Mechanism Of Mark4 On Browning Of White Adipose Tissue By Regulating Of Adipocytes Autophagy

Mark4 is microtubules affinity regulated kinase 4, which belongs to serine/threonine kinase(MARKs) family and regulates a variety of physiological processes. Previous studies have foundmicelacking of Mark4 exhibitappetite improved, activity and metabolic rateincreased, accompanied by brown fat activity up-regulated, while overexpression of Mark4 promoted fat deposition.Autophagy is a major cellular degradation pathway,it can remove the excess or dysfunctional proteins and organelles to maintain cellular homeostasis and provide an internal source of nutrients for energy generation.Studies have confirmed that inhibition of adipose-specific autophagy leads to increased numbers of adipocytes with features of brown adipocytesand therefore favors fatty acid oxidation and increases insulin sensitivity either in vitro or in vivo.Browning of white adipose tissue means increase the number of beige adipocytes(with features of brown adipocytes) in ingunal white adipose tissue thereby increase energy expenditure,knock-down Mark4 gene also up-regulate the brown fat activity.Thus, we speculated that Mark4-autophagy-browning of white adipose tissue had certain linkages, whether Mark4 could influence autophagy had not been reported. To clarify the effect of Mark4 gene regulation adipocytes autophagy and white adipose tissue browning,we over-expressed and knock-down Mark4 gene in 3T3-L1 adipocytesand further validated the role of Mark4 onautophagy by constructing serum starvation and rapa-induced autophagy models, the results as follows:1.Study on the role of Mark4 in regulatingserum-starvation-induced adipocytes autophagy. The results showed adipocytesautophagy model wassuccessfully constructedby serum starvation for8 h. Under this conditionwe found autophagy marker genesBeclin1andATG7 increased significantly, accompanied by promoting autophagy marker protein LC3 Atransformedinto LC3B andinhibitingtheprotein level of P62(P<0.05).The number ofautophagic vacuoles and intracellular green fluorescent protein GFP-LC3 expressionincreased significantly(P<0.05), howeverinterfering Mark4 group showed opposite results.We also found that overexpression Mark4 significantly increasedthephosphorylation level of AMPK, reduced thephosphorylation levelofAKT, along with increased LC3 A conversion to LC3 B but reduced P62 protein levels(P<0.05), howeverinterference group has the opposite result, indicating Mark4 promote autophagy by activatingAMPK signalingpathway and inhibiting AKTsignaling pathway.2.Study on the role of Mark4 inregulating rapa-induced adipocytes autophagy.As can be seen from the results, 100 nM rapa treat adipocytes 12 h was used to construct adipocyte autophagy model successfully.Under this conditionwe found theexpression levelautophagy marker genesBeclin1 and ATG7increased significantly, accompanied by promoting autophagy marker protein LC3 A transformed into LC3 B and inhibitingtheprotein level of P62(P<0.05).We also found the number of autophagic vacuolesandintracellular green fluorescent protein GFP-LC3 expression wereincreased(P<0.05), howeverinterfering Mark4 group showed opposite results. OverexpressionofMark4 significantly reduced thephosphorylation levels of m TOR and S6K(P<0.05), interference group had the opposite result, indicating Mark4 promoted autophagy by inhibiting mTOR signaling pathway independent on AMPK/AKTsignaling pathways.3.Study on the role of Mark4 in regulating the browning of white adipose tissue through inhibiting autophagy. As we can see from the results cold stimulation up-regulated mRNA level of thermogenesis genes UCP1, PGC1 a, Prdm16 and Cideain WAT, but lower Mark4、Beclin1and ATG7 expression level(P<0.05). Though Mark4 expression level decreased in BAT, thethermogenesis genes UCP1, PGC1 a, Prdm16, Cidea and autophagy marker genes Beclin1 and ATG7 were increased(P<0.05). Mice on HFD were significantly increased Mark4 expression level no matter in WAT or BAT(P<0.05). The mRNA expression levelof autophagy genes were significantly up-regulated but thermogenesis genes had no difference in WAT(P<0.05). Interestingly, both autophagy genes and thermogenesis genes were up-regulated in BAT. Overexpression Mark4 increase thermogenesis genes expression via inhibiting autophagy, but the interfering Mark4 group and 3-MA have a synergistic effect then promoted thermogenesis genes expression significantly(P<0.05). Oil Red O staining results showed interfering Mark4 group with 3-MA treatment make lipid droplets smaller and less which was a feature of browning of WAT, the other groups had the opposite results.In summary, this study weestablished serum starvation and rapa inducedadipocyte autophagy models successfully, then demonstrated Mark4 promoted autophagy by regulation AMPK/AKT/m TOR signalling pathways and clarified the regulation mechanism. Further researchconfirmed Mark4 inhibitedthe expressionof browning of white adipose tissue related-genes by promoting autophagy.