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The production of bioactive peptides from biologically inactive precursors involves extensive post-translational processing, including enzymatic cleavage by proteolytic peptidases. Endoproteolytic prohormone-convertases initially cleave the precursors of many neuropeptides at specific amino acid sequences to generate intermediates with basic amino acid extensions on their C-termini. Subsequently, the related exopeptidases, carboxypeptidases D and E (CPD and CPE), are responsible for removing these amino acids before the peptides achieve biological activity. We investigated the effect of photoperiod on the processing of the neuropeptide precursor pro-opiomelanocortin (POMC) and its derived neuropeptides, α-melanocyte-stimulating hormone (MSH) and β-endorphin (END), within the hypothalamus of the seasonal Siberian hamster (Phodopus sungorus). We thus compared hypothalamic distribution of CPD, CPE, α-MSH and β-END using immunohistochemistry and measured the enzyme activity of CPE and concentrations of C-terminally cleaved α-MSH in short-day (SD; 8 : 16 h light/dark) and long-day (LD; 16 : 8 h light/dark) acclimatised hamsters. Increased immunoreactivity (-IR) of CPE, as well as higher CPE activity, was observed in SD. This increase was accompanied by more β-END-IR cells and substantially higher levels of C- terminally cleaved α-MSH, as determined by radioimmunoassay. Our results suggest that exoproteolytic cleavage of POMC-derived neuropeptides is tightly regulated by photoperiod in the Siberian hamster. Higher levels of biological active α-MSH- and β-END in SD are consistent with the hypothesis that post-translational processing is a key event in the regulation of seasonal energy balance., (© 2012 British Society for Neuroendocrinology.)

Maternal diet ingested during gestation can profoundly alter production and action of hypothalamic neuropeptides involved in feeding and body weight regulation. In this study, we set out to simulate, in a rat model, modifications to feeding habit often observed in pregnant women. Gestating dams were fed a restricted normal diet with the opportunity to complete their energy requirements with either a high-fat (HF) or a high-carbohydrate (HC) diet. Growth and hypothalamic feeding peptides were measured in the offspring at 3 (weaning) and 20 weeks of age. At weaning, body weight was lower in HC pups than in HF pups or control (Ca) pups born to dams fed control diet ad libitum. Expression of neuropeptide Y (NPY) and AgRP mRNA in the arcuate nucleus were increased in HC pups vs Ca and HF pups. By 20 weeks of age, body weight differentials had disappeared, and there was no differences in NPY and AgRP gene expression, although POMC expression was lower in HC rats than in HF rats. NPY and orexin peptide concentrations in the paraventricular nucleus at this age were higher in HC rats than in Ca and HF rats. In HC rats, there was also a greater positive gradient of peptide concentration between the zone of release and the zone of synthesis for NPY and orexin. The early up-regulation of orexigenic peptides in HC rats may be a compensatory adjustment to low body weight. This persisting overactive orexigenic drive might have deleterious metabolic effects in an obesogenic environment at adulthood., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Central regulation of energy balance in seasonal mammals such as the Siberian hamster is dependent on the precise integration of short-term satiety information arising from the gastrointestinal tract with long-term signals on the status of available energy reserves (e.g. leptin) and prevailing photoperiod. Within the central nervous system, the brainstem nucleus of the solitary tract (NTS) and the parabrachial nucleus (PBN) are major relay nuclei that transmit information from the gastrointestinal tract to higher forebrain centres. We extended studies on the seasonal programming of the hypothalamus to examine the effect of the photoperiod on neuropeptidergic circuitries of this gut-brain axis. In the NTS and PBN we performed gene expression and immunoreactivity (-ir) studies on selected satiety-related neuropeptides and receptors: alpha-melanocyte stimulating hormone, melanocortin-3 receptor, melanocortin-4 receptor (MC4-R), growth hormone secretagogue-receptor, cocaine- and amphetamine-regulated transcript, preproglucagon (PPG), glucagon-like peptide 1 (GLP-1), cholecystokinin (CCK), peptide YY, galanin, neurotensin, and corticotrophin releasing hormone (CRH). Gene expression of PPG and MC4-R, and -ir of CCK and GLP-1, in the NTS were up-regulated after 14 weeks in long-day photoperiod (16 h light:8 h dark) compared to short-days (8 h light:16 h dark), whereas CRH-ir and NT-ir were increased in short-days within the PBN. We suggest that brainstem neuroendocrine mechanisms contribute to the long-term regulation of body mass in the Siberian hamster by a photoperiod-related modulation of satiety signalling.

In the adult brain, leptin regulates energy homeostasis primarily via hypothalamic circuitry that affects food intake and energy expenditure. Evidence from rodent models has demonstrated that during early postnatal life, leptin is relatively ineffective in modulating these pathways, despite the high circulating levels and the presence of leptin receptors within the central nervous system. Furthermore, in recent years, a neurotrophic role for leptin in the establishment of energy balance circuits has emerged. The precise way in which leptin exerts these effects, and the site of leptin action, is unclear. To provide a detailed description of the development of energy balance systems in the postnatal rat in relation to leptin concentrations during this time, endogenous leptin levels were measured, along with gene expression of leptin receptors and energy balance neuropeptides in the medial basal hypothalamus, using in situ hybridization. Expression of leptin receptors and both orexigenic and anorexigenic neuropeptides increased in the arcuate nucleus during the early postnatal period. At postnatal day 4 (P4), we detected dense leptin receptor expression in ependymal cells of the third ventricle (3V), which showed a dramatic reduction over the first postnatal weeks, coinciding with marked morphological changes in this region. An acute leptin challenge robustly induced suppressor of cytokine signaling-3 expression in the 3V of P4 but not P14 animals, revealing a clear change in the location of leptin action over this period. These findings suggest that the neurotrophic actions of leptin may involve signaling at the 3V during a restricted period of postnatal development.

Previously, 40% food restriction of male Siberian hamsters over 21 days in short-day (SD) photoperiod induced characteristic changes in expression of hypothalamic arcuate nucleus energy balance genes; mRNAs for neuropeptide Y, agouti-related peptide, and leptin receptor were upregulated, and those of proopiomelanocortin and cocaine- and amphetamine-regulated transcript were depressed. The present study examined the effect of refeeding hamsters for 6 days (approximately 50% recovery of weight differential) or 19 days (resumption of appropriate weight trajectory). Hyperphagia continued throughout refeeding, but differences in fat pad weights and leptin levels had disappeared after 19 days. Cocaine- and amphetamine-regulated transcript gene expression was depressed by prior restriction in both refed groups. The depressive effect of prior restriction on proopiomelanocortin gene expression had disappeared after 19 days of refeeding. There was no effect of prior food restriction on neuropeptide Y or agouti-related peptide gene expression. Expression of the anorexigenic brain-derived neurotrophic factor was downregulated in the ventromedial nucleus after SD exposure for 12 wk. In the SD food restriction study, there were effects of photoperiod on brain-derived neurotrophic factor gene expression but not of prior food restriction. Hypothalamic energy balance genes in the hamster respond asynchronously to return to a seasonally appropriate body weight. The achievement of this weight rather than the weight at which caloric restriction was imposed is the critical factor. The differential responses of hypothalamic energy balance genes to food restriction and refeeding are poorly characterized in any species, a critical issue given their potential relevance to human weight loss strategies that involve caloric restriction.

Nutritional factors have a critical influence during prenatal life on the development and regulation of networks involved in body weight and feeding regulation. To establish the influence of the macronutrient type on feeding regulatory mechanisms and more particularly on stimulatory pathways (galanin and orexins), we fed female rats on either a high-carbohydrate (HC), a high-fat (HF), or a well-balanced control diet during gestation and lactation, and measured peptide expression in the hypothalamus and important hormones (leptin, insulin) in their pups at weaning. HF weanlings were 30% lighter than control and HC pups (P<0.001). They were characterized by reduced plasma glucose and insulin levels (P<0.01 or less). Their galanin and orexin systems were upregulated as shown by the significant augmentation of mRNA expression in the paraventricular nucleus and lateral hypothalamus, respectively. Inhibitory peptides like corticotropin-releasing hormone and neurotensin were not affected by this dietary treatment during early life. There was, therefore, a more intense drive to eat in HF pups, perhaps to compensate for the lower body weight at weaning. HF diets during early life had meanwhile some positive consequences: the lower metabolic profile might be beneficial in precluding the development of obesity and metabolic syndrome later in life. This is however valid only if the orexigenic drive is normalized after weaning.

The SD (Sprague-Dawley) rat model of DIO (diet-induced obesity) is reported to exhibit a clear segregation into susceptible and resistant subpopulations shortly after transfer to a HE (high energy) diet. This does not appear to be the case for rats sourced in the U.K., where body weight gain on obesogenic HE diet is normally distributed, as might be anticipated for a polygenic trait in an outbred population. Many of the energy balance effects of dietary manipulation in this model (e.g. supplementation of HE diet with the liquid diet, Ensure; energy intake and defence of body weight following withdrawal of obesogenic diet) appear to be characteristics of the diets being manipulated rather than subject traits. The activities of energy balance-related hypothalamic signals are affected by diet and the development of DIO, but may not be able to differentiate between different diets and the relative levels of obesity that develop.

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

In young (35- to 56-day-old) and middle-aged (9-mo-old) wild-type (+/+) and melanocortin-4 receptor (MC4R)-deficient (+/-, -/-) mice, expressions of neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin (POMC), and cocaine-and-amphetamine-regulated transcript (CART) were analyzed in the arcuate nucleus (ARC) and adjacent regions comprising the dorsomedial (DMN) and ventromedial (VMN) nucleus. In the ARC of young mice, NPY and AGRP expression increased and POMC and CART expression decreased with body fat content. Adjusting for the influence of body fat content by ANCOVA showed that the levels of NPY, POMC, and CART were highest and of AGRP lowest in young -/- mice. In the middle-aged mice, feedback from body fat content was weakened. For -/- mice ANCOVA revealed higher NPY and AGRP, lower POMC, and unchanged CART expression levels relative to young -/- mice. In the DMN and VMN, POMC and AGRP signals were absent at each age. CART was expressed in the DMN independent of age, fat content, and genotype. For NPY expression, an age-dependent induction was found in the DMN and VMN; it was absent in the young but present in the middle-aged mice, showing close positive correlations between body fat content and the numbers of NPY-labeled cells which were further enhanced in -/- mice. Thus MC4R deficiency augments age-induced NPY expression in the DMN and VMN with no feedback from body fat content. Negative feedback control by body fat content on ARC neuropeptide expression is present in young animals but vanishes with age and is modulated by MC4R deficiency.

Neuromedin U (NMU) has been associated with the regulation of food-intake and energy balance in rats. The objective of this study was to identify the sites of gene expression for NMU and the NMU receptor-2 (NMU2R) in the mouse and rat hypothalamus and ascertain the effects of nutritional status on the expression of these genes. In situ hybridization studies revealed that NMU is expressed in several regions of the mouse hypothalamus associated with the regulation of energy balance. Analysis of NMU expression in the obese ob/ob mouse revealed that NMU mRNA levels were elevated in the dorsomedial hypothalamic (DMH) nucleus of obese ob/ob mice compared to lean litter-mates. In addition, NMU mRNA levels were elevated in the DMH of mice fasted for 24 h relative to ad libitum fed controls. The pattern of expression of NMU and NMU2R were more widespread in the hypothalamus of mice than rats. These data provide the first detailed anatomical analysis of the NMU and NMU2R expression in the mouse and advance our knowledge of expression in the rat. The data from the obese rodent models supports the hypothesis that NMU is involved in the regulation of nutritional status.

The Siberian hamster, Phodopus sungorus, is a powerful model of physiological body weight regulation. This seasonal model offers the potential to distinguish between the compensatory neuroendocrine systems that defend body weight against imposed negative energy balance, and those that are involved in the programming of the level of body weight that will be defended-a seasonally appropriate body weight. Of the known, studied, components of the hypothalamic energy balance system, the anorexogenic peptide, cocaine- and amphetamine-regulated transcript (CART), is the only candidate where gene expression changes in a manner consistent with a role in initiating or sustaining photoperiod-induced differences in body weight trajectory. Siberian hamsters effect a reversible biannual switch in leptin sensitivity in which only short day (SD)-acclimated hamsters that have undergone a reduction in body weight, adiposity and plasma leptin are sensitive to peripheral exogenous leptin. The suppressor of cytokine signalling protein, SOCS3, appears to be the molecular correlate of this seasonal sensitivity.

The expression of precursor-protein convertase (PC)1, PC2 and paired basic amino acid cleaving enzyme four mRNA was studied by in situ hybridisation in regions of the hypothalamus involved in energy regulation in relation to obese (ob) gene mutation and energy deficit. PC1 gene was differentially expressed in hypothalamic nuclei of mice from different genetic backgrounds or energetic status, whereas no differences in expression were observed for either the PC2 or paired basic amino acid cleaving enzyme four genes. In obese ob/ob mice, PC1 mRNA levels were increased in the paraventricular nucleus, decreased in the lateral hypothalamus and unchanged in the ventromedial nucleus and arcuate nucleus relative to lean controls. In response to intraperitoneal injection of murine leptin, PC1 mRNA levels in obese ob/ob mice decreased in the arcuate nucleus, increased in the lateral hypothalamus and were unchanged in both the paraventricular nucleus and ventromedial nucleus. In mice deprived of food for 24 h, PC1 mRNA levels were reduced in the ventromedial nucleus, increased in the lateral hypothalamus and unchanged in the paraventricular nucleus and arcuate nucleus relative to ad libitum-fed controls. Overall, whilst the data show effects related to leptin and energetic status, they do not support a strong and consistent link between PC1 gene expression and energy balance. This suggests that if PC1 is important to the control of energy balance then protein expression and activity, rather than gene expression may be the more critical parameters of regulation. The relationship between PC1 and candidate energy balance-related genes in the lateral hypothalamus was investigated by dual in situ hybridisation. PC1 mRNA was localised in prepro-orexin mRNA expressing neurons in the lateral hypothalamus, which suggests a functional relationship.

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

Hypothalamic orexin gene expression has not been reported in the ruminant. Here, we describe the localization of preproorexin mRNA in the ovine lateral hypothalamic area (LHA) and zona incerta (ZI) using in situ hybridization. The hypothalamic localization of the orexin gene expression was similar in sheep to rodent models. Since appetite in sheep is seasonally (photoperiodically) regulated, we compared the amounts of preproorexin mRNA between long- (LD) and short-day (SD) photoperiods in both freely feeding (food intake is 20% higher in LD than SD) and food-restricted sheep (50% liveweight maintenance for 11 weeks). Gene expression was higher in SDs than in LDs but was not affected by chronic food restriction. In a second study, hypothalamic orexin gene expression in castrate sheep was not affected by a 4-day fast, irrespective of gonadal steroid (estradiol) replacement, and was not affected by the gonadal steroid per se. The results demonstrate the sensitivity of orexin gene expression to photoperiod, but up-regulation occurs in SDs when the appetite is characteristically low and no sensitivity to imposed changes in food intake. This supports the concept that orexins may not have a primary role in appetite regulation and correction of negative energy balance but since the sheep breed only in SDs, their role in seasonal reproductive activation deserves further study.

Seasonal animals are able both to programme changes in body weight in response to annual changes in photoperiod (anticipatory regulation) and to correct changes in body weight caused by imposed energetic demand (compensatory regulation). Experimental evidence from the Siberian hamster suggests that seasonally appropriate body weight is continually reset according to photoperiodic history, even when actual body weight is driven away from this target weight by manipulation of energy intake. These characteristics constitute the "sliding set point" of seasonal body weight regulation. To define the mechanisms and molecules underlying anticipatory body weight regulation, we are investigating the involvement of hypothalamic systems with an established role in the compensatory defence of body weight. Weight loss or restricted growth induced by short days (SD) results in low circulating leptin compared with long day (LD) controls. However, this chronic low leptin signal is read differently from acute low leptin resulting from food deprivation; leptin receptor gene expression in the hypothalamic arcuate nucleus (ARC) is lower in SD, whereas food deprivation increases expression levels, suggesting changes in sensitivity to leptin feedback. SD alterations in mRNA levels for a number of hypothalamic neuropeptide and receptor genes appear counter-intuitive for a SD body weight trajectory. However, early increases in ARC cocaine-and amphetamine-regulated transcript (CART) gene expression in SDs could be involved in driving body weight loss or growth restriction. The sites of photoperiod interaction with energy balance neuronal circuitry and the neurochemical encoding of body weight set point require full characterisation. Study of anticipatory regulation in seasonal animals offers new insight into body weight regulation across mammalian species, including man.

In small rodents there is compelling evidence of a lipostatic system of body mass regulation in which peripheral signals of energy storage are decoded in the hypothalamus. The ability of small mammals to defend an appropriate mass against imposed energy imbalance has implicated hypothalamic neuroendocrine systems in body mass regulation. The effect of the neuropeptide systems involved in this regulation is primarily compensatory. However, small mammals can also effect changes in the level of body mass that they will defend, as exemplified by seasonal species. Regulatory control over fat mass may be relatively loose in humans; the sizes of long-term storage depots may not themselves be regulated, but rather may be a consequence of temporal variations in the matching of supply and demand. Whether food intake is regulated to match energy demand, or to match demand and to regulate storage, it is clear that physiological defects or genetic variation in hypothalamic and peripheral feedback systems will have profound implications for fat storage. Study of mechanisms implicated in energy homeostasis in laboratory rodents is likely to continue to identify targets for pharmacological manipulation in the management of human obesity.

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.