A Study of the Regulatory Mechanism of the CB1/PPARγ2/PLIN1/HSL Pathway for Fat Metabolism in Cattle

Abstract

Fat metabolism is closely related to the economic characteristics of beef cattle. Therefore, regulating fat deposition and increasing intramuscular fat deposition are among the main goals of breeders. In this study, we aim to explore the regulatory role of CB1 gene on PPARγ2/PLIN1/HSL pathway in fat metabolism, and to further explore the differential expression of regulatory factors of this pathway in Shandong black cattle and Luxi cattle. In this study, CB1 overexpression stimulated lipid synthesis in adipocytes to some extent by increasing the levels of FASN and ACSL1. CB1 inhibitors reduce the lipid content in adipocytes and reduce the expression of GLUT1 and Insig1. In addition, overexpression of CB1 decreased the expression of PPARγ2 and led to an increase in PLIN1 expression and a decrease in HSL expression in adipocytes. We also found that the CB1/PPARγ2/PLIN1/HSL was differentially expressed in the different breeds of cattle and was involved in the regulation of fat metabolism, which affected the fatty acid content in the longissimus dorsi muscle of the two breeds. In short, CB1 participates in lipid metabolism by regulating HSL in the PPARγ2 and PLIN1 pathways, and improves lipid formation in adipocytes. In conclusion, CB1/PPARγ2/PLIN1/HSL pathway may be involved in the regulation of lipid metabolism.

Keywords: CB1; HSL; cattle; fat metabolism; pathway.

FIGURE 1

The regulatory network of fat metabolism in adipocytes. The regulatory network of fat metabolism includes five complete regulatory pathways: the excitatory G protein-coupled receptor pathway, inhibitory G protein-coupled receptor pathway, tyrosine kinase receptor pathway, Gq/PLC/PKC pathway and JAK/STAT pathway. Each color represents a pathway, and the most regulated genes are PLIN1 and HSL, which are the terminal regulators of multiple integrated pathways.

FIGURE 2

The effect of a CB1 overexpression or inhibitor on the growth of preadipocytes (3T3-L1). (A) Preadipocytes growth diagram of CB1 simulant or inhibitor treatment for 48 h. (B) CB1 expression was measured 24 h after initiation of CB1 overexpression or application of the inhibitor treatment, and the quantitative data showed multiple changes compared to the NC. (C) Western blotting was used to detect CB1 overexpression- or inhibitor-induced changes in cell growth-related protein levels. In the experiment, according to the size of maker, we cut PVDF membrane and incubate different protein antibodies, respectively. In the results, the bands are presented independently. (D) The quantitative data of the Western blotting are presented as fold changes compared to the NC data after ImageJ quantification; a, b, c indicate significant differences among the different treatments (n = 3; P < 0.05).

FIGURE 3

Effects of CB1 overexpression or inhibition on the lipid metabolism of adipocytes. (A) CB1 overexpression decreased and the CB1 inhibitor increased the levels of the HSL protein, as shown by IHC. Blue represents nucleus, Red represents HSL positive expression. (B–F) are the same as Figure 2.

FIGURE 4

Relative expression levels of the CB1/PPARγ2/PLIN1/HSL genes in different tissues. (A–D) qRT-PCR was used to detect the expression levels of CB1, PPARγ2, PLIN1 and HSL in different tissues. There was a difference in the expression between Shandong black cattle and Luxi cattle; B represents Shandong black cattle; L represents Luxi cattle; a and b were significantly different because of the different treatments (n = 3; P < 0.05).

FIGURE 5

Relative expression of the four proteins in tissues. (A) Relative expression of the four proteins in tissues as shown by IHC. (B) The protein levels of CB1, PPARγ2, PLIN1 and HSL were detected by Western blotting. (C) The quantitative data from the Western blot analyses, as determined by Image J, indicated that the protein levels were changed, and the significant differences were the same as those described above.

FIGURE 6

Fatty acid and Flavor substance determination. (A) SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; IMF, Intramuscular fat content. (B) Relative contents of various fatty acid classes in Longissimus dorsi between Shandong black cattle and Luxi cattle, detailed information in Abbreviations. (C) Relative contents of volatile flavor compounds in Longissimus dorsi between Shandong black cattle and Luxi cattle. The significant differences were the same as those described above.