The Effect Of INSIG Gene On Lipid Metabolism In Goat Mammary Gland Epithelial Cells

Insulin induced gene(INSIGs), located in the membrane of endoplasmic reticulum(ER), which including two subtypes of INSIG1 and INSIG2, play an important role in the regulation of lipid metabolism. There are some differences of biological function and expression regulation mechanism between these two isoforms in different tissues. However, there are rare reports about the regulatory function of INSIGs gene on lipid metabolism in goat mammary gland cells(GMEC).To investigate the role of INSIGs in goat mammary gland, samples of mammary gland tissue during different lactation periods of Xinong Saanen dairy goat were collected, and q RT-PCR was used to measure the m RNA expression profiles of INSIG1 and INSIG2. Then, we constructed the recombinant adenovirus containing INSIG1 or INSIG2 gene, and obtained the overexpression adenovirus Ad-INSIG1 and Ad-INSIG2 with high titer. In addition, the effective si RNA were designed to knockdown the m RNA expression of INSIG1 or INSIG2. After knockdown and overexpression of INSIGs gene, we measured the expression change of lipid-related genes, and accumulation of triacylglycerol(TAG), total cholesterol(TC) and lipid droplets. This study provided the theoretical foundation for the function study of INSIGs in goat mammary lipid metabolism. The main results of this study are as follows:(1) In the mammary gland of dairy goat, the expression of INSIG1 and INSIG2 are similar during different lactating stages. Both of them get the highest expression in the early-lactating stage, and get the lowest expression in dry lactation period.(2) With transmembrane protein structure features, both of INSIG1 and INSIG2 contain five transmembrane structures, and the conservative areas of both two subtypes were concentrated in the transmembrane region, and they shared a 71% similarity in nucleotide sequence, a 80% similarity in amino acids sequence.(3) The INSIG1 overexpression recombinant adenovirus vector was successfully constructed, and the overexpression recombinant adenovirus Ad-INSIG1 with a high titer of 2×108 U/m L was obtained. Compared with Ad-GFP infected group, the m RNA expression of INSIG1 increased about 500-fold after incubating with Ad-INSIG1 for 48 h. No obvious changes were observed on the m RNA expression of SREBP1 and SREBP cleavage-activating protein(SCAP). However, there was a significant(P < 0.05) decrease in the expression of genes related to fatty acid de novo synthesis and desaturasion(ACCα; FASN; SCD1), TAG synthesis(GPAM; DGAT2), and TAG hydrolysis(ATGL). Under the same condition, no significant difference was observed in the cellular TAG content, but the content of total cholesterol decreased significantly(P < 0.05), the accumulation of lipid droplet slightly decreased.(4) The INSIG2 overexpression recombinant adenovirus vector was successfully constructed, and the overexpression adenovirus Ad-INSIG2 with a high titer of 3×108 U/m L was obtained. Compared with Ad-GFP infected group, the m RNA expression of INSIG2 increased about 50-fold after incubating with Ad-INSIG2 for 48 h. No obvious changes were observed in the m RNA expression of SREBP1 and SCAP. However, there was a significant(P < 0.05) decrease in expression of genes related to fatty acid de novo synthesis and desaturasion(ACCα, SCD1), TAG synthesis(DGAT2; AGPAT6), and TAG hydrolysis(HSL). Under the same condition, there was a significant decrease in the accumulation of cellular TAG, TC and lipid droplet accumulation(P < 0.05).(5) Two pairs of si RNA sequences(si INSIG1-100 and si INSIG1-547; si INSIG2 and si INSIG2-175) were designed for INSIG1 and INSIG2 gene, respectively. After transfected for 48 h, the INSIG1 or INSIG2 gene expression were downregulated about 80% by si INSIG1-547 and si INSIG2-175 in GMEC, respectively. When co-transfected the two si RNAs simultaneously, the expression of INSIGs were significantly reduced by 80%. While knockdown of either INSIG1 or INSIG2, lipid metabolism related genes expression, TAG content and lipid droplets accumulation all had no obvious changes, while the cellular content of TC increased significantly(P < 0.05). Knockdown of both INSIG1 and INSIG2 significantly stimulated the expression of lipid-related genes, resulted in the increase of cellular TAG, TC content, and lipid droplets formation(P < 0.05).This study shows that INSIG1 and INSIG2 could regulate the expression of lipid-related genes in GMEC, the overexpression of INSIGs can inhibit cell lipid accumulation, and only when simultaneous knockdown of both INSIGs isoforms could increase lipid accumulation, but not in INSIG1 or INSIG2 individual suppressed cells. Compared with INSIG1, INSIG2 may have a bigger influence on TAG metabolism.