the return to LD physiology involves reversal of SD expression levels of key hypothalamic genes to their LD values and thereby implicate genes required for LD physiology. Male Siberian hamsters were kept in either LD or SD for up to 39 weeks during which time SD hamster body weight decreased before increasing, after more than 20 weeks, back to LD values. Brain tissue was collected between 14 and 39 weeks for in situ hybridization to determine hypothalamic gene expression. In VE cells lining the third ventricle, expression of nestin, vimentin, Crbp1 and Gpr50 were down-regulated at 18 weeks in SD photoperiod, but expression was not restored to the LD level in photorefractory hamsters. Dio2, Mct8 and Tsh-r expression were altered by SD photoperiod and were fully restored, or even exceeded values found in LD hamsters in the refractory state. In hypothalamic nuclei, expression of Srif and Mc3r mRNAs was altered at 18 weeks in SD, but were similar to LD expression values in photorefractory hamsters. We conclude that in refractory hamsters not all VE cell functions are required to establish LD physiology. However, thyroid hormone signalling from ependymal cells and reversal of neuronal gene expression appear to be essential for the SD refractory response.

Effects of manipulating hypothalamic triiodothyronine concentrations on seasonal body weight and torpor cycles in Siberian hamsters.

Siberian hamsters display photoperiodically regulated annual cycles in body weight, appetite, and reproduction. Previous studies have revealed a profound up-regulation of type 3 deiodinase (DIO3) mRNA in the ventral ependyma of the hypothalamus associated with hypophagia and weight loss in short-day photoperiods. DIO3 reduces the local availability of T(3), so the aim of this study was to test the hypothesis that decreased hypothalamic T(3) availability underlies the short-day-induced catabolic state. The experimental approach was to determine whether a local increase in T(3) in the hypothalamus of hamsters exposed to short days could reverse the behavioral and physiological changes induced by this photoperiod. In study 1, microimplants releasing T(3) were placed bilaterally into the hypothalamus. This treatment rapidly induced a long-day phenotype including increased appetite and body weight within 3 wk of treatment and increased fat mass and testis size by the end of the 10-wk study period. In study 2, hypothalamic T(3) implants were placed into hamsters carrying abdominal radiotelemetry implants. Again body weight increased significantly, and the occurrence of winter torpor bouts was dramatically decreased to less than one bout per week, whereas sham-implanted hamsters entered torpor up to six times a week. Our findings demonstrate that increased central T(3) induces a long-day metabolic phenotype, but in neither study was the molt cycle affected, so we infer that we had not disrupted the initial detection of photoperiod. We conclude that hypothalamic thyroid hormone availability plays a key role in seasonal regulation of appetite, body weight, and torpor.

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.

Histaminergic regulation of seasonal metabolic rhythms in Siberian hamsters.

We investigated whether histaminergic tone contributes to the seasonal catabolic state in Siberian hamsters by determining the effect of ablation of histaminergic neurons on food intake, metabolic rate and body weight. A ribosomal toxin (saporin) conjugated to orexin-B was infused into the ventral tuberomammillary region of the hypothalamus, since most histaminergic neurons express orexin receptors. This caused not only 75-80% loss of histaminergic neurons in the posterior hypothalamus, but also some loss of other orexin-receptor expressing cells e.g. MCH neurons. In the long-day anabolic state, lesions produced a transient post-surgical decrease in body weight, but the hamsters recovered and maintained constant body weight, whereas weight gradually increased in sham-lesioned hamsters. VO(2) in the dark phase was significantly higher in the lesioned hamsters compared to shams, and locomotor activity also tended to be higher. In a second study in short days, sham-treated hamsters showed the expected seasonal decrease in body weight, but weight remained constant in the lesioned hamsters, as in the long-day study. Lesioned hamsters consumed more during the early dark phase and less during the light phase due to an increase in the frequency of meals during the dark and decreased meal size during the light, and their cumulative food intake in their home cages was greater than in the control hamsters. In summary, ablation of orexin-responsive cells in the posterior hypothalamus blocks the short-day induced decline in body weight by preventing seasonal hypophagia, evidence consistent with the hypothesis that central histaminergic mechanisms contribute to long-term regulation of body weight.

Prokineticin 2 is a hypothalamic neuropeptide that potently inhibits food intake.

OBJECTIVE: Prokineticin 2 (PK2) is a hypothalamic neuropeptide expressed in central nervous system areas known to be involved in food intake. We therefore hypothesized that PK2 plays a role in energy homeostasis. RESEARCH DESIGN AND METHODS: We investigated the effect of nutritional status on hypothalamic PK2 expression and effects of PK2 on the regulation of food intake by intracerebroventricular (ICV) injection of PK2 and anti-PK2 antibody. Subsequently, we investigated the potential mechanism of action by determining sites of neuronal activation after ICV injection of PK2, the hypothalamic site of action of PK2, and interaction between PK2 and other hypothalamic neuropeptides regulating energy homeostasis. To investigate PK2's potential as a therapeutic target, we investigated the effect of chronic administration in lean and obese mice. RESULTS: Hypothalamic PK2 expression was reduced by fasting. ICV administration of PK2 to rats potently inhibited food intake, whereas anti-PK2 antibody increased food intake, suggesting that PK2 is an anorectic neuropeptide. ICV administration of PK2 increased c-fos expression in proopiomelanocortin neurons of the arcuate nucleus (ARC) of the hypothalamus. In keeping with this, PK2 administration into the ARC reduced food intake and PK2 increased the release of alpha-melanocyte-stimulating hormone (alpha-MSH) from ex vivo hypothalamic explants. In addition, ICV coadministration of the alpha-MSH antagonist agouti-related peptide blocked the anorexigenic effects of PK2. Chronic peripheral administration of PK2 reduced food and body weight in lean and obese mice. CONCLUSIONS: This is the first report showing that PK2 has a role in appetite regulation and its anorectic effect is mediated partly via the melanocortin system.

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.

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.

The neuroendocrine timing of puberty.

Puberty is the attainment of fertility, a process encompassing morphological, physiological and behavioural development. The increased hypothalamic secretion of the gonadotrophin-releasing hormone decapeptide (GnRH) is essential for the activation of the pituitary-gonadal axis at puberty. The GnRH secretory network initially develops and is temporarily active during species-specific periods of fetal/neonatal development, so puberty is the secondary reactivation of an existing system. From a neurobiological perspective, the timing of puberty is therefore a function of changes in the neural systems controlling GnRH release. The large variability between individuals in the onset and progression of puberty indicates that the timing of puberty is not simply a function of chronological age. Rather, the neurotransmitter and neuromodulatory systems that impact upon the GnRH secretory network convey information about metabolic fuels, energy stores and somatic development and, for many species, information about season and social environment. The clear links demonstrated between metabolic fuel availability and reproductive function in many animal models provides evidence that the earlier onset of pubertal development observed in girls in certain US study populations is likely to relate to the increasing prevalence of overweight and obesity in adolescents.

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.

Decrease of food intake by MC4-R agonist MTII in Siberian hamsters in long and short photoperiods.

We investigated the role of the hypothalamic melanocortin system in the regulation of food intake in the Siberian hamster, which shows a profound seasonal decrease in food intake and body weight in short photoperiod (SP). In male hamsters maintained in long photoperiod (LP), intracerebroventricular injection of melanotan II (MTII) just before lights off significantly decreased food intake relative to vehicle treatment over the 6-h observation period. Similar effects were observed in age-matched hamsters after exposure to a short daylength for 9 wk, when body weight had significantly decreased. There was no clear difference in either the magnitude of response or the dose required for half-maximal inhibition of food intake in hamsters in SP compared with those in LP. MTII significantly increased grooming in both LP and SP. Our results indicate that the melanocortin system is a potent short-term regulator of food intake. However, the lack of differential response or sensitivity to MTII treatment in the obese (LP) vs. lean (SP) states does not support the hypothesis that changes in this melanocortin pathway underlie the long-term decrease in food intake that occurs in this seasonal model.