Abstract
Intramuscular fat (IMF) is considered as the fat deposited between muscle fibers. The extracellular matrix microenvironment of adipose tissue is of critical importance for the differentiation, remodeling and function of adipocytes. Therefore, in this study we extracted the muscle tissue centrifugal fluid (MTF) of the longissimus dorsi of Erhualian pigs to mimic the microenvironment of intramuscular pre-adipocytes. MTF of pigs with low intramuscular fat level can inhibit pig intramuscular pre-adipocytes differentiation. Then, proteomics technology (iTRAQ) was used to analyze the MTF with different IMF content, and it was found that individuals with high IMF had low ACAT2 (Acyl-CoA: cholesterol acyltransferases 2) levels, while individuals with low IMF had high ACAT2 levels. Significant changes took place in the pathways involved in coenzyme A, which are closely related to fat and cholesterol metabolism. Therefore, we speculate that ACAT2, as an important element involved in cholesterol metabolism, may become a potential molecular marker for the mechanism of pig intramuscular preadipocytes differentiation. Overexpression of ACAT2 in pig intramuscular pre-adipocytes can inhibit their differentiation, while adding ACAT2 inhibitor avasimibe can rescue the process. Knockdown of srebp2 or ldlr, which are two key genes closely related to ACAT2 and cholesterol metabolism, can inhibit pig intramuscular pre-adipocytes differentiation. Overall, our results suggest that ACAT2 is a novel negative regulator of intramuscular adipocyte differentiation through regulation of pparγ, cebpα signaling and srebp2/ldlr signaling involved in cholesterol metabolism.
Highlights
Intramuscular fat (IMF), the fat deposited between muscle fibers, has negative effects on health, closely related to insulin resistance and obesity [1]
We detected the mRNA and protein expression of pparγ and cebpα and Oil Red O staining to confirm the function of Muscle Tissue Fluid (MTF) on intramuscular pre-adipocytes differentiation
The muscle tissue fluid of individuals with very high intramuscular fat content had no effect on the differentiation of intramuscular pre-adipocytes (Figure S2C–H)
Summary
Intramuscular fat (IMF), the fat deposited between muscle fibers, has negative effects on health, closely related to insulin resistance and obesity [1]. Our previous studies have shown that muscle conditioned medium, as a simulation of the extracellular microenvironment, can reduce intramuscular adipocyte differentiation and lipid accumulation [4,5]. ACAT1 and ACAT2 are two key genes encoding cholesterol esterification enzymes that convert free cholesterol to cholesteryl esters for storage [14,15]. Both are membrane-spanning proteins located in the ER [16]. Studies have shown that ACATs over-expressed in mouse cells can inhibit the differentiation of pre-adipocytes, make mature lipid droplets size smaller and increase the free cholesterol level on the surface of the lipid droplets, thereby hindering the function of adipocytes [23]. Our data demonstrated that increased ACAT2 activity reduced the levels of SREBP2, LDLR, PPARγ and CEBPα, impaired differentiation of intramuscular pre-adipocytes
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