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. 2021 Feb 24;9(1):e001905. doi: 10.1136/bmjdrc-2020-001905

Figure 2.

Figure 2

Chronic glucolipotoxicity impairs glucose and free fatty acid metabolism and KATP and Ca channels NCI-H716 cells were cultured under 5 or 25 mM glucose with or without 500 µM palmitate for up to 72 hours. (A) MTT assay for measuring cellular toxicity at various time points. (B) Protein levels of SGLT1 and GLUT2 were measured by western blotting. (C) Glucose uptake was measured using 2-NBDG, a non-metabolized fluorescent analog. (D) Cellular NADPH levels were measured. (E) Protein levels of CD36 and PPARα were measured by western blotting. (F) Cellular triglyceride levels and (G) ATP levels were estimated. (H) Protein levels of SUR1 and Kir6.2 were measured by western blotting. (I) cAMP was measured. (J) Protein levels of pCAMK2, EPAC, and PKA were measured by western blotting. 5 mM G=5 mM glucose, 5 mM GP=5 mM glucose+500 µM palmitate, 25 mM G=25 mM glucose, 25 mM GP=25 mM glucose+500 µM palmitate. Data are presented as the mean±SD (n=5). Values with different superscript letters are significantly different at p<0.05, compared with the control. EPAC, exchange proteins directly activated by cAMP; GLUT2, glucose transporter 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NADPH, nicotinamide-adenine dinucleotide phosphate; NBDG, 6-deoxy-6[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose; NS, not significant; PKA, protein kinase A; SGLT1, sodium-glucose cotransporter 1; SUR1, sulfonylurea receptor 1.