Insulin-induced gene 2 (INSIG2) is a recently identified gene that is implicated in the regulation of cholesterol metabolism and lipogenesis in mammals. Although the data in goats emphasizes a role for INSIG2 in milk fat synthesis, the regulatory mechanism in buffalo is not clear. In this study, we analyzed the protein abundance of INSIG2 at peak lactation and dry-off period in buffalo mammary tissue. The results indicated that, relative to the peak lactation, the protein abundance of INSIG2 in the dry-off period was higher. To determine the function of INSIG2 in milk fat synthesis, INSIG2 was overexpressed and knocked down by lentiviral transfection in buffalo mammary epithelial cells (BuMECs). The response to overexpressing INSIG2 included down-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 coupled with a decrease in content of triacylglycerol (TAG). However, in response to knockdown of INSIG2, the significant increase in content of TAG along with marked up-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 suggests that INSIG2 negatively affects milk fat synthesis in BuMECs. No significant difference in mRNA abundance of GPAM and DGAT2 in response to overexpression or interference of INSIG2 indicates that they might also be influenced by other regulatory factors. Taken together, our results provide strong support for the negative effect of INSIG2 on milk fat synthesis in BuMECs.
Keywords: Buffalo; INSIG2; interference and overexpression; milk fat synthesis.