Fig. 8: Lepr overexpression can rescue Lepr-KO rats’ hepatocytes from metformin-induced pyroptosis.

A To measure glucose uptake, upon isolation from HFD-fed Lepr WT rats and Lepr-KO rats, hepatocytes were subject to Lepr transfection. n = 10. At 30 min post transfection, in the presence of metformin, the hepatocytes were incubated with 100 mg/mL 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) in glucose-free medium for 1 h. The fluorescence was measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively. *p < 0.05 vs. HFD-fed Lepr-KO rat-derived hepatocytes in the presence of Lepr transfection and metformin; ^#p < 0.05 vs. HFD-fed ZDF rat-derived hepatocytes in the presence of Lepr transfection and metformin. B Enzyme-linked immunosorbent assay (ELISA). n = 12. Culture medium was collected and centrifuged to remove cell debris (the original medium in each well of a 12-well plate was 1 ml). a, b hepatocytes isolated from HFD-fed Lepr WT rats were transfected with Lepr construct (b) or control vector (a) in the absence of metformin; c-d, hepatocytes isolated from HFD-fed Lepr WT rats were transfected with Lepr construct (d) or control vector (c), followed by metformin treatment for 96 h; e, f hepatocytes isolated from HFD-fed Lepr-KO rats were transfected with Lepr construct (f) or control vector (e) in the absence of metformin; g-h, hepatocytes isolated from HFD-fed Lepr-KO rats were transfected with Lepr construct (h) or control vector (g), followed by metformin treatment for 96 h. *p < 0.05 vs. h in IL-1β detection; ^#p < 0.05 vs. h in IL-18 detection. a–h are the same as those in C–G. C Western blotting analysis for proteins (as indicated) in hepatocytes isolated from HFD-fed Lepr-knockout (Lepr-KO) rats and Lepr wild type (Lepr WT) rats at 96 h post transfection in the presence and absence of metformin. D immunostaining on IL-1β was performed at 96 h post transfection in either the presence or the absence of metformin. Scale bars, 25 μm. E Detection of telomerase activity in a–h. n = 16. *p < 0.05 vs. h. F Telomere length reflected by telomere fluorescence intensities were measured by flow-fluorescence in situ hybridization (flow-FISH). G Quantification of F. n = 16. *p < 0.05 vs. h.