Activation of skeletal carbohydrate-response element binding protein (ChREBP)-mediated de novo lipogenesis increases intramuscular fat content in chickens

Abstract

The intracellular lipids in muscle cells of farm animals play a crucial role in determining the overall intramuscular fat (IMF) content, which has a positive impact on meat quality. However, the mechanisms underlying the deposition of lipids in muscle cells of farm animals are not yet fully understood. The purpose of this study was to determine the roles of carbohydrate-response element binding protein (ChREBP) and fructose in IMF deposition of chickens. For virus-mediated ChREBP overexpression in tibialis anterior (TA) muscle of chickens, seven 5-d-old male yellow-feather chickens were used. At 10 d after virus injection, the chickens were slaughtered to obtain TA muscles for analysis. For fructose administration trial, sixty 9-wk-old male yellow-feather chickens were randomly divided into 2 groups, with 6 replicates per group and 5 chickens per replicate. The chickens were fed either a basal diet or a basal diet supplemented with 10% fructose (purity ≥ 99%). At 4 wk later, the chickens were slaughtered, and breast and thigh muscles were collected for analysis. The results showed that the skeletal ChREBP mRNA levels were positively associated with IMF content in multiple species, including the chickens, pigs, and mice (P < 0.05). ChREBP overexpression increased lipid accumulation in both muscle cells in vitro and the TA muscles of mice and chickens in vivo (P < 0.05), by activation of the de novo lipogenesis (DNL) pathway. Moreover, activation of ChREBP by dietary fructose administration also resulted in increased IMF content in mice and notably chickens (P < 0.05). Furthermore, the lipidomics analysis revealed that ChREBP activation altered the lipid composition of chicken IMF and tented to improve the flavor profile of the meat. In conclusion, this study found that ChREBP plays a pivotal role in mediating the deposition of fat in chicken muscles in response to fructose-rich diets, which provides a novel strategy for improving meat quality in the livestock industry.

Keywords: Carbohydrate-response element binding protein; Chicken; Fructose; Intramuscular fat; Meat quality.

Fig. 1

ChREBP-β overexpression in muscle cells promotes lipid accumulation. (A) The retroviral-infected C2C12 myoblasts. Scale bar, 100 μm. (B to C) The analysis of ChREBP expression levels in C2C12 myoblasts by qRT-PCR and Western blotting. (D) Oil red O staining of C2C12 myoblasts overexpressing GFP or ChREBP-β under adipogenic induction for 7 d. Scale bar, 100 μm. (E) TG content in C2C12 myoblasts overexpressing GFP or ChREBP-β for 7 d of adipogenic induction. (F to H) qRT-PCR analysis of mRNA levels of (F) adipogenic genes, (G) lipogenic genes, and (H) myogenic genes. (I) Western blotting analysis for the expression of PPARγ, FAS, and MyHC proteins. GFP = green fluorescent protein; ChREBP-α = carbohydrate-response element binding protein-α; ChREBP-β = carbohydrate-response element binding protein-β; TG = triglyceride; Pparg/PPARγ = peroxisome proliferator activated receptor gamma; Cebpa = CCAAT enhancer binding protein alpha; Adipoq = adiponectin; Fabp4 = fatty acid binding protein 4; ChREBP = carbohydrate-response element binding protein; Fasn/FAS = fatty acid synthase; Acaca = acetyl-CoA carboxylase alpha; Acly = ATP citrate lyase; Glut4 = glucose transporter 4; Elovl6 = ELOVL fatty acid elongase 6; Srebp1c = sterol regulatory element binding transcription factor 1; MyoG = myogenin; MyoD1 = myogenic differentiation 1; MyhC = myosin heavy chain; a-tubulin = alpha-tubulin; All the results are shown as the means ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 2

RNA sequencing (RNA-seq) analysis reveals ChREBP-β overexpression in C2C12 myoblasts activates lipogenic pathway but inhibits myogenic pathway. (A) Volcano plot comparison of gene expression. (B) Cluster analysis of all differential genes. (C) Gene set enrichment analysis (GSEA) of differentially expressed genes. (D and E) GSEA analysis of the significantly upregulated (regulation of lipid biosynthetic process) and downregulated (muscle cell development) pathways. (F and G) Heat map of genes related with lipid synthesis and muscle development. (H and I) qRT-PCR analysis of lipogenic genes and myogenic genes. GFP = green fluorescent protein; ChREBP-β = carbohydrate-response element binding protein-β; ES = enrichment score; NES = normalized enrichment scores; FDR = false discovery rate; Pdh1 = pyruvate dehydrogenase 1; Scd1 = stearoyl-coenzyme A desaturase 1; Gadph = glyceraldehyde-3-phosphate dehydrogenase; Lpcat1 = lysophosphatidylcholine acyltransferase 1; Fasn = fatty acid synthase; Acaca = acetyl-CoA carboxylase alpha; Acly = ATP citrate lyase; Mlxipl = MLX interacting protein-like; Myf5 = myogenic factor 5; Six4 = SIX homeobox 4; MyoD1 = myogenic differentiation 1; Six1 = SIX homeobox 1; Sirt1 = sirtuin 1; MyoG = myogenin; Notch1 = notch receptor 1; Mef2a = myocyte enhancer factor 2A; MyhC = myosin heavy chain. All data were collected from C2C12 cells overexpressing GFP or ChREBP-β without induction (n = 3). All the results are shown as the means ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 3

AAV-mediated overexpression of ChREBP-β in mice TA muscles facilitates DNL pathway and increases TG accumulation. (A) Diagram of experimental design. (B) qRT-PCR analysis of ChREBP expression in TA muscles (n = 6 to 7). (C) Western blotting analysis of Flag-ChREBP-β expression in TA muscles (n = 3). (D and E) Relative mRNA levels of lipogenic genes and pan-adipocyte genes in TA muscles (n = 6 to 7). (F) TG content in TA muscles (n = 6). (G) The weight of TA muscles (n = 10). (H) Hematoxylin and eosin (H&E) staining of paraffin sections of TA muscles. Scale bar, 100 μm. (I) Quantification of average muscle fiber area from images depicted in Fig. 3H. AAV = adeno-associated virus; TA = tibialis anterior; Flag-GFP = FLAG-tagged green fluorescent protein; Flag-ChREBP-β = FLAG-tagged carbohydrate-response element binding protein-β; ChREBP = carbohydrate-response element binding protein; Fasn = fatty acid synthase; Acaca = acetyl-CoA carboxylase alpha; Acly = ATP citrate lyase; Pparg = peroxisome proliferator activated receptor gamma; Cebpa = CCAAT enhancer binding protein alpha; Adipoq = adiponectin; Fabp4 = fatty acid binding protein 4; TG = triglyceride. GFP treatment = AAV-Flag-green fluorescent protein; ChREBP treatment = AAV-Flag-ChREBP-β. All the results are shown as the means ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 4

Untargeted lipidomic analysis reveals dietary fructose supplementation remodels intramuscular fat (IMF) composition in chickens. (A) Percentages of lipid subclasses in TA muscles of control and fructose addition groups (n = 7). (B) Volcano plot comparison of lipid levels (n = 7). (C) Heat map analysis of all content-differential lipid molecules (n = 7). (D) The numbers of content-increased lipid molecules in fructose-fed group (n = 7). (E to I) The representative content-upregulated lipid molecules in each lipid subclass (n = 7). TG = triglyceride; DG = diglyceride; PS = phosphatidylserine; PE = phosphatidylethanolamine; PC = phosphatidylcholine; SM = sphingomyelin; Cer = ceramide. Control treatment = basal diet; Fructose treatment = basal diet supplemented with 10% fructose (purity ≥ 99%). All the results are shown as the means ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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