Dihydrokaempferol Derivatives Inhibit Lipid Deposition To Improve Insulin Resistance Via Regulating AMPK/PGC-1? Pathway In Skeletal Muscle

In the worldwide,the incidence of diabetes mellitus(DM)is growing rapidly.DM has become the third largest major chronic non infectious diseases after malignant tumor and cardiovascular disease,which has been a serious threat to human health.Type 2 diabetes mellitus(T2DM)is the most common type of diabetes,accounting for more than 90% of diabetes patients.It is widely believed that insulin resistance(IR)is an important pathogenesis of T2 DM and exists the whole process of the occurrence and development of DM.Skeletal muscle is the main glucose and fat metabolism tissue in the human and plays an important role in maintaining the glucose homeostasis.The excessive accumulation offat in the non-fatty tissues such as muscle,liver,pancreas and so on was namely ectopic lipid deposition.More and more research evidences have indicated that ectopic lipid deposition in skeletal muscle has a very strongassociationwith occurrence and development of insulin resistance.Mitochondria is the main place of the oxidative metabolism of fat,glucose and other energy substrate.Therefore,enhancing skeletal muscle cells' mitochondrial function to improve skeletal muscle ectopic fat deposition has become the important way of prevention and treatment of insulin resistance and related metabolic diseases.In recent years,dietary flavonoids compounds have shown significant effect on the prevention and treatment of diabetes in vitro and animals studies.Some experimental studies confirmed dihydrokaempferol derivatives such as kaempferol(KMF),dihydromyricetin(DHM),myricetin(MYC)and quercetin(QUC)had a positive effect on skeletal muscle insulin resistance,but the exact mechanism is not very clear and needs more investigation.AMP-activated protein kinase(AMPK)is a receptor for sensing celluar energy change,AMPK can promote the biosynthesis of mitochondria,mitochondrial fuction,and increase the metabolism of fatty acid oxidation through activating its downstream transcription activating factor peroxisome proliferator activator receptor gamma coactivator-1alpha(PGC-1?)in a direct or indirect way.DHM,MYC and QUC had been shown to increase the activity of AMPK.Therefore,we speculate that dihydrokaempferol derivatives such as KMF,DHM,MYC and QUC can inhibit lipid deposition in skeletal muscle by regulating AMPK/ PGC-1 ? pathway,which plays a effect of improving insulin resistance.In this study,we first investigated the effect of four derivatives of dihydrokaempferol on lipid deposition in skeletal muscle with insulin resistance model of mouse C2C12 myotubeswith palmitic acid(PA).Lipid accumulation and triglyceride(TG)contents in C2C12 myotubes were evaluated by oil O dye staining and TG kit respectively;2-NBDG assay was used to detect glucose uptake under insulin stimulation;AMPK,PGC-1? protein expression were detected by Western blot,discussing the effect of four dihydrokaempferol derivatives on PA induced lipid deposition and insulin resistance in C2C12 myotubes and the role of AMPK/PGC-1 pathway in the study.Then,we established a mouse model of insulin resistance by using high fat feedstuff to feed C57BL/6J mice,and observed the effect of DHM intervention on TG contents?insulin sensitivity and AMPK,PGC-1? protein expression in skelete muscle,and further verifyed the vitro experimental research results.The main results and conclusions are as follows:(1)After PA treatment,lipid deposition and TG significantlyincreased in C2C12 myotubes,glucose uptake significantly decreased under insulin stimulation in C2C12 myotubes.DHM,MYC and QUC administration were significantly inhibit PA induced lipid deposition,increased TG leveland decreased glucose uptake.The inhibitory effect of DHM was more significantly than others,indicating that DHM and other derivatives of dihydrokaempferol can inhibit the high fat induced lipid deposition and insulin resistance in skeletal muscle cells.(2)After PA treatment,p-AMPK and PGC-1 ? were decreased significantly in C2C12 myotubes.DHM,MYC,QUC treatment could significantly increased the levels of p-AMPK and PGC-1?.The inhibitory effect of DHM was more significantly than others,suggesting that DHM and other derivatives of dihydrokaempferol inhibite high fat lipid induced lipid deposition in skeletal muscle cells and insulin resistance may be associated with AMPK/PGC-1? pathway.(3)AMPK activity inhibitor Compound C significantly weaken the inhibitory effect of DHM on PA induced lipid deposition,increased TG content,decreased PGC-1? protein level and decreased glucose uptake in C2C12 myotubes,indicating that DHM upregulates the expression of PGC-1 ? by activing AMPK and reduces high fat induced lipid deposition in C2C12 myotubes,to further improve insulin resistance.(4)Homeostasis model of assessment for insulin resistence index(HOMA-IR)was increased significantly in high-fat diet fed C57BL/6J mice compared with the low-fat diet group.In the soleus,TG content were increased significantly,the protein expression levels of p-AMPK and PGC-1? were decreased significantly.HOMA-IRand TG level in soleus muscle in DHM(100mg/kg.bw)intervention group were decreased significantly compared with the model group.Meanwhile,the protein levels of p-AMPK and PGC-1 ? were elevated significantly in DHM(100mg/kg.bw)intervention group,further suggesting that DHM reduces high fat induced lipid deposition in skeletal muscle to play the role of improving insulin resistance through activating AMPK/PGC-1?pathway in skeletal muscle.In summary,dihydrokaempferol derivatives such as DHM,MYC,QUC can inhibit high fat induced lipid deposition in skeletal muscle by regulating AMPK/ PGC-1? signaling pathway,thereby improving insulin resistance.