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 substrate­1 (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 high­fat diet and KKay mice fed a high­fat 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.

Involvement of substance p/neurokinin-1 receptor in the analgesic and anticancer activities of minimally toxic fraction from the traditional Chinese medicine Liu-Shen-Wan in vitro.

Liu-Shen-Wan (LSW), an ancient preparation used to treat localized infection with pain, was recently reported to possess anticancer activity. The mechanism responsible for LSW's analgesic and anticancer activity is unclear. In the present study, we obtained a LSW supernatant (LSWS) fraction from ultrasound-assisted ethanol extraction (yield 15.9%) which proved to be safer than LSW in terms of hepatotoxicity. The LSWS (1 and 10 µg/mL) exhibited a potent inhibitory effect on the bradykinin-evoked rapid release of substance P from dorsal root ganglion (DRG) cells. At concentrations of 0.1 µg/mL and higher, the LSWS resulted in a concentration-related growth inhibitory effect on HepG2, a representative cancer cell lines. The LSWS significantly down-regulated the neurokinin-1 (NK-1) receptor expression in both HepG2 and bradykinin-treated DRG cells. In addition to the NK-1 receptor-dependent growth inhibition in HepG2 cells (0.1-100 µg/mL), the LSWS induced mitochondria-mediated apoptosis at a higher concentration (1-100 µg/mL). In conclusion, we recently isolated a safer LSW fraction which maintained its analgesic and anticancer activity, and found that the substance P/NK-1 receptor system was partly responsible for these effects. Our findings will be useful for developing more effective and less toxic LSW preparations.

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

AIM: 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 AMPKalpha 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-[(3)H]-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.

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.

AIM: 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 microg/mL, 24 h) were treated with APS. The oral glucose tolerance test was measured to reflex insulin sensitivity with the calculated homeostasis model assessment (HOMA-IR) 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 (GSK3beta), 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.