miRNA-374 Regulates Dexamethasone-induced Differentiation of Primary Cultures of Porcine Adipocytes

Abstract

To investigate the effect of glucocorticoid on adipocytes metabolism and miRNAs that may be involved in adipocyte differentiation, primary porcine preadipocytes were treated with 10⁻⁶ M dexamethasone and RU486 (a glucocorticoid receptor antagonist) for 48 h. PPAR-γ (peroxisome proliferators-activated receptor-γ), and C/EBP-β (CCTTA enhancer binding protein-β) gene and protein expression were measured. The expressions of miRNAs predicted to directly target C/EBP-β were determined, and the functions of the potential miRNAs were verified. The results showed that the triglyceride content in cultured adipocytes increased significantly after 10⁻⁶ M dexamethasone treatment for 48 h, whereas the cell viability did not differ among the four groups (Control: 10⁻⁶ M dexamethasone; 10⁻⁶ M RU486: 10⁻⁶ M dexamethasone+10⁻⁶ M RU486) (p>0.05). Cells treated with dexamethasone for 48 h significantly upregulated perilipin and PPAR-γ gene expression, and PPAR-γ protein expression was also significantly increased. However, C/EBP-β mRNA and protein expression levels were significantly decreased. Both miR-374a and miR-374b, targeting the C/EBP-β 3′-UTR (3′-untranslated region), were significantly increased. Dual luciferase activity assay results indicated that miR-374a/b was directly recognised and bound to the 3′-UTR of C/EBP-β and thereby suppressed C/EBP-β gene expression. The present study showed that 10⁻⁶ M dexamethasone promotes lipid accumulation in primary cultures of porcine preadipocytes. PPAR-γ and C/EBP-β protein abundance showed differences after 48 h dexamethasone treatment; miR-374a/b may be involved in regulating of C/EBP-β expression. These results provide new targets for further regulation of porcine lipid metabolism.

Key words

miRNAs - C/EBP-β - porcine adipocyte - dexamethasone

• References

• 1 Peckett AJ, Wright DC, Riddell MC. The effects of glucocorticoids on adipose tissue lipid metabolism. Metabolism 2011; 60: 1500-1510
• 2 Djurhuus CB, Gravholt CH, Nielsen S, Mengel A, Christiansen JS, Schmitz OE, Moller N. Effects of cortisol on lipolysis and regional interstitial glycerol levels in humans. Am J Physiol Endocrinol Metab 2002; 283: E172-E177
• 3 Krsek M, osicka MR, Nedvidkova J, Kvasnickova H, Hana V, Marek J, Haluzik M, Lai EW, Pacak K. Increased lipolysis of subcutaneous abdominal adipose tissue and altered noradrenergic activity in patients with Cushing’s syndrome: an in-vivo microdialysis study. Physiol Res 2006; 55: 421-428
• 4 Wiper-Bergeron N, Wu D, Pope L, Schild-Poulter C, Hache RJ. Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex. EMBO J 2003; 22: 2135-2145
• 5 Caprio M, Feve B, Claes A, Viengchareun S, Lombes M, Zennaro MC. Pivotal role of the mineralocorticoid receptor in corticosteroid-induced adipogenesis. FASEB J 2007; 21: 2185-2194
• 6 Hausman GJ. Dexamethasone induced preadipocyte recruitment and expression of CCAAT/enhancing binding protein alpha and peroxisome proliferator activated receptor-gamma proteins in porcine stromal-vascular (S-V) cell cultures obtained before and after the onset of fetal adipogenesis. Gen Comp Endocrinol 2003; 133: 61-70
• 7 Hausman GJ. The influence of dexamethasone and insulin on expression of CCAAT/enhancer binding protein isoforms during preadipocyte differentiation in porcine stromal-vascular cell cultures: evidence for very early expression of C/EBPalpha. J Anim Sci 2000; 78: 1227-1235
• 8 Zhang H, Wu J, Wang B, Lu Z, Yang G. Regulation of SOCS-3, OB, GLUT4 and PPARgamma gene expression by insulin and dexamethasone in porcine primary adipocyte. Sheng Wu Gong Cheng Xue Bao 2008; 24: 1354-1360
• 9 Romao JM, Jin W, Dodson MV, Hausman GJ, Moore SS, Guan le L. MicroRNA regulation in mammalian adipogenesis. Exp Biol Med (Maywood) 2011; 236: 997-1004
• 10 Lefterova MI, Lazar MA. New developments in adipogenesis. Trends Endocrinol Metab 2009; 20: 107-114
• 11 Blanchette-Mackie EJ, Dwyer NK, Barber T, Coxey RA, Takeda T, Rondinone CM, Theodorakis JL, Greenberg AS, Londos C. Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes. J Lipid Res 1995; 36: 1211-1226
• 12 Cai Y, Yu X, Hu S, Yu J. A brief review on the mechanisms of miRNA regulation. Genomics Proteomics Bioinformatics 2009; 7: 147-154
• 13 Chua JH, Armugam A, Jeyaseelan K. MicroRNAs: biogenesis, function and applications. Curr Opin Mol Ther 2009; 11: 189-199
• 14 Davis-Dusenbery BN, Hata A. Mechanisms of control of microRNA biogenesis. J Biochem 2010; 148: 381-392
• 15 Xu P, Vernooy SY, Guo M, Hay BA. The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr Biol 2003; 13: 790-795
• 16 Huang Y, Cai X, Hu Y. The important role of microRNAs in lipid metabolism. Metabolism 2012; 62: e1-e2
• 17 Nakanishi N, Nakagawa Y, Tokushige N, Aoki N, Matsuzaka T, Ishii K, Yahagi N, Kobayashi K, Yatoh S, Takahashi A, Suzuki H, Urayama O, Yamada N, Shimano H. The up-regulation of microRNA-335 is associated with lipid metabolism in liver and white adipose tissue of genetically obese mice. Biochem Biophys Res Commun 2009; 385: 492-496
• 18 Bai L, Pang WJ, Yang YJ, Yang GS. Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes. Mol Cell Biochem 2008; 307: 129-140
• 19 Li B, Zerby HN, Lee K. Heart fatty acid binding protein is upregulated during porcine adipocyte development. J Anim Sci 2007; 85: 1651-1659
• 20 Wang T, Li M, Guan J, Li P, Wang H, Guo Y, Shuai S, Li X. MicroRNAs miR-27a and miR-143 Regulate Porcine Adipocyte Lipid Metabolism. Int J Mol Sci 2011; 12: 7950-7959
• 21 Ramírez-Zacarías JL, Castro-Muñozledo F, Kuri-Harcuch W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochem 1992; 97: 493-497
• 22 Dionisi M, Alexander SP, Bennett AJ. Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ) in vitro. Lipids Health Dis 2012; 11: 51
• 23 Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da Piedade I, Gunsalus KC, Stoffel M, Rajewsky N. Combinatorial microRNA target predictions. Nat Genet 2005; 37: 495-500
• 24 John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA targets. PLoS Biol 2004; 2: e363
• 25 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120: 15-20
• 26 Shi R, Chiang VL. Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 2005; 39: 519-525
• 27 Mendoza-Figueroa T, Hernandez A, De Lourdes Lopez M, Kuri-Harcuch W. Intracytoplasmic triglyceride accumulation produced by dexamethasone in adult rat hepatocytes cultivated on 3T3 cells. Toxicology 1988; 52: 273-286
• 28 Wu CH, Chien HF, Chang CY, Chen SH, Huang YS. Response of amoeboid and differentiating ramified microglia to glucocorticoids in postnatal rats: a lectin histochemical and ultrastructural study. Neurosci Res 2001; 40: 235-244
• 29 Pantoja C, Huff JT, Yamamoto KR. Glucocorticoid signaling defines a novel commitment state during adipogenesis in vitro. Mol Biol Cell 2008; 19: 4032-4041
• 30 Mendoza FT, Hernandez A, De LLM, Kuri HW. Intracytoplasmic triglyceride accumulation produced by dexamethasone in adult rat hepatocytes cultivated on 3T3 cells. Toxicology 1988; 52: 273-286
• 31 Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR, Koder A, Evans RM. PPAR gamma is required for placental, cardiac, and adipose tissue development. Mol Cell 1999; 4: 585-595
• 32 Rosen ED, Sarraf P, Troy AE, Bradwin G, Moore K, Milstone DS, Spiegelman BM, Mortensen RM. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell 1999; 4: 611-617
• 33 Bickel PE, Tansey JT, Welte MA. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores. Biochim Biophys Acta 2009; 1791: 419-440
• 34 Liu MF, Xu GH. Function of PAT family proteins in the lipid metabolism. Sheng Li Ke Xue Jin Zhan 2006; 37: 103-107
• 35 Yamaguchi T. PAT family: lipid droplet-associated proteins that regulate fat storage and lipolysis. Seikagaku 2007; 79: 162-166
• 36 Arimura N, Horiba T, Imagawa M, Shimizu M, Sato R. The peroxisome proliferator-activated receptor gamma regulates expression of the perilipin gene in adipocytes. J Biol Chem 2004; 279: 10070-10076
• 37 Nagai S, Shimizu C, Umetsu M, Taniguchi S, Endo M, Miyoshi H, Yoshioka N, Kubo M, Koike T. Identification of a functional peroxisome proliferator-activated receptor responsive element within the murine perilipin gene. Endocrinology 2004; 145: 2346-2356
• 38 Wiper-Bergeron N, Salem HA, Tomlinson JJ, Wu D, Hache RJ. Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPbeta by GCN5. Proc Natl Acad Sci USA 2007; 104: 2703-2708
• 39 Rangwala SM, Lazar MA. Transcriptional control of adipogenesis. Annu Rev Nutr 2000; 20: 535-559
• 40 Yeh WC, Cao Z, Classon M, McKnight SL. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev 1995; 9: 168-181
• 41 Rosen ED. The transcriptional basis of adipocyte development. Prostaglandins Leukot Essent Fatty Acids 2005; 73: 31-34
• 42 Tomlinson JJ, Boudreau A, Wu D, Atlas E, Hache RJ. Modulation of early human preadipocyte differentiation by glucocorticoids. Endocrinology 2006; 147: 5284-5293
• 43 Vosa U, Vooder T, Kolde R, Fischer K, Valk K, Tonisson N, Roosipuu R, Vilo J, Metspalu A, Annilo T. Identification of miR-374a as a prognostic marker for survival in patients with early-stage nonsmall cell lung cancer. Genes Chromosomes Cancer 2011; 50: 812-822
• 44 Delic D, Dkhil M, Al-Quraishy S, Wunderlich F. Hepatic miRNA expression reprogrammed by Plasmodium chabaudi malaria. Parasitol Res 2011; 108: 1111-1121
• 45 Huang Y, Guerrero-Preston R, Ratovitski EA. Phospho-DeltaNp63alpha-dependent regulation of autophagic signaling through transcription and micro-RNA modulation. Cell Cycle 2012; 11: 1247-1259
• 46 Wang YX, Zhang XY, Zhang BF, Yang CQ, Chen XM, Gao HJ. Initial study of microRNA expression profiles of colonic cancer without lymph node metastasis. J Dig Dis 2010; 11: 50-54
• 47 Wang C, Yang C, Chen X, Yao B, Zhu C, Li L, Wang J, Li X, Shao Y, Liu Y, Ji J, Zhang J, Zen K, Zhang CY, Zhang C. Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility. Clin Chem 2011; 57: 1722-1731
• 48 Pan S, Zheng Y, Zhao R, Yang X. MicroRNA-130b and microRNA-374b mediate the effect of maternal dietary protein on offspring lipid metabolism in Meishan pigs. Br J Nutr 2012; 1-8