Comparison of the effects of intracerebroventricular administration of glucagon-like peptides 1 and 2 on hypothalamic appetite regulating factors and sleep-like behavior in chicks.

Glucagon-like peptide (GLP)-1 and GLP-2, proglucagon-derived brain-gut peptides, function as anorexigenic neuropeptides in mammals. We previously showed that central administration of GLP-1 and GLP-2 potently suppressed food intake in chicks. GLP-1 and GLP-2 specifically activate their receptors GLP-1 receptor (GLP1R) and GLP-2 receptor (GLP2R), respectively in chickens. In adult chickens, GLP1R and GLP2R are expressed in different brain regions. These findings raise the hypothesis that both GLP-1 and GLP-2 function as anorexigenic peptides in the chicken brain but the mechanisms underlying the anorexigenic effects are different between them. In the present study, we compared several aspects of GLP-1 and GLP-2 in chicks. GLP1R mRNA levels in the brain stem and optic lobes were significantly higher than in other parts of the brain, whereas GLP2R mRNA was densely expressed in the telencephalon. Intracerebroventricular administration of either GLP-1 or GLP-2 significantly reduced the mRNA levels of corticotrophin releasing factor and AMP-kinase (AMPK) α1. The mRNA level of proopiomelanocortin was significantly increased, and those of AMPKα2 and GLP2R were significantly decreased by GLP-2, whereas the mRNA level of pyruvate dehydrogenase kinase 4 was significantly increased, and that of GLP1R was significantly decreased by GLP-1. Intracerebroventricular administration of either GLP-1 or GLP-2 induced sleep-like behavior in chicks. Our findings suggest that the anorexigenic peptides GLP-1 and GLP-2 induce similar behavioral changes in chicks, but the mechanism may differ between them.

Enzymatically-Synthesized Glycogen Induces Cecal Glucagon-Like Peptide-1 Production and Suppresses Food Intake in Mice.

It is well known that dietary fiber stimulates the release of satiety hormones such as glucagon-like peptide-1 (GLP-1), which in turn suppresses appetite. In order to evaluate appetite regulating role of enzymatically synthesized glycogen (ESG, one of the resistant starch), we examined the effects of dietary supplementation of ESG on food intake and cecal proglucagon gene expression in normal and high fat diet-fed mice. Twenty four male ICR mice were weighed and assigned to four groups: normal diet group; normal diet containing 25% ESG group; high-fat diet (HFD) group; HFD containing 25% ESG group. Each group was fed the relevant diets for 3 wk. All data were analyzed by a two-way ANOVA with the main effects of HFD and ESG. ESG significantly decreased food intake and increased the weight of the cecum and cecal content. Plasma total short chain fatty acids concentration was significantly elevated by ESG. The mRNA levels of proglucagon in the cecum and plasma total GLP-1 concentration were significantly increased by ESG. The mRNA levels of appetite regulating neuropeptides such as neuropeptide Y, agouti-related protein, proopiomelanocortin, and cocain- and amphetamine-regulating transcript in the hypothalamus were not influenced by ESG. There is no significant interaction between diet and ESG in any parameters. These results suggest that ESG-induced upregulation of GLP-1 production in the cecum suppresses food intake in mice and that fecal fermentation may be involved in the anorexigenic effect.

Effects of fasting and re-feeding on the expression of CCK, PYY, hypothalamic neuropeptides, and IGF-related genes in layer and broiler chicks.

Cholecystokinin (CCK) and peptide YY (PYY) have been investigated as gut hormones that send satiation signals to the brain in mammals. There is evidence that chicken PYY mRNA expression was the highest in the pancreas compared to other tissues. We recently suggested that insulin-like growth factor (IGF)-1 and its binding proteins (IGFBPs) may be involved in the appetite regulation system in chicks. In the present study, in order to evaluate the possible roles of CCK, PYY, and IGF-related proteins in the appetite regulation system in chicks, we analyzed changes in the mRNA levels of these genes in response to fasting and re-feeding in layer and hyperphagic broiler chicks. In layer chicks, 12 h of fasting reduced the mRNA levels of intestinal CCK, PYY, Y2 receptor, and pancreatic PYY, and these changes were reversed by 12 h of re-feeding. On the other hand, in broiler chicks 12 h of fasting reduced the mRNA levels of intestinal PYY and Y2 receptor, but not intestinal CCK and pancreatic PYY, and these changes were reversed by 12 h of re-feeding. Hypothalamic NPY mRNA significantly increased by 12 h of fasting in both chicks, and these changes were reversed by re-feeding. Also, 12 h of fasting significantly increased the mRNA levels of hypothalamic agouti-related protein and reduced the mRNA levels of hepatic IGF-1 only in broiler chicks, and 12 h of re-feeding did not change these. IGFBP-1 and -2 mRNA levels were markedly increased by 12 h of fasting in both chicks, and these changes were reversed by re-feeding. IGFBP-3 mRNA levels were increased by 12 h of fasting only in layer chicks, while re-feeding reduced the mRNA levels of IGFBP-3 in both types of chicks. These results suggest that several peripheral hormones, such as pancreatic PYY and intestinal CCK, may not play important roles in the regulation of food intake in broiler chicks.

Central administration of insulin-like growth factor-2 suppresses food intake in chicks.

Insulin-like growth factor (IGF)-2 is a multifunctional hormone with structural and functional similarity to IGF-1 in mammals and chickens. We previously showed that intracerebroventricular administration of IGF-1 suppresses food intake in chicks. Also, central administration of IGF-2 suppresses food intake in rats. In the present study, we evaluated whether IGF-2 is involved in the regulation of food intake in chicks. We also examined the effects of fasting on the mRNA levels of IGF binding proteins (IGFBPs) in the liver and hypothalamus, because IGFBPs bind IGF-1 and -2 in plasma and block their binding to the receptors, and locally expressed IGFBPs also influence IGFs binding to the receptors in mammals. Intracerebroventricular administration of IGF-2 significantly suppressed food intake in chicks. The mRNA levels of IGFBPs in the hypothalamus were not affected by six hours of fasting. On the other hand, six hours of fasting markedly increased the mRNA levels of hepatic IGFBP-1 and -2 (5.47- and 6.95-fold, respectively). The mRNA levels of IGFBP-3 were also significantly increased (1.36-fold) by six hours of fasting, whereas the mRNA levels of IGF-2, IGFBP-4, and -5 were unchanged. These findings suggest that circulating IGF-2 may be involved in satiety signals, but its physiological role may be regulated by IGFBPs production in the liver in chicks.

The Extract of Soybean Protein Increases Slow-Myosin Heavy Chain Expression in C2C12 Myotubes.

Skeletal muscle is composed of four types of fibers in mammals; oxidative slow-twitch type I, oxidative fast-twitch IIA, and glycolytic fast-twitch IIB and IIX/D. In this study using C2C12 myotubes, an extract of soybean protein significantly upregulated mRNA level of myosin heavy chain 7 (Myh7), the predominant isoform expressed in oxidative slow-twitch type I and downregulated mRNA levels of Myh4, the predominant isoform expressed in glycolytic fast-twitch IIB. Similarly, its hydrolysate prepared using digestive enzyme also significantly increased Myh7 expression. In contrast, no significant change was observed in Myh4 mRNA level after the hydrolysate treatment. These findings suggest that dietary intake of the soybean protein extract may increase oxidative slow-twitch fiber in skeletal muscle.

Hypothalamic Akt-mediated signaling regulates food intake in chicks.

The central anorexigenic mechanism seems to be similar in mammals and chicks, because the appetite-suppressive action of a number of peptide hormones is similar in both species. Accumulating evidence in mammals has revealed that hypothalamic Akt-mediated signaling factors (for instance, mTOR and FOXO1) are significantly involved in the regulation of food intake. However, the role of hypothalamic Akt in feeding regulation is yet to be determined in chickens. In this study, we showed that pAkt (Thr308)/Akt, pFOXO1/FOXO1, and pS6 levels were significantly increased in the hypothalami of chicks refed 1 h after a 24 h-fast in correlation to increases in the plasma concentrations of insulin, one of the activators of the Akt-mediated signaling pathways. In addition, central administration of insulin increased the phosphorylation of Akt, FOXO1, and S6 in chicken hypothalami. Furthermore, intracerebroventricular injections of both phosphoinositide 3-kinase inhibitor LY294002 and mTOR inhibitor rapamyacin enhanced the food intake of chicks. These findings suggest that hypothalamic Akt-mediated signaling pathways contribute to the regulation of food intake in chicks.

Effects of dietary heme iron and exercise training on abdominal fat accumulation and lipid metabolism in high-fat diet-fed mice.

Animal by-products can be recycled and used as sources of essential nutrients. Water-soluble heme iron (WSHI), a functional food additive for supplementing iron, is produced by processing animal blood. In this study, we investigated the effects of dietary supplementation of 3% WSHI and exercise training for 4 weeks on the accumulation of abdominal fat and lipid metabolism in mice fed high-fat diet. Exercise-trained mice had significantly less perirenal adipose tissue, whereas WSHI-fed mice tended to have less epididymal adipose tissue. In addition, total weight of abdominal adipose tissues was significantly decreased in the Exercise + WSHI group. Dietary WSHI significantly increased the messenger RNA (mRNA) levels of lipoprotein lipase and hormone-sensitive lipase. WSHI-fed mice also tended to show increased mRNA levels of adipose triglyceride lipase in their epididymal adipose tissue. Dietary WSHI also significantly decreased the mRNA levels of fatty acid oxidation-related enzymes in the liver, but did not influence levels in the Gastrocnemius muscle. Exercise training did not influence the mRNA levels of lipid metabolism-related enzymes in the epididymal adipose tissue, liver or the Gastrocnemius muscle. These findings suggest that the accumulation of abdominal fat can be efficiently decreased by the combination of dietary WSHI and exercise training in mice fed high-fat diet.

Identification, expression analysis, and functional characterization of peptide YY in chickens (Gallus gallus domesticus).

Peptide YY (PYY) functions as a postprandial satiety signal in mammals. However, the genomic information and physiological roles of chicken PYY have not yet been clarified, although PYY peptide was isolated from chicken intestines in 1992. In this study, we identified a full-length complementary DNA (cDNA) sequence encoding the chicken PYY precursor. The deduced amino acid sequence of chicken PYY was completely consistent with the previously identified peptide sequence. PYY mRNA was abundantly expressed in the small intestine compared with the large intestine. PYY mRNA levels in the jejunum were significantly higher during ad libitum feeding compared with fasting, suggesting that intestinal PYY expression is altered in response to nutritional status in chicks. Intravenous administration of PYY significantly suppressed food intake in chicks. Furthermore, neuropeptide Y receptor Y2, a possible target of PYY, was expressed in various brain regions including the appetite-regulating centers in chicks. This is the first evidence that the intestinal hormone PYY may function as an anorexigenic hormone in chicks.

Correlation analysis of hypothalamic mRNA levels of appetite regulatory neuropeptides and several metabolic parameters in 28-day-old layer chickens.

Various lines of evidence suggest that appetite-related neuropeptides in the hypothalamus are regulated by adiposity signals such as leptin and insulin in mammals. In the present study, we examined age-dependent changes in the weight of abdominal fat and hypothalamic mRNA levels of neuropeptide Y (NPY, an orexigenic neuropeptide) and proopiomelanocortin (POMC, a precursor of anorexigenic neuropeptides) in growing chickens at 7, 14, 21 and 28 days of age. Hypothalamic NPY mRNA levels were significantly (P < 0.05) decreased after 14 days of age, whereas hypothalamic POMC mRNA levels were significantly (P < 0.05) increased at 28 days of age. The percentage of abdominal fat was significantly increased after 14 days of age in chickens. We next examined the correlation of hypothalamic NPY and POMC mRNA levels and several parameters at 28 days of age. There were no significant correlations between hypothalamic mRNA levels of NPY or POMC and the percentage of abdominal fat. These findings suggest that the gene expressions of NPY and POMC do not depend on adiposity in chickens, at least in 28-day-old layer chickens.

Enzymatically synthesized glycogen reduces lipid accumulation in diet-induced obese rats.

Based on a recent study indicating that enzymatically synthesized glycogen (ESG) possesses a dietary, fiber-like action, we hypothesized that ESG can reduce the risk of obesity. In this study, the antiobesity effects of ESG were investigated in a model of diet-induced obesity. Male Sprague-Dawley rats were divided into 4 groups and fed a normal or high-fat diet, with or without 20% ESG, for 4 weeks. Body weight, food intake, lipid deposition in the white adipose tissues and liver, fecal lipid excretion, and plasma lipid profiles were measured. At week 3, the body fat mass was measured using an x-ray computed tomography system, which showed that ESG significantly suppressed the high-fat diet-induced lipid accumulation. Similar results were observed in the weight of the adipose tissue after the experiment. Moreover, ESG significantly suppressed the lipid accumulation in the liver but increased fecal lipid excretion. The plasma concentrations of triacylglycerol and nonesterified fatty acid were lowered after a high-fat diet, whereas the total bile acid concentration was increased by ESG. However, the hepatic messenger RNA (mRNA) levels of enzymes related to lipid metabolism were not affected by ESG. Conversely, the mRNA levels of long-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase were up-regulated by ESG in the muscle. These results suggest that the combined effects of increased fecal lipid excretion, increased mRNA levels of enzymes that oxidize fatty acids in the muscle, and increased total bile acid concentration in the plasma mediate the inhibitory effect of ESG on lipid accumulation.

Effect of licorice flavonoid oil on cholesterol metabolism in high fat diet rats.

Dietary licorice fravonoid oil (LFO) significantly decreased hepatic cholesterol and plasma lipoprotein cholesterol levels in high-fat diet rats. It significantly suppressed hydroxymethylglutaryl-CoA synthase activity and increased cholesterol 7α-hydroxylase activity. The low density lipoprotein receptor mRNA level was significantly increased by LFO. These results suggest that dietary LFO improves cholesterol metabolism in obese animals.

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

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

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.

Corticotropin-releasing factor is a downstream mediator of the beta-melanocyte-stimulating hormone-induced anorexigenic pathway in chicks.

Proopiomelanocortin (POMC, a precursor of anorexigenic neuropeptides) neurons in hypothalamus suppresses food intake in both mammals and chickens. In mammals, several lines of evidence suggest that POMC-derived anorexigenic peptides upregulate mRNA levels of anorexigenic peptides such as corticotropin-releasing factor (CRF) and thyrotropin-releasing factor and downregulate mRNA levels of orexigenic peptides such as orexin and melanin-concentrating hormone. However, the POMC-induced anorexigenic pathway in chickens has not been well characterized. In the present study, we investigated how POMC neurons regulate mechanisms of food intake using an anorexigenic peptide, beta-melanocyte-stimulating hormone (beta-MSH), derived from the post-transcriptional cleavage of POMC. Central administration of beta-MSH in chicks significantly suppressed food intake, and importantly, this suppression was accompanied by a significant upregulation of CRF mRNA levels. Furthermore, the CRF type 2 receptor antagonist alpha-helical CRF significantly reversed the anorexigenic action of beta-MSH. These findings indicate that CRF and its receptor, CRF type 2 receptor, act as the major mediators in beta-MSH-induced anorexigenic action in chicks. beta-MSH significantly increased orexin mRNA levels and did not alter mRNA levels of thyrotropin-releasing factor and melanin-concentrating hormone in chicks, suggesting that the beta-MSH-induced anorexigenic pathway in chicks is different from that in mammals. Increases in orexin mRNA levels were accompanied by significant decreases in plasma glucose concentration, suggesting that orexin mRNA might be stimulated by beta-MSH-induced hypoglycemia. Thus, this study demonstrates the direct evidence that CRF is a critical downstream target in the beta-MSH-induced anorexigenic pathway in chicks.

The molecular mechanism underlying the reduction in abdominal fat accumulation by licorice flavonoid oil in high fat diet-induced obese rats.

Licorice (Glycyrrhiza glabra) has been widely used in traditional medicines, and its flavonoid oil (LFO) decreases abdominal adipose tissue weight in mammals. In the present study, we investigated the molecular mechanisms underlying the decrease in abdominal adipose tissue weight by LFO. LFO significantly decreased the mRNA levels of rate-limiting enzymes in the hepatic fatty acid synthetic pathway, whereas LFO significantly increased the mRNA levels of a rate-limiting enzyme in the hepatic fatty acid oxidative pathway. LFO significantly decreased the mRNA levels of sterol regulatory element-binding protein-1c (SREBP-1c) (a transcription factor that promotes hepatic fatty acid synthesis), whereas the mRNA levels of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) (a transcription factor that promotes hepatic fatty acid oxidation) was significantly increased. All our findings suggest that the decrease in abdominal adipose tissue weight by LFO is mediated by the transcriptional regulation of SREBP-1c and PPAR-alpha in the liver. Thus, we infer that the natural ingredient LFO is a promising candidate for use as a feed additive to reduce abdominal fat accumulation in domestic animals.

Investigation of the anti-obesity action of licorice flavonoid oil in diet-induced obese rats.

Licorice flavonoid oil (LFO), which contains hydrophobic flavonoids from Glycyrrhiza glabra LINNE, is a new ingredient for functional foods. In this study, we investigated the anti-obesity action of LFO in diet-induced obese rats. The addition of 2% LFO in a high-fat diet significantly decreased the weight of abdominal adipose tissue and the levels of hepatic and plasma triglycerides. We found that the enzymatic activities of acetyl-CoA carboxylase and fatty acid synthase, the rate-limiting enzymes in the fatty acid synthetic pathway, were significantly decreased by LFO, whereas the enzymatic activity of acyl-CoA dehydrogenase, the rate-limiting enzyme in the fatty acid oxidative pathway, was significantly increased. All our findings suggest that the anti-obesity action of LFO is controlled by regulation of the rate-limiting enzymes in the fatty acid synthetic and oxidative pathways in the liver.

Central administration of glucagon suppresses food intake in chicks.

Food intake in chickens is regulated in a manner similar to that in mammals. Corticotropin-releasing factor (CRF), which increases the plasma corticosterone concentration, plays an important role as a mediator of many appetite-suppressive peptides in the central nervous system in both species. Central administration of glucagon suppresses food intake in rats. However, the anorexigenic action of glucagon in chicks has not yet been identified. In the present study, we investigated the effects of central administration of glucagon on food intake in chicks. Intracerebroventricular administration of glucagon in chicks significantly suppressed food intake and significantly induced hyperglycemia. In contrast, peripheral administration of the same dose of glucagon did not influence food intake and plasma glucose concentration. These results suggest that glucagon functions in chicks as an appetite-suppressive peptide in the central nervous system. Intracerebroventricular administration of glucagon in chicks also significantly increased CRF mRNA expression and plasma corticosterone concentration, suggesting that CRF acts as a downstream molecule for a glucagon-induced appetite-suppressive pathway in chicks. It is likely that the induction of hyperglycemia by central administration of glucagon is involved in its anorexigenic action, because peripheral administration of glucose in chicks suppressed food intake. These results suggest that CRF- and/or hyperglycemia-mediated pathways are involved in the anorexigenic action of glucagon in chicks.