Zoe A. Archer

Hypothalamic gene expression in sheep for cocaine- and amphetamine-regulated transcript, pro-opiomelanocortin, neuropeptide Y, agouti-related peptide and leptin receptor and responses to negative energy balance.

Hypothalamic pathways involved in the regulation of energy balance have not been widely studied in ruminants to date. Here, we used in situ hybridisation to study the gene expression of a number of leptin-sensitive receptors and neuropeptides in the ovine hypothalamus. Gene expression was first localised for cocaine- and amphetamine-regulated transcript (CART) and agouti-related peptide (AGRP). We then examined in adult male castrated sheep the effects of acute negative energy balance induced by a 4-day fast on the amounts of these mRNAs and those for leptin receptor (OB-Rb), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC). CART mRNA was localised in the arcuate nucleus (ARC), paraventricular nucleus, median eminence and ventromedial hypothalamic nucleus, and extensive co-localisation with POMC mRNA was demonstrated in the ARC. AGRP mRNA was localised in the ARC. Fasting up-regulated gene expression for OB-Rb and for the orexigenic neuropeptides NPY and AGRP in the ARC. There was a trend towards down-regulation of gene expression for the anorexigenic neuropeptide CART and no effect on POMC in the ARC, although these results are inconclusive. The presence or absence of oestradiol-containing subcutaneous implants did not influence gene expression or the effects of fasting. The hypothalamic changes were consistent with responses to the observed reduction in circulation leptin and suggest that the peripheral feedback and central mechanisms for restoring the energy balance may be largely conserved across monogastric and ruminant species.

Programming of hypothalamic neuropeptide gene expression in rats by maternal dietary protein content during pregnancy and lactation.

Epidemiological studies show a link between low birthweight and increased obesity. In contrast, slow growth during the lactation period reduces obesity risk. The present study investigates the potential underlying mechanisms of these observations. Rats were established as follows: (i) control animals [offspring of control dams fed a 20% (w/v) protein diet], (ii) recuperated animals [offspring of dams fed an isocaloric low-protein (8%, w/v) diet during pregnancy and nursed by control dams], and (iii) postnatal low protein animals (offspring of control dams nursed by low-protein-fed dams). Serum and brains were collected from fed and fasted animals at weaning. Expression of hypothalamic energy balance genes was assessed using in situ hybridization. Recuperated pups were smaller at birth, but caught up with controls by day 21 and gained more weight than controls between weaning and 12 weeks of age (P<0.05). At 21 days, they were hypoleptinaemic compared with controls in the fed state, with generally comparable hypothalamic gene expression. Postnatal low protein offspring had significantly lower body weights than controls at weaning and 12 weeks of age (P<0.001). At 21 days, they were hypoglycaemic, hypoinsulinaemic and hypoleptinaemic. Leptin receptor gene expression in the arcuate nucleus was increased in postnatal low protein animals compared with controls. Consistent with hypoleptinaemia, hypothalamic gene expression for the orexigenic neuropeptides NPY (neuropeptide Y) and AgRP (Agouti-related peptide) was increased, and that for the anorexigenic neuropeptides POMC (pro-opiomelanocortin) and CART (cocaine- and amphetamine-regulated transcript) was decreased. These results suggest that the early nutritional environment can affect the development of energy balance circuits and consequently obesity risk.

Circulating Ghrelin Levels and Central Ghrelin Receptor Expression are Elevated in Response to Food Deprivation in a Seasonal Mammal (Phodopus sungorus)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). However, the functional interaction of ligand and receptor is not very well understood. We demonstrate that GHSR mRNA is up‐regulated after food deprivation (48 h) in the hypothalamic arcuate nucleus and ventromedial nucleus of the seasonal Siberian hamster, Phodopus sungorus. This increase is accompanied by a two‐fold elevation of circulating ghrelin concentration. Chronic changes in feeding state imposed by food restriction over a period of 12 weeks during long day‐length induced increased GHSR gene expression, whereas food restriction for 6 weeks had no effect. Phodopus sungorus reveals remarkable seasonal changes in body weight, fat mass and circulating leptin levels. Ghrelin is generally regarded as having opposing effects on appetite and body weight with respect to those exhibited by leptin. However, our study revealed that seasonal adaptations were not accompanied by changes in either GHSR gene expression or circulating ghrelin concentration. Therefore, we suggest that ghrelin only plays a minor role in modulating long‐term seasonal body weight cycles. Our findings imply that ghrelin predominantly acts as a short‐term regulator of feeding.

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.

Orexin gene expression and regulation by photoperiod in the sheep hypothalamus.

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.

PC1/3 and PC2 Gene Expression and Post-Translational Endoproteolytic Pro-Opiomelanocortin Processing is Regulated by Photoperiod in the Seasonal Siberian Hamster (Phodopus sungorus)

A remarkable feature of the seasonal adaptation displayed by the Siberian hamster (Phodopus sungorus) is the ability to decrease food intake and body weight (by up to 40%) in response to shortening photoperiod. The regulating neuroendocrine systems involved in this adaptation and their neuroanatomical and molecular bases are poorly understood. We investigated the effect of photoperiod on the expression of prohormone convertases 1 (PC1/3) and 2 (PC2) and the endoproteolytic processing of the neuropeptide precursor pro‐opiomelanocortin (POMC) within key energy balance regulating centres of the hypothalamus. We compared mRNA levels and protein distribution of PC1/3, PC2, POMC, adrenocorticotrophic hormone (ACTH), α‐melanocyte‐stimulating hormone (MSH), β‐endorphin and orexin‐A in selected hypothalamic areas of long day (LD, 16 : 8 h light : dark), short day (SD, 8 : 16 h light : dark) and natural‐day (ND, photoperiod depending on time of the year) acclimated Siberian hamsters. The gene expression of PC2 was significantly higher within the arcuate nucleus (ARC, P < 0.01) in SD and in ND (versus LD), and is reflected in the day length profile between October and April in the latter. PC1/3 gene expression in the ARC and lateral hypothalamus was higher in ND but not in SD compared to the respective LD controls. The immunoreactivity of PC1/3 cleaved neuropeptide ACTH in the ARC and PC1/3‐colocalised orexin‐A in the latyeral hypothalamus were not affected by photoperiod changes. However, increased levels of PC2 mRNA and protein were associated with higher abundance of the mature neuropeptides α‐MSH and β‐endorphin (P < 0.01) in SD. This study provides a possible explanation for previous paradoxical findings showing lower food intake in SD associated with decreased POMC mRNA levels. Our results suggest that a major part of neuroendocrine body weight control in seasonal adaptation may be effected by post‐translational processing mediated by the prohormone convertases PC1/3 and PC2, in addition to regulation of gene expression of neuropeptide precursors.

Brain responses to obesogenic diets and diet-induced obesity.

Rodent models of diet-induced obesity (DIO) mimic common human obesity more accurately than obese single-gene mutation lines, such as the ob/ob mouse. Sprague-Dawley rats sourced in the UK develop obesity when fed a high-energy diet, but susceptibility to DIO is normally distributed, as might be anticipated for a polygenic trait in an outbred population, in contrast to reports in the literature using ostensibly the same strain of rats sourced in the USA. Nevertheless, the responses of these rats to solid and liquid obesogenic diets are very similar to those reported elsewhere, and this model of DIO has much to commend it as a vehicle for the mechanistic study of susceptibility to DIO, development and reversal of obesity on solid and liquid diets and the response of peripheral and central energy balance systems to the development of obesity and to the obesogenic diets themselves. In general, hypothalamic energy-balance-related systems respond to obesogenic diets and developing obesity with activity changes that appear designed to counter the further development of the obese state. However, these hypothalamic changes are apparently unable to maintain body weight and composition within normal limits, suggesting that attributes of the obesogenic diets either evade the normal regulatory systems and/or engage with reward pathways that override the homeostatic systems. Since diets are a risk factor in the development of obesity, it will be important to establish how obesogenic diets interact with energy-balance pathways and whether there is potential for diets to be manipulated with therapeutic benefit.

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 °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.

Introduction of a high-energy diet acutely up-regulates hypothalamic cocaine and amphetamine-regulated transcript, Mc4R and brown adipose tissue uncoupling protein-1 gene expression in male Sprague-Dawley rats.

Obesity is an escalating problem in Western societies. Susceptibility to weight gain within an obesogenic environment is variable. It remains unclear how the range of weight gain responses are generated. It is possible that an individuals immediate and/or sustained appetite for apparently palatable foods, or metabolic adaptations to a new diet could be important. The present study therefore examined the short‐ to medium‐term effects of a high‐energy (HE) diet on bodyweight, food intake, and energy balance‐related signalling systems. Sprague‐Dawley rats were fed either chow or an HE diet for 12 h, 24 h, 48 h or 14 days. Blood hormones and metabolites were assayed, and expression of uncoupling protein‐1 (UCP‐1) and hypothalamic energy‐balance related genes were determined by Northern blotting or in situ hybridisation, respectively. Short‐term exposure (12 h, 24 h, 48 h) to the HE diet had no effect on grams of food consumed, but caloric intake was increased. Exposure to HE diet for 14 days (medium term) established a bodyweight differential of 7.7 g, and animals exhibited a transient increase in caloric intake of 5 days duration. Terminal levels of leptin, insulin, glucose and non‐esterified fatty acids (NEFAs) were all increased in HE‐fed animals. UCP‐1 mRNA was elevated in interscapular brown adipose tissue from HE‐fed rats only at 12 h. Cocaine and amphetamine‐regulated transcript (CART) and Mc4R gene expression in the hypothalamus were increased after 12 h and 24 h on an HE diet, respectively. The rats appear to passively over‐consume calories as a result of consuming a similar weight of a more energy dense food. This evokes physiological responses, which adjust caloric intake over several days. Circulating NEFA and insulin concentrations, UCP‐1, Mc4R and CART gene expression are increased as an immediate consequence of consuming HE diet, and may be involved in countering hypercaloric intake. Circulating leptin is increased in the HE‐fed animals after 48 h, reflecting their increasing adiposity.

Photoperiod regulates genes encoding melanocortin 3 and serotonin receptors and secretogranins in the dorsomedial posterior arcuate of the Siberian hamster.

The mechanism(s) involved in the regulation of the seasonal‐appropriate body weight of the Siberian hamster are currently unknown. We have identified photoperiodically regulated genes including VGF in a sub‐region of the arcuate nucleus termed the dorsomedial posterior arcuate (dmpARC). Gene expression changes in this nucleus so far account for a significant number of those reported as photoperiodically regulated and are therefore likely to contribute to seasonal physiological responses of the hamsters. The present study aimed to identify additional genes expressed in the dmpARC regulated by photoperiod that could be involved in regulating the activity of this nucleus with respect to seasonal physiology of the Siberian hamster. Using laser capture microdissection coupled with a microarray analysis and a candidate gene approach, we have identified several photoperiodically regulated genes in the dmpARC that are known to have roles in secretory and intracellular signalling pathways. These include secretogranin (sg) III and SgVI (secretory pathway), melanocortin 3 receptor (MC3‐R) and serotonin (5‐HT) receptors 2A and 7 (signalling pathway), all of which increase in expression under a short photoperiod. The spatial relationship between receptor signalling and potential secretory pathways was investigated by dual in situ hybridisation, which revealed that 5‐HT2A and 5‐HT7 receptors are expressed in neurones expressing VGF mRNA and that a sub‐population (approximately 40%) of these neurones express MC3‐R. These gene expression changes in dmpARC neurones may reflect the functional requirement of these neurones for seasonal physiological responses of the hamster.