Multi-omics Reveals The Mechanism Of Thermal Adaptation Of Adipose And Muscle Tissues Improved By Lipid Droplets

During livestock and poultry production,heat stress damage to the organism caused by hot summer weather can reduce livestock and poultry production,increase livestock and poultry mortality,and cause major losses to the global livestock economy every year.Heat stress(HS)not only causes physiological metabolic disorders in live animals,but also affects the glycolysis rate and final p H of meat after slaughter,ultimately damaging carcass traits and meat quality.Because of the thick subcutaneous fat and the lack of functional sweat glands on the skin surface,pigs are vulnerable to high temperature environments.In pork,intramuscular fat exists mainly in the form of lipid droplet(LD),implying that LD plays an important role in maintaining pork quality.The LD is far from being an "inert storage" for lipid storage in cells,as its single phospholipid membrane surface,rich in functional regulatory proteins and its internal envelope of different lipid components,has been shown to play an important role in biological processes such as lipid metabolism,stress resistance,immune regulation and signal transduction in cells.Recent studies have found that pig adipose tissue has a specific hyperthermic response mechanism,but the molecular mechanism remains unclear.In the previous study,we found that HS affected systemic lipid metabolic processes by causing disruption of cholesterol metabolism in liver tissues of Tongcheng pigs(Lian et al 2022).To further investigate the effect of HS on meat lipid metabolism and the role played by LD in the process of heat stress response of the organism.We increased the content of LD in animal tissues by high-fat diet(HFD)and constructed heat treatment models for normal mice and obese mice,respectively.We used molecular biology and multi-omics techniques to reveal the effects of heat treatment on lipid metabolism in adipose tissue(iBAT)and muscle tissue(SMT)of mice,respectively,and analyzed the role played by LD in the process of alleviating the heat stress response of the organism.The main results are as follows:1.Heat treatment(40℃,4h)caused heat stress damage to iBAT in normal mice(1)Heat treatment of mice at 40℃ for 4 hours resulted in a systemic non-specific heat stress response in the organism,including: high expression of HSPA8,SREBF2 and FABP4 proteins in liver tissue;causing high levels of heat shock protein 70(HSP70),cortisol and total cholesterol levels in the serum.(2)HS disrupted the structure and expression of genes related to lipid metabolism in mouse iBAT.HS caused an increase in the level of ROS in iBAT(P < 0.001),resulting in oxidative stress damage in adipocytes.Transmission electron microscopy revealed that HS damaged the structure of mitochondria,leading to the appearance of oversized lipid droplets(P < 0.01).In RNA-seq and q PCR results,we found that HS repressed thermogenic genes(Prdm16,Adipor2,Acsl1 and Dio2,etc.)and lipid metabolismrelated genes(Hsd17b7,Ucp3,Gpat4,Agpat2,Hmgcs1,Ddhd2,Srebf1,Abcd2,Dgat1,Acat2,Plin5 and Ehhadh,etc.)in iBAT of normal mice(P < 0.05).(3)HS disrupted lipid metabolism disorders in iBAT of normal mice.Lipidomics data analysis shows that HS decreases the total lipid composition in iBAT of mice.HS disrupts the composition of fatty acid acyl chains in glycerol esters and glycerophospholipids.In addition,HS decreases the CL content in iBAT,leading to a breakdown of mitochondrial structure.HS decreases the thermogenic consumption of fatty acids by mitochondria in iBAT by β-oxidation,ultimately increasing the free FA used for cholesterol synthesis of cholesteryl esters.(4)A combined RNA-seq and Lipidomics analysis of mouse iBAT revealed that HS reduced glycerophospholipids(PC,PE.)components in iBAT by suppressing the expression of genes(Dgat1,Gpat3,Acot2,Ddha2,Agpat2,Ehhadh and Plin5,etc.)involved in regulating glycerophospholipid synthesis in iBAT(P < 0.05).The formation of ultralarged-LD not only accommodated excess harmful cholesterol esters but also attenuated the effects of glycerophospholipid absence on cellular homeostasis.2.LD improved the adaptation of iBAT to hyperthermic environment in obese mice(1)Heat treatment(40℃,4h)did not cause HS damage in iBAT of obese mice.Increasing lipid droplet content by high-fat diet improved the expression of ROS response capacity genes(Fosl2,Acot2 and Apold1)and antioxidant genes(Pla2g7 and Ehhadh)in iBAT of obese mice(P < 0.05).Furthermore,heat treatment did not affect the serum levels of heat shock factor,cortisol and lipids in obese mice.HS did not affect the levels of HSP70 and ROS in iBAT(P > 0.05).More importantly,transmission electron microscopy showed that heat treatment did not cause damage to the subcellular structures of iBAT.(2)In RNA-seq analysis,it was found that heat treatment dynamically regulated the expression of lipid metabolism and signal transduction-related genes(Pnpla3,Mttp,Aacs,Irs2,Ehhadh,Acat2,Acot2,etc.)in iBAT of obese mice by activating the p53 signaling pathway(P < 0.05)to protect them from HS damage.In the lipidomics analysis,it was found that heat treatment not only increased the total lipid composition in iBAT in obese mice,it also selectively improved the composition of fatty acid acyl chains in glycerol esters(DAG)and glycerophospholipids(PE,PC,PS and CL).Consequently,it improved the adaptation of iBAT to hyperthermic environments by enhancing the signaling capacity in response to the heat stimulus.(3)In addition,heat treatment significantly increased the amount of oxidized lipids of LD of brown adipocytes in obese mice.Heat treatment induced lipids oxidation in iBAT of obese mice.The excess oxidized lipids were stored in LD,reducing the damage to brown adipocytes.More importantly,LD could also improve adaptation to hyperthermic environment by alleviating oxidative stress damage in iBAT of obese mice in response to HS.3.LD improved adaptation of SMT to hyperthermic environment in obese mice(1)Heat treatment(40℃,4h)promoted high expression of HSP70 in SMT of obese mice(P < 0.001),but had no effect on the levels of TNFR1,Cyto C and ROS(P > 0.05),implying that heat treatment did not lead to heat stress injury in SMT.Heat treatment decreased the concentration of blood glucose and promoted the synthesis of myoglycogen(P < 0.05),suggesting that heat treatment improved the glucose metabolism capacity of SMT.(2)The combined analysis of RNA-seq and metabolomics revealed that heat treatment induced the maintenance of gene expression at the decaying stage of HSF1 in SMT of obese mice.The induction of HSP70 protein synthesis not only reduced the expression of genes related to ROS production and lipid oxidation(Ncf1,Mmp9)in SMT of obese mice;it also activated the sphingolipid metabolic process in SMT of obese mice.These results suggested that the expression of HSP70 as the core protein improved the adaptation ability of SMT to HS in obese mice.(3)Lipidomics analysis revealed that heat treatment improved the composition of Cer,SM and PE in the SMT of obese mice.Heat treatment decreased the content of Cer and increased the level of SM and PE in SMT of obese mice(P < 0.05),implying that heat treatment promoted Cer breakdown in SMT of obese mice.(4)To further determine the role of LD in skeletal muscle cells of obese mice during heat treatment.We performed lipidomics analysis of the lipid composition in LD and found heat treatment increased the content of Cer,PE and CE in LD.HS decreased the content of SM and DAG.These results suggested heat treatment promoted the transfer and accumulation of Cer to LD and inhibited the degradation process of Cer in LD.In summary,HS damaged the structure and function of mitochondria of adipocytes and reduced the thermogenic capacity of lipid metabolism of iBAT in mouse.LD accommodated harmful lipids in the adipocyte and improved the adaptability of adipocytes to heat stress.HFD increased the LD content and adaptability to hyperthermic environment of SMT in mice.In particular,when obese mice were subjected to heat treatment,LD accommodated harmful Cer and CE,which not only improved insulin resistance in the SMT of obese mice,but also prevented damage to skeletal muscle cells from lipotoxicity.These results suggested that lipid droplets can improve the adaptation of adipose and muscle tissues to hyperthermic environments.LD is of great relevance for healthy pig production and for finding measures to mitigate heat stress injury.