Molecular mechanism of ectopic lipid accumulation induced by methylglyoxal via activation of the NRF2/PI3K/AKT pathway implicates renal lipotoxicity caused by diabetes mellitus

Figures

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  Fig 1. Experimental data from the diabetic mice model of control, HFD, and HFD+MG groups (n = 8 in each group). (a) Body weight changes during the 15-week experiment (b) Plasma sugar level (c) Plasma triglyceride levels (d) Plasma cholesterol levels (e) H&E examination of kidney tissues (f) Pathological scores of kidney fatty changes (g) Pathological scores of renal tubular degeneration (h) Pathological scores of mononuclear cell infiltration (i) The immunohistochemical staining and quantification of CD36 in the control, HFD, and HFD+MG groups. green▲, lipid change; black ↑, tubular degeneration; yellow ↑, monocyte infiltration. *vs. the control group, #vs. the HFD group (#p<0.05, **p<0.01, ***p<0.001).The pathology report (no: 107–20040) was provided by National Applied Research Laboratories (Taipei, Taiwan).

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  Fig 2. Effect of MG on the cell viability, cell cycle, and apoptosis of NRK52E cells. (a) MTT assay of NRK52E cells at 24 and 48 h after MG treatment (0~800 μM). (b) Quantification of cell viability at 48 h affected by MG compared to the control (100%). NRK52E cells were seeded onto 24-well plates at a density of 2.5×104 cells/well and treated with MG (0~800 μM) for 24~48 h, after which an MTT assay was carried out. Data obtained from triplicate experiments were treated statistically and are expressed as the mean±SD (n = 3); asterisk superscripts indicate a significant difference with respect to the control (*** p<0.001). (c) Flow cytometric analysis affected by MG treatment at 48 h. (d) Quantification of the cell cycle at 48 h. The abscissa represents the propidium iodide (PI) fluorescence intensity, and the ordinate is cell counts compared to the control. (e) Apoptotic analysis of NRK52E cells by MG treatment at 48 h. (f) Quantification of the population of apoptotic cells. NRK52E cells were treated as described above with MG at 0~700 μM for 48 h. Data were statistically treated, and the results are expressed as the mean±SD (n = 3). Percentages of the cell cycle and apoptosis are…

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  Fig 3. Cell cycle- and apoptosis-related proteins affected by MG. (a) Western blotting of cyclin D, CDK4, and cleaved caspase-3. β-Actin was used as an internal control. (b) Quantified bar diagrams. NRK52E cells were treated with 500 μM MG for 6, 24, and 48 h. (c) Western blotting and quantification of p21. β-Actin was used as an internal control. Proteins were collected from cell lysates and subjected to Western blotting. Data were statistically treated, and results are expressed as the mean±SD (n = 3). Different levels of significance are expressed as * p<0.05 and ** p<0.01 compared to the control, #p<0.05 compared to 24h.

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  Fig 4. Expression of intracellular glyoxalase 1 (GLO-1) affected by MG. NRK52E cells were treated with 0~700 μM MG for 0~48 h. (a) Time-dependent stimulation of GLO-1 activity by 500 μM MG in NRK52E cells for 0~24 h. (b) GLO-1 activity in NRK52E cells affected by 300, 500, and 700 μM MG. GLO-1 activity was measured with a Glyoxalase I Activity Assay Kit (Biovision). GLO-1 activity is expressed as units/mg protein. (c) Western blotting of the GLO-1 protein after incubation with MG for 6, 24, and 48 h. (d) Quantified diagram showing the ratio of GLO-1/β-actin vs. incubation time for 0~48 h. Triplicate data were statistically treated, and results are expressed as the mean±SD (n = 3). Different levels of significance are expressed as * p<0.05 and *** p<0.001 compared to the control.

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  Fig 5. Time- and dose-dependent intracellular reactive oxidative stress responses and Nrf2 expression affected by MG. (a) Time-dependent reactive oxygen species (ROS) production per cell. NRK52E cells seeded onto six-well plates at a density of 1.5×106 cells/well, were treated with MG (500 μM) for 0.5~4 h. Harvested cells were treated with fluorescent DHE dye and subjected to a flow cytometric fluorescence analysis. (b) Dose-dependent ROS production per cell treated with MG (0~700 μM) for 30 min. Triplicate data were statistically treated, and results are expressed as the mean±SD (n = 3). ROS levels were measured with a Muse® Oxidative Stress Kit (Luminex). (c) Western blotting of Nrf2 from NRK52E cells treated with MG (500 μM) for 15, 30, and 60 min. (d) Quantified bar diagrams of data obtained from Western blotting of Nrf2 (n = 3). (e) The Nrf2 transcriptional activity in NRK52E cells treated with MG 500 μM at 30 and 60 min. Different levels of significance are expressed as *p<0.05, ** p<0.01, and *** p<0.001 compared to the control.

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  Fig 6. PI3K/Akt/AMPK pathways in NRK52E cells affected by MG. NRK52E cells, treated with MG (500 μM), were incubated for 0.5, 2, 6, 24, and 48 h. Cell lysates were collected and subjected to a Western blot analysis. (a) Western blotting of phosphorylated (p)-PI3K/PI3K, p-AKT/AKT, and an internal control protein (β-actin). (b) Quantified data of Western blot of p-PI3K/PI3K and p-AKT/AKT. (c) Western blotting of p-AMPK and AMPK. (d) Image of quantification of Western blot of p-AMPK/AMPK, with β-actin as an internal control. The image was quantified with ImagePro software. Data were statistically treated, and results are expressed as the mean±SD (n = 3). Different levels of significance are expressed as *p<0.05, **p<0.01, and ***p<0.001 compared to the control.

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  Fig 7. Intracellular lipid accumulation affected by MG. (a) Lipid droplet distribution under Nanolive 3D Cell Explorer. NRK52E cells, cultured in 35-mm μ-Dishes (from ibidi) were treated with MG (500 μM) for 24 h and subjected to Nanolive 3D Cell Explorer (Nanolive) and STEVE® software analyses. Differential diffraction illustrates intracellular lipid droplet distribution. (b) Confocal microscopy of Nile red fluorescence with DAPI stain in NRK52E cells. Cells were treated with MG (500 μM) for 48 h, fixed, stained with Nile red and DAPI, and observed with an inverted microscope (magnification, 200x). Areas were integrated with Image Pro software, and results are expressed as the mean±SD (n = 10). (c) NRK52E cells were incubated with different dosages (0, 300, 500, and 700 μM) of MG for 48 h and subjected to a flow cytometric analysis at 2000 cells per event. Abscissa: the intensity of Nile red. Ordinate: the cell count. With higher intracellular lipid accumulation, Nile red signals further shifted to the right. d) Nile red flow cytometric data were statistically analyzed, and results are expressed as the mean±SD (n = 3). (e) NRK52E cells were treated with MG (500 μM) for 48 h. Harvested…

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  Fig 8. Lipid metabolism-related proteins affected by MG. NRK52E cells, treated with MG (500 μM), were incubated for 6, 24, and 48 h. Cell lysates were collected and subjected to a Western blot analysis. (a) Western blot pattern and image quantification of SREBP1, ACC, and FAS. (b) Western blot pattern and image quantification of SREBP2 and HMGCR. (c) Western blot pattern and image quantification of PPARα and PPARγ internal control protein. (d) Western blots indicating the protein expressions of FATP2, and CD36. Internal control proteins were HDAC for nuclei and β-actin for the cytosol. Images were quantified with ImagePro software. Data were statistically analyzed, and results are expressed as the mean±SD (n = 3). Different levels of significance are expressed as * p<0.05, ** p<0.01, and *** p<0.001 compared to the control.

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  Fig 9. The expression of FAS was modulated by Glo1 inducers on NRK52E. NRK52E cells treated with Mang or RH with/without MG (500 μM) were incubated for 48 h. Cell lysates were collected and subjected to a Western blot analysis (a) and image quantification (b) for the Glo1 and FAS (**p<0.01 vs. control). Western blot analysis for the Glo1 and FAS (**p<0.01 vs. control).

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  Fig 10. Graphic summary of potential signaling pathway involved in MG-induced lipotoxicity in vitro and in vivo. (FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; HFD, high-fat diet; FATP2, fatty acid transport protein 2; HMGCR, HMG-CoA reductase).