VDAC-dependent mitochondrial metabolism and cell proliferation differ between VDAC knockout and transient inhibition of VDAC expression

Abstract

VDAC1, 2, and 3, are major controllers of mitochondrial metabolism. We previously showed that single and double VDAC knockdown decreased mitochondrial membrane potential (DJm) and that knockdown of the minor isoform VDAC3 decreased cellular ATP and NADH 48 h after transfection in HepG2 hepatocarcinoma cells. Here, we studied the effects of VDAC double knockouts (KO) on mitochondrial metabolism and proliferation. We hypothesized that compensatory mechanisms after VDAC KO induce metabolic changes different from shortterm inhibition of VDAC expression. We used wild-type (wt) and CRISPRCas9 VDAC double KO (VDAC 1-2, VDAC 1-3 and VDAC 2-3 KO) HepG2 cells. We assessed DJm, NADH and mitochondrial morphology by confocal microscopy; respiration by Resipher; triacylglycerides (TG) and cholesterol esters (CE) by thin layer chromatography; and proliferation rate by doubling time DJm, NADH, respiration, mitochondrial morphology, proliferation, and neutral lipids were almost identical in 2-3 VDAC KO cells (VDAC1 present) compared to wt. Absence of VDAC1 (VDAC 1-2/1-3 KO) was associated with changes in metabolism and proliferation. In VDAC 1-2 KO cells (VDAC3 present), DJm and NADH were similar, basal respiration lower, and cell proliferation slower. Both VDAC 1-2 and VDAC 1-3 KO (VDAC2 present) displayed higher TG and CE content than wt. VDAC 1-3 KO cells had similar DJm and respiration, higher NADH and proliferation rates than wt, and giant mitochondria with DJm and NADH 2.0 -fold higher than non-giant counterparts. VDAC1 alone sustains mitochondrial metabolism and proliferation. VDAC1 absence is associated with TG/CE accumulation, and lower rate of proliferation in cells containing only VDAC3, or higher if VDAC2 is the only isoform. These data suggest possible compensatory mechanisms triggered by the lack of specific VDAC isoforms.