Fig 1.
Effect of uric acid (UA) on 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) uptake and Akt phosphorylation in H9c2 cardiomyocytes.
(A, B, D, E) Flow cytometry of cells pretreated with UA (0, 5, 10, and 15 mg/dl) or 0, 30 min and 1, 8, 16 and 24 h, then assayed for insulin-stimulated 2-NBDG uptake. (C, F) Western blot analysis of phosphorylated and total Akt levels. *P<0.05 vs. 0, 30 min, and 1 h. #P<0.05 vs. 0, 5, and 10 mg/dl. GAPDH was a normalization control.
Fig 2.
(A-C) Effect of HUA on 2-NBDG uptake in H9c2 cardiomyocytes. Cells were pretreated with HUA, then underwent basal or insulin-stimulated 2-NBDG uptake assay detected by fluorescence microscopy (A) and analyzed by flow cytometry (B). (C) *P<0.01 vs. control, #P<0.01 vs. insulin and HUA. (D-E) Effect of HUA on ROS generation in H9c2 cardiomyocytes. Cells were co-incubated with HUA and stained with DCFH-DA for fluorescence microscopy (D) and analyzed by flow cytometry (E). (F) *P<0.01 vs. control, #P<0.01 vs. HUA and N-acetyline cystein (NAC). (G-I) Effect of HUA on 2-NBDG uptake in H9c2 cardiomyocytes via oxidative stress. Cells were pretreated with HUA with or without NAC, then underwent basal or insulin-stimulated 2-NBDG uptake assay detected by fluorescence microscopy (G) and flow cytometry (H). (I) *P<0.01 vs. NAC, #P<0.01 vs. NAC+HUA. Data are mean±SD from 3 separate experiments.
Fig 3.
Effect of HUA on 2-NBDG uptake in primary cardiomyocytes via oxidative stress.
Cells were pretreated with HUA with or without NAC, then underwent basal or insulin-stimulated 2-NBDG uptake assay detected by fluorescence microscopy (A) and analyzed by flow cytometry (B) (C). *P<0.01 vs. control, #P<0.01 vs. insulin.
Fig 4.
(A-E) Effect of HUA on IR (A), phospho-IR (Tyr1361) and phospho-IGF1R (Tyr1161) (B), total and phosphorylated levels of IRS1(Ser307) (C), Akt (D) and JNK (E) in H9c2 cardiomyocytes. Western blot analysis of cells pretreated with HUA with or without NAC, then examined for basal or insulin-inhibited expression. Data are mean±SD from 3 separate experiments. (C) *P<0.01 vs. control and HUA+insulin; #P<0.05 vs. HUA. (D) *P<0.01 vs. control and HUA; #P<0.01 vs. NAC+HUA. (E) *P<0.01 vs. control; #P<0.05 vs. HUA. (F-G) Effect of SP600125 on HUA-mediated phospho-JNK, phospho-IRS1(Ser307) and phospho-Akt in H9c2 cardiomyocytes.
Fig 5.
(A-B) Effect of SP600125 on HUA inhibiting 2-NBDG uptake stimulated by insulin in H9c2 cardiomyocytes. (B) *P<0.01 vs. control; #P<0.01 vs. insulin. (C) Effect of HUA on GLUT4 expression in H9c2 cardiomyocytes. (C) *P<0.01 vs. control and HUA; #P<0.01 vs. insulin.
Fig 6.
(A-B) Glucose tolerance test (GTT) (A) and insulin tolerance test (ITT) (B) in an acute hyperuricemic mice model (with HUA level). Data are mean±SD from 3 separate experiments. *P < 0.05, **p < 0.01 vs. control. (C-E) Western blot analysis of IR (C), phospho-IRS1 (Ser307) (D) and phospho-Akt (E) level in cardiac tissues. *P<0.01 vs. control. #P<0.01 vs. control. (F) Schematic representation of HUA-mediated insulin resistance in cardiomyocytes. Increased HUA-induced oxidative stress activates phospho-IRS1 (Ser307) level, which impairs Akt (Ser 437) phosphorylation, thus increasing acute insulin resistance in cardiomyocytes with HUA treatment.