Effects Of Mark4on Mitochondrial Biosynsis And Inflammatory Factors In3T3-L1Adipocytes

MAP/Microtubule affinity-regulating kinase4(Mark4) is one of the family members ofMARKs. It has been reported that this kinase, like its other family members, could participatein many physiological processes other than regulating microtubule stability or cell polarity,such as formation of vascular bundles, apoptosis, glucose metabolism, cell reproduction,immune respond, energy homeostasis and the development of the nervous system and so on.Evidences of Mark4negatively regulating energy metabolism and insulin sensitivity havebeen offered, however, the specific mechanism of that regulation is still elusive. Besides, therole Mark4played in insulin resisitance relative processes like mitochondrial biosynsis or thesecretion of inflammatory factors is also uncertain. Thus we made exploration in the functionof Mark4hoping to make contribution to the treatment of relative human diseases assomewhat theoretical guidance.Specifically in this study, we transfected3T3-L1preadipocytes respectively with theover-expression plasmid vector of Mark4and its interference plasmid vector. Then four daysafter adding differentiation inducements, we studied if Mark4has affected the fat metabolism,mitochondrial biosynsis, and expression of key proinflammatory factors in3T3-L1cells;besides, this paper also explored the mechanism of these effects via detecting relatedsignaling pathways and studying the potential transcription factor of Mark4. In summary,main results obtained are listed as follow:1. Mark4promoted lipid metabolism in3T3-L1cells. A) Stained with Bodipy and OilRed O, more lipid droplets were found in3T3-L1cells transfacted with over-expressionMark4vector; on the contrary, the deposition was inhibited in cells transfected with Mark4interference plasmid vector. B) Over-expressed Mark4significantly promoted the expressionof CEBPα, PPARγ as well as lipogenic genes FAS and ACC α in3T3-L1adipocytes, togetherwith the expression of hormone sensitive lipase HSL.2. Effects of Mark4on the3T3-L1cells in mitochondria biosynsis. Over-expression ofMark4inhibited mitochondria biosynsis in3T3-L1adipocytes: A) Stained with JC-1dye, itwas found that over-expression of Mark4impaired mitochondrial membrane potential in 3T3-L1adipocytes, and this impairment was repaired when Mark4was blocked. B)Over-expression of Mark4significantly increased the expression of Cyt-C (P<0.01); and theexpression of Cyt-C in Mark4interference group was decreased (P<0.01). C) In the groupover-expressed Mark4, key genes of mitochondrial synthesis were significantly decreased atboth transcription and translation levels, like TFAM and PGC-1α (P<0.01); so did theexpression of key genes in oxidative respiratory, such as UCP2, MDH1, CPT1, CAT, COX2,the exprssion of MDH1and CPT1were significantly decreased at translation level. D)Over-expression of Mark4significantly inhibited the activation of AKT and mTOR, butincreased ERK1/2and JNKs phosphorylation.3. Effects of Mark4on proinflammatory factors in3T3-L1cells. At the level oftranscription, over-expression of Mark4promoted the expression of proinflammatory factors:IL1β, IL6and MCP1in3T3-L1cells, as well as the the expression of Leptin, whilesignificantly reduced the expression of PKA. Besides, analysis of Mark4gene promoter wasmanaged. PPARγ was predicted to be a potential transcription factor of Mark4withbioinformatics softwares. Via luciferase reporter system, binding site of PPARγ might be atthe promoter region of Mark4,but need to be confirmed.In summary, Mark4is involved lipid metabolism, mitochondrial biosynsis and affectedthe expression of inflammatory factors in3T3-L1adipocytes. Besides, PPAR γ, AKT, mTOR,JNKs and ERK1/2pathways may play important roles in this regulation induced by Mark4.