Insulin-induced gene 1 and 2 isoforms synergistically regulate triacylglycerol accumulation, lipid droplet formation, and lipogenic gene expression in goat mammary epithelial cells

Abstract

The insulin-induced genes INSIG1 and INSIG2 (INSIG) are known to regulate adipogenesis in nonruminants. Although data in bovine mammary tissue underscore a role for INSIG1 during lactation, regulatory mechanisms of INSIG action in ruminant mammary lipid metabolism are not well known. In the present study, INSIG1 and INSIG2 were overexpressed or silenced through adenoviral transfection to evaluate their role in lipid metabolism in goat mammary epithelial cells (GMEC). The INSIG were overexpressed using an adenovirus system with recombinant green fluorescent protein as the control. Downregulation of INSIG was performed via small interfering RNA targeting INSIG with a scrambled small interfering RNA as a negative control. The GMEC were treated with these constructs for 48 h before analyses. Responses to overexpressing INSIG1 or INSIG2 included downregulation of SREBF1, ACACA, FASN, SCD1, GPAM, DGAT2, ATGL, and HSL coupled with a decrease in content of triacylglycerol (TAG), total cholesterol (TC), and lipid droplet accumulation. The marked decrease in content of TAG and TC in response to overexpression of INSIG2, along with a modest decrease in content of TAG when INSIG1 was overexpressed, suggested that TAG synthesis is mainly regulated by INSIG2, whereas TC synthesis is equally regulated by INSIG2 and INSIG1. The lack of difference in mRNA expression of genes related to lipid metabolism, content of TAG, and accumulation of lipids in response to interference alone of INSIG1 or INSIG2 indicated that INSIG proteins play a biological role in the maintenance of lipid homeostasis. However, in response to simultaneous interference of INSIG1 and INSIG2, the marked increase in content of TAG and TC and accumulation of lipids along with significant upregulation of SREBF1, ACACA, SCD1, AGPAT6, and DGAT2 suggested that INSIG1 and INSIG2 synergistically regulate milk fat synthesis in GMEC. These results highlight an essential role of INSIG in regulating lipid synthesis in dairy goat mammary cells and underscore the complexity of mammary lipid synthesis in ruminants.