bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒05‒15
37 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Sci Adv. 2022 May 13. 8(19): eabl8716
      Several subunits in the matrix domain of mitochondrial complex I (CI) have been posited to be redox sensors for CI, but how elevated levels of reactive oxygen species (ROS) impinge on CI assembly is unknown. We report that genetic disruption of the mitochondrial NADPH-generating enzyme, isocitrate dehydrogenase 2 (IDH2), in Drosophila flight muscles results in elevated ROS levels and impairment of assembly of the oxidative phosphorylation system (OXPHOS). Mechanistically, this begins with an inhibition of biosynthesis of the matrix domain of CI and progresses to involve multiple OXPHOS complexes. Despite activation of multiple compensatory mechanisms, including enhanced coenzyme Q biosynthesis and the mitochondrial unfolded protein response, ferroptotic cell death ensues. Disruption of enzymes that eliminate hydrogen peroxide, but not those that eliminate the superoxide radical, recapitulates the phenotype, thereby implicating hydrogen peroxide as the signaling molecule involved. Thus, IDH2 modulates the assembly of the matrix domain of CI and ultimately that of the entire OXPHOS.
  2. FASEB J. 2022 May;36 Suppl 1
      Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax ) in isolated human airway smooth muscle (hASM) cells using 1-methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax , the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil-immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP-mediated disruption of the mitochondrial proton gradient does not affect SDHmax , while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.
  3. FASEB J. 2022 May;36 Suppl 1
      RATIONALE: It is recognized that the kinetics and efficiency of mitochondrial O2 consumption for ATP production depend on the choice of respiratory substrates. Various substrates differentially generate the reducing equivalents NADH and FADH2 via the tricarboxylic acid (TCA) cycle that feed electrons to the electron transport chain (ETC) driving oxidative phosphorylation (OxPhos). However, the contributions of these substrates and their combinations have not been systematically characterized in either the heart or the kidney, the two major energy consuming organs in the body.METHOD: Bioenergetic responses (respiration and membrane potential) of mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague-Drawly rats were characterized with different substrate combinations and different ADP perturbations. To better understand these distinct substrate and tissue-specific mitochondrial bioenergetics, thermodynamically-constrained mechanistic computational models were developed by integrating the kinetics and regulation of mitochondrial substrate transport and oxidation, TCA cycle, and ETC/OxPhos, incorporating published data and the novel mitochondrial bioenergetics data from our laboratory. Intrinsic model parameters such as Michaelis-Menten constants were assumed identical and fixed for the heart and kidney, while extrinsic model parameters such as maximal enzymatic rates were separately estimated based on experimental data for the heart and kidney mitochondrial bioenergetics.
    RESULTS: Consistent with the data, model simulations of the ADP-induced state 3 responses of the heart and kidney cortex and OM mitochondria yielded dramatically different values, which for a given organ were also very distinct for different substrates. In addition, heart mitochondria energized with succinate without rotenone (blocker of complex I) and saturated [ADP] failed to produce a robust state 3 response. This was in contrast to kidney cortex and OM mitochondria for which responses to succinate±rotenone were similar, indicating that reverse electron transfer via complex I played less prominent regulatory role in the kidney than the heart. In addition, oxaloacetate, a TCA cycle intermediate, was shown to accumulate faster in the heart compared to the kidney, thereby potently inhibiting succinate oxidation more in the heart than the kidney. As such, the model was able to quantitatively characterize how various substrate combinations account for the differential contributions of different substrate oxidation pathways within the mitochondria towards OxPhos and ATP synthesis in the heart and kidney cortex and OM.
    CONCLUSION: Modeling of these data provided a deeper quantitative understanding of key determinants of kinetic and molecular mechanisms involved in the differential regulation of substrate-dependent mitochondrial bioenergetics in the heart and kidney cortex and OM.
  4. FASEB J. 2022 May;36 Suppl 1
      Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within the organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 unexpectedly caused perinatal lethality in mice, suggesting that the regulation of mitochondrial phosphorylation is essential in mammalian development. Pptc7-/- mice exhibit severe metabolic deficiencies, including hypoglycemia and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7-/- tissues have decreased mitochondrial function concomitant with a post-transcriptional downregulation of mitochondrial proteins. Multiple elevated mitochondrial protein phosphorylation sites in Pptc7-/- tissues suggest novel functional connections between Pptc7-mediated dephosphorylation and these observed metabolic consequences. Interestingly, these modifications occur on components of the import machinery of the mitochondria and within the mitochondrial targeting sequences of select nuclear-encoded precursor proteins. Collectively, our data reveal an unappreciated role for a matrix-localized phosphatase in the post-translational regulation of the mitochondrial proteome and organismal metabolic homeostasis.
  5. FASEB J. 2022 May;36 Suppl 1
      Caloric restriction (CR) in laboratory rodents prevents obesity, promotes healthy aging, and increases resilience against several pathological stimuli. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and retention capacity, preventing Ca2+ -induced mitochondrial permeability transition. Dietary restriction has been demonstrated to increase kidney resistance against damaging stimuli such as ischemia/reperfusion, but if these effects are related to similar mitochondrial adaptations had not yet been uncovered. Here, we characterized changes in mitochondrial function in response to six months of CR in rats, measuring oxidative phosphorylation, redox balance and calcium homeostasis. CR promoted an increase in mitochondrial oxygen consumption rates under non-phosphorylating (state 4) and uncoupled (state 3U) conditions. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2 O2 release was enhanced in CR rats, although levels of carbonylated proteins and methionine sulfoxide were unchanged. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+ -induced mitochondrial permeability transition. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials, or the amounts of the Mitochondrial Calcium Uniporter (MCU). Instead, increased mitochondrial calcium uptake rates correlate with a loss of Mitochondrial Calcium Uptake 2 (MICU2), an MCU modulator, in CR kidneys. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet-sensitive regulatory role determining mitochondrial calcium homeostasis. Together, our results remark the highly organ-specific bioenergetic, redox, and ionic transport effects of CR. Specifically, we describe the regulation of the expression of MICU2, and its effects on mitochondrial calcium transport, as a novel and interesting aspect of the metabolic responses to dietary interventions.
  6. FASEB J. 2022 May;36 Suppl 1
      Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Butyrate is a short-chain fatty acid produced by resident bacteria in the colon and serves as the primary energy source for non-cancerous colonocytes. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate, and produces ATP. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not by PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 inhibits hypoxia-inducible factor-1 alpha (HIF1α) and suppresses glycolysis. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.
  7. Nat Commun. 2022 May 12. 13(1): 2614
      The interaction of germline variation and somatic cancer driver mutations is under-investigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondrially-encoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 (IDH1), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1-mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.
  8. Int J Mol Sci. 2022 May 04. pii: 5111. [Epub ahead of print]23(9):
      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. NextGen-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.
    Keywords:  coenzyme Q; proline dehydrogenase; reducing equivalent; substrate-level phosphorylation
  9. Elife. 2022 May 13. pii: e74552. [Epub ahead of print]11
      Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.
    Keywords:  cell biology; human
  10. FASEB J. 2022 May;36 Suppl 1
      Mitochondria undergo permeability transition (PT) resulting in the opening of the non-selective PT pores (PTPs) in the inner mitochondrial membrane in response to energy and oxidative stresses associated with Ca2+ overload and ROS accumulation. The mitochondrial PTPs are permeable to ions and solutes with a molecular mass <1.5 kD that increases the colloidal osmotic pressure in the matrix leading to mitochondrial swelling. Calcium retention capacity (CRC) reflects the maximum amount of Ca2+ mitochondria can uptake to provoke the PTP opening. Quantification of CRC is important to study the effects of various pathological stimuli and the efficacy of pharmacological agents on the metabolism and function of mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the advantages/disadvantages of the fluorescent technique in isolated mitochondria vs. permeabilized cells. The cells were permeabilized using digitonin or saponin, and the CRC was assessed using the Ca2+ -sensitive fluorescence probe Calcium Green-5N. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. The CRC of saponin-permeabilized cells was higher than the CRC of digitonin-permeabilized cells. In addition, the mitochondrial CRC of saponin-permeabilized cells was higher than isolated mitochondria using the same number of cells. In conclusion, this study demonstrates that the fluorescent technique for CRC analysis in saponin-permeabilized cells has more advantages than isolated mitochondria.
  11. Cancer Res. 2022 May 11. pii: canres.3910.2021. [Epub ahead of print]
      Micropeptides are a recently discovered class of molecules that play vital roles in various cellular processes, including differentiation, proliferation, and apoptosis. Here, we sought to identify cancer-associated micropeptides and to uncover their mechanistic functions. A micropeptide named short trans-membrane protein 1 (STMP1) that localizes at the inner mitochondrial membrane was identified to be upregulated in various cancer types and associated with metastasis and recurrence of hepatocellular carcinoma. Both gain- and loss-of-function studies revealed that STMP1 increased dynamin-related protein 1 (DRP1) activation to promote mitochondrial fission and enhanced migration of tumor cells. STMP1 silencing inhibited in vivo tumor metastasis in xenograft mouse models. Overexpression of STMP1 led to redistribution of mitochondria to the leading edge of cells and enhanced lamellipodia formation. Treatment with a DRP1 inhibitor abrogated the promotive effect of STMP1 on mitochondrial fission, lamellipodia formation, and tumor cell migration in vitro and metastasis in vivo. Furthermore, STMP1 interacted with myosin heavy chain 9 (MYH9), the subunit of non-muscle myosin II, and silencing MYH9 abrogated STMP1-induced DRP1 activation, mitochondrial fission, and cell migration. Collectively, this study identifies STMP1 as a critical regulator of metastasis and a novel unit of the mitochondrial fission protein machinery, providing a potential therapeutic target for treating metastases.
  12. FASEB J. 2022 May;36 Suppl 1
      Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient-derived fibroblasts and iPSC-derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome-mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.
  13. Cell Rep. 2022 May 10. pii: S2211-1247(22)00567-8. [Epub ahead of print]39(6): 110800
      Tumors are heterogeneous cellular environments with entwined metabolic dependencies. Here, we use a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. We identify metabolic genes and processes recurrently altered in cancer cells across tumor types, highlighting pan-cancer upregulation of deoxythymidine triphosphate (dTTP) production. In contrast, the tryptophan catabolism rate-limiting enzymes IDO1 and TDO2 are highly overexpressed in stroma, raising the hypothesis that kynurenine-mediated suppression of antitumor immunity may be predominantly constrained by the stroma. Oxidative phosphorylation is the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the Warburg effect may be less pronounced in cancer cells in vivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism.
    Keywords:  CP: Cancer; CP: Metabolism; metabolism; tumor microenvironment
  14. Oncogenesis. 2022 May 09. 11(1): 24
      Lung cancer is the leading cause of cancer-related death worldwide despite the success of therapies targeting oncogenic drivers and immune-checkpoint inhibitors. Although metabolic enzymes offer additional targets for therapy, the precise metabolic proteome of lung adenocarcinomas is unknown, hampering its clinical translation. Herein, we used Reverse Phase Protein Arrays to quantify the changes in enzymes of glycolysis, oxidation of pyruvate, fatty acid metabolism, oxidative phosphorylation, antioxidant response and protein oxidative damage in 128 tumors and paired non-tumor adjacent tissue of lung adenocarcinomas to profile the proteome of metabolism. Steady-state levels of mitochondrial proteins of fatty acid oxidation, oxidative phosphorylation and of the antioxidant response are independent predictors of survival and/or of disease recurrence in lung adenocarcinoma patients. Next, we addressed the mechanisms by which the overexpression of ATPase Inhibitory Factor 1, the physiological inhibitor of oxidative phosphorylation, which is an independent predictor of disease recurrence, prevents metastatic disease. We highlight that IF1 overexpression promotes a more vulnerable and less invasive phenotype in lung adenocarcinoma cells. Finally, and as proof of concept, the therapeutic potential of targeting fatty acid assimilation or oxidation in combination with an inhibitor of oxidative phosphorylation was studied in mice bearing lung adenocarcinomas. The results revealed that this therapeutic approach significantly extended the lifespan and provided better welfare to mice than cisplatin treatments, supporting mitochondrial activities as targets of therapy in lung adenocarcinoma patients.
  15. FASEB J. 2022 May;36 Suppl 1
      Non-cancerous colonocytes use butyrate, which is a short-chain fatty acid derived from the fermentation of dietary fiber, as a primary energy source. In contrast, cancerous colonocytes use glucose as their preferred energetic substrate and undergo enhanced glycolysis or the Warburg effect. It has been reported that increased production of pro-inflammatory cytokines such as interleukin1 beta and tumor necrosis factor alpha (TNFalpha) promote glycolysis. Previously, we have shown that interleukin1 beta increased glycolysis and decreased butyrate oxidation. However, the mechanism underlying how this pro-inflammatory cytokine increased glycolysis was unclear. Here, we have discovered that Uncoupling Protein 2 (UCP2) appears to mediate the elevation in glycolysis induced by interleukin1 beta. UCP2 is a mitochondrial protein that transports the protons back into the mitochondrial matrix, thus dissipating the proton gradient and reducing ATP production. UCP2 is upregulated in various cancers such as breast, prostate, skin, and colorectal cancers. We hypothesize that interleukin1 beta promotes glycolysis by upregulating UCP2 and activating Akt in colorectal cancer cells. We have found colorectal cancer cells treated with interleukin1 beta showed elevated UCP2 expression and increased Akt activation. Next, we utilized the Seahorse XFe24 analyzer to check whether interleukin1 beta affects proton leak and ATP production in colorectal cancer cells. Preliminary data suggests that interleukin1 beta increases proton leak and decreases ATP production in mitochondria, which is consistent with the upregulation of UCP2 and demonstrates a functional consequence. To study whether UCP2 mediates the interleukin1 beta effect toward increasing glycolysis, we created a stable UCP2 knockdown in colorectal cancer cells and tested whether colorectal cancer cells that had diminished UCP2 from the RNAi knockdown did not show an increase in glycolysis when treated with interleukin1 beta as compared to RNAi mock colorectal cancer cells. Our preliminary data allude to a role for UCP2 in mediating the interleukin1 beta induced increase in glycolysis observed in colorectal cancer cells.
  16. FASEB J. 2022 May;36 Suppl 1
      Activation of stem cell proliferation is a critical event in tissue regeneration. The metabolic switch in adult stem cells from the oxidative to the glycolytic mode of carbon utilization is essential for rapid proliferative bursts, but its impact on self-renewal is unclear. During the glycolytic mode of glucose utilization, glutamine-derived carbons drive the mitochondrial TCA cycle. While glutamine is required for stem cell proliferation burst, its role in maintaining stem cell self-renewal property remains unclear. Here, we show that withdrawal or chemical inhibition of mitochondrial glutamine metabolism blunted adult muscle stem cell proliferation, but also reactivated the transcription of self-renewal-associated transcripts, such as Pax7, to reduce stem cell heterogeneity and build the self-renewing stem cell population. Thus, surprisingly, glutamine withdrawal preserved and accentuated the self-renewing stem cell population. This effect of glutamine is mediated via reductive carboxylation of alpha-ketoglutarate. Mechanistically, we extensively show that glutamine inhibited cell-cycle linked self-renewing network during the G2-M phase of cell-cycle to drive the exit from self-renewal during the terminal mitosis phase before differentiation. Thus, we propose that glutamine metabolism plays an unexpected role in building the progenitor population that is uniquely primed for differentiation during tissue regeneration.
  17. FASEB J. 2022 May;36 Suppl 1
      NAD+ is an essential coenzyme found in all living cells. NAD+ concentrations decline during aging, but whether this reflects impaired production or accelerated consumption remains unclear. Here we employed isotope tracing and mass spectrometry to probe NAD+ metabolism across tissues in aged mice. In 25-month-old mice, we observe modest tissue NAD+ depletion (median decrease ~30%) without significant changes in circulating NAD+ precursors. Isotope tracing showed unimpaired synthesis of circulating nicotinamide from tryptophan, and maintained flux of circulating nicotinamide into tissue NAD+ pools. Although absolute NAD+ biosynthetic flux was maintained in most tissues of aged mice, fractional tissue NAD+ labeling from infused labeled nicotinamide was modestly accelerated, consistent with increased activity of NAD+ consuming enzymes. Long-term calorie restriction partially mitigated age-associated NAD+ decline despite decreasing NAD+ synthesis, suggesting that calorie restriction reduces NAD+ consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, age-related decline in NAD+ is relatively subtle and driven by increased NAD+ consumer activity rather than impaired production.
  18. Commun Biol. 2022 May 12. 5(1): 453
      Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1-8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson's disease.
  19. FASEB J. 2022 May;36 Suppl 1
      RATIONALE: Reactive oxygen species (ROS; e.g. O2 · - and H2 O2 ) play important roles in both physiological and pathophysiological processes. ROS in low concentrations contribute to physiological processes, such as cellular redox signaling and phagocytosis, whereas ROS in high concentrations are toxic to the cell causing tissue injury contributing to the pathogenesis of cardiovascular and chronic renal diseases, including salt-sensitive hypertension. Mitochondria, which produce ROS as byproducts of aerobic respiration via both forward electron transfer (FET) and reverse electron transfer (RET) are known to be one of the major cellular sources of ROS. Although it is recognized that the RET mechanism in which electrons flow back from complex II to complex I contribute significantly to ROS production in cardiac mitochondria, the mechanisms of ROS production and the role of RET in kidney mitochondria has remained poorly understood.METHOD: We evaluated the relative contributions of FET and RET towards overall ROS production in mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague-Dawley rats. H2 O2 emission was measured by a spectrofluorometer in isolated mitochondria in the presence of either Succinate (Suc) simulating RET or succinate+rotenone (Suc+Rot) simulating FET. Furthermore, we measured mitochondrial rates of H2 O2 production along with respiration and membrane potential under three respiratory states namely (i) leak state (state 2; after substrate addition), (ii) oxidative phosphorylation (OxPhos) state (state 3; after ADP addition), and (iii) maximum respiratory state (state 5; after the addition of the uncoupler FCCP).
    RESULTS: It was found that mitochondria isolated from the heart and kidney cortex produced the least and the most ROS, respectively. The rate of ROS production in the presence of Suc+Rot compared to Suc alone decreased significantly in the heart and to a lesser extent in OM, indicating significant contribution of RET to overall ROS production in the heart and slightly in the OM. In contrast, there was not significant difference in ROS production rates in the presence of Suc and Suc +Rot in mitochondria from kidney cortex, showing that RET is not predominant in the kidney cortex. Also, we observed significant reduction in the ROS production rate in state 4 compared to state 2 in the heart mitochondria compared to kidney cortex and OM. A possible explanation for these differential results is that oxaloacetate (OAA), produced by the tricarboxylic acid cycle, accumulates resulting in succinate dehydrogenase (SDH) inhibition more rapidly in the heart than in the kidney affecting mitochondrial ROS production, respiration, and bioenergetics.
    CONCLUSION: RET mechanism contributes to mitochondrial ROS production significantly in the heart and slightly in the kidney OM, but not in the kidney cortex. OAA accumulation contributes to SDH inhibition significantly in the heart than in the kidney.
    Keywords:  Forward electron transfer; Mitochondrial bioenergetics; Oxidative stress; ROS production; Reverse electron transfer
  20. Cell Mol Life Sci. 2022 May 08. 79(6): 284
      BACKGROUND AND AIMS: Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of colorectal cancer (CRC). To overcome treatment resistance, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new inhibitor of NCLX.METHODS: We examined whether curcumin and pharmacological compounds induced the inhibition of NCLX-mediated mitochondrial calcium (mtCa2+) extrusion, the role of redox metabolism in this process. We evaluated their anti-tumorigenic activity in vitro and in a xenograft mouse model. We analyzed NCLX expression and associations with survival in The Cancer Genome Atlas (TCGA) dataset and in tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC.
    RESULTS: In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis. NCLX inhibition with pharmacological and molecular approaches reproduced the effects of curcumin. NCLX inhibitors decreased CRC tumor growth in vivo. Both transcriptomic analysis of TCGA dataset and immunohistochemical analysis of tissue microarrays demonstrated that higher NCLX expression was associated with MSI status, and for the first time, NCLX expression was significantly associated with recurrence-free survival.
    CONCLUSIONS: Our findings highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic schedule of patients with MSI CRC.
    Keywords:  Calcium signaling; Colorectal cancer; Curcumin; Mitochondria; NCLX
  21. FASEB J. 2022 May;36 Suppl 1
      Mitochondria undergo coordinated rounds of fusion and fission that are critical for maintaining the functional integrity of this essential organelle. While a growing number of proteins have been identified as important regulators of mitochondrial dynamics, the direct role of membrane lipid composition during the fusion and fission processes is poorly understood. To address these shortcomings, we devised a protein-engineering platform that allows for the acute remodeling of structural phospholipids within the outer mitochondrial membrane (OMM) of intact cells. Specifically, we modified a bacterial phospholipase C (Bacillus cereus (Bc)PI-PLC) to initiate the rapid hydrolysis of phosphatidylinositol (PI) and locally generate diacylglycerol (DAG); an important intracellular signaling molecule and metabolic precursor that is used in diverse lipid biosynthetic pathways. Spatial restriction of enzyme activity was achieved using a chemically inducible system consisting of a rapamycin-dependent dimerization module (FKBP-BcPI-PLC) along with an OMM targeting sequence tagged with the FKBP-rapamycin binding domain (OMM-FRB). Using these unique molecular tools, we show that recruitment of FKBP-BcPI-PLC to the OMM not only causes the expected local accumulation of DAG, but also initiates the rapid and uniform fragmentation of the mitochondrial network. Mitochondrial fission induced by FKBP-BcPI-PLC is accompanied by profound swelling of the mitochondrial matrix along with vesiculation of the inner mitochondrial membrane (IMM) and a general loss of cristae, which all occur within minutes of tethering FKBP-BcPI-PLC to the OMM. Expression of dominant-negative constructs targeting essential GTPases known to regulate OMM fission suggest that both dynamin-related protein 1 (Drp1) and dynamin 2 (Dnm2) work together to drive efficient BcPI-PLC-induced mitochondrial division. However, results using a validated Drp1 knockout cell line show that the loss of Drp1 alone is sufficient to prevent the mitochondrial fragmentation initiated by FKBP-BcPI-PLC recruitment, indicating that Drp1 likely functions upstream or independent of Dnm2 in this context. Interestingly, unlike the induced OMM fission, removal of Drp1 from cells does not prevent the matrix swelling or OMM constrictions observed in response to acute generation of DAG within the OMM. Ongoing experiments are now focused on characterizing new methods to sequentially metabolize the DAG generated within the OMM as well as investigate how local lipid composition influences the binding and oligomerization of membrane-shaping proteins that may function in concert with Drp1 to regulate mitochondrial remodeling. Overall, these studies establish a direct relationship between lipid metabolism within the OMM and clinically relevant morphological changes that are known to manifest in mitochondrial-associated diseases.
  22. Am J Cancer Res. 2022 ;12(4): 1436-1455
      Tricarboxylic acid (TCA) cycle, also called Krebs cycle or citric acid cycle, is an amphoteric pathway, contributing to catabolic degradation and anaplerotic reactions to supply precursors for macromolecule biosynthesis. Oxoglutarate dehydrogenase complex (OGDHc, also called α-ketoglutarate dehydrogenase) a highly regulated enzyme in TCA cycle, converts α-ketoglutarate (αKG) to succinyl-Coenzyme A in accompany with NADH generation for ATP generation through oxidative phosphorylation. The step collaborates with glutaminolysis at an intersectional point to govern αKG levels for energy production, nucleotide and amino acid syntheses, and the resources for macromolecule synthesis in cancer cells with rapid proliferation. Despite being a flavoenzyme susceptible to electron leakage contributing to mitochondrial reactive oxygen species (ROS) production, OGDHc is highly sensitive to peroxides such as HNE (4-hydroxy-2-nonenal) and moreover, its activity mediates the activation of several antioxidant pathways. The characteristics endow OGDHc as a critical redox sensor in mitochondria. Accumulating evidences suggest that dysregulation of OGDHc impairs cellular redox homeostasis and disturbs substrate fluxes, leading to a buildup of oncometabolites along the pathogenesis and development of cancers. In this review, we describe molecular interactions, regulation of OGDHc expression and activity and its relationships with diseases, specifically focusing on cancers. In the end, we discuss the potential of OGDHs as a therapeutic target for cancer treatment.
    Keywords:  2-oxoglutarate dehydrogenase; cancer metabolism; reactive oxygen species; tricarboxylic acid cycle; α-ketoglutarate dehydrogenase complex
  23. FASEB J. 2022 May;36 Suppl 1
      Previously, we reported that airway inflammation mediated by pro-inflammatory cytokines such as TNFα induces hypercontractility and increased ATP consumption in human airway smooth muscle (hASM). We also found that TNFα induces an increase in maximum O2 consumption rate as well as mitochondrial biogenesis and an increase in mitochondrial volume density. However, when normalized to mitochondrial volume, maximum O2 consumption rate per mitochondrion is reduced in hASM after TNFα exposure. Unfortunately, standard respirometry (e.g., Seahorse) measures only the averaged maximum O2 consumption rate of a large number of cells. In the present study, we used a quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase reaction (SDHmax ) together with mitochondrial volume density in individual hASM cells. SDH is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC), and SDHmax reflects the maximum respiratory capacity of individual mitochondrion. We hypothesized that TNFα decreases SDHmax per mitochondrion in hASM cells. hASM cells were dissociated from bronchial biopsies obtained during thoracic surgeries in patients with no history of asthma or chronic obstructive pulmonary disease. The hASM cells were treated with TNFα (20 ng/ml) or vehicle (DMSO) for 24 h, and SDHmax was measured using a solution containing 80 mM succinate (maximum substrate concentration) and 1.5 mM nitro blue tetrazolium (NBT) as the reaction indicator. As the SDH reaction proceeded, the hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using an oil-immersion ×60/1.4 NA objective on a Nikon Eclipse A1 laser scanning confocal system. The change in optical density (OD), reflecting the accumulation of NBT diformazan reaction product in the hASM cell, was measured every 15 s over a 10 min period. Based on the Beer-Lambert equation, the slope of the change in OD was used to determine SDHmax expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the same hASM cells were also loaded with MitoTracker Green (500 nM) and imaged in 3D. We found that mitochondria were more fragmented after TNFα exposure although mitochondrial volume density increased. When SDHmax was normalized to mitochondrial volume, we found that SDHmax per mitochondrion decreased in hASM cells, consistent with the reduction in maximum O2 consumption per mitochondrion that we previously observed. Thus, the increased energetic demand induced by inflammation (TNFα) are met by mitochondrial fragmentation leading to biogenesis and an increase in mitochondrial volume density rather than an increase in mitochondrial respiratory capacity.
  24. Mol Cancer Res. 2022 May 13. pii: molcanres.0085.2022. [Epub ahead of print]
      Ceramide kinase (CERK) is the mammalian lipid kinase from which the bioactive sphingolipid, ceramide-1-phosphate (C1P), is derived. CERK has been implicated in several pro-malignant phenotypes with little known as to mechanistic underpinnings. In this study, the mechanism of how CERK inhibition decreases cell survival in mutant (Mut) KRAS non-small cell lung cancer (NSCLC), a major lung cancer subtype, was revealed. Specifically, NSCLC cells possessing a KRAS mutation were more responsive to inhibition, downregulation, and genetic ablation of CERK compared to those with wild-type (WT) KRAS regarding a reduction in cell survival. Inhibition of CERK induced ferroptosis in Mut KRAS NSCLC cells, which required elevating VDAC-regulated mitochondria membrane potential (MMP) and the generation of cellular reactive oxygen species (ROS). Importantly, through modulation of VDAC, CERK inhibition synergized with the first-line NSCLC treatment, cisplatin, in reducing cell survival and in vivo tumor growth. Further mechanistic studies indicated that CERK inhibition impacted MMP and cell survival by limiting AKT activation and translocation to mitochondria, and thus, blocking VDAC phosphorylation and tubulin recruitment. Implications: Our findings depict how CERK inhibition may serve as a new key point in combination therapeutic strategy for NSCLC, specifically precision therapeutics targeting NSCLC possessing a KRAS mutation.
  25. Clin Transl Med. 2022 May;12(5): e852
      BACKGROUND: Glutaminolysis is a critical metabolic process that promotes cancer cell proliferation, including hepatocellular carcinoma (HCC). Delineating the molecular control of glutaminolysis could identify novel targets to ameliorate this oncogenic metabolic pathway. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a regulator of mitochondrial protein acetylation, in modulating the acetylation and activity of glutaminase to regulate HCC development.METHODS: Cell proliferation was determined by MTT, 2D and soft agar clone formation assays and orthotopic tumour assays in nude mice. GLS1/2 acetylation and activities were measured in cells and tumours to analyse the correlation with GCN5L1 expression and mTORC1 activation.
    RESULTS: Hepatic GCN5L1 ablation in mice markedly increased diethylnitrosamine (DEN)-induced HCC, and conversely, the transduction of mitochondrial-restricted GCN5L1 protected wild-type mice against HCC progression in response to DEN and carbon tetrachloride (CCl4 ) exposure. GCN5L1-depleted HepG2 hepatocytes enhanced tumour growth in athymic nude mice. Mechanistically, GCN5L1 depletion promoted cell proliferation through mTORC1 activation. Interestingly, liver-enriched glutaminase 2 (GLS2) appears to play a greater role than ubiquitous and canonical tumour-enriched glutaminase 1 (GLS1) in promoting murine HCC. Concurrently, GCN5L1 promotes acetylation and inactivation of both isoforms and increases enzyme oligomerisation. In human HCC tumours compared to adjacent tissue, there were variable levels of mTORC1 activation, GCN5L1 levels and glutaminase activity. Interestingly, the levels of GCN5L1 inversely correlated with mTORC1 activity and glutaminase activity in these tumours.
    CONCLUSIONS: Our study identified that glutaminase activity, rather than GLS1 or GLS2 expression, is the key factor in HCC development that activates mTORC1 and promotes HCC. In the Kaplan-Meier analysis of liver cancer, we found that HCC patients with high GCN5L1 expression survived longer than those with low GCN5L1 expression. Collectively, GCN5L1 functions as a tumour regulator by modulating glutaminase acetylation and activity in the development of HCC.
    Keywords:  GCN5L1; HCC; glutaminase; mTORC1; mitochondria acetylation
  26. Theranostics. 2022 ;12(7): 3534-3552
      Rationale: Malignant ascites in peritoneal metastases is a lipid-enriched microenvironment and is frequently involved in the poor prognosis of epithelial ovarian cancer (EOC). However, the detailed mechanisms underlying ovarian cancer (OvCa) cells dictating their lipid metabolic activities in promoting tumor progression remain elusive. Methods: The omental conditioned medium (OCM) was established to imitate the omental or ascites microenvironment. Mass spectrometry, RT-qPCR, IHC, and western blot assays were applied to evaluate human fatty acid desaturases expressions and activities. Pharmaceutical inhibition and genetic ablation of SCD1/FADS2 were performed to observe the oncogenic capacities. RNA sequencing, lipid peroxidation, cellular iron, ROS, and Mito-Stress assays were applied to examine ferroptosis. OvCa patient-derived organoid and mouse model of peritoneal metastases were used to evaluate the combined effect of SCD1/FADS2 inhibitors with cisplatin. Results: We found that two critical fatty acid desaturases, stearoyl-CoA desaturase-1 (SCD1) and acyl-CoA 6-desaturase (FADS2), were aberrantly upregulated, accelerating lipid metabolic activities and tumor aggressiveness of ascites-derived OvCa cells. Lipidomic analysis revealed that the elevation of unsaturated fatty acids (UFAs) was positively associated with SCD1/FADS2 levels and the oncogenic capacities of OvCa cells. In contrast, pharmaceutical inhibition and genetic ablation of SCD1/FADS2 retarded tumor growth, cancer stem cell (CSC) formation and reduced platinum resistance. Inhibition of SCD1/FADS2 directly downregulated GPX4 and the GSH/GSSG ratio, causing disruption of the cellular/mitochondrial redox balance and subsequently, iron-mediated lipid peroxidation and mitochondrial dysfunction in ascites-derived OvCa cells. Conclusions: Combinational treatment with SCD1/FADS2 inhibitors and cisplatin synergistically repressed tumor cell dissemination, providing a promising chemotherapeutic strategy against EOC peritoneal metastases.
    Keywords:  lipid desaturases; lipid metabolism; ovarian cancer; oxidative stress; peritoneal metastases
  27. Sci Adv. 2022 May 13. 8(19): eabm6638
      Exploiting cancer vulnerabilities is critical for the discovery of anticancer drugs. However, tumor suppressors cannot be directly targeted because of their loss of function. To uncover specific vulnerabilities for cells with deficiency in any given tumor suppressor(s), we performed genome-scale CRISPR loss-of-function screens using a panel of isogenic knockout cells we generated for 12 common tumor suppressors. Here, we provide a comprehensive and comparative dataset for genetic interactions between the whole-genome protein-coding genes and a panel of tumor suppressor genes, which allows us to uncover known and new high-confidence synthetic lethal interactions. Mining this dataset, we uncover essential paralog gene pairs, which could be a common mechanism for interpreting synthetic lethality. Moreover, we propose that some tumor suppressors could be targeted to suppress proliferation of cells with deficiency in other tumor suppressors. This dataset provides valuable information that can be further exploited for targeted cancer therapy.
  28. FASEB J. 2022 May;36 Suppl 1
      In most organisms, proteasomes are enriched in cell nuclei under optimal physiological conditions, indicating an important need for protein degradation in this organelle. However, stress conditions can result in proteasome nuclear export, possibly reflecting changes in demand for protein degradation. To understand this adaptive response, it is crucial to know when and how proteasome localization and activity are altered upon changing cellular needs. In yeast, carbon starvation triggers a reversible re-localization of proteasomes to cytosolic granules known as proteasome storage granules (PSGs). These PSGs behave like liquid-liquid phase separated structures and highlight a link between cellular metabolism and proteasome localization. We hypothesized that mitochondrial respiration is important in this process. To test this, we manipulated the glycolytic and respiratory activity in yeast using different carbon sources as well as chemical inhibitors of mitochondrial function and monitored proteasome localization by fluorescent microscopy. Growing cells in carbon sources that necessitate respiration prior to starvation, caused a strong reduction in proteasome storage granule formation upon carbon starvation. This suggests that the mitochondrial activity of cells is a determining factor in proteasome localization. Consistent with this, upon chemical inhibition of mitochondrial function, we observed proteasomes re-localize to cytosolic granules independent of starvation stress. Interestingly, the stress response kinase Snf1, which is not required for proteasome re-localization upon carbon starvation, was required for proteasome re-localization following mitochondrial inhibition. In addition, MAP kinases of the cell wall integrity cascade were required for proteasome granule formation, specifically following respiratory but not glycolytic growth, upon carbon starvation or mitochondrial inhibition. In all, our data point to a model where mitochondria regulate proteasome localization via kinases that sense the cells metabolic state.
  29. Cancers (Basel). 2022 Apr 26. pii: 2155. [Epub ahead of print]14(9):
      Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors; it is often diagnosed at an advanced stage and is hardly treatable. These issues are strictly linked to the absence of early diagnostic markers and the low efficacy of treatment approaches. Recently, the study of the metabolic alterations in cancer cells has opened the way to important findings that can be exploited to generate new potential therapies. Within this scenario, mitochondria represent important organelles within which many essential functions are necessary for cell survival, including some key reactions involved in energy metabolism. These organelles remodel their shape by dividing or fusing themselves in response to cellular needs or stimuli. Interestingly, many authors have shown that mitochondrial dynamic equilibrium is altered in many different tumor types. However, up to now, it is not clear whether PDAC cells preferentially take advantage of fusion or fission processes since some studies reported a wide range of different results. This review described the role of both mitochondria arrangement processes, i.e., fusion and fission events, in PDAC, showing that a preference for mitochondria fragmentation could sustain tumor needs. In addition, we also highlight the importance of considering the metabolic arrangement and mitochondria assessment of cancer stem cells, which represent the most aggressive tumor cell type that has been shown to have distinctive metabolic features to that of differentiated tumor cells.
    Keywords:  PDAC; cancer stem cells; metabolism; mitochondrial dynamics; molecular target
  30. FASEB J. 2022 May;36 Suppl 1
      Coenzyme Q (CoQ, ubiquinone) is a ubiquitous redox-active lipid that is required for oxidative phosphorylation, acts as a lipophilic antioxidant, and is a critical cofactor for numerous other processes, such as uridine biosynthesis and fatty acid oxidation. Primary defects in CoQ biosynthesis cause a variety of phenotypes, from nephropathy and myopathy to fatal multiorgan disease. Secondary defects in CoQ levels have been implicated in the pathophysiology of many common conditions, such as neurodegenerative diseases, cardiomyopathies, diabetes, and aging. Unfortunately, exogenous oral supplementation of CoQ aimed at alleviating or preventing disease is difficult due to its large molecular weight and hydrophobicity. Only 2% of orally administered CoQ reaches the bloodstream, and less than 15% of the CoQ in the blood becomes incorporated into mitochondria in tissues; therefore, is often impossible for patients reach an effective dose, even when taking grams of CoQ daily. More efficient means of delivering exogenous CoQ to mitochondria, its main site of action, need to be developed. Here, we describe the synthesis and evaluation of an initial set of compounds designed to deliver CoQ in larger quantities selectively to mitochondria. These compounds incorporate a mitochondria-targeting triphenylphosphonium (TPP) moiety attached to CoQ through a reversible linker, and are designed to accumulate in mitochondria and be cleaved to release native CoQ. Our results indicate that select versions of these compounds can successfully be delivered to mitochondria in a cell model and be cleaved to produced CoQ, encouraging further development. Our ongoing efforts include assessing the efficacy of these compounds in rescuing CoQ-deficient cells, and further optimizing design features to further promote effective cleavage and mitochondrial localization while minimizing toxicity.
  31. Cancers (Basel). 2022 May 05. pii: 2297. [Epub ahead of print]14(9):
      Oxidative phosphorylation is an active metabolic pathway in cancer. Atovaquone is an oral medication that inhibits oxidative phosphorylation and is FDA-approved for the treatment of malaria. We investigated its potential anti-cancer properties by measuring cell proliferation in 2D culture. The clinical formulation of atovaquone, Mepron, was given to mice with ovarian cancers to monitor its effects on tumor and ascites. Patient-derived cancer stem-like cells and spheroids implanted in NSG mice were treated with atovaquone. Atovaquone inhibited the proliferation of cancer cells and ovarian cancer growth in vitro and in vivo. The effect of atovaquone on oxygen radicals was determined using flow and imaging cytometry. The oxygen consumption rate (OCR) in adherent cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption and ATP production were inhibited by atovaquone. Imaging cytometry indicated that the majority of the oxygen radical flux triggered by atovaquone occurred in the mitochondria. Atovaquone decreased the viability of patient-derived cancer stem-like cells and spheroids implanted in NSG mice. NMR metabolomics showed shifts in glycolysis, citric acid cycle, electron transport chain, phosphotransfer, and metabolism following atovaquone treatment. Our studies provide the mechanistic understanding and preclinical data to support the further investigation of atovaquone's potential as a gynecologic cancer therapeutic.
    Keywords:  metabolism; mitochondria; oxidative phosphorylation
  32. FASEB J. 2022 May;36 Suppl 1
      Numerous metabolic changes occur in skeletal muscle with aging. At the crossroads of these changes lies the cellular energy highway, the mitochondrial reticulum. Aging is associated with altered patterns of mitochondrial dynamics and morphology, which are thought to be primarily driven by mitochondrial fragmentation. This fragmentation phenomenon may also lead to altered mitochondrial respiration with aging. We used a mouse model and measured parameters of respiration in muscle mitochondrial preparations to evaluate the hypothesis of aging-related effects on metabolism and energy substrate partitioning. Mixed skeletal muscle of C57/Bl6 mice (n=32) was excised from single hind-limbs of young males (YM) (n=8), old males (OM) (n=8), young females (YF) (n=8) and old females (OF) (n=8). Tissues were homogenized in an isotonic solution supplemented with 0.15% protease inhibitor. Mitochondria were isolated by differential centrifugation and their concentrations were determined by BCA analysis. Isolated mitochondria (0.1mg of mitochondria/mL of respiration medium) were then respired using a Clarke-Type Electrode at 25°C with 100 nM of ADP and either 10 mM Pyruvate+ 2.5 mM Malate (P+M) or 40 uM Palmitoyl-L-Carnitine+ 1 mM Malate (PC+M) as substrates. Respiratory Control Ratios (RCR) were calculated as the quotient of State 3 (ADP-stimulated respiration) and State 4 (recovery rate post ATP synthesis) respiratory rates (State 3/State 4) and compared between substrate and age. ADP to oxygen ratios (P:O) were calculated by dividing the amount of ADP added to the respiratory chamber by the oxygen consumption rate (nmol of O2 /mg of mitochondria/minute) of state 3 respiration. RCR in both P+M and PC+M significantly decreased with aging [P+M (p<0.05): YM (4.1±0.9) vs. OM (2.7±1.1); YF (5.1±1.4) vs. OF (3.2±2)] [PC+M (p<0.05): YM (4.2±0.9) vs. OM (2.4±0.7); YF (3.4.±0.5) vs. OF (2.2±0.9)]. P:O in both P+M and PC+M did not significantly change with aging [P+M: (p=0.69): YM (2.9±0.3) vs. OM (2.9±0.2); YF: (p=0.07): (3±0.1) vs. OF (2.9±0.1)] [PC+M:(p=0.65); YM (2.9±0.4) vs. OM (2.9±0.3); (p=0.22): YF (2.9±0.2) vs. OF (2.7±0.3)]. While there were no significant changes in the P:O the age related decreases in the RCR were characterized by a lower capacity to respire derivatives of both CHO and FA. Decreased State 3 respiration and RCR typically corresponds to loss of respiratory capacity and loosening of coupling efficiency. Because exercise increases mitochondrial function and biogenesis it may be prudent to study older animals after exercise training. We plan to further investigate the structural changes in the mitochondrial reticulum and proteome that may be altering mitochondrial function in aging.
  33. Cell Rep. 2022 May 10. pii: S2211-1247(22)00559-9. [Epub ahead of print]39(6): 110792
      Reduced p62 levels are associated with the induction of the cancer-associated fibroblast (CAF) phenotype, which promotes tumorigenesis in vitro and in vivo through inflammation and metabolic reprogramming. However, how p62 is downregulated in the stroma fibroblasts by tumor cells to drive CAF activation is an unresolved central issue in the field. Here we show that tumor-secreted lactate downregulates p62 transcriptionally through a mechanism involving reduction of the NAD+/NADH ratio, which impairs poly(ADP-ribose)-polymerase 1 (PARP-1) activity. PARP-1 inhibition blocks the poly(ADP-ribosyl)ation of the AP-1 transcription factors, c-FOS and c-JUN, which is an obligate step for p62 downregulation. Importantly, restoring p62 levels in CAFs by NAD+ renders CAFs less active. PARP inhibitors, such as olaparib, mimick lactate in the reduction of stromal p62 levels, as well as the subsequent stromal activation both in vitro and in vivo, which suggests that therapies using olaparib would benefit from strategies aimed at inhibiting CAF activity.
    Keywords:  AP-1; CP: Cancer; NAD(+)/NADH; PARP inhibitors; SQSTM1; cancer metabolism; cancer-associated fibroblasts; olaparib; p62; poly(ADP-ribose)-polymerase 1; stroma
  34. Cell Death Dis. 2022 May 11. 13(5): 448
      The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.
  35. Acta Biochim Biophys Sin (Shanghai). 2022 Mar 25. 54(3): 301-310
      Hepatocellular carcinoma (HCC) is the most common primary liver tumor and one of the leading causes of cancer-related death worldwide. Chemotherapeutic agents/regimens such as cisplatin (DDP) are frequently used for advanced HCC treatment. However, drug resistance remains a major hindrance and the underline mechanisms are not fully understood. In this study, we investigated the expression pattern and function of mitochondrial fission factor (Mff) in cisplatin-resistant HCC. We found that Mff is highly expressed in cisplatin-resistant HCC tissues and cell lines. Knockdown of Mff suppresses cell proliferation and promotes cell apoptosis of HCC/DDP cells. In addition, knockdown of Mff sensitizes Huh-7/DDP cells to cisplatin treatment, inhibits cell proliferation, migration and invasion, and enhances cell apoptosis. Confocal imaging showed that knockdown of Mff inhibits the mitochondrial fission and downregulates the expression of GTPase dynamin-related protein 1 (Drp1) in cisplatin-resistant Huh-7/DDP cells. Moreover, xenograft tumor model revealed that knockdown of Mff sensitizes Huh-7/DDP xenograft tumor to cisplatin treatment . In summary, our findings suggest that Mff regulates mitochondrial Drp1 expression and promotes cisplatin resistance in HCC, which provides a potential therapeutic target for the treatment of resistant HCC.
    Keywords:  cisplatin resistance; dynamin-related protein 1; hepatocellular carcinoma; mitochondrial fission factor
  36. FASEB J. 2022 May;36 Suppl 1
      Several aggressive human malignant tumors are characterized by an intense glycolytic rate, over-expression of lactic acid dehydrogenase A (LDHA), and subsequent lactate accumulation, all of which contribute toward an acidic peri-cellular immunosuppressive tumor microenvironment (TME). While recent focus has been directed at how to inhibit LDHA, it is now becoming clear that the multiple isozymes of LDH must simultaneously be inhibited to suppress lactic acid and halt glycolysis. In this study, we explore the biochemical and genomic consequences of the administration of triple LDH isozyme inhibitor (A, B &C) (GNE-140) in MDA-MB-231 triple-negative breast cancer cells (TNBC) cells. The findings confirm that GNE-140 does, in fact, fully block the production of lactic acid, which also results in a block of glucose utilization and severe impedance of the glycolytic pathway. Without a fully functional glycolytic pathway, breast cancer cells continue to thrive, sustain viability, produce ample energy, and maintain mitochondrial potential (ΔΨM). The only observable negative consequence of GNE-140 in this study was the attenuation of cell division, evident in both 2D and 3D cultures and occurring in fully viable cells. Of important note, the cytostatic effects were not reversible by the addition of exogenous lactic acid. The effects of GNE-140 on the whole transcriptome were mild (12 up-regulated differential expressed genes (DEGs) / 77 down-regulated DEGs) out of the 48,226 evaluated, and downregulated DEGS collectively centered around a loss of genes related to mitosis, cell cycle, G0/G1; G1/S transition, and DNA replication. These data were observed by digital fluorescence cytometry and flow cytometry, both corroborating a G0/G1 phase blockage. In conclusion, the findings in this work suggest that there is an unknown element linking LDH enzyme activity to the cell cycle and that this factor is completely independent of lactic acid. The data also establish that complete inhibition of LDH in cancer cells does not affect cell viability or energy production.
  37. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Exposure to an obesogenic gestational environment during early development increases offspring risk of developing obesity and cardiometabolic diseases. Skeletal muscle of obese and insulin resistant individuals show signs of mitochondrial dysfunction including reduced oxidative capacity and increased oxidative stress. Subsequent oxidative damage is caused by reactive oxygen species (ROS) generated primarily by mitochondrial Complex I (CI). Previous work in our non-human primate model shows decreased oxidative capacity in fetal skeletal muscle from obesogenic pregnancies, a phenotype we have confirmed is retained into adolescence. The purpose of this study was to evaluate how this reduced oxidative capacity correspond with ROS production in skeletal muscle of lean adolescent offspring.EXPERIMENTAL DESIGN: Lean (body fat <25%) adult female Japanese macaques were maintained on a control diet (CD) or Western-style diet (WD) prior to and throughout pregnancy and lactation. Dams chronically consuming WD with body fat >30% were classified as obese and included in this study. Male and female offspring were weaned to CD and moved to independent housing at 7 months of age. Muscle samples were collected from offspring gastrocnemius (gastroc) and soleus at 40 months. Offspring data were analyzed by Student's t-test.
    RESULTS: Exposure to maternal WD during pregnancy showed moderate but significant reductions in oxidative damage and a modest (but insignificant) decrease in ROS generation in gastroc of offspring from obese pregnancies. In the soleus, ROS production was significantly reduced in these offspring. Both nuclear- and mitochondrial-encoded genes for CI were decreased with maternal obesity/WD pregnancy, indicating transcriptional regulation of CI in exposed offspring. Finally, CI protein abundance was also decreased in primary myotubes from these offspring in lieu of additional metabolic challenges, providing further evidence of lasting metabolic reprogramming in skeletal muscle.
    CONCLUSIONS: We find that early life exposure to maternal WD-induced obesity leaves a lasting impression on offspring skeletal muscle metabolism that persists into young adulthood. It is possible that transcriptional suppression of CI activity is a protective adaptation to mitigate oxidative damage caused by mitochondrial ROS. While long-term consequences of altered oxidation patterns relating to ROS flux require further investigation, the loss of CI content and diminished oxidative capacity in these lean offspring likely drive their increased susceptibility for cardiometabolic diseases - particularly when faced with metabolic stress and environmental insults throughout the lifespan.