Fig. 4: Isolated Lipid Droplet associated Mitochondria (LDM) from the liver exhibit reduced respiration, complex I + III and II + III coupled activities, TCA cycle capacity, ATP levels, and bioenergetics.

a–i LDM and Cytoplasmic Mitochondria (CM) from rat liver. Data are presented as mean values ± SD. Significance was calculated by two tailed un-paired Student t-test. a, b LDMs are compromised for State II and State III respiration. State II and III respiration rates are driven by succinate (a) and glutamate/malate (b) in LDM and CM isolated from rat liver. State II respiration quantifies respiration driven by proton leak (no ATP synthesis). State III quantifies respiration driven by ATP synthesis. (n = 4) ****p < 0.0001. c Quantification of the activities of complexes I, II, III, and IV of the Electron Transport Chain (ETC) in LDM and CM (n = 4). d LDM has reduced TCA flux. Quantification of citrate synthase activity by DTNB absorption assay in isolated LDM and CM (n = 4) ***p = 0.0003. e, f LDM trails CM significantly in the channeling of electrons. Super complex I + III and II + III coupled activities were performed using an equal amount of LDM and CM samples. (n = 6) **p = 0.001, ****p < 0.0001. g ATP levels diminished in LDM. Quantification of the ATP levels in isolated LDM and CM. (n = 6) *p = 0.02. h CM has a more efficient proton gradient than LDM. Confocal imaging of LDM and CM stained with TMRE, a fluorescent probe that accumulates and stains negatively charged mitochondria (scale bar, 20 μm). Lower panel shows quantification of TMRE fluorescence in LDM and CM. **p = 0.002. i LDM from rat liver has high MFN2 expression. A representative Western blot probed with MFN2 antibody is shown in the upper panel, and the lower panel is a Ponceau S stain of the Western blot to serve as a loading control. Lower panel shows quantification of the MFN2 protein expression signal from i. (n = 6) **p = 0.005.