Mechanism Of Dihydromyricetin In Reducing Lipid Deposition In 3T3-L1 Cells By Activating CaMKK2-AMPK Pathway

Obesity is the medical state that the body’s excessive fat deposition exceeds the normal state.Obesity will cause various diseases,especially cardiovascular disease,type 2 diabetes,fatty liver,osteoarthritis and some cancers.The obesity are caused by following three main reasons: excessive food intake,physical activity lack and genetic susceptibility;and others are caused by genes,endocrine disorders,medications,and mental disorders.The key to prevent obesity is to reduce body fat deposition.Dihydromyricetin(DHM)is a flavanonol with the functions of anti-oxidation,anti-inflammatory,hypolipidemic,anti-fatty liver,and so on.It has great value to medicinal.However,the mechanism of the funtion of DHM on obesity has not been studied so far.Calcium ion is indispensable in the physiological activities of various cells and organisms.Calcium ion regulates the body through regulating intracellular and extracellular potential balance and activities related proteases,such as Ca MKK2 protein.Ca MKK2 as a calmodulin kinase kinase activates AMPK under the regulation of calcium ions.AMPK pathway is a key pathway of cell metabolism regulation.Activation of the AMPK pathway will inhibit the differentiation and lipid accumulation of preadipocytes and promote triglyceride degradation in adipocytes.3T3-L1 cell line as a model in this study,research the function and mechanism of DHM on fat in vitro.At first detected the absorption efficiency of DHM by HPLC.Then observed the effect of DHM on cytoplasmic calcium ion concentration by calcium ion probe and Leica living cell workstation.After that the toxicity and proliferation of DHM on 3T3-L1 cells were examined to rule out cell fat deposition due to increasing or decreasing in cell activity and quantity.The mechanism of DHM was predicted by oil red O staining,q PCR and western blot to detect the effects of DHM on lipid deposition-related phenotypes,m RNA and protein expression.Through the literature searching and KEGG pathway analysis,Ca MKK2 was found as upstream factor of DHM action.After Ca MKK2 was interfered,the cells were treated with DHM,observed the lipid deposition and detected the expression of related proteins.Finally,the mechanism of DHM to reduce fat deposition by activating Ca MKK2-AMPK pathway was elucidated,which will improve the targets of glucose and lipid metabolism in the future and established theoretical basis for the development of new drugs for treating obesity.The results of this study are as follows: 1.Effect of DHM on proliferation and cell viability in 3T3-L1 cellsAfter treatment of 3T3-L1 cells with different concentrations of DHM,the cell viability and proliferation were detected by flow cytometry and Ed U.It was found that DHM did not affect the proliferation and cell viability of 3T3-L1 cells below 80 μM.2.Effects of different concentrations of DHM on lipid accumulation in 3T3-L1 cellsAfter induced differentiation of 3T3-L1 cells,treated by different concentrations of DHM.The results showed that 60 μM DHM inhibited 3T3-L1 cell differentiation and lipid deposition without affecting cell viability.3.Effect of DHM on 3T3-L1 cells’ differentiationDHM inhibited the expression of m RNA and protein related to induce differentiation of 3T3-L1 cells,and promoted the protein β-catenin expression to prevent the accumulation of lipids in cells.4.Effect of DHM on lipolysis of differentiated 3T3-L1 cellsDHM promotes m RNA expression of PKA,HSL and ATGL in adipocytes to promotes lipid droplets degradation in mature adipocytes,and reduces fat deposition.5.Absorption efficiency of DHM and its effect on cytoplasmic calcium ionsThe results of HPLC showed that the absorption efficiency of DHM in 3T3-L1 cells showed a negative logarithmic linear trend.DHM in medium was completely absorbed by cells after treating 3 h.The calcium ion fluorescent probe signal showed that DHM could increase the cytoplasmic calcium ion content.6.Effect of DHM on CAMKK2 and AMPK in 3T3-L1 cellsResults of q PCR showed that DHM promoted Ca MKK2 m RNA expression,and Western blot showed that DHM promoted AMPK phosphorylation.7.Effect of DHM on the expression of lipid-related protein in 3T3-L1 cells after interference with Ca MKK2After STO-609 interfered with Ca MKK2,AMPK phosphorylation was inhibited,and DHM had no effect on the expression of differentiation,adipogenic and lipolysis-related proteins.DHM no longer affected lipid deposition in 3T3-L1 cells.Conclution: DHM increases the cytoplasmic calcium concentration and up-regulate the m RNA expression level of Ca MKK2 to promote AMPK phosphorylation to inhibit 3T3-L1 cell differentiation,reduce lipogenesis and promote the lipolysis in cells,thereby regulating intracellular lipid metabolism.