Inhibition of mitochondrial respiration overcomes hepatocellular carcinoma chemoresistance

The clinical management of advanced hepatocellular carcinoma (HCC) is challenging due to its resistance to chemotherapy. In our work, we demonstrate that an antiparasitic drug atovaquone at clinically relevant concentrations is active against chemoresistant HCC. We show that atovaquone inhibits proliferation and induces apoptosis in not only HCC parental cells but also cells exposed to long time culture of chemotherapeutic agents. Consistently, the combination of atovaquone with cisplatin or doxorubicin achieved remarkably greater efficacy than single drug alone. Mechanistically, atovaquone overcomes HCC chemoresistance via supressing mitochondrial respiration and inducing oxidative stress. Atovaquone but not cisplatin or doxorubicin is ineffective in mitochondrial respiration-deficient ρ0, confirming mitochondria as a specific upstream target of atovaquone. Interestingly, we show that prolonged exposure of HCC cells to chemotherapeutic agents induces higher level of mitochondrial respiration, suggesting that tumors which develop chemoresistance after chemotherapy might be more dependent on mitochondrial respiration than primary tumors and explaining the sensitivity of chemoresistant HCC cells to atovaquone. We further show that atovaquone at tolerable does significantly inhibits chemoresistant HCC growth in mice throughout the duration of treatment. In line with in vitro data, we observe the increased oxidative stress in atovaquone-treated tumors. Our findings highlight the dependency of chemoresistant HCC on mitochondrial respiration and demonstrate that atovaquone is a potential drug to overcome HCC chemoresistance.     

Atovaquone enhances doxorubicin’s efficacy via inhibiting mitochondrial respiration and STAT3 in aggressive thyroid cancer

The clinical management of anaplastic thyroid carcinoma and follicular thyroid carcinoma is challenging and requires an alternative therapeutic strategy. Although atovaquone is an FDA-approved anti-malarial drug, studies has recently demonstrated its anti-cancer activities. In line with these efforts, our study shows that atovaquone is an attractive candidate for thyroid cancer treatment. We show that atovaquone significantly inhibits growth, migration and survival in a concentration-dependent manner in 8505C and FTC113 cells. Mechanistically, atovaquone inhibits mitochondrial complex III activity, leading to mitochondrial respiration inhibition and reduction of ATP production in thyroid cancer cells. The inhibitory effects of atovaquone is reversed in mitochondrial respiration-deficient 8505C ρ0 cells, confirming mitochondrial respiration as the mechanism of atovaquone’s action in thyroid cancer. In addition, atovaquone suppresses phosphorylation of STAT3 in thyroid cancer wildype but not ρ0 cells, demonstrating that STAT3 phosphorylation inhibition by atovaquone is a consequence of mitochondrial respiration inhibition. Notably, we further demonstrate that atovaquone significantly augments doxorubicin’s inhibitory effects via suppressing mitochondrial respiration and STAT3. Our findings suggest that atovaquone can be repurposed for thyroid cancer treatment. Our work also highlights that targeting mitochondrial respiration may represent potential therapeutic strategy in thyroid cancer.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Increased expression of transglutaminase 2 drives glycolytic metabolism in renal carcinoma cells

Transglutaminase 2 (TGase 2) expression and glycolysis are increased in most renal cell carcinoma (RCC) cell lines compared to the HEK293 kidney cell line. Although increased glycolysis and altered tricarboxylic acid cycle are common in RCC, the detailed mechanism by which this phenomenon occurs remains to be elucidated. In the present study, TGase 2 siRNA treatment lowered glucose consumption and lactate levels by about 20–30 % in RCC cells; conversely, high expression of TGase 2 increased glucose consumption and lactate production together with decreased mitochondrial aconitase (Aco 2) levels. In addition, TGase 2 siRNA increased mitochondrial membrane potential and ATP levels by about 20–30 % and restored Aco 2 levels in RCC cells. Similarly, Aco 2 levels and ATP production decreased significantly upon TGase 2 overexpression in HEK293 cells. Therefore, TGase 2 leads to depletion of Aco 2, which promotes glycolytic metabolism in RCC cells.     

Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism

Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (rho(-)) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.     

Preservation of NADH ubiquinone-oxidoreductase activity by Src kinase-mediated phosphorylation of NDUFB10

The tyrosine kinase Src is upregulated in several cancer cells. In such cells, there is a metabolic reprogramming elevating aerobic glycolysis that seems partly dependent on Src activation. Src kinase was recently shown to be targeted to mitochondria where it modulates mitochondrial bioenergetics in non-proliferative tissues and cells. The main goal of our study was to determine if increased Src kinase activity could also influence mitochondrial metabolism in cancer cells (143B and DU145 cells). We have shown that 143B and DU145 cells produce most of the ATP through glycolysis but also that the inhibition of OXPHOS led to a significant decrease in proliferation which was not due to a decrease in the total ATP levels. These results indicate that a more important role for mitochondria in cancer cells could be ensuring mitochondrial functions other than ATP production. This study is the first to show a putative influence of intramitochondrial Src kinase on oxidative phosphorylation in cancer cells. Indeed, we have shown that Src kinase inhibition led to a decrease in mitochondrial respiration via a specific decrease in complex I activities (NADH-ubiquinone oxidoreductase). This decrease is associated with a lower phosphorylation of the complex I subunit NDUFB10. These results suggest that the preservation of complex I function by mitochondrial Src kinase could be important in the development of the overall phenotype of cancer.     

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.     

Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation

Bone remodeling is a continuous physiological process that requires constant generation of new osteoblasts from mesenchymal stem cells (MSCs). Differentiation of MSCs to osteoblast requires a metabolic switch from glycolysis to increased mitochondrial respiration to ensure the sufficient energy supply to complete this process. As a consequence of this increased mitochondrial metabolism, the levels of endogenous reactive oxygen species (ROS) rise. In the current study we analyzed the role of forkhead box O3 (FOXO3) in the control of ROS levels in human MSCs (hMSCs) during osteogenic differentiation. Treatment of hMSCs with H₂O₂ induced FOXO3 phosphorylation at Ser294 and nuclear translocation. This ROS-mediated activation of FOXO3 was dependent on mitogen-activated protein kinase 8 (MAPK8/JNK) activity. Upon FOXO3 downregulation, osteoblastic differentiation was impaired and hMSCs lost their ability to control elevated ROS levels. Our results also demonstrate that in response to elevated ROS levels, FOXO3 induces autophagy in hMSCs. In line with this, impairment of autophagy by autophagy-related 7 (ATG7) knockdown resulted in a reduced capacity of hMSCs to regulate elevated ROS levels, together with a reduced osteoblast differentiation. Taken together our findings are consistent with a model where in hMSCs, FOXO3 is required to induce autophagy and thereby reduce elevated ROS levels resulting from the increased mitochondrial respiration during osteoblast differentiation. These new molecular insights provide an important contribution to our better understanding of bone physiology.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.     

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.     

Modulation of mammalian sperm motility by quercetin

The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3-4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548-11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin-treated cells show high motility, which continues for additional 2-3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin-treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial complex III in larval stage of Echinococcus multilocularis as a potential chemotherapeutic target and in vivo efficacy of atovaquone against primary hydatid cysts

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc₁ complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC₅₀). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug.     

Osteoclast precursors display dynamic metabolic shifts toward accelerated glucose metabolism at an early stage of RANKL-stimulated osteoclast differentiation.

Mature osteoclasts have an increased citric acid cycle and mitochondrial respiration to generate high ATP production and ultimately lead to bone resorption. However, changes in metabolic pathways during osteoclast differentiation have not been fully illustrated. We report that glycolysis and oxidative phosphorylation characterized by glucose and oxygen consumption as well as lactate production were increased during receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 and bone marrow-derived macrophage cells. Cell proliferation and differentiation varied according to glucose concentrations (0 to 100 mM). Maximal cell growth occurred at 20 mM glucose concentration and differentiation occurred at 5 mM concentration. Despite the similar growth rates exhibited when cultured cells were exposed to either 5 mM or 40 mM glucose, their differentiation was markedly decreased in high glucose concentrations. This finding suggests the possibility that osteoclastogenesis could be regulated by changes in metabolic substrate concentrations. To further address the effect of metabolic shift on osteoclastogenesis, we exposed cultured cells to pyruvate, which is capable of promoting mitochondrial respiration. Treatment of pyruvate synergistically increased osteoclastogenesis through the activation of RANKL-stimulated signals (ERK and JNK). We also found that osteoclastogenesis was retarded by blocking ATP production with either the inhibitors of mitochondrial complexes, such as rotenone and antimycin A, or the inhibitor of ATP synthase, oligomycin. Taken together, these results indicate that glucose metabolism during osteoclast differentiation is accelerated and that a metabolic shift towards mitochondrial respiration allows high ATP production and induces enhanced osteoclast differentiation.     

K-ras(G12V) transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis

Increased aerobic glycolysis and oxidative stress are important features of cancer cell metabolism, but the underlying biochemical and molecular mechanisms remain elusive. Using a tetracycline inducible model, we show that activation of K-ras(G12V) causes mitochondrial dysfunction, leading to decreased respiration, elevated glycolysis, and increased generation of reactive oxygen species. The K-RAS protein is associated with mitochondria, and induces a rapid suppression of respiratory chain complex-I and a decrease in mitochondrial transmembrane potential by affecting the cyclosporin-sensitive permeability transition pore. Furthermore, pre-induction of K-ras(G12V) expression in vitro to allow metabolic adaptation to high glycolytic metabolism enhances the ability of the transformed cells to form tumor in vivo. Our study suggests that induction of mitochondrial dysfunction is an important mechanism by which K-ras(G12V) causes metabolic changes and ROS stress in cancer cells, and promotes tumor development.     

Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function

Parkin is an ubiquitin‐protein ligase (E3), mutations of which cause juvenile onset – autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2‐DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (±2‐fold change; p<0.05) using 2‐DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2‐DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.     

Glycolysis–respiration relationships in a neuroblastoma cell line

Background

Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods

We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results

Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions

These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance

Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

In EXOG‐depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain

Depletion of mitochondrial endo/exonuclease G‐like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease G‐like‐depleted cells, and this is marked by a decrease in mitochondrial reserve capacity. Neurohormonal stimulation with phenylephrine (PE) did not have an additive effect on the hypertrophic response induced by endo/exonuclease G‐like depletion. Interestingly, PE‐induced atrial natriuretic peptide (ANP) gene expression was completely abolished in endo/exonuclease G‐like‐depleted cells, suggesting a reverse signaling function of endo/exonuclease G‐like. Endo/exonuclease G‐like depletion initially resulted in increased mitochondrial OCR, but this declined upon PE stimulation. In particular, the reserve capacity of the mitochondrial respiratory chain and maximal respiration were the first indicators of perturbations in mitochondrial respiration, and these marked the subsequent decline in mitochondrial function. Although pathological stimulation accelerated these processes, prolonged EXOG depletion also resulted in a decline in mitochondrial function. At early stages of endo/exonuclease G‐like depletion, mitochondrial ROS production was increased, but this did not affect mitochondrial DNA (mtDNA) integrity. After prolonged depletion, ROS levels returned to control values, despite hyperpolarization of the mitochondrial membrane. The mitochondrial dysfunction finally resulted in cell death, which appears to be mainly a form of necrosis. In conclusion, endo/exonuclease G‐like plays an essential role in cardiomyocyte physiology. Loss of endo/exonuclease G‐like results in diminished adaptation to pathological stress. The decline in maximal respiration and reserve capacity is the first sign of mitochondrial dysfunction that determines subsequent cell death.     

Mitofusin 1 and optic atrophy 1 shift metabolism to mitochondrial respiration during aging

Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re‐enter the proliferative cycle. This study investigated whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were taken in quiescent NHFs from younger (newborn, 3‐day, 5‐month, and 1‐year) and older (58‐, 61‐, 63‐, 68‐, and 70‐year) healthy donors as well as NHFs from the same individual at different ages (29, 36, and 46 years). Results show significant changes in cellular metabolism during aging of quiescent NHFs: Old NHFs exhibit a significant decrease in glycolytic flux and lactate levels, and increase in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from the Seahorse XF Cell Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more toward fusion. Genetic knockdown of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more toward glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation‐induced increase in doubling time of NHFs. In summary, results show that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during chronological lifespan, and MFN1 and OPA1 regulate this process.