Methylmalonate impairs mitochondrial respiration supported by NADH-linked substrates: involvement of mitochondrial glutamate metabolism.

The neurodegeneration that occurs in methylmalonic acidemia is proposed to be associated with impairment of mitochondrial oxidative metabolism resulting from methylmalonate (MMA) accumulation. The present study evaluated the effects of MMA on oxygen consumption by isolated rat brain mitochondria in the presence of NADH-linked substrates (α-ketoglutarate, citrate, isocitrate, glutamate, malate, and pyruvate). Respiration supported either by glutamate or glutamate plus malate was significantly inhibited by MMA (1-10 mM), whereas no inhibition was observed when a cocktail of NADH-linked substrates was used. Measurements of glutamate transport revealed that the inhibitory effect of MMA on respiration maintained by this substrate is not due to inhibition of its mitochondrial uptake. In light of this result, the effect of MMA on the activity of relevant enzymes involved in mitochondrial glutamate metabolism was investigated. MMA had minor inhibitory effects on glutamate dehydrogenase and aspartate aminotransferase, whereas α-ketoglutarate dehydrogenase was significantly inhibited by this metabolite (K(i) = 3.65 mM). Moreover, measurements of α-ketoglutarate transport and mitochondrial MMA accumulation indicated that MMA/α-ketoglutarate exchange depletes mitochondria from this substrate, which may further contribute to the inhibition of glutamate-sustained respiration. To study the effect of chronic in vivo MMA treatment on mitochondrial function, young rats were intraperitoneally injected with MMA. No significant difference was observed in respiration between isolated brain mitochondria from control and MMA-treated rats, indicating that in vivo MMA treatment did not lead to permanent mitochondrial respiratory defects. Taken together, these findings indicate that the inhibitory effect of MMA on mitochondrial oxidative metabolism can be ascribed to concurrent inhibition of specific enzymes and lower availability of respiratory substrates.

Safranine as a fluorescent probe for the evaluation of mitochondrial membrane potential in isolated organelles and permeabilized cells.

The mitochondrial electrical membrane potential (Δψ) is the main component of the proton motive force (Δp) generated across the inner mitochondrial membrane during electron flow through the respiratory chain. Among the techniques available to assess Δψ, methods that rely on the spectrophotofluorometric responses of dyes are widely employed for whole suspensions of isolated mitochondria or permeabilized cells. Safranine is one of the dyes currently used most often for this purpose. Safranine is a lipophilic cationic dye that undergoes optical shifts upon its potential-dependent distribution between the external medium and the intramitochondrial compartment and on its stacking to inner mitochondrial membrane anionic sites. The association between the optical changes of safranine and the membrane potential allows unknown Δψ values to be estimated from an equation describing their relationship. Here, we describe the use of safranine as a fluorescent indicator of Δψ in isolated mitochondria and digitonin-permeabilized cells. We present suitable conditions to employ safranine as a Δψ indicator.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia.

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.

Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis.

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.

3-nitropropionic acid-induced mitochondrial permeability transition: comparative study of mitochondria from different tissues and brain regions.

The adult rat striatum is particularly vulnerable to systemic administration of the succinate dehydrogenase inhibitor 3-nitropropionic acid (3NP), which is known to induce degeneration of the caudate-putamen, as occurs in Huntington's disease. The aim of the present study was to compare the susceptibility of isolated mitochondria from different rat brain regions (striatum, cortex, and cerebellum) as well as from the liver, kidney, and heart to mitochondrial permeability transition (MPT) induced by 3NP and Ca(2+). In the presence of micromolar Ca(2+) concentrations, 3NP induces MPT in a dose-dependent manner, as estimated by mitochondrial swelling and a decrease in the transmembrane electrical potential. A 3NP concentration capable of promoting a 10% inhibition of ADP-stimulated, succinate-supported respiration was sufficient to stimulate Ca(2+)-induced MPT. Brain and heart mitochondria were generally more sensitive to 3NP and Ca(2+)-induced MPT than mitochondria from liver and kidney. In addition, a partial inhibition of mitochondrial respiration by 3NP resulted in more pronounced MPT in striatal mitochondria than in cortical or cerebellar organelles. A similar inhibition of succinate dehydrogenase activity was observed in rat tissue homogenates obtained from various brain regions as well as from liver, kidney, and heart 24 hr after a single i.p. 3NP dose. Mitochondria isolated from forebrains of 3NP-treated rats were also more susceptible to Ca(2+)-induced MPT than those of control rats. We propose that the increased susceptibility of the striatum to 3NP-induced neurodegeneration may be partially explained by its susceptibility to MPT, together with the greater vulnerability of this brain region to glutamate receptor-mediated Ca(2+) influx.