Rutin Promotes White Adipose Tissue “browning” And Brown Adipose Tissue Activation Partially Through The Calmodulin-dependent Protein Kinase Kinase β/AMP Activated Protein Kinase Pathway

Objective:To investigate the effect of rutin on adipose" browning" and the mechanism of AMPK pathway that regulates adipose" browning",in order to provide new theoretical basis for reducing obesity and improving metabolism.Method:In vivoSix-week-old male C57BL/6 mice were divided into normal diet group(ND,n=6),high-fat diet group(HFD,n=6)and high-fat diet group containing 0.1%Rutin(HFD+Rutin,n=6).After 12 w,mice were anesthetized with 1%sodium pentobarbital.Blood was obtained by cardiac puncture,and plasma metabolic indexes were measured.Inguinal adipose tissue(iWAT),epididymal adipose tissue(eWAT),and brown adipose tissue(BAT)were isolated for Western blot,RT-PCR,and histological analysis.In vitroAMPK inhibitor CC,AMPK agonist AICAR,CaMKKβ inhibitor STO-609,and calcium chelator BAPTA-AM were given in co-culture with Rutin at the differentiation maturity stage of 3T3-L1 cells.Cells were collected for the detection of energy depletion factors,lipogenic genes,lipid accumulation,mitochondrial membrane potential and other indicators.Results:(1)Compared with the HFD group,Rutin intervention reduced body weight,adipose tissue and liver weight in obese mice(P<0.05),decreased plasma metabolic index(P<0.05),reduced adipocyte size;increased thermogenic markers and P-AMPK contents(P<0.05),reduced WAT adipogenic genes expression(P<0.05),increased AP2 contents in BAT(P<0.05).(2)Rutin promoted 3T3-L1 cells thermogenic markers and P-AMPK expression(P<0.001),decreased adipogenic genes contents(P<0.05),reduced lipid droplet accumulation and increased mitochondrial membrane potential.(3)3T3-L1 cells treated with the AMPK inhibitor CC showed that AMPK inhibition resulted in downregulation of thermogenic proteins(P<0.05).The AMPK agonist AICAR increased the expression of thermogenic markers and P-AMPK in 3T3-L1 cells(P<0.05).(4)In Hela cells,Rutin induced the upregulation of thermogenic proteins(P<0.01).when the CaMKKβ inhibitor STO-609 was used to treat 3T3-L1 cells,the effect of rutin on promoting the expression of thermogenic and P-AMPK proteins was diminished(P<0.05).Treatment of 3T3-L1 cells with the calcium chelator BAPTA-AM showed reduced expression of thermogenic and P-AMPK proteins compared to the Rutin group(P<0.05).Conclusion:(1)Rutin improves HFD-induced disorders of lipid metabolism,obesity,and non-alcoholic fatty liver disease.(2)Rutin promotes adipose "browning",which in turn alleviates metabolic disorders.(3)Rutin stimulates WAT "browning" and BAT activation by a mechanism related to the CaMKKβ-AMPK pathway.