Mechanism Of M~6A Methyltransferase Complex In Regulating Energy Metabolism In Brown Adipose Tissue

Obesity is caused by an imbalance in energy metabolism.Obesity can lead to a variety of diseases such as type 2 diabetes mellitus（T2DM）,fatty liver and cardiovascular diseases^[1-4].Fat plays an extremely important role in energy metabolism.There are three main types of adipose tissue:Brown adipose tissue（BAT）,White adipose tissue（WAT）and Beige adipose cells.White adipose tissue stores large amounts of triglycerides and promotes obesity.Both brown adipose tissue and beige adipose cells express thermogenic genes such as Uncoupling protein 1（UCP1）,which can oxidize fatty acids and release them as heat.Therefore,increasing the energy consumption of brown adipose tissue is one of the effective measures to alleviate obesity.It is of great significance to study the molecular mechanism of brown adipose tissue development.The results showed that Positive regulatory domain containing 16（PRDM16）and Peroxisome Proliferator-activated receptorγ（PPARγ）are key transcription factors that determine the fate of brown fat Receptors^[5,6].Peroxisome proliferator-activated receptor-gamma coactivator-1α（PGC-1α）can trigger mitochondrial biogenesis,oxidative metabolism,and thermogenesis by inducing the expression of UCP1 and many other genes^[7].Whether their m RNA m⁶A modification can regulate BAT development and energy expenditure is largely unknown.N⁶-methyladenine（m⁶A）is one of the most common m RNA modifications in eukaryotes.m⁶A Methyltransferase complex includes methyltransferase-like 3（METTL3）,methyltransferase-like 14（METTL14）and Wilms’tumor 1-associating protein（WTAP）.METTL3 is a key m⁶A methyltransferase that has been shown to regulate early embryonic development^[8],neurogenesis^[9],stem cell differentiation^[10,11],and non-alcoholic steatohepatitis^[12].WTAP is a regulatory protein of m⁶A m RNA modification.WTAP has been shown to regulate X chromosome imprinting and cell proliferation^[13].Our study found that METTL3 and WTAP were highly expressed in mouse Interscapular brown adipose tissue（i BAT）,and significantly increased during the development of i BAT after birth,similar to the expression pattern of UCP1.This suggests that METTL3 and WTAP may be involved in the postnatal development of i BAT.In order to study the effects of METTL3 and WTAP on the development of BAT in mice after birth,we have constructed BAT-specific knockout of Mettl3（Mettl3-BKO）and BAT-specific knockout of Wtap（Wtap-BKO）mice using the Cre/Lox P system.The results showed that both Mettl3-BKO and Wtap-BKO mice showed large and white i BAT phenotypes at the fifth to tenth day after birth,suggesting that METTL3 and WTAP are essential for the postnatal development of i BAT in mice.In addition,Mettl3-BKO and Wtap-BKO mice had impaired energy expenditure and were more sensitive to cold stimulation at 4℃.BAT-specific knockout of Mettl3 further promoted obesity and systemic insulin resistance induced by high fat diet（HFD）.These data suggest that METTL3 is an important regulator of postnatal development and energy homeostasis of i BAT.In terms of molecular mechanism,RNA-Seq analysis of Mettl3-BKO and Wtap-BKO mouse i BAT showed that most genes related to BAT maturation,respiratory electron transport chain,adaptive heat production and energy expenditure were significantly down-regulated.Me RIP-seq analysis showed that Mettl3 knockout with BAT specificity reduced the m⁶A modification of Prdm16,Pparg,and Ucp1 m RNA.Knockdown of Mettl3 in primary brown precursor adipocytes significantly decreased luciferase activity at Prdm16,Pparg,and Ucp1 m⁶A modification sites.WTAP has strong interaction with METTL3.METTL3 protein levels in i BAT of Wtap-BKO mice were significantly decreased,but m RNA levels were not.The protein synthesis inhibitor cycloheximide（CHX）was treated in brown adipocytes with WTAP knockdown in vitro,and the stability of METTL3 protein was significantly reduced.Moreover,Wtap-BKO dependence on the proteasome system reduces the stability of METTL3 protein in i BAT.WTAP can regulate m⁶A modification by regulating the stability of METTL3 protein.BAT specific overexpression of METTL3 can partially save the phenotype of BAT dysplasia and thermogenesis in Wtap-BKO mice.These data indicate that METTL3regulates the development and maturation of BAT in mice after birth by regulating the expressions of Prdm16,Pparg and Ucp1.WTAP regulates BAT development and energy metabolism by regulating the stability of METTL3 protein.The results provide a new drug target for BAT energy consumption in the treatment of obesity.