Leptin Regulates Lipid Metabolism And Energy Metabolism In Weaned Piglets By Promoting The Expression Of Lipid Droplet Coating Protein Perilipin5

Leptin is a protein hormone encoded by the obesity gene(ob)and is mainly synthesized and secreted by white adipose tissue.Leptin plays a vital role in regulating lipid deposition and controlling body weight.Leptin is therefore used as a treatment for obesity and complications.Leptin functions in the body mainly through the Leptin receptor signaling pathway.Plin5 also participates in both lipogenesis and lipolysis processes.It is reported that Plin5 can promote the expression of lipolytic genes;however,some studies also showed that Plin5 can promote the expression of lipid synthesis genes.Meanwhile,Plin5 also affects the beta oxidation process of fatty acids and mitochondrial function;and Leptin receptor signaling can cause changes in the expression of PPAR family genes.At the same time,Plin family genes expression are regulated by transcription factors such as PPARγand PPARα.Research also showed that Leptin can also cause fat mass and obesity associated(FTO)upregulation in vivo.In addition to regulating fat metabolism,FTO is an important enzyme for inhibiting N6-methyladenosine(m~6A)methylation modification via mediating the m~6A methyl demethylation process.It has not been reported whether Leptin or FTO can regulate the expression of Plin5.In this study,q PCR,Western blotting,immunofluorescence,m~6A-IP and other methods were used to explore the effect and mechanism of the m~6A methylation modification of lipid droplet coating protein Perilipin5 by Leptin through regulating FTO,thereby promoting Perilipin5 expression and regulating lipid metabolism energy and energy metabolism.Our results provide evidence and ideas for further research on the interaction between Leptin and Plin5 and the prevention of fat metabolism diseases in weaned piglets caused by obesity.The test obtained the following results:1. Leptin promotes lipolysis and up-regulates the levels of Plin5 and downstream genes in piglets.Leptin significantly reduced body weight gain,body fat rate and blood lipid-related indicators of piglets(P<0.05),and body temperature increased significantly(P<0.05).Leptin treatment significantly increased leptin(P<0.05)and leptin receptor(P<0.05)expression in piglets inguinal subcutaneous fat tissue.Meanwhile,we found the m RNA(P<0.05)and protein(P<0.05)levels of Plin5 became obviously higher in white adipose tissue,and its downstream genes HSL,ATGL,ABHD5 m RNA levels increased(P<0.05).The lipolysis gene LPL m RNA expression was enhanced(P<0.05),while lipogenesis genes ACC1 and Fasn m RNA levels were reduced significantly(P<0.05);Mitochondrial oxidation-related genes and mitochondrial complex-related genes were upregulated(P<0.05);Adipocytes had smaller size(P<0.05)and reduced lipid droplets amount(P<0.05).These results indicate that Leptin up-regulates Plin5 and its downstream lipolytic genes expression,and promotes mitochondria function and lipolysis.2. Leptin inhibits m~6A methylation of Plin5 by up-regulating FTO in adipocytes.Leptin treatment significantly up-regulated the m RNA levels of FTO and methylation recognition enzyme Ythdf2(P<0.05).Dot Blot and total m~6A methylation measurement showed that the total RNA m~6A methylation levels of cells were significantly down-regulated(P<0.05),and m~6A-IP result showed Plin5 m~6A levels were significantly down-regulated(P<0.05).Overexpression of FTO significantly reduced the m~6A methylation level of Plin5(P<0.05),while interference vector of FTO reversed this change(P<0.05).Leptin and FTO vector co-treatment aggravated the decrease the methylation level of total m RNA(P<0.05),while interference fragment reversed the reduction of m~6A methylation(P<0.05).m~6A methylation of Plin5 m RNA had consistent change with total m~6A methylation levels(P<0.05).The above results indicate that Leptin treatment inhibits the m~6A methylation level of Plin5 m RNA,which is caused by the up-regulation of FTO.3. FTO up-regulates Plin5 protein level by inhibiting m~6A methylation of Plin5.Overexpression of FTO significantly increased the protein level of Plin5(P<0.01),but the m RNA level of Plin5 did not change significantly(P>0.05),and the m~6A methylation level of Plin5 m RNA was also significantly reduced(P<0.05).Methylation inhibitor Cycloleucine(CL)significantly reduced the m~6A methylation level of Plin5 m RNA(P<0.05),and the methylation agonist betaine(Bet)significantly increased(P<0.01)Plin5 m RNA m~6A methylation level with no significant change of Plin5 m RNA level(P>0.05).CL significantly increased the protein level of Plin5(P<0.05).After methylation site mutation,the effect of FTO overexpression on m~6A m RNA of Plin5 m RNA is not significant(P>0.05),and the m RNA level of Plin5(P>0.05)and protein level(P>0.05)are not significantly changed.The above results show that FTO up-regulates Plin5 protein expression by inhibiting the m~6A methylation modification of Plin5.4. Plin5 promotes lipid droplet degradation and activates downstream lipolysis genes and mitochondrial function genes expression in the basal state.In the normal state,Plin5 overexpression in mature adipocytes significantly reduced lipid droplets size(P<0.05),lipolytic genes LPL,CPT1a and Pin5 downstream genes ATGL,HSL and ADBH5 had increased m RNA and protein level(P<0.05).The m RNA levels of the thermogenic genes PGC1a,UCP1,and ATP5a1 were significantly increased(P<0.05).The levels of mitochondrial function genes TFAM,Ndufb8,SDHb,UQCRC2,Cox4i1(P<0.05),and ATP production were also significantly increased(P<0.05).The ratio of NAD+/NADH was significantly increased(P<0.05).The above results show that the up-regulation of Plin5 can promote the function of lipolytic enzymes on the surface of lipid droplets,accelerate the decomposition of lipid droplets,promote the decomposition of triglycerides and the operation of mitochondrial respiration chain,increase heat production and energy metabolism.5. In the state of lipotoxicity,Plin5 promotes lipogenesis metabolism and fatty acidβ-oxidation,and accelerates the consumption of free fatty acids.First,a fatty acid lipotoxicity model was constructed by sodium palmitate treatment.There was no significant difference in m RNA levels of apoptosis-related genes such as Caspase3,Caspase9,Bax,Bcl-2(P>0.05),and no remarkable increase was found in the number of apoptotic cells(P>0.05)compared to control group.Under the condition of fatty acid content became higher(P<0.01),and lipid droplet accumulation increased significantly(P<0.05),indicating that the lipotoxicity model was successfully constructed.Overexpression of Plin5significantly reduced intracellular fatty acid content(P<0.05),and the levels ofβ-oxidative rate-limiting enzyme ACSL2 and triglyceride synthetase DGAT2,Fasn,and ACC1 were significantly increased(P<0.05).The levels of mitochondrial complex genes and the NADP+/NADPH ratio were also significantly reduced(P<0.05).The above results show that Plin5 can alleviate metabolic disorders caused by excessive free fatty acids and accelerate the consumption of free fatty acids.In the state of lipotoxicity,Plin5overexpression could promote lipid synthesis and enhance mitochondrialβ-oxidation.This test demonstrated that Leptin can inhibit the m~6A methylation level of Plin5 by up-regulating the expression of FTO,and then up-regulate the expression of Plin5.In the basic state,Plin5 can activate the downstream lipolytic genes,leading to the breakdown of lipid droplets,thereby promoting fatty acid catabolism and production of heat.While in the lipotoxic state,Plin5 can increase the consumption of free fatty acids,and protect the lipid metabolism homeostasis.This study initially revealed the role of Leptin in Plin5’s regulation of body lipid metabolism and energy metabolism,providing a theoretical basis for in-depth study of fat metabolism and function,and further providing research ideas for the treatment of obesity and related metabolic syndrome.