In order to evaluate the role of insulin in chicken, an insulin immuno-neutralization was performed. Fed chickens received 1 or 3 i.v. injections of anti-insulin serum (2-h intervals), while fed or ...fasted controls received normal serum. Measurements included insulin signaling cascade (at 1 h in liver and muscle), metabolic or endocrine plasma parameters (at 1 and 5 h), and qRT-PCR analysis (at 5 h) of 23 genes involved in endocrine regulation, metabolisms, and transcription. Most plasma parameters and food intake were altered by insulin privation as early as 1 h and largely at 5 h. The initial steps of insulin signaling pathways including insulin receptor (IR), IR substrate-1 (IRS-1), and Src homology collagen and downstream elements: phosphatidylinositol 3-kinase (PI3K), Akt, GSK3, ERK2, and S6 ribosomal protein) were accordingly turned off in the liver. In the muscle, IR, IRS-1 tyrosine phosphorylation, and PI3K activity remained unchanged, whereas several subsequent steps were altered by insulin privation. In both tissues, AMPK was not altered. In the liver, insulin privation decreased Egr1, PPAR gamma, SREBP1, THRSP alpha (spot 14), D2-deiodinase, glucokinase (GK), and fatty acid synthase (whereas D3-deiodinase and IGF-binding protein 1 transcripts were up-regulated. Liver SREBP1 and GK and plasma IGFBP1 proteins were accordingly down- and up-regulated. In the muscle, PPAR beta delta and atrogin-1 mRNA increased and Egr1 mRNA decreased. Changes in messengers were partly mimicked by fasting. Thus, insulin signaling in muscle is peculiar in chicken and is strictly dependent on insulin in fed status. The 'diabetic' status induced by insulin immuno-neutralization is accompanied by impairments of glucagon secretion, thyroid axis, and expression of several genes involved in regulatory pathways or metabolisms, evidencing pleiotropic effects of insulin in fed chicken.
AMP-activated protein kinase (AMPK) is a fuel sensor in glucose, lipid, and cholesterol metabolism. Using RT-PCR and Western blot, AMPK subunits mRNAs (alpha1/2, beta1/2, and gamma1/2) and proteins ...(alpha1/2 and beta1/2) can be found in the hen preovulatory follicles and precisely in both granulosa and theca cells. These preovulatory follicles are organized in a hierarchy according to their size (F5/6 to F1). The smallest number (F1) corresponds to the largest size and the latest mature stage. Phosphorylation of AMPKalpha on Thr172 and of acetyl-CoA carboxylase on Ser79 are higher in F4 and F3 than in F1 granulosa cells. However, they are not affected in F4-F1 theca cells. Treatment with 1 mM 5-amino-imidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, dose dependently increased phosphorylation of AMPKalpha on Thr172 in primary F3/4 and F1 granulosa cells. In the absence of FSH, AICAR treatment increased progesterone, P450 side chain cleavage and steroidogenic acute regulatory (StAR) production in both F3/4 and F1 granulosa cells. However, in the presence of FSH, AICAR treatment for 36 h increased progesterone secretion, StAR protein levels and reduced extracellular signal-regulated kinase (ERK)1/2 phosphorylation in F3/4 granulosa cells. Opposite data were observed in F1 granulosa cells. Adenovirus-mediated expression of dominant-negative AMPK totally restored the effects of AICAR on FSH-induced progesterone secretion, StAR protein production, and ERK1/2 phosphorylation in F3/4 and F1 granulosa cells. Using a specific inhibitor of ERK1/2 (U0126), we also showed that this kinase is a negative regulator of the FSH-induced progesterone secretion in F3/4 and F1 granulosa cells, suggesting that AICAR-mediated AMPK activation modifies FSH-induced progesterone secretion differently through the ERK1/2 signaling pathway in hen F3/4 and F1 granulosa cells.
In hens, the ovarian follicles committed to ovulation are arranged in an ordered follicular hierarchy. In standard broiler breeders hens genetically selected for high growth rate the reproductive ...function is clearly dysfunctional. Feed restriction is needed during reproductive development to limit the formation of excessive numbers of ovarian yellow follicles arranged in multiple hierarchies. To determine whether leptin is involved in the nutritional and reproductive interactions controlling follicular hierarchy in hens, blood leptin levels and ovarian expression of the leptin receptor mRNA were determined during follicle maturation in three chicken lines; a slow growing broiler "Label" genotype without reproductive dysfunction, a fast growing "Standard" genotype fed ad libitum or restricted and a fast growing "Experimental" line with intermediate reproductive performance levels. Whereas expression of the leptin receptor mRNA did not change in the theca, it clearly decreased with follicular differentiation in the granulosa of slow growing hens. In fast growing standard hens fed ad libitum and presenting significant reproductive dysfunction, the decrease was disrupted and dramatic up-regulation of granulosa cell expression of the leptin receptor was observed. On the other hand, feed restriction decreased the overall level of expression of the leptin receptor mRNA and restored the decrease with follicular growth. The level of expression of the leptin receptor probably modulates the action of leptin on follicular differentiation. Since blood leptin and other metabolic factors were not affected by the genotype or by nutritional state, the factors involved in the regulation of leptin receptor gene expression remain to be determined. This study demonstrates the involvement of leptin in the nutritional control of reproduction in birds. Leptin action on the ovary probably controls follicular hierarchy through the regulation of steroidogenesis.
In mammals, insulin regulates S6K1, a key enzyme involved in the control of protein synthesis, via the well-documented phosphoinositide-3′kinase (PI3K) pathway. Conversely, S6K1 is activated by ...insulin in avian muscle despite the relative insulin insensitivity of the PI3K pathway in this tissue. Mitogen-activated protein kinase (MAPK) cascade is another insulin sensitive pathway. The aim of this study was to explore the potential involvement of the ERK1/2 MAPK pathway in the control of p70 S6 kinase (S6K1) in avian species. Firstly, we characterized ERK1/2 MAPK in various chicken tissues. ERK2 was the only isoform detected in avian species whatever the tissue studied. We also showed that ERK2 is activated in vivo by insulin in chicken muscle. The regulation and the role of ERK2 in insulin signaling were next investigated in chicken hepatoma cells (LMH) and primary myoblasts. Insulin stimulation led to ERK2 and S6K1 phosphorylation, and concomitantly increased kinase activity. U0126, an inhibitor of the ERK MAPK pathway, completely abolished insulin-induced S6K1 phosphorylation and activity in chicken myoblasts, whereas its effect was only partial in LMH cells. In conclusion, these results show that ERK1/2 MAPK is involved in the control of S6K1 by insulin in chicken cells, particularly myoblasts.
The phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase able to inhibit significant actors of cell signaling (i.e. phosphatidylinositol-3′kinase and ...mitogen-activated protein kinase pathways). The aim of this study was to characterize PTEN and to investigate its regulation during ontogenesis in chicken muscle. Pectoralis major muscle was sampled on day 18 of the embryonic period (E18), at hatching (d0) and in fed chickens at 2, 7 and 43 days after hatching (d2, d7 and d43). We first cloned the totality of chicken PTEN cDNA; its translation into a putative protein showed more than 95% sequence identity with that characterized in mammals (humans, mice). PTEN was expressed under two major transcripts in the majority of tissues, including muscles where the expression of PTEN mRNA increased with age (
P
<
0.05). Surprisingly, the protein levels of PTEN (protein characterized with an apparent molecular weight of 55
kDa) and its activity were considerably decreased between the E18 and d43 stages (approximately 8–10-fold reduction,
P
<
0.001). An association between these decreases and higher phosphorylation levels of two potential indirect downstream targets of phosphatase (i.e. AKT and ERK) was observed only in the early growth phases. It was concluded that phosphatase PTEN was expressed in chicken muscle and that its expression was regulated during ontogenesis.
Decades of genetic selection have generated 2 different, highly specialized types of chickens in which 1 type, known as the layer-type chicken, expresses high laying performance while the other type, ...known as the broiler-type chicken, is dedicated to the production of fast-growing birds. Selected lines for the latter type often express disorders in their reproductive performance including early sexual maturation and accelerated, non-reversible seasonal decline of their semen production and mating behavior. The aim of the present study was to characterize some metabolic markers of the Sertoli cell populations. Sertoli cells are somatic cells known to support, coordinate, nourish, and protect the germ cell populations from onset to the end of their meiotic process. Comparisons of gonadal development between males of the 2 genetic types taken at their pre-pubertal period indicated that the testes of layer-type chickens are significantly less developed than in broiler-type males taken at the same age. In addition, cultures of purified Sertoli cells from the 2 types revealed in vitro a higher proliferative capacity when issued from layer compared to broiler-type chickens. This was associated with a higher expression of the genes involved in the beta-oxidation of fatty acids (CPT1; PPARβ) as well as a 4-fold increase in the Lactate Dehydrogenase-A expression and activity. In contrast, Sertoli cells from broiler-type chickens presented an elevated activity of citrate synthase and mitochondria, suggesting a better efficacy of aerobic metabolism in Sertoli cells from broiler compared to layer-type chickens. Moreover, the testis from broiler-type chickens seems to be more sensitive to oxidative stress due to the lower global antioxidant capacity compared to layer-type chickens.In conclusion, these results suggest that the metabolic activity of testicular tissues is different in the layer and broiler breeder chickens. The aerobic metabolism more prevalent in broiler-type chickens could be a factor to reduce the male fertility such as germ cell quality.
Abstract
Glucose transport into cells is the first limiting step for the regulation of glucose homeostasis. In mammals, it is mediated by a family of facilitative glucose transporters (GLUTs) ...(encoded by SLC2A* genes), with a constitutive role (GLUT1), or insulin-sensitive transporters (GLUT4, GLUT8, and GLUT12). Compared to mammals, the chicken shows high levels of glycemia and relative insensitivity to exogenous insulin. To date, only GLUT1, GLUT8, and GLUT12 have been described in chicken skeletal muscles but not fully characterized, whereas GLUT4 was reported as lacking. The aim of the present study was to determine the changes in the expression of the SLC2A1, SLC2A8, and SLC2A12 genes, encoding GLUT1, GLUT8, and GLUT12 proteins respectively, during ontogenesis and how the respective expression of these three genes is affected by the muscle type and the nutritional or insulin status of the bird (fed, fasted, or insulin immunoneutralized). SLC2A1 was mostly expressed in the glycolytic pectoralis major (PM) muscle during embryogenesis and 5 d posthatching while SLC2A8 was mainly expressed at hatching. SLC2A12 expression increased regularly from 12 d in ovo up to 5 d posthatching. In the mixed-type sartorius muscle, the expression of SLC2A1 and SLC2A8 remained unchanged, whereas that of SLC2A12 was gradually increased during early muscle development. The expression of SLC2A1 and SLC2A8 was greater in oxidative and oxidoglycolytic muscles than in glycolytic muscles. The expression of SLC2A12 differed considerably between muscles but not necessarily in relation to muscle contractile or metabolic type. The expression of SLC2A1, SLC2A8, and SLC2A12 was reduced by fasting and insulin immunoneutralization in the PM muscle, while in the leg muscles only SLC2A12 was impaired by insulin immunoneutralization. Our findings clearly indicate differential regulation of the expression of three major GLUTs in skeletal muscles, with some type-related features. They provide new insights to improve the understanding of the fine regulation of glucose utilization in chicken muscles.
Meat type chickens have limited capacities to cope with high environmental temperatures, this sometimes leading to mortality on farms and subsequent economic losses. A strategy to alleviate this ...problem is to enhance adaptive capacities to face heat exposure using thermal manipulation (TM) during embryogenesis. This strategy was shown to improve thermotolerance during their life span. The aim of this study was to determine the effects of TM (39.5 °C, 12 h/24 vs 37.8 °C from d7 to d16 of embryogenesis) and of a subsequent heat challenge (32 °C for 5 h) applied on d34 on gene expression in the Pectoralis major muscle (PM). A chicken gene expression microarray (8 × 60 K) was used to compare muscle gene expression profiles of Control (C characterized by relatively high body temperatures, Tb) and TM chickens (characterized by a relatively low Tb) reared at 21 °C and at 32 °C (CHC and TMHC, respectively) in a dye-swap design with four comparisons and 8 broilers per treatment. Real-time quantitative PCR (RT-qPCR) was subsequently performed to validate differential expression in each comparison. Gene ontology, clustering and network building strategies were then used to identify pathways affected by TM and heat challenge.
Among the genes differentially expressed (DE) in the PM (1.5 % of total probes), 28 were found to be differentially expressed between C and TM, 128 between CHC and C, and 759 between TMHC and TM. No DE gene was found between TMHC and CHC broilers. The majority of DE genes analyzed by RT-qPCR were validated. In the TM/C comparison, DE genes were involved in energy metabolism and mitochondrial function, cell proliferation, vascularization and muscle growth; when comparing heat-exposed chickens to their own controls, TM broilers developed more specific pathways than C, especially involving genes related to metabolism, stress response, vascularization, anti-apoptotic and epigenetic processes.
This study improved the understanding of the long-term effects of TM on PM muscle. TM broilers displaying low Tb may have lower metabolic intensity in the muscle, resulting in decreased metabolic heat production, whereas modifications in vascularization may enhance heat loss. These specific changes could in part explain the better adaptation of TM broilers to heat.
To improve the early perinatal conditions of broiler chicks, alternative hatching systems have beendeveloped. On-farm hatching (OFH) with an enriched microbial and stimulating environment by ...thepresence of an adult hen is a promising solution. Day-old chicks were allotted within five hatchingand rearing conditions: OFH, conventional hatchery (CH), CH and post-hatching treatment withantibiotics (CH + AB), as well as both hatching systems with an adult hen at hatching (OFH + H,CH + H). To challenge the robustness of chickens, they were exposed on D27 to suboptimal rearingconditions by combining for 4 h transport in boxes in a new room at a lower temperature andfasting. On their return to the original room, the chicken density was increased, and birds wereorally vaccinated with the Gumboro vaccine. The impacts of these conditions on hatchability, chickquality score, performance, health and robustness were determined. The OFH chick body weights(BWs) were significantly greater than those of CH chicks at hatching.Whereas there was no effectof hatching conditions, the presence of hens decreased the hatchability rate, the quality score ofOFH chicks and increased mortality at hatching. Treatment of CH chicks with antibiotics (CH + AB)temporarily decreased chicken BW at D19, but the feed conversion ratio (FCR) was not modified.At D19, OFH chicks had the highest BWcompared to the other groups, and the presence of hens athatching harmed chicken BWregardless of the hatching condition and FCR. An interaction betweenthe effect of experimental rearing conditions and chicken sex was observed later for BW. In males,the OFH chickens were the heaviest compared to the other groups at D34 but not at D56. Thepresence of hens negatively impacted CH chicken BW at D56. In females, there was no effect ofhatching condition on the BWs at D34 and D56, and the presence of hens had a positive impact onOFH chicken BW. There was no effect of hatching conditions on health parameters. In conclusion,the OFH system was a hatching system at least equivalent to the CH system. The presence of thehen at hatching and during the chick start-up phase on performance interacted with the hatchingcondition and the sex of the chickens.
Genetic selection has significantly improved the muscle development of fast-growing broiler chickens in the last 50 yr. However, improvement in muscle growth has coincided with relatively poor ...development of visceral systems, resulting in impaired ability to cope with high environmental temperatures. The aim of this study was to elucidate the effects of thermal manipulation (TM) during different periods of embryogenesis on chick hatchability, BW and thermoregulation upon hatching, on their ability to cope with thermal challenge at 42 d of age, and on carcass and breast meat traits. Control embryos were incubated at 37.8 degrees C. The TM embryos were incubated at 37.8 degrees C and treated for 3 h at 39.5 degrees C on the following days of embryogenesis: E8 to E10 early (EA), E16 to E18 late (LA), and both E8 to E10 and E16 to E18 (EA-LA). Body weight and body temperature (T(b)) were measured at hatching and throughout the growth period as well as during exposure of 42-d-old chickens to a thermal challenge at 35 degrees C for 6 h. The LA and EA chicks exhibited significantly lower T(b) than control chicks (37.9 vs. 38.2 degrees C) at hatching, but during the growth period, differences in T(b) between treated and control chicks decreased with age. Significant hyperthermia (over 44 degrees C) was monitored in all groups during the thermal challenge, but mortality was higher in treated than in control chickens. No effect of treatments on BW was found during the entire growth period. However, breast yield was higher in LA chickens than in controls at slaughter. The EA and EA-LA treatments slightly decreased the ultimate pH of breast meat, whereas the LA treatment had no effect. In conclusion, none of the TM conditions tested in the present study were able to improve long-term thermotolerance in chickens. Late treatment favored breast muscle growth without affecting ultimate pH and drip loss of breast meat.
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