Journal: The Journal of Biological Chemistry

Article Title: 2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes

doi: 10.1074/jbc.M116.767384

Figure Lengend Snippet: Characterization of TNFα-induced IR. A, relative mRNA levels of proinflammatory markers. *, p < 0.01 by unpaired Student's t test. B, representative Western-blotting analysis of the insulin (Ins) signaling pathway following insulin stimulation. p, phospho; t, total. C, relative mRNA level of Glut4. *, p < 0.01 by unpaired Student's t test. D, insulin-stimulated GLUT4-myc translocation. *, p = 0.047 by two-way ANOVA with Bonferroni's multiple comparisons post hoc test. E, insulin-stimulated glucose uptake. *, p = 0.041 by two-way ANOVA with Bonferroni's multiple comparisons post hoc test. ACTB, β-actin. F, representative periodic acid-Schiff staining for intracellular glycogen content. Scale bar = 100 μm, ×40 magnification. G, glucose oxidase assay for quantitation of intracellular glycogen content. *, p < 0.01 by unpaired Student's t test. Data are expressed as mean ± S.D. n = 4–5 independent experiments in HL-1 cardiomyocytes.

Article Snippet: When the cells reached a 30–50% confluent state (± 4 days of culture), cardiac differentiation was induced by replacement of Essential 8 medium with cardiomyocyte differentiation medium (PSC cardiomyocyte differentiation kit (Gibco) containing differentiation medium A, differentiation medium B, and maintenance medium).

Techniques: Western Blot, Translocation Assay, Staining, Glucose Oxidase Assay, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: 2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes

doi: 10.1074/jbc.M116.767384

Figure Lengend Snippet: 2-AG activates the AMPK signaling pathway in cardiomyocytes. A, phospho (p-) and total (t-) ACC and AMPK protein levels upon 2AG treatment in HL-1 and primary cardiomyocytes. veh, vehicle. B, phospho and total ACC and AMPK protein levels upon 2-AG treatment for 1 h, preceded by treatment with AM251 or AM630 for 1 h in HL-1 cardiomyocytes. C, relative mRNA levels of Cb1r and Cb2r in cardiac tissues obtained from db/+ (n = 4) and db/db (n = 6) mice. *, p < 0.01 by unpaired Student's t test. D, relative mRNA levels of Cb1r and Cb2r in HL-1 cardiomyocytes upon 2-AG treatment for 1 h, preceded by treatment with AM251 or AM630 for 1 h in HL-1 cardiomyocytes. *, p < 0.01 by one-way ANOVA with Tukey's multiple comparisons post hoc test. Ctl, control. E, phospho and total AMPK protein levels upon 2-AG treatment for 1 h, preceded by treatment with KN, STO, or OZ for 1 h in LKB1-deficient HeLa cells. F, phospho and total AMPK protein levels upon 2-AG treatment for 1 h, preceded by treatment with CC, KN, STO, or OZ for 1 h in primary rat cardiomyocytes. G, glucose uptake in primary rat cardiomyocytes upon 2-AG treatment for 1 h, preceded by treatment with CC, KN, STO, or OZ for 1 h. ACTB, β-actin. *, p < 0.01 by one-way ANOVA with Tukey's multiple comparisons post hoc test. Data are expressed as mean ± S.D. n = 4–5 independent experiments.

Article Snippet: When the cells reached a 30–50% confluent state (± 4 days of culture), cardiac differentiation was induced by replacement of Essential 8 medium with cardiomyocyte differentiation medium (PSC cardiomyocyte differentiation kit (Gibco) containing differentiation medium A, differentiation medium B, and maintenance medium).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: 2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes

doi: 10.1074/jbc.M116.767384

Figure Lengend Snippet: 2-AG reverses inflammatory stress-induced IR. A, representative Western-blotting analysis of the insulin (Ins) signaling pathway following insulin stimulation. p, phospho; t, total. B, insulin-stimulated GLUT4-myc translocation. *, p < 0.01; #, p = 0.043; two-way ANOVA with Bonferroni's multiple comparisons post hoc test. Ctl, control. C, insulin-stimulated glucose uptake. *, p < 0.01 by two-way ANOVA with Bonferroni's multiple comparisons post hoc test. ACTB, β-actin. D, relative mRNA levels of the proinflammatory markers Glut4 and Ppargc1a. *, p < 0.01 by one-way ANOVA with Tukey's multiple comparisons post hoc test. E, glucose oxidase assay for quantitative determination of intracellular glycogen content in cardiomyocytes subjected to 1-h pretreatment with STO or CC, followed by 16 h of TNFα and/or 2-AG treatment as indicated. *, p < 0.01 by one-way ANOVA with Tukey's multiple comparisons post hoc test. Data are expressed as mean ± S.D. n = 4–5 independent experiments in primary rat cardiomyocytes.

Article Snippet: When the cells reached a 30–50% confluent state (± 4 days of culture), cardiac differentiation was induced by replacement of Essential 8 medium with cardiomyocyte differentiation medium (PSC cardiomyocyte differentiation kit (Gibco) containing differentiation medium A, differentiation medium B, and maintenance medium).

Techniques: Western Blot, Translocation Assay, Glucose Oxidase Assay

Journal: The Journal of Biological Chemistry

Article Title: 2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes

doi: 10.1074/jbc.M116.767384

Figure Lengend Snippet: Knockdown of AMPK attenuates the ameliorative effect of 2-AG on IR. HL-1 cardiomyocytes were infected with shRNA scramble (shScr) or AMPKβ2 (shAMPKβ2) and selected for puromycin resistance, followed by treatments as indicated. A, representative Western-blotting analysis of the insulin (Ins) signaling pathway following insulin stimulation. p, phospho; t, total. B, relative mRNA levels of the proinflammatory markers Glut4 and Ppargc1a. *, p = 0.03, 0.05, 0.041, 0.022, and 0.026, respectively, by one-way ANOVA with Tukey's multiple comparisons post hoc test. C, insulin-stimulated GLUT4-myc translocation. *, p = 0.044; #, p < 0.01; two-way ANOVA with Bonferroni's multiple comparisons post hoc test. D, insulin-stimulated glucose uptake. ACTB, β-actin. *, p = 0.047; #, p < 0.01; two-way ANOVA with Bonferroni's multiple comparisons post hoc test. Data are expressed as mean ± S.D. n = 5 independent experiments.

Article Snippet: When the cells reached a 30–50% confluent state (± 4 days of culture), cardiac differentiation was induced by replacement of Essential 8 medium with cardiomyocyte differentiation medium (PSC cardiomyocyte differentiation kit (Gibco) containing differentiation medium A, differentiation medium B, and maintenance medium).

Techniques: Infection, shRNA, Western Blot, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: 2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes

doi: 10.1074/jbc.M116.767384

Figure Lengend Snippet: The ameliorative effect of 2-AG on inflammatory stress-induced insulin resistance in hESC-derived cardiomyocytes. A, relative mRNA level of MYH6 and TNNT2. *, p < 0.01 by unpaired Student's t test. B, relative mRNA level of proinflammatory markers and PGC1α. *, p = 0.037, 0.041, 0.044, and 0.041, respectively, by one-way ANOVA with Tukey's multiple comparisons post hoc test. Ctl, control. C, representative Western-blotting analysis of the insulin (Ins) signaling pathway following insulin stimulation. p, phospho; t, total; A, AM251; S, STO-609; C, compound C. D, insulin-stimulated glucose uptake. ACTB, β-actin. *, p = 0.03 and 0.044, respectively, by one-way ANOVA with Tukey's multiple comparisons post hoc test. Data are expressed as mean ± S.D. n = 5 independent experiments.

Article Snippet: When the cells reached a 30–50% confluent state (± 4 days of culture), cardiac differentiation was induced by replacement of Essential 8 medium with cardiomyocyte differentiation medium (PSC cardiomyocyte differentiation kit (Gibco) containing differentiation medium A, differentiation medium B, and maintenance medium).

Techniques: Derivative Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: 2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes

doi: 10.1074/jbc.M116.767384

Figure Lengend Snippet: Proposed model of 2-AG action in stressed cardiomyocytes. Inflammation (induced by TNFα) and lipid oversupply (FFA) perturbs insulin sensitivity and restricts glucose uptake in cardiomyocytes in the long term, leading to DCM. The endocannabinoid 2-AG, via CB1R and CaMKKβ, activates the AMPK signaling pathway to inhibit inflammation, restore insulin sensitivity, and facilitate glucose uptake in cardiomyocytes, implying that 2-AG treatment can be a viable therapeutic approach to restore cardiometabolic homeostasis via energy balance in IR.

Article Snippet: When the cells reached a 30–50% confluent state (± 4 days of culture), cardiac differentiation was induced by replacement of Essential 8 medium with cardiomyocyte differentiation medium (PSC cardiomyocyte differentiation kit (Gibco) containing differentiation medium A, differentiation medium B, and maintenance medium).

Techniques: