Effects Of M~6A Modification In High-Fat Diet-Induced The Disorders Of Glucose And Lipid Metabolism In Broilers And Mice

Glycolipid metabolism in poultry is different from that in mammals,with the liver as the main organ for fat synthesis in poultry.At the same time,the blood glucose level of poultry is higher than that of mammals,whereas,the insulin sensitivity of the thigh is lower.The mechanisms responsible for these actions needs to be understood.Broilers grow and develop rapidly,and high fat diet are usually added to their diet to maintain their energy metabolism.Studies in mammals have shown that epigenetic modifications are closely related to the occurrence and development of various diseases.As the most common RNA modification,m~6A modification plays an important role in glycolipid metabolism.In this study,a combined analysis of MeRIP-seq and RNA clarified that the expression level of METTL3 in thigh changed the level of FOXO1 m~6A modification in animals fed with high-fat diets.These results provide a new insight into the role of m~6A modification in the regulation of glucose and lipid metabolism of thigh and broaden the understanding of the regulatory mechanism of m~6A modification induced by high-fat diets.The main contents are as follows:1.To study the effects of high-fat diet on glycolipid metabolism and m~6A modification in broilers.Broilers were randomly divided into 2 groups:control group fed with a normal diet（Con）;high-fat diet（HF）（fat levels:1-21 d,3.99%;22-42 d,4.92%）.The results showed that the contents of TG and TCHO in the liver and thigh of the high-fat broilers were significantly higher than those in the control group（P<0.05）.The expression levels of some glucose and lipid metabolism-related genes in the liver and thigh of the high-fat group were significantly different from those of the control group（P<0.05）.In the high-fat group,the expression of METTL3 in liver tissue of broilers was significantly increased,the expression of FTO was significantly decreased（P<0.05）,and the level of m~6A modification was significantly increased（P<0.05）.The expression levels of METTL3 and YTHDF2 in the thigh of broilers fed high-fat diet were also significantly increased（P<0.05）,whereas the m~6A modification level was significantly decreased（P<0.05）.High-fat diet had no significant effect on the expression of m~6A methylation-related genes in the adipose tissue of broilers（P>0.05）and on the level of m~6A modification（P>0.05）.In conclusion,high-fat diet led to the disorder of glucose and lipid metabolism of broilers and dysregulated the m~6A methylation modification level in the liver and thigh tissue.The regulation of m~6A methylation in broiler liver tissue could be associated with the up-regulation in METTL3 expression,whereas,the regulation of m~6A methylation in the thigh tissue may be mainly by down-regulating the expression of METTL3.2.To study the effects of high-fat diets on glucose and lipid metabolism and m~6A modification in mice.Forty 3-week-old male C57BL/6 mice with similar body weights were raised in SPF environment and were randomly divided into two groups having 20mice in each group,which were fed with a basal diet（ND group）or a high-fat diet（HFD group）,respectively.The results showed that after 4 weeks of feeding,the body weight,body fat percentage,and fasting blood glucose level of the mice in the HFD group were significantly higher than those in the ND group（P<0.05）.The GLU content of the serum was significantly higher than that in the ND group（P<0.05）.The expressions of methyltransferases METTL3 and METTL14 in the liver tissue of mice in the HFD group were significantly increased（P<0.05）,and the m~6A modification level was significantly increased（P<0.05）.The expression of methyltransferase METTL3 in thigh of mice of high-fat group was significantly decreased（P<0.05）,and the level of m~6A modification was significantly decreased.This indicated that the regulation of m~6A methylation modification in liver tissue of mice by high-fat diets may be mainly through up-regulation of the expression levels of METTL3 and METTL14 in liver tissue.The regulation of m~6A methylation modification in thigh may be mainly through down-regulation of the expression level of METTL3 expression.3.Association analysis of MeRIP-seq and RNA in thigh of mice treated with high-fat diet.The analyzed samples were the thigh of mice from the ND group and the HFD group.RNA-seq analysis found that 547 genes were altered in the thigh of mice fed with a high-fat diet,in which 438 genes were up-regulated and 109 genes were down-regulated.The results of KEGG pathway analysis and GO enrichment analysis showed that compared with the ND group,the changed genes in transcripts in the thigh of the HFD group were involved with hypertrophic cardiomyopathy,AMPK signaling pathway,long-chain fatty acid binding,insulin-like growth factor binding,and fatty acid binding,etcetera.The MeRIP-seq analysis showed that the motif occurred methylation modification was GGACU/A sequence.In the m~6A methylation region,the CDS region accounted for47.6%.The enrichment analysis of differentially methylated genes showed that these genes were involved in thigh contraction,ATP metabolism,glycogen metabolism,glucose homeostasis,MAPK cascade and other metabolic processes,as well as the Fox O signaling pathway,PPAR signaling pathway,PI3K-AKT signaling pathway,AMPK signaling pathway and other regulatory pathways.The differential genes were screened,and the m~6A modification visualization analysis was carried out.The level of m~6A modification of FOXO1 was increasingly changed in the high-fat group,and FOXO1 participate in the PI3K-AKT signaling pathway.The m~6A visualization analysis showed a significant increase in the m~6A modification level of FOXO1 in the mice of high-fat group.It is understood that the high-fat diet may regulate the glucose and lipid metabolism of thigh through the METTL3/FOXO1/PI3K-AKT pathway.4.To study the effect of betaine on glucose and lipid metabolism and m~6A methylation in high-fat diet-fed mice.Forty 3-week-old male C57BL/6 mice with similar body weights were selected and randomly divided into two groups with 20 mice in each group.After the successful establishment of the obesity model in the high-fat group mice,the mice in the ND group and the HFD group were randomly divided into two treatments including 1%betaine added to drinking water and normal drinking water,namely the betaine group（BET group）and the high-fat added betaine group（HFBET group）.The results showed that betaine could alleviate the obesity and insulin resistance in mice caused by high-fat diets and improved the thermogenic ability of mice under cold stimulation（P<0.05）.The expression of METTL3 protein in thigh of mice in HFBET group was significantly increased（P<0.05）,and the level of m~6A modification was significantly increased（P<0.05）.Visual analysis of FOXO1 m~6A modification showed that the level of FOXO1 m~6A modification in the thigh of mice in the HFBET group was significantly reduced.Pull down analysis of FOXO1 binding proteins showed the enriched proteins were involved in oxidative phosphorylation,diabetic cardiomyopathy and other related metabolism,while the related binding proteins were mainly LPL,ATP2A3,ATP5D and ATP5K.The results suggest that betaine can mediate the m~6A modification level of FOXO1 and that FOXO1 m~6A modification is involved in glucose and lipid metabolism in mouse thigh.5.To determine the regulatory mechanism of m~6A modification on glycolipid metabolism in C2C12 cells.C2C12 cells were first treated with insulin to construct an in vitro model.Sh RNA and lentivirus were constructed to knockdown and overexpress METTL3,respectively.The results showed that insulin treatment of C2C12 cells significantly decreased METTL3 protein level（P<0.05）and activated PI3K-AKT signaling pathway（P>0.05）.C2C12 cells were treated with Sh METTL3.The results showed that Sh METTL3 significantly reduced METTL3 protein level（P<0.05）,increased glucose uptake capacity（P<0.05）,promoted GLUT4 translocation to the cell membrane,and activated PI3K-AKT signaling pathway.Lentiviral overexpression of METTL3significantly increased the expression level of METTL3 protein（P<0.05）,increased m~6A modification level（P<0.05）,and FOXO1 protein phosphorylation level（P<0.05）,and decreased the glucose uptake（P<0.05）of C2C12 cells.In this experiment,the protein levels of related pathways enriched in the earlier experiments were detected by knocking down and overexpressing METTL3,and the results showed that m~6A modification may regulate the glucose and lipid metabolism of C2C12 cells through the METTL3/FOXO1/PI3K-AKT pathway.6.Association analysis of gut microbiota and m~6A modification.The gut microbiota structure of the Con group and HF group broilers were analyzed.Through 16S r RNA analysis,the results showed that there were 525 unique OTUs in the cecum content of broilers fed with a high-fat diet.Beta diversity analysis showed the composition of the two groups was different.High-fat diet decreased the abundance of Bacteroidetes and significantly increased the abundance of Verrucomicrobia at the phylum level.The abundances of microbial communities at the genus level was detected.The results showed that feeding high-fat diets significantly decreased the abundance of alistipes,lachnospiraceae,and clostridiales in the cecum microbiota of broilers in the high-fat group,but significantly increased the abundance of Bacteroides,Akkermansia,Faecalibacterium,and intestinimonas.The gut microbiota of the mice in the ND group and the HFD group showed that there were 2532 OTUs in the mice of the high-fat group.The abundance of actinobacyeria was significantly reduced.Genus-level analysis showed that the abundance of Lactobacillus,Desulfovibrionaceae,and lachnospiraceae was significantly increased in the HFD group mice.However,the abundance of muribaculaceae,dubosiella,and bifidobacterium was significantly decreased in mice of HFD group.The gut microbiota of mice of the HFD group and HFBET group were analyzed.After betaine supplementation,the abundance of Lactobacillus was significantly reduced in mice.Similarly,the abundances of Lachnospiraceae,Ruminiclostridium and Dubosiella was significantly increased.The abundance of Bifidobacterium was negatively correlated with the level of m~6A modification,whereas,the abundance of Lachnospiraceae,Bacteroides and Clostridium were positively correlated with the level of m~6A modification in the thigh tissue.These results indicated that the gut microbiota may be involved in the regulation of glucose and lipid metabolism by m~6A modification in high-fat diet-fed mice.In conclusion,high-fat diets altered the glucose and lipid metabolism in broilers and mice.The regulatory mechanism of m~6A methylation in the liver and thigh of high-fat diet-fed broilers and mice was different.Under high-fat diet feeding,m~6A methylation regulated thigh glucose and lipid metabolism through the METTL3/FOXO1/PI3K-AKT pathway.Bifidobacterium,Lachnospiraceae,Bacteroides and Clostridium were involved in the regulation of thigh glucose and lipid metabolism by m~6A modification under high-fat diet.