Effects And Mechanisms Of Pentamethylquercetin On Driving White Adipose Tissues Browning/Beiging

With the better understanding of adipose tissue plasticity,promoting white adipose tissue(WAT)browning/beiging has been regarded as a promising strategy for combating obesity and obesity-related disordes.Thus,drug research and development on this field has drawn much more attention than ever before.Pentamethylquercetin(PMQ),as a typical member of natural polymethoxylated flavonoids,is originally found in sea buckthorn and the rhizome of Kaempferia parviflora.As restricted by natural resources,it is studied little.We adopted a semi-synthetic method to obtain PMQ through methylation modification of quercetin,which solved the problem of pharmaceutical sources bottlenecks.Previous studies have demonstrated that PMQ possesses health beneficial effects including anti-metabolic syndrome,anti-diabetes,anti-myocardial hypertrophy and anti-thrombotic.Although it has been proven that PMQ could induce brown-like transition in WAT depots,the underlying molecular mechanisms on this process are still need to be further explored.Therefore,in this study,we investigated the effectiveness and mechanisms of PMQ on promoting browning/beiging in monosodium glutamate-induced obese(MSGIO)mice and 3T3-L1 adipocytesPart Ⅰ PMQ promotes browning/beiging of inguinal WAT in MSGIO miceObjective:To study the effect of PMQ on inguinal WAT browning/beiging in MSGIO mice.Methods:MSGIO mice model was established.At 5 weeks of age,the male MSGIO mice were selected and randomized into five groups as follows:Control group,MSGIO mice group,PMQ group(MSGIO mice given at 5,10,20 mg/kg,respectively).All mice were administrated by gastric gavage once a day for 19 consecutive weeks.Oral glucose tolerance test(OGTT)was performed at 22 weeks of age.Body weight,body length and waist circumference were measured at the end of experiment.Then blood was collected from the orbital vein for separating serum.After sacrificed by decapitation,adipose tissues and gastrocnemius muscles of mice were dissected and stored at-80 ℃.The serum levels of fasting glucose,iinsulin,triglyceride as well as cholesterol were determined respectively and the homeostasis model assessment-insulin resistance(HOMA-IR)was calculated.Inguinal WAT embedded in paraffin were sectioned and stained with hematoxylin and eosin or with UCP-1 primary antibodies.The mRNA expression levels of brown-specific genes and beige-cell markers in inguinal WAT were measured by RT-PCR.The UCP-1 protein levels of inguinal WAT were examined by Western blot analysis.Results:(1)MSGIO mice exhibited significant obesity.Compared with control group,body weight,waist circumference,lee index,fat mass and fat index(fat mass(g)/BW(g))in MSGIO mice were increased by 45%,29%,33%,282%and 179%,respectively.MSGIO mice had emerged gluco-lipid metabolic disorders.The serum levels of fasting glucose,TG and TC in MSGIO mice were increased by 51%,120%and 96%,respectively when compared to those of the control mice;MSGIO mice had hyperinsulinemia,insulin resistance and impaired glucose tolerance.Serum insulin levels,HOMA-IR index and AUC of-OGTT in MSGIO group were elevated by 3.3 folds,5.6 folds and 60%,respectively compared to control mice.These results confirm that MSGIO mice model is one of representative obese type 2 diabetic mice models.(2)Compared to control group,inguinal WAT in MSGIO mice possessed larger adipocytes,which appeared as unilocular large lipid droplets.The expression levels of brown/beige fat specific genes were down-regulated in the inguinal WAT of MSGIO mice when compared with control mice.The expression of brown-specific genes Ucp-1,Pgc-1αCidea and Cox-7al were decreased by 46%,42%,34%and 26%respectively.Beige-cell markers including Tbx-1,Tmem26 and CD137 were also decreased by 60%,49%.56%respectively.Moreover,UCP-1+ adipocytes as well as protein abundance of UCP-1(decreased by 45%)in the inguinal WAT were also markedly decreased compared to control group.These data indicate that the potentialities of inducing white adipose tissues browning/beiging in obese type 2 diabetes mice are reduced.(3)PMQ treatment dose-dependently lowered body weight,waist circumstance.Lee index,fat mass and%(fat mass of BW),of which PMQ(20 mg/kg)decreased these parameters by 16%,8%,4%,20%and 10%when compared to MSGIO group.Serum levels of fasting glucose,TG,TC and insulin as well as HOMA-IR index and AUC of OGTT were reduced upon PMQ intervention in a dose-dependent manner compared with MSGIO group mice.PMQ 20 mg/kg could make these parameters at or near to the level of control group.These test results revealed that the efficacy of PMQ treatment meets the primary and secondary endpoint requirements of weight-loss drugs.(4)Compared to MSGIO mice,a decrease in the mean surface area of cells with a multilocular brown phenotype in the inguinal fat pads was observed after PMQ’s administration.Moreover,PMQ treatment dose-dependently elevated mRNA levels for brown-specific genes(Ucp-1,Pgc-1α,Cidea,Cox-7al),beige-cell markers(Tbx-1,Tmem26,CD137).Meanwhile,UCP-1 protein levels were up-regulated after PMQ intervention.All of above indicators in PMQ 20 mg/kg group were raised up to the level of control group.These results demonstrate that PMQ could promote browning/beiging phenotype in inguinal WAT from MSGIO mice.Part Ⅱ The possible mechanisms of PMQ in inducing white adipose browning/beigingObjective:To study the mechanisms of PMQ in the induction of white fat browning/beiging in vivo and in vitro.Methods:The expression levels of AMPK-PGC-1α-FNDC5 in gastrocnemius muscle were detected by RT-PCR and Western blot.Serum irisin levels were determined by ELISA analysis.In C2C12 myotubes exposed to PMQ at concentrations ranging from 1 to 10 μM,the levels of AMPK-PGC-1α-FNDC5 and MCM irisin concentration were measured by Western blot and ELISA.Myotubes conditioned medium(MCM)were collected and FNDC5 antibody at a concentration of 1.25 μg/mL was added to the MCM-PMQ10.The experimental groups are as follows:MCM-Vehicle,MCM-PMQ1/3/10,MCM-PMQ10+anti-FNDC5.All of MCM were used to treat 3T3-L1 cells and the expression levels of BAT signature genes,beige-cell markers and UCP-1 in 3T3-L1 adipocytes were analyzed by RT-PCR and Western blot.After PGC-1α was suppressed by siRNA-PGC-1α,C2C12 myotubes were incubated with or without PMQ 10 μM.The levels of PGC-1α-FNDC5 expression,irisin concentration as well as UCP-1 protein expression of 3T3-L1 adipocytes after MCM exposure were tested by the methods of Western blot and ELISA.C2C12 myotubes were pretreated with AMPK inhibitor(Compound C).and exposed to DMSO or PMQ 10 μM for 16 h.Then the expression changes of AMPK-PGC-1α-FNDC5 were measured by Western blot.Results:(1)Compared with control group,on mRNA and protein levels in gastrocnemius muscle of MSGIO mice,the levels of FNDC5 expression were respectively decreased by 34%and 45%,and the levels of PGC-1α expression were respectively reduced by 38%and 34%.MSGIO mice had significantly lower serum irisin levels(decrease by 52%)than those of control mice.Furthermore,the phosphorylation levels of AMPK in muscle of MSGIO mice were decreased by 49%.These findings suggest that the levels of AMPK-PGC-1α-FNDC5 signaling expression in gastrocnemius muscle and serum irisin from obese type 2 diabetes mice are dramatically down-regulated.(2)PMQ up-regulated the mRNA and protein levels of PGC-1α and FNDC5 in skeletal muscle as well as serum irisin levels in a dose-dependent manner when compared with MSGIO mice.Meanwhile,PMQ dose-dependently increased AMPK phosphorylation levels in gastrocnemius muscle of MSGIO mice.PMQ 20 mg/kg treatment completely restored the impaired AMPK-PGC-1α-FNDC5 signaling in muscle and the reduced serum irisin levels to control group level.These data thus demonstrate that PMQ can activate AMPK-PGC-1α-FNDC5 signaling pathway in muscle and stimulate irisin secretion.(3)PMQ stimulated the FNDC5 expression and irisin secretion in a concentration-dependent manner in C2C12 myotubes.Conditioned medium collected from C2C12 myotubes exposed to PMQ 1/3/10μM(MCM-PMQ1/3/10)enhanced the expression of brown specifc genes(Ucp-1,Pgc-1α.Cidea.Cox-7al)and beige-cell markers(Tbx-1,Tmem26,CD 137)accompanied with UCP-1 protein levels in a concentration-dependent manner in 3T3-L1 adipocytes compared to CM-vehicle.We next used FNDC5 antibody to neutralize the irisin in MCM-PMQ10.The stimulatory effects of MCM-PMQ10 on these browning/beiging fators in 3T3-L1 adipocytes were antagonized partially or abrogated by Anti-FNDC5.These data altogether show that FNDC5/irisin plays a pivotal role in the PMQ mediated browning of white adipocytes.(4)PMQ dose-dependently elevated PGC-1α protein levels.The decrease in PGC-and FNDC5 induced by siRNA-PGC-could not be reversed by PMQ.In addition,PMQ did not cancel the decrease in irisin concentration induced by siRNA-PGC-1α.More importantly,protein levels of UCP-1 which up-regulated by MCM-PMQ in 3T3-L1 adipocytes were abolished by siRNA-PGC-1α.Collectively,these results suggest that PMQ increases UCP-1 expression in adipocytes via up-regulation of PGC-1α/FNDC5/irisin.(5)In C2C12 myotubes.PMQ increased P-AMPK levels in a dose-dependent manner.It was found that compound C pretreatment dramatically reduced protein levels of P-AMPK,PGC-1α and FNDC5 which enhanced by PMQ incubation in C2C12 myotubes.Overall,these results suggest that PMQ stimulates PGC-1α-FNDC5 pathway via activating AMPK.Conclusion:(1)White adipose tissues from obese type 2 diabetes mice(MSGIO)have a lower potential ability to induce brown/beige cell formation.(2)PMQ could induce WAT browning/beiging and generate weight-loss effects in MSGIO mice.(3)PMQ promotes WAT browning/beiging through activating AMPK-PGC-1α-FNDC5 axis in skeletal muscle and irisin production.