The effect of physical exercise on orexigenic and anorexigenic peptides and its role on long-term feeding control.

Over the past decades, life-styles changing have led to exacerbated food and caloric intake and a reduction in energy expenditure. Obesity, main outcome of these changes, increases the risk for developing type 2 diabetes, cardiovascular disease and metabolic syndrome, the leading cause of death in adult and middle age population. Body weight and energy homeostasis are maintained via complex interactions between orexigenic and anorexigenic neuropeptides that take place predominantly in the hypothalamus. Overeating may disrupt the mechanisms of feeding control, by decreasing the expression of proopiomelanocortin (POMC) and α-melanocyte stimulating hormone (α-MSH) and increasing orexigenic neuropeptide Y (NPY) and agouti-related peptide (AgRP), which leads to a disturbance in appetite control and energy balance. Studies have shown that regular physical exercise might decrease body-weight, food intake and improve the metabolic profile, however until the currently there is no consensus about its effects on the expression of orexigenic/anorexigenic neuropeptides expression. Therefore, we propose that the type and length of physical exercise affect POMC/αMSH and NPY/AgRP systems differently and plays an important role in feeding behavior. Moreover, based on the present reports, we hypothesize that increased POMC/αMSH overcome NPY/AgRP expression decreasing food intake in long term physical exercise and that results in amelioration of several conditions related to overweight and obesity.

Role of the Paraventricular Nucleus of the Hypothalamus in the Sympathoexcitatory Effects of Leptin.

Leptin binds to receptors in multiple hypothalamic nuclei to increase sympathetic nerve activity; however, the neurocircuitry is unclear. Here, using anesthetized male Sprague-Dawley rats, we investigated the role of the paraventricular nucleus of the hypothalamus. Intracerebroventricular injection of leptin slowly increased lumbar sympathetic nerve activity (LSNA), heart rate, mean arterial pressure, and baroreflex control of LSNA and heart rate. Inhibition of the paraventricular nucleus with muscimol completely reversed leptin's effects. Blockade of paraventricular melanocortin 3/4 receptors with SHU9119 or ionotropic glutamate receptors with kynurenate, alone or together, each partially reversed the effects of leptin, implicating increased activation of glutamate and melanocortin 3/4 receptors. Conversely, although blockade of neuropeptide Y Y1 receptors in the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate, these responses were prevented by intracerebroventricular or arcuate nucleus injections of leptin, suggesting that, at least in part, leptin also increases sympathetic nerve activity by suppression of tonic neuropeptide Y inhibitory inputs from the arcuate nucleus. Injection of the melanocortin 3/4 receptor agonist melanotan-II into the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate only after blockade of neuropeptide Y Y1 receptors. Therefore, we conclude that leptin increases LSNA in part via increased glutamatergic and α-melanocyte-stimulating hormone drive of paraventricular sympathoexcitatory neurons, the latter of which requires simultaneous withdrawal of tonic neuropeptide Y inhibition.

A comparative study of the central effects of melanocortin peptides on food intake in broiler and layer chicks.

Broiler chicks eat more food than layer chicks. However, the causes of the difference in food intake in the neonatal period between these strains are not clear. In this study, we examined the involvement of proopiomelanocortin (POMC)-derived melanocortin peptides α-, ß- and γ-melanocyte-stimulating hormones (MSHs) in the difference in food intake between broiler and layer chicks. First, we compared the hypothalamic mRNA levels of POMC between these strains and found that there was no significant difference in these levels between broiler and layer chicks. Next, we examined the effects of central administration of MSHs on food intake in these strains. Central administration of α-MSH significantly suppressed food intake in both strains. Central administration of ß-MSH significantly suppressed food intake in layer chicks, but not in broiler chicks, while central administration of γ-MSH did not influence food intake in either strain. It is therefore likely that the absence of the anorexigenic effect of ß-MSH might be related to the increased food intake in broiler chicks.

Sexual behaviour and neuronal activation in the vomeronasal pathway and hypothalamus of food-deprived male rats.

As feeding and mating are mutually-exclusive goal-orientated behaviours, we investigated whether brief food deprivation would impair the display of sexual behaviour of male rats. Analysis of performance in a sexual incentive motivation test revealed that, similar to fed males, food-deprived males preferred spending time in the vicinity of receptive females rather than nonreceptive females. Despite this, food-deprived males were more likely to be slow to mate than normally-fed males, and a low dose of the satiety peptide α-melanocyte-stimulating-hormone attenuated the effect of hunger. Using Fos immunocytochemistry, we compared neuronal activity in the vomeronasal projection pathway in response to oestrous cues from receptive females between food-deprived and fed males. As in fed males, more Fos expression was seen in the rostral part of the bed nucleus of the stria terminalis and in the medial preoptic area in food-deprived males, confirming that food-deprived males can recognise and respond to female oestrous cues. However, although there was also an increase in Fos expression in the bed nucleus of the accessory tract and in the posteromedial amygdala in fed males, no increases were seen in these areas in food-deprived rats. We also found selective attenuation in the activation of lateral posterior paraventricular nucleus (lpPVN) oxytocin neurones in food-deprived males. Taken together, the data show that, although food-deprived males can still become sexually motivated, copulation is delayed, and this is accompanied by variations in neuronal activity in the vomeronasal projection pathway. We propose that, in hungry rats, the lpPVN oxytocin neurones (which project to the spinal cord and are involved in maintaining penile erection) facilitate the transition from motivation to intromission, and their lack of activation impairs intromission, and thus delays mating.

Melanocortin-induced PKA activation inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells.

α-Melanocyte-stimulating hormone (α-MSH)-induced activation of the melanocortin-4 receptor in hypothalamic neurons increases energy expenditure and inhibits food intake. Active hypothalamic AMP-activated protein kinase (AMPK) has recently been reported to enhance food intake, and in vivo experiments suggested that intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of AMPK activity. However, it is not clear whether α-MSH affects AMPK via direct intracellular signaling cascades or if the release of paracrine factors is involved. Here, we used a murine, hypothalamic cell line (GT1-7 cells) and monitored AMPK phosphorylation at Thr(172), which has been suggested to increase AMPK activity. We found that α-MSH dephosphorylated AMPK at Thr(172) and consequently decreased phosphorylation of the established AMPK substrate acetyl-coenzyme A-carboxylase at Ser(79). Inhibitory effects of α-MSH on AMPK were blocked by specific inhibitors of protein kinase A (PKA) or ERK-1/2, pointing to an important role of both kinases in this process. Because α-MSH-induced activation of ERK-1/2 was blunted by PKA inhibitors, we propose that ERK-1/2 serves as a link between PKA and AMPK in GT1-7 cells. Furthermore, down-regulation of liver kinase B-1, but not inhibition of calcium-calmodulin-dependent kinase kinase-ß or TGFß-activated kinase-1 decreased basal phosphorylation of AMPK and its dephosphorylation induced by α-MSH. Thus, we propose that α-MSH inhibits AMPK activity via a linear pathway, including PKA, ERK-1/2, and liver kinase B-1 in GT1-7 cells. Given the importance of the melanocortin system in the formation of adipositas, detailed knowledge about this pathway might help to develop drugs targeting obesity.

α-melanocyte-stimulating hormone modulates lipopolysaccharide plus interferon-γ-induced tumor necrosis factor-α expression but not tumor necrosis factor-α receptor expression in cultured hypothalamic neurons.

In a previous work we showed that the melanocortin alpha-melanocyte-stimulating hormone (α-MSH) exerts anti-inflammatory action through melanocortin 4 receptor (MC4R) in vivo in rat hypothalamus. In this work, we examined the effect of α-MSH on the expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) and their receptors in primary cultured rat hypothalamic neurons. We also investigated α-MSH's possible mechanism/s of action. α-MSH (5 µM) decreased TNF-α expression induced by 24h administration of a combination of bacterial lipopolysaccharide (LPS, 1 µg/ml) plus interferon-γ (IFN-γ, 50 ng/ml). Expression of TNF-α and IL-1ß receptors TNFR1, TNFR2 and IL-1RI, was up-regulated by LPS+IFN-γ whereas α-MSH did not modify basal or LPS+IFN-γ-induced-TNFRs or IL-1RI expression. Both α-MSH and LPS+IFN-γ treatments increased CREB activation. α-MSH did not modify NF-κB activation induced by LPS+IFN-γ in hypothalamic neurons. In conclusion, our data show that α-MSH reduces TNF-α expression in hypothalamic neurons by a mechanism which could be mediated by CREB. The regulation of inflammatory processes in the hypothalamus by α-MSH might help to prevent neurodegeneration resulting from inflammation.

Evaluation of 3,4-dihydroquinazoline-2(1H)-thiones as inhibitors of alpha-MSH-induced melanin production in melanoma B16 cells.

Novel 3,4-dihydroquinazoline-2(1H)-thiones (QNTs) 1 were found to be potent inhibitors of alpha-MSH-induced melanin production. The effect of QNTs to inhibit melanin formation in B16 melanoma cells was screened in the presence of alpha-MSH. In defining the mechanism of activity, the effects on tyrosinase activity, on tyrosinase synthesis and on the depigmentation of melanin were evaluated. QNTs did not affect the catalytic activity of tyrosinase, but rather acted as an inhibitor of tyrosinase synthesis.

Endogenous ACTH, not only alpha-melanocyte-stimulating hormone, reduces food intake mediated by hypothalamic mechanisms.

ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH) are both consecutively processed from proopiomelanocortin (POMC), which is synthesized in hypothalamic arcuate neurons innervating the paraventricular nuclei (PVN). POMC secretion/synthesis is regulated by energy availability. ACTH and alpha-MSH bind with equal affinity to melanocortin-4 receptors and elicit similar effects on signal transduction in-vitro. Endogenous alpha-MSH thus far is believed to be the major physiological agonist and to act in an anorexigenic manner. Until now, it was fully unknown whether endogenous ACTH is also involved in the regulation of appetite and food intake. In this study in rats, we now show that icv ACTH as well as alpha-MSH possess anorexigenic effects in the PVN or areas in close proximity in vivo and that the effect of ACTH is direct and not mediated via alpha-MSH. We investigated the roles of endogenous ACTH and alpha-MSH by PVN application of the respective antibodies under different physiological conditions. In satiated rats with high levels of ACTH and alpha-MSH in the PVN, antibody administration increased food intake and body weight gain; hungry animals were unaffected. Finally, repeated injections of ACTH antibodies into PVN resulted in persistently increased food intake during the light period. These data now provide robust evidence that endogenous ACTH without further processing acts in the PVN or areas in close proximity to reduce food intake under conditions of feeding-induced satiety.

Reduced levels of neurotransmitter-degrading enzyme PRCP promote a lean phenotype. [corrected].

The level of neurotransmitters present in the synaptic cleft is a function of the delicate balance among neurotransmitter synthesis, recycling, and degradation. While much is known about the processes controlling neurotransmitter synthesis and release, the enzymes that degrade peptide neurotransmitters are poorly understood. A new study in this issue of the JCI reveals the important role of neuropeptide degradation in regulating obesity (see the related article beginning on page 2291). Wallingford et al. provide evidence that, in mice, the enzyme prolylcarboxypeptidase (PRCP) degrades alpha-melanocyte-stimulating hormone (alpha-MSH) to an inactive form that is unable to inhibit food intake. Their studies indicate that PRCP expression promotes obesity, while inhibitors of the enzyme counteract obesity.

Prolylcarboxypeptidase regulates food intake by inactivating alpha-MSH in rodents.

The anorexigenic neuromodulator alpha-melanocyte-stimulating hormone (alpha-MSH; referred to here as alpha-MSH1-13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of alpha-MSH1-13 maturation and inactivation is incompletely understood. Here we have provided insight into alpha-MSH1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of alpha-MSH1-13, producing alpha-MSH1-12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where alpha-MSH1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active alpha-MSH1-13 levels.

MCH and feeding behavior-interaction with peptidic network.

Numerous works associate the MCH peptide, and the hypothalamic neurons that produce it, to the feeding behavior and energy homeostasis. It is commonly admitted that MCH is an orexigenic peptide, and MCH neurons could be under the control of arcuate NPY and POMC neurons. However, the literature data is not always concordant. In particular questions about the intrahypothalamic circuit involving other neuropeptides and about the mechanisms through which MCH could act are not yet clearly answered. For example, which receptors mediate a MCH response to NPY or alpha-MSH, does MCH act alone, is there any local anatomical organization within the tuberal LHA? A review of the current literature is then needed to help focus attention on these unresolved and often neglected issues.

Energy expenditure: a critical determinant of energy balance with key hypothalamic controls.

Energy stores are regulated through complex neural controls exerted on both food intake and energy expenditure. These controls are insured by interconnected neurons that produce different peptides or classic neurotransmitters, which have been regrouped into anabolic' and catabolic' systems. While the control of energy intake has been addressed in numerous investigations, that of energy expenditure has, as yet, only received a moderate interest, even though energy expenditure represents a key determinant of energy balance. In laboratory rodents, in particular, a strong regulatory control is exerted on brown adipose tissue (BAT), which represent an efficient thermogenic effector. BAT thermogenesis is governed by the sympathetic nervous system (SNS), whose activity is controlled by neurons comprised in various brain regions, which include the paraventricular hypothalamic nucleus (PVH), the arcuate nucleus (ARC) and the lateral hypothalamus (LH). Proopiomelanocortin neurons from the ARC project to the PVH and terminate in the vicinity of the melanocortin-4 receptors, which are concentrated in the descending division of the PVH, which comprise neurons controlling the SNS outflow to BAT. The LH contains neurons producing melanin-concentrating hormone or orexins, which also are important peptides in the control of energy expenditure. These neurons are not only polysynaptically connected to BAT, but also linked to brains regions controlling motivated behaviors and locomotor activity and, consequently, their role in the control of energy expenditure could go beyond BAT thermogenesis.

A comparative study of the central effects of specific proopiomelancortin (POMC)-derived melanocortin peptides on food intake and body weight in pomc null mice.

Functional disruption of either MC3R or MC4R results in obesity, implicating both in the control of energy homeostasis. The ligands for these receptors are derived from the prohormone proopiomelancortin (POMC), which is posttranslationally processed to produce a set of melanocortin peptides with a range of activities at the MC3R and MC4R. The relative importance of each of these peptides alpha-MSH, gamma3-MSH, gamma2-MSH, gamma-lipotropin (gamma-LPH) and, in man but not in rodents, beta-MSH] in the maintenance of energy homeostasis is, as yet, unclear. To investigate this further, equimolar amounts (2 nmol) of each peptide were centrally administered to freely feeding, corticosterone-supplemented, Pomc null (Pomc-/-) mice. After a single dose at the onset of the dark cycle, alpha-MSH had the most potent anorexigenic effect, reducing food intake to 35% of sham-treated animals. beta-MSH, gamma-LPH, and gamma3- and gamma2-MSH all reduced food intake but to a lesser degree. The effects of peptide administration over 3 d were also assessed. Only alpha-MSH significantly reduced body weight, affecting both fat and lean mass. Other peptides had no significant effect on body weight. Pair-feeding of sham-treated mice to those treated with alpha-MSH resulted in identical changes in total weight, fat and lean mass indicating that the effects of alpha-MSH were primarily due to reduced food intake rather than increased energy expenditure. Although other melanocortins can reduce food intake in the short-term, only alpha-MSH can reduce the excess fat and lean mass found in Pomc-/- mice, mediated largely through an effect on food intake.

alpha-Melanocyte-stimulating hormone and oxytocin: a peptide signalling cascade in the hypothalamus.

alpha-Melanocyte-stimulating hormone (alpha-MSH) and oxytocin share remarkable similarities of effects on behaviour in rats; in particular, they both inhibit feeding behaviour and stimulate sexual behaviour. Recently, we showed that alpha-MSH interacts with the magnocellular oxytocin system in the supraoptic nucleus; alpha-MSH induces the release of oxytocin from the dendrites of magnocellular neurones but it inhibits the secretion of oxytocin from their nerve terminals in the posterior pituitary. This effect of alpha-MSH on supraoptic nucleus oxytocin neurones is remarkable for two reasons. First, it illustrates the capacity of magnocellular neurones to differentially regulate peptide release from dendrites and axons and, second, it emphasises the putative role of magnocellular neurones as a major source of central oxytocin release, and as a likely substrate of some oxytocin-mediated behaviours. The ability of peptides to differentially control secretion from different compartments of their targets indicates one way by which peptide signals might have a particularly significant effect on neuronal circuitry. This suggests a possible explanation for the striking way in which some peptides can influence specific, complex behaviours.

Hypothalamic regulatory pathways and potential obesity treatment targets.

With an ever-growing population of obese people as well as comorbidities associated with obesity, finding effective weight loss strategies is more imperative than ever. One of the challenges in curbing the obesity crisis is designing successful strategies for long-term weight loss and weight-loss maintenance. Currently, weight-loss strategies include promotion of therapeutic lifestyle changes (diet and exercise), pharmacological therapy, and bariatric surgery. This review focuses on several pharmacological targets that activate central nervous system pathways that normally limit food intake and body weight. Though it is likely that no single therapy will prove effective for everyone, this review considers several recent pre-clinical targets, and several compounds that have been in human clinical trials.

Evidence from glut2-null mice that glucose is a critical physiological regulator of feeding.

A role for glucose in the control of feeding has been proposed, but its precise physiological importance is unknown. Here, we evaluated feeding behavior in glut2-null mice, which express a transgenic glucose transporter in their beta-cells to rescue insulin secretion (ripglut1;glut2-/- mice). We showed that in the absence of GLUT2, daily food intake was increased and feeding initiation and termination following a fasting period were abnormal. This was accompanied by suppressed regulation of hypothalamic orexigenic and anorexigenic neuropeptides expression during the fast-to-refed transition. In these conditions, however, there was normal regulation of the circulating levels of insulin, leptin, or glucose but a loss of regulation of plasma ghrelin concentrations. To evaluate whether the abnormal feeding behavior was due to suppressed glucose sensing, we evaluated feeding in response to intraperitoneal or intracerebroventricular glucose or 2-deoxy-D-glucose injections. We showed that in GLUT2-null mice, feeding was no longer inhibited by glucose or activated by 2-deoxy-D-glucose injections and the regulation of hypothalamic neuropeptide expression by intracerebroventricular glucose administration was lost. Together, these data demonstrate that absence of GLUT2 suppressed the function of central glucose sensors, which control feeding probably by regulating the hypothalamic melanocortin pathway. Furthermore, inactivation of these glucose sensors causes overeating.

Anorexigenic hormones leptin, insulin, and alpha-melanocyte-stimulating hormone directly induce neurotensin (NT) gene expression in novel NT-expressing cell models.

Neurotensin (NT) is implicated in the regulation of energy homeostasis, in addition to its many described physiological functions. NT is postulated to mediate, in part, the effects of leptin in the hypothalamus. We generated clonal, immortalized hypothalamic cell lines, N-39 and N-36/1, which are the first representative NT-expressing cell models available for the investigation of NT gene regulation and control mechanisms. The cell lines express the Ob-Rb leptin receptor neuropeptide Y (NPY)-Y1, Y2, Y4, Y5 receptors, melanocortin 4 receptor, insulin receptor, and the NT receptor. NT mRNA levels are induced by approximately 1.5-fold to twofold with leptin, insulin, and alpha-melanocyte stimulating hormone treatments but not by NPY. Leptin-mediated induction of NT gene expression was biphasic at 10(-11) and 10(-7) M. The leptin responsive region was localized to within -381 to -250 bp of the 5' regulatory region of the NT gene. Furthermore, we demonstrated direct leptin-mediated signal transducers and activators of transcription (STAT) binding to this region at 10(-11) m, but not 10(-7) m leptin, in chromatin precipitation assays. Leptin-induced NT regulation was attenuated by dominant-negative STAT3 protein expression. These data support the hypothesis that NT may have a direct role in the neuroendocrine control of feeding and energy homeostasis.

Regulation of oxytocin secretion.

A baby sucks at a mother's breast for comfort and, of course, for milk. Milk is made in specialized cells of the mammary gland, and for a baby to feed, the milk must be released into a collecting chamber from where it can be extracted by sucking. Milk "let-down" is a reflex response to the suckling and kneading of the nipple--and sometimes in response to the sight, smell, and sound of the baby--and is ultimately affected by the secretion of oxytocin. Oxytocin has many physiological roles, but its only irreplaceable role is to mediate milk let-down: oxytocin-deficient mice cannot feed their young; the pups suckle but no milk is let down, and they will die unless cross-fostered. Most other physiological roles of oxytocin, including its role in parturition, are redundant in the sense that the roles can be assumed by other mechanisms in the absence of oxytocin throughout development and adult life. Nevertheless, physiological function in these roles can be altered or impaired by acute interventions that alter oxytocin secretion or change the actions of oxytocin. Here we focus on the diverse stimuli that regulate oxytocin secretion and on the apparent diversity of the roles for oxytocin.

Sensing the fat: fatty acid metabolism in the hypothalamus and the melanocortin system.

Recent evidence has demonstrated that circulating long chain fatty acids act as nutrient abundance signals in the hypothalamus. Moreover, pharmacological inhibition of fatty acid synthase (FAS) results in profound decrease in food intake and body weight in rodents. These anorectic actions are mediated by the modulation of hypothalamic neuropeptide systems, such as melanocortins. In this review, we summarize what is known about lipid sensing and fatty acid metabolism in the hypothalamus. Understanding these molecular mechanisms could provide new pharmacological targets for the treatment of obesity and appetite disorders, as well as novel concepts in the nutritional design.