Effect Of P62 On Adipogenesis Of Human Adipose Derived Stromal Cells Induced By Oleic Acid Through Autophagy

Objective:Obesity is related to the excessive adipose accumulation and(or)abnormal distribution,which is the imbalance of energy intake and consumption under the genetic background.Obesity is closely related with metabolic diseases such as hypertension and diabetes and becomes a very serious public health problem in nowadays.Cores of obesity are the growth in number and enlargement in size.Over proliferation of adipocytes is largely due to abnormal differentiation.To explore the molecular mechanism of adipose differentiation which controls the number of adipocytes is very important to prevent and treat the obesity and its related diseases.Adipose differentiation includes two phases : First,the mesenchymal stem cells orientate to pre-adipocytes(orientation stage);then,pre-adipocytes terminally differentiate to mature fat cells(terminal differentiation stage).The key factor to control adipose differentiation is peroxisome proliferator activated receptor γ(PPARγ)which is specific to fat.Expression of PPARγ increases continuously from the initiation of differentiation to the mature adipocyte,so it is the identification marker of adipose differentiation.Most current researches apply mouse 3T3-L1 pre-adipocyte as the cell model to investigate the terminal differentiation while the adipose stromal cells are seldom chose.So we choose human adipose derived stromal cells(h ADSCs)as the cell model to investigate the adipose differentiation,which is closer to human biology and has a guiding significance to the prevention and treatment of disease.At present,the union of insulin,dexamethasone and 3-isobutyl-1-methylxanthine(IBMX)is the common adipose differentiation reagent.IBMX is a chemical substance and can not present very well the stimulation of obestiy in human body.Obesity is closely related to high-fat diet and oleic acid(OA)is common in free fatty acid(FFA)of animal and vegetable oil and it is also the most abundant in blood FFA.So it substitutes IBMX to induce the adipose,which can simulate the body’s physiological model to a certain extent.p62 is a multifunctional protein involved in both autophagy-lysosome and ubiquitin–proteasome systems.Ubiquitin proteins bind to the ubiquitin associated domains of p62 to form the complex which is degraded by ubiquitin – proteasome system.These ubiquitin proteins can also bind to LC3 interactingregion targeted to LC3,which is degraded by autophagy-lysosome system.So p62 is the important mediate factor in both systems.The researches on p62 are most related to tumors.More reports on the obesity and p62 are about the energy metabolism.Cell in 2011 reported that the p62 knockout mice developed the obesity and insulin resistance,fat accumulation in white adipose tissue,slow basic lipid hydrolysis,and increase of lipid synthesis.Whether the p62 regulates the number of adipocytes from adipose differentiation and affects the incidence of obesity is rarely reported.Autophagy is one of the main ways to clear the abnormal proteins and damaged organelles by lysosomes in eukaryotic cells,which plays a vital role in maintaining cell homeostasis.Its morphologic character is the autophagosome formed by the substrate and double membrane.The basic process of autophagy is the initiation,closure of membrane to become the autophagosome,fusion of autophagosome and lysosomes,then degradation of the substrate in lysosomes.LC3 is the key molecule in formation of autophagosome which is regarded as the marker of autophagy.Reports show that when autophagy starts,p62 combines with substrate to form polymer,then mediates it to combine with autophagic vacuole membrane,thus forms autophagosome and transfers to lysosome for degradation.So p62 can mediate the autophagy and meanwhile is the substrate of autophagy.Given the important role of p62 in autophagy system,this topic will discuss the role and relationship of p62 and autophagy in h ADSCs adipose differentiation.The mitophagy is a kind of a selective autophagy.The specific receptor in autophagic vacuole membrane can be identified by damaged mitochondria and targeted degradation happens,which is very important to maintain normal number and function of mitochondria.Adipose differentiation process is accompanied by the formation of lipid droplets and the synthesis of triglycerides,and mitochondria is the core of the fatty acid oxidation,so this topic will further discuss the relationship of mitophaghy and p62 after identifying the basic function of autophagy and p62 on h ADSCs adipose differentiation.It will illustrate the molecular mechanism of human adipose differentiation and provide new way to find new drug targets for obesity.We will isolate and culture the h ADSCs extracted from thigh liposuction,and use oleic acid as physical stimulation factor to do adipose induction,then build p62 sh RNA slowvirus vector,observe the effect of p62 absence on self-renewal ability and adipogenesis of h ADSCs.After that,we explore the role and relations of p62 and autophagy in adipogenesis of h ADSCs,then further investigate the role and relations of p62 and mitophagy.Methods: 1.Cells were isolated from the human adipose liquid extracted from thigh liposuction throughⅠcollagenase digestion.Then isolated cells were identified: Cell surface markers CD49 d,CD105,CD31 and CD34 were detected using immunofluorescence staining method;h ADSCs were induced to adipocyte by the union of insulin,dexamethasone and IBMX,then lipid droplets were stained by oil red O;h ADSCs were induced to nerve cells by the union of b FGF and ratinoic acid.2.h ADSCs were induced to adipose by 50μM,80μM and 100μM oleic acid with insulin and dexamethasone,then the adipogenesis was observed by oil red O staining,the protein expression level of PPARγ was examined by Western-Blot.The best concentration of oleic acid was screened through the above methods.3.self-duplication ability was detected of shp62 h ADSCs: cell growth curve was examined by CCK-8 assay.4.The effect of shp62 on h ADSCs adipogenesis was observed.5.The autophagy in h ADSCs adipogenesis was observed: Punctiform accumulation of autophagy marker molecular LC3 was observed by immunofluorescence staining.LC3 was detected by Western-Blot.6.The effect of inhibition of autophagy by 10 m M 3-MA on h ADSCs adipogenesis at early,middle and late stage.7.The relation of p62 and autophagy was observed:The autophagosome was observed by MDC staining;LC3 Ⅱ / Ⅰ was examined by Western-Blot;Adipose differentiation was detected by quantitation of triglyeride and PPARγm RNA level after the inhibition of autophagy by 3-MA.8.The number and fission-fussion of mitochondria were observed: mt DNA(Nd1 gene)copy number was measured by q PCR;Drp-1,Mfn-1 and mitochondrial membrane protein TOM20 were detected by Western-Blot.9.The relation of p62 and mitophagy was observed:Autophagosome-mitochondria complex was labeled by fluorescent co-localization of LC3 and Mitotracker Red;mitochondria LC3 and cytoplasm(without mitochondria)LC3 were detected by isolating mitochondria;Expression of PPARγ was examined by Western-Blot after the treatment of 5μM Cs A.Results: 1.The isolated cells showed typical long spindle,fish pattern with a polarity under the microscope.Cell surface molecules detection showed that CD49 d and CD105 were positive while CD31(endothelial cell marker)and CD34 were negative;after adipogenesis,many lipid droplets appeared in cell plasma and were stained red by oil red O.After neural induction,cell bulging out the slender protrusions that have rich network connection.The results showed that the isolated cells were h ADSCs.2.Oleic acid union with insulin and dexamethasone successfully induced h ADSCs to adipocytes: Lipid droplets were visible in cytoplasm and were stained red by oil red O.Oil red O positive cells rate and protein expression level of PPARγ were highest at 80μM among three concentrations.3.p62 deletion decreased the self-duplication ability of h ADSCs: Cell growth curve showed that the slow growth and proliferation of shp62 h ADSCs.4.p62 deletion promoted the h ADSCs adipogenesis: Oil red O positive cells,lipid content,m RNA and protein levels of PPARγ significantly increased.5.Autophagy occurred during h ADSCs adipose differentiation: Immunofluorescence test results showed that the accumulation of LC3-Ⅱ dots was weak at undifferentiated stage(d0),increased significantly at early differentiation(d1-3),and gradually fell to the level before differentiation.Western Blot results showed that LC3 Ⅱ / Ⅰ in early differentiation increased gradually and began to decline during middle and late stage.6.Inhibition of autophagy by 10 m M 3-MA at early stage significantly reduced adipose differentiation: Oil red O positive cells and protein levels of PPARγ decreased.There was no statistically significant difference of adipogenesis rate with inhibition at middle and late stage.The results showed that autophagy mainly play vital a role in early differentiation.7.shp62 promoted adipogenesis by increasing autophagy: MDC positive cells and LC3Ⅱ/Ⅰincreased,which showed shp62 increase autophagy.Triglyeride and PPARγ m RNA level decreased,which showed that the adipogenesis resulted from shp62 decreased after the inhibition of autophagy by 3–MA,which showed shp62 promoted adipogenesis by increasing autophagy.8.The number of mitochondria increased during adipogenesis of h ADSCs: expression level of Nd1 and TOM20 increased.The fission mitochondria increased in early stage of h ADSCs adipogenesis: expression level of Drp-1 increased,while Mfn-1 had no difference.9.shp62 promoted mitophagy:co-localization of LC3 and Mitotracker Red increased;Mitochondria LC3 Ⅱ / Ⅰ increased and cytoplasm(without mitochondria)LC3 Ⅱ / Ⅰ were detected by isolating mitochondria;shp62 adipogenesis was inhibited after the inhibition of autophagy by 5μM Cs A,which showed that shp62 might promote adipogenesis by increasing mitophagy.Conclusion: 1.Union of OA,insulin and dexamethasone induced the adipogenesis of h ADSCs;p62 deletion increased the adipogenesis of h ADSCs induced by OA.2.Autophagy played a vital role in early stage of h ADSCs adipogenesis;p62 deletion might promote adipogenesis by increasing autophagy.3.The contents of mitochondria increased significantly after h ADSCs adipogenesis;p62 deletion might promote adipogenesis by increasing mitophagy.