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 IN-SIG2, 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.

De novo assembly and analysis of the transcriptome of the Dermacentor marginatus genes differentially expressed after blood-feeding and long-term starvation

“…The unigenes involved in cholesterol metabolism were insulin induced protein (DN55975), high-density lipoprotein (DN55629), Niemann-Pick type C1 (DN34326) and Niemann-Pick type C2 (DN53284) during blood-feeding, and their expression profile indicated lipid metabolism was very active in newly molted ticks. In non-ruminants, the INSIG1 gene modulates cholesterol metabolism, lipogenesis, and glucose homeostasis with a cellular localization on endoplasmic reticulum membrane [ 64 ]. High density lipoprotein transports diglyceride from the fat body cell into the hemolymph, and is involved in the transport of cholesterol from the gut into the hemolymph in insects [ 65 , 66 ].…”

De Novo Assembly and Analysis of Transcriptome of the Dermacentor Marginatus Genes Differentially Expressed After Blood Feeding and Long-term Starvation

“…The unigenes involved in cholesterol metabolism were insulin induced protein (DN55975), high-density lipoprotein (DN55629), Niemann-Pick type C1 (DN34326) and Niemann-Pick type C2 (DN53284) during blood feeding, and their expression pro le indicated lipid metabolism was very active in newly molted ticks. In nonruminants The INSIG1 gene modulates cholesterol metabolism, lipogenesis, and glucose homeostasis with a cellular localization on endoplasmic reticulum membrane [61]. High density lipoprotein transports diglyceride from the fat body cell into the hemolymph, and is involved in the transport of cholesterol from the gut into the hemolymphin insects [62,63].…”

Activation of G Protein-coupled Receptor 30 Promotes Proliferation of Goat Mammary Epithelial Cells via MEK/Erk&PI3K/Akt Signaling Pathway

“…Goat mammary gland tissues were surgically isolated from a 2-year-old lactating Guanzhong dairy goat at 36 d of lactation as described previously [22], which were reared in Shaanxi Province (P. R. China). The mammary parenchyma was obtained under sterile conditions, placed in sterilized PBS with penicillin, streptomycin, and amphotericin B at 100 U/mL, 100 µg/mL, and 25 ng/mL (Sigma, St. Louis, MO, USA), and immediately transported to the laboratory.…”