Palmitic Acid Results In Mitochondrial Dysfunction And Cell Apoptosis By Inhibiting Fatty Acid β-oxidation In Sertoli Cells

Background:In recent years,male fertility has shown a significant downward trend in the whole world.A large number of clinical epidemiology and animal experiments have found that obesity is significantly negatively correlated with male fertility.Obesity brings about the transfer and deposition of a large number of free fatty acids(FFA)into non-adipose tissues and cells,inducing cell stress and inflammation,causing chronic tissue and cell damage and organ function decline,which is called lipotoxicity.Palmitic acid(PA)is the main component of fatty acids in body and the main saturated fatty acid causing lipotoxicity.Sertoli cells(SCs)plays an important role in the development of germ cells(GCs)by providing physical barrier and nutritional support to spermatogenesis.The energy metabolism homeostasis of SCs is very important to maintain the function of SCs and the development of GCs.SCs provides energy through the β-oxidation of fatty acid,and lactic acid produced by glycolysis is the main source of energy for GCs.PA has been shown to affect male fertility by impairs the function of SCs.However,whether PA damages the function of SCs by disrupting energy metabolism homeostasis needs further research to confirm.Methods:Advances in high-throughput metabolomics and lipidomics measurement platforms provide powerful tools to gain insights into complex biological systems.This research aimed to explore the potential molecular mechanisms of PA regulating energy metabolism in SCs based on metabolomics and lipidomics.Treated with 0.5 mM PA or Bovine Serum Albumin(BSA)after 24 h,SCs and culture media were collected for metabolomics and lipidomics assays.The data were normalized separately before further processing.The quantitative information obtained by the above methods was then integrated,and duplicate data were eliminated to ensure uniqueness of metabolites and lipids.After removing the duplicated substances,multivariate analysis,including principal component analysis(PCA),OrthoPLS-DA(OPLSDA)and univariate analysis,including independent samples t-test and fold change were performed on polar metabolites and lipidosomes.Then RT-PCR was used to detect mRNA levels of smpd3 and cls1,which encoded Ceramide synthase and Cardiolipin synthase,respectively.SCs were stained with MitoSOX-Red and MitoTracker-Green.Changes in protein levels of apoptosis-related molecules Bcl-2,Bax and Cleaved Caspase-3 were detected by Western Blot,combined with flow cytometry to detect cell apoptosis rate,which collectively reflects the level of apoptosis.Results:1.Glucose metabolism-related metabolites were not significantly changed after PA treatment,and the pool of amino acids and the levels of most individual amino acids involved in the tricarboxylic acid(TCA)cycle also were not changed.However,medium-and long-chain acylcamitines accumulated in SCs exposed to PA.2.PA treatment of SCs significantly altered the lipidome,including significant decreases in cardiolipin and glycolipids as well as remarkable increases in ceramide and lysophospholipids.3.Mitochondrial ROS levels increased in SCs treated with PA.The mRNA levels of smpd3 and cls1 increased.An increased apoptosis rate was founded in SCs exposed to PA.Cleaved Caspase-3 and Bax protein expression levels increased,while Bcl-2 protein expression levels decreased.Conclusion:In summary,the results showed that fatty acid β-oxidation of SCs is disrupted after PA treatment.Although SCs are still able to provide energy for GCs under PA stimulation,their own source of energy supply is inhibited,causing mitochondrial damage,leading to apoptosis.