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Background: The thyroid hormone triiodothyronine (T3) is known to affect energy balance. Recent evidence points to an action of T3 in the hypothalamus, a key area of the brain involved in energy homeostasis, but the components and mechanisms are far from understood. The aim of this study was to identify components in the hypothalamus that may be involved in the action of T3 on energy balance regulatory mechanisms., Methods: Sprague Dawley rats were made hypothyroid by giving 0.025% methimazole (MMI) in their drinking water for 22 days. On day 21, half the MMI-treated rats received a saline injection, whereas the others were injected with T3. Food intake and body weight measurements were taken daily. Body composition was determined by magnetic resonance imaging, gene expression was analyzed by in situ hybridization, and T3-induced gene expression was determined by microarray analysis of MMI-treated compared to MMI-T3-injected hypothalamic RNA., Results: Post mortem serum thyroid hormone levels showed that MMI treatment decreased circulating thyroid hormones and increased thyrotropin (TSH). MMI treatment decreased food intake and body weight. Body composition analysis revealed reduced lean and fat mass in thyroidectomized rats from day 14 of the experiment. MMI treatment caused a decrease in circulating triglyceride concentrations, an increase in nonesterified fatty acids, and decreased insulin levels. A glucose tolerance test showed impaired glucose clearance in the thyroidectomized animals. In the brain, in situ hybridization revealed marked changes in gene expression, including genes such as Mct8, a thyroid hormone transporter, and Agrp, a key component in energy balance regulation. Microarray analysis revealed 110 genes to be up- or downregulated with T3 treatment (± 1.3-fold change, p<0.05). Three genes chosen from the differentially expressed genes were verified by in situ hybridization to be activated by T3 in cells located at or close to the hypothalamic ventricular ependymal layer and differentially expressed in animal models of long- and short-term body weight regulation., Conclusion: This study identified genes regulated by T3 in the hypothalamus, a key area of the brain involved in homeostasis and neuroendocrine functions. These include genes hitherto not known to be regulated by thyroid status.

Male C57BL/6 mice fed ad libitum on control diet but allowed access to a palatable high fat diet (HFD) for 2 h a day during the mid-dark phase rapidly adapt their feeding behaviour and can consume nearly 80% of their daily caloric intake during this 2 h-scheduled feed. We assessed food intake microstructure and meal pattern, and locomotor activity and rearing as markers of food anticipatory activity (FAA). Schedule fed mice reduced their caloric intake from control diet during the first hours of the dark phase but not during the 3-h period immediately preceding the scheduled feed. Large meal/binge-like eating behaviour during the 2-h scheduled feed was characterised by increases in both meal number and meal size. Rearing was increased during the 2-h period running up to scheduled feeding while locomotor activity started to increase 1 h before, indicating that schedule-fed mice display FAA. Meal number and physical activity changes were sustained when HFD was withheld during the anticipated scheduled feeding period, and mice immediately binged when HFD was represented after a week of this "withdrawal" period. These findings provide important context to our previous studies suggesting that energy balance systems in the hypothalamus are not responsible for driving these large, binge-type meals. Evidence of FAA in HFD dark phase schedule-fed mice implicates anticipatory processes in binge eating that do not involve immediately preceding hypophagia or regulatory homeostatic signalling., (Copyright © 2014. Published by Elsevier Ltd.)

In the Siberian hamster, seasonal weight loss occurs gradually over many weeks during autumn and winter. This is driven by a regulatory mechanism that is able to integrate duration of exposure to short days (SDs) with the size of body energy reserves. After food restriction in SDs, followed by ad libitum refeeding, body weight of the hamster does not return to its former level; rather, it increases to a level defined by the length of time spent in SDs. In this report, we show that components of the thyroid hormone system that are involved in seasonal weight loss change expression in response to 48 h of starvation. Eight weeks in an SD photoperiod induced weight loss in the Siberian hamster. In the hypothalamus of these hamsters, type II deiodinase expression was decreased and type III deiodinase expression was induced, but there was no change in hypothalamic neuropeptide Y or thyrotropin-releasing hormone gene expression. For the first time, we show that the thyroid hormone transporter monocarboxylate transporter 8 is expressed in tanycytes and is increased in response to an SD photoperiod. Food restriction (48 h of starvation) reversed the direction of gene expression change for type II and III deiodinase and monocarboxylate transporter 8 induced by SD photoperiods. Furthermore, fasting increased neuropeptide Y expression and decreased thyrotropin-releasing hormone expression. VGF, a gene upregulated in SDs in the dorsal region of the medial posterior area of the arcuate nucleus, was not changed by starvation. These data point to a mechanism whereby energy deprivation can interact with an SD photoperiod on hypothalamic tanycytes to regulate components of the thyroid hormone system involved in photoperiodic regulation of seasonal physiology.

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.

The proportion of the child and adolescent population that is in appropriate energy balance is declining throughout the developed world, and childhood obesity is a particular problem in the UK relative to other northern European countries. Assessment of the underlying causes of obesity, and the different routes to its development, may assist in the definition of successful intervention strategies. The network of peripheral and central (brain) regulatory systems that underlie energy balance and body weight and composition can, for the most part, only be approached experimentally through the study of appropriate laboratory animal models. This problem is particularly acute when the target is overweight and obesity in the young. Some of the mechanisms underlying the development of energy imbalance and specifically the onset of overweight and obesity in the young, and the metabolic health consequences of obesity, can be addressed by examination of experimental rodent models in which mutation of a single gene causes early-onset extreme obesity, genetic susceptibility to obesity is revealed in an obesogenic environment or early-life nutritional experience programmes susceptibility to obesity or metabolic problems in later life. These studies highlight genes that are essential to normal body-weight regulation in rodents and man, the impact of diet and diet-induced obesity on regulatory systems in the young and the potential sensitivity of developing regulatory systems to nutritional experiences in utero and during early life.

Hypothalamic energy balance genes have been examined in the context of seasonal body weight regulation in the Siberian hamster. Most of these long photoperiod (LD)/short photoperiod (SD) comparisons have been of tissues collected at a single point in the light-dark cycle. We examined the diurnal expression profile of hypothalamic genes in hamsters killed at 3-h intervals throughout the light-dark cycle after housing in LD or SD for 12 wk. Gene expression of neuropeptide Y, agouti-related peptide, proopiomelanocortin, cocaine- and amphetamine-regulated transcript, long-form leptin receptor, suppressor of cytokine signaling-3, melanocortin-3 receptor, melanocortin-4 receptor, and the clock gene Per1 as control were measured by in situ hybridization in hypothalamic nuclei. Effects of photoperiod on gene expression and leptin levels were generally consistent with previous reports. A clear diurnal variation was observed for Per1 in the suprachiasmatic nucleus in both photoperiods. Temporal effects on expression of energy balance genes were restricted to long-form leptin receptor in the arcuate nucleus and ventromedial nucleus, where similar diurnal expression profiles were observed, and melanocortin-4 receptor in the paraventricular nucleus; these effects were only observed in LD hamsters. There was no variation in serum leptin concentration. The 24-h profiles of hypothalamic energy balance gene expression broadly confirm photoperiodic differences that were observed previously, based on single time point comparisons, support the growing consensus that these genes have a limited role in seasonal body weight regulation, and further suggest limited involvement in daily rhythms of food intake.

Obesity prevalence is increasing worldwide, leading to increased morbidity, mortality and health care costs due to its role as a key risk factor in many diseases. Early life growth and nutrition has been implicated in determining susceptibility to obesity in both childhood and adulthood; however, the mechanisms underlying this link are poorly understood. A variety of animal models have been established to try and uncover the developmental programming effects of maternal early life nutrition on energy balance regulation and the mechanisms behind them.

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.

Weight loss in humans requires, except during an illness, some form of imposed restriction on food intake or increase in energy expenditure. This necessitates overcoming powerful peripheral and central signals that serve to protect against negative energy balance. The identification of the systems and pathways involved has come from mouse models with genetic and targeted mutations, e.g., ob/ob and MC4 R(-/-) as well as rat models of obesity. Study of seasonal animals has shown that they undergo annual cycles of body fattening and adipose tissue loss as important adaptations to environmental change, yet these changes appear to involve mechanisms distinct from those known already. One animal model, the Siberian hamster, exhibits marked, but reversible, weight loss in response to shortening day length. The body weight is driven by a decrease in food intake with the magnitude of the loss of body weight being directly related to the length of time of exposure to short photoperiod. The most important facet of this response is that the point of energy balance is continuously re-adjusted during the transition in body weight reflecting an apparent 'sliding set point'. Studies have focused on identifying the neural basis of this mechanism. Initial studies of known genes (e.g., NPY, POMC, and AgRP) both through the measurement of gene expression in the arcuate nucleus as well as following intracerebroventricular (i.c.v.) injection indicated that the systems involved are not those involved in restoring energy balance following energy deficits. Instead, a novel mechanism of regulation is implied. Recent studies have begun to explore the neural basis of the seasonal body weight response. A discrete and novel region of the posterior arcuate nucleus, the dorsal medial posterior arcuate nucleus (dmpARC) has been identified, where a battery of gene expression changes for signalling molecules (vgf and histamine H3 receptor) and transcription factors (RXRgamma and RAR) occur in association with seasonal changes in body weight. This work provides the basis of a potentially novel mechanism of energy balance regulation.

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.

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.

Previous studies on mice with melanocortin-4 receptor gene (MC4r) knockout have focused on obese adults. Because humans with functional MC4r mutations show early-onset obesity, we determined the onset of excessive fat deposition in 10- to 56-day-old mice, taking into account sex and litter influences. Total body fat content of MC4r-/- on day 35 and MC4r+/- on day 56 significantly exceeds that of MC4r+/+. Plasma leptin levels increase in proportion to fat mass. According to cumulative food intake and energy expenditure measurements from day 21 to 35, onset of excessive fat deposition in MC4r-/- is fueled by hyperphagia and counteracted partially by hypermetabolism. In 35- to 56-day-old mice, arcuate nucleus neuropeptide Y (NPY) mRNA decreases and pro-opiomelanocortin (POMC) mRNA increases with fat content and plasma leptin levels independently of genotype. Taking into account fat content by ANCOVA reveals, however, increases in both NPY mRNA and POMC mRNA due to melanocortin-4 receptor (MC4R) deficiency. We conclude that hyperphagia, not hypometabolism, is the primary disturbance initiating excessive fat deposition in MC4R-deficient mice at weaning and that the overall changes in NPY and POMC expression tend to antagonize the onset of excessive fat deposition.

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 basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.

This paper outlines the concepts and objectives behind the Fifth Framework Programme 'Diet and Obesity', scheduled to run from February 2001 to January 2004 with financial support from the European Commission. The objective is to identify the causes of weight gain on an energy-dense, high fat diet, and apply findings in the diagnosis, prevention and management of obesity. Molecular, physiological, behavioural and clinical approaches will be applied to the study of the mechanisms underlying diet preferences and susceptibility to weight gain, in both humans and laboratory rodents. This strategy is based on the observation that not all individuals habitually eating a high fat diet are obese; some have a similar body mass index (BMI) to low fat consumers despite the consumption of substantially more fat (and energy). The project will investigate why some individuals preferentially select a high fat diet, and how genetic background interacts with diet to confer susceptibility or resistance to obesity. Improved diagnosis of individuals at risk may allow treatment and preventive measures, including advice during pregnancy, to be more targeted. The effect of early life nutrition on dietary preferences, susceptibility to obesity, and the programming of brain signalling systems will be investigated in parallel clinical and rodent studies.

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.

A new dimension to the regulation of energy balance has come from the identification of the ob (obese) gene and its protein product, leptin. Leptin is produced primarily in white adipose tissue, but synthesis also occurs in brown fat and the placenta. Several physiological functions have been described for leptin the inhibition of food intake, the stimulation/maintenance of energy expenditure, as a signal of energy reserves to the reproductive system, and as a factor in haematopoiesis. The production of leptin by white fat is influenced by a number of factors, including insulin and glucocorticoids (which are stimulatory), and fasting, cold exposure and beta-adrenoceptor agonists (which are inhibitory). A key role in the regulation of leptin production is envisaged for the sympathetic nervous system, operating through beta 3-adrenoceptors. The leptin receptor gene is expressed in a wide range of tissues, and several splice variants are evident. A long form variant (Ob-Rb) with an intracellular signalling domain is found particularly in the hypothalamus. Leptin exerts its central effects through neuropeptide Y, and through the glucagon-like peptide-1 and melanocortin systems, but it may also interact with other neuroendocrine pathways. The role and function of the leptin system in agricultural animals has not been established, but it offers a potential new target for the manipulation of body fat.