The Effect Of Several Effective Components Of Chinese Herbs On Insulin Sensitivity In 3T3-L1 Adipocyte

Objective1.Huangqi,Huanglian and Puhuang are the main composition of the Chinese herb formula YIQISANJU.To study the effect of Astragalus polysaccharides(APS), Berberine(BER) and Pollen Typha Total Flavone(PTF),which are the main chemical components of Chinese herbs Huangqi,Huanglian and Puhuang,on the glucose and lipid metabolism of insulin resistant adipocyte.To find if these components have the effect of increase the insulin sensitivity.2.To explore the effect of APS,BER and PTF on differentiation of preadipocyte and the related gene PPARγand C/EBPαmRNA expression.To compare the different mechanism between theirs and TZDs'.3.On the base of the effect of APS,BER and PTF on increasing insulin sensitivity,to study their effect on the insulin signal transduction pathway and the secretion of related adipocyte cytokines.To explore the possible mechanism of their affection insulin sensitivity.4.To explore the character of insulin resistance and its treatment principle by Traditional Chinese Medicine(TCM) theory.Method2.Effect of several Chinese herb components on glucose and lipid metabolism of insulin resistant adipocyteThe Establishment and identification of the insulin resistance cellular model in 3T3-L1 adipocytes:We cultured the mature 3T3-L1 adipocytes with low glucose DMEM which contained 1%BSA and 5.5mmol/L glucose in 24 apertures plates.12 hours later the adipocytes were grouped and treated.There were two groups which were the normal glucose and normal insulin group and the high glucose and high insulin group.Each group had seven samples.24 hours after the treatment,the ³H-glucose uptake rates were detected to identify the model.Glucose consumption test and XTT test in normal 3T3-L1 adipocytes:We set up five groups,the control group(CON),the APS group,the BER group,the PTF group and the ROS group,every group had five concentrations except the CON 24 hours after the treatment,the glucose concentration in medium were detected by glucoseoxidase method,be subtracted by the glucose concentration of blank apertures in which there were no cells.Then we obtained the glucose consumption quantity.After the detected curture medium had been shift out in consumption test, we detected the cellular vitality by XTT.Then we chose the best concentration of every group by the outcomes of both of the two test.Glucose uptaking test in insulin resistant adipocytes:We set up six groups, the control group(CON),the model group(MOD),the APS group,the BER group, the PTF group and the ROS group.24 hours after the treatment,the glucose uptake rates were detected by glucose uptaking test both under basal situation and insulin stimulating situation.Free Fatty Acid(FFA) efflux test in insulin resistant adipocytes:We set up six groups,CON,MOD,the APS group,the BER group,the PTF group and the ROS group.24 hours after the treatment,supemate fluid were collected and FFA concentration were detected by colorimetric method.2.Effect of several Chinese herb components on the differentiation and related gene expression of 3T3-L1 pre-adipocyte.Differentiation of pre-adiopocyte treated by Chinese herb components:We set up six groups,CON,MOD,the APS group,the BER group,the PTF group and the ROS group.The pro-adipocytes were treated with medicine when had been induced differentiation.The medicine and culture medium were replaced synchronistically.At the 8th day after been treated,the adipocytes were stained by oil red O,then been pictured.The stained cells were treated with dimethyl carbinol. OD₅₇₀ was detected at wavelength of 570nm.Differentiation related gene expression treated by Chinese herb components:We set up eight groups,CON,MOD,the APS group,the BER group, thePTF group and the ROS group.The pro-adipocytes were treated with medicine when had been induced differentiation.The medicine and culture medium were replaced synchronistically.At the 8th day after been treated,the cellular total RNA were extracted.Expression of PPARγand C/EBPαmRNA were detected by Realtime- Polymerase Chain Reaction(RT-PCR).3.Effect of several Chinese herb components on the insulin signal transduction pathway and the secretion of related adipocyte cytokines.Glucose uptaking test in insulin resistant adipocytes with Wortmannnin intervention.We set up six groups,CON,MOD,the APS group,the BER group, the PTF group and the ROS group.24 hours after the treatment,all groups were intervened by Wortmannnin for 30 min,then the glucose uptake rates were detected by glucose uptaking test.Expression of key phosphorated protein of insulin signal transduction:We set up six groups,CON,MOD,the APS group,the BER group,the PTF group and the ROS group.24 hours after the treatment,the whole cellular protein in adipocytes was extracted.The quantity of Try phosphorated insulin receptorβprotein,Ser phosphorated Akt protein and Try phosphorated c-Cbl protein in the whole cellular protein were detected respectively by western blot after SDS-PAGE.4.Resistin and TNFαsecretion of insulin resistant adipocytes:We set up six groups,CON,MOD,the APS group,the BER group,the PTF group and the ROS group.24 hours after the treatment,supernate fluid were collected and resistin and TNFαconcentration were detected by ELISA.Results1.Effect of several Chinese herb components on glucose and lipid metabolism in insulin resistant adipocyteThe establishment and identification of the insulin resistance cellular model in 3T3-L1 adipocytes:The glucose uptake in normal glucose and normal insulin group was acted as control(100%),the glucose uptake rate declined 59.5%in the high glucose and high insulin group.Glucose consumption test and XTT test in 3T3-L1 adipocytes:Several Chinese herbal extracts at the proper concentration could all promote glucose consumption in normal adipocytes.APS had the largest glucose consumption at the concentration of 0.4g/L,increased 11%than the CON.BER had the largest glucose consumption at the concentration of 40μmol/L,increased 20%than the CON. PTF had the largest glucose consumption at the concentration of 0.2g/L,increased 27%than CON.The XTT results showed that,at the concentration from 0.05g/L to 0.4g/L,APS had no toxic effect on cellular vitality.At the concentration of up to 10μmol/L,BER XTT value was reduced significantly,implying toxic effects on the cell,but had no effect on cellular vitality under the concentration of 5μmol/L.From the concentration of 0.05g/L to 0.4g/L,PTF had no toxic effect on cellular vitality. According to the results of the glucose consumption and XTT test,we chose a most suitable concentration for every Chinese herb component,hwhich are APS 0.4 g/L,BER5μmol/L,PTF0.2g/L and ROS 5μmol/L.Under those concentrations, they can increase the glucose consumption without toxic effect on the cell.Glucose uptaking test in insulin resistant adipocytes in basal situation:The glucose uptake in MOD group was acted as base value(100%),the uptake of glucose of MOD group decreased 46.8%compare to CON group in basal situation.APS,BER and PTF could all promote ³H-glucose uptake in insulin resistance adipocytes in basal situation.APS group had the highest uptake rate which was 309.8%(P＜0.01).The glucose uptake rate of BER group and PTF group were 207.6%(P＜0.05) and 298.3%(P＜0.01).The glucose uptake rate of ROS group was385.0%(P＜0.01).Glucose uptaking test in insulin resistant adipocytes with insulin stimulation:The glucose uptake in MOD group was acted as base value(100%),the uptake of glucose of MOD group decreased 59.5%compare to CON group with insulin stimulation.APS,BER and PTF could all promote ³H-glucose uptake in insulin resistance adipocytes with insulin stimulation.BER group had the highest uptake rate which was 144.8%(P＜0.01).The glucose uptake rate of APS group and PTF group were 126.8%(P＜0.01) and 121.7%(P＜0.01).The glucose uptake rate of ROS group was194.9%(P＜0.01).Free Fatty Acid(FFA) efflux test in insulin resistant adipocytes:With the culture environment of high glucose and high insulin,the FFA ettlux of MOD group increased 17.5%compare to CON group.The FFA concentrations in the cell supernate fluid were all decreased after treated by herb components.The FFA efflux of APS group,BER group,PTF group and ROS group decreased 8.7%,7.1%,16.4%and 9.7%(P＜0.01 or P＜0.05) compared to MOD group.3.Effect of several Chinese herb components on the differentiation and related gene expression of 3T3-L1 pre-adipocyte.differentiation of pre-adiopocyte treated by Chinese herb components: APS,PTF and ROS promoted the differentiation of 3T3-L1 pre-adipocytes significantly(compared to the CON,P＜0.01).BER suppressed the differentiation of of 3T3-L1 pre-adipocytes,reduced the accumulation of lipid in adipocytes (compared to the CON,P＜0.01).The differentiation degree of Chinese herbal components groups were all lower than ROS(P＜0.01).Expression of PPARγand C/EBPαmRNA in 3T3-L1 adipocytes:APS and PTF up-regulated the expression of PPARγmRNA in 3T3-L1 adipocytes (compared to the CON,P＜0.01).BER down-regulated the expression of PPARγmRNA in 3T3-L1 adipocytes(compared to the CON,P＜0.01).APS and PTF up-regulated the expression of C/EBPa mRNA in 3T3-L1 adipocytes(compared to the CON,P＜0.01).BER down-regulated the expression of C/EBPa mRNA in 3T3-L1 adipocytes(compared to the CON,P＜0.01)..The expression of PPARγand C/EBPαmRNA in all Chinese herbal components groups were all lower than ROS(P＜0.01).3.Effect of several Chinese herb components on the insulin signal transduction pathway and the secretion of related adipocyte cytokines.Glucose uptaking test in insulin resistant adipocytes with Wortmannnin intervention:The glucose uptake rate of MOD group decreased 47.8%compared to CON group both with Wortmannnin intervention.After the treatment of Chinese herbal components and intervention of Wortmannnin,the glucose uptake of APS group,BER group,PTF group and ROS group increased 86.8%,190.8%,187.4%and 201.1% compared with MOD group.When compare the glucose uptake rate before and after the intervention of Wortmannnin,we found that in CON,MOD,APS and ROS group,the glucose uptakes after the intervention decreased 60.3%,45.6%,78.3%å’Œ76.9%than that before the Wortmannin intervention.While in BER group and PTF group,there is no statistical difference in glucose uptake before and after Wortmannin intervention.Expression of key phosphorated protein in insulin signal transduction pathway with Westernblot:The expression of Tyr phosphorated InsRβin MOD group was acted as base value.The expression of Tyr phosphorated InsRβin MOD group down regulated significantly than CON group(P＜0.01).Compared to MOD group,Chinese herbal groups and ROS group can up regulate the expression of Tyr phosphorated InsRβin insulin resistant adipocyte significantly(P＜0.01).The expression of Ser phosphorated Akt in MOD group was acted as base value. The expression of Ser phosphorated Akt in MOD group down regulated significantly than CON group(P＜0.01).Compared to MOD group,Chinese herbal groups and ROS group can up regulate the expression of Ser phosphorated Akt in insulin resistant adipocyte significantly(P＜0.01).The expression of Tyr phosphorated c-Cbl in MOD group was acted as base value.The expression of Tyr phosphorated c-Cbl in MOD group down regulated significantly than CON group(P＜0.01).Compared to MOD group,Chinese herbal groups and ROS group can up regulate the expression of Tyr phosphorated c-Cbl in insulin resistant adipocyte significantly(P＜0.01).Resistin and TNFαsecretion of insulin resistant adipocytes:TNFαsecretion of MOD group increased 57.6%than that of CON group.After the treatment of APS and ROS,TNF a secretion decreased 19.9%(P＜0.05)and 17.4% (P＜0.01) compared with MOD group.While BER and PTF seemed has no effect on the secretion of TNFα.Resistin secretion of MOD group had no statistical difference with that of CON group.After the treatment of APS,PTF and ROS,Resistin secretion decreased 27.9%(P＜0.01),13.7%(P＜0.05) and 30.9(P＜0.01) compared with MOD group.While BER seemed has no effect on the secretion of Rsistin.Conclusion1,3T3-L1 adipocytes could be induced to insulin resistance model successfully when thery were cultured in high concentration insulin and glucose medium,in which condition their ³H-glucose uptake rates were inhibited distinctly.This insulin resistance model could be used in medicine screening and the mechanism study.2,On the base of little toxic effect to adipocytes,APS,BER and PTF could all improve glucose metabolism both under basal and insulin stimulate situation.And at the same time lipid metabolism is improved as well.These results revealed that they might improve insulin resistance in adipocytes.3,APS promoted differentiation of of 3T3-L1 pre-adipocytes,up-regulated the expression of PPARγand C/EBPαmRNA in 3T3-L1 adipocytes,behave the effect similar to PPARγagonists.BER suppressed the differentiation of of 3T3-L1 pro-adipocytes,reduced accumulation of lipid in adipocytes,down-regulated the expression of PPARγand C/EBPαmRNA in 3T3-L1 adipocytes,behave the effect similar to PPARγinhibitor.They could not gain weight and fat.These results would provide new ideas for prevention and cure to insulin resistance,broaden the approach for insulin resistance prevention and treatment with the method of TCM or integrated medicine.4,APS,BER and PTF can improve insulin sensitivity through insulin signal transduction pathway.APS,BER and PTF can all up regulate the expression of Tyr phosphorated InsRβand activate the down stream signal transduction pathway in the cell.APS improves insulin sensitivity at least partly through PI-3Kpathway.While BER and PTF more likely stimulate through c-Cbl/CAP pathway to improve insulin sensitivity5,Chinese herbal components can adjust insulin resistance positive cytokine like TNFα,Resistin secretion in insulin resistant adipocyte.6,Cellular experiment result improves Chinese herbal main components of YIQISANJU have the effect of improve insulin sensitivity with different pathway. Part of the mechanism was explored on cellular and molecular level.