Abstract:Adiponectin can improve insulin sensitivity, regulate fatty acid metabolism and participate in the cell proliferation and differentiation. Based on the highly conservative region of adiponectin receptor 1 gene (AdipoR1) sequences from bovine (Bos taurus), human (Homo sapiens) and mouse (Mus musculus) in GenBank, the specific primers were designed. A full-length 2 032 bp cDNA sequence of AdipoR1 gene(GenBank accession GQ918145) was isolated and cloned in mammary gland of Xinong Saanen dairy goats (Capra hircus) using RT-PCR and RACE. AdipoR1 gene contained 185 bp 5' untranslated regions(UTR), 719 bp 3' UTR, and 1 128 bp coding sequences (CDS) which encode 375 amino acid (AA). AA sequence alignment showed that the AA of goat AdipoR1 had higher similarity (>95%) with bovine, pig(Sus scrofa), mouse and human. The protein structure analysis showed that the predicted molecular weight was 42.44 kD, the isoelectric point was 7.19, and 7 transmembrane domains and no signal peptide were included in the entire sequence. Furthermore, expression analysis showed that AdipoR1 gene had the most abundant expression in lung, followed by the small intestine and the minimal expression was observed in heart. However, the expression of AdipoR1 in remaining eight tissues changed more gently. Expression analysis in the two different stages of mammary tissue revealed that there was significantly different (P<0.05) between peak lactation and dry period. Goat mammary epithelial cells were treated with different concentrations of insulin and prolactin, and the result showed that the mRNA level of AdipoR1 in group treated with insulin decreased and there was the most obvious effect at insulin concentration of 50 nmol/L (P<0.01); the mRNA level of AdipoR1 in group treated with prolactin increased and there was the most obvious effect at prolactin concentration of 100 mg/mL (P<0.05). These results indicated that AdipoR1 had certain regulative effects in goat mammary epithelial cells. This work provides basic information to further reveal the function of AdipoR1 in goat mammary gland.
Bjursell, M., A. Ahnmark, Y. M. Bohlooly, L. William-Olsson, M. Rhedin, X. R. Peng, K. Ploj, A. K. Gerdin, G. Arnerup, A. Elmgren, A. L. Berg, J. Oscarsson, and D. Linden. 2007. Opposing effects of adiponectin receptors 1 and 2 on energy metabolism. Diabetes 56(3):583-593.Bluher, M., M. Fasshauer, S. Kralisch, M. R. Schon, K. Krohn, and R. Paschke. 2005. Regulation of adiponectin receptor R1 and R2 gene expression in adipocytes of C57BL/6 mice. Biochemical and biophysical research communications 329(3):1127-1132.Chinetti, G., C. Zawadski, J. C. Fruchart, and B. Staels. 2004. Expression of adiponectin receptors in human macrophages and regulation by agonists of the nuclear receptors PPARalpha, PPARgamma, and LXR. Biochemical and biophysical research communications 314(1):151-158.Civitarese, A. E., C. P. Jenkinson, D. Richardson, M. Bajaj, K. Cusi, S. Kashyap, R. Berria, R. Belfort, R. A. DeFronzo, L. J. Mandarino, and E. Ravussin. 2004. Adiponectin receptors gene expression and insulin sensitivity in non-diabetic Mexican Americans with or without a family history of Type 2 diabetes. Diabetologia 47(5):816-820.Coope, A., M. Milanski, E. P. Araujo, M. Tambascia, M. J. Saad, B. Geloneze, and L. A. Velloso. 2008. AdipoR1 mediates the anorexigenic and insulin/leptin-like actions of adiponectin in the hypothalamus. FEBS letters 582(10):1471-1476.Cui, X. B., C. Wang, L. Li, D. Fan, Y. Zhou, D. Wu, Q. H. Cui, F. Y. Fu, and L. L. Wu. 2012. Insulin decreases myocardial adiponectin receptor 1 expression via PI3K/Akt and FoxO1 pathway. Cardiovascular research 93(1):69-78.Guo, Z., Z. Xia, V. G. Yuen, and J. H. McNeill. 2007. Cardiac expression of adiponectin and its receptors in streptozotocin-induced diabetic rats. Metabolism: clinical and experimental 56(10):1363-1371.Inukai, K., Y. Nakashima, M. Watanabe, N. Takata, T. Sawa, S. Kurihara, T. Awata, and S. Katayama. 2005. Regulation of adiponectin receptor gene expression in diabetic mice. American journal of physiology. Endocrinology and metabolism 288(5):E876-882.Neumeier, M., J. Weigert, A. Schaffler, T. Weiss, S. Kirchner, S. Laberer, J. Scholmerich, and C. Buechler. 2005. Regulation of adiponectin receptor 1 in human hepatocytes by agonists of nuclear receptors. Biochemical and biophysical research communications 334(3):924-929.Nilsson, L., N. Binart, Y. M. Bohlooly, M. Bramnert, E. Egecioglu, J. Kindblom, P. A. Kelly, J. J. Kopchick, C. J. Ormandy, C. Ling, and H. Billig. 2005. Prolactin and growth hormone regulate adiponectin secretion and receptor expression in adipose tissue. Biochemical and biophysical research communications 331(4):1120-1126.Ohtani, Y., T. Yonezawa, S. H. Song, T. Takahashi, A. Ardiyanti, K. Sato, A. Hagino, S. G. Roh, and K. Katoh. 2011. Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells. Animal science journal = Nihon chikusan Gakkaiho 82(1):99-106.Tsuchida, A., T. Yamauchi, Y. Ito, Y. Hada, T. Maki, S. Takekawa, J. Kamon, M. Kobayashi, R. Suzuki, K. Hara, N. Kubota, Y. Terauchi, P. Froguel, J. Nakae, M. Kasuga, D. Accili, K. Tobe, K. Ueki, R. Nagai, and T. Kadowaki. 2004. Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity. The Journal of biological chemistry 279(29):30817-30822.Yamauchi, T., J. Kamon, Y. Ito, A. Tsuchida, T. Yokomizo, S. Kita, T. Sugiyama, M. Miyagishi, K. Hara, M. Tsunoda, K. Murakami, T. Ohteki, S. Uchida, S. Takekawa, H. Waki, N. H. Tsuno, Y. Shibata, Y. Terauchi, P. Froguel, K. Tobe, S. Koyasu, K. Taira, T. Kitamura, T. Shimizu, R. Nagai, and T. Kadowaki. 2003. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423(6941):762-769.Yamauchi, T., Y. Nio, T. Maki, M. Kobayashi, T. Takazawa, M. Iwabu, M. Okada-Iwabu, S. Kawamoto, N. Kubota, T. Kubota, Y. Ito, J. Kamon, A. Tsuchida, K. Kumagai, H. Kozono, Y. Hada, H. Ogata, K. Tokuyama, M. Tsunoda, T. Ide, K. Murakami, M. Awazawa, I. Takamoto, P. Froguel, K. Hara, K. Tobe, R. Nagai, K. Ueki, and T. Kadowaki. 2007. Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nature medicine 13(3):332-339.Yoon, M. J., G. Y. Lee, J. J. Chung, Y. H. Ahn, S. H. Hong, and J. B. Kim. 2006. Adiponectin increases fatty acid oxidation in skeletal muscle cells by sequential activation of AMP-activated protein kinase, p38 mitogen-activated protein kinase, and peroxisome proliferator-activated receptor alpha. Diabetes 55(9):2562-2570.Hu S L, Luo J, Wang H, Zhao W S, Li J, Sun Y T, Zhu J J and Shi H B, 2012. Cloning and tissues expression of insulin- induced gene 2 (INSIG2) in Xinong Saanen dairy goats(Capra hircus). Journal of Agricultural Biotechnology, 20(9): 1048~1054(胡仕良, 罗军, 王慧, 赵旺生, 李君, 孙雨婷, 朱江江, and 石恒波. 2012. 西农萨能羊胰岛素诱导基因2(INSIG2)的克隆及组织表达分析. 农业生物技术学报 (09):1048-1054.)Teng Y L, Luo J, Li J, Zhao W S, Wang Z, Wang W, and Hu S L, 2010. Cloning and tissue expression analys is of adipose triglyceride lipase gene (ATGL) of Xinong Saanen dairy goats. Journal of Agricultural Biotechnology, 18(6): 1134~1142(滕炎玲, 罗军, 李君, 赵旺生, 王桢, 王维, and 胡仕良. 2010. 西农萨能奶山羊甘油三酯水解酶基因(ATGL)的cDNA克隆与组织表达分析. 农业生物技术学报 (06):1134-1142)Wang Z, Luo J, Wang W, Zhao W S and Lin X Z, 2010. Characterization and culture of isolated primary dairy goat mammary gland epithelial cells. Chinese Journal of Biotechnology, 26(8): 1123~1127(王桢, 罗军, 王伟, 赵旺生, and 林先滋. 2010. 奶山羊乳腺上皮细胞的分离、培养及鉴定. 生物工程学报 (08):1123-1127)