Regulation of mitochondrial function by voltage dependent anion channels in ethanol metabolism and the Warburg effect

Abstract

Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Figure 1. Metabolism of ethanol

See text for details.

Figure 2. Ethanol limits suppresses respiration and accessibility to adenylate kinase in the mitochondrial intermembrane space

In A, succinate-supported respiration of untreated and ethanol-treated rat hepatocytes was measured before and after sequential addition of 8 and 80 μM digitonin to permeabilize the plasma membrane and MOM, respectively. Plasmalemmal permeabilization allowed entry of succinate and stimulation of respiration, which was less in ethanol-treated hepatocytes. After MOM permeabilization with high digitonin, respiration of ethanol-treated and untreated hepatocytes was not different. In B, untreated and ethanol-treated rat hepatocytes were exposed to 8 μM digitonin, centrifuged and resuspended to remove cytosolic enzymes. AK was measured with and without 80 μM digitonin to permeabilize MOM, as indicated. After ethanol, AK was decreased. Suppression of AK activity was restored by high digitonin, which showed that AK inhibition was due to decreased access of substrates for AK into the IMS. Adapted from [41].

Figure 3. Decreased entry of 3 kDa rhodamine-dextran into the mitochondrial intermembrane space after ethanol treatment

Hepatocytes were pretreated with vehicle (Control) or 50 mM ethanol subjected to a RhoDex entrapment protocol. Note that ethanol pretreatment decreases mitochondrial retention of red-fluorescing RhoDex. Adapted from [41].

Figure 4. Suppression of ureagenic respiration by ethanol and acetaldehyde

Respiration by cultured rat hepatocytes was measured before and after sequential addition of ureagenic substrates (NH₄Cl, ornithine, lactate) and KCN, as indicated, in the presence and absence of ethanol (left panel) or acetaldehyde (right panel). Note inhibition of respiration by ethanol and acetaldehyde. Adapted from [42].

Figure 5. Modulation of mitochondrial membrane potential by free tubulin and protein kinase

A. Pseudocolor of the fluorescence of TMRM was used to monitor changes of mitochondrial Ψ in HepG2 human hepatoma cells. Ψ decreased after microtubule depolymerization with colchicine (Col) and nocodazole (Ncz). Paclitaxel (Ptx), microtubule stabilizer, hyperpolarized mitochondria even in the presence of nocodazole. Dibutyrl cAMP (cAMP), a PKA agonist, also depolarized mitochondria, whereas H89, a PKA inhibitor, hyperpolarized mitochondria even in the presence of dibutyrl cAMP. Adapted from [52].

Figure 6. Inverse relationship between free to polymerized tubulin and mitochondrial membrane potential

Mitochondrial Ψ and free to polymerized tubulin were measured after exposure of HepG2 cells to colchicine (Col), myxothiazol (Myxo), nocodazole (Ncz), paclitaxel (Ptx) and rotenone (Rot) in various combinations. Adapted from [52].

Figure 7. Schemes of voltage dependent anion channel closure

In A, ethanol metabolism by ADH and P450 2E1 generates AcAld, which promotes VDAC closure in MOM. P450 2E1 also generates ROS to cause lipid peroxidation and formation of MDA, which also closes VDAC. Simultaneously in vivo, a proton-conducting uncoupling pathway opens in MIM. These two events together promote increased mitochondrial respiration and selective, more rapid oxidation and detoxification of AcAld and MDA by ALDH. In B, rotenone, colchicine and nocodazole increase free tubulin by depolmerizing microtubules. Tubulin binds to and inhibits VDAC, which decreases uptake of respiratory substrates and produces a relative mitochondrial depolarization. Paclitaxel stabilizes microtubules, which promotes VDAC opening and mitochondrial hyperpolarization. VDAC phosphorylation by PKA augments tubulin-dependent VDAC closure, whereas GSK3β promotes opening. High free tubulin in tumor cells causes a relative closure of VDAC, which suppresses mitochondrial metabolism and contributes to the Warburg phenomenon of aerobic glycolysis.