Altered expression of SOCS3 in the hypothalamic arcuate nucleus during seasonal body mass changes in the field vole, Microtus agrestis.

We have previously shown that cold-acclimated (8 degrees C) male field voles (Microtus agrestis) transferred from short day (SD, 8 h light) to long day (LD, 16 h light) photoperiod exhibit an increase in body mass lasting 4 weeks, after which they stabilise at a new plateau approximately 7.5 g (24.8%) higher than animals maintained in SD. By infusing voles with exogenous leptin, we have also demonstrated that SD voles respond to the hormone by reducing body mass and food intake, whereas LD animals increasing body mass are resistant to leptin treatment. In the present study, we investigated whether seasonal changes in body mass could be linked to modulation of the leptin signal by suppressor of cytokine signalling-3 (SOCS3). We used in situ hybridisation to examine hypothalamic arcuate nucleus (ARC) expression of SOCS3, neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) genes in 90 voles exposed to either SD or LD for up to 11 weeks. LD voles increasing body mass had significantly higher levels of SOCS3 mRNA than SD or LD voles with a stable body mass. There were no associated changes in expression of NPY, AgRP, POMC and CART genes. These results suggest that voles that regulate body mass at either the lower (SD) or upper (LD) plateau remain sensitive to leptin action, whereas SOCS3-mediated leptin resistance is a short-term mechanism that enables animals to move between the stable body mass plateaus. Our data provide evidence that expression of SOCS3 in the ARC is involved in the modulation of the strength of the leptin signal to facilitate seasonal cycles in body mass and adiposity.

Hypothalamic energy balance gene responses in the Sprague-Dawley rat to supplementation of high-energy diet with liquid ensure and subsequent transfer to chow.

Energy dense, high fat, high sugar, foods and beverages in our diet are a major contributor to the escalating global obesity problem. Here, we examine the physiological and neuroendocrine effects of feeding rats a solid high-energy (HE) diet with or without a liquid supplement (Ensure) and the consequence of subsequently transferring animals back to chow (C). Outbred Sprague-Dawley rats were fed C until 49-56 days of age, and then transferred a HE diet for 3 weeks before allocation to one of two weight-matched groups. Over the next 10 weeks, one group remained on HE diet, whereas the other had access to the liquid diet, chocolate Ensure (EN), in addition to HE diet (HE + EN). Half the rats from each group were then killed, and the remainder were returned to C for 3 weeks. Supplementation of the HE diet with EN accelerated weight gain and increased daily energy intake, adipose tissue mass, and circulating leptin levels. Transferring animals back to C caused a decrease in bodyweight in the HE + EN group, whereas HE animals were weight stable. Both groups also exhibited voluntary hypophagia, although the magnitude and duration of this response was greater in HE + EN animals. The only effect of Ensure on the hypothalamic genes studied was on tyrosine kinase B expression in the ventromedial hypothalamic nucleus (VMH), which was increased in rats given the supplement. Withdrawal of the obesogenic diets decreased gene expression for cocaine-and-amphetamine regulated transcript (CART) and dynorphin (DYN) in the arcuate nucleus (ARC), and DYN and brain-derived neurotrophic factor (BDNF) in the VMH, whereas neuropeptide Y (NPY) gene expression in the ARC was increased. These changes were independent of previous dietary history. EN supplementation generates distinct physiological responses, yet has a minimal effect on hypothalamic neuropeptide or receptor gene expression, possibly due to the development of leptin resistance. Withdrawal of obesogenic diets induces changes in the gene expression consistent with NPY, CART and BDNF attempting to oppose weight gain on either HE or HE + EN.

Localization of leptin receptor (Ob-R) messenger ribonucleic acid in the rodent hindbrain.

The behavioral and neuroendocrine effects of the adipose tissue-derived circulating protein, leptin, appear to be mediated by the hypothalamus. We have investigated whether the leptin receptor gene is expressed in hindbrain regions known to be involved in the processing of satiety and energetic signals of peripheral origin. In the mouse, gene expression was detected in the nucleus of the solitary tract, lateral parabrachial nucleus, and medullary reticular nucleus and diffusely elsewhere by in situ hybridization. Receptor messenger RNA in these neuronal areas consisted largely, if not exclusively, of the long splice variant, Ob-Rb. Presumed short receptor splice variants were abundantly expressed in the leptomeninges and the choroid plexus of the fourth ventricle. Similar levels of leptin receptor gene expression were present in the hindbrain of lean and obese (ob/ob) mice. The leptin receptor gene was expressed comparatively weakly in the nucleus of the solitary tract of the rat and was not detectable in the lateral parabrachial nucleus. However, by contrast with the mouse, a high level of receptor gene expression was observed in the cerebellum of the rat. A number of rodent hindbrain sites expressing the leptin receptor gene are activated by circulating leptin and may form a monitoring/signaling pathway to complement more direct hypothalamic interactions.

Seasonally inappropriate body weight induced by food restriction: effect on hypothalamic gene expression in male Siberian hamsters.

Male Siberian hamsters undergo physiological weight change in changing photoperiod. Weight loss was induced by food restriction in long days to mimic short-day weight loss, or by food restriction superimposed on short-day weight loss, to test the hypothesis that the hypothalamus differentiates between weight change induced by imposed negative energy balance (inappropriate body weight) and seasonal, appropriate, body weight change, even when these are of similar magnitude. Short-day weight loss was accompanied by reduced POMC and leptin receptor (OB-Rb) mRNA in the arcuate nucleus but elevated cocaine- and amphetamine-regulated transcript. Melanocortin 3-receptor gene expression was reduced in the arcuate nucleus but elevated in the ventromedial nucleus compared with ad libitum-fed long-day controls. Weight loss in long-day restricted animals generated a gene expression profile typical of negative energy balance with low cocaine- and amphetamine-regulated transcript mRNA and elevated OB-Rb. Melanocortin 3-receptor mRNA levels were indistinguishable in short-day and long-day food-restricted hamsters. The hypothalamic correlates of food restriction in short days included up-regulated anabolic neuropeptides and increased OB-Rb mRNA. Low plasma leptin is integrated differently in short-day and long-day restricted animals, and seasonally-inappropriate body weight in either photoperiod engages the compensatory neuropeptide systems involved in the defense of body weight.

Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, photoperiodic regulation of puberty may be modulated by age, by photoperiodic history, and by changes in leptin signaling and the activity of the leptin-sensitive hypothalamic melanocortin system (POMC, MC3-R). A role for CART in photoperiodic regulation of growth is suggested, because the changes in CART gene expression preceded significant divergence of growth trajectories in the opposite photoperiods.

Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold-exposed lean mice.

Leptin receptor gene expression has been measured in arcuate and ventromedial hypothalamic nuclei. Receptor mRNA in both hypothalamic areas was higher in obese mice than in lean littermates. Twice daily leptin administration for 7 days profoundly affected food intake, reduced leptin receptor mRNA in the arcuate nucleus, and had a similar effect on neuropeptide Y gene expression. A single leptin injection was ineffective. Exposure of lean mice to cold for 24 h caused an induction of leptin receptor and NPY mRNA which was normalized when animals were returned to the warm. Regulation of receptor gene expression may be an important component in the reading of the leptin signal.

Leptin interacts with glucagon-like peptide-1 neurons to reduce food intake and body weight in rodents.

The adipose tissue hormone, leptin, and the neuropeptide glucagon-like peptide-1 (7-36) amide (GLP-1) both reduce food intake and body weight in rodents. Using dual in situ hybridization, long isoform leptin receptor (OB-Rb) was localized to GLP-1 neurons originating in the nucleus of the solitary tract. ICV injection of the specific GLP-1 receptor antagonist, exendin(9-39), at the onset of dark phase, did not affect feeding in saline pre-treated controls, but blocked the reduction in food intake and body weight of leptin pre-treated rats. These findings suggest that GLP-1 neurons are a potential target for leptin in its control of feeding.

B219/OB-R 5'-UTR and leptin receptor gene-related protein gene expression in mouse brain and placenta: tissue-specific leptin receptor promoter activity.

Leptin receptor (OB-R) splice variants either encode proteins with different 3' cytoplasmic domains or have different 5' untranslated regions (UTR), indicative of dual promoters. The B219/OB-R promoter transcribes only OB-R transcripts, whereas the OB-R/GRP promoter initiates transcription of both OB-R and another protein of unknown function, called the leptin receptor gene-related protein (OB-RGRP). We compared expression of B219/OB-R 5'-UTR and OB-RGRP mRNAs by in situ hybridization. We thus assessed, by inference, the contributions of the two promoters to the leptin receptor transcript pool, in murine brain or in placenta, a tissue with abundant leptin receptor mRNA. Expression of B219/OB-R 5'-UTR mRNA (and thus by inference B219/OB-R promoter activity) in brain was similar in both distribution and relative intensity to OB-R mRNA. OB-RGRP mRNA (and thus by inference OB-R/GRP promoter activity) was widely distributed in murine brain, with elevated expression in the hypothalamic regions that express the leptin receptor mRNA, including the paraventricular nucleus. B219/OB-R 5'-UTR mRNA, but not OB-RGRP mRNA, was upregulated in hypothalamus of obese ob/ob mice. In placenta, B219/OB-R 5'-UTR mRNA was restricted to the maternal interface, and transcription of both long and short leptin receptor splice variants in the main body of the tissue thus proceeds via the OB-R/GRP promoter, strongly indicative of tissue-specific promoter usage.

The differential regulation of CART gene expression in a pituitary cell line and primary cell cultures of ovine pars tuberalis cells.

The cocaine-amphetamine regulated transcript (CART) encodes for a protein which has an important role in the regulation of appetite and body weight. To date, no details of the molecular events and signal transduction pathways which regulate this gene are available. We report the identification of CART gene expression in the GH3 pituitary cell line. We have used activators of the cAMP or protein kinase C (PKC) signal transduction pathways to show that, in GH3 cells, CART is transcriptionally up-regulated by activators of the cAMP signal transduction pathway. We also identify CART gene expression in ovine pars tuberalis (PT) tissue and primary cell cultures. In PT cells in contrast to GH3 cells, CART gene expression is upregulated by activators of the PKC signal transduction pathway. Cultured cells have provided a valuable resource for the detailed analysis of specific regulatory mechanisms underlying transcriptional or translational regulation of genes, signal transduction events and many other cellular processes. GH3 and PT cells may therefore provide a resource for the further detailed molecular analysis of the events regulating CART gene expression and processing.

Localization of the melatonin-related receptor in the rodent brain and peripheral tissues.

Previous studies have provided a limited examination of the expression of the orphan melatonin-related receptor in the pituitary and hypothalamus of human and sheep and retinal tissue in the sheep. The present study reports evidence of conservation of expression in regions of the hypothalamus (dorsal medial hypothalamus, lateral hypothalamus, arcuate nucleus), the epithelial layer lining the third ventricle and the paraventricular thalamic nucleus of the mouse, rat and hamster. An extensive and detailed analysis of melatonin-related receptor mRNA expression in the mouse central nervous system and peripheral tissues is presented. Mapping the distribution throughout the entire mouse brain has revealed new sites of expression in a number of brain nuclei, including preoptic areas, parabrachial nuclei and widespread distribution in the olfactory bulb. Reverse transcriptase-polymerase chain reaction was performed with RNA isolated from peripheral tissues revealing expression of the melatonin-related receptor mRNA in the mouse kidney, adrenal gland, intestine, stomach, heart, lung, skin, testis and ovary. These results suggest a conserved function in neuroendocrine regulation and a potential role in coordinating physiological responses in the central nervous system and peripheral tissues.

Leptin receptor and neuropeptide Y gene expression in the sheep brain.

Leptin, a protein secretory product of adipocytes, is important in appetite control, energy balance and reproduction. In rodents, the physiological effects of leptin are centrally mediated, in part via the neuropeptide Y (NPY) system in the hypothalamus. The role of leptin in ruminants, where appropriate nutrition and reproductive status are of major economic concern, is largely unknown. To elucidate the function of leptin in sheep we have investigated putative sites of action for leptin in the brain and pituitary gland using both in-situ hybridization to detect expression of the signalling form of the leptin receptor (OB-Rb) and in-vitro autoradiography using (125I)leptin to detect sites of specific leptin binding. OB-Rb gene expression occurred in the hippocampus, cerebral cortex, preoptic area, stria terminalis and choroid plexus, and within the hypothalamus in the paraventricular (PVN), ventromedial (VHM) and arcuate (ARC) nuclei. OB-Rb gene expression in the ovine pituitary gland was not detected by in-situ hybridization. Sites of OB-Rb and NPY gene expression were compared using both in-situ hybridization on adjacent sections containing the arcuate and ventromedial nuclei, and dual in-situ hybridization on sections containing these areas. In serial sections, OB-Rb expression was found to correspond closely with that of NPY over the arcuate nuclei. Using dual in-situ hybridization, NPY expressing neurones in the arcuate nuclei were also positive for OB-Rb gene expression. Therefore, it appears that leptin may partly act via OB-Rb located on NPY neurones in the sheep hypothalamus as in the rodent.

The effect of leptin on luteinizing hormone release is exerted in the zona incerta and mediated by melanin-concentrating hormone.

The adipose hormone, leptin, not only restrains appetite, but also influences energy expenditure. One such influence is to promote sexual maturation and fertility. The neuromodulatory circuits that mediate this effect are not well known but the present study suggests that one mediator could be melanin-concentrating hormone (MCH). We show that the long-form receptor (Ob-Rb) is expressed in the zona incerta of the rat and that administration of leptin (both 0.5 microg and 1.0 microg/side) into this area of ovariectomized, oestrogen-primed rats stimulated the release of luteinizing hormone (LH) within 1 h, the effect enduring for a further 1 h. Injections of leptin into the arcuate nucleus induced a smaller, transient rise in LH while injections into the paraventricular and ventromedial nuclei were without effect. MCH neurones are present in the zona incerta and administration of this hormone into the medial preoptic area (mPOA) stimulates LH release, therefore we investigated the possibility that MCH might mediate this effect of leptin. An injection of MCH antiserum into mPOA prevented the rise in LH normally induced by leptin injected into the zona incerta. In addition, melanocortin receptor antagonists ([D-Arg8]ACTH(4-10) and [Ala6]ACTH(4-10)), previously shown to inhibit the stimulatory effect of MCH on LH release, also inhibited the effect of leptin. We propose that one route by which leptin may promote reproductive activity is by enhancing MCH release from fibres within the mPOA. Speculative mechanisms for the action of MCH include the following possibilities: MCH may be acting on the specific MCH receptor which in turn interacts with a melanocortin or melanocortin-like receptor; MCH may bind directly to one of the melanocortin receptors; or melanocortin antagonists may interact with the MCH receptor.

Association of leptin receptor (OB-Rb), NPY and GLP-1 gene expression in the ovine and murine brainstem.

Appetite-related neuropeptide systems have not been studied extensively in the ruminant, although there have been a number of recent studies of the hypothalamus. Since some leptin signaling is integrated in the rodent brainstem, and leptin modulates neuropeptidergic activity, we now describe leptin receptor (long splice variant, OB-Rb), neuropeptide Y (NPY) and glucagon-like peptide-1 (GLP-1) gene expression in the ovine brainstem. Leptin receptor mRNA was localized to the spinal trigeminal tract and nucleus, nucleus of the solitary tract (NTS), area postrema and dorsal motor nucleus of the vagus. NPY gene expression was abundant in the ovine medulla, occurring in two bilateral 'bands' that encompassed the NTS region and ran ventrolaterally. GLP-1 mRNA was confined largely to the NTS. The distribution of OB-Rb mRNA overlapped with that of NPY and GLP-1 gene expression, suggesting the possibility of interaction between leptin and these brainstem neuropeptide systems. However, in an extension of earlier work, co-expression studies in the murine brainstem revealed only a small number of neurons that expressed both NPY and leptin receptor mRNA, despite the widespread and abundant expression of the former. Thus the majority of NPY synthesis in the brainstem may not be directly regulated by leptin. The sheep brainstem had similar anatomical distribution of OB-Rb, NPY and GLP-1 gene expression to the rodent, consistent with a role for this region in peripheral leptin feedback signaling and brainstem-hypothalamo communication.

Hypothalamic neuropeptide Y content and mRNA expression in weanling rats subjected to dietary manipulations during fetal and neonatal life.

Hypothalamic neuropeptide Y (NPY) is present very early during the fetal life and is rapidly functional in the regulation of feeding behavior after birth. In the present experiment, we tried to determine the influence that the diet type ingested by dams during gestation and lactation would have on the growth and hypothalamic and pancreatic peptides of their progeny immediately after weaning. The dams were fed on either a high-carbohydrate (HC), a high-fat (HF) or a control diet ad libitum. At 3 days of age, the HC pups weighed significantly more than the two other groups (P < 0.02 vs. C and P < 0.002 vs. HF). At weaning, the HF rats were significantly lighter than the two other groups (P < 0.001). Food intake was significantly lower in the HF rats than in the two other groups 3 days (P < 0.002) and 5 days after weaning (P < 0.02). Plasma glucose of the HF rats was significantly lower than that of the control rats (P < 0.05) and of the HC rats (P < 0.01). Immunoreactive insulin in the HF rats was also significantly lower than that in the control rats (-53%; P < 0.001) and in the HC rats (-47%; P < 0.001). NPY content and mRNA expression in the arcuate nucleus were not significantly different between the three groups. NPY concentration only varied in the ventromedian nucleus. In the control rats, it was significantly lower than that of the HC rats (-35%; P < 0.01) and that of the HF rats (-32%; P < 0.002). These data demonstrated that the regulatory mechanisms of feeding behavior in offspring are completely and differentially modified by the macronutrient content of the diets ingested by their mother. Both peripheral and central mediators were strongly implicated. These modifications could have long-term repercussions on body weight and composition.

Leptin (ob) mRNA and hypothalamic NPY in food-deprived/refed Syrian hamsters.

Food deprivation in the laboratory rat decreases plasma leptin and insulin, elevates glucocorticoid concentration, and increases the activity of the neuropeptide Y (NPY) system and feeding drive. In contrast, Syrian hamsters fail to modify feeding behaviour in response to various food scarcity paradigms. Two components of the neuroendocrine-hormonal response to food deprivation, adipose tissue-derived leptin and hypothalamic NPY, are investigated in the Syrian hamster. ob (leptin) mRNA was less abundant in subcutaneous than abdominal adipose tissue, but not to the extent observed in other rodents. Food deprivation for 48 h reduced ob mRNA in inguinal and retroperitoneal white adipose tissue; gene expression was partially restored by refeeding. In contrast, in epididymal fat there was no effect on ob mRNA. NPY concentrations in hypothalamic nuclei were also unaffected by feeding state. The predicted amino acid sequence of leptin from the Syrian hamster was over 90% homologous with Djungarian hamster and mouse sequences, and the leptin receptor gene (OB-R), and specifically the long intracellular splice variant, OB-Rb, was expressed in the same forebrain and hypothalamic regions that have been described in laboratory mice and rats, including hypothalamic arcuate, dorsomedial, and ventromedial nuclei. The failure of food deprivation to affect NPY and feeding behaviour in Syrian hamsters is unlikely to be due to defects in the leptin system, although there may be region-specific differences in the regulation of leptin signaling in laboratory rats and Syrian hamsters.

Inhibin betaB expression in murine adipose tissue and its regulation by leptin, insulin and dexamethasone.

Inhibin betaB (INHBB; coding for the activin betaB subunit) has previously been identified in both human and rodent adipose tissue and using Taqman real-time PCR with specific primers we confirm the expression of INHBB mRNA in rodent adipose tissue. Expression of INHBB in murine epididymal adipose tissue was higher than in any of the other tissues studied and appears to be regulated by changes in energy balance and leptin. It was increased fourfold in the epididymal fat depot of ob/ob mice compared with the same fat depot in lean mice. The i.p. administration of leptin in obese ob/ob mice decreases the expression of INHBB. In human adipose tissue, INHBB is reduced by weight loss. In keeping with this, we demonstrate that INHBB expression in murine adipose tissue is decreased in fasting and increased upon refeeding. We show that INHBB is expressed in both the mature adipocyte and the stromal vascular fraction of adipose tissue. INHBB increases with the differentiation of pre-adipocytes into mature adipocytes in the 3T3-L1 cell line. In differentiated 3T3-L1 adipocytes, where receptors to activin have been previously reported, insulin increases the expression of INHBB, while dexamethasone decreases the expression of INHBB when compared with untreated control cells. Taken together, these results suggest that the regulation of INHBB expression in adipose tissue may play a physiological role in energy balance or the insulin insensitivity associated with obesity.

Photoperiod regulates arcuate nucleus POMC, AGRP, and leptin receptor mRNA in Siberian hamster hypothalamus.

Siberian hamsters decreased body weight by 30% during 18 wk in short day (SD) vs. long day (LD) controls. Subsequent imposed food deprivation (FD; 24 h) caused a further 10% decrease. In the hypothalamic arcuate nucleus (ARC), SDs reduced proopiomelanocortin (POMC) gene expression and agouti-related protein (AGRP) mRNA was elevated, changes that summate to reduced catabolic drive through the melanocortin receptors. There was no effect of photoperiod on neuropeptide Y (NPY), melanin concentrating hormone, orexin, or corticotropin-releasing factor mRNAs. Superimposed FD increased AGRP gene expression and caused a localized elevation of NPY mRNA in the ARC. Both adipose tissue leptin and ARC leptin receptor (OB-Rb) mRNAs were downregulated in SDs, whereas FD increased OB-Rb gene expression. Thus OB-Rb mRNA is differentially regulated by acute and chronic changes in plasma leptin in this species. In a separate experiment in LDs, AGRP gene expression was increased by 24 or 48 h FD, whereas POMC mRNA was downregulated in the caudal ARC. AGRP and NPY mRNAs were extensively coexpressed in the ARC, and their differential regulation by photoperiod and FD is suggestive of transcript-specific regulation at the level of individual neurons.

Short-day weight loss and effect of food deprivation on hypothalamic NPY and CRF mRNA in Djungarian hamsters.

The effect of food deprivation on hypothalamic neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) gene expression in the Djungarian hamster was quantified by in situ hybridization. Hamsters housed in short days (SD) for 18 wk decreased body weight by 40% and exhibited 200% increases in both NPY and CRF mRNA when deprived of food for 24 h. Prior gonadectomy in long days (LD) affected neither basal gene expression nor the induction of gene expression by food deprivation. Gene expression in juvenile LD hamsters similar in body weight to SD animals was relatively insensitive to food deprivation of either 24- or 48-h duration or to subsequent refeeding. In juvenile hamsters, food deprivation for 24 but not 48 h decreased ob (obese) gene expression in inguinal but not epididymal white adipose tissue; ob mRNA levels were restored by refeeding. All food-deprived hamsters had reduced plasma insulin concentrations, but plasma cortisol was only elevated in SD food-deprived animals. NPY gene expression was also increased after daily dexamethasone injections in adult LD hamsters. These results suggest that the neuroendocrine consequences of food deprivation in SD Djungarian hamsters are determined by some factor other than absolute body mass such as the size of adipose tissue reserves.

Anorexia in rats infected with the nematode, Nippostrongylus brasiliensis: experimental manipulations.

Nippostrongylus brasiliensis induces a biphasic anorexia in laboratory rats, the first phase coincident with lung invasion (ca day 2) and the second when the worms mature in the intestine (ca day 8). Using the anthelminthic, mebendazole (MBZ), N. brasiliensis infections of the rat were eliminated between the first and second anorexic episodes. This intervention prevented the expression of the second phase of anorexia. Rats exposed to a second infection with N. brasiliensis, 3 weeks after the primary infection, exhibited only a first phase anorexic response which was not influenced by MBZ termination of the primary infection. The lower cumulative food intake and weight gain of all infected rats after 8 days of infection were accompanied by elevated plasma insulin and, in some individuals, by elevated plasma leptin, compared with uninfected controls and previously-infected MBZ-treated rats. Messenger RNA levels for neuropeptide Y were higher in the hypothalamic arcuate nucleus of 8-day infected rats than in recovering MBZ-treated animals. Inoculation of rats with heat-killed N. brasiliensis larvae failed to induce anorexia and did not alter the severity of biphasic anorexia on subsequent injection of viable larvae. The first anorexic episode is therefore dependent upon viable migrating larvae. The second phase of anorexia clearly requires the continuing presence of the parasite beyond the lung phase. Viable migrating larvae are also required to confer 'resistance' to reinfection.

Photoperiodic effects on body mass, energy balance and hypothalamic gene expression in the bank vole.

We examined the effect of increasing photoperiod, at a constant low temperature, on the body mass and energy budget of the bank vole Clethrionomys glareolus. Simultaneously, we determined the hypothalamic gene expression of neuropeptides and receptors known to be involved in short-term energy balance. Despite an increase in body mass (approximately 10% of initial mass), we found no significant changes in any energetic parameters (food intake, energy assimilation rate, resting metabolic rate and total daily energy expenditure by doubly-labelled water). Apparent energy assimilation efficiency was higher in voles exposed to long-days (LD) compared to short-days (SD). Surprisingly, gene expression of corticotrophin releasing factor (CRF; in the paraventricular nucleus), and the melanocortin-3 receptor (in the arcuate nucleus), both known to be involved in appetite suppression and elevation of energy expenditure in short-term energy balance, were higher in voles kept in LD compared to SD. CRF expression was also elevated in females compared to males. These paradoxical data suggest an alternative mechanism for the control of seasonal body mass changes compared to short-term body mass changes, and between male and female voles. Furthermore, they highlight the need for studies to perform simultaneous measurements at both the molecular and whole animal levels.