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. 2023 Sep 26;48(2):215–230. doi: 10.4093/dmj.2022.0332

Fig. 3.

Fig. 3.

Extracellular vimentin promotes glucose uptake and free fatty acid (FFA) uptake via changes in expression of glucose transporters and fatty acid transporter. (A) 3T3-L1-derived adipocytes were treated with recombinant vimentin (20 µg/mL for 24 hours), insulin (1 µM for 30 minutes) or both vimentin (20 µg/mL for 24 hours) and insulin (1 µM for 30 minutes). 2-Deoxyglucose (2-DG) uptake was measured (n=4). (B) 3T3-L1-derived adipocytes were treated with or without recombinant vimentin (20 µg/mL) for 24 hours and FFA uptake was measured using fluorometric fatty acid dye-loading solution (TF2-C12 Fatty Acid) uptake assay (n=3). (C) Western blot analyses for CD36, glucose transporter type 4 (GLUT4), and GLUT1 were performed using fractionated lysates (plasma membrane and cytosol) of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using either caveolin-1 (BD Biosciences) as a plasma membrane marker, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytosol marker (n=3). (D) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for CD36, GLUT1, and GLUT4 were performed using RNA from 3T3-L1-derived adipocytes cultured with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (n=5, n=6, n=4). (E) Western blot analyses for GLUT1 and GLUT4 using total lysates of 3T-3L1-derived adipocytes treated with or with recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using GAPDH, as an internal control (n=3). (F) Western blot analyses for GLUT1 and GLUT4 were performed using total lysates of 3T3-L1-derived adipocytes with or with recombinant vimentin (20 µg/mL) for 36 hours. Band quantification was performed using α-tubulin (AbFrontier), an internal control (n=3). (G) qRT-PCR analysis for hypoxia-inducible factor 1α (Hif-1α) was performed using RNA from 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (n=4). RFU, relative fluorescence unit. aP<0.05, bP<0.01, cP<0.001.