The reaction catalyzed by succinate-CoA ligase in the mitochondrial matrix yields a high-energy phosphate when operating towards hydrolysis of the thioester bond of succinyl-CoA, known as mitochondrial substrate-level phosphorylation (mSLP). The catabolism of several metabolites converge to succinyl-CoA but through different biochemical pathways. Among them, threonine, serine and methionine catabolize to succinyl-CoA through the common intermediate, 2-ketobutyrate. During the course of this pathway 2-ketobutyrate will become succinyl-CoA through propionyl-CoA catabolism, obligatorily passing through an ATP-consuming step substantiated by propionyl-CoA carboxylase. Here, by recording the directionality of the adenine nucleotide translocase while measuring membrane potential we tested the hypothesis that catabolism of 2-ketobutyrate negates mSLP due to the ATP-consuming propionyl-CoA carboxylase step in rotenone-treated, isolated mouse liver and brain mitochondria. 2-Ketobutyrate produced a less negative membrane potential compared to NADH or FADH2-linked substrates, which was sensitive to inhibition by rotenone, atpenin and arsenate, implying the involvement of complex I, complex II and a dehydrogenase—most likely branched chain keto-acid dehydrogenase, respectively. Co-addition of 2-ketobutyrate with NADH- or FADH2-linked substrates yielded no greater membrane potential than in the presence of substrates alone. However, in the presence of NADH-linked substrates, 2-ketobutyrate prevented mSLP in a dose-dependent manner. Our results imply that despite that 2-ketobutyrate leads to succinyl-CoA formation, obligatory metabolism through propionyl-CoA carboxylase associated with ATP expenditure abolishes mSLP. The provision of metabolites converging to 2-ketobutyrate may be a useful way for manipulating mSLP without using pharmacological or genetic tools.
The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. Next, Gen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.
Alterations in metabolism are a hallmark of cancer. It is unclear if oxidative phosphorylation (OXPHOS) is necessary for tumour cell survival. In this study, we investigated the effects of severe hypoxia, site-specific inhibition of respiratory chain (RC) components, and uncouplers on necrotic and apoptotic markers in 2D-cultured HepG2 and MCF-7 tumour cells. Comparable respiratory complex activities were observed in both cell lines. However, HepG2 cells exhibited significantly higher oxygen consumption rates (OCR) and respiratory capacity than MCF-7 cells. Significant non-mitochondrial OCR was observed in MCF-7 cells, which was insensitive to acute combined inhibition of complexes I and III. Pre-treatment of either cell line with RC inhibitors for 24–72 h resulted in the complete abolition of respective complex activities and OCRs. This was accompanied by a time-dependent decrease in citrate synthase activity, suggesting mitophagy. High-content automated microscopy recordings revealed that the viability of HepG2 cells was mostly unaffected by any pharmacological treatment or severe hypoxia. In contrast, the viability of MCF-7 cells was strongly affected by inhibition of complex IV (CIV) or complex V (CV), severe hypoxia, and uncoupling. However, it was only moderately affected by inhibition of complexes I, II, and III. Cell death in MCF-7 cells induced by inhibition of complexes II, III, and IV was partially abrogated by aspartate. These findings indicate that OXPHOS activity and viability are not correlated in these cell lines, suggesting that the connection between OXPHOS and cancer cell survival is dependent on the specific cell type and conditions.
Alterations in metabolism is a hallmark of cancer. It is unclear, however, if oxidative phosphorylation (OXPHOS) is required for tumor cell survival. We investigated the effect of severe hypoxia, site-specific inhibition of respiratory chain (RC) components, and uncouplers on the survival of HepG2 and MCF-7 2D cultured cells. Comparable respiratory complex activities were observed in both cell lines, but HepG2 cells exhibited much higher oxygen consumption rates (OCR) and respiratory capacity than the MCF-7 cells. Significant non-mitochondrial OCR was found in MCF-7 cells that was insensitive to acute combined inhibition of complexes I and III. However, pre-treatment of either cell line with RC inhibitors for 24-72 hours abolished respective complex activities and OCRs completely, and this was associated with a time-dependent decrease in citrate synthase activity, suggesting mitophagy. HepG2 cells viability was mostly unaffected by any pharmacological treatment or severe hypoxia as temporally recorded from high-content automated microscopy. Conversely, MCF-7 cells viability exhibited strong sensitivity to CIV or CV inhibition, severe hypoxia, and uncoupling, but were only moderately affected by CI, CII and CIII inhibition. CII, CIII and CIV-inhibitor mediated MCF-7 cell death were partially abrogated by aspartate. The data show that OXPHOS activity and viability are uncorrelated in these cell lines indicating that a linkage of OXPHOS to cancer cell survival must be cell- and condition-defined.
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