Effectof FTO, METTL3Gene Expression On MRNAm~6Amathylationand Lipid Metabolism Inporcinesubcutaneousadipocytes

Excessive lipid deposition in livestock not only reduces the quality of meat products but also threats the health of humanbeings, so investigation of the regulatory mechanisms of body fat deposition and regulating body fat distribution through nutrition has become one of the problems to be solved in pig husbandry. The fatmass and obesity-associated gene (FTO) is one of the genes known to contribute to polygenic obesity. In fact it was the first one to be identified by genome wide association studies (GWAS). While initially investigated as one of the genes deleted in the fused toes mouse mutant, general interest was triggered by its robust association with obesity in numerous independent populations. A cluster of single nucleotide polymorphism (SNPs) in the first intron of the FTO gene was found to correlate with an increase in body mass index (BMI) in both children and adults, regardless of gender. Global FTO overexpression in mice leads to obesity in a gene dosage dependent manner while FTO-deficient mice exhibit a reduction in both lean and fat mass. So far, there are few reaearches about relationship between FTO expression and fat metabolism has been reported. On the molecular level, FTO was identified as a2-oxoglutarate dependent demethylase which is able to demethylate in vivo and affects a specific subset of m6A modified mRNAs, implicating a role for m6A in fat metabolism. Related to FTO is METTL3, a70-kDA subunit that forms part of the N6-adenosine methyltransferase, and it is thought that METTL3is responsible for methylating regular adenosines into m6A. Using castrated male fatty Jinghua pigs, lean Landrace pigs and LR×JH F2Changjin pigs at180days of age as model, the tissue-specific and the breed differences of FTO and METTL3, the effects of FTO and METTL3on the mRNA m6A level, the regulation on the fat metabolism and the expression of fat metabolism related genes were studied by using m6A blot, real-time PCR, RNA interference (RNAi) and overexpression techniques. Furthermore, the regulations of FTO expression and m6A level by cycloleucine and trimethylglycine were also studied. Results are as bellow:Exp.I Comparative study of obesity phenotypes in Landrace, Changjin pigs and Jinhua pigsBoth subcutaneous and visceral fat percentage of Jinhua pigs were significantly higher than Landrace and Changjin pigs, subcutaneous percentage of Jinhua pigs was2.2and1.75times of Landrace and Changjin pigs, while visceral fat percentage was4.35and1.57times, respectively. Comparing with Landrace, Changjin pigs had a higher visceral fat (p<0.05). Intramuscular fat content was greatest in fatty JH pigs (3.49%), and least in lean LR pigs (1.22%, P<0.05). HE staining of adipose tissue sections showed that both the subcutaneous and visceral adipocyte area were biggest in Jinhua pigs and smallest in Landrace pigs. These three pig lines are fit for models to elucidate the underlying mechanism of lipid metabolism on the basis of obesity phenotypes differences.Exp.Ⅱ The study on tissue-specific and breed differences of FTO and METTL3, adipose tissue m6A level in procineAdipose tissue, liver, hypothalamus regulats animal lipid storage, synthesis and feeding behavior, respectively. Tissue-specific study results showed that the FTO mRNA was highly expressed in hypothalamus, to a lesser degree in subcutaneous adipose tissues, and minimum in muscle tisssue. METTL3mRNA was highly expressed in adipose tissue, liver and hypothalamus tissues. These results indicated that expression of FTO and METTL3maybe related to fat or energy metabolism in porcine.Breed differences of gene expression showed that FTO was highest in Jinhua pigs and lowest in Changjin pigs at the level of transcription. FTO gene expression was significantly higher in Jinhua and Landrace pigs than in Changjin pigs. Jinhua pigs showed higher mRNA level of FTO than Landrace and Changjin pigsin AF, RAD, MAD and PAD (p<0.05). In the liver and hypothalamus tissues, FTO mRNA levels of Jinhua pig were significantly higher than Landrace and Chagnjin pigs (p<0.05), whereas no significant difference was obtained between Landrace and Changjin pigs. Breed differences in FTO protein levels consisted with the resultes in mRNA levels.On the other hand, METTL3expression in adipose tissues was highest in Landrace pigs and lowest in Changjin pigs at the level of transcription. In BF, RAD and GOM, METTL3gene expression was significantly higher in Landrace pigs than in Chagnjin and Jinhua pigs (p<0.05). There was no significant difference among breeds in liver or hypothalamus. Breed differences in METTL3protein levels consisted with the resultes in mRNA levels.The results of m6A blot showed that m6A level in mRNA of BF, AF, RAD, MAD and PAD was highest in Landrace pig and lowest in Jinhua pigs. Results above suggested that mRNA m6A level may play an important role in pig fat or energy metabolism.Exp. III Effect of mRNA m6A on fat deposition in porcine subcutaneous adipocytsIn vitro culture system of porcine adipocytes was successfully established. Using the RNA interference (RNAi) and overexpression techniques to explore the effect of FTO, METTL3on the m6A level and fat metabolism. FTO overexpression in porcine adipocyte significantly decreased the m6A levels (p<0.05) and (p=0.011), increased the intracellular triglyceride content (p<0.05). Meanwhile, mRNA expression of the adipogenic differentiation associated genes c/EBPp (p<0.05) and PPARy, fatty acids de novo synthesis genes FAS (p<0.05) and ACC was elevated, the lipolysis genes ATGL (p<0.05), HSL (p<0.05) expression was attenuated. FTO RNAi significantly decreased the m6A level and intracellular triglyceride content (p<0.05), increased glycerol content inculture medium. Expression of the adipogenic differentiation associated gene c/EBPβ (p<0.05) and PPARy (p<0.01), fatty acids de novo synthesis genes FAS (p<0.05) and ACC (p<0.01) was reduced, the lipolysis gene ATGL (p<0.05), HSL (p<0.05) expression were elevated, at the transcriptional level. These results suggested that the FTO expression can positively regulated mRNA m6A methylation, promote fat deposition and reduce fat decomposition in porcine subcutaneous adipocytes.METTL3overexpression significantly increased mRNA m6A level of porcine adipocyte (p<0.05), reduced the intracellular triglyceride content (p<0.05) with glycerol content increase in the medium (p<0.05). The adipogenic differentiation associated gene PPARy (p<0.01), fatty acids de novo synthesis genes FAS (p<0.05), ACC were downregulated, while lipolysis genes ATGL, HSL expression showed an uptrend. METTL3shRNA lentiviral infection had no significant effect on mRNA m6A level,, intracellular triglyceride content or glycerol content of the medium, indicating that there may be alternative ways to m6A catalytic reaction in porcine subcutaneous adipocytes. Consistency of mRNA m6A levels and fat metabolism changes indicated that mRNA m6A may play a role for the regulation of fat metabolism. Exp. IV Exogenous regulation of mRNA m6A methylation and its effect on fat depositionTo investigate the effect of mRNA m6A on fat metabolism, exogenous methylation inhibitor cycloleucine and methyl donors betaine were added in. Results showed that cycloleucine treatment significantly inhibited mRNA m6A in porcine subcutaneous adipocytes in a dose-dependent manner. The methyl donor showed that betaine can significantly improve mRNA m6A methylation in a dose-dependent effect in porcine subcutaneous adipocytes. Cycloleucine treatment significantly promoted fat deposition of porcine subcutaneous adipocytes (p<0.01), and significantly reduced the content of glycerol in the medium (p<0.05). Betaine treatment significantly reduced the intracellular triglyceride content(p<0.05), while increasing the content of glycerol in the medium. Meanwhile, cycloleucine treatment significantly increased the adipogenic differentiation associated gene c/EBPβPPARy gene expression (p<0.05), increased the fatty acid de novo synthesis genes FAS, ACC (p<0.05), but significantly downregulated the lipolysis genes ATGL (p<0.05), HSL(p<0.01) expression. Betaine treatment significantly reduced the adipogenic differentiation associated gene c/EBPβ (p<0.01), PPARy (p<0.05) gene expression, downregulated the fatty acids de novo synthesis genes FAS, ACC expression, while increasing lipolysis gene ATGL, HSL (p<0.01) expression. These findings suggested that the change of m6A methylation can regulate fat metabolism in porcine subcutaneous adipocytes. The effect of betaine on reducing fat deposition and promoting lipolysis may be related to the regulation of mRNA m6A methylatioIn summary, mRNA m6A methylation of porcine subcutaneous adipocytes negatively regulated fat deposition, and mRNA m6A methylation could be regulated by demethylase FTO, m6A methyltransferase METTL3, exogenous methylation inhibitor and methyl donors.