Agouti Gene-related Protein Research Articles

Effects of fishmeal substitution by four fermented soybean meals on growth， antioxidant capacity and inflammation responses of turbot juveniles (Scophthalmus maximus L.)

A 10-week feeding trial was conducted to examine the effects of fishmeal (FM) substitution by different types of FSBM on growth performance, antioxidant capacity and inflammatory responses of turbot juveniles (Scophthalmus maximus L.). Four strains of microorganisms Saccharomycopsis fibuligera isolate Y27 (SF), Bacillus tequilensis KCTC 13622 (BT), Bacillus subtilis strain D7XPN1 (BS) and Bacillus aryabhattai B8W22 (BA) with high-efficiency degradation of glucoside of soybean isoflavones (SI) were obtained through screening and isolation, and were used to ferment soybean meal (SBM) to enhance the utilization of SBM. Six isonitrogenous (50.05% crude protein) and isolipidic (10.46% crude lipid) diets were formulated. The FM diet (the control diet) was formulated with 55% fishmeal. Five experimental diets (Diets SBM45, SF45, BT45, BS45 and BA45) were formulated by replacing 45% of FM inclusion level of control diet using SBM or either FSBM produced by the above four strains, respectively. Results showed that there was a significant decrease in feed intake (FI) of fish fed the SBM45 diet than those of the control and either FSBM diets (P = 0.000), and no significant difference was found among the control, SF45, BT45 and BS45 groups (P > 0.05). Meanwhile, fish fed the diet BA45 showed a significantly higher FI compared with the control group (P = 0.000). The appetite-stimulating factor (agouti gene-related protein, agrp1) gene expression was significantly up-regulated in fish fed the diet SF45 compared with the SBM45 group (P = 0.058). Meanwhile, the appetite-suppressive factors (proopiomelanocorein, pomc and cocaine and amphetamine regulated transcript, cart) gene expression was significantly down-regulated in fish fed the diet SF45 compared with the SBM45 group (P = 0.009, 0.006). The foregut α-amylase and trypsin activity were significantly promoted in fish fed the diet BS45 compared with those of the SBM45 and BA45 treatments (P = 0.000). There was a significant decrease in apparent digestibility coefficient of dry matter in fish fed the SBM45 diet compared with fish fed the control diet and fish fed either FSBM-based diet (P = 0.000), however, no significant difference was found among the control, SF45, BS45 and BA45 groups (P > 0.05). Moreover, no significant difference was observed in survival rate among dietary treatments (P > 0.05). Fish fed the diet BT45 had significantly lower feed efficiency than that of the control group (P = 0.048), meanwhile no significant difference was found among the control, SBM45, SF45, BS45 and BA45 treatments (P > 0.05). Fish fed the SBM45 diet had significantly lower specific growth rate compared with fish fed the control diet and fish fed either FSBM-based diet (P = 0.000). Furthermore, the antioxidant capacity significantly increased in fish fed the control and four FSBM diets compared with the SBM45 group (P < 0.05). The serum, liver and hindgut inflammatory responses of fish fed the control and four FSBM diets were significantly lower than those of the SBM45 group (P < 0.05). In conclusion, fish fed four FSBM diets could promote growth, enhance antioxidant capacity and reduce inflammatory responses of turbot juveniles compared with those of the SBM45 group.

Effects of dietary carbohydrate to lipid ratios on growth, biochemical indicators, lipid metabolism, and appetite in Chinese perch (Siniperca chuatsi).

An 8-week feeding trial was conducted to evaluate the effects of dietary carbohydrate to lipid (CHO:L) ratios on growth performance, body composition, serum biochemical indexes, lipid metabolism, and gene expression of central appetite regulating factors in Chinese perch (Siniperca chuatsi) (mean initial weight: 12.86 ± 0.10g). Five isonitrogenous and isoenergetic diets (fish meal, casein as main protein sources) were formulated to contain different graded CHO:L ratio diets ranging from 0.12, 0.86, 1.71, 3.29, and 7.19. Each diet was assigned to triplicate groups of 18 experimental fish. Our results revealed that final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), and protein efficiency ratio (PER) increased with dietary CHO:L ratio from 0.12 to 1.71 and then decreased with further increases in dietary CHO:L ratio. A two-slope broken-line regression analysis based on WGR showed that the optimal dietary CHO:L level for maximum growth performance of fish was 1.60. Crude lipid and crude protein content in the liver and glycogen concentration in the muscle and liver were significantly influenced by the dietary CHO:L ratios (P < 0.05). The lowest crude lipid content in the liver was observed in fish fed the diet with a CHO:L ratio of 1.71(P < 0.05). Dietary CHO:L ratios significantly induced the glucose concentration of serum (P < 0.05). The relative expression levels of genes involved in lipid metabolism, such as srebp1 and fas in the liver, showed a trend of first decreased and then increased with the increase of dietary CHO:L ratio levels. Appropriate CHO:L ratio in the diet can effectively reduce the accumulation of liver fat. We observed in fish fed the 1.71 CHO:L ratio diet showed higher feed intake, up-regulated mRNA expression of neuropeptide Y (npy) and agouti gene-related protein (agrp), and down-regulated mRNA expression of cocaine- and amphetamine-regulated transcript (cart) and pro-opiomelanocorticoid (pomc) significantly as compared to control group. Thus, these results provide the theoretical basis for feed formulation to determine the appropriate CHO:L ratio requirement of Chinese perch.

The Ciji-Hua'ai-Baosheng II Formula Attenuates Chemotherapy-Induced Anorexia in Mice With H22 Hepatocellular Carcinoma.

Background: Ciji-Hua’ai-Baosheng II Formula (CHB-II-F) is a traditional Chinese medicine formula, which specifically targets different aspects of chemotherapy-induced adverse effects in patients with cancer. In our clinical application, CHB-II-F significantly alleviated chemotherapy-induced anorexia (loss of appetite) and improved the quality of life for patients with tumor during and after chemotherapy. However, the mechanism of CHB-II-F in alleviation of chemotherapy-induced anorexia remains to be further investigated.Aim of Study: To explore the therapeutic effect and mechanism of CHB-II-F on chemotherapy-induced anorexia in the mice model of H22 hepatoma.Materials and Methods: A total of 72 Kunming mice of SPF grade were inoculated subcutaneously with H22 hepatoma cells into the right anterior armpit of the mice. After 1 week of seeding, mice were injected intraperitoneally with a high dose of 5-fluorouracil (200 mg/kg 5-FU) to establish the model of chemotherapy. The mice were randomly divided into six groups: untreated group, 5-FU group, 5-FU plus Yangzheng Xiaoji capsule (YZXJC) group, and three groups of 5-FU plus different concentrations of CHB-II-F. All the mice in each group were treated for 14 days. The body weight, food intake, tumor volume, and tumor weight of mice were measured, and pathological examinations of tumor tissue, stomach, and duodenum were carried out. Expressions of serum Leptin, Neuropeptide Y (NPY), epidermal cell growth factor (EGF), Motilin (MTL), Orexin A (OXA), Gastrin (GAS), Ghrelin, Prostaglandin E2 (PGE2), and jejunum superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined. The protein and mRNA levels of proopiomelanocortin (POMC), Orexin receptor 1 (OX1R), neuropeptide Y (NPY), cocaine and amphetamine regulated transcript peptide (CART), Agouti gene-related protein (AgRP), Leptin receptor (Ob-R), and Ghrelin receptor (GHSR) were examined in hypothalamus, and the protein levels of substance P (SP) and 5-hydroxytryptamine (5-HT) in duodenum were measured.Results: The combination of CHB-II-F and 5-FU could enhance the inhibitory effect of 5-FU on tumor. The tumor inhibition rates of 5-FU group, YZXJC group, CHB-II-F(H) group, CHB-II-F(M) group, and CHB-II-F(L) group were 58.88, 28.08, 54.96, 37.69, and 28.61%, respectively. Compared with untreated group and 5-FU group, CHB-II-F significantly increased the body weight and food intake of tumor-bearing mice; increased the content of NPY, Orexin A, Ghrelin, GAS, MTL, EGF, and PGE2 in serum and the activity of SOD in jejunum; and decreased the content of Leptin in serum and the content of MDA in jejunum. Compared with untreated group and 5-FU group, CHB-II-F also enhanced the expression of OX1R, GHSR, NPY, and AgRP protein and gene and decreased the expression of Ob-R, POMC, and CART protein and gene in hypothalamus of mice, and the gene expression was consistent with the protein expression. In addition, CHB-II-F decreased the expression of 5-HT and SP protein in duodenum.Conclusion: In the murine model of H22 hepatocellular carcinoma (HCC) receiving chemotherapy, CHB-II-F enhances the inhibitory effect of 5-FU on tumor, significantly improves the pathological injury of gastrointestinal tract caused by chemotherapy, and regulates the secretion of gastrointestinal hormones. It may alleviate chemotherapy-induced anorexia by affecting appetite regulatory factors in the feeding area of hypothalamus central nervous system and peripheral appetite regulatory factors.

Probiotics Improve Eating Disorders in Mandarin Fish (Siniperca chuatsi) Induced by a Pellet Feed Diet via Stimulating Immunity and Regulating Gut Microbiota.

Eating disorders are directly or indirectly influenced by gut microbiota and innate immunity. Probiotics have been shown to regulate gut microbiota and stimulate immunity in a variety of species. In this study, three kinds of probiotics, namely, Lactobacillus plantarum, Lactobacillus rhamnosus and Clostridium butyricum, were selected for the experiment. The results showed that the addition of three probiotics at a concentration of 108 colony forming unit/mL to the culture water significantly increased the ratio of the pellet feed recipients and survival rate of mandarin fish (Siniperca chuatsi) under pellet-feed feeding. In addition, the three kinds of probiotics reversed the decrease in serum lysozyme and immunoglobulin M content, the decrease in the activity of antioxidant enzymes glutathione and catalase and the decrease in the expression of the appetite-stimulating regulator agouti gene-related protein of mandarin fish caused by pellet-feed feeding. In terms of intestinal health, the three probiotics reduced the abundance of pathogenic bacteria Aeromonas in the gut microbiota and increased the height of intestinal villi and the thickness of foregut basement membrane of mandarin fish under pellet-feed feeding. In general, the addition of the three probiotics can significantly improve eating disorders of mandarin fish caused by pellet feeding.

Promotion of pellet-feed feeding in mandarin fish (Siniperca chuatsi) by Bdellovibrio bacteriovorus is influenced by immune and intestinal flora

The feeding preference of the mandarin fish, Siniperca chuatsi, for live bait fish over pellet feed increases the costs and risks of breeding. Animal feeding behavior is directly or indirectly influenced by intestinal flora and innate immunity. Probiotics have been shown to regulate intestinal flora and stimulate innate immunity in a variety of species. In this study, during the pellet-feed feeding process of mandarin fish, Bdellovibrio bacteriovorus at a concentration of 108 PFU (plaque forming unit)/mL was added to the water body for 6 weeks, and the effects on the survival, growth, appetite, intestinal flora, innate immunity, antioxidant capacity, and intestinal histology of mandarin fish were studied. The results showed that addition of B. bacteriovorus at a concentration of 108 PFU/mL to aquaculture water could significantly promote mandarin fish pellet-feed feeding, improve the survival rate and growth performance of mandarin fish that were fed pellet feed. Compared with the control group, addition of B. bacteriovorus promoted the expression of appetite-regulating genes nerve peptide Y (Npy) and agouti gene-related protein (Agrp). Intestinal microflora analysis and intestinal histological analysis results revealed that B. bacteriovorus reduce the abundance of pathogenic bacteria Aeromonas in the intestinal flora, reduce damage to the intestinal mucosa and epithelial cells, and increase the thickness of the intestinal basement membrane and height of intestinal villi. Similarly, an increase in the serum lysozyme and immunoglobulin M (IgM) contents was observed after B. bacteriovorus administration. Moreover, B. bacteriovorus improved the activity of antioxidant enzymes, reduced glutathione (GSH) and catalase (CAT). In general, addition of B. bacteriovorus at a concentration of 108 PFU/mL to aquaculture water could significantly promote pellet-feed feeding of mandarin fish and improve the growth performance and survival rates of mandarin fish under pellet-feed feeding conditions. This promoting effect may be achieved by B. bacteriovorus stimulation of innate immunity and regulation of intestinal flora.

Brain JNK and metabolic disease.

Obesity, which has long since reached epidemic proportions worldwide, is associated with long-term stress to a variety of organs and results in diseases including type 2 diabetes. In the brain, overnutrition induces hypothalamic stress associated with the activation of several signalling pathways, together with central insulin and leptin resistance. This central action of nutrient overload appears very rapidly, suggesting that nutrition-induced hypothalamic stress is a major upstream initiator of obesity and associated diseases. The cellular response to nutrient overload includes the activation of the stress-activated c-Jun N-terminal kinases (JNKs) JNK1, JNK2 and JNK3, which are widely expressed in the brain. Here, we review recent findings on the regulation and effects of these kinases, with particular focus on the hypothalamus, a key brain region in the control of energy and glucose homeostasis. JNK1 blocks the hypothalamic-pituitary-thyroid axis, reducing energy expenditure and promoting obesity. Recently, opposing roles have been identified for JNK1 and JNK3 in hypothalamic agouti gene-related protein (AgRP) neurons: while JNK1 activation in AgRP neurons induces feeding and weight gain and impairs insulin and leptin signalling, JNK3 (also known as MAPK10) deletion in the same neuronal population produces very similar effects. The opposing roles of these kinases, and the unknown role of hypothalamic JNK2, reflect the complexity of JNK biology. Future studies should address the specific function of each kinase, not only in different neuronal subsets, but also in non-neuronal cells in the central nervous system. Decoding the puzzle of brain stress kinases will help to define the central stimuli and mechanisms implicated in the control of energy balance. Graphical abstract.

Post-natal Growth Retardation Associated With Impaired Gut Hormone Profiles, Immune and Antioxidant Function in Pigs.

The factors that cause post-natal growth retardation (PGR) in pigs are complicated; however, metabolic and immune system impairment seem to be involved. The purpose of this study was to investigate the changes of blood parameters, hormone profiles, antioxidant capacity, and immune responses in PGR pigs. Blood and small intestinal mucosa samples were collected from 42-days-old PGR and healthy pigs. The results showed that compared with the healthy group, the relative weight of spleen and kidney were greater, but the liver was lighter in PGR pigs (P < 0.05). The PGR pigs had increased serum alanine transaminase, urea nitrogen, blood ammonia, IgG, and complement 4, but decreased glucose and albumin (P < 0.05). The higher levels of serum leptin (LEP) and thyroxin (T4), and the lower levels of insulin-like growth factor-1 (IGF-1), 5-hydroxytryptamine (5-HT), somatostatin (SS), and agouti gene-related protein (AgRP) were observed in PGR pigs (P < 0.05). Consistent with the serum levels of hormones, the mRNA levels of gut hormones and their receptors were also altered in intestinal mucosa from PGR pigs (P < 0.05). The PGR pigs exhibited higher plasma concentrations of interleukin-1β (IL-1β), IL-6, IL-8, and transformed growth factor beta (TGFβ) (P < 0.05). However, the mRNA expressions of several cytokines were lower in the small intestinal mucosa of PGR pigs (P < 0.05). Abnormal antioxidant indexes in serum of PGR pigs were observed, which was in accordance with the reduced mRNA expression of several anti-oxidative genes in the small intestinal mucosa of PGR pigs (P < 0.05). These data demonstrate that an abnormal gut hormone system, immune dysfunction, and decreased antioxidant capacity may contribute to PGR in pigs. These changes could provide a valuable target in the regulation of post-natal growth retardation in animals and humans.

Chlorella additive increased growth performance, improved appetite and immune response of juvenile crucian carp Carassius auratus

This study was designed to investigate effects of dietary Chlorella meal (CM) additive on growth performance, immune responses and appetite regulation of juvenile crucian carp Carassius auratus (initial body weight: 1.27 ± 0.03 g). Four experimental diets were formulated to contain 0% (CM0), 1% (CM1), 2% (CM2) and 4% Chlorella meal (CM4), respectively. Each diet was randomly assigned to triplicate groups with 40 juvenile fish per fibreglass tank for 8 weeks. Weight gain rate, specific growth rate and feed intake increased with increasing dietary CM levels. In contrast, FCR (feed conversion rate) declined with dietary CM levels. No significant differences were observed in moisture, crude protein, crude lipid and ash contents of muscle and liver tissues. Dietary CM addition increased activities of acid and alkaline phosphatase in liver and kidney. Dietary CM up-regulated the mRNA expression levels of NKEF-B, MCHII and IgM in kidney, and increased the mRNA levels of NPY and agouti gene-related protein in the brain, but down-regulated mRNA levels of MC4R, LEP, LEPR, CART1, CART2 and CCK8 genes. Based on these observations above, this study indicated that dietary CM additive increased growth performance, immune responses and appetite of crucian carp. The results, for the first time, demonstrate a role for the central nervous system in the control of food intake in fish fed dietary Chlorella meal.

POMC Neurons: Feeding, Energy Metabolism, and Beyond.

The central melanocortin system is a well-established neuronal pathway involved in regulating energy metabolism. Pro-opiomelanocortin (POMC) neurons, agouti gene-related protein (AgRP) neurons, and their downstream cells expressing the melanocortin-3 (MC3R) and melanocortin-4 receptors (MC4R) are three key components of the central melanocortin pathway. This chapter focuses on the Pomc gene and the POMC neural system. First, I summarize the established role of this system in inhibiting food intake. Second, in light of new cutting-edge techniques, our understanding of how POMC neurons function to regulate energy homeostasis has been refined during the last few years. I describe some recent advances and discuss bidirectional effects of POMC neurons on feeding. Finally, the physiological significance beyond energy metabolism, in particular for reward and sex, is also discussed.

Inhibitory neurotransmitter serotonin and excitatory neurotransmitter dopamine both decrease food intake in Chinese perch (Siniperca chuatsi).

Aminergic neurotransmitters play important roles in the regulation of food intake. However, their effects on feeding in fish have been less explored and still unclear. In the present study, the effects of serotonin (5-HT) and dopamine (DA) on food intake were evaluated through intraventricular (ICV) administration in Chinese perch (Siniperca chuatsi) and the mRNA expression levels of neuropeptide Y (NPY), agouti gene-related protein (AgRP), and pro-opiomelanocortin (POMC) were detected. At 1h post-injection, 5-HT significantly decreased food intake in a dose-dependent manner. The mRNA expression of NPY and AgRP were significantly decreased (p<0.05), whereas the mRNA expression of POMC was significantly increased (p<0.05), suggesting the involvement of NPY, AgRP, and POMC in inhibitory action of 5-HT on food intake in Chinese perch. DA significantly decreased (p<0.05) food intake and AgRP mRNA expression at 1h post-injection, indicating the inhibitory effect of DA on food intake might be mediated through AgRP. This might shed new light on the regulation of food intake in Chinese perch.

Leptin regulates energy intake but fails to facilitate hibernation in fattening Daurian ground squirrels (Spermophilus dauricus)

Body fat storage before hibernation affects the timing of immergence in Daurian ground squirrels (Spermophilus dauricus). Leptin is an adipose signal and plays vital role in energy homeostasis mainly by action in brain. To test the hypothesis that leptin plays a role in facilitating the process of hibernation, squirrels were administrated with recombinant murine leptin (1μg/day) through intracerebroventricular (ICV) injection for 12 days during fattening. From day 7 to 12, animals were moved into a cold room (5±1°C) with constant darkness which functioned as hibernaculum. Energy intake, body mass and core body temperature (Tb) were continuously monitored throughout the course of experiment. Resting metabolic rate (RMR) was measured under both warm and cold conditions. At the end of leptin administration, we measured the serum concentration of hormones related to energy regulation, mRNA expression of hypothalamic neuropeptides and uncoupling protein 1 (UCP1) levels in brown adipose tissue (BAT). Our results showed that during leptin administration, the cumulative food intake and increase of body mass were suppressed while Tb and RMR were unaltered. The proportion of torpid squirrels was not different between two groups. At the end of leptin administration, the expressions of hypothalamic neuropeptide Y and agouti gene-related protein were suppressed. There were no differences in UCP1 mRNA expression or protein content in BAT between groups. Our data suggest that leptin can affect energy intake via hypothalamic neuropeptides, but is not involved in the initiation of hibernation in fattening Daurian ground squirrels.

Expression and colocalization patterns of calbindin-D28k, calretinin and parvalbumin in the rat hypothalamic arcuate nucleus

Calcium binding proteins (CaBPs) form a diverse group of molecules that function as signal transducers or as intracellular buffers of Ca2+ concentration. They have been extensively used to histochemically categorize cell types throughout the brain. One region which has not yet been characterized with regard to CaBP expression is the hypothalamic arcuate nucleus, which plays a vital role in neuroendocrine control and the central regulation of energy metabolism. Using in situ hybridization and immunofluorescence, we have investigated the cellular distribution of the three CaBPs, calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) in the rat arcuate nucleus. Both mRNA and immunoreactivity was detected in the arcuate nucleus for CB – located in the medial aspects – and CR – located ventrolaterally. No PV mRNA was detected in the arcuate nucleus. Immunofluorescence results for PV were ambiguous; while one antibody detected a group of cell somata, a different antibody failed to visualize any arcuate nucleus cell profiles. Using double-labeling, neither of the examined CaBPs were observed in cells immunoreactive for the signaling molecules agouti gene-related protein, tyrosine hydroxylase, neurotensin, growth hormone-releasing hormone, somatostatin, enkephalin, dynorphin or galanin. We did, however, observe CB- and CR-immunoreactivity, in two distinct populations of neurons immunoreactive for the melanocortin peptide α-melanocyte-stimulating hormone. These data identify distinct subpopulations of arcuate neurons defined by their expression of CaBPs and provide further support for differentiation between subpopulations of anorexigenic melanocortin neurons.

Long term exendin-4 treatment reduces food intake and body weight and alters expression of brain homeostatic and reward markers.

Repeated administration of the long-acting glucagon-like peptide 1 receptor agonist exendin-4 (EX-4) has been shown to reduce food intake and body weight and do so without a rebound increase in food intake after treatment termination. The current study examines the neural mechanisms underlying these actions. After 6 weeks of maintenance on a standard chow or a high-fat (HF) diet, male Sprague Dawley rats were treated with EX-4 (3.2 μg/kg, i.p., twice a day) or vehicle for 9 consecutive days. Food intake and body weight (BW) were monitored daily. Expression of the genes for the hypothalamic arcuate nucleus (ARC) peptides proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti gene-related protein was determined. Expression of the dopamine precursor tyrosine hydroxylase (TH) gene in the ventral tegmental area and genes for dopamine receptors 1 (D1R) and dopamine receptor 2 in the nucleus accumbens were also determined. Pair-fed groups were included to control for the effects of reduced food intake and BW. Treatment with EX-4 significantly decreased food intake and BW over the 9-day period in both the standard chow and HF groups. HF feeding decreased POMC without changing NPY/agouti gene-related protein gene expression in the ARC. Treatment with EX-4 increased POMC and decreased NPY expression independent of the reduction of food intake and BW. Mesolimbic TH and D1R gene expression were decreased significantly in chronic HF diet-fed rats, and these changes were reversed in both EX-4 and pair-fed conditions. These results suggest a role for increased POMC and decreased NPY expression in the ARC in the effects of EX-4 on food intake and BW. Our findings also suggest that EX-4 induced the recovery of mesolimbic TH and D1R expression in HF diet-fed rats may be secondary to HF intake reduction and/or weight loss.

Influence of abnormally high leptin levels during pregnancy on metabolic phenotypes in progeny mice

Maternal obesity increases the risk of obesity in offspring, and obesity is accompanied by an increase in blood leptin levels. The "yellow" mutation at the mouse agouti locus (A(y)) increases blood leptin levels in C57BL preobese pregnant mice without affecting other metabolic characteristics. We investigated the influence of the A(y) mutation or leptin injection at the end of pregnancy in C57BL mice on metabolic phenotypes and the susceptibility to diet-induced obesity (DIO) in offspring. In both C57BL-A(y) and leptin-treated mice, the maternal effect was more pronounced in male offspring. Compared with males born to control mothers, males born to A(y) mothers displayed equal food intake (FI) but decreased body weight (BW) gain after weaning, equal glucose tolerance, and enhanced FI-to-BW ratios on the standard diet but the same FI and BW on the high-fat diet. Males born to A(y) mothers were less responsive to the anorectic effect of exogenous leptin and less resistant to fasting (were not hyperphagic and gained less weight during refeeding after food deprivation) compared with males born to control mothers. However, all progeny displayed equal hypothalamic expression of Agouti gene-related protein (AgRP), neuropeptide Y (NPY), and proopiomelanocortin (POMC) and equal plasma leptin and glucose levels after food deprivation. Leptin injections in C57BL mice on day 17 of pregnancy decreased BW in both male and female offspring but inhibited FI and DIO only in male offspring. Our results show that hyperleptinemia during pregnancy has sex-specific long-term effects on energy balance regulation in progeny and does not predispose offspring to developing obesity.

Role of GABA Release From Leptin Receptor-Expressing Neurons in Body Weight Regulation

It is well established that leptin regulates energy balance largely through isoform B leptin receptor-expressing neurons (LepR neurons) in the brain and that leptin activates one subset of LepR neurons (leptin-excited neurons) while inhibiting the other (leptin-inhibited neurons). However, the neurotransmitters released from LepR neurons that mediate leptin action in the brain are not well understood. Previous results demonstrate that leptin mainly acts on γ-aminobutyric acid (GABA)ergic neurons to reduce body weight, and that leptin activates proopiomelanocortin neuron activity by reducing GABA release onto these neurons, suggesting a body weight-promoting role for GABA released from leptin-inhibited neurons. To directly examine the role of GABA release from LepR neurons in body weight regulation, mice with disruption of GABA release specifically from LepR neurons were generated by deletion of vesicular GABA transporter in LepR neurons. Interestingly, these mice developed mild obesity on chow diet and were sensitive to diet-induced obesity, which were associated with higher food intake and lower energy expenditure. Moreover, these mice showed blunted responses in both food intake and body weight to acute leptin administration. These results demonstrate that GABA plays an important role in mediating leptin action. In combination with the previous studies that leptin reduces GABA release onto proopiomelanocortin neurons through leptin-inhibited neurons and that disruption of GABA release from agouti gene-related protein neurons, one subset of LepR-inhibited neurons, leads to a lean phenotype, our results suggest that, under our experimental conditions, GABA release from leptin-excited neuron dominates over leptin-inhibited ones.

Obesity, metabolic syndrome, adipocytes and vascular function: A holistic viewpoint

1. Obesity is a metabolic disease of pandemic proportions largely arising from positive energy balance, a consequence of sedentary lifestyle, conditioned by environmental and genetic factors. Several central and peripheral neurohumoral factors (the major ones being the anorectic adipokines leptin and adiponecin and the orexigenic gut hormone ghrelin) acting on the anorectic (pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript) and orexigenic (neuropeptide Y and agouti gene-related protein) neurons regulate energy balance. These neurons, mainly in the arcuate nucleus of the hypothalamus, project to parts of the brain modulating functions such as wakefulness, autonomic function and learning. A tilt in the anorectic-orexigenic balance, perhaps determined genetically, leads to obesity. 2. Excess fat deposition requires space, created by adipocyte (hypertrophy and hyperplasia) and extracellular matrix (ECM) remodelling. This process is regulated by several factors, including several adipocyte-derived Matrix metalloproteinases and the adipokine cathepsin, which degrades fibronectin, a key ECM protein. Excess fat, also deposited in visceral organs, generates chronic low-grade inflammation that eventually triggers insulin resistance and the associated comorbidities of metabolic syndrome (hypertension, atherosclerosis, dyslipidaemia and diabetes mellitus). 3. The perivascular adipose tissue (PVAT) has conventionally been considered non-physiological structural tissue, but has recently been shown to serve a paracrine function, including the release of adipose-derived relaxant and contractile factors, akin to the role of the vascular endothelium. Thus, PVAT regulates vascular function in vivo and in vitro, contributing to the cardiovascular pathophysiology of the metabolic syndrome. Defining the mechanism of PVAT regulation of vascular reactivity requires more and better controlled investigations than currently seen in the literature.

Evidence of hypothalamic degeneration in the anorectic anx/anx mouse

Mice homozygous for the anorexia (anx) mutation are characterized by poor food intake and death by three to five weeks after birth. By P21 these mice display lower density of hypothalamic neuropeptides, including Agouti gene-related protein (AGRP). The AGRP/neuropeptide Y (NPY) system of the anx/anx mice develops normally until postnatal day (P) 12, then the normal increase in fiber density ceases, in some areas even distinctly decreases. This overlaps with activation of microglia, indicating an inflammatory and/or degenerative process. Here we studied, by in situ hybridization and immunohistochemistry (IHC), the expression of major histocompatibility complex (MHC) class I-related molecules and markers for cellular reactivity in hypothalamus of anx/anx mice. MHC class I transcript and -related proteins were found in arcuate nucleus (Arc), presumably both in neurons and glia, the latter also in areas innervated by AGRP (NPY) neurons. In the anx/anx hypothalamus, using TUNEL labeling, significantly higher number of apoptotic cells were found compared with +/+ mice, and active caspase 6 immunoreactivity was detected in degenerating NPY-fibers as well as signs of "microglia-associated cell death". In addition, Y1 receptor-labeled processes and soma of pro-opiomelanocortin (POMC) neurons, were markedly decreased at P21. These results support the hypothesis of degeneration of hypothalamic arcuate neuron populations in the anx/anx mice, whereby the AGRP system may be first affected, the changes in the POMC system being secondary in this process.

Intragastric administration of evodiamine suppresses NPY and AgRP gene expression in the hypothalamus and decreases food intake in rats

Evodiamine (Evo), an alkaloidal component extracted from the fruit of Evodiae fructus (Evodia rutaecarpa Bentham, Rutaceae), decreases the body weight of rats through a poorly defined mechanism. The hypothalamus is one of the areas in the brain linked to the control of food intake and energy expenditure. We postulate that Evo mediates this activity by modulating feeding-related peptides in the hypothalamus. We investigated the effects of Evo on food intake, body weight, the mRNA expression and peptide level of feeding-related peptides in the hypothalamus, in male rats. The juvenile rats of 5 weeks old were used at the start of the experiment. Evo (40 mg/kg or 4 mg/kg) was administered intragastrically for 25 days, and food intake and body weight of rats were recorded daily. Blood samples were collected for leptin radioimmunoassay (RIA). Real-Time PCR was used to analyze the mRNA expression. Western Blotting and immunohistochemistry were used to analyze the peptide. Our results show that intragastric administration of Evo (40 mg/kg) decreased rate of food intake and body weight increase following rat growth, reduced orexigenic neuropeptide Y (NPY) and agouti-gene related protein (AgRP) mRNA levels and NPY peptide level in the arcuate nucleus (ARC) of the hypothalamus, but it increases the circulating level of leptin. Intragastric administration of a smaller dose of Evo (4 mg/kg) was ineffective. These data suggest that Evo decreases food intake, and therefore body weight, partly by down-regulating NPY and AgRP mRNA expression and peptide expression in the ARC of the hypothalamus.

Hyperphagia and obesity produced by arcuate injection of NPY–saporin do not require upregulation of lateral hypothalamic orexigenic peptide genes

Neuropeptide Y (NPY) conjugated with a ribosomal inactivating toxin, saporin (SAP), is a toxin that targets NPY receptor-expressing cells. Injection of NPY–SAP into the rat arcuate nucleus (Arc) and basomedial hypothalamus (BMH) destroys two populations of NPY-receptor-expressing neurons important for the control of food intake and body weight, NPY and pro-opiomelanocortin (POMC) and cocaine and amphetamine related transcript (CART) neurons, and produces profound hyperphagia and obesity. Here, we investigated the contribution of lateral hypothalamus (LHA) orexigenic peptides, orexins and melanocortin concentrating hormone (MCH), to these lesion effects. We microinjected NPY–SAP into two sites on each side of the Arc, causing a loss of NPY and POMC/CART neurons that was limited to the Arc. Lesioned rats rapidly became hyperphagic and obese. However, MCH and prepro-orexin mRNA expression were not increased in the LHA in the lesioned rats, but were decreased at some levels of the LHA or were unchanged. NPY–SAP-induced obesity therefore differs from dietary obesity and from obesity associated with leptin or leptin receptor deficiency in which MCH gene expression is increased. The Arc NPY–SAP lesion produces obesity and hyperphagia that does not require overexpression of hypothalamic neuropeptides currently considered to provide major stimulatory drive for food intake: NPY, agouti gene-related protein, MCH or orexins. The source of the seemingly unregulated stimulatory drive for feeding in these animals has not been identified, but may be associated with hindbrain or endocrine mechanisms.

Bsx, a Novel Hypothalamic Factor Linking Feeding with Locomotor Activity, Is Regulated by Energy Availability

We recently reported that the hypothalamic homeobox domain transcription factor Bsx plays an essential role in the central nervous system control of spontaneous physical activity and the generation of hyperphagic responses. Moreover, we found Bsx to be a master regulator for the hypothalamic expression of key orexigenic neuropeptide Y and agouti gene-related protein. We now hypothesized that Bsx, which is expressed in the dorsomedial and arcuate nucleus (ARC) of the hypothalamus, is regulated by afferent signals in response to peripheral energy balance. Bsx expression was analyzed using in situ hybridization in fed vs. fasted (24 h) and ghrelin vs. leptin-treated rats, as well as in mice deficient for leptin or the ghrelin signaling. Ghrelin administration increased, whereas ghrelin receptor antagonist decreased ARC Bsx expression. Leptin injection attenuated the fasting-induced increase in ARC Bsx levels but had no effect in fed rats. Dorsomedial hypothalamic nucleus Bsx expression was unaffected by pharmacological modifications of leptin or ghrelin signaling. Obese leptin-deficient (ob/ob) mice, but not obese melanocortin 4 receptor-knockout mice, showed higher expression of Bsx, consistent with dependency from afferent leptin rather than increased adiposity per se. Interestingly, exposure to a high-fat diet triggered Bsx expression, consistent with the concept that decreased leptin signaling due to a high-fat diet induced leptin resistance. Our data indicate that ARC Bsx expression is specifically regulated by afferent energy balance signals, including input from leptin and ghrelin. Future studies will be necessary to test if Bsx may be involved in the pathogenesis of leptin resistance.