Molecular Mechanism Of Flavonoid From Seed Of Hippophae Rhamnoides L.Enhances Glucose Uptake In Adipocytes And Myotubes

Flavonoid-enriched extract from the seed of Hippophae rhamnoides L.(FSH).The past decade has witnessed abundant studies of FSH.Our lab has established the optimal extraction process of FSH,analyzed the main components of FSH,clarified its material basis and formulated the quality control standard.The accumulated evidence of animal experiments indicated that FSH exhibited hypoglycemic activity and ameliorated insulin resistance,but its cellular and molecular mechanisms are still unclear.To gain a comprehensive picture of the regulations of glucose homeostasis by FSH.We sought in the present study to observe the effects of FSH on cell signaling pathway albout glucose uptake in C2C12 myotubes and 3T3-L1 adipocytes as well as the underlying molecular targets.Our results showed that 50-200?g/mL FSH could significantly enhance glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes.The effect of FSH with time and dose dependent manner on the glucose uptake was detected by 2-NBDG.FSH can trigger AMPK signaling pathway,up-regulate p-AMPK and induce GLUT4 translocation to cell membranes.PPAR? inhibition is tightly associated with AMPK activity.It has been indicated that PPAR? is inhibited by FSH treament.In order to confirm the positive regulation of AMPK pathway by FSH under basal state,AMPK inhibitor Compound C were used to attenuate the action of FSH,indicating that FSH enhance glucose uptake through AMPK signaling pathway.Insulin promotes glucose uptake in muscle and adipose tissue(AT)by facilitating GLUT4 translocation,which plays an important role in whole body glucose homeostasis.Cells were pretreated with FSH for 24 h,and then incubated with 1 OOnM insulin for another 30min.Insulin signal pathway was triggered by insulin.The results show that FSH down-regulate the expression of IRS 1 in two kinds of cells,inhibit the expression of tyrosine and serine phosphorylation in insulin receptor substrate,decrease the expression of PI3K and inhibit the phosphorylation of AKT and AS 160.FSH inhibited the phosphorylation levels of ERK,JNK and P38 in both cells.FSH could significantly reduce the expression of PKC? protein in C2C12 myotubes,and had no effect on CAP protein level.However,FSH can up-regulate CAP protein level in adipocytes.These results indicate that FSH can inhibit IRS-mediated PI3-K/AKT/AS 160 signaling pathways and MAPK signaling pathway.The effects of FSH co-treated with insulin promoted the glucose uptake and enhanced the level of p-AMPK,indicating that Insulin pathway inhibition does not affect FSH-enhanced glucose uptake.FSH could trigger the AMPK signaling pathway and enhance glucose uptake when combine with insulin.It has been well established that inflammation is tightly associated with insulin resistant.The activation of monocytes-macrophages and adipocytes can secrete multiple inflammatory factors.We treated RAW264.7 monocyte-macrophages with LPS(100 ng/mL)to establish an in vitro inflammatory model.The results show that FSH inhibited the expression of NF-?B,P38 MAPK pathway and CXCL10 and IL-6 mRNAin RAW264.7 cells,and promoted the expression of TNF-a mRNA.FSH could also inhibit NF-?B signaling pathway and its mRNA in 3T3-L1 adipocytes and promote the expression of TNF-a mRNA.FSH may ameliorate insulin resistance through inhibiting the NF-?B,P38 MAPK signaling pathway,reducing the expression of some inflammatory factors.In conclusion,FSH can enhance glucose uptake through AMPK-mediated pathway.FSH may ameliorate insulin resistance by inhibiting NF-?B,P38 MAPK signaling pathway and the mRNA expression of CXCL10 and IL-6.