Effect of intracerebroventricular alpha-MSH on food intake, adiposity, c-Fos induction, and neuropeptide expression

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a hypothalamic neuropeptide proposed to play a key role in energy homeostasis. To investigate the behavioral, metabolic, and hypothalamic responses to chronic central alpha-MSH administration, alpha-MSH was infused continuously into the third cerebral ventricle of rats for 6 days. Chronic alpha-MSH infusion reduced cumulative food intake by 10.7% (P < 0.05 vs. saline) and body weight by 4.3% (P < 0.01 vs. saline), which in turn lowered plasma insulin levels by 29.3% (P < 0.05 vs. saline). However, alpha-MSH did not cause adipose-specific wasting nor did it alter hypothalamic neuropeptide mRNA levels. Central alpha-MSH infusion acutely activated neurons in forebrain areas such as the hypothalamic paraventricular nucleus, as measured by a 254% increase in c-Fos-like immunoreactivity (P < 0.01 vs. saline), as well as satiety pathways in the hindbrain. Our findings suggest that, although an increase of central melanocortin receptor signaling acutely reduces food intake and body weight, its anorectic potency wanes during chronic infusion and causes only a modest decrease of body weight.     

Orexins (hypocretins): novel hypothalamic peptides with divergent functions.

The hypothalamus is the most important region in the control of food intake and body weight. The ventromedial "satiety center" and lateral hypothalamic "feeding center" have been implicated in the regulation of feeding and energy homeostasis by various studies of brain lesions. The discovery of orexin peptides, whose neurons are localized in the lateral hypothalamus and adjacent areas, has given us new insight into the regulation of feeding. Dense fiber projections are found throughout the brain, especially in the raphe nucleus, locus coeruleus, paraventricular thalamic nucleus, arcuate nucleus, and central gray. Orexins mainly stimulate food intake, but by the virtue of wide immunoreactive projections throughout the brain and spinal cord, orexins interact with various neuronal pathways to potentiate divergent functions. In this review, we summarize recent progress in the physiological, neuroanatomical, and molecular studies of the novel neuropeptide orexins (hypocretins).     

Morphological evidence for neural interactions between leptin and orexin in the hypothalamus

Both leptin and orexin have been recently discovered as peptides involved in feeding regulation. The morphological evidence of neural interaction between leptin and orexin, one considered to inhibit food intake and the other to stimulate it in the central nervous system (CNS), was studied by use of double immunostaining method. The leptin receptor-like immunoreactive (LR-LI) neurons in the hypothalamic arcuate nucleus and ventromedial nucleus were innervated by orexin-like immunoreactive (OX-LI) neurons. The distribution of LR-LI neurons in the hypothalamus was very similar to that of OX-LI neurons. These results may suggest that leptin and orexin are intimately correlated with each other and that they reciprocally regulate feeding at the hypothalamic level.     

Increased hypothalamic melanin concentrating hormone gene expression during energy restriction involves a melanocortin-independent, estrogen-sensitive mechanism

Increased expression of melanin concentrating hormone (MCH), an orexigenic neuropeptide produced by neurons in the lateral hypothalamic area (LHA), is implicated in the effect of energy restriction to increase food intake. Since melanocortins inhibit Mch gene expression, this effect of energy restriction to increase Mch signaling may involve reduced hypothalamic melanocortin signaling. Consistent with this hypothesis, we detected increased hypothalamic Mch mRNA levels in agouti (Ay) mice (by 102%; P < 0.05), a model of genetic obesity resulting from impaired melanocortin signaling, compared to wild-type controls. If reduced melanocortin signaling mediates the effect of energy restriction, hypothalamic Mch gene expression in Ay mice should not be increased further by energy restriction, since melanocortin signaling is impaired in these animals regardless of nutritional state. We therefore investigated the effects of energy restriction on hypothalamic Mch gene expression in both Ay mice and in wild-type mice with diet-induced obesity (DIO). Responses in these mice were compared to those induced by administration of 17beta-estradiol (E2) at a dose previously shown to reduce food intake and Mch expression in rats. In both Ay and DIO mice, energy restriction increased hypothalamic Mch mRNA levels (P < 0.05 for each) via a mechanism that was fully blocked by E2. However, E2 did not lower levels of Mch mRNA below basal values in Ay mice, whereas it did so in DIO mice. Thus, the effect of energy restriction to increase hypothalamic Mch gene expression involves an E2-sensitive mechanism that is not altered by impaired melanocortin signaling. By comparison, impaired melanocortin signaling increases hypothalamic Mch gene expression via a mechanism that is insensitive to E2. These findings suggest that while both energy restriction and reduced melanocortin signaling stimulate hypothalamic Mch gene expression, they do so via distinct mechanisms.     

C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism

C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.     

Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment

We previously demonstrated that C75, a specific and potent inhibitor of fatty acid synthase (FAS), reduced food intake and decreased body weight in mice. In the present study, we determined that these effects were not due to conditioned taste aversion. To investigate the mechanism of C75 action, we examined FAS brain expression. FAS was expressed in a number of brain regions, including arcuate and paraventricular nuclei (PVN) within regions that comprise the arcuate-PVN pathway in mouse and human. Although C75 and fasting significantly downregulated liver FAS, FAS levels remained high in hypothalamus, indicating that FAS levels were regulated differently in brain from those in liver. Double fluorescence in situ for FAS and neuropeptide Y (NPY) showed that FAS co-localized with NPY in neurons in the arcuate nucleus. NPY immnuoreactivity after C75 treatment was decreased in axon terminals that innervate the PVN and lateral hypothalamus. Collectively, these results demonstrate that FAS is present and active in neurons and suggests that C75 may alter food intake via interactions within the arcuate-PVN pathway mediated by NPY.     

Daily Intraparaventricular Orexin‐A Treatment Induces Weight Loss in Rats

The neuropeptide orexin (hypocretin) increases energy expenditure partially through increasing spontaneous physical activity. The ability of exogenous orexin to alter body weight has never been established, however. We sought to determine whether orexin‐A microinjected into the paraventricular nucleus of the hypothalamus (PVN) induced weight loss in rats. Chronic guide cannulae were implanted into rats, aimed at the PVN. Rats were given daily microinjections of orexin (0.5 nmol) or vehicle into the PVN for 6 days; food intake and body weight were measured daily. In a separate group of rats, we injected orexin‐A and vehicle intra‐PVN and measured daily activity levels. Daily orexin treatment induced weight loss: orexin‐A‐treated rats lost significantly more weight than their vehicle‐injected counterparts without a significant difference in food intake. Rats were significantly more active after intra‐PVN orexin compared to vehicle. These results support the concept that orexinergic agents have the potential to produce negative energy balance through increasing physical activity. This presents a promising, untapped potential resource for weight loss.     

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.     

Alpha-melanocyte stimulating hormone plays an important role in the regulation of food intake by the central melanocortin system in chicks

Proopiomelanocortin (POMC, a precursor of melanocortin peptides) neurons in the hypothalamus play an important role in the central regulation of food intake in mammals. There is evidence that human melanocortin peptides alpha-, beta- and gamma2-melanocyte-stimulating hormone (α-, β- and γ2-MSH) significantly decreased food intake in chickens. However, the amino acid sequences of β- and γ2-MSH of chickens are different from those of humans whereas the amino acid sequence of α-MSH is conserved between these species. In the present study, we examined the effects of the central administration of α-, chicken β-, and chicken γ2-MSH on food intake in chicks. Central administration of α-MSH significantly suppressed food intake in chicks. In contrast, β- and γ2-MSH did not influence food intake in chicks. Central administration of HS014, a melanocortin 4 receptor antagonist, significantly reversed the anorexigenic action of α-MSH, suggesting that this action is mediated by the melanocortin 4 receptor in chicks as well as in mammals. These results suggest that α-MSH may play an important role in the regulation of food intake by the central melanocortin system in chicks.     

Ghrelin microinjection into forebrain sites induces wakefulness and feeding in rats

Ghrelin, a gut-brain peptide, is best known for its role in the stimulation of feeding and growth hormone release. In the brain, orexin, neuropeptide Y (NPY), and ghrelin are parts of a food intake regulatory circuit. Orexin and NPY are also implicated in maintaining wakefulness. Previous experiments in our laboratory revealed that intracerebroventricular injections of ghrelin induce wakefulness in rats. To further elucidate the possible role of ghrelin in the regulation of arousal, we studied the effects of microinjections of ghrelin into hypothalamic sites, which are implicated in the regulation of feeding and sleep, such as the lateral hypothalamus (LH), medial preoptic area (MPA), and paraventricular nucleus (PVN) on sleep in rats. Sleep responses, motor activity, and food intake after central administration of 0.04, 0.2, or 1 mug (12, 60, or 300 pmol) ghrelin were recorded. Microinjections of ghrelin into the LH had strong wakefulness-promoting effects lasting for 2 h. Wakefulness was also stimulated by ghrelin injection into the MPA and PVN; the effects were confined to the first hour after the injection. Ghrelin's non-rapid-eye-movement sleep-suppressive effect was accompanied by attenuation in the electroencephalographic (EEG) slow-wave activity and changes in the EEG power spectrum. Food consumption was significantly stimulated after microinjections of ghrelin into each hypothalamic site. Together, these results are consistent with the hypothesis that forebrain ghrelinergic mechanisms play a role in the regulation of vigilance, possibly through activating the components of the food intake- and arousal-promoting network formed by orexin and NPY.     

Neuropeptide Y content in the hypothalamic paraventricular nucleus responds to fasting and refeeding in broiler chickens

To examine the neural mechanism by which hypothalamic neuropeptide Y (NPY) regulates energy homeostasis and feeding behavior in commercial broilers, we measured NPY content in several hypothalamic regions of birds that were fasted and then refed. After fasting for 48 and 72 h, body weight significantly decreased, and food intake significantly increased during the subsequent refeeding. The lost body weight was not restored to ad libitum feeding levels even after 3 days of refeeding. Plasma glucose concentration and body fat content significantly decreased and plasma non-esterified fatty acid (NEFA) concentration significantly increased after 48- and 72-h fasting. Refeeding for 24 h restored plasma metabolites and body fat content to pre-fasting levels. NPY content in the paraventricular nucleus (PVN) and infundibular nucleus significantly increased during fasting, and NPY content of the PVN was restored to pre-fasting levels after 24-h refeeding. However, there was no significant change in the NPY content of the lateral hypothalamic area during fasting or refeeding. The present results of changes in the hypothalamic NPY content during fasting and refeeding support the hypothesis that NPY plays a central role in regulation of energy homeostasis, with especially important effect on feeding behavior and body weight in broiler chickens.     

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.     

Role of the non-opioid dynorphin peptide des-Tyr-dynorphin (DYN-A2−17) in food intake and physical activity,and its interaction with orexin-A.

Food intake and physical activity are regulated by multiple neuropeptides, including orexin and dynorphin (DYN). Orexin-A (OXA) is one of two orexin peptides with robust roles in regulation of food intake and spontaneous physical activity (SPA). DYN collectively refers to several peptides, some of which act through opioid receptors (opioid DYN) and some whose biological effects are not mediated by opioid receptors (non-opioid DYN). While opioid DYN is known to increase food intake, the effects of non-opioid DYN peptides on food intake and SPA are unknown. Neurons that co-express and release OXA and DYN are located within the lateral hypothalamus. Limited evidence suggests that OXA and opioid DYN peptides can interact to modulate some aspects of behaviors classically related to orexin peptide function. The paraventricular hypothalamic nucleus (PVN) is a brain area where OXA and DYN peptides might interact to modulate food intake and SPA. We demonstrate that injection of des-Tyr-dynorphin (DYN-A₂₋₁₇, a non opioid DYN peptide) into the PVN increases food intake and SPA in adult mice. Co-injection of DYN-A₂₋₁₇ and OXA in the PVN further increases food intake compared to DYN-A₂₋₁₇ or OXA alone. This is the first report describing the effects of non-opioid DYN-A₂₋₁₇ on food intake and SPA, and suggests that DYN-A₂₋₁₇ interacts with OXA in the PVN to modulate food intake. Our data suggest a novel function for non-opioid DYN-A₂₋₁₇ on food intake, supporting the concept that some behavioral effects of the orexin neurons result from combined actions of the orexin and DYN peptides.     

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-, β- 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 β-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 β-MSH might be related to the increased food intake in broiler chicks.     

Melanocortin activation of nucleus of the solitary tract avoids anorectic tachyphylaxis and induces prolonged weight loss

To examine the role of the brain stem melanocortin system in long-term energy regulation, we assessed the effects of overproduction of proopiomelanocortin (POMC) in the caudal brain stem of F344xBN rats with adult-onset obesity. Recombinant adeno-associated viral vector encoding POMC gene was delivered to the nucleus of solitary tract (NTS) in the hindbrain, and food intake, body weight, glucose and fat metabolism, brown adipose tissue thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors were assessed. POMC delivery resulted in sustained reduction in food intake and body weight over 42 days and improved insulin sensitivity. At death, in recombinant adeno-associated viral vector-POMC-treated rats vs. control rats, alpha-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic alpha-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyceride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyceride and nonesterified fatty acids were reduced by 35% and 34%, respectively; phosphorylation of acetyl-CoA carboxylase was elevated by 63% in soleus muscle; brown adipose tissue uncoupling protein 1 increased by 30%; and melanocortin 3 receptor expression declined by 60%, whereas neuropeptide Y, agouti-related protein, and MC4 receptor mRNA levels were unchanged in the NTS. In conclusion, POMC overexpression in the NTS produces a characteristic unabated hypophagia that is uniquely different from the anorexic tachyphylaxis following POMC overexpression in the hypothalamus. The sustained anorectic response may result from absence of compensatory elements in the NTS, such as increased agouti-related protein expression, suggesting melanocortin activation of the brain stem may be a viable strategy to alleviate obesity.     

Leptin downregulates heat shock protein-70 (HSP-70) gene expression in chicken liver and hypothalamus

Heat shock protein (HSP)-70 is expressed in normal and stressed cells but is highly stress-inducible. Although leptin has long been suggested to be involved in the regulation of stress response, its interaction with the HSP-70 gene is still unknown, under both unstressed and stressed conditions. The present study has aimed to investigate the effect of leptin on HSP-70 gene expression in normal chicken liver, hypothalamus, and muscle. Continuous infusion of recombinant chicken leptin (8 μg/kg per hour) at a constant rate of 3 ml/h for 6 h in 3-week-old broiler chickens significantly (P < 0.05) decreased food intake and HSP-70 mRNA levels in liver and hypothalamus, but not in muscle. In an attempt to discriminate between the effect of leptin and of leptin-reduced food intake on HSP-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses in two broiler chicken lines genetically selected for low (LL) or high (FL) abdominal fat pad size. Food deprivation for 16 h did not affect HSP-70 gene expression in any of the studied tissues indicating that the effect of leptin was independent of the inhibition of food intake. Regardless of the nutritional status, HSP-70 mRNA levels were significantly (P < 0.05) higher in the hypothalamus of FL compared with LL chickens consistent with higher mRNA levels for hypothalamic corticotropin-releasing factor. To assess, whether the effects of leptin were direct or indirect, we carried out in vitro studies. Leptin treatments did not affect HSP-70 mRNA levels in a leghorn male hepatoma cell line or quail myoblast cell line suggesting that the effect of leptin on HSP-70 gene expression is mediated through the central nervous system. Furthermore, HSP-70 gene expression was gender-dependent with significantly (P < 0.05) higher levels in male than in female chickens. This work was supported by a research grant (G.0402.05) from the FWO-Flanders (Belgium). No conflicts of interest would prejudice impartiality.     

Neuropeptide Y effect on food intake in broiler and layer chicks

Broiler chicks eat more food than layer chicks. In this study, we examined the involvement of orexigenic peptide neuropeptide Y (NPY) in the difference in food intake between broiler and layer chicks (Gallus gallus). First, we compared the hypothalamic mRNA levels of NPY and its receptors (Y1 and Y5 receptors) between these strains at 1, 2, 4, and 8 days of age. Daily food intake was significantly higher in broiler chicks than layer chicks after 2 days of age. However, the hypothalamic NPY mRNA level was significantly lower in broiler chicks than layer chicks except at 8 days of age. In addition, the mRNA levels of NPY receptors were also significantly lower in broiler chicks than layer chicks at 2 and 4 days of age (Y1 receptor) or 2 days of age (Y5 receptor). These results suggest that the differences in the expressions of hypothalamic NPY and its receptors do not cause the increase in food intake in broiler chicks. To compare the orexigenic effect of NPY between broiler and layer chicks, we next examined the effects of central administration of NPY on food intake in these strains. In both strains, central administration of NPY significantly increased food intake at 2, 4 and 8 days of age. All our findings demonstrated that the increase in food intake in broiler chicks is not accompanied with the over-expression of NPY or its receptor.     

Orexin 和P物质对顺铂诱发大鼠异食癖的影响

目的:观察食欲素(Orexin)和P物质(SP)对顺铂诱发大鼠异食癖的影响。方法:雄性Wistar大鼠被随机分为对照组和顺铂处理组,顺铂处理组给予大鼠顺铂(3或6 mg/kg,腹腔注射),对照组给予等量生理盐水。记录顺铂大鼠摄食高岭土量、摄食量的改变;Real-time PCR法观察顺铂对大鼠下丘脑Orexin和延髓中SP前体-前速激肽原A(PPT-A)m RNA表达的影响;分别和联合应用SP受体(NK1受体)拮抗剂阿瑞匹坦和Orexin-A对顺铂大鼠异食癖和摄食量的作用。结果:皮下注射3 mg/kg(低剂量组)的顺帕后大鼠高岭土摄入量和摄食量与对照组相比无明显差异(P0.05),而注射6 mg/kg(高剂量组)顺铂后,大鼠高岭土摄入量与对照组和低剂量组相比显著增加(P0.05);高剂量的顺铂作用12 h时,大鼠延髓内PPT-A的m RNA表达有轻微增加,但无统计学差异(P0.05),24 h后,延髓内PPT-A的m RNA表达量显著增加(P0.05)。在此后持续观察的5天中,顺铂可持续引起延髓中PPT-A的mRNA表达增高,在第5天时PPT-A的m RNA仍维持166.23±16.92%的高表达。高剂量顺铂抑制大鼠下丘脑中Orexin的mRNA表达,24 h时Orexin降低幅度最明显,为对照组的34.81±7.22%(P0.05)。此后检测的5天,Orexin浓度均低于对照组;将阿瑞匹坦和orexin联合应用,大鼠高岭土摄入量较单独应用阿瑞匹坦或orexin明显减少,摄食量显著增加(P0.05)。结论:P物质和orexin通路对顺铂化疗大鼠的异食癖和摄食量调控具有协同作用。