Effects of hypothalamic neurodegeneration on energy balance

Normal aging in humans and rodents is accompanied by a progressive increase in adiposity. To investigate the role of hypothalamic neuronal circuits in this process, we used a Cre-lox strategy to create mice with specific and progressive degeneration of hypothalamic neurons that express agouti-related protein (Agrp) or proopiomelanocortin (Pomc), neuropeptides that promote positive or negative energy balance, respectively, through their opposing effects on melanocortin receptor signaling. In previous studies, Pomc mutant mice became obese, but Agrp mutant mice were surprisingly normal, suggesting potential compensation by neuronal circuits or genetic redundancy. Here we find that Pomc-ablation mice develop obesity similar to that described for Pomc knockout mice, but also exhibit defects in compensatory hyperphagia similar to what occurs during normal aging. Agrp-ablation female mice exhibit reduced adiposity with normal compensatory hyperphagia, while animals ablated for both Pomc and Agrp neurons exhibit an additive interaction phenotype. These findings provide new insight into the roles of hypothalamic neurons in energy balance regulation, and provide a model for understanding defects in human energy balance associated with neurodegeneration and aging.     

AMPK: a nutrient and energy sensor that maintains energy homeostasis

AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor. Once activated by falling energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism while conserving ATP by switching off biosynthetic pathways. AMPK also regulates metabolic energy balance at the whole-body level. For example, it mediates the effects of agents acting on the hypothalamus that promote feeding and entrains circadian rhythms of metabolism and feeding behaviour. Finally, recent studies reveal that AMPK conserves ATP levels through the regulation of processes other than metabolism, such as the cell cycle and neuronal membrane excitability.     

Immortalized neurons for the study of hypothalamic function

The hypothalamus is a vital part of the central nervous system: it harbors control systems implicated in regulation of a wide range of homeostatic processes, including energy balance and reproduction. Structurally, the hypothalamus is a complex neuroendocrine tissue composed of a multitude of unique neuronal cell types that express a number of neuromodulators, including hormones, classical neurotransmitters, and specific neuropeptides that play a critical role in mediating hypothalamic function. However, neuropeptide and receptor gene expression, second messenger activation, and electrophysiological and secretory properties of these hypothalamic neurons are not yet fully defined, primarily because the heterogeneity and complex neuronal architecture of the neuroendocrine hypothalamus make such studies challenging to perform in vivo. To circumvent this problem, our research group recently generated embryonic- and adult-derived hypothalamic neuronal cell models by utilizing the novel molecular techniques of ciliary neurotrophic factor-induced neurogenesis and SV40 T antigen transfer to primary hypothalamic neuronal cell cultures. Significant research with these cell lines has demonstrated their value as a potential tool for use in molecular genetic analysis of hypothalamic neuronal function. Insights gained from hypothalamic immortalized cells used in conjunction with in vivo models will enhance our understanding of hypothalamic functions such as neurogenesis, neuronal plasticity, glucose sensing, energy homeostasis, circadian rhythms, and reproduction. This review discusses the generation and use of hypothalamic cell models to study mechanisms underlying the function of individual hypothalamic neurons and to gain a more complete understanding of the overall physiology of the hypothalamus.     

Determinants affecting physical activity levels in animal models

Weight control is dependent on energy balance. Reduced energy expenditure (EE) associated with decreased physical activity is suggested to be a major underlying cause in the increasing prevalence of weight gain and obesity. Therefore, a better understanding of the biological determinants involved in the regulation of physical activity is essential. To facilitate interpretation in humans, it is helpful to consider the evidence from animal studies. This review focuses on animal studies examining the biological determinants influencing activity and potential implications to human. It appears that physical activity is influenced by a number of parameters. However, regardless of the parameter involved, body weight appears to play an underlying role in the regulation of activity. Furthermore, the regulation of activity associated with body weight appears to occur only after the animal achieves a critical weight. This suggests that activity levels are a consequence rather than a contributor to weight control. However, the existence of an inverse weight-activity relationship remains inconclusive. Confounding the results are the multifactorial nature of physical activity and the lack of appropriate measuring devices. Furthermore, many determinants of body weight are closely interlocked, making it difficult to determine whether a single, combination, or interaction of factors is important for the regulation of activity. For example, diet-induced obesity, aging, lesions to the ventral medial hypothalamus, and genetics all produce hypoactivity. Providing a better understanding of the biological determinants involved in the regulation of activity has important implications for the development of strategies for the prevention of weight gain leading to obesity and subsequent morbidity and mortality in the human population.     

Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns

It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.     

C75, a fatty acid synthase inhibitor, reduces food intake via hypothalamic AMP-activated protein kinase

Energy homeostasis and feeding are regulated by the central nervous system. C75, a fatty acid synthase (FAS) inhibitor, causes weight loss and anorexia, implying a novel central nervous system pathway(s) for sensing energy balance. AMP-activated protein kinase (AMPK), a sensor of peripheral energy balance, is phosphorylated and activated when energy sources are low. Here, we identify a role for hypothalamic AMPK in the regulation of feeding behavior and in mediating the anorexic effects of C75. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, increased food intake, whereas compound C, an inhibitor of AMPK, decreased food intake. C75 rapidly reduced the level of the phosphorylated AMPK alpha subunit (pAMPKalpha) in the hypothalamus, even in fasted mice that had elevated hypothalamic pAMPKalpha levels. Furthermore, AICAR reversed both the C75-induced anorexia and the decrease in hypothalamic pAMPKalpha levels. C75 elevated hypothalamic neuronal ATP levels, which may contribute to the mechanism by which C75 decreased AMPK activity. C75 reduced the levels of pAMPKalpha and phosphorylated cAMP response element-binding protein (pCREB) in the arcuate nucleus neurons of the hypothalamus, suggesting a mechanism for the reduction in NPY expression seen with C75 treatment. These data indicate that modulation of FAS activity in the hypothalamus can alter energy perception via AMPK, which functions as a physiological energy sensor in the hypothalamus.     

AMP-activated protein kinase plays a role in the control of food intake

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that acts as an intracellular energy sensor maintaining the energy balance within the cell. The finding that leptin and adiponectin activate AMPK to alter metabolic pathways in muscle and liver provides direct evidence for this role in peripheral tissues. The hypothalamus is a key regulator of food intake and energy balance, coordinating body adiposity and nutritional state in response to peripheral hormones, such as leptin, peptide YY-(3-36), and ghrelin. To date the hormonal regulation of AMPK in the hypothalamus, or its potential role in the control of food intake, have not been reported. Here we demonstrate that counter-regulatory hormones involved in appetite control regulate AMPK activity and that pharmacological activation of AMPK in the hypothalamus increases food intake. In vivo administration of leptin, which leads to a reduction in food intake, decreases hypothalamic AMPK activity. By contrast, injection of ghrelin in vivo, which increases food intake, stimulates AMPK activity in the hypothalamus. Consistent with the effect of ghrelin, injection of 5-amino-4-imidazole carboxamide riboside, a pharmacological activator of AMPK, into either the third cerebral ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake. These results suggest that AMPK is regulated in the hypothalamus by hormones which regulate food intake. Furthermore, direct pharmacological activation of AMPK in the hypothalamus is sufficient to increase food intake. These findings demonstrate that AMPK plays a role in the regulation of feeding and identify AMPK as a novel target for anti-obesity drugs.     

Electrophysiological effects of MCH on neurons in the hypothalamus

Melanin concentrating hormone (MCH) has been implicated in many brain functions and behaviors essential to the survival of animals. The hypothalamus is one of the primary targets where MCH-containing nerve fibers and MCH receptors are extensively expressed and its actions in the brain are exerted. Since the identification of MCH receptors as orphan G protein coupled receptors, the cellular effects of MCH have been revealed in many non-neuronal expression systems (including Xenopus oocytes and cell lines), however, the mechanism by which MCH modulates the activity in the neuronal circuitry of the brain is still under investigation. This review summarizes our current knowledge of electrophysiological effects of MCH on neurons in the hypothalamus, particularly in the lateral hypothalamus. Generally, MCH exerts inhibitory effects on neurons in this structure and may serve as a homeostatic regulator in the lateral hypothalamic area. Given the contrast between the limited data on cellular functions of MCH in the hypothalamus versus a fast growing body of evidence on the vital role of MCH in animal behavior, further investigations of the former are warranted.     

Hypothalamic gene transfer of BDNF promotes healthy aging in mice

The aging process and age‐related diseases all involve perturbed energy adaption and impaired ability to cope with adversity. Brain‐derived neurotrophic factor (BDNF) in the hypothalamus plays important role in regulation of energy balance. Our previous studies show that recombinant adeno‐associated virus (AAV)‐mediated hypothalamic BDNF gene transfer alleviates obesity, diabetes, and metabolic syndromes in both diet‐induced and genetic models. Here we examined the efficacy and safety of a built‐in autoregulatory system to control transgene BDNF expression mimicking the body's natural feedback systems in middle‐aged mice. Twelve‐month‐old mice were treated with either autoregulatory BDNF vector or yellow fluorescence protein (YFP) control, maintained on normal diet, and monitored for 28 weeks. BDNF gene transfer prevented the development of aging‐associated metabolic declines characterized by: preventing aging‐associated weight gain, reducing adiposity, reversing the decline of brown fat activity, increasing adiponectin while reducing leptin and insulin in circulation, improving glucose tolerance, increasing energy expenditure, alleviating hepatic steatosis, and suppressing inflammatory genes in the hypothalamus and adipose tissues. Moreover, BDNF treatment reduced anxiety‐like and depression‐like behaviors. These safety and efficacy data provide evidence that hypothalamic BDNF is a target for promoting healthy aging.     

Autophagy and phagocytosis-like cell cannibalism exert opposing effects on cellular survival during metabolic stress

Understanding mechanisms controlling neuronal cell death and survival under conditions of altered energy supply (e.g., during stroke) is fundamentally important for the development of therapeutic strategies. The function of autophagy herein is unclear, as both its beneficial and detrimental roles have been described. We previously demonstrated that loss of AMP-activated protein kinase (AMPK), an evolutionarily conserved enzyme that maintains cellular energy balance, leads to activity-dependent degeneration in neuronal tissue. Here, we show that energy depletion in Drosophila AMPK mutants results in increased autophagy that convincingly promotes, rather than rescues, neurodegeneration. The generated excessive autophagic response is accompanied by increased TOR and S6K activity in the absence of an AMPK-mediated negative regulatory feedback loop. Moreover, energy-depleted neurons use a phagocytic-like process as a means to cellular survival at the expense of surrounding cells. Consequently, phagocytosis stimulation by expression of the scavenger receptor Croquemort significantly delays neurodegeneration. This study thus reveals a potentially novel strategy for cellular survival during conditions of extreme energy depletion, resembling xeno-cannibalistic events seen in metastatic tumors. We provide new insights into the roles of autophagy and phagocytosis in the neuronal metabolic stress response and open new avenues into understanding of human disease and development of therapeutic strategies.     

Pharmacological inhibition of AMP-activated protein kinase provides neuroprotection in stroke

The restoration of energy balance during ischemia is critical to cellular survival; however, relatively little is known concerning the regulation of neuronal metabolic pathways in response to central nervous system ischemia. AMP-activated protein kinase (AMPK), a master sensor of energy balance in peripheral tissues, is phosphorylated and activated when energy balance is low. We investigated whether AMPK might also modulate neuronal energy homeostasis during ischemia. We utilized two model systems of ischemia, middle cerebral artery occlusion in vivo and oxygen-glucose deprivation in vitro, to delineate changes in AMPK activity incurred from a metabolic stress. AMPK is highly expressed in cortical and hippocampal neurons under both normal and ischemic conditions. AMPK activity, as assessed by phosphorylation status, is increased following both middle cerebral artery occlusion and oxygen-glucose deprivation. Pharmacological inhibition of AMPK by either C75, a known modulator of neuronal ATP levels, or compound C reduced stroke damage. In contrast, activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside exacerbated damage. Mice deficient in neuronal nitric-oxide synthase demonstrated a decrease in both stroke damage and AMPK activation compared with wild type, suggesting a possible interaction between NO and AMPK activation in stroke. These data demonstrate a role for AMPK in the response of neurons during metabolic stress and suggest that in ischemia the activation of AMPK is deleterious. The ability to manipulate pharmacologically neuronal energy balance during ischemia represents an innovative approach to neuroprotection.     

HYPOTHesizing about central comBAT against obesity

The hypothalamus is a brain region in charge of many vital functions. Among them, BAT thermogenesis represents an essential physiological function to maintain body temperature. In the metabolic context, it has now been established that energy expenditure attributed to BAT function can contribute to the energy balance in a substantial extent. Thus, therapeutic interest in this regard has increased in the last years and some studies have shown that BAT function in humans can make a real contribution to improve diabetes and obesity-associated diseases. Nevertheless, how the hypothalamus controls BAT activity is still not fully understood. Despite the fact that much has been known about the mechanisms that regulate BAT activity in recent years, and that the central regulation of thermogenesis offers a very promising target, many questions remain still unsolved. Among them, the possible human application of knowledge obtained from rodent studies, and drug administration strategies able to specifically target the hypothalamus. Here, we review the current knowledge of homeostatic regulation of BAT, including the molecular insights of brown adipocytes, its central control, and its implication in the development of obesity.

     

Anorexia in cancer: role of feeding-regulatory peptides

Anorexia is one of the most common symptoms in advanced cancer and is a frequent cause of discomfort for cancer patients and their families. The pathogenesis of cancer anorexia is multi-factorial and involves most of the hypothalamic neuronal signalling pathways modulating energy homeostasis. It is considered to be the result of a failure of usual appetite and satiety signals. Loss of appetite can arise from decreased taste and smell of food, as well as from dysfunctional hypothalamic signalling pathways and cytokine production. Cytokines in particular, appear to play a key role in energy balance through persistent activation of the melanocortin system and inhibition of the neuropeptide Y pathway. The imbalance between anorexigenic and orexigenic peptides leads to suppression of appetite, and increased satiety and satiation associated with marked weight loss and decline in physical performance. High levels of serotonin also appear to contribute to these effects and recent findings implicate corticotropin-releasing factor in the pathogenesis of cancer anorexia as well. Despite significant advances in our understanding of the regulation of food intake and energy expenditure, few effective therapies are available. A better appreciation of the molecular and neuronal mechanisms that control body weight homeostasis may lead to the development of new therapies for improving the survival and quality of life of these patients.     

The food-contaminant deoxynivalenol modifies eating by targeting anorexigenic neurocircuitry

Physiological regulations of energy balance and body weight imply highly adaptive mechanisms which match caloric intake to caloric expenditure. In the central nervous system, the regulation of appetite relies on complex neurocircuitry which disturbance may alter energy balance and result in anorexia or obesity. Deoxynivalenol (DON), a trichothecene, is one of the most abundant mycotoxins found on contaminated cereals and its stability during processing and cooking explains its widespread presence in human food. DON has been implicated in acute and chronic illnesses in both humans and farm animals including weight loss. Here, we provide the first demonstration that DON reduced feeding behavior and modified satiation and satiety by interfering with central neuronal networks dedicated to food intake regulation. Moreover, our results strongly suggest that during intoxication, DON reaches the brain where it modifies anorexigenic balance. In view of the widespread human exposure to DON, the present results may lead to reconsider the potential consequences of chronic DON consumption on human eating disorders.     

Mouse models for the central melanocortin system

Obesity is characterized by an excess storage of body fat and promotes the risk for complex disease traits such as diabetes mellitus and cardiovascular diseases. The obesity prevalence in Europe is rising and meanwhile ranges from 10 to 20% in men and 15–25% in women. Body fat accumulation occurs in states of positive energy balance and is favored by interactions among environmental, psychosocial and genetic factors. Energy balance is regulated by a complex neuronal network of anorexigenic and orexigenic neurons which integrates peripheral and central hormonal and neuronal signals relaying information on the metabolic status of organs and tissues in the body. A key component of this network is the central melanocortin pathway in the hypothalamus that elicits metabolic and behavioral adaptations for the maintenance of energy homeostasis. Genetic defects in this system cause obesity in mice and humans. In this review we emphasize mouse models with spontaneous natural mutations as well as targeted mutations that contributed to our understanding of the central melanocortin system function in the control of energy balance.     

Overnight Fasting Regulates Inhibitory Tone to Cholinergic Neurons of the Dorsomedial Nucleus of the Hypothalamus

The dorsomedial nucleus of the hypothalamus (DMH) contributes to the regulation of overall energy homeostasis by modulating energy intake as well as energy expenditure. Despite the importance of the DMH in the control of energy balance, DMH-specific genetic markers or neuronal subtypes are poorly defined. Here we demonstrate the presence of cholinergic neurons in the DMH using genetically modified mice that express enhanced green florescent protein (eGFP) selectively in choline acetyltransferase (Chat)-neurons. Overnight food deprivation increases the activity of DMH cholinergic neurons, as shown by induction of fos protein and a significant shift in the baseline resting membrane potential. DMH cholinergic neurons receive both glutamatergic and GABAergic synaptic input, but the activation of these neurons by an overnight fast is due entirely to decreased inhibitory tone. The decreased inhibition is associated with decreased frequency and amplitude of GABAergic synaptic currents in the cholinergic DMH neurons, while glutamatergic synaptic transmission is not altered. As neither the frequency nor amplitude of miniature GABAergic or glutamatergic postsynaptic currents is affected by overnight food deprivation, the fasting-induced decrease in inhibitory tone to cholinergic neurons is dependent on superthreshold activity of GABAergic inputs. This study reveals that cholinergic neurons in the DMH readily sense the availability of nutrients and respond to overnight fasting via decreased GABAergic inhibitory tone. As such, altered synaptic as well as neuronal activity of DMH cholinergic neurons may play a critical role in the regulation of overall energy homeostasis.     

Past, present and future strategies to study the genetics of body weight regulation.

Genetic advances have made remarkable progress towards our understanding of body weight regulation. Much of our current knowledge has come from the cloning and characterisation of the genes responsible for obesity syndromes in the mouse, and the identification of homologous mutations causing rare forms of obesity in humans. Gene targeting experiments in mice have been instrumental in confirming the importance of many genes in the aetiology of obesity, and the existence of a fundamental physiological pathway that controls energy balance is becoming clear. The genetic determinants that underlie common forms of human obesity are largely polygenic, with most genes producing small effects. Thus, elucidating the many genetic determinants of obesity is a current challenge for modern geneticists. Despite the inherent difficulties, progress has been made through linkage/association studies and a genetic map of quantitative trait loci for human obesity is beginning to emerge. Obesity research is now very much in a transition period. Not so long ago, access to high throughput screening, as well as microarray and proteomic techniques, was prohibitively expensive and available only to the few. In recent years, these technologies have become more accessible to the larger scientific community and, in this paper, we will discuss how such technological advances are likely to drive the next wave of progress in obesity research. For example, large-scale mutagenesis screens in rodents coupled with high throughput screening are likely to emerge as important technologies for identifying genes previously unexpected to be involved in body weight regulation. Furthermore, applications of microarray and proteomic techniques will further refine our understanding of currently known peptides as well as identify novel pathways and molecules which are involved in energy homeostasis.     

Roles for melanocortins and leptin in adipose tissue apoptosis and fat deposition

Leptin has a wide range of effects on physiological functions related to the regulation of body energy balance. Many of leptin's effects are mediated through neuropeptide-containing neurons and neuropeptide receptors in the hypothalamus. The melanocortin system includes both agonist (alpha-melanocyte stimulating hormone, alphaMSH) and antagonist peptides (agouti related peptide, AGRP). Increased melanocortin receptor stimulation following leptin administration plays an important role in leptin-induced hypophagia and increased sympathetic nervous system activity and is partly responsible for leptin-induced weight loss. However, melanocortins do not appear to mediate some of the more striking centrally-mediated effects of leptin on adipose tissue, including adipose tissue apoptosis, that lead to the extensive depletion of fat.