Jingping Ouyang

Astragalus polysaccharide improves insulin sensitivity in KKAy mice: Regulation of PKB/GLUT4 signaling in skeletal muscle

ETHNOPHARMACOLOGICAL RELEVANCE Astragalus polysaccharide (APS) is an important bioactive component of Astragalus membranaceus Bunge (Leguminosae) that has been used in traditional Chinese medicine for treating diabetes. AIM OF THE STUDY To study the mechanisms by which APS ameliorates diabetes, we examined whether treatment with APS improves insulin sensitivity in insulin-resistant mice and whether this is associated with an improvement of dysregulated protein kinase B and glucose transporter 4 expressions in skeletal muscle. METHODS APS (700 mg kg(-1)day(-1)) or vehicle was administered to 12-week-old diabetic KKAy and nondiabetic C57BL/6J mice for 8 weeks. Changes in body weight, blood glucose level, insulin resistance index, and oral glucose tolerance were routinely evaluated. The expressions of protein kinase B and glucose transporter 4 in skeletal muscle tissues were determined with Western blot. RESULTS KKAy mice developed persistent hyperglycemia, impaired glucose tolerance and insulin resistance. Insulin-stimulated protein kinase B phosphorylation and glucose transporter 4 translocation were significantly decreased in KKAy compared to age-matched C57BL/6J mice. APS treatment ameliorated hyperglycemia and insulin resistance. Although the content of protein kinase B and glucose transporter 4 in KKAy skeletal muscle were not affected by APS, insulin-induced protein kinase B Ser-473 phosphorylation and glucose transporter 4 translocation in skeletal muscle were partially restored by APS treatment. In contrast, APS did not have any effect on C57BL/6J mice. CONCLUSIONS These results indicate that APS can regulate part of the insulin signaling in insulin-resistant skeletal muscle, and that APS could be a potential insulin sensitizer for the treatment of type 2 diabetes.

Astragalus polysaccharides decreased the expression of PTP1B through relieving ER stress induced activation of ATF6 in a rat model of type 2 diabetes

Protein tyrosine phosphatase 1B (PTP1B) was considered as a potential therapeutic target of type 2 diabetes (T2DM) because of its negative regulation of insulin signaling. It located on the cytosolic surface of endoplasmic reticulum (ER) and played an essential role in the ER stress signaling. Activating transcription factor 6 (ATF6) was an ER stress regulated transmembrane transcription factor that activated the transcription of ER molecular chaperones. We hypothesized that the expression of PTP1B may be regulated by ATF6 when ER stress happened. Our previous studies showed that Astragalus polysaccharide (APS) increased the insulin sensitivity through decreasing the overexpression of PTP1B in T2DM animal models. In this study, we intended to investigate the possible mechanisms involved in this effect. A rat model of T2DM was established using high fat diet associated with intraperitoneal injection of 25 mg/kg streptozocin; 25 mmol/l D-glucose and 5x10(-7) mol/l insulin were used as in vitro investigations to mimic T2DM-like environment. 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and pCI-Flag-ATF6(N)(2-366) plasmid were treated separately on human hepatocyte line HL-7702 to observe the effect of ATF6 on the expression of PTP1B. The results suggested that APS not only restored the glucose homeostasis but also reduced the ER stress in this rat model of T2DM; ATF6 was involved in mediating the expression of PTP1B when ER stress happened; APS decreased the expression of PTP1B at least partly through inhibiting the activation of ATF6.

Hypoglycemic effect of polysaccharide enriched extract of Astragalus membranaceus in diet induced insulin resistant C57BL/6J mice and its potential mechanism

Our previous studies found that Astragalus polysaccharide (APS) exerts insulin-sensitizing and hypoglycemic activities in type 2 diabetic (T2DM) rats. The present study was designed to further confirm the hypoglycemic effect of APS and to investigate its possible mechanism underlying the improvement of insulin resistance in vivo and in vitro. Diet-induced insulin resistant C57BL/6J mice treated with or without APS (orally, 700 mg/kg/d) for 8 weeks were analyzed and compared. Simultaneously, an insulin resistant C(2)C(12) cell model and an ER stressed HepG2 cell model were established and incubated with or without APS (200 microg/ml) for 24h respectively. Systematic insulin sensitivity was measured with an insulin-tolerance test (ITT) and an homeostasis model assessment (HOMA IR) index. Metabolic stress variation was analyzed for biochemical parameters and pathological variations. The expression and activity of protein tyrosine phosphatase 1B (PTP1B), which plays a very important role in insulin signaling and in the ER stress response, was measured by immunoprecipitation and Western blot. The ER stress response was analyzed through XBP1 transcription and splicing by real-time PCR. APS could alleviate insulin resistance and ER stress induced by high glucose in vivo and in vitro, respectively. The hyperglycemia, hypolipemia, and hyperinsulinemia status were controlled with APS therapy. Insulin action in the liver of insulin resistant mice was restored significantly with APS administration. APS enhanced adaptive capacity of the ER and promoted insulin signaling by the inhibition of the expression and activity of PTP1B. Furthermore, the anti-obesity effect and hypolipidemia effects of APS were probably due partly to decreasing the leptin resistance of mice, which would positively couple with the normalization of plasma insulin levels. We have shown that APS has beneficial effects on insulin resistance and hyperglycemia. The mechanism is related to the alleviation of ER stress and insulin resistance under hyperglycemia conditions.

Astragalus polysaccharide reduces hepatic endoplasmic reticulum stress and restores glucose homeostasis in a diabetic KKAy mouse model

AbstractAim:To examine the potential effects of Astragalus polysaccharide (APS) on hepatic endoplasmic reticulum (ER) stress in vivo and in vitro and its link with hypoglycemia activity, thus establishing the mechanism underlying the hypoglycemic action of APS.Methods:The obese and type 2 diabetic KKAy mouse model, which is the yellow offspring of the KK mice expressed Ay gene (700 mg.kg−1 .d−1, 8 weeks) and a high glucose-induced HepG2 cell model (200 μg/mL, 24 h) were treated with APS. The oral glucose tolerance test was measured to reflex insulin sensitivity with the calculated homeostasis model assessment (HOMAIR) index. XBP1 (XhoI site-binding protein 1) transcription and splicing, an indicator of ER stress, was analyzed by RT-PCR and real-time PCR. The expression and activation of glycogen synthase kinase 3 beta (GSK3β), an insulin signaling protein, was measured by Western blotting.Results:APS can alleviate ER stress in cultured cells in vivo. The hyperglycemia status, systemic insulin sensitivity, fatty liver disease, and insulin action in the liver of diabetic mice were partly normalized or improved in response to APS administration.Conclusion:Our results indicate that APS enables insulin-sensitizing and hypoglycemic activity at least in part by enhancing the adaptive capacity of the ER, which can further promote insulin signal transduction. Thus, APS has promising application in the treatment of type 2 diabetes.

Astragalus polysaccharides alleviates glucose toxicity and restores glucose homeostasis in diabetic states via activation of AMPK

AbstractAim:To establish the mechanism underlying the improvement of glucose toxicity by Astragalus polysaccharide (APS), which occurred via an AMP activated protein kinase (AMPK)-dependent pathway.Methods:In vivo and in vitro effects of APS on glucose homeostasis were examined in a type 2 diabetes mellitus (T2DM) rat model. The T2DM rat model was duplicated by a high-fat diet (58% fat, 25.6% carbohydrate, and 16.4% protein) and a small dose of streptozotocin (STZ, 25 mg/kg, ip). After APS therapy (700 mg·kg−1·d−1, ig) for 8 weeks, blood glucose, glycosylated hemoglobin, and serum insulin were measured. Insulin sensitivity was evaluated by the comprehensive analysis of oral glucose tolerance tests (OGTT) and HOMA IR index. Hepatic glycogen was observed by the PAS staining method. The expression and activity of skeletal muscle AMPKα and acetyl-CoA carboxylase (ACC), and the phosphorylation of hepatic glycogen synthase (GS), the glycogen synthase (GS),were measured by Western blotting. Glucose uptake was measured with the 2-deoxy-[3H]-D-glucose method in C2C12 cells.Results:The hyperglycemia status, insulin sensitivity, glucose uptake, and activation level of AMPK in diabetic rats were improved in response to APS administration. APS could also alleviate glucose toxicity in cultured mouse cells by the activation of AMPK.Conclusion:APS can alleviate glucose toxicity by increasing liver glycogen synthesis and skeletal muscle glucose translocation in the T2DM rat model, via activation of AMPK.

Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation.

Aim:To establish the mechanism responsible for the stimulation of glucose uptake by Astragalus polysaccharide (APS), extracted from Astragalus membranaceus Bunge, in L6 myotubes in vitro.Methods:APS-stimulated glucose uptake in L6 myotubes was measured using the 2-deoxy-[3H]-D-glucose method. The adenine nucleotide contents in the cells were measured by HPLC. The phosphorylation of AMP-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) was examined using Western blot analysis. The cells transfected with 4P mutant AS160 (AS160-4P) were constructed using gene transfer approach.Results:Treatment of L6 myotubes with APS (100−1600 μg/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 μg/mL) for 36 h. The APS-stimulated glucose uptake was significantly attenuated by pretreatment with Compound C, a selective AMPK inhibitor or in the cells overexpressing AS160-4P. Treatment of L6 myotubes with APS strongly promoted the activation of AMPK. We further demonstrated that either Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) or liver kinase B1 (LKB1) mediated APS-induced activation of AMPK in L6 myotubes, and the increased cellular AMP: ATP ratio was also involved. Treatment of L6 myotubes with APS robustly enhanced the phosphorylation of AS160, which was significantly attenuated by pretreatment with Compound C.Conclusion:Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.

Promotion of PDGF-induced endothelial cell migration by phosphorylated VASP depends on PKA anchoring via AKAP

Vasodilator-stimulated phosphoprotein (VASP), an important substrate of PKA, plays a critical role in remodeling of actin cytoskeleton and actin-based cell motility. However, how PKA accurately transfers extracellular signals to VASP and then how phosphorylation of VASP regulates endothelial cell migration have not been clearly defined. Protein kinase A anchoring proteins (AKAPs) are considered to regulate intracellular-specific signal targeting of PKA via AKAP-mediated PKA anchoring. Thus, our study investigated the relationship among AKAP anchoring of PKA, PKA activity, and VASP phosphorylation, which is to clarify the exact role of VASP and its upstream regulatory mechanism in PKA-dependent migration. Our results show that chemotactic factor PDGF activated PKA, increased phosphorylation of VASP at Ser157, and enhanced ECV304 endothelial cell migration. However, phosphorylation site-directed mutation of VASP at Ser157 attenuated the chemotactic effect of PDGF on endothelial cells, suggesting phosphorylation of VASP at Ser157 promotes PKA-mediated endothelial cell migration. Furthermore, disrupting PKA anchoring to AKAP or PKA activity significantly attenuated the PKA activity, VASP phosphorylation, and subsequent cell migration. Meanwhile, disrupting PKA anchoring to AKAP abolished PDGF-induced lamellipodia formation and special VASP accumulation at leading edge of lamellipodia. These results indicate that PKA activation and PKA-mediated substrate responses in VASP phosphorylation and localization depend on PKA anchoring via AKAP in PDGF-induced endothelial cell migration. In conclusion, AKAP anchoring of PKA is an essential upstream event in regulation of PKA-mediated VASP phosphorylation and subsequent endothelial cell migration, which contributes to explore new methods for controlling endothelial cell migration related diseases and angiogenesis.

The role of quantitative changes in the epxression of insulin receptor substrate-1 and nuclear ubiquitin in abnormal glycometabolism in the livers of KKay mice and the relative therapeutic mechanisms of Astragalus polysaccharide.

Ubiquitin and the ubiquitination pathway are important regulators of insulin signaling. The insulin receptor substrate‑1 (IRS-1), an ubiquitin-interacting adaptor protein, serves as the key docking protein in insulin signaling. The effects of this dynamic interaction and the changes in ubiquitin expression on hepatic insulin signaling, as well as the relative therapeutic effects of Astragalus polysaccharide (APS) have not yet been elucidated. In this study, we aimed to investigate the abnormal changes which occur in the levels of IRS-1 and ubiquitin in the livers of mice (mice with insulin resistance and diabetes), and to elucidate the possible mechanisms responsible for these changes. A control group (CG), an insulin resistance group (IG) and a diabetes group (DG) were respectively composed of 12-week-old C57BL/6J mice fed a normal diet, C57BL/6J mice fed a high‑fat diet and KKay mice fed a high‑fat diet, and treatment groups were composed of corresponding groups treated with APS (CG + A, IG + A, DG + A). All the mice were age-matched and grouped at random. After eight weeks, the mouse models were successfully established and the related physiological or biochemical indexes were detected using corresponding methods. Ubiquitin expression in the liver was detected by immunohistochemisty, and western blot analysis was used to detect the expression of IRS-1 and ubiquitin. The results revealed that the expression of IRS-1 in the DG was significantly lower compared to that in the CG and IG; however, the nuclear expression of ubiquitin and the ubiquitination levels of IRS-1, including body weight and blood glucose and triglyceride levels in the DG were significantly higher compared to those in the CG or IG (P<0.05). There was a significant improvement in the ubiquitination levels in DG + A, including the blood glucose and triglyceride levels compared with the DG (P<0.05). From the stage of insulin resistance to the stage of diabetes, the reduced expression of IRS-1 and its enhanced ubiquitination levels combined with the overexpression of nuclear ubiquitin contributed to the abnormal glycometabolism and the disruption of insulin signaling. APS showed beneficial effects, such as lowering body weight, as well as blood glucose and triglyceride levels, and these effects correlated with the downregulation of the ubiquitination levels of IRS-1 and the nuclear expression of ubiquitin.