Methylglyoxal attenuates isoproterenol-induced increase in uncoupling protein 1 expression through activation of JNK signaling pathway in beige adipocytes

Figures

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  Fig. 1. MG represses isoproterenol-stimulated Ucp1 expression in differentiated immortalized iWAT-derived pre-adipocytes. Adipocytes were treated with 1 mM MG (A), or 0.5, 1, or 2 mM MG (B) for 30 min, followed by stimulation with 1 μM isoproterenol (Iso) for 3 h. mRNA expression levels of Ucp1 in the cells were then determined by real-time PCR. Data are presented as the mean ± SEM (error bars), with n = 3–4 per group. (C) C3H10T1/2 fibroblasts were transfected with the Ucp1 promoter reporter plasmid, pUCP1-pro-Luc. After that, the cells were treated with 1 mM MG for 30 min, followed by stimulation with 1 μM isoproterenol for 6 h. Cells were lysed and luciferase assay was then performed. Data are presented as the mean ± SEM (error bars), with n = 5 per group. 20 μM BBGC (D), or 12.5, 25 or 50 mM DHA (E) were pre-treated in adipocytes for 30 min, followed by stimulation with 1 μM isoproterenol for 3 h. Then, mRNA expression levels of Ucp1 in the cells were determined by real-time PCR. Data are presented as the mean ± SEM (error bars), with n = 3–4 per group. One-way ANOVA followed by Dunnett's (A,D) or Sidak's (B,C,E) post-hoc tests were done to determine statistical significance. *, P <…

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  Fig. 2. MG does not suppress the isoproterenol-stimulated PKA lipolytic pathway. (A) Adipocytes were treated with 20 μM BBGC or 1 mM MG for 30 min before stimulation with 1 μM isoproterenol (Iso) for 30 min. Phosphorylation of HSL (p-HSL), protein levels of HSL, phosphorylation of perilipin (p-perilipin), protein levels of perilipin, phosphorylation of CREB (p-CREB), protein levels of CREB, and protein levels of β-actin were determined using anti-phospho HSL Ser660, anti-HSL, anti-phospho-PKA substrate, anti-perilipin, anti-phospho CREB Ser133, anti-CREB, and anti-β-actin antibodies respectively. (B) Phosphorylation levels of CREB were quantified by measuring the intensity of the immunoreactive bands using ImageJ. The ratio of p-CREB/CREB in the non-stimulated vehicular control group was assigned a relative value of 100. Data are presented as the mean ± SEM (error bars), with n = 5 per group. (C,D) Adipocytes were treated with 20 μM BBGC or 1 mM MG for 30 min before stimulation with 1 μM isoproterenol (Iso) for 3 h. NEFA (C) and glycerol (D) levels in the culture medium were measured and normalized to cellular protein levels. Data are presented as the mean ± SEM (error bars), with n =…

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  Fig. 3. MG represses isoproterenol-induced Ucp1 expression in a JNK-dependent manner. Adipocytes were treated with 1 mM MG for 5, 15, 30, or 60 min (A) or 20 μM BBGC for 30 min (B). Phosphorylation of p38 (p-p38), protein levels of p38, phosphorylation of JNK (p-JNK), protein levels of JNK, phosphorylation of ERK (p-ERK), and protein levels of ERK were determined using anti-phospho p38 Thr180/Tyr182, anti-p38, anti-phospho JNK Thr183/Tyr185, anti-JNK, anti-phospho ERK Thr202/Tyr204, and anti-ERK antibodies, respectively. (C,D) Adipocytes were pre-treated with 10 μM SB203580 (SB), a p38 inhibitor, or 10 μM SP600125 (SP), a JNK inhibitor, for 30 min. The cells were then treated with either 1 mM MG (C) or 20 μM BBGC (D) for an additional 30 min before stimulation with 1 μM isoproterenol (Iso) for 3 h. mRNA expression levels of Ucp1 in the cells were determined by real-time PCR. Data are presented as the mean ± SEM (error bars), with n = 3–4 per group. One-way ANOVA followed by Sidak's post-hoc test was done to determine statistical significance for (C,D). *, P < 0.05; **, P < 0.01 between the non-stimulated vehicular control group and the respective groups. #, P < 0.05; ##, P < 0.01.

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  Fig. 4. Effect of NAC on MG-induced activation of JNK signaling. (A) Adipocytes were pre-treated with 2 μM H2DCFDA with or without 10 mM NAC for 30 min, followed by treatment with either 500 μM H2O2 or 1 mM MG for 3 h. Intracellular ROS assay was then performed. Data are presented as the mean ± SEM (error bars), with n = 4 per group. (B) Adipocytes were pre-treated with 10 mM NAC, a ROS inhibitor, for 30 min before treatment with 1 mM MG for 1 h. Phosphorylation of JNK (p-JNK) and protein levels of JNK were determined as described in Fig. 3 (A,B). (C) Phosphorylation levels of JNK were quantified by measuring the intensity of the immunoreactive bands using ImageJ. The ratio of p-JNK/JNK in the non-stimulated vehicular control group was assigned a relative value of 100. Data are presented as the mean ± SEM (error bars), with n = 6 per group. (D) After pretreatment of cells with 10 mM NAC and 1 mM MG for 30 min each, cells were stimulated with 1 μM isoproterenol (Iso) for 3 h, and mRNA expression levels of Ucp1 were determined by real-time PCR. Data are presented as the mean ± SEM (error bars), with n = 3–4 per group. One-way ANOVA followed by Sidak's post-hoc test was done to determine…