Study On The Mechanism Of Fat Deposition Action Of AdipoQ And AdipoR1/2 In Goat

This study is to detect the functions of AdipoQ, AdipoR1 and AdipoR2 in regulating fat deposition, and lay a fundation for improving the meat quality through regulating the fat acid synthesis by AdipoQ in goat. Here, we cloned the full-length coding sequence of Nanjiang Yellow goat AdipoQ, AdipoRl and AdipoR 2 genes. Semiquantitative RT-PCR was used to detect their expression patterns in eleven tissues, including ovary, spleen, rumen, liver, brain, heart, gastrocnemius muscle, biceps femoris muscle, longissimus dorsi muscle, mesenteric adipose and subcutaneous adipose. Then, qPCR was used to quantify their mRNA relative expressions in longissimus dorsi muscle at 3 days,30 days, 60 days,90 days and 120 days.High concentration glucose culture medium (25 mM/L) was used to induce the differentiation of SMSCs into adipocytes, and cells were stained with Oil Red O to examine the formation of lipids, qPCR was employed to detect the relative expressions of ACC, FAS, C/EBPα, PPARγ, SREBP-1, AdipoQ, AdipoR1 and AdipoR2 at 0 day and five time points (2 days,4 days,6 days,8 days and 10 days) under the conditions of high and low concentration glucose.We constructed the pEGFP-N1-AD recombinant plasmid and overexpressed the AdipoQ gene in SMSCs. The adiponectin protein subcellular localization in cell was detected by fluorescence microscope, and qPCR was used to quantify the relative expressions of ACC, FAS, C/EBPα, PPARγ, SREBP-1, AdipoQ, AdipoR1 and AdipoR2 genes. Results were showed as follows:(1) We obtained the full-length coding sequences of goat AdipoQ, AdipoR1 and AdipoR2 genes (Accession number:JX573539, KC286912 and JX573540) which are 720 bp,1128 bp and 1176 bp. These genes encode 239,375 and 391 amino acids, respectively. The AdipoQ gene shares 85%,97% and 88% similarity with the orthologous gene of humans, cattle and pig. AdipoRl gene shares 87%,97% and 90% similarity, and AdipoR2 gene shares 86%,97% and 88% similarity with the orthologous gene of humans, cattle and pig, which indicated that these three genes were conserved in species.(2) The semiquantitative RT-PCR indicated that the AdipoQ expression was predominant in mesenteric adipose and subcutaneous adipose, weak in gastrocnemius muscle, longissimus dorsi muscle and biceps femoris muscle, and no expression in other tissues. The AdipoRl mRNA was expressed in almost all tissues, with a high expression in skeletal muscles, rumen and adipose tissues, whereas its expressions in other tissues were relatively weak. Similarly, the AdipoR2 was also expressed in all tissues and most abundant in adipose tissues and skeletal muscles, weakly expressed in other tissues.(3) The qPCR results showed that AdipoQ was expressed in a up-down-up trend in both male and female goat, with the lowest (P<0.05) level and the highest (P<0.05) level at 3 days and 120 days in male goat, and 3 days and 60 days in female goat. The expression of AdipoRl in male goat was in a down-up trend which reached the highest (P<0.05) level at 60 days and decreased to the lowest (P<0.05) level at 90 days. In female goat, it was expressed in a down-up-down trend, with the lowest (P<0.05) level at 30 days and increased to the highest (P<0.01) level at 60 days. The expression of AdipoR2 was in a down-up trend in male goat and a down-up-down trend in female goat, while both reached the lowest (P<0.01) level at 30 days and the highest (P<0.01) level at 90 days.(4) High concentration glucose culture medium was used to induce the goat SMSCs to synthesise fatty acid. We observed some lipid droplets at 6 days and more lipid droplets at 10 days in cells stained with Oil Red O, while almost no lipid droplets was observed in low glucose group. The qPCR results indicated that the expressions of ACC, FAS, C/EBPa, PPARy and SREBP-1 were significantly up-regulated by high glucose. In this process, the expressions of AdipoQ, AdipoRl and AdipoR2 were also up-regulated, which means these three genes also play roles in fatty acid synthesis and adipocyte differentiation.(5) In SMSCs overexpressed with AdipoQ gene, the AdipoQ fusion protein was only observed in cytoplasm, which suggested its important functions in cytoplasm. The qPCR results showed that the expressions of ACC, FAS, SREBP-1, AdipoR1 and AdipoR2 were significantly up-regulated, whereas the expressions of C/EBPa, PPARy and AdipoRl had no significant changes. These results indicated that AdipoQ increased the expressions of ACC, FAS, SREBP-1 and AdipoR2, and promoted fatty acid synthesis.