Downregulated Adipose Tissue Expression of Browning Genes With Increased Environmental Temperatures.

CONTEXT: Climate change and global warming have been hypothesized to influence the increased prevalence of obesity worldwide. However, the evidence is scarce. OBJECTIVE: We aimed to investigate how outside temperature might affect adipose tissue physiology and metabolic traits. METHODS: The expression of genes involved in thermogenesis/browning and adipogenesis were evaluated (through quantitative polymerase chain reaction) in the subcutaneous adipose tissue (SAT) from 1083 individuals recruited in 5 different regions of Spain (3 in the North and 2 in the South). Plasma biochemical variables and adiponectin (enzyme-linked immunosorbent assay) were collected through standardized protocols. Mean environmental outdoor temperatures were obtained from the National Agency of Meteorology. Univariate, multivariate, and artificial intelligence analyses (Boruta algorithm) were performed. RESULTS: The SAT expression of genes associated with browning (UCP1, PRDM16, and CIDEA) and ADIPOQ were significantly and negatively associated with minimum, average, and maximum temperatures. The latter temperatures were also negatively associated with the expression of genes involved in adipogenesis (FASN, SLC2A4, and PLIN1). Decreased SAT expression of UCP1 and ADIPOQ messenger RNA and circulating adiponectin were observed with increasing temperatures in all individuals as a whole and within participants with obesity in univariate, multivariate, and artificial intelligence analyses. The differences remained statistically significant in individuals without type 2 diabetes and in samples collected during winter. CONCLUSION: Decreased adipose tissue expression of genes involved in browning and adiponectin with increased environmental temperatures were observed. Given the North-South gradient of obesity prevalence in these same regions, the present observations could have implications for the relationship of the obesity pandemic with global warming.

Liver Brain Interactions: Focus on FGF21 a Systematic Review.

Fibroblast growth factor 21 is a pleiotropic hormone secreted mainly by the liver in response to metabolic and nutritional challenges. Physiologically, fibroblast growth factor 21 plays a key role in mediating the metabolic responses to fasting or starvation and acts as an important regulator of energy homeostasis, glucose and lipid metabolism, and insulin sensitivity, in part by its direct action on the central nervous system. Accordingly, pharmacological recombinant fibroblast growth factor 21 therapies have been shown to counteract obesity and its related metabolic disorders in both rodents and nonhuman primates. In this systematic review, we discuss how fibroblast growth factor 21 regulates metabolism and its interactions with the central nervous system. In addition, we also state our vision for possible therapeutic uses of this hepatic-brain axis.

Crosstalk between Melanin Concentrating Hormone and Endocrine Factors: Implications for Obesity.

Melanin-concentrating hormone (MCH) is a 19aa cyclic peptide exclusively expressed in the lateral hypothalamic area, which is an area of the brain involved in a large number of physiological functions and vital processes such as nutrient sensing, food intake, sleep-wake arousal, memory formation, and reproduction. However, the role of the lateral hypothalamic area in metabolic regulation stands out as the most relevant function. MCH regulates energy balance and glucose homeostasis by controlling food intake and peripheral lipid metabolism, energy expenditure, locomotor activity and brown adipose tissue thermogenesis. However, the MCH control of energy balance is a complex mechanism that involves the interaction of several neuroendocrine systems. The aim of the present work is to describe the current knowledge of the crosstalk of MCH with different endocrine factors. We also provide our view about the possible use of melanin-concentrating hormone receptor antagonists for the treatment of metabolic complications. In light of the data provided here and based on its actions and function, we believe that the MCH system emerges as an important target for the treatment of obesity and its comorbidities.

Hypothalamic Actions of SIRT1 and SIRT6 on Energy Balance.

Sirtuins are NAD+ dependent deacetylases that regulate a large number of physiological processes. These enzymes are highly conserved and act as energy sensors to coordinate different metabolic responses in a controlled manner. At present, seven mammalian sirtuins (SIRT 1-7) have been identified, with SIRT1 and SIRT6 shown to exert their metabolic actions in the hypothalamus, both with crucial roles in eliciting responses to dampen metabolic complications associated with obesity. Therefore, our aim is to compile the current understanding on the role of SIRT1 and SIRT6 in the hypothalamus, especially highlighting their actions on the control of energy balance.

Conversion from Duodenal Switch to Single Anastomosis Duodenal Switch to Deal with Postoperative Malnutrition.

BACKGROUND: Duodenal switch (DS) is considered one of the most effective bariatric techniques for long-term weight and comorbidity control. After these operations, some patients may get severe complications related to malnutrition and a few of them may need surgical revision. Lengthening the common channel (CC) is usually the solution: changing the Roux anastomosis or with a side-to-side anastomosis (kissing X). We propose that when simplified construction of the DS is used, conversion to single anastomosis DS (SADI-S/OADS) is an easy and safe choice. OBJECTIVES: To evaluate the safety and effectiveness of conversion from DS to SADI-S in cases of malnutrition. METHODS: We report three patients with severe malnutrition after a DS at 9, 74, and 84 months. One of them had also liver failure related to alcohol abuse and malnutrition. Laparoscopic reoperations included a new ileo-ileal anastomosis and takedown of the Roux-en-Y anastomosis with the aim of lengthening the CC. RESULTS: All three patients were successfully converted by laparoscopy. After a median follow-up of 54.6 months [32-76 months], all of them had moderate weight regain and returned to normal biochemical nutritional parameters. Two patients with type 2 diabetes (T2DM) before DS had complete remission before conversion; one of them had recurrence of T2DM after conversion. The patient with liver failure improved significantly after conversion. CONCLUSIONS: Conversion from DS to SADI-S/OADS is a simple operation with excellent results in resolving malnutrition in those patients. However, weight regain and recurrence of comorbidities may arise.

mTOR Pathway is Involved in Energy Homeostasis Regulation as a Part of the Gut-Brain Axis.

Mammalian, or mechanic, target of rapamycin (mTOR) signaling is a crucial factor in the regulation of the energy balance that functions as an energy sensor in the body. The present review explores how the mTOR/S6k intracellular pathway is involved in modulating the production of different signals such as ghrelin and nesfatin-1 in the gastrointestinal tract to regulate food intake and body weight. The role of gastric mTOR signaling in different physiological processes was studied in depth through different genetic models that allow the modulation of mTOR signaling in the stomach and specifically in gastric X/A type cells. It has been described that mTOR signaling in X/A-like gastric cells has a relevant role in the regulation of glucose and lipid homeostasis due to its interaction with different organs such as liver and adipose tissue. These findings highlight possible therapeutic strategies, with the gut-brain axis being one of the most promising targets in the treatment of obesity.

Regulation of Chemerin and CMKLR1 Expression by Nutritional Status, Postnatal Development, and Gender.

Chemerin (also known as tazarotene-induced gene 2 and retinoic acid receptor responder 2) has been identified as an adipokine that exerts effects on many biological processes, including adipogenesis, angiogenesis, inflammation, immune responses, and food intake. This variety of effects has led to its implication in obesity and co-morbidities including diabetes and a risk of cardiovascular disease. The biological effects are mostly mediated by a so-called G protein-coupled receptor, chemokine-like receptor 1 (CMKLR1). Given the association of chemerin with obesity and related diseases, we decided to study in detail the regulation of chemerin and CMKLR1 expression in white adipose tissue (WAT). Specifically, we focused on their expression levels in physiological and pathophysiological settings involved in energy balance: e.g., fasting, postnatal development, and gender. We used Sprague Dawley rats with different nutritional statuses, levels of hormonal deficiency, and states of development as well as ob/ob (leptin-deficient) mice. We analysed the protein expression of both the ligand and receptor (chemerin and CMKLR1) in gonadal WAT by western blotting. We found that chemerin and CMKLR1 protein levels were regulated in WAT by different conditions associated with metabolic changes such as nutritional status, sex steroids, pregnancy, and food composition. Our data indicate that regulation of the expression of this new adipokine and its receptor by nutritional status and gonadal hormones may be a part of the adaptive mechanisms related to altered fat mass and its metabolic complications.

Visceral and subcutaneous adipose tissue express and secrete functional alpha2hsglycoprotein (fetuin a) especially in obesity.

The secretion of the hepatokine alpha-2-Heremans-Schmid glycoprotein/Fetuin A, implicated in pathological processes including systemic insulin resistance, by adipose tissue has been recently described. Thus, we have recently identified its presence in white adipose tissue secretomes by mass spectrometry. However, the secretion pattern and function of adipose-derived alpha-2-Heremans-Schmid glycoprotein are poorly understood. The aim of this study is to evaluate the expression and secretion of total and active phosphorylated alpha-2-Heremans-Schmid glycoprotein by adipose tissue from visceral and subcutaneous localizations in animals at different physiological and nutritional status including anorexia and obesity. Alpha-2-Heremans-Schmid glycoprotein expression and secretion in visceral adipose tissue and subcutaneous adipose tissue explants from animals under fasting and exercise training, at pathological situations such as anorexia and obesity, and from human obese individuals were assayed by immunoblotting, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We reveal that visceral adipose tissue expresses and secretes more alpha-2-Heremans-Schmid glycoprotein than subcutaneous adipose tissue, and that this secretion is diminished after fasting and exercise training. Visceral adipose tissue from anorectic animals showed reduced alpha-2-Heremans-Schmid glycoprotein secretion; on the contrary, alpha-2-Heremans-Schmid glycoprotein is over-secreted by visceral adipose tissue in the occurrence of obesity. While secretion of active-PhophoSer321α2HSG by visceral adipose tissue is independent of body mass index, we found that the fraction of active-alpha-2-Heremans-Schmid glycoprotein secreted by subcutaneous adipose tissue increments significantly in situations of obesity. Functional studies show that the inhibition of adipose-derived alpha-2-Heremans-Schmid glycoprotein increases insulin sensitivity in differentiated adipocytes. In conclusion, visceral adipose tissue secretes more alpha-2-Heremans-Schmid glycoprotein than subcutaneous adipose tissue and this secretion is more sensitive to nutritional and physiological changes. The over-secretion of alpha-2-Heremans-Schmid glycoprotein by visceral adipose tissue, the increased secretion of the active phosphorylated form by subcutaneous adipose tissuein obese animals, and the adipose-derived alpha-2-Heremans-Schmid glycoprotein capacity to inhibit the insulin pathway suggest the participation of adipose-derived alpha-2-Heremans-Schmid glycoprotein in the deleterious effects of obesity.

Nesfatin-1 in human and murine cardiomyocytes: synthesis, secretion, and mobilization of GLUT-4.

Nesfatin-1, a satiety-inducing peptide identified in hypothalamic regions that regulate energy balance, is an integral regulator of energy homeostasis and a putative glucose-dependent insulin coadjuvant. We investigated its production by human cardiomyocytes and its effects on glucose uptake, in the main cardiac glucose transporter GLUT-4 and in intracellular signaling. Quantitative RT-PCR, Western blots, confocal immunofluorescence microscopy, and ELISA of human and murine cardiomyocytes and/or cardiac tissue showed that cardiomyocytes can synthesize and secrete nesfatin-1. Confocal microscopy of cultured cardiomyocytes after GLUT-4 labeling showed that nesfatin-1 mobilizes this glucose transporter to cell peripherals. The rate of 2-deoxy-D-[(3)H]glucose incorporation demonstrated that nesfatin-1 induces glucose uptake by HL-1 cells and cultured cardiomyocytes. Nesfatin-1 induced dose- and time-dependent increases in the phosphorylation of ERK1/2, AKT, and AS160. In murine and human cardiac tissue, nesfatin-1 levels varied with diet and coronary health. In conclusion, human and murine cardiomyocytes can synthesize and secrete nesfatin-1, which is able to induce glucose uptake and the mobilization of the glucose transporter GLUT-4 in these cells. Nesfatin-1 cardiac levels are regulated by diet and coronary health.

Gut microbiota composition in male rat models under different nutritional status and physical activity and its association with serum leptin and ghrelin levels.

BACKGROUND: Several evidences indicate that gut microbiota is involved in the control of host energy metabolism. OBJECTIVE: To evaluate the differences in the composition of gut microbiota in rat models under different nutritional status and physical activity and to identify their associations with serum leptin and ghrelin levels. METHODS: In a case control study, forty male rats were randomly assigned to one of these four experimental groups: ABA group with food restriction and free access to exercise; control ABA group with food restriction and no access to exercise; exercise group with free access to exercise and feed ad libitum and ad libitum group without access to exercise and feed ad libitum. The fecal bacteria composition was investigated by PCR-denaturing gradient gel electrophoresis and real-time qPCR. RESULTS: In restricted eaters, we have found a significant increase in the number of Proteobacteria, Bacteroides, Clostridium, Enterococcus, Prevotella and M. smithii and a significant decrease in the quantities of Actinobacteria, Firmicutes, Bacteroidetes, B. coccoides-E. rectale group, Lactobacillus and Bifidobacterium with respect to unrestricted eaters. Moreover, a significant increase in the number of Lactobacillus, Bifidobacterium and B. coccoides-E. rectale group was observed in exercise group with respect to the rest of groups. We also found a significant positive correlation between the quantity of Bifidobacterium and Lactobacillus and serum leptin levels, and a significant and negative correlation among the number of Clostridium, Bacteroides and Prevotella and serum leptin levels in all experimental groups. Furthermore, serum ghrelin levels were negatively correlated with the quantity of Bifidobacterium, Lactobacillus and B. coccoides-Eubacterium rectale group and positively correlated with the number of Bacteroides and Prevotella. CONCLUSIONS: Nutritional status and physical activity alter gut microbiota composition affecting the diversity and similarity. This study highlights the associations between gut microbiota and appetite-regulating hormones that may be important in terms of satiety and host metabolism.

FNDC5/irisin is not only a myokine but also an adipokine.

Exercise provides clear beneficial effects for the prevention of numerous diseases. However, many of the molecular events responsible for the curative and protective role of exercise remain elusive. The recent discovery of FNDC5/irisin protein that is liberated by muscle tissue in response to exercise might be an important finding with regard to this unsolved mechanism. The most striking aspect of this myokine is its alleged capacity to drive brown-fat development of white fat and thermogenesis. However, the nature and secretion form of this new protein is controversial. The present study reveals that rat skeletal muscle secretes a 25 kDa form of FNDC5, while the 12 kDa/irisin theoretical peptide was not detected. More importantly, this study is the first to reveal that white adipose tissue (WAT) also secretes FNDC5; hence, it may also behave as an adipokine. Our data using rat adipose tissue explants secretomes proves that visceral adipose tissue (VAT), and especially subcutaneous adipose tissue (SAT), express and secrete FNDC5. We also show that short-term periods of endurance exercise training induced FNDC5 secretion by SAT and VAT. Moreover, we observed that WAT significantly reduced FNDC5 secretion in fasting animals. Interestingly, WAT of obese animals over-secreted this hormone, which might suggest a type of resistance. Because 72% of circulating FNDC5/irisin was previously attributed to muscle secretion, our findings suggest a muscle-adipose tissue crosstalk through a regulatory feedback mechanism.

Muscle tissue as an endocrine organ: comparative secretome profiling of slow-oxidative and fast-glycolytic rat muscle explants and its variation with exercise.

The notion that skeletal muscle is a secretory organ capable to release proteins that can act locally in an autocrine/paracrine manner or even in an endocrine manner to communicate with distant tissues has now been recognized. Under this context, a new paradigm has arisen implicating the muscle in metabolism regulation. Considering the evidences that give exercise a protective role against illnesses associated to physical inactivity, it becomes of especial relevance to characterize muscle secreted proteins. In the present study we show for the first time the secretome characterization and the comparative 2-DE secretome analysis among fast-glycolytic (gastrocnemius) and slow-oxidative (soleus) rat muscle explants and its variation after exercise intervention. We have identified 19 differently secreted proteins when comparing soleus and gastrocnemius secretomes, and 10 in gastrocnemius and 17 in soleus distinctive secreted proteins after 1 week of endurance exercise training. Among identified proteins, DJ-1 was found to be more abundant in fast-glycolytic fiber secretomes. On the contrary, FABP-3 was elevated in slow-oxidative fiber secretomes, although its secretion from gastrocnemius muscle increased in exercised animals. These and other secreted proteins identified in this work may be considered as potential myokines.

Obesidomics: contribution of adipose tissue secretome analysis to obesity research.

Obesity is presently reaching pandemic proportions and it is becoming a major health concern in developed and developing countries due to its comorbidities like type II diabetes, cardiovascular pathologies, and some cancers. The discovery of the adipose tissue role as an endocrine gland able to secrete adipokines that affects whole-body energy homeostasis has become a key break-through toward a better molecular understanding of obesity. Among the known adipokines involved in the regulation of energy metabolism very few have been clearly seen as central regulators of insulin sensitivity, metabolism, and energy homeostasis. Thus, the discovery and characterization of new adipocyte-derived factors is still in progress. Proteomics technology has emerged as a useful tool to analyze adipose tissue secretion (secretome) dynamics giving a wider picture into the molecular events that control body weight. Besides the identification of new secreted proteins, the advantage of using this approach is the possibility to detect post-translational modifications and protein interactions that generally cannot be predicted by genome studies. In this review, we summarize the recent efforts to identify new bioactive adipokines by proteomics especially in pathological situations such as obesity.

Preproghrelin expression is a key target for insulin action on adipogenesis.

This study aimed to investigate the role of preproghrelin-derived peptides in adipogenesis. Immunocytochemical analysis of 3T3-L1 adipocyte cells showed stronger preproghrelin expression compared with that observed in 3T3-L1 preadipocyte cells. Insulin promoted this expression throughout adipogenesis identifying mTORC1 as a critical downstream substrate for this profile. The role of preproghrelin-derived peptides on the differentiation process was supported by preproghrelin knockdown experiments, which revealed its contribution to adipogenesis. Neutralization of endogenous O-acyl ghrelin (acylated ghrelin), unacylated ghrelin, and obestatin by specific antibodies supported their adipogenic potential. Furthermore, a parallel increase in the expression of ghrelin-associated enzymatic machinery, prohormone convertase 1/3 (PC1/3) and membrane-bound O-acyltransferase 4 (MBOAT4), was dependent on the expression of preproghrelin in the course of insulin-induced adipogenesis. The coexpression of preproghrelin system and their receptors, GHSR1a and GPR39, during adipogenesis supports an autocrine/paracrine role for these peptides. Preproghrelin, PC1/3, and MBOAT4 exhibited dissimilar expression depending on the white fat depot, revealing their regulation in a positive energy balance situation in mice. The results underscore a key role for preproghrelin-derived peptides on adipogenesis through an autocrine/paracrine mechanism.

Secretome analysis of rat adipose tissues shows location-specific roles for each depot type.

Obesity prevalence is reaching pandemic proportions becoming a major public health threat for many industrialized nations. It is especially worrying as it causes a higher risk of premature death due to associated diseases such as type 2 diabetes, cardiovascular disease, and some cancers. Current evidence shows biological and genetic differences between adipose tissues depending on its anatomical location. Particularly, upper body/visceral fat distribution in obesity is closely linked to metabolic complications. In this report, we characterize for the first time the secretome of rat adipose tissue explants from different anatomical localizations and its differential analysis. Visceral, subcutaneous, and gonadal fat specific secretomes and differentially secreted proteins among the three fat depots were analyzed by 2-DE and MS. Reference maps for location-specific adipose tissue secretomes are shown and the 45 most significant differences are listed. Identified proteins include classical adipokines and novel secreted proteins. Interestingly, our results show that the type of proteins and their role in different biological processes diverge significantly when comparing the set of proteins identified from visceral, subcutaneous and gonadal fat explants. This study emphasizes and supports the differential role of adipose tissue in accordance to its anatomical localization.

Obestatin as a regulator of adipocyte metabolism and adipogenesis.

The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3α/ß, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPα, C/EBPß, C/EBPδ and PPARγ promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome.

Orexin-A regulates growth hormone-releasing hormone mRNA content in a nucleus-specific manner and somatostatin mRNA content in a growth hormone-dependent fashion in the rat hypothalamus.

The orexins or hypocretins are two neuropeptides involved in the regulation of diverse biological processes such as feeding, sleep and neuroendocrine function. Recent findings suggest a possible functional interaction between orexins, somatostatin and growth hormone-releasing hormone (GHRH) in the rat hypothalamus. In order to understand the possible functional linkage between orexins and these neuropeptides, we determined the effects of intracerebroventricular orexin-A administration on hypothalamic somatostatin and GHRH mRNA levels. Furthermore, we examined whether growth hormone (GH) mediates these interactions by using two animal models that showed GH deficiency: hypophysectomized rats and dwarf Lewis rats. Using in situ hybridization, our data showed that GHRH mRNA levels in the paraventricular nucleus of the hypothalamus are decreased after orexin-A treatment, without changes in the arcuate nucleus of the hypothalamus. On the other hand, orexin-A treatment induces a GH-dependent stimulatory effect on somatostatin mRNA content in the periventricular nucleus of the hypothalamus. Finally, we demonstrated, for the first time, that hypophysectomized rats and dwarf Lewis rats, two classical models of GH deficiency with alterations in sleep patterns, showed a marked reduction in the GHRH mRNA levels in the paraventricular nucleus of the hypothalamus. These data improve our understanding of the interactions among the different systems involved in the control and pathophysiology of food intake, sleep and GH secretion.

Ghrelin: from a GH-secretagogue to the regulation of food intake, sleep and anxiety.

Grhelin is an endogenous ligand for the growth hormone secretagogue receptor from the stomach. It is a 28-aminoacid peptide of which the serine 3 residue is n-octanoylated. Ghrelin strongly stimulates GH secretion in vivo as well as in vitro. This endogenous ligand promotes the production of orexigenic neuropeptides (NPY and AgRP) in the hypothalamic arcuate nuclei and activates the neurons that produce these orexigenic peptides, resulting in an increase in feeding and body weight. Ghrelin has other significant actions, including control of acid secretion, influences on sleep and on the regulation of anxiety.

Neuropeptide Y, but not agouti-related peptide or melanin-concentrating hormone, is a target peptide for orexin-A feeding actions in the rat hypothalamus.

We examined the effects of orexin A on the mRNA levels of neuropeptide Y, agouti-related peptide, melanin-concentrating hormone, prepro-orexin and orexin receptors in the rat hypothalamus. Adult male rats were treated centrally (i.c.v.) with a single dose of orexin A (3 nmol). After 2, 6 and 12 h, neuropeptide Y, agouti-related peptide, melanin-concentrating hormone, and prepro-orexin mRNA levels were measured by semiquantitative RT-PCR and in situ hybridization; orexin receptors mRNA content was quantified by semiquantitative RT-PCR. We found that orexin A increased neuropeptide Y expression in the arcuate nucleus of the rat hypothalamus. This stimulatory effect was transient, being observed 2 h after the treatment, and disappearing after longer periods (6 and 12 h). In contrast, no change was demonstrated in hypothalamic agouti-related peptide, melanin-concentrating hormone, prepro-orexin or orexin receptors mRNA levels at any time evaluated. Our results suggest that neuropeptide Y synthesized in the arcuate nucleus of the hypothalamus, but not agouti-related peptide and melanin-concentrating hormone pathways, is likely involved in orexin-induced feeding behavior, and raise the possibility that this functional linkage may also be involved in other actions mediated by orexins such as locomotor activity and sympathetic function.