Amino acids are known to be anabolic factors that affect protein metabolism, but the response of animals to daily amino acid changes is little understood. We aimed to test the effects of feeding ...birds with alternations of diets varying in lysine content on the expression of genes related to proteolysis in chicken muscle. Cyclic feeding programs with 2 diets, each given for 24 h during 48-h cycles, were carried out from 10 d of age. Three programs were used: 1) control treatment with continuous distribution of a complete diet containing standard medium lysine level (ML; 11.9 g/kg); 2l alternation of diets with high (HL) and low (LL) lysine levels; 3) alternation of ML and LL diets, where LL = 70%, ML = 100%, HL = 130% of standard lysine level. The Pectoralis major muscles were sampled after 2 wk of cyclic feeding. Measurements included the expression patterns of 6 genes involved in proteolysis, and mammalian target of rapamycin and Forkhead box-O transcription factor (FoxO) signaling. Cathepsin B, m-calpain, and E3 ubiquitin ligases Muscle Ring Finger-1 and Muscle Atrophy F box were significantly overexpressed in chickens transiently fed the LL diet, whereas the mRNA levels of 20S proteasome C2 subunit and ubiquitin remained unchanged. Modifications of E3 ubiquitin ligase expression can be partly explained by significant changes in FoxO phosphorylation with cyclic dietary treatments. Our results suggest timing-sensitive regulation of proteolysis in chicken muscle according to dietary treatment and a high metabolism capacity to compensate for changes in amino acid supply, which might be used for nutritional purposes. PUBLICATION ABSTRACT
Amino acids are known to be anabolic factors that affect protein metabolism, but the response of animals to daily amino acid changes is little understood. We aimed to test the effects of feeding ...birds with alternations of diets varying in lysine content on the expression of genes related to proteolysis in chicken muscle. Cyclic feeding programs with 2 diets, each given for 24 h during 48-h cycles, were carried out from 10 d of age. Three programs were used: 1) control treatment with continuous distribution of a complete diet containing standard medium lysine level (ML; 11.9 g/kg); 2) alternation of diets with high (HL) and low (LL) lysine levels; 3) alternation of ML and LL diets, where LL = 70%, ML = 100%, HL = 130% of standard lysine level. The Pectoralis major muscles were sampled after 2 wk of cyclic feeding. Measurements included the expression patterns of 6 genes involved in proteolysis, and mammalian target of rapamycin and Forkhead box-O transcription factor (FoxO) signaling. Cathepsin B, m-calpain, and E3 ubiquitin ligases Muscle Ring Finger-1 and Muscle Atrophy F box were significantly overexpressed in chickens transiently fed the LL diet, whereas the mRNA levels of 20S proteasome C2 subunit and ubiquitin remained unchanged. Modifications of E3 ubiquitin ligase expression can be partly explained by significant changes in FoxO phosphorylation with cyclic dietary treatments. Our results suggest timing-sensitive regulation of proteolysis in chicken muscle according to dietary treatment and a high metabolism capacity to compensate for changes in amino acid supply, which might be used for nutritional purposes.
To explore the mechanisms leading to excessive adiposity in chicken, we investigated the regulation of fatty acid oxidation depending on genotype-related body fatness and diet composition. mRNA ...expression and/or activity of proteins involved in mitochondrial energy metabolism were measured in liver and
gastrocnemius muscle of genetically lean or fat chickens reared on a low-fat/high-protein diet or an isoenergetic high-fat/low-protein diet (HF/LP). Muscle expressions of the muscle isoform of carnitine-palmitoyltransferase 1 (M-CPT1) and PPARβ/δ were higher in fat than in lean chickens. This was also observed in liver, although only with the HF/LP diet for M-CPT1. This could stimulate mitochondrial fatty acid oxidation in fat chickens. Up-regulations of liver and muscle CPT-1 hepatic isoform, and muscle cytochrome-c-oxidase mRNA expressions, and of β-hydroxyacyl-CoA-dehydrogenase activities suggest higher fatty acid utilization with the HF/LP diet. PPARβ/δ and PGC-1α could control fatty acid oxidation in muscle and liver, respectively. Regulation of avian uncoupling protein (avUCP) mRNA was tissue-dependent. Predominantly expressed in muscle, it was stimulated in fat and in HF/LP-fed chickens, where it could be associated to the special need in muscle anti-oxidant pathways of fatter animals. In liver it was lower in fat than in lean chickens, and its potential function remains to be clarified.
In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and ...no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.
The acute effect of leptin on the regulation of food intake was investigated in layer and broiler chickens. In an initial study, we observed that a single intraperitoneal injection of recombinant ...chicken leptin (1
mg/kg BW) dramatically reduced (38%) food intake in 56-day-old layer chickens, more moderately reduced (15%) food intake in 9-day-old layer chicks, and had no significant effect in 9-day-old broiler chicks. In a subsequent study, body weight and plasma concentrations of leptin were measured weekly in layer and broiler chicks from day 1 to 35 of age and brain leptin receptor and neuropeptide Y (NPY) mRNA expression were analyzed at 1, 9, and 35 days of age. At day 1 of age, peripheral concentrations of leptin were significantly greater in layer than broiler chicks. Subsequently, despite increases in body weight and differences in growth rates between layer and broiler chicks from day 8 to day 35 of age, peripheral concentrations of leptin were constant and similar in both genotypes. Leptin receptor and NPY mRNA were expressed in brain from day 1 in chicks of both genotypes and increased significantly to day 35 of age. These observations provide evidence that the inhibitory effect of leptin on the regulation of food intake in growing chicks is an age dependent process. Furthermore, acquisition of the anorectic effect of leptin is likely to be associated with greater expression of the leptin receptor and NPY mRNAs than to changes in blood levels of leptin. Finally, this study provides evidence that chickens selected for high growth rates may be less sensitive or responsive to peripheral concentrations of leptin than chickens with low growth rates (layers), suggesting that the faster growth of broiler chicks may be related to a lessened responsiveness to anorexigenic factors.
In chickens, leptin is expressed mainly in the liver, where its receptor gene expression has also been reported, and in adipose tissue. In view of the key role played by the liver in lipogenesis in ...avian species, the hepatic expression of leptin may have physiological significance. In this study, we showed that leptin is constitutively expressed and secreted in a chicken-derived hepatoma cell line (LMH). Although insulin regulates leptin expression in vivo, incubation of LMH cells in the presence of 100 nM insulin for 24 or 48 h had no effect on leptin expression or its secretion in the culture medium. In addition, we developed a specific chicken leptin receptor real-time reverse transcription (RT)-PCR, and downregulation of leptin receptor gene expression by homologous and heterologous signals was demonstrated, as relative leptin receptor mRNA levels were significantly decreased after exposure of LMH cells to recombinant chicken leptin or porcine insulin. In conclusion, our results indicate that leptin is probably able to desensitize its own response in the chicken liver. Finally, the ability of insulin and leptin to regulate chicken leptin receptor gene expression suggests a direct role of leptin in the control of hepatic metabolism.
Early-age thermal conditioning (TC) by exposing young chicks to 40 C for 24 h reduces body temperature (Tb) and has been showed by others to improve long-term resistance of broilers to heat stress. ...Uncoupling oxidative phosphorylation in pectoral muscle mitochondria might be related to heat production. Fertile eggs were hatched under video control, and 161 pedigree chicks froml2 sires and 22 dams were immediately allocated to two groups (T, a group composed of 81 chicks exposed to TC at 5 d of age, and N, a control group of 80 nonexposed chicks). Body weights and Tb were measured at 2 and 7 d of age. Five pairs (one N and one T) of full sib chicks from families that exhibited the largest difference of Tb variation from 2 to 7 d of age between the two treatments were chosen for pectoral muscle sampling. Avian uncoupling protein (avUCP) messenger RNA expression was measured by reverse transcript-PCR coupled to southern blot in the pectoral muscle of 7-d-old broiler chicks. At 7 d of age, there were no BW differences between treatments and Tb was significantly reduced by TC (-0.13 C on average). Heritability of Tb variation between 2 and 7 d was 0.38 +/- 0.20 (SE) for T chicks and 0.35 +/- 0.17 for N chicks without a significant genetic correlation between the two environments. Expression of avUCP mRNA was significantly (85%) lower in T chicks than in N chicks. Uncoupling protein mRNA expression in pectoral muscle and Tb are quickly adjusted in broiler chicks 24 h after early thermal conditioning.
The avian uncoupling protein 3 (UCP3), mainly expressed in muscle tissue, could be involved in fatty acid (FA) metabolism, limitation of reactive oxygen species production, and/or nonshivering ...thermogenesis. We recently demonstrated that UCP3 mRNA expression was increased by isoproterenol (Iso), a β-agonist, in chicken Pectoralis major. This upregulation was associated with changes in FA metabolism and variations in the activation of AMP-activated protein kinase (AMPK) and in the expression of the transcription factors peroxisome proliferator-activated receptor (PPAR)α, PPARβ/δ, and PPARγ coactivator-1α (PGC-1α). The aim of the present study was to elucidate the mechanisms involving AMPK and PPARα in UCP3 regulation in primary cultures of chick myoblasts. Avian UCP3 mRNA expression, associated with p38 mitogen-activated protein kinase (p38 MAPK) activation, was increased by Iso and/or FAs. The PKA pathway mediated the effects of Iso on UCP3 expression. FA stimulation also led to AMPK activation. Furthermore, the direct involvement of AMPK on UCP3 regulation was shown by using 5-aminoimidazole-4-carboxyamide ribonucleoside and Compound C. The use of the p38 MAPK inhibitor SB202190, which was associated with AMPK activation, also dramatically enhanced UCP3 mRNA expression. Finally the PPARα agonist WY-14643 strongly increased UCP3 mRNA expression. This study highlights the control of UCP3 expression by the β-adrenergic system and FA in chick myoblasts and demonstrates that its expression is directly regulated by AMPK and by PPARα. Overexpression of avian UCP3 might modulate energy utilization or limit oxidative stress when mitochondrial metabolism of FA is triggered by catecholamines.
So far, there has been no evidence for any direct pancreatic effect of leptin in the chicken. The present study was aimed at detecting chicken leptin receptor (cOb-R) expression in isolated chicken ...islets of Langerhans and to examine the direct effect of leptin on insulin secretion after stimulation by acetylcholine (1 micro M) + glucose (14 mM) from isolated perfused chicken pancreas. We will show that i) full length cOb-R mRNA was expressed in isolated pancreatic islets of chickens, ii) recombinant chicken leptin (10 nM) or diazoxide (100 micro M) rapidly (within 2 min) and significantly suppressed insulin secretion induced by acetylcholine stimulation without any change in volume outflow rate, iii) tolbutamide (100 micro M) introduced 10 min after leptin and perfused for 10 min fully reversed the suppressive effect of leptin on pre-established acetylcholine-induced insulin release. In conclusion, we found that leptin has a profound inhibitory influence upon insulin secretion in perfused chicken pancreas. The results suggest that leptin inhibits insulin secretion by acting before or at the level of K ATP channels in chicken pancreatic beta-cells. Further studies are warranted to clarify the specific inhibitory mechanism.
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