The Effect Of Starvation On Lipolysis And Lipophagy Of Lipid Droplets In Mice Cervical Epithelial Cells

Lipid droplets(LDs)are specific,complex,and multi-functional organelles originating from the endoplasmic reticulum,and found in almost all types of cells.With the development of modern science and technology,there is increasingly understanding on structure,functions,proteomics,and related diseases of LDs.Lipid such as triglycerides and cholesterol esters in LDs would be hydrolyzed by both neutral lipases including triglyceride lipase(ATGL)and hormone-sensitive esterase(HSL)and degraded by lysomal enzymes within autophagosomes after LDs can be engulfed by autophagosomes.Metabolic abnormalities and dysfunction of LDs are closely related to various diseases such as obesity,fatty liver,atherosclerosis,type 2diabetes,inflammation and cancer.The cervix is an important organ connected to the uterus and vagina in the reproductive system.The cervical closure ensures the normal development of fetus during pregnancy,and the opening of cervix has a direct impact on the expulsion of fetus at term delivery.The opening and closure of the cervix requires a continuous supply of energy.Whether LDs in cervical epithelial cells are one of the sources supplementing energy are not yet clear.The study aimed to demonstrate the change of metabolism of LDs in murine cervical epithelial cells(MCECs)under starvation.The MCECs were firstly collected,digested and isolated,and then in vitro cultured in DMEM,and the positive rate of MCECs were determined with keratin in MCECs detected by immunofluorescence.The results showed that the purity of MCECs was more than 98%.The normal culture medium was replaced with Hank’s balanced salt solution(HBSS)to induce starvation,and the MCECs were starved for 0h,6h,12 h,and 24 h,respectively.To observe the morphological changes between fatty acids(FAs),LDs and mitochondria(Mito)in MCECs with fluorescence chase.FAs were labeled with Bodipy558/568(Red C12)before treatment with HBSS,and LDs labeled with Bodipy493/503 and Mito with MitoTracker Green.After starvation for 24 hours,the morphology of fluorescent LDs,FAs and Mito was observed under confocal microscopy,and statistically analyzed.The results suggested that with the development of starvation,the content of FAs in the cytoplasm were gradually increased,and the distance between Mito and LDs was gradually shortened,and numerous FAs were trafficked into Mito.At 24 h after starvation,the number of Mito was decreased and LDs became larger.To determine TG content in the cells through triglyceride quantitative detection kit,and after treatment with HBSS,the results showed that the amount of TG was significantly reduced.After detecting the expression of ATGL,HSL,PKA and AMPK related to LDs metabolism,and the expression of autophagy marker protein LC3II/LC3 I by WesternBlotting,the results indicated that the expression levels of ATGL,HSL,PKA and AMPK were significantly down-regulated with the development of starvation,but the expression of LC3II/LC3 I was significantly up-regulated.After H89(PKA inhibitor)was added in medium,the result suggested that the expression of ATGL and HSL was markedly reduced,but the expression of LC3Ⅱ/LC3Ⅰdramatically increased,and AMPK not significant.In the experiment for detecting ATP by luminescence viability kit,the results had shown that the level of ATP was markedly decreased at 6h after starvation.Finally,Mito membrane potential was detected by flow cytometry.The results showed that the Mito membrane potential was gradually decreased,and the damage of Mito was more and more severe.Conclusively,the above results indicated that neutral lipids within LDs in MCECs were degraded under starvation,and FFAs from neutral lipids released into cytoplasm are increased.The expression of AMPK and PKA,ATGL and HSL was inhibited,and the role of PKA in stimulating ATGL and HSL to induce the lipolysis of LDs was decreased.Autophagosomes play a major role in the LDs catabolism in starved MCECs,while upstream regulatory neutral lipases are mostly inhibited under starvation.Excessive fatty acids were trafficked into the mitochondria to cause damage on mitochondria,decreased membrane potential and a decrease in the level of ATP.Autophagosomes are involved not only in the catabolism of LDs,but also in the removal of damaged organelles induced by excess fatty acids.FAs derived from LDs was not mainly used for the production of ATP.