Comparative Biochemistry and Physiology, Part A 169 (2014) 90–95
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Effects of tonic immobility (TI) and corticosterone (CORT) on energy status and protein metabolism in pectoralis major muscle of broiler chickens Yujing Duan, Wenyan Fu, Song Wang, Yingdong Ni ⁎, Ruqian Zhao Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e
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Article history: Received 26 October 2013 Received in revised form 16 December 2013 Accepted 25 December 2013 Available online 31 December 2013 Keywords: Tonic immobility Corticosterone Protein metabolism Energy status Pectoralis major muscle Broilers
a b s t r a c t Tonic immobility (TI), which can be divided into short (STI) or long (LTI) duration, is a character related to fear. Our previous study has demonstrated LTI phenotype and chronic corticosterone (CORT) administration retarded growth of breast muscle in broiler chickens. In order to investigate the mechanism behind the negative effects of LTI and CORT on growth, the level of mRNA transcription of several key genes linked to energy and protein metabolism was measured in muscle. LTI broilers showed lower levels of ATP, energy charge (EC) (p b 0.01), and lower muscle glycogen content (p b 0.05) but higher level of ADP (p = 0.08) than STI birds. CORT treatment elevated EC level (p b 0.05) and reduced liver glycogen content (p b 0.05). Real-time PCR results showed that STI chickens had higher mRNA expression of PPAR α (p = 0.06) and AMPK α (p = 0.09) than LTI. CORT significantly down-regulated α-enolase mRNA expression in breast muscle compared to control (p b 0.05). Neither TI nor CORT altered gene expression in Akt/mTOR/p70s6k cascade pathway in muscle (p N 0.05). However, western blot results showed that LTI chickens exhibited higher protein content of total Akt (p = 0.05) and phosphorylated Akt (p = 0.06) than STI. CORT treatment decreased the total protein content of Akt (p = 0.09) and p70s6k (p = 0.08). These results suggest that the retardation of muscle growth by LTI and chronic CORT administration parallels a strong alternation in energy status but slight changes of Akt/mTOR/p70s6k cascade, indicating that a decrease in muscle growth induced by LTI and CORT might not be mediated through mTOR-dependent signaling pathways. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Animals show a tonic immobility (TI) response when they are facing danger, attacking or fear. TI is an unlearnt catatonic state considered as the final stage in a chain of anti-predator behavior patterns (Jones, 1986; Mills and Faure, 1991). It has been reported that a strong TI response always associates with high level of endogenous corticosterone (CORT) in mammals (Kalin et al., 1998). Moreover, stressful stimuli can increase the duration of TI in several species (Miranda et al., 2006; Zamudio et al., 2009). Microinjection of CORT into unilateral nucleus
Abbreviations: ADP, adenosine diphosphate; Akt, serine/threonine protein kinase B; AMP, adenosine monophosphate; AMPK α, AMP-activated protein kinase alpha; ATP, adenosine triphosphate; Atrogin-1, muscle atrophy F box; CORT, corticosterone; EC, energy charge; HPLC, high performance liquid chromatography; LTI, long tonic immobility; mTOR, mammalian target of rapamycin; MuRF1, muscle RING finger-1; p70s6k, ribosomal protein S6 kinase; PMC, pectoralis major muscle; PPAR α, peroxisome proliferator-activated receptor alpha; STI, short tonic immobility; TI, tonic immobility. ⁎ Corresponding author at: Key Laboratory of Animal Physiology and Biochemistry, Nanjing Agricultural University, Nanjing 210095, China. Tel.: + 86 2584399020; fax: + 86 2584398669. E-mail address: [email protected] (Y. Ni). 1095-6433/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.12.019
pontis oralis (Sandoval-Herrera et al., 2011) and systemic injection of CORT (Zamudio et al., 2009) can increase TI duration in rats. Our previous study found that the growth of breast muscle was severely retarded in long TI (LTI) broilers and chronic stress mimicked by administration of CORT in drinking, and CORT treatment significantly suppressed the capacity of protein production (Wang et al., 2013b). Reduced body weight was observed in LTI chickens (El-Lethey et al., 2003) and similar phenotype could be found upon CORT treatment animals, which usually combined with suppressed protein synthesis capacity (Dong et al., 2007). We assume that LTI and CORT may cause the suppressed protein synthesis or augmented protein breakdown in this study. Previous studies have revealed that the Akt/mTOR/p70s6k signaling pathway is essential for muscle growth through regulating protein metabolism (Elliott et al., 2012; Hornberger et al., 2004). In this pathway, Akt activation can lead to activation of mTOR and its downstream target, p70s6k, resulting in protein synthesis. On the other hand, protein degradation in skeletal muscle is essentially mediated by the activity of the ubiquitin-proteasomal pathway (Sandri, 2008), which is mainly induced by the transcriptional activation of muscle atrophy F box (Atrogin-1) and muscle ring finger protein-1 (MuRF1) (Glass, 2003; Sandri et al., 2004). However, information is limited on the changes of
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gene expression involved in protein synthesis and proteolysis in muscles with different TI phenotypes, chronic stress or their interactions. Animals stay in a state of nervousness and fear when in danger and followed by showing TI, and several behavior experiments have concluded that LTI duration exhibited more fear than short TI (STI) duration (Hazard et al., 2008). And when in fight response linked to anger and fear, energy mobilized by triggering adaptive biological processes is required (Moons et al., 2010). In chickens, CORT administration significantly increased proteolysis and gluconeogenesis (Gao et al., 2008) and fasting resulted in depletion of glycogen, ATP and ADP in breast muscles before or immediately after slaughter (Wang et al., 2013a). A major energy sensor in skeletal muscle AMP-activated protein kinase α (AMPK α), whose phosphorylation activated degree relies on muscle contraction rate and muscular glycogen content, regulates key metabolic pathways. Moreover, peroxisome proliferator-activated receptor α (PPAR α) also regulates energy metabolism in the whole body (Grimaldi, 2007), especially in muscle (Horowitz et al., 2000). However, few studies have been conducted in the mechanisms of energy change in STI or LTI chickens. Therefore, the objective of this study was to investigate the mechanisms on how LTI or chronic CORT stress caused the retardation of PMC growth. To clarify this phenomenon, we mainly focus on energy metabolic status and Akt/mTOR/p70S6k pathway. The results may help to better understand the effect of TI and CORT on breast muscle and may be valuable in the enhancement of both welfare and productivity on broiler chickens.
2. Materials and methods 2.1. Ethics statement This study was approved by the Animal Ethics of Nanjing Agricultural University and the sampling procedures complied with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398 set by the Ministry of Science and Technology, China and “the Regulation regarding the Management and Treatment of Experimental Animals” (2008) No.45 set by the Jiangsu Provincial People's Government.
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2.3. RNA isolation, cDNA synthesis and real-time PCR Total RNA was extracted from PMC samples with Trizol Reagent (15596026, Invitrogen). Quantity of the RNA was measured by NanoDrop ND-1000 Spectrophotometer (Thermo, USA). The ratios of absorption (260/280 nm) of all samples were between 1.8 and 2.0. Aliquots of RNA samples were subjected to electrophoresis through a 1.4% agaroseformaldehyde gel to verify their integrity. Then two micrograms of total RNA was treated with RNase-Free DNase (M6101, Promega, USA) and reverse-transcribed according to manufacturer's instructions. 2 μL of diluted cDNA (1:40, vol/vol) was used for real-time PCR which was detected in Mx3000P (Stratagene, USA). β-Actin, which is not affected by the experimental factors (TI & CORT), was chosen as the reference gene. All the primers chosen to study the expression of genes related to protein and energy metabolism, as listed in Table 1, were synthesized by Generay (Shanghai, China). The method of 2−△△Ct was used to analyze the real-time PCR results and gene mRNA levels were expressed as the fold change relative to the mean value of STI control group (Livak and Schmittgen, 2001).
2.4. Muscle preparation and western blotting 50 mg frozen PMC was minced and homogenized in 1 mL of ice-cold homogenization buffer RIPA containing the protease inhibitor cocktail Complete EDTA-free and PhosSTOP (Roche, Penz-berg, Germany). The
Table 1 Primer sequences for real-time PCR amplification. Target genes
Reference/ Genbank accession
PCR products (bp)
Primer sequences
Akt
NM001005838.1
130
mTOR
XM417614.4
p70S6K
NM001030721.1
199
MuRF1
XM424369
163
Atrogin-1
NM001030956
124
α-enolase
D37900.1
156
PPAR α
NM001001464
149
AMPK α
NM001039605.1
215
β-actin
L08165
300
F: 5′-CATTCCCGCC ATTATGAATGAAGTA-3′ R: 5′-CTTGTAGCCAATGA ATGTGCCATC-3′ F: 5′-GAAGTCCTGCGCGA GCATAAG-3′ R: 5′-TTTGTGTCCATCAGCC TCCAGT-3′ F: 5′-AAGTTGAAATAGGA GGGC-3′ R: 5′-GAAGATGTCACTGC GAAT-3′ F: 5′-CGACATCTACAAGC AGGAGT-3′ R: 5′-TGAGCACCGAAGAC CTT-3′ F: 5′-CACGGAAGGAGCAG TATGGT-3′ R: 5′-AGGTCTCTGGGTTG TTGGCT-3′ F: 5′-GGATGGAACGGAGA ACA-3′ R: 5′-GCAGGAACAGGCAG AA-3′ F: 5′-TTGTCGCTGCCATCAT TTG-3′ R: 5′-GAGAAGTTTCGGGA AGAGGA-3′ F: 5′-GGGACCTGAAACCA GAGAACG-3′ R: 5′-ACAGAGGAGGGCAT AGAGGATG-3′ F: 5′-TGCGTGACATCAAG GAGAAG-3′ R: 5′-TGCCAGGGTACATT GTGGTA-3′
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2.2. Animals and experimental design Experimental design and animal management have been well documented in the previous publication (Wang et al., 2013b). In brief, a total of 600 broiler (Gallus gallus) breeder eggs (Ross 308) were incubated under standard conditions. Newly hatched chickens received a starter diet (12.5 ME/kg; 21% CP) from 1 d to 20 d and a finish diet (12.8 ME/kg; 19.5% CP) from 21 d to 42 d and water was provided ad libitum throughout the experiment. The light was continuous during the first week and decreased gradually to 18 L:6D until 21 d. To establish STI and LTI phenotype groups, chickens received twice TI test on d10 and d21. The TI test was described in detail by Wang et al. At last, eighty chickens showing the shortest TI duration (29.6 ± 2.3 s) and 80 scoring the longest duration (246.2 ± 26.8 s) were classified into STI and LTI groups, respectively. The remaining chickens with intermediate TI duration were excluded from the study. On d27, the chickens of STI and LTI groups were respectively divided into control and CORTtreated subgroups. Chickens in CORT groups of both STI and LTI phenotypes (40 from LTI and 40 from STI) were supplied water supplemented with 5 mg/L CORT (C2505, SIGMA, USA), in contrast, those in control groups (40 per phenotype) were supplied water supplemented with equivalent volume of the solvent (absolute ethanol) for the following 15 d. On d42, all chickens were sacrificed by decapitation, and PMC samples were rapidly frozen in liquid nitrogen and then stored at − 70 °C until further analyzed.
Akt, serine/threonine protein kinase B; mTOR, mammalian target of rapamycin; p70s6k, ribosomal protein S6 kinase; MuRF1, muscle RING finger 1; Atrogin-1, muscle atrophy F box; PPAR α, peroxisome proliferator-activated receptor alpha; AMPK α, AMP-activated protein kinase alpha.
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homogenates were centrifuged at 12,000 rpm for 20 min at 4 °C and then collected the cytosolic fraction. Protein concentration was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Thirty micrograms of protein extract from each sample was then loaded onto 7.5% SDS-PAGE gels and the separated proteins were transferred onto the nitrocellulose membranes (Bio Trace, Pall Co, USA). After transfer, membranes were blocked for 2 h at room temperature in blocking buffer and then membranes were incubated with the following primary antibodies: phosphorylated Ser 473-Akt1 (1:5000; ab81283 Abcam), Akt (1:1000; AP250059, Bioworld, USA), phosphorylated Ser 2448mTOR (1:5000; ab109268, Abcam), p70S6K (1:500; bs253635, Bioworld USA), GAPDH (1:10000; AP0066, Bioworld, USA), in dilution buffer for night at 4 °C. After several washes in Tris-buffered-saline with Tween (TBST), membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Bioworld, USA) in dilution buffer for 2 h at room temperature. After several washes, bands were visualized by enhanced chemiluminescence's (ECL) using the LumiGlo substrate (Super Signal West Pico Trial Kit, Pierce, USA), and the signals were recorded by an imaging System (Bio-Rad, USA), and analyzed with Quantity One software (Bio-Rad, USA).
2.5. Measurement of ATP, ADP and AMP levels in muscle Concentrations of AMP, ADP and ATP in muscle were determined according to previous publications with some modifications (Jia et al., 2013). In short, tissue extracts were prepared from frozen PMC using 0.6 M perchloric acid, and then centrifuged at 16,000 g for 15 min at 4 °C. The standards of ATP (FLAAS), ADP (A5285) and AMP (01930) were purchased from Sigma. High performance liquid chromatography (HPLC) was performed with a reverse-phase column (99603, C18, 5 mm, 25064.6 mm, Dikma Technologies Inc., USA) and the column temperature was set at 25 °C. For measurements of metabolites, a mobile phase consisting of 215 mM KH2PO4, 1.2 mM tetrabutylammonium bisulfate, 1% acetonitrile (pH 6.0) was used and the flow rate was maintained at 0.8 mL/min by a HPLC pump (600E, Waters). Eluted samples were detected at 260 nm with a dual λ absorbance detector (2478, Waters). Calibration curves were prepared by a six-point standard (0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625 mg/mL) of ATP, ADP and AMP in 0.6 M perchloric acid, respectively.
2.6. Glycogen content in muscle and liver The glycogen content was measured by a commercial kit (A043, Jiancheng Bioengineering Institute, Nanjing, China) (Wang et al., 2013a).
2.7. Statistical analysis The results are presented as means ± SEM. The general linear model was conducted to evaluate the effects of CORT and TI, as well as their interactions. All analyses were performed using SPSS 18.0 software. P values less than 0.05 were considered statistically significant.
3. Results 3.1. The content of glycogen in muscle and liver As shown in Fig. 1, LTI broilers exhibited lower content of muscle glycogen compared to STI chickens (p b 0.05) and CORT treatment significantly decreased liver glycogen content (p b 0.05). There was no interaction between TI and CORT on glycogen content in muscle or liver (p N 0.05).
3.2. The content of ATP, ADP, AMP and EC in muscle As shown in Table 2, LTI broilers demonstrated significantly lower ATP and EC content in breast muscle compared to STI chickens (p b 0.01), whereas LTI broilers showed an increased tendency of ADP level (p = 0.08). CORT treatment markedly elevated EC content (p b 0.05). There was a significant interaction of TI and CORT in muscle AMP content (p b 0.01).
3.3. Expression of genes related to protein and energy metabolism As shown in Table 3, neither TI nor CORT altered the gene transcription of Akt, mTOR, p70s6k, MURF1 and Atrogin-1 in PMC (p N 0.05). With respect to energy metabolic genes, PPAR α (p = 0.06) and AMPK α (p = 0.09) mRNA expression were moderately down-regulated in LTI chickens compared to STI broilers. CORT treatment significantly decreased mRNA expression of α-enolase in breast muscle (p b 0.05).
3.4. Protein expression in Akt/mTOR/p70s6k signaling pathway As shown in Fig. 2, CORT administration moderately decreased the total protein levels of Akt (p = 0.09) and p70s6k (p = 0.08) in PMC. Total Akt (p = 0.05) and phosphorylation Akt (p = 0.06) levels were higher in LTI broilers compared to STI chickens, while the ratio of phosphorylation Akt to total amount of Akt and p-mTOR did not show significant changes between the two TI phenotype chickens.
Fig. 1. Effect of TI and CORT on glycogen content in PMC (A) and liver (B) of broiler chickens. Con = control; CORT = corticosterone; STI = short tonic immobility duration; LTI = long tonic immobility duration; TI = tonic immobility. Data are expressed by means ± SEM (n = 6).
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Table 2 Effect of TI and CORT on the contents of ATP, ADP, AMP and EC in PMC of broiler chickens. Items
CONa
(mg/g)
STId
ATP ADP AMP ECf
0.16 3.18 0.02 0.52
CORTb LTIe
± ± ± ±
0.01 0.11 0.01 0.00
0.12 3.48 0.01 0.51
STI ± ± ± ±
0.02 0.21 0.00 0.00
0.18 2.77 0.01 0.53
LTI ± ± ± ±
0.01 0.19 0.00 0.00
0.13 3.49 0.03 0.52
± ± ± ±
0.01 0.50 0.01 0.00
TIc
CORT
TI*CORT
effect
effect
effect
p 0.01 NS NS p 0.01
NS NS NS p 0.05
NS NS p 0.01 NS
a Con = control; bCORT = corticosterone; cTI = tonic immobility; dSTI = short tonic immobility duration; eLTI = long tonic immobility duration; fEC = energy charge. EC = ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]). Values are means ± SEM. n = 6/group.
4. Discussion 4.1. The protein metabolism induced by LTI and CORT administration Skeletal muscle is a major glucocorticoid target tissue (Kuo et al., 2013) and glucocorticoids can inhibit protein synthesis and promote proteolysis in skeletal muscle (Kuo et al., 2012). Corticosterone decreases the rates of growth (Lin et al., 2004a,b) and protein accretion (Shoji, 1989) caused mainly by both increasing catabolism and reducing synthesis of protein. In a good agreement, our results showed that protein synthesis capacity estimated by the RNA:protein ratio was significantly lower in the breast muscle of CORT-treated than untreated control broilers (data under peer-reviewed). It's reported that glucocorticoid reduced protein synthesis via inhibiting the activity of mTOR and then blocking phosphorylation of p70S6K and 4E-BP1 (Shah et al., 2000). In this study, both Akt and p70s6k protein contents showed a tendency to decrease in PMC by CORT administration, which may contribute to induce the retardation of protein synthesis and muscle growth. It's speculated that CORT-induced muscle degradation was, at least partially, mediated through the Akt/mTOR/p70s6k signaling pathway in breast muscle of broilers. When considering the process of protein degradation, Atrogin-1 and MuRF1, two key proteins linked to muscle protein breakdown (Glass, 2003; Sandri et al., 2004), their gene expressions were up-regulated in most glucocorticoids-induced muscle atrophy. Our real-time PCR results showed that, chronic CORT administration did not affect Atrogin-1 or MuRF1 mRNA expression in PMC muscles of broilers. However, it cannot exclude the post-transcriptional regulation in the biological process of CORT-induced decrease of protein synthesis capacity and muscle growth in broilers, which still awaits further study. However, contrary to our expectation, LTI phenotype chickens with lower muscle growth had a potential higher level of Akt and phosphorylation Akt in muscle compared to STI counterparts showing significantly higher muscle mass. Till now, it's difficult to explain these
controversial results, and there's no relevant information available to support and explain this phenomenon. Except a trend increase of Akt protein content in PMC of LTI broilers, other proteins as well as their corresponding genes expression in mTOR signaling pathway did not show a difference between LTI and STI chickens. The two key genes regulating muscle protein breakdown, Atrogin-1 and MuRF1 mRNA expression did not change between these two TI phenotype chickens. It's very important to note that TI phenotype did not affect the protein synthesis capacity of PMC (data under review). Taken together, it can be concluded that the lower muscle weight in LTI chickens was not related to muscle protein metabolism in PMC. Moriarty reported that GABA systems are involved in TI and related behaviors in quails (Moriarty, 1995). It's reasonable to speculate that the difference in central nervous system controlling energy metabolism and appetite may responsible for the different growth performance between LTI and STI. Unfortunately, in this study, broilers were fed ad lib and the food intake was not recorded. In addition, no significant interactions of TI duration and chronic CORT treatment were observed in gene expression and the content of their proteins involved in protein metabolic pathway. Consistently, our previous data showed that there was also no interaction between TI and CORT in protein synthesis capacity in PMC muscles. Interestingly, Schütz et al. reported that a significant quantitative trait locus (QTL) for TI duration in chickens was found on chromosome 1 that coincided with a QTL for egg weight and growth in the same animals (Schutz et al., 2004). Therefore, our study will provide new insights into the genetic control of fearfulness and growth in poultry production. 4.2. The energy expender induced by LTI and CORT administration In this study, CORT decreased liver glycogen content and significantly down-regulated α-enolase mRNA expression in PMC. As a ratelimiting enzyme in glycolysis, α-enolase acts mainly through adjusting energy producing process of cells to maintain ATP level and ensure
Table 3 Effects of TI and CORT on genes expression in PMC of broiler chickens. Items
CONa
CORTb
TIc
CORT
TI*CORT
STId
LTIe
STI
LTI
effect
effect
effect
Protein synthesis Akt mTOR p70s6k
1.00 ± 0.18 1.00 ± 0.24 1.00 ± 0.07
0.79 ± 0.05 0.79 ± 0.05 1.01 ± 0.10
0.89 ± 0.13 1.10 ± 0.16 1.22 ± 0.10
0.70 ± 0.05 0.79 ± 0.09 1.07 ± 0.07
NS NS NS
NS NS NS
NS NS NS
Proteolysis MuRF1 Atrogin-1
1.00 ± 0.13 1.00 ± 0.39
0.85 ± 0.13 0.76 ± 0.14
1.55 ± 0.61 0.79 ± 0.35
0.85 ± 0.08 0.75 ± 0.11
NS NS
NS NS
NS NS
Energy metabolism PPAR α AMPK α α-enolase
1.00 ± 0.15 1.00 ± 0.19 1.00 ± 0.08
0.67 ± 0.07 0.77 ± 0.07 0.90 ± 0.08
0.93 ± 0.22 1.05 ± 0.13 0.96 ± 0.05
0.72 ± 0.06 0.86 ± 0.08 0.74 ± 0.07
0.07 0.09 NS
NS NS p 0.05
NS NS NS
a Con = control; bCORT = corticosterone; cTI = tonic immobility; dSTI = short tonic immobility duration; eLTI = long tonic immobility duration. Values are means ± SEM. n = 6/group.
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Fig. 2. Effect of TI and CORT on Akt/mTOR/p70s6k signaling pathway in PMC of broiler chickens. Quantification of the densitometry of total-Akt (A), phospho–Akt (Ser473) (B), the phosphorylation ratio of Akt to total Akt (C), phospho-mTOR (Ser2448) (D) and total-p70s6k (E). Immunoblots were used to detect the indicated proteins (F). These results were averaged from at least six immunoblots. Con = control; CORT = corticosterone; STI = short tonic immobility duration; LTI = long tonic immobility duration; CSTI = corticosterone short tonic immobility duration; CLTI = corticosterone long tonic immobility; TI = tonic immobility. Data were normalized to GAPDH optical density and expressed by means ± SEM (n = 6).
physiology functions, especially in muscle. However, our results showed that the level of ATP, ADP and AMP in breast muscle was not changed by CORT treatment. Moreover, the concentration of lactic acid in blood also showed no significant difference between control and CORT-treated chickens (Wang et al., 2013b). The down-regulation of α-enolase gene expression might be resulting from a lack of available oxygen to the muscle tissues. In addition, α-enolase could be considered as a marker of pathological stress in a high number of diseases, performing multiple functions, mainly as plasminogen receptor (Diaz-Ramos et al., 2012). In this study, we found that CORT treatment significantly decreased the contents of glycogen in liver tissues but not in PMC muscles. The stable of glycogen storage in breast muscle was not associated with a significant increase of energy status in PMC estimated by energy charge (EC) after a chronic CORT administration. However, the decreased glycogen content in liver, a key site of energy metabolism in avian species (Hermier, 1997), also represents the negative role of CORT on energy
synthesis and storage. Similarly, glucocorticoids have been reported to induce muscular glycogen mobilization (Hazard et al., 2011) then resulting in an increased use of energy stores (Coderre et al., 1991; Sapolsky et al., 2000) during the stress response. As reported previously, LTI chickens showed significantly lower body weight and muscle mass (Wang et al., 2013b). In this study, we found a significant decrease of muscle glycogen in LTI broilers compared to STI chickens. However, there was no obvious difference of glycogen in liver between two TI phenotype chickens. Consistently, the level of ATP contents as well as energy charge in STI breast muscle showed significantly higher than LTI chickens, indicating the energy status in STI muscles is increased. As reported previously, chickens submitted to the restraint test showed a decreased abundance of glycogen, resulting in an increase in the glycolysis activities and a decrease of muscular energetic stores (Hazard et al., 2011). Thus, all these results suggested that LTI chickens were in shortage of energy and chickens upon CORT
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treatment were in a state of low energy stores. Moreover, the significant interaction between TI and CORT treatment on AMP revealed that TI could influence the redistribution of energy. As an alternative explanation, the fear response itself also costs energy. Taken together, paralleling a retarded growth and muscle development, CORT as well as LTI induced a decrease of glycogen storage and a lower energy storage, however, there was no significant interaction of these two factors on energy metabolism in muscle. Our results indicated that STI chickens expressed higher AMPK α in mRNA level compared to LTI chickens. Because AMPK can stimulate fat oxidation and glucose uptake when the rate of ATP utilization is increased, once activated, it arouses energy producing pathways while switches off consuming pathways (Winder, 2001). The expression of PPAR α, a member of the nuclear hormone receptor superfamily, also showed a tendency to increase in muscle of STI chickens compared to LTI. In view of the fact that PPAR α was highly expressed in skeletal muscles with a high capacity of fatty acid oxidation, ketone body synthesis, and glucose sparing (Ferre, 2004). Therefore, the moderate increase of AMPK α as well as PPAR α might indicate a higher energy transfer from acid oxidation into muscle in STI chickens, which still deserves further study. In conclusion, the retardation of muscle growth by LTI and chronic CORT administration parallels a significant decrease in energy storage but slight changes of Akt/mTOR/p70s6k cascade, indicating that a decrease in muscle growth induced by LTI and CORT might not be mediated through mTOR-dependent signaling pathways.
Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201003011), the Fundamental Research Funds for the Central Universities (KYZ201212) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Effects of tonic immobility (TI) and corticosterone (CORT) on energy status and protein metabolism in pectoralis major muscle of broiler chickens Yujing Duan, Wenyan Fu, Song Wang, Yingdong Ni ⁎, Ruqian Zhao Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e
i n f o
Article history: Received 26 October 2013 Received in revised form 16 December 2013 Accepted 25 December 2013 Available online 31 December 2013 Keywords: Tonic immobility Corticosterone Protein metabolism Energy status Pectoralis major muscle Broilers
a b s t r a c t Tonic immobility (TI), which can be divided into short (STI) or long (LTI) duration, is a character related to fear. Our previous study has demonstrated LTI phenotype and chronic corticosterone (CORT) administration retarded growth of breast muscle in broiler chickens. In order to investigate the mechanism behind the negative effects of LTI and CORT on growth, the level of mRNA transcription of several key genes linked to energy and protein metabolism was measured in muscle. LTI broilers showed lower levels of ATP, energy charge (EC) (p b 0.01), and lower muscle glycogen content (p b 0.05) but higher level of ADP (p = 0.08) than STI birds. CORT treatment elevated EC level (p b 0.05) and reduced liver glycogen content (p b 0.05). Real-time PCR results showed that STI chickens had higher mRNA expression of PPAR α (p = 0.06) and AMPK α (p = 0.09) than LTI. CORT significantly down-regulated α-enolase mRNA expression in breast muscle compared to control (p b 0.05). Neither TI nor CORT altered gene expression in Akt/mTOR/p70s6k cascade pathway in muscle (p N 0.05). However, western blot results showed that LTI chickens exhibited higher protein content of total Akt (p = 0.05) and phosphorylated Akt (p = 0.06) than STI. CORT treatment decreased the total protein content of Akt (p = 0.09) and p70s6k (p = 0.08). These results suggest that the retardation of muscle growth by LTI and chronic CORT administration parallels a strong alternation in energy status but slight changes of Akt/mTOR/p70s6k cascade, indicating that a decrease in muscle growth induced by LTI and CORT might not be mediated through mTOR-dependent signaling pathways. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Animals show a tonic immobility (TI) response when they are facing danger, attacking or fear. TI is an unlearnt catatonic state considered as the final stage in a chain of anti-predator behavior patterns (Jones, 1986; Mills and Faure, 1991). It has been reported that a strong TI response always associates with high level of endogenous corticosterone (CORT) in mammals (Kalin et al., 1998). Moreover, stressful stimuli can increase the duration of TI in several species (Miranda et al., 2006; Zamudio et al., 2009). Microinjection of CORT into unilateral nucleus
Abbreviations: ADP, adenosine diphosphate; Akt, serine/threonine protein kinase B; AMP, adenosine monophosphate; AMPK α, AMP-activated protein kinase alpha; ATP, adenosine triphosphate; Atrogin-1, muscle atrophy F box; CORT, corticosterone; EC, energy charge; HPLC, high performance liquid chromatography; LTI, long tonic immobility; mTOR, mammalian target of rapamycin; MuRF1, muscle RING finger-1; p70s6k, ribosomal protein S6 kinase; PMC, pectoralis major muscle; PPAR α, peroxisome proliferator-activated receptor alpha; STI, short tonic immobility; TI, tonic immobility. ⁎ Corresponding author at: Key Laboratory of Animal Physiology and Biochemistry, Nanjing Agricultural University, Nanjing 210095, China. Tel.: + 86 2584399020; fax: + 86 2584398669. E-mail address: [email protected] (Y. Ni). 1095-6433/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.12.019
pontis oralis (Sandoval-Herrera et al., 2011) and systemic injection of CORT (Zamudio et al., 2009) can increase TI duration in rats. Our previous study found that the growth of breast muscle was severely retarded in long TI (LTI) broilers and chronic stress mimicked by administration of CORT in drinking, and CORT treatment significantly suppressed the capacity of protein production (Wang et al., 2013b). Reduced body weight was observed in LTI chickens (El-Lethey et al., 2003) and similar phenotype could be found upon CORT treatment animals, which usually combined with suppressed protein synthesis capacity (Dong et al., 2007). We assume that LTI and CORT may cause the suppressed protein synthesis or augmented protein breakdown in this study. Previous studies have revealed that the Akt/mTOR/p70s6k signaling pathway is essential for muscle growth through regulating protein metabolism (Elliott et al., 2012; Hornberger et al., 2004). In this pathway, Akt activation can lead to activation of mTOR and its downstream target, p70s6k, resulting in protein synthesis. On the other hand, protein degradation in skeletal muscle is essentially mediated by the activity of the ubiquitin-proteasomal pathway (Sandri, 2008), which is mainly induced by the transcriptional activation of muscle atrophy F box (Atrogin-1) and muscle ring finger protein-1 (MuRF1) (Glass, 2003; Sandri et al., 2004). However, information is limited on the changes of
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gene expression involved in protein synthesis and proteolysis in muscles with different TI phenotypes, chronic stress or their interactions. Animals stay in a state of nervousness and fear when in danger and followed by showing TI, and several behavior experiments have concluded that LTI duration exhibited more fear than short TI (STI) duration (Hazard et al., 2008). And when in fight response linked to anger and fear, energy mobilized by triggering adaptive biological processes is required (Moons et al., 2010). In chickens, CORT administration significantly increased proteolysis and gluconeogenesis (Gao et al., 2008) and fasting resulted in depletion of glycogen, ATP and ADP in breast muscles before or immediately after slaughter (Wang et al., 2013a). A major energy sensor in skeletal muscle AMP-activated protein kinase α (AMPK α), whose phosphorylation activated degree relies on muscle contraction rate and muscular glycogen content, regulates key metabolic pathways. Moreover, peroxisome proliferator-activated receptor α (PPAR α) also regulates energy metabolism in the whole body (Grimaldi, 2007), especially in muscle (Horowitz et al., 2000). However, few studies have been conducted in the mechanisms of energy change in STI or LTI chickens. Therefore, the objective of this study was to investigate the mechanisms on how LTI or chronic CORT stress caused the retardation of PMC growth. To clarify this phenomenon, we mainly focus on energy metabolic status and Akt/mTOR/p70S6k pathway. The results may help to better understand the effect of TI and CORT on breast muscle and may be valuable in the enhancement of both welfare and productivity on broiler chickens.
2. Materials and methods 2.1. Ethics statement This study was approved by the Animal Ethics of Nanjing Agricultural University and the sampling procedures complied with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398 set by the Ministry of Science and Technology, China and “the Regulation regarding the Management and Treatment of Experimental Animals” (2008) No.45 set by the Jiangsu Provincial People's Government.
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2.3. RNA isolation, cDNA synthesis and real-time PCR Total RNA was extracted from PMC samples with Trizol Reagent (15596026, Invitrogen). Quantity of the RNA was measured by NanoDrop ND-1000 Spectrophotometer (Thermo, USA). The ratios of absorption (260/280 nm) of all samples were between 1.8 and 2.0. Aliquots of RNA samples were subjected to electrophoresis through a 1.4% agaroseformaldehyde gel to verify their integrity. Then two micrograms of total RNA was treated with RNase-Free DNase (M6101, Promega, USA) and reverse-transcribed according to manufacturer's instructions. 2 μL of diluted cDNA (1:40, vol/vol) was used for real-time PCR which was detected in Mx3000P (Stratagene, USA). β-Actin, which is not affected by the experimental factors (TI & CORT), was chosen as the reference gene. All the primers chosen to study the expression of genes related to protein and energy metabolism, as listed in Table 1, were synthesized by Generay (Shanghai, China). The method of 2−△△Ct was used to analyze the real-time PCR results and gene mRNA levels were expressed as the fold change relative to the mean value of STI control group (Livak and Schmittgen, 2001).
2.4. Muscle preparation and western blotting 50 mg frozen PMC was minced and homogenized in 1 mL of ice-cold homogenization buffer RIPA containing the protease inhibitor cocktail Complete EDTA-free and PhosSTOP (Roche, Penz-berg, Germany). The
Table 1 Primer sequences for real-time PCR amplification. Target genes
Reference/ Genbank accession
PCR products (bp)
Primer sequences
Akt
NM001005838.1
130
mTOR
XM417614.4
p70S6K
NM001030721.1
199
MuRF1
XM424369
163
Atrogin-1
NM001030956
124
α-enolase
D37900.1
156
PPAR α
NM001001464
149
AMPK α
NM001039605.1
215
β-actin
L08165
300
F: 5′-CATTCCCGCC ATTATGAATGAAGTA-3′ R: 5′-CTTGTAGCCAATGA ATGTGCCATC-3′ F: 5′-GAAGTCCTGCGCGA GCATAAG-3′ R: 5′-TTTGTGTCCATCAGCC TCCAGT-3′ F: 5′-AAGTTGAAATAGGA GGGC-3′ R: 5′-GAAGATGTCACTGC GAAT-3′ F: 5′-CGACATCTACAAGC AGGAGT-3′ R: 5′-TGAGCACCGAAGAC CTT-3′ F: 5′-CACGGAAGGAGCAG TATGGT-3′ R: 5′-AGGTCTCTGGGTTG TTGGCT-3′ F: 5′-GGATGGAACGGAGA ACA-3′ R: 5′-GCAGGAACAGGCAG AA-3′ F: 5′-TTGTCGCTGCCATCAT TTG-3′ R: 5′-GAGAAGTTTCGGGA AGAGGA-3′ F: 5′-GGGACCTGAAACCA GAGAACG-3′ R: 5′-ACAGAGGAGGGCAT AGAGGATG-3′ F: 5′-TGCGTGACATCAAG GAGAAG-3′ R: 5′-TGCCAGGGTACATT GTGGTA-3′
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2.2. Animals and experimental design Experimental design and animal management have been well documented in the previous publication (Wang et al., 2013b). In brief, a total of 600 broiler (Gallus gallus) breeder eggs (Ross 308) were incubated under standard conditions. Newly hatched chickens received a starter diet (12.5 ME/kg; 21% CP) from 1 d to 20 d and a finish diet (12.8 ME/kg; 19.5% CP) from 21 d to 42 d and water was provided ad libitum throughout the experiment. The light was continuous during the first week and decreased gradually to 18 L:6D until 21 d. To establish STI and LTI phenotype groups, chickens received twice TI test on d10 and d21. The TI test was described in detail by Wang et al. At last, eighty chickens showing the shortest TI duration (29.6 ± 2.3 s) and 80 scoring the longest duration (246.2 ± 26.8 s) were classified into STI and LTI groups, respectively. The remaining chickens with intermediate TI duration were excluded from the study. On d27, the chickens of STI and LTI groups were respectively divided into control and CORTtreated subgroups. Chickens in CORT groups of both STI and LTI phenotypes (40 from LTI and 40 from STI) were supplied water supplemented with 5 mg/L CORT (C2505, SIGMA, USA), in contrast, those in control groups (40 per phenotype) were supplied water supplemented with equivalent volume of the solvent (absolute ethanol) for the following 15 d. On d42, all chickens were sacrificed by decapitation, and PMC samples were rapidly frozen in liquid nitrogen and then stored at − 70 °C until further analyzed.
Akt, serine/threonine protein kinase B; mTOR, mammalian target of rapamycin; p70s6k, ribosomal protein S6 kinase; MuRF1, muscle RING finger 1; Atrogin-1, muscle atrophy F box; PPAR α, peroxisome proliferator-activated receptor alpha; AMPK α, AMP-activated protein kinase alpha.
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homogenates were centrifuged at 12,000 rpm for 20 min at 4 °C and then collected the cytosolic fraction. Protein concentration was determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Thirty micrograms of protein extract from each sample was then loaded onto 7.5% SDS-PAGE gels and the separated proteins were transferred onto the nitrocellulose membranes (Bio Trace, Pall Co, USA). After transfer, membranes were blocked for 2 h at room temperature in blocking buffer and then membranes were incubated with the following primary antibodies: phosphorylated Ser 473-Akt1 (1:5000; ab81283 Abcam), Akt (1:1000; AP250059, Bioworld, USA), phosphorylated Ser 2448mTOR (1:5000; ab109268, Abcam), p70S6K (1:500; bs253635, Bioworld USA), GAPDH (1:10000; AP0066, Bioworld, USA), in dilution buffer for night at 4 °C. After several washes in Tris-buffered-saline with Tween (TBST), membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Bioworld, USA) in dilution buffer for 2 h at room temperature. After several washes, bands were visualized by enhanced chemiluminescence's (ECL) using the LumiGlo substrate (Super Signal West Pico Trial Kit, Pierce, USA), and the signals were recorded by an imaging System (Bio-Rad, USA), and analyzed with Quantity One software (Bio-Rad, USA).
2.5. Measurement of ATP, ADP and AMP levels in muscle Concentrations of AMP, ADP and ATP in muscle were determined according to previous publications with some modifications (Jia et al., 2013). In short, tissue extracts were prepared from frozen PMC using 0.6 M perchloric acid, and then centrifuged at 16,000 g for 15 min at 4 °C. The standards of ATP (FLAAS), ADP (A5285) and AMP (01930) were purchased from Sigma. High performance liquid chromatography (HPLC) was performed with a reverse-phase column (99603, C18, 5 mm, 25064.6 mm, Dikma Technologies Inc., USA) and the column temperature was set at 25 °C. For measurements of metabolites, a mobile phase consisting of 215 mM KH2PO4, 1.2 mM tetrabutylammonium bisulfate, 1% acetonitrile (pH 6.0) was used and the flow rate was maintained at 0.8 mL/min by a HPLC pump (600E, Waters). Eluted samples were detected at 260 nm with a dual λ absorbance detector (2478, Waters). Calibration curves were prepared by a six-point standard (0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625 mg/mL) of ATP, ADP and AMP in 0.6 M perchloric acid, respectively.
2.6. Glycogen content in muscle and liver The glycogen content was measured by a commercial kit (A043, Jiancheng Bioengineering Institute, Nanjing, China) (Wang et al., 2013a).
2.7. Statistical analysis The results are presented as means ± SEM. The general linear model was conducted to evaluate the effects of CORT and TI, as well as their interactions. All analyses were performed using SPSS 18.0 software. P values less than 0.05 were considered statistically significant.
3. Results 3.1. The content of glycogen in muscle and liver As shown in Fig. 1, LTI broilers exhibited lower content of muscle glycogen compared to STI chickens (p b 0.05) and CORT treatment significantly decreased liver glycogen content (p b 0.05). There was no interaction between TI and CORT on glycogen content in muscle or liver (p N 0.05).
3.2. The content of ATP, ADP, AMP and EC in muscle As shown in Table 2, LTI broilers demonstrated significantly lower ATP and EC content in breast muscle compared to STI chickens (p b 0.01), whereas LTI broilers showed an increased tendency of ADP level (p = 0.08). CORT treatment markedly elevated EC content (p b 0.05). There was a significant interaction of TI and CORT in muscle AMP content (p b 0.01).
3.3. Expression of genes related to protein and energy metabolism As shown in Table 3, neither TI nor CORT altered the gene transcription of Akt, mTOR, p70s6k, MURF1 and Atrogin-1 in PMC (p N 0.05). With respect to energy metabolic genes, PPAR α (p = 0.06) and AMPK α (p = 0.09) mRNA expression were moderately down-regulated in LTI chickens compared to STI broilers. CORT treatment significantly decreased mRNA expression of α-enolase in breast muscle (p b 0.05).
3.4. Protein expression in Akt/mTOR/p70s6k signaling pathway As shown in Fig. 2, CORT administration moderately decreased the total protein levels of Akt (p = 0.09) and p70s6k (p = 0.08) in PMC. Total Akt (p = 0.05) and phosphorylation Akt (p = 0.06) levels were higher in LTI broilers compared to STI chickens, while the ratio of phosphorylation Akt to total amount of Akt and p-mTOR did not show significant changes between the two TI phenotype chickens.
Fig. 1. Effect of TI and CORT on glycogen content in PMC (A) and liver (B) of broiler chickens. Con = control; CORT = corticosterone; STI = short tonic immobility duration; LTI = long tonic immobility duration; TI = tonic immobility. Data are expressed by means ± SEM (n = 6).
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Table 2 Effect of TI and CORT on the contents of ATP, ADP, AMP and EC in PMC of broiler chickens. Items
CONa
(mg/g)
STId
ATP ADP AMP ECf
0.16 3.18 0.02 0.52
CORTb LTIe
± ± ± ±
0.01 0.11 0.01 0.00
0.12 3.48 0.01 0.51
STI ± ± ± ±
0.02 0.21 0.00 0.00
0.18 2.77 0.01 0.53
LTI ± ± ± ±
0.01 0.19 0.00 0.00
0.13 3.49 0.03 0.52
± ± ± ±
0.01 0.50 0.01 0.00
TIc
CORT
TI*CORT
effect
effect
effect
p 0.01 NS NS p 0.01
NS NS NS p 0.05
NS NS p 0.01 NS
a Con = control; bCORT = corticosterone; cTI = tonic immobility; dSTI = short tonic immobility duration; eLTI = long tonic immobility duration; fEC = energy charge. EC = ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]). Values are means ± SEM. n = 6/group.
4. Discussion 4.1. The protein metabolism induced by LTI and CORT administration Skeletal muscle is a major glucocorticoid target tissue (Kuo et al., 2013) and glucocorticoids can inhibit protein synthesis and promote proteolysis in skeletal muscle (Kuo et al., 2012). Corticosterone decreases the rates of growth (Lin et al., 2004a,b) and protein accretion (Shoji, 1989) caused mainly by both increasing catabolism and reducing synthesis of protein. In a good agreement, our results showed that protein synthesis capacity estimated by the RNA:protein ratio was significantly lower in the breast muscle of CORT-treated than untreated control broilers (data under peer-reviewed). It's reported that glucocorticoid reduced protein synthesis via inhibiting the activity of mTOR and then blocking phosphorylation of p70S6K and 4E-BP1 (Shah et al., 2000). In this study, both Akt and p70s6k protein contents showed a tendency to decrease in PMC by CORT administration, which may contribute to induce the retardation of protein synthesis and muscle growth. It's speculated that CORT-induced muscle degradation was, at least partially, mediated through the Akt/mTOR/p70s6k signaling pathway in breast muscle of broilers. When considering the process of protein degradation, Atrogin-1 and MuRF1, two key proteins linked to muscle protein breakdown (Glass, 2003; Sandri et al., 2004), their gene expressions were up-regulated in most glucocorticoids-induced muscle atrophy. Our real-time PCR results showed that, chronic CORT administration did not affect Atrogin-1 or MuRF1 mRNA expression in PMC muscles of broilers. However, it cannot exclude the post-transcriptional regulation in the biological process of CORT-induced decrease of protein synthesis capacity and muscle growth in broilers, which still awaits further study. However, contrary to our expectation, LTI phenotype chickens with lower muscle growth had a potential higher level of Akt and phosphorylation Akt in muscle compared to STI counterparts showing significantly higher muscle mass. Till now, it's difficult to explain these
controversial results, and there's no relevant information available to support and explain this phenomenon. Except a trend increase of Akt protein content in PMC of LTI broilers, other proteins as well as their corresponding genes expression in mTOR signaling pathway did not show a difference between LTI and STI chickens. The two key genes regulating muscle protein breakdown, Atrogin-1 and MuRF1 mRNA expression did not change between these two TI phenotype chickens. It's very important to note that TI phenotype did not affect the protein synthesis capacity of PMC (data under review). Taken together, it can be concluded that the lower muscle weight in LTI chickens was not related to muscle protein metabolism in PMC. Moriarty reported that GABA systems are involved in TI and related behaviors in quails (Moriarty, 1995). It's reasonable to speculate that the difference in central nervous system controlling energy metabolism and appetite may responsible for the different growth performance between LTI and STI. Unfortunately, in this study, broilers were fed ad lib and the food intake was not recorded. In addition, no significant interactions of TI duration and chronic CORT treatment were observed in gene expression and the content of their proteins involved in protein metabolic pathway. Consistently, our previous data showed that there was also no interaction between TI and CORT in protein synthesis capacity in PMC muscles. Interestingly, Schütz et al. reported that a significant quantitative trait locus (QTL) for TI duration in chickens was found on chromosome 1 that coincided with a QTL for egg weight and growth in the same animals (Schutz et al., 2004). Therefore, our study will provide new insights into the genetic control of fearfulness and growth in poultry production. 4.2. The energy expender induced by LTI and CORT administration In this study, CORT decreased liver glycogen content and significantly down-regulated α-enolase mRNA expression in PMC. As a ratelimiting enzyme in glycolysis, α-enolase acts mainly through adjusting energy producing process of cells to maintain ATP level and ensure
Table 3 Effects of TI and CORT on genes expression in PMC of broiler chickens. Items
CONa
CORTb
TIc
CORT
TI*CORT
STId
LTIe
STI
LTI
effect
effect
effect
Protein synthesis Akt mTOR p70s6k
1.00 ± 0.18 1.00 ± 0.24 1.00 ± 0.07
0.79 ± 0.05 0.79 ± 0.05 1.01 ± 0.10
0.89 ± 0.13 1.10 ± 0.16 1.22 ± 0.10
0.70 ± 0.05 0.79 ± 0.09 1.07 ± 0.07
NS NS NS
NS NS NS
NS NS NS
Proteolysis MuRF1 Atrogin-1
1.00 ± 0.13 1.00 ± 0.39
0.85 ± 0.13 0.76 ± 0.14
1.55 ± 0.61 0.79 ± 0.35
0.85 ± 0.08 0.75 ± 0.11
NS NS
NS NS
NS NS
Energy metabolism PPAR α AMPK α α-enolase
1.00 ± 0.15 1.00 ± 0.19 1.00 ± 0.08
0.67 ± 0.07 0.77 ± 0.07 0.90 ± 0.08
0.93 ± 0.22 1.05 ± 0.13 0.96 ± 0.05
0.72 ± 0.06 0.86 ± 0.08 0.74 ± 0.07
0.07 0.09 NS
NS NS p 0.05
NS NS NS
a Con = control; bCORT = corticosterone; cTI = tonic immobility; dSTI = short tonic immobility duration; eLTI = long tonic immobility duration. Values are means ± SEM. n = 6/group.
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Fig. 2. Effect of TI and CORT on Akt/mTOR/p70s6k signaling pathway in PMC of broiler chickens. Quantification of the densitometry of total-Akt (A), phospho–Akt (Ser473) (B), the phosphorylation ratio of Akt to total Akt (C), phospho-mTOR (Ser2448) (D) and total-p70s6k (E). Immunoblots were used to detect the indicated proteins (F). These results were averaged from at least six immunoblots. Con = control; CORT = corticosterone; STI = short tonic immobility duration; LTI = long tonic immobility duration; CSTI = corticosterone short tonic immobility duration; CLTI = corticosterone long tonic immobility; TI = tonic immobility. Data were normalized to GAPDH optical density and expressed by means ± SEM (n = 6).
physiology functions, especially in muscle. However, our results showed that the level of ATP, ADP and AMP in breast muscle was not changed by CORT treatment. Moreover, the concentration of lactic acid in blood also showed no significant difference between control and CORT-treated chickens (Wang et al., 2013b). The down-regulation of α-enolase gene expression might be resulting from a lack of available oxygen to the muscle tissues. In addition, α-enolase could be considered as a marker of pathological stress in a high number of diseases, performing multiple functions, mainly as plasminogen receptor (Diaz-Ramos et al., 2012). In this study, we found that CORT treatment significantly decreased the contents of glycogen in liver tissues but not in PMC muscles. The stable of glycogen storage in breast muscle was not associated with a significant increase of energy status in PMC estimated by energy charge (EC) after a chronic CORT administration. However, the decreased glycogen content in liver, a key site of energy metabolism in avian species (Hermier, 1997), also represents the negative role of CORT on energy
synthesis and storage. Similarly, glucocorticoids have been reported to induce muscular glycogen mobilization (Hazard et al., 2011) then resulting in an increased use of energy stores (Coderre et al., 1991; Sapolsky et al., 2000) during the stress response. As reported previously, LTI chickens showed significantly lower body weight and muscle mass (Wang et al., 2013b). In this study, we found a significant decrease of muscle glycogen in LTI broilers compared to STI chickens. However, there was no obvious difference of glycogen in liver between two TI phenotype chickens. Consistently, the level of ATP contents as well as energy charge in STI breast muscle showed significantly higher than LTI chickens, indicating the energy status in STI muscles is increased. As reported previously, chickens submitted to the restraint test showed a decreased abundance of glycogen, resulting in an increase in the glycolysis activities and a decrease of muscular energetic stores (Hazard et al., 2011). Thus, all these results suggested that LTI chickens were in shortage of energy and chickens upon CORT
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treatment were in a state of low energy stores. Moreover, the significant interaction between TI and CORT treatment on AMP revealed that TI could influence the redistribution of energy. As an alternative explanation, the fear response itself also costs energy. Taken together, paralleling a retarded growth and muscle development, CORT as well as LTI induced a decrease of glycogen storage and a lower energy storage, however, there was no significant interaction of these two factors on energy metabolism in muscle. Our results indicated that STI chickens expressed higher AMPK α in mRNA level compared to LTI chickens. Because AMPK can stimulate fat oxidation and glucose uptake when the rate of ATP utilization is increased, once activated, it arouses energy producing pathways while switches off consuming pathways (Winder, 2001). The expression of PPAR α, a member of the nuclear hormone receptor superfamily, also showed a tendency to increase in muscle of STI chickens compared to LTI. In view of the fact that PPAR α was highly expressed in skeletal muscles with a high capacity of fatty acid oxidation, ketone body synthesis, and glucose sparing (Ferre, 2004). Therefore, the moderate increase of AMPK α as well as PPAR α might indicate a higher energy transfer from acid oxidation into muscle in STI chickens, which still deserves further study. In conclusion, the retardation of muscle growth by LTI and chronic CORT administration parallels a significant decrease in energy storage but slight changes of Akt/mTOR/p70s6k cascade, indicating that a decrease in muscle growth induced by LTI and CORT might not be mediated through mTOR-dependent signaling pathways.
Acknowledgments This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201003011), the Fundamental Research Funds for the Central Universities (KYZ201212) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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