| AimMammalian target of rapamycin (mTOR) is a key factor of insulin signaling pathway, which involves in regulating of cell metabolism, proliferation and growth. In the insulin resistance (IR) condition, mTOR and its downstream substrate S6 kinase1 (ribosomes protein S6 kinase 1, S6K1) are excessively activated, which has an negative feedback regulation on insulin signaling pathways. Our previous study had demonstrated that cardamonin (CAR) might play a role in ameliorating insulin resistance and smooth muscle hyperplasia of major vessels in fructose-induced rats, possibly via a mechanism that involves the modulation of insulin/mTOR signaling. In this study, the activity of the key enzymes of glucose metabolism and the key molecules of insulin signaling pathway are measured to investigate the mechanism of CAR on glucose metabolism.Methods1 Cell cultureVascular smooth muscle cells were primary cultured by the planted method. Passage cells are cultured with high glucose and high insulin medium (2.5×10-2 mol·L-1 glucose and 100 U·L-1 insulin) to mimics insulin resistance.2 ProtocolControl group and solvent group (0.1 % Dimethyl Sulfoxide , DMSO) were set in cells cultured with normal medium. Control group, solvent group, pioglitazone group (10-5 mol·L-1), RAP group (10-7 mol·L-1) and CAR group (10-6, 10-5 mol·L-1) were set in cells in the mimic IR medium. Control group, solvent control, RAP group (10-7 mol·L-1) and CAR group (10-6, 10-5 mol·L-1) were set in cells treated with PA. 3 Measurements and methodology3.1 The glucose concentration in the medium was measured with hexokinase method.3.2 Cell proliferation was measured with MTT method (λ=490).3.3 Hexokinase activity was assayed by glucose-6-phosphate dehydrogenase coupling colorimetric.3.4 Glycogen concentration was determined by sulfuric acid-anthracene ketone method.3.5 Examination of protein expression of IRS-1, p-IRS-1, Akt, p-Akt, mTOR, p-mTOR, S6K1, p-S6K1, Glut4, GSK-3β, p-GSK–3βand hexokinase were performed by Western blotting method.Results1 Cultured with high glucose and high insulin for 72 h, glucose consumption, hexokinase activity and glycogen concentration of vascular smooth muscle cells (VSMCs)were significantly decreased (P<0.01);2 Treated with CAR for 48 h, the glucose consumption,hexokinase activity and glycogen concentration of VSMCs in IR statue were increased(P<0.01);3 The expression of hexokinase was increased but the expression and the activity of glycogen synthase kinase 3βwere inhibited by CAR in the IR VSMCs. However, the expression of glucose transporter protein 4 was not affected by CAR;4 The expression and activity of insulin receptor substrate 1 and the phosphorylation of protein kinase B (Akt) were increased by CAR;5 The phosphorylation of mTOR and S6K1 were significantly inhibited by CAR.Conclusions1 The glucose consumption and improve glucose metabolisms were enhanced by CAR through regulating the activity of enzyme of glucose metabolism;2 The negative feedback regulation of insulin signaling pathways was eliminated by CAR through inhibiting the activity of mTOR and S6K1.
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