Study Of Macrophage Recruitment During Lipolysis And Effect Of Echnoside A On Macrophage Accumulation

Excess calories intake and insufficient exercise lead to the increased occurrence of obesityduring recent decades. Obesity is associated with an array of additional heath problems,including increased risk of type2diabetes, atherosclerosis, fatty liver disease, degenerativedisorders like aged dementia and cancers. Obesity constitutes a serious threat to all populationin the world. Recent research found, adipose tissue is not only for energy storage, alsofunctions as an endocrine organ. Deregulation of adipokines secreted by adipose tissue resultsin macrophages infiltration and adipose tissue inflammation.Triglycerol content is relative low in adipose tissue in lean individuals; in consequence, thebasal lipolysis rate determined by adipocyte triglyceride content is low. Obesity increasedadipocyte size and therefore caused basal lipolysis with higher fatty acid release. Fasting orfood restriction resulted in mobilization of adipocyte triglyceride and stimulated demandlipolysis. Macrophage accumulation was found in adipose tissue either in obesity developmentor early body weight loss, throwing us the question how does lipolysis lead to macrophagerecruitment? We therefore performed a series of experiments in vivo and in vitro to clarify themechanism by which lipolysis affect macrophage recruitment in adipose tissue. The resultsshowed that PGE2might be the mediator linking lipolysis and macrophage recruitment, andperhaps participate in macrophage infiltration during obesity.Triterpene glycoside derived from sea cucumber alleviated obesity and obesity-relateddisease, which is proved by our previous work. However the underlying mechanism oftriterpene glycoside is still not clear. Echnoside A (EA), the most abundant component oftriterpene glycoside with defined structure, was used to further examine its role on improvingobesity. The regulatory effects on adipose tissue macrophage accumulation and COXexpression were measured especially in the current study. 3T3L1adipocyte was differentiated and treated with forskolin to induce lipolysis, and thenfree fatty acid (FFA), MCP-1, nitric oxide, arachidonic acid (AA) and its metabolites produced bylipolysis was determined. Transwell migration assay was used to examine the effect of abovesubstances on macrophage chemotaxis. We finally found out FFA and PGE2co-stimulatedmacrophage migration during lipolysis, and PGE2exhibited stronger effect to inducemacrophage recruitment. As the most critical precursor of PGE2, AA availability could alterPGE2production. Our results showed that cytosolic phospholipase A2(cPLA2) gene expressionwas upregulated, and phosphorylated by ERK1/2during lipolysis. CPLA2was also observed tomove from cytosol to plasma membrane. All these data indicated that cPLA2was activated bylipolysis, which means AA liberation was increased by deacylation of membrane phospholipids.Moreover, LPCAT gene expression was not altered by lipolysis, suggesting lipolysis did not affectLPCAT-mediated AA reacylation. Except for the increased AA production, COX2transcriptionwas dramatically stimulated by foskolin. As the rate-limiting enzyme of PGE2synthesis, COX2up-regulation could partly explain why PGE2level was increased during lipolysis.FAT/CD36is a transmembrane protein with two transmembrane regions and a largeextracellular loop. It is a multifunctional scavenger receptor participated in lipid uptake and cellsignaling pathway. Our previous work indicated that CD36acts as the regulator of lipolysis, sothe role of CD36on lipolysis induced macrophage recruitment was examined in this study.CD36was firstly knockdown by SiRNA, and then both FFA and PGE2level was compared inadipocyte with normal CD36and silencing CD36expression. The data indicated that CD36knockdown suppressed macrophage migration with stimulation of lipolysis, decreased PGE2production, but with no influence on FFA release, which further confirmed PGE2may facilitatemacrophage recruitment. In CD36knockdown adipocytes, cPLA2expression andphosphorylation was both decreased compared with normal adipocytes during lipolysis, leadingto a decrease of AA. In addition, COX2expression of CD36knockdown adipocyte was also lower,suggesting that CD36knockdown also inhibited PGE2synthesis. Based on these results, CD36KDattenuated macrophage migration by PGE2inhibition during lipolysis in vitro.To further elucidate whether CD36participated in macrophage recruitment during lipolysis,CD36null mice was used here. Both wild type (WT) and CD36null mice were fasted overnight to stimulate fat mobilization, and macrophage accumulation in adipose tissue was examined.The results showed that fasting mainly recruited anti-inflammatory M2macrophage in adiposetissue, and caused an increase of PGE2. Compared with WT mice, CD36-/-mice exhibited lessadipose tissue macrophage accumulation, increased PGE2level and COX1gene expression, buthigher serum FFA was observed in CD36-/-mice. Notably, COX1mRNA expression was found tohighly correlate with the change of f4/80expression. All these data suggested COX1-mediatedPGE2production, played a critical role in adipose tissue macrophage recruitment.We next examined whether EA could improve obesity via COX pathway in high-fat diet-fedmice. The results indicated that EA reduced body weight and body fat mass, as well as PGE2content and COX1mRNA expression of adipose tissue. Macrophage infiltration in adiposetissue was also suppressed by EA administration. In addition, EA stimulated adiponectinsecretion, promoted PPAR and its downstream gene transcription, and enhancedphosphorylation of AMPK. EA treatment might activate AMPK and PPAR pathway, thenresulted in enhancement of fatty acid catabolism. All these results indicated that EA alleviatedobesity through inhibition of COX-mediated macrophage accumulation, and stimulation of fattyacid oxidation.In conclusion, PGE2is the main mediator linking lipolysis and macrophage accumulation,and perhaps involve in obesity induced macrophage infiltration, broadening our understandingof the importance of the macrophage in metabolism regulation. Recruited macrophagesfunction in a negative feedback loop to uptake excess FFA released during lipolysis, protectfrom lipotoxity. Moreover, EA suppressed macrophage infiltration and adipose tissueinflammation via down-regulation of COX1expression and PGE2production, which providesscientific support for its application in functional food.