| Diabetes mellitus is a kind of common and multiple disease induced by several factors such as heredity, environment, immunity and so on, it severely endangers human health. At present, there are about 250 million type 2 diabetic patients in the world, and this data will be increased to 380 million in 2025. China is one of the three countries which have the most diabetic patients,90% are type 2 diabetes(T2DM) which is becoming the third "killer" of the health of mankind along with cancer, cardiovascular and cerebrovascular diseases because of its complications involved in several important organs. With the epidemic of population aging, westernization of the Asian diet and infaust life style such as prolonged sitting, the prevalence of T2DM increases rapidly, so it is of great importance to investigate the pathogenesis and search the effective treatment targets of T2DM.Our previous studies suggested that astragalus polysaccharides(APS), a most polysaccharide component of an aquenous extract of astragalus membranaceus roots, possesses a favourable hypoglycemic and insulin sensitization effect, what’s worth to be mentioned is that it has little toxicity and side effect compared with chemical antidiabetic drug. Decreased expression of protein tyrosine phosphatase 1B(PTP1B) is involved in its insulin sensitization effect, but the exact mechanisms involved haven’t been well studied. In this study, high fat diet associated with small dose injection of streptozotocin(STZ,25mg/kg) were treated on SD rat to establish a model of T2DM, combining with investigations in vitro to further study the effects of APS on the insulin resistance of T2DM rats and the mechanisms of APS decreased the expression of PTP1B. The study was divided into three parts as follows. Aim:Insulin resistance was the most characteristic feature of T2DM. Endoplasmic reticulum stress(ERS) was considered as the molecular link between obesity, insulin resistance and T2DM. APS is a most polysaccharide component of an aquenous extract of astragalus membranaceus roots, In this study, a rat model of T2DM was established, APS was treated on T2DM rat, the effects of APS on the level of blood glucose, insulin sensitivity, ERS and insulin signaling of T2DM rats were detected to study the effects of APS on the insulin resistance of T2DM rats and its possible mechanisms.Methods:High fat diet associated with small dose injection of streptozotocin (STZ,25mg/kg) was treated on SD rats to establish a model of T2DM. Animals were divided into four groups as follows:control group(C group), control+APS group(C+APS group), T2DM group(DM group) and T2DM+APS group(DM+APS group). C+APS group and DM+APS group were treated with APS(700mg/kg/day), meanwhile C group and DM group were treated with equal volume of sodium chloride by gastric perfusion for 8 weeks. Fast blood glucose(FBG), random blood glucose(RBG), oral glucose tolerance test(OGTT), glycosylated hemoglobin(GHb), serum insulin and HOMA-IR were detected to investigate the effect of APS on the blood glucose and insulin sensitivity; the expressions of phosphorylated IRE1 and activated ATF6(p50-ATF6) which were important proteins involved in ERS in the liver of T2DM rats were detected by western blotting to investigate the effects of APS on the ERS of the liver; the expression of PTP1B in the liver of T2DM rats were detected by western blotting and RT-PCR to investigate the effect of APS on insulin signaling. Results:(1)The RBG, FBG, blood glucose at all time points in OGTT of DM group were all higher than C group, but the aforesaid indexes in DM+APS group were all significantly lower than DM group, there was no significant difference between C group and C+APS group; the level of GHb in DM group was higher than C group, and APS decreased the level of GHb of DM group;(2)No significant difference was observed between each group on the level of serum insulin, but HOMA-IR in DM group was higher than C group, HOMA-IR in DM+APS group was lower than DM group, there was no significant difference between C group and C+APS group;(3)The expressions of phosphorylated IRE1 and activated ATF6(p50-ATF6) in DM group were both higher than C group, and the expression of p90-ATF6 was lower, while the expression of phosphorylated IRE1 and p50-ATF6 in DM+APS group was lower than DM group, but the expression of p90-ATF6 was higher than C group, no significant difference was observed between C group and C+APS group;(4)The mRNA and protein expression of PTP1B in DM group were both higher than C group, APS decreased both the mRNA and protein expression of PTP1B, but had no significant impact on normal rats.Conclusions:(1)APS lowered the blood glucose, improved the glucose tolerance and increased the insulin sensitivity of high fat diet associated with small dose injection of STZ induced T2DM rats;(2)APS significantly relieved the ERS in the liver of T2DM rats;(3)APS decreased the mRNA and protein expression of PTP1B in the liver of T2DM rats, this would be the important mechanisms of those effects mentioned above of APS. Aim:PTP1B as an important negative regulatory factor of insulin signaling located on the endoplasmic reticulum(ER) membrane and played an important role in IRE1 mediated ERS signaling, and this role depended on the tyrosine phosphates activity of PTP1B, but PTP1B didn’t influence the phosphorylation of IRE1 directly. Activating transcription factor 6(ATF6) was an important ERS signaling protein which and played a role in activating the transcription of various transcription factors and binding proteins and enzymes for protein folding involved in ERS. We presumed that the expression of PTP1B was regulated by ATF6. In this study, thapsigargin(TG), high concentration of D-glucose and insulin were used to establish ER stressed and insulin resistant cell models respectively in order to investigate the regulation of the activation of ATF6 induced by ERS on the expression of PTP1B.Methods:This study was divided into three groups:(1)pCI-Flag-ATF6(N)(2-366) and pCI-blank plasmid were treated on HL-7702 cells by transient transfection, the mRNA and protein expression of PTP1B were detected by RT-PCR and western blotting respectively; (2)TG, high concentration of insulin(5×10-7mol/L) and different concentration of glucose were treated on the cells, the expression of p50-ATF6 was detected by western blotting to determine the concentration of D-glucose and insulin that could induce ERS; (3)ER stressed and insulin resistant cell models were established by TG, high glucose and high insulin respectively, AEBSF was pretreated as an inhibitor of the activation of ATF6, the mRNA and protein expression of PTP1B were detected by RT-PCR and western blotting respectively, the groups were divided as follows:①Vehicle group, TG group, AEBSF+TG group, AEBSF group②Vehicle group, HG group, AEBSF+HG group, AEBSF group③Control group, Ins group, AEBSF+Ins group, AEBSF group Results:(1)The expression of activated ATF6(p50-ATF6) and the mRNA expression of PTP1B were both higher in cells transiently transfected with pCI-Flag-ATF6(N)(2-366) plasmid than pCI-Blank group, but no such effect was observed on p90-ATF6 and the protein expression of PTP1B;(2)The expression of p50-ATF6 in 25mmol/L D-glucose,35mmol/L D-glucose, 5×10-7mol/L insulin and 2μmol/L TG group were higher than control group, but the was no significant difference between these groups;(3)The mRNA and protein expression of PTP1B were both increased in TG group, HG group and Ins group, but pretreatment with AEBSF decreased the increased mRNA and protein expression of PTP1B, AEBSF had no significant impact on the expression of PTP1B in normal cells.Conclusions:(1)In HL-7702 cells, overexpression of p50-ATF6 increased the mRNA expression of PTP1B, but no such effect was observed on the protein expression of PTP1B;(2)TG, high glucose and high insulin all induced ERS in HL-7702 cells;(3)The activation of ATF6 regulated the protein and mRNA expression of PTP1B in TG induced ER stressed cells and high glucose and insulin induced insulin resistant cells.Aim:APS decreased the level of blood glucose, increased the insulin sensitivity and relieved the ERS of T2DM rats. While the expression of PTP1B was regulated by the activation of ATF6 in ER stressed and insulin resistant cell models. So this study was designed to indentify whether APS inhibited the expression of PTP1B through preventing the activation of ATF6.Methods:(1)The cytotoxicity of APS on HL-7702 cells was detected by MTT; (2)cells were induced by 25mmol/L D-glucose for 24h, and then different concentration of APS(0.05mg/ml、0.1mg/ml、0.2mg/ml、0.4mg/ml、0.8mg/ml、1.6mg/ml) were treated on the cells, the expression of p50-ATF6 and PTP1B were detected by western blotting; (3)0.4mg/ml APS was used on 25mmol/L D-glucose induced cells, cells were divided into C group, HG group, APS+HG group and APS group, the intracellular location of ATF6 was detected by immunofluorescence.Results:(1)APS at 0-1.6mg/ml had little cytotoxicity on HL-7702 cells, no significant difference of cell proliferation between each group was observed;(2)APS at 0.1-0.8mg/ml decreased the increased expression of p50-ATF6 induced by 25mmol/L D-glucose, and the expression of p50-ATF6 in 0.2-0.8mg/ml APS treated group were lower than 0.1mg/ml APS group, meanwhile, the expression of PTP1B in 0.2-0.8mg/ml APS treated group were all lower than control group and 0.4mg/ml APS treated group was significantly lower than 0.2mg/ml and 0.8mg/ml treated groups; there was a significant positive correlation between the expression of p50-ATF6 and PTP1B in 0.2-0.8mg/ml APS treated cells;(3)In control group, the red fluorescence(labeled p90-ATF6 and p50-ATF6) distributed dispersedly in punctiform form in the cytoplasm, little coloring was observed in the cell nucleus; the red fluorescence distributed around the cell nucleus increased, as well as in the cell nucleus in HG group; the red fluorescence distributed around and in the cell nucleus decreased, and the red fluorescence distributed dispersedly in cytoplasm increased in APS+HG group than in HG group; no significant difference of the distribution of the red fluorescence was observed between APS group and control group, the red fluorescence mainly distributed dispersedly in the cytoplasm. Conclusions:(1)ATF6 simultaneously decreased the expression of PTP1B and inhibited the activation of ATF6 and its transloction to cell nucleus after avtivation induced by high glucose;(2)APS inhibited the overexpression of PTP1B at least partly through preventing the activation of ATF6 induced by high glucose. |
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