Prolonged hyperglycemia related to diabetes and its complications leads to multiple cellular disorders, the central one being the dysfunction of mitochondria. Voltage-dependent anion channels (VDAC) of the outer mitochondrial membrane control the metabolic, ionic, and energy cross-talk between mitochondria and the rest of the cell and serve as the master regulators of mitochondrial functions. Here, we have investigated the effect of pharmacological suppression of VDAC1 by the newly developed inhibitor of its oligomerization, VBIT-4, in the primary culture of mouse lung endotheliocytes and downregulated expression of VDAC1 in human skin fibroblasts on the progression of mitochondrial dysfunction upon hyperglycemic stress. The cells were grown in high-glucose media (30 mM) for 36 h. In response to hyperglycemia, the mRNA level of VDAC1 increased in endotheliocytes and decreased in human skin fibroblasts. Hyperglycemia induced overproduction of mitochondrial ROS, an increase in the susceptibility of the organelles to mitochondrial permeability transition (MPT) pore opening and a drop in mitochondrial membrane potential, which was accompanied by a decrease in cell viability in both cultures. Treatment of endotheliocytes with 5 µM VBIT-4 abolished the hyperglycemia-induced increase in susceptibility to spontaneous opening of the MPT pore and ROS generation in mitochondria. Silencing of VDAC1 expression in human skin fibroblasts exposed to high glucose led to a less pronounced manifestation of all the signs of damage to mitochondria. Our data identify a mitochondria-related response to pharmacological and genetic suppression of VDAC activity in vascular cells in hyperglycemia and suggest the potential therapeutic value of targeting these channels for the treatment of diabetic vasculopathies.

The present study evaluates the cytotoxicity of a previously synthesized conjugate of betulinic acid (BA) with the penetrating cation F16 on breast adenocarcinoma (MCF-7) and human fibroblast (HF) cell lines, and also shows the mechanism underlying its membranotropic action. It was confirmed that the conjugate exhibits higher cytotoxicity compared to native BA at low doses also blocking the proliferation of both cell lines and causing cell cycle arrest in the G₀/G₁ phase. We show that the conjugate indeed has a high potential for accumulation in mitochondria, being visualized in these organelles, which is most pronounced in cancer cells. The effect of the conjugate was observed to be accompanied by ROS hyperproduction in both cancerous and healthy cells, despite the lower base level of ROS in the latter. Along with this, using artificial liposomes, we determined that the conjugate is able to influence the phase state of lipid membranes, make them more fluid, and induce nonspecific permeabilization contributing to the overall cytotoxicity of the tested agent. We conclude that the studied BA–F16 conjugate does not have significant selective cytotoxicity, at least against the studied breast cancer cell line MCF-7.

The present work shows the cytotoxic effects of novel conjugates of ursolic, oleanolic, maslinic, and corosolic acids with the penetrating cation F16 on cancer cells (lung adenocarcinoma A549 and H1299, breast cancer cell lines MCF-7 and BT474) and non-tumor human fibroblasts. It has been established that the conjugates have a significantly enhanced toxicity against tumor-derived cells compared to native acids and also demonstrate selectivity to some cancer cells. The toxic effect of the conjugates is shown to be due to ROS hyperproduction in cells, induced by the effect on mitochondria. The conjugates caused dysfunction of isolated rat liver mitochondria and, in particular, a decrease in the efficiency of oxidative phosphorylation, a decrease in the membrane potential, and also an overproduction of ROS by organelles. The paper discusses how the membranotropic- and mitochondria-targeted effects of the conjugates may be related to their toxic effects.

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 μM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload.

Background: Amyotrophic lateral sclerosis (ALS) is a progressive multisystem disease characterized by limb and trunk muscle weakness that is attributed, in part, to abnormalities in mitochondrial ultrastructure and impaired mitochondrial functions. This study investigated the time course of structural and functional rearrangements in skeletal muscle mitochondria in combination with motor impairments in Tg (copper-zinc superoxide dismutase enzyme (SOD1) G93A) dl1/GurJ (referred to as SOD1-G93A/low) male mice, a familial ALS model, as compared with non-transgenic littermates. Methods: The neurological status and motor functions were assessed weekly using the paw grip endurance method and the grid suspension test with two-limb and four-limb suspension tasks. Transmission electron microscopy followed by quantitative analysis was performed to study ultrastructural alterations in the quadriceps femoris. Functional analysis of skeletal muscle mitochondria was performed using high-resolution Oxygraph-2k (O2K) respirometry and methods for assessing the calcium retention capacity index and the content of lipid peroxidation products in freshly isolated preparations. Results: Based on the behavioral phenotyping data, specific age groups were identified: postnatal day 56 (P56) (n = 10–11), 84 (P84) (n = 10–11), and 156 (P154) (n = 10–12), representing the pre-symptomatic, early-symptomatic and late-symptomatic stages of ALS progression in SOD1-G93A/low mice, respectively. Electron microscopy showed mosaic destructive changes in subsarcolemmal mitochondria in fibers of the quadriceps femoris from 84-day-old SOD1-G93A/low mice. Morphometric analysis revealed an elevation in the mean size of the mitochondria in SOD1-G93A mice at P84 and P154. In addition, the P154 transgenic group demonstrated a decrease in sarcomere width and the number of mitochondria per unit area. At the symptomatic stage, SOD1-G93A mice exhibited a decreased respiratory control ratio, ADP-stimulated, and uncoupled respiration rates of mitochondria isolated from the quadriceps femoris muscle, as measured by high-resolution respirometry. In parallel, the mitochondria showed lower calcium retention capacity and increased levels of lipid peroxidation products compared with the control. Conclusions: Taken together, these results indicate stage-dependent changes in skeletal muscle mitochondrial ultrastructure and functions associated with defective oxidative phosphorylation, impaired calcium homeostasis, and oxidative damage in the SOD1-G93A/low mouse model, which appears to be a promising direction for the development of combination therapies for ALS.

This paper demonstrates the membranotropic effect of modified levopimaric acid diene adducts on liver mitochondria and lecithin liposomes. We found that the derivatives dose-dependently reduced the efficiency of oxidative phosphorylation of mitochondria due to inhibition of the activity of complexes III and IV of the respiratory chain and protonophore action. This was accompanied by a decrease in the membrane potential in the case of organelle energization both by glutamate/malate (complex I substrates) and succinate (complex II substrate). Compounds 1 and 2 reduced the generation of H₂O₂ by mitochondria, while compound 3 exhibited a pronounced antioxidant effect on glutamate/malate-driven respiration and, on the other hand, caused ROS overproduction when organelles are energized with succinate. All tested compounds exhibited surface-active properties, reducing the fluidity of mitochondrial membranes and contributing to nonspecific permeabilization of the lipid bilayer of mitochondrial membranes and swelling of the organelles. Modified levopimaric acid diene adducts also induced nonspecific permeabilization of unilamellar lecithin liposomes, which confirmed their membranotropic properties. We discuss the mechanisms of action of the tested compounds on the mitochondrial OXPHOS system and the state of the lipid bilayer of membranes, as well as the prospects for the use of new modified levopimaric acid diene adducts in medicine.