The Effect Of PINK1/Parkin Pathway In White Adipose Tissue In Obese Mice And In PA-treated 3T3-L1 Cells And Its Mechanism Study

BackgroundObesity is a chronic metabolic disease caused by energy imbalance,which is often accompanied by type 2 diabetes,non-alcoholic fatty liver disease,cardiovascular and cerebrovascular diseases,sleep apnea syndrome and other diseases.And several kinds of tumors are closely related with obesity.Meanwhile,obesity has a certain degree of relativity with the increase of all-cause mortality.In recent years,obesity has become a tremendous challenge to global public health.White adipose tissue,the clearly important energy storage organ,plays a crucial role in the development of nutritional obesity.As an important organelle that regulates energy metabolism,mitochondrion provides energy for the lipid storage,lipid mobilization and hormone secretion of adipocytes.Therefore,its homeostasis and metabolic renewal are particularly significant.However,due to the particularity of its function,mitochondria are vulnerable to oxidative stress in energy excess.It has been reported that excess energy causes elevated production of excess reactive oxygen species(ROS)in white adipose tissue which can damage mitochondria to some extent,affecting the function of adipocytes and aggravating obesity.Another article has described the outcomes of damaged mitochondria:some,which underwent a repair system,could be fused with other defective mitochondria to generate new mitochondria with intact function,while some,which could not be repaired,would be digested by lysosomes and then be recycled through the mechanism called mitophagy.Mitophagy mediated by PINK1(PTEN-induced putative kinase 1)and Parkin helps maintain a certain number of mitochondria in the cell with high quality,so as to guarantee the cellular function.When the mitochondria are in the normal state,PINK1 will be cleaved by presenilins-associated rhomboid-like protein(PARL)on the inner mitochondrial membrane.When the dysfunctional mitochondria lost membrane potential,PINK1 will accumulate on the surface of defective mitochondria,then Parkin and autophagy-related proteins are recruited.Parkin is an E3 ubiquitin ligase that recruits microtubule-associated protein 1 light chain 3(LC3)through ubiquitination of proteins.Then the autophagosomes containing damaged mitochondria(mitophagosomes)are produced and then degraded by lysosomes.Eventually the aging or damaged mitochondria are eliminated.Overall,the PINK 1/Parkin pathway can maintain the overall function of cellular mitochondria through mitophagy and exert protective effect on cells.However,it has not been clarified whether the pathway is involved in protecting adipocytes when obtaining excessive energy and undergoing metabolic stress.Therefore,studying the PINK1/Parkin signaling is very important to clarify the mechanism of adipocytes maintaining the basic function under energy-excessive condition.It may provide a new way of thinking in the treatment of obesity.Objectives1.To determine the PINK1/Parkin expression profile in adipose tissue in obese mice induced by high-fat diet.2.To clarify the effect of PINK1/Parkin on the mitochondrial function of 3T3-L1 cells treated by palmitic acid.3.To identify the effect of PINK1/Parkin on the cellular death of 3T3-L1 cells treated by palmitic acid.MethodsA.Animal Models1.In this study,4 weeks old male C57BL/6J mice were used.After adapting to feeding environment,45 mice were randomly divided into control diet group(CTRL)(n=15)and high fat diet group(HFD)(n= 30),and were fed for another 12 weeks.Body weights were measured and recorded weekly.2.lntraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin resistance test(IPITT)were performed on the 11th week and the 12th week respectively.3.At the end of feeding,mice were tested for body fat content with dual energy X-ray to confirm the establishment of the obese-mice model.After that,the mice were anesthetized and blood samples were taken from the apex to determine the biochemical indexes such as blood lipids.Subcutaneous white adipose tissue in the abdomen and visceral white adipose tissue in the epididymis were obtained.4.The morphology of mitochondria and the ultrastructure of autophagosomes in adipose tissue were observed by transmission electron microscopy.5.Mitophagy-related proteins were detected by qPCR and Western blot.B.Cell culture1.3T3-L1 cells were cultured in high glucose DMEM medium containing 10%fetal bovine serum(FBS).2.Lipofectamine 2000 was used to transfect cells with PINK1/Parkin small interfering RNA.3.The cells were stimulated with different concentrations of palmitic acid for 48h,then the expression of various target genes and proteins in different conditions were detected by qPCR and Western blot.4.Apoptosis detection:Roche TUNEL assay kit was used to detect apoptosis under different treatment conditions.5.Immunofluorescence staining:Cells treated with different stimulation conditions were planted on the cell slides.After fixation,the cells were incubated with primary and secondary antibodies.Then the slides were sealed and observed under the microscope.6.Detection of mitochondrial functi,on:Cells treated with different stimulation conditions were detected by ROS detection kit,ATP detection kit and JC-1 detection kit.Results1.The PINK1/Parkin signaling pathway is activated in adipose tissue in obese mice.2.The PINK 1/Parkin signaling pathway prevents PA-induced mitochondrial dysfunction in 3T3-L1 cells.3.The PINK1/Parkin signaling pathway inhibits PA-induced 3T3-L1 cell death.ConclusionsThe PINK1/Parkin signaling pathway protects the cellular function from metabolic stress through maintaining the function of intracellular mitochondria.