AMPK Subunit Expression Regulates Intramuscular Fat Content and Muscle Fiber Type in Chickens

Ye YANG,Jiao SONG,Ruiqi FU,Yanfa SUN,Jie WEN

1.College of Animal Science,Yangtze University,Jingzhou 434025,China;

2.State Key Laboratory of Animal Nutrition,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100094,China

AMPK Subunit Expression Regulates Intramuscular Fat Content and Muscle Fiber Type in Chickens

Ye YANG1*,Jiao SONG1,Ruiqi FU2,Yanfa SUN2,Jie WEN2

1.College of Animal Science,Yangtze University,Jingzhou 434025,China;

2.State Key Laboratory of Animal Nutrition,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100094,China

The objective of this study was to assess the role of AMPK in intramuscular fat(IMF)and fiber type in chicken muscle.The chickens were slaughtered and their muscles were collected at the ages of 4,8,and 16 weeks so as to determine the IMF contents,as well as the expression levels of AMPK subunits,regulators of adipogenesis.In addition,the myosin heavy chains(MyHCs)in thigh muscle tissues were also measured.The results showed that the IMF contents in 16-week old chickens were higher than those in 4 and 8-week-old chickens(P＜0.05). The expression levels of fatty acid synthase(FAS)and fatty aicd translocase CD36 (FAT/CD36)mRNA were increased significantly in samples collected at the ages of 4 and 16 weeks(P＜0.05).The expression levels of MyHC IIa and IIb differed significantly among all the developmental stages(P＜0.05).The AMPKα2,AMPKγ1, and AMPKγ3 mRNA levels were dramatically decreased with the increase of age (P＜0.05).To examine the role of AMPK in adipogenesis regulation,the SV cells were cultured in an adipogenesis medium and treated with AICAR and Compound C respectively,the specific activator and inhibit of AMPK.The Compound C induced dramatically a greater expression of C/EBPβ,SREBP1 and PPARγ(P＜0.05).In conclusion,the expression of AMPKα2,AMPKγ1,and AMPKγ3 mRNA is significantly correlated with the adipogenesis in skeletal muscle of chickens.

Chicken;Adenosine monophosphate-activated protein kinases;Intramuscular fat;Muscle fiber

I ntramuscular fat(IMF)content and muscle fiber type are important indicators of meat quality,influencing the flavor,texture,and visual appeal of the meat[1-3].Differences in IMF content are often related to the muscle fiber type composition.For example,the breast muscle of chicken is composed exclusively of type IIb gly colytic fibers,with a lipid content of 1%-2%,while the breast muscle of duck is composed of only 15%type IIb glycolytic fibers,with a lipid content of 2%-3%[4].Therefore,IMF accumulation depends on the metabolic activity of adipocytes,as well as the muscle fiber type.

Skeletal muscle consists of both fast and slow contracting muscle fibers.In chickens,fast fibers are generally more abundant[5].Different types of fast fibers can be identified on the basis of the expression of the various MyHC isoforms:IIa,IIx,and IIb.However,unlike in mammals,type IIx fibers have not been described in avian muscle[6].

Intramuscular fat content is affected largely by the extent of adipogenesis within the muscle.Thus,the factors that regulate adipogenesis have certain effect on the degree of IMF deposition and meat quality.Adipogenesis is a well-regulated process regulated by many important transcription factors,such as CCAAT/enhancer-binding protein factors(C/EBP),sterol regulatory element-binding protein 1 (SREBP1),and peroxisome proliferator-activated receptors(PPAR)[7].

Further studies have shown that adipogenesis is also regulated by calcium signaling pathways.Adenosine monophosphate-activated protein kinases(AMPKs)are known to play a major role in fiber type determination as well as IMF accumulation[8].AMPK promotes fatty acid oxidation and inhibits lipid synthesis in cells through phosphorylation and the inhibition of acetyl-CoA carboxylase activity[9].The studies also confirmed that the role of functional AMPK pathway in energy homeostasis in chickens is similar to the described role of AMPK in mammals[10].AMPK s are serine/threonine protein kinases that exist as heterotrimeric complexes comprising a catalytic α-subunit as well as regulatory β-and γ-subunits.The objective of this study was to determine the association of AMPK subunit expression with IMF content and muscle fiber type in chickens.

Materials and Methods

All animal procedures and care were performed in accordance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology(Beijing,China).

Animals and sample collection

A total of ninety female 1-day BJY birds(Institute of Animal Sciences, Chinese Academy of Agricultural Sciences,Beijing,China)were raised starting from 1 day of age.The starter ration(1-21 d)containing 20%of pro-tein and 12.01 MJ/kg of energy differed slightly from that used in the growth phase(＞22 d),which contained 19%of ontained(＞r ration(1-crude protein and 12.55 MJ/kg of energy. The feed and water were provided ad libitum during the experiment.A total of 12 chickens were slaughtered respectively at each of the ages of 4,8 and 16 weeks.Subsequently,the thigh muscles were collected.The right thigh muscles of the 12 chickens collected at each of the ages were collected and stored at-20℃for IMF determination as described by Zhao et al[1].The left thigh muscles of 6 chickens at each of the ages were stored at -80℃for RNA extraction.

Cell isolation and culture

All the chemicals for cell culture were purchased from the Sigma-Aldrich Corporation(St.Louis,MO) unless noted otherwise.

The 4-7 day old Beijing Fatty chickens were slaughtered and then their pectoral muscles(PM)were isolated aseptically and finely minced after removing all the visible connective tissues.The muscle stromal-vascular (SV)cells were obtained according to the procedures modified from previous report[11].

The tissues of PM were digested 30-40 min by 0.1%collagenase type I (GIBCO,Grand Island,NY,USA)and the digesta was centrifuged at 1 000 r/min for 8 min.Then the cell pellet was digested 15-20 min by 0.25% trypsin(GIBCO,Grand Island,NY, USA).The digesta was passed through 200,400 and 600-mesh filters to isolate aseptically the digested cells and centrifuged at 1 000 r/min for 5 min.The isolated cells were rinsed with Dulbeco’s modified Eagle’s medium with F12(DMEM/F12,1:1, GIBCO,Grand Island,NY,USA),centrifuged at 1 000×g for 5 min,re-suspended in 15 ml growth medium containing 84%DMEM/F12,15%fetal bovine serum(FBS,GIBCO,Grand Island,NY,USA),1%HEPES,100 U/ml penicillin and 100 U/ml streptomycin,plated in 6 well culture plate at 37℃and humidified in 5%CO2atmosphere.The cell cultures were aspirated from the plate 1 h after the plating and the fresh growth medium was added to each of the plates as described by Hausman and Poulos[11].

At 30%confluence,SV cells were incubated in an adipogenic medium composed of 10%FBS/DMEM supplemented with insulin(10 g/ml),dexamethasone(1 μM)and 3-isobutyl-1-methylxanthine(IBMX,115 ng/ml)for 24 h.Then the cells were treated with AMPK activator AICAR(2 mmol/L, AICAR)or AMPK inhibitor Compound C(20 μmol/L,COM).The control (CON)was absent from activator or inhibitor.The cells were collected at 24 h after initiating incubation for RNA extraction and mRNA analysis.

Total RNA preparation and quantitative RT-PCR(qPCR)

The total RNA was extracted using TRIzol reagent(Invitrogen,USA) according to the manufacturer’s instructions.After the DNase I (Promega,Beijing,China)treatment, the RNA concentrations were measured by spectrophotometry(optical density at 260 and 280 nm),and the integrity was verified by 1%agarose gel electrophoresis.All the purified RNA samples were diluted with RNase-free water to 1 μg/μl and stored at-80℃for qRT-PCR assays.

The gene specific primers were designed by Primer Premier 5.0, based on the corresponding chicken sequences,to be intron spannings in order to avoid co-amplification of genomic DNA(Table 1).The primers were synthesized by the Huada Biology Company(Beijing Genome Institute,Beijing,China).Theβ-actin was used as an endogenous control to normalize the reverse transcription reactions.The reverse transcription of 2 μg RNA to first-strand cDNA was performed using a kit according to the manufacturer’s instructions (Promega,Beijing,China).

The real-time PCR amplification of AMPK subunits(α1,α2,β1,β2,γ1, γ2 and γ3),FAT/CD36,FAS,C/EBPβ, PPARγ,SREBP1 and MyHC IIa and IIb transcripts were performed with SYBR Green I RT-PCR Master Mix Plus(Takara,Dalian,China)using ABI PRISM 7500 apparatus(Applied Biosystems,USA).A total volume of 20 μl[10 μl 2×SYBR Green I real-time PCR Master Mix(ABI),1 μl forward primer(10 pmol),1 μl reverse primer (10 pmol),2 μl cDNA,0.4 μl 50×ROX Reference Dye II and 5.6 μl dH2O]was pre-heated at 95℃for 10 min followed by 40 cycles of 95℃for 15 s and 63℃for 45 s.The dissociation analysis of amplification products was performed after each PCR to confirm that only one PCR product was amplified and detected.

The data was analyzed with ABI 7500 SDS software(ABI)with the baseline set automatically by the software,and the values of average dCT (normalized usingβ-actin)were exported into Excel for the calculation of relative mRNA expression.The 2-ΔΔCtmethod of quantification[12]was used to calculate the relative expression levels of each gene.The target and internal control genes of 4-week-old BJY chickens were measured as the calibrator.The results were expressed as the relative mRNA expression levels which were log(2-ΔΔCt)at each of the ages from triplicate analysis.

Statistical analysis

All the data was analyzed by ANOVA of SAS software(version 8.0). The differences between the means were assessed using Duncan’s multiple range tests,and P＜0.05 was considered as significant.

Results

The thigh IMF contents and gene expression levels were shown in Table 2.The IMF content and expression levels of FAS and FAT/CD36 were increased with the increased age from 4 to 16 weeks.There were no significant differences in IMF content,FAS or FAT/CD36 mRNA expression between the ages of 4 weeks and 8 weeks(P＞0.05).But the IMF contents in 16-weekold chickens were significantly higher than those in 8-week-old chickens(P＜0.05,Table 2).It was suggested that the accumulation of IMF from 4thto 8thweek exceeded that from 8thto 16thweek in chicken thigh muscle(6%to 22%).So the period from 8thto 16thweek is the most important stage for IMF deposition in BJY chickens.

As shown in Table 2,the expression of MyHC IIa gene was significantly increased with age increased from 4 to 16 weeks(P＜0.05).But the MyHC IIb mRNA expression was significantly decreased with age increased from 4 to 16 weeks(P＜0.05).

Table 1 Gene accession numbers and primer sequences1

The mRNA abundances of AMPK s were shown in Table 3. There were no significant differences in expression level of AMPK subunits among different ages except AMPKα2, AMPKγ1 and AMPKγ3(P＞0.05).The increased age significantly decreased the AMPKα2,AMPKγ1 and AMPKγ3 mRNA levels(P＜0.05).

To examine the role of AMPK in adipogenesis regulation,the SV cells were cultured in an adipogenesis medium and treated with AICAR and Compound C respectively,the specific activator and inhibit of AMPK.The SV cells were treated for 24 h,and then the expression levels of C/EBPβ, PPARγ and SREBP1 were determined.After 24 h differentiation,the AICAR treatment inhibited the expression of C/EBPβ,SREBP1 and PPARγ, but the Compound C treatment dramatically induced a greater expression of C/EBPβ,SREBP1 and PPARγ(P＜0.05,Table 4).

Discussion

IMF content is the main factor affecting meat quality,such as tenderness,juiciness and flavor,and it varies with aging.In this study,the IMF content at the age of 16 weeks was significantly higher than those at the ages of 4 and 8 weeks(P＜0.05).It suggested that the increase of IMF content from 8thto 16thweek(22%)was higher than that from 4thto 8thweek(6%),which was consistent with Li et al.’s[13]study result.

Intramuscular fat content is affected largely by the extent of adipogenesis within the muscle.The FAS and FAT/CD36 genes play essential roles in adipogenesis.High muscle lipid content is accompanied by an abundance of the fatty acid transporter FAT/CD36 in muscle[14],which is consistent with the findings of this study.

IMF accumulation is also associated with muscle growth[4].Skeletal muscle consists of slow-contractingoxidative(type I)and fast-contracting glycolytic(type II)muscle fibers.Glycolytic fibers predominate in avian muscle,with oxidative fibers accounting for＜10%of total muscle fibers[15]. Thus,this study mainly describes the expression of type II fibers.The MyHC IIa and IIb expressions differed significantly among different ages in this study.Karlsson et al.[16]found that type II fibers,especially IIb(fast-contracting glycolytic)fibers,contained less IMF. Thigh muscles of chickens have higher IMF levels than breast muscles because the breast muscle of chickens is mainly composed of type IIb glycolytic fibers[17].In this study,higher IMF content was associated with lower MyHC IIb expression in 16-week-old chickens.

Table 2 Regulation of adipogenesis and muscle type1

Table 3 Relative mRNA expression of AMPK subunits in chicken muscle1

Table 4 Effect of AMPK on the adipogenesis gene expression1

AMPK is a key player in adipogenesis[8]as well as in fiber type transformation[18].Adipogenesis is a well-regulated process regulated by many important transcription factors,such as C/EBPβ,SREBP1 and PPARγ[7].In this study,AMPK activation could induce the expression of C/EBPβ, SREBP1 and PPARγ.AMPK is also considered to regulate adipogenesis through the FAT/CD36 pathway by playing an important role in the regulation of FAT/CD36 distribution[19]. AMPK activation by AICAR increases the FAT/CD36 plasma membrane translocation[20],leading to decreased FAT/CD36 expression at the cell surface[21].The results of this study indicate that decreased FAT/CD36 expression can lead to decreased IMF content.

There are some other evidences supporting the inhibition of fatty acid synthesis in white adipose tissues via AMPK activation[22].AMPK can regulate lipid metabolism through several mechanisms including the inhibition of preadipocyte differentiation and lipid accumulation[23].AMPK promotes fatty acid oxidation and inhibits lipid synthesis in cells through phosphorylation and the inhibition of acetyl-CoA carboxylase activity[9].AMPK is primarily activated by phosphorylation of the α2 subunit[24].AMPKγ3 also has an important role not only in glucose uptake and glycogen synthesis,but also in oxidation and lipid accumulation in the muscle.The activated AMPKγ3 mutation in muscle can increase oleate oxidation and prevent triglyceride accumulation[25].

AMPK expression also differed between fiber types[26].Lee-Young et al.[27]reported that AMPK phosphorylation was more pronounced in type II fibers than in type I fibers.Therefore, the correlation between IMF content and fiber type may be related to AMPK activation in muscle.

In conclusion,this study confirms that AMPK subunits,especially AMPKα2,AMPKγ1 and AMPKγ3, are significantly correlated with adipogenesis in chicken muscle.

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Responsible editor:Tingting XU

Responsible proofreader:Xiaoyan WU

Supported by National Natural Science Foundation of China(31472117);Natural Science Foundation of Hubei Province of China(2011CDB012);Project of State Key Laboratory of Animal Nutrition in China(2004DA125184F1012).

*Corresponding author.E-mail:yangyecaas@sina.com

Received:March 2,2015 Accepted:April 13,2015