Mitochondrial Electron Transport Chain Activity Research Articles

RNAi screens identify HES4 as a regulator of redox balance supporting pyrimidine synthesis and tumor growth.

NADH/NAD+ redox balance is pivotal for cellular metabolism. Systematic identification of NAD(H) redox regulators, although currently lacking, would help uncover unknown effectors critically implicated in the coordination of growth metabolism. In this study, we performed a genome-scale RNA interference (RNAi) screen to globally survey the genes involved in redox modulation and identified the HES family bHLH transcription factor HES4 as a negative regulator of NADH/NAD+ ratio. Functionally, HES4 is shown to be crucial for maintaining mitochondrial electron transport chain (ETC) activity and pyrimidine synthesis. More specifically, HES4 directly represses transcription of SLC44A2 and SDS, thereby inhibiting mitochondrial choline oxidation and cytosolic serine deamination, respectively, which, in turn, ensures coenzyme Q reduction capacity for DHODH-mediated UMP synthesis and serine-derived dTMP production. Accordingly, inhibition of choline oxidation preserves mitochondrial serine catabolism and ETC-coupled redox balance. Furthermore, HES4 protein stability is enhanced under EGFR activation, and increased HES4 levels facilitate EGFR-driven tumor growth and predict poor prognosis of lung adenocarcinoma. These findings illustrate an unidentified mechanism, underlying pyrimidine biosynthesis in the intersection between serine and choline catabolism, and underscore the physiological importance of HES4 in tumor metabolism.

The interplay between sex, time of day, fasting status, and their impact on cardiac mitochondrial structure, function, and dynamics

Mitochondria morphology and function, and their quality control by mitophagy, are essential for heart function. We investigated whether these are influenced by time of the day (TOD), sex, and fed or fasting status, using transmission electron microscopy (EM), mitochondrial electron transport chain (ETC) activity, and mito-QC reporter mice. We observed peak mitochondrial number at ZT8 in the fed state, which was dependent on the intrinsic cardiac circadian clock, as hearts from cardiomyocyte-specific BMAL1 knockout (CBK) mice exhibit different TOD responses. In contrast to mitochondrial number, mitochondrial ETC activities do not fluctuate across TOD, but decrease immediately and significantly in response to fasting. Concurrent with the loss of ETC activities, ETC proteins were decreased with fasting, simultaneous with significant increases of mitophagy, mitochondrial antioxidant protein SOD2, and the fission protein DRP1. Fasting-induced mitophagy was lost in CBK mice, indicating a direct role of BMAL1 in regulating mitophagy. This is the first of its kind report to demonstrate the interactions between sex, fasting, and TOD on cardiac mitochondrial structure, function and mitophagy. These studies provide a foundation for future investigations of mitochondrial functional perturbation in aging and heart diseases.

PSV-10 Effects of Rumen-Bypass Protein Supplement on Growth Performance, Hepatic Mitochondrial Function and Immune Gene Expression of Beef Steers with Divergent Residual Feed Intake Phenotype

Abstract We examined the effects of rumen-bypass protein supplement on growth performance, hepatic mitochondrial Electron Transport Chain (ETC) activities, and mRNA expression of innate and adaptive immune genes of beef steers with divergent residual feed intake (RFI) phenotype. Thirty-nine crossbred beef steers (average BW = 492 ± 36 kg) were used in a generalized randomized block design with a 2 × 2 factorial arrangement of treatments for a 42-d experimental period. The factors evaluated were 1) RFI classification [low-RFI (-2.12kg/d) vs. high-RFI (2.02kg/d)], and 2) feed additive: rumen-bypass protein supplement (RCP; 227 g· steer-1· day-1) vs. Control diet (CON; 0 g/d), resulting in four treatments: Low-RFI-CON (n = 10), low-RFI-RCP (n = 9), high-RFI-CON (n = 10), and high-RFI-RCP (n = 10). The rumen-bypass protein supplement (84% CP) is a blend of hydrolyzed feather meal, porcine blood meal, and DL-methionine hydroxy analogue. The beef steers were stratified by BW and randomly assigned to treatments and housed in four pens equipped with two GrowSafe feed bunks each to measure individual DMI. Body weight was measured every 7 days. Liver tissue was sampled on d 42 from all animals using a liver biopsy needle for mRNA expression analysis of 84 immune genes and analysis of mitochondrial ETC activities of complexes I – V. No effects of supplemental feed additive or RFI × feed additive interactions were observed (P > 0.05) for ADG and DMI. Compared with high-RFI, low-RFI beef steers tended to have less DMI (13.6 vs 12.9 kg/d; P = 0.09) but ADG was similar for the two RFI groups. Feed additive × RFI interactions were observed (P ≤ 0.05) for mitochondrial activities of Complexes IV, V, and mRNA expressions of some immune genes. Supplemental RCP increased Complex IV (39.2 nmol/min/mg) activity and reduced that of Complex V (94.1 nmol· min-1· mg-1) in high-RFI beef steers whereas the activity of Complex IV (13.1 nmol· min-1· mg-1) was reduced and that of Complex V (120 nmol· min-1· mg-1) was increased in low-RFI beef steers. Supplemental RCP increased (P < 0.05) the mRNA expression of TLR2 [fold change (FC) = 1.17 vs 0.98], TLR3 (FC = 1.49 vs 0.97), NFKB1 (FC = 1.02 vs 0.90), and IL23A (FC = 1.35 vs 1.07) in high-RFI beef steers but the expression of these genes were not altered in low-RFI beef steers. In conclusion, RCP supplementation did not affect the growth performance and the observed effects on hepatic immune gene expression and activities of mitochondrial ETC complexes are dependent on the RFI phenotype of the beef steers. Further research should explore the mechanism responsible for the interactions between RCP supplementation and RFI status.

Lactate activates the mitochondrial electron transport chain independently of its metabolism

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary Tcells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.

222-OR: Metformin Reduces Fasting Glycemia in Well-Controlled Type 2 Diabetes by Promoting Aerobic Glycolysis Independent of Decreasing Endogenous Glucose Production

It is well established that metformin improves glycemic control in individuals with poorly-controlled type 2 diabetes (T2D) by reducing rates of endogenous glucose production (EGP) mediated by inhibition of hepatic gluconeogenesis. However, the mechanism by which metformin reduces blood glucose levels in well-controlled individuals with T2D is still unknown. To address this question, we examined rates of EGP, lactate turnover and hepatic triglyceride (TAG), ATP and glycogen content in near-normoglycemic individuals with T2D (n=10, HbA1c 7±1%) with (M+; 1000 mg bid x 2 wks) and without (M-; 2 wks) metformin treatment using the PINTA method with [2H7]glucose and [3-13C]lactate tracer infusions in combination with 1H- /31P- /13C- magnetic resonance spectroscopy. Metformin treatment did not affect rates of EGP, but reduced fasting plasma glucose concentrations by 30% (p<0.01 vs. M-), which could be attributed to increased rates of glucose clearance (+18%, p<0.005 vs. M-) resulting in increased lactate production (+14%, p<0.01 vs. M-). These changes were associated with decreased hepatic ATP content (-20%, p<0.05 vs. M-), increased hepatic glycogen content (+40%, p<0.05 vs. M-) and no changes in hepatic TAG content. Conclusion: In contrast to the glucose-lowering effect of metformin in poorly-controlled T2D, which is due to reductions in gluconeogenesis, this study demonstrates that in well-controlled T2D metformin reduces fasting glycemia - independent of reductions in EGP - primarily by increasing rates of aerobic glycolysis as reflected by increased peripheral glucose clearance and lactate production as well as reduced hepatic ATP content, which is consistent with metformin-induced inhibition of mitochondrial electron transport chain activity. Disclosure T.Sarabhai: None. T.E.Lamoia: None. S.Friesl: None. M.Jonuscheit: None. K.Petersen: Advisory Panel; Novo Nordisk, Research Support; Merck Sharp & Dohme Corp., Gilead Sciences, Inc. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. M.Roden: Advisory Panel; Eli Lilly and Company, Consultant; TARGET PharmaSolutions, Inc., Research Support; Boehringer-Ingelheim, Novo Nordisk, Novartis, Sanofi.

Cardiomyocyte ZKSCAN3 regulates remodeling following pressure‐overload

Autophagy is important for protein and organelle quality control. Growing evidence demonstrates that autophagy is tightly controlled by transcriptional mechanisms, including repression by zinc finger containing KRAB and SCAN domains 3 (ZKSCAN3). We hypothesize that cardiomyocyte-specific ZKSCAN3 knockout (Z3K) disrupts autophagy activation and repression balance and exacerbates cardiac pressure-overload-induced remodeling following transverse aortic constriction (TAC). Indeed, Z3K mice had an enhanced mortality compared to control (Con) mice following TAC. Z3K-TAC mice that survived exhibited a lower body weight compared to Z3K-Sham. Although both Con and Z3K mice exhibited cardiac hypertrophy after TAC, Z3K mice exhibited TAC-induced increase of left ventricular posterior wall thickness at end diastole (LVPWd). Conversely, Con-TAC mice exhibited decreases in PWT%, fractional shortening (FS%), and ejection fraction (EF%). Autophagy genes (Tfeb, Lc3b, and Ctsd) were decreased by the loss of ZKSCAN3. TAC suppressed Zkscan3, Tfeb, Lc3b, and Ctsd in Con mice, but not in Z3K. The Myh6/Myh7 ratio, which is related to cardiac remodeling, was decreased by the loss of ZKSCAN3. Although Ppargc1a mRNA and citrate synthase activities were decreased by TAC in both genotypes, mitochondrial electron transport chain activity did not change. Bi-variant analyses show that while in Con-Sham, the levels of autophagy and cardiac remodeling mRNAs form a strong correlation network, such was disrupted in Con-TAC, Z3K-Sham, and Z3K-TAC. Ppargc1a also forms different links in Con-sham, Con-TAC, Z3K-Sham, and Z3K-TAC. We conclude that ZKSCAN3 in cardiomyocytes reprograms autophagy and cardiac remodeling gene transcription, and their relationships with mitochondrial activities in response to TAC-induced pressure overload.

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson’s Disease

Multiple evidences suggest that mitochondrial dysfunction is implicated in the pathogenesis of Parkinson's disease via the selective cell death of dopaminergic neurons, such as that which occurs after prolonged exposure to the mitochondrial electron transport chain (ETC) complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP). However, the effects of chronic MPTP on the ETC complexes and on enzymes of lipid metabolism have not yet been thoroughly determined. To face these questions, the enzymatic activities of ETC complexes and the lipidomic profile of MPTP-treated non-human primate samples were determined using cell membrane microarrays from different brain areas and tissues. MPTP treatment induced an increase in complex II activity in the olfactory bulb, putamen, caudate, and substantia nigra, where a decrease in complex IV activity was observed. The lipidomic profile was also altered in these areas, with a reduction in the phosphatidylserine (38:1) content being especially relevant. Thus, MPTP treatment not only modulates ETC enzymes, but also seems to alter other mitochondrial enzymes that regulate the lipid metabolism. Moreover, these results show that a combination of cell membrane microarrays, enzymatic assays, and MALDI-MS provides a powerful tool for identifying and validating new therapeutic targets that might accelerate the drug discovery process.

Comprehensive Analysis of Mitochondrial Dynamics Alterations in Heart Diseases

The most common alterations affecting mitochondria, and associated with cardiac pathological conditions, implicate a long list of defects. They include impairments of the mitochondrial electron transport chain activity, which is a crucial element for energy formation, and that determines the depletion of ATP generation and supply to metabolic switches, enhanced ROS generation, inflammation, as well as the dysregulation of the intracellular calcium homeostasis. All these signatures significantly concur in the impairment of cardiac electrical characteristics, loss of myocyte contractility and cardiomyocyte damage found in cardiac diseases. Mitochondrial dynamics, one of the quality control mechanisms at the basis of mitochondrial fitness, also result in being dysregulated, but the use of this knowledge for translational and therapeutic purposes is still in its infancy. In this review we tried to understand why this is, by summarizing methods, current opinions and molecular details underlying mitochondrial dynamics in cardiac diseases.

Fecal microbiota transplantation and replenishment of short-chain fatty acids protect against chronic cerebral hypoperfusion-induced colonic dysfunction by regulating gut microbiota, differentiation of Th17 cells, and mitochondrial energy metabolism

BackgroundLittle is known about the association between gut microbiota and intestinal injury under a state of chronic cerebral hypoperfusion (CCH). Here, the effects of gut microbiota and short-chain fatty acids (SCFAs), as important metabolic products, on intestinal function and potential mechanisms after CCH were investigated.MethodsRats were subjected to bilateral common carotid artery occlusion (BCCAo) to induce CCH. The gut microbiota and metabolites of SCFAs were assessed by 16S rRNA sequencing and targeted metabolomics, respectively. Transcriptomic analysis of colon tissues was also conducted. Subsequently, potential molecular pathways and differentially expressed genes were verified by western blot, immunoprecipitation, and immunofluorescence analyses. Furthermore, the integrity of the colonic barrier was evaluated by hematoxylin and eosin and mucin 2 staining and expression levels of tight junction proteins. Besides, colonic inflammation was further assessed by flow cytometry and expression levels of inflammatory cytokines. In addition, colonic mitochondrial dysfunction was analyzed via membrane potential, reactive oxygen species, electron transport chain (ETC) activities, adenosine triphosphate content, and mitochondrial ultrastructure.ResultsCCH modified gut microbial composition and microbial metabolism of SCFAs, which may be associated with inhibition of mitochondrial ETC activities and oxidative phosphorylation, leading to dysregulation of mitochondrial energy metabolism. Furthermore, CCH induced differentiation of pathogenic Th17 cells, promoted the formation of complexes of interferon regulatory factor 4 and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation of STAT3. This was associated with an impairment of colonic barrier function and chronic colonic inflammation. In contrast, FMT and SCFA replenishment ameliorated CCH-induced gut microbial dysbiosis by increasing the intestinal content of Ruminococcus_sp_N15_MGS_57 and modulating microbial metabolism of SCFAs by increasing acetic acid contents associated with an improvment of the balance between Tregs and Th17 cells, mitochondrial ETC activities, and oxidative phosphorylation to prevent colonic inflammation and dysregulation of mitochondrial energy metabolism.ConclusionThese findings indicate that FMT and SCFA replenishment present a promising therapeutic strategy against colonic dysfunction under a state of chronic cerebral ischemia.

Antidepressants that increase mitochondrial energetics may elevate risk of treatment-emergent mania

Preclinical evidence suggests that antidepressants (ADs) may differentially influence mitochondrial energetics. This study was conducted to investigate the relationship between mitochondrial function and illness vulnerability in bipolar disorder (BD), specifically risk of treatment-emergent mania (TEM). Participants with BD already clinically phenotyped as TEM+ (n = 176) or TEM− (n = 516) were further classified whether the TEM associated AD, based on preclinical studies, increased (Mito+, n = 600) or decreased (Mito−, n = 289) mitochondrial electron transport chain (ETC) activity. Comparison of TEM+ rates between Mito+ and Mito− ADs was performed using generalized estimating equations to account for participants exposed to multiple ADs while adjusting for sex, age at time of enrollment into the biobank and BD type (BD-I/schizoaffective vs. BD-II). A total of 692 subjects (62.7% female, 91.4% White, mean age 43.0 ± 14.0 years) including 176 cases (25.3%) of TEM+ and 516 cases (74.7%) of TEM- with previous exposure to Mito+ and/or Mito- antidepressants were identified. Adjusting for age, sex and BD subtype, TEM+ was more frequent with antidepressants that increased (24.7%), versus decreased (13.5%) mitochondrial energetics (OR = 2.21; p = 0.000009). Our preliminary retrospective data suggests there may be merit in reconceptualizing AD classification, not solely based on monoaminergic conventional drug mechanism of action, but additionally based on mitochondrial energetics. Future prospective clinical studies on specific antidepressants and mitochondrial activity are encouraged. Recognizing pharmacogenomic investigation of drug response may extend or overlap to genomics of disease risk, future studies should investigate potential interactions between mitochondrial mechanisms of disease risk and drug response.

Targeting DNA methylation can reduce cardiac injury associated with ischemia reperfusion: One step closer to clinical translation with blood-borne assessment.

Ischemia reperfusion (I/R) injury is one of the main clinical challenges for cardiac surgeons. No effective strategies or therapy targeting the molecular and cellular mechanisms to reduce I/R exists to date, despite altered gene expression and cellular metabolism/physiology. We aimed to identify whether DNA methylation, an unexplored target, can be a potential site to curb I/R-associated cell death by using the left anterior descending artery occlusion model in male Wistar rats. I/R rat heart exhibited global DNA hypermethylation with a corresponding decline in the mitochondrial genes (PGC-1α, TFAM, POLG, ND1, ND3, ND4, Cyt B, COX1, and COX2), antioxidant genes (SOD2, catalase, and Gpx2) and elevation in apoptotic genes (Casp3, Casp7, and Casp9) expression with corresponding changes in their activity, resulting in injury. Targeting global DNA methylation in I/R hearts by using its inhibitor significantly reduced the I/R-associated infarct size by 45% and improved dysferlin levels via modulating the genes involved in cell death apoptotic pathway (Casp3, Casp7, and PARP), inflammation (IL-1β, TLR4, ICAM1, and MyD88), oxidative stress (SOD1, catalase, Gpx2, and NFkB) and mitochondrial function and its regulation (MT-ND1, ND3, COX1, ATP6, PGC1α, and TFAM) in the cardiac tissue. The corresponding improvement in the genes’ function was reflected in the respective hearts via the reduction in apoptotic TUNEL positive cells and ROS levels, thereby improving myocardial architecture (H&E staining), antioxidant enzymes (SOD, catalase activity) and mitochondrial electron transport chain activities and ATP levels. The analysis of blood from the I/R animals in the presence and absence of methylation inhibition exhibited a similar pattern of changes as that observed in the cardiac tissue with respect to global DNA methylation level and its enzymes (DNMT and TET) gene expression, where the blood cardiac injury markers enzymes like LDH and CK-MB were elevated along with declined tissue levels. Based on these observations, we concluded that targeting DNA methylation to reduce the level of DNA hypermethylation can be a promising approach in ameliorating I/R injury. Additionally, the blood-borne changes reflected I/R-associated myocardial tissue alteration, making it suitable to predict I/R-linked pathology.

Inner-Membrane-Bound Gold Nanoparticles as Efficient Electron Transfer Mediators for Enhanced Mitochondrial Electron Transport Chain Activity.

Electron transfer through the mitochondrial electron transport chain (ETC) can be critically blocked by the dysfunction of protein complexes. Redox-active molecules have been used to mediate the electron transfer in place of the dysfunctional complexes; however, they are limited to replacing complex I and are known to be toxic. Here we report artificial mitochondrial electron transfer pathways that enhance ETC activity by exploiting inner-membrane-bound gold nanoparticles (GNPs) as efficient electron transfer mediators. The hybridization of mitochondria with GNPs, driven by electrostatic interaction, is successfully visualized in real time at the level of a single mitochondrion. By observing quantized quenching dips via plasmon resonance energy transfer, we reveal that the hybridized GNPs are bound to the inner membrane of mitochondria irrespective of the presence of the outer membrane. The ETC activity of mitochondria with GNPs such as membrane potential, oxygen consumption, and ATP production is remarkably increased in vitro.

Glutamine Metabolism Mediates Sensitivity to Respiratory Complex II Inhibition in Acute Myeloid Leukemia.

This work highlights the effects of direct CII inhibition in mediating selective AML cell death and provides insights into glutamine anaplerosis as a metabolic adaptation that can be therapeutically targeted.

Inhibition of ATR Reverses a Mitochondrial Respiratory Insufficiency.

Diseases that affect the mitochondrial electron transport chain (ETC) often manifest as threshold effect disorders, meaning patients only become symptomatic once a certain level of ETC dysfunction is reached. Cells can invoke mechanisms to circumvent reaching their critical ETC threshold, but it is an ongoing challenge to identify such processes. In the nematode Caenorhabditis elegans, severe reduction of mitochondrial ETC activity shortens life, but mild reduction actually extends it, providing an opportunity to identify threshold circumvention mechanisms. Here, we show that removal of ATL-1, but not ATM-1, worm orthologs of ATR and ATM, respectively, key nuclear DNA damage checkpoint proteins in human cells, unexpectedly lessens the severity of ETC dysfunction. Multiple genetic and biochemical tests show no evidence for increased mutation or DNA breakage in animals exposed to ETC disruption. Reduced ETC function instead alters nucleotide ratios within both the ribo- and deoxyribo-nucleotide pools, and causes stalling of RNA polymerase, which is also known to activate ATR. Unexpectedly, atl-1 mutants confronted with mitochondrial ETC disruption maintain normal levels of oxygen consumption, and have an increased abundance of translating ribosomes. This suggests checkpoint signaling by ATL-1 normally dampens cytoplasmic translation. Taken together, our data suggest a model whereby ETC insufficiency in C. elegans results in nucleotide imbalances leading to the stalling of RNA polymerase, activation of ATL-1, dampening of global translation, and magnification of ETC dysfunction. The loss of ATL-1 effectively reverses the severity of ETC disruption so that animals become phenotypically closer to wild type.

CEST-2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants.

Mitochondrial dysfunction can either extend or decrease Caenorhabditis elegans lifespan, depending on whether transcriptionally regulated responses can elicit durable stress adaptation to otherwise detrimental lesions. Here, we test the hypothesis that enhanced metabolic flexibility is sufficient to circumvent bioenergetic abnormalities associated with the phenotypic threshold effect, thereby transforming short‐lived mitochondrial mutants into long‐lived ones. We find that CEST‐2.2, a carboxylesterase mainly localizes in the intestine, may stimulate the survival of mitochondrial deficient animals. We report that genetic manipulation of cest‐2.2 expression has a minor lifespan impact on wild‐type nematodes, whereas its overexpression markedly extends the lifespan of complex I‐deficient gas‐1(fc21) mutants. We profile the transcriptome and lipidome of cest‐2.2 overexpressing animals and show that CEST‐2.2 stimulates lipid metabolism and fatty acid beta‐oxidation, thereby enhancing mitochondrial respiratory capacity through complex II and LET‐721/ETFDH, despite the inherited genetic lesion of complex I. Together, our findings unveil a metabolic pathway that, through the tissue‐specific mobilization of lipid deposits, may influence the longevity of mitochondrial mutant C. elegans.

Molecular Hydrogen Enhances Proliferation of Cancer Cells That Exhibit Potent Mitochondrial Unfolded Protein Response.

Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.

Abstract P5-05-06: Metformin concentration is a deciding factor of its pro- or anti-tumor function in triple negative breast cancer

Abstract We have previously reported that triple negative (TN) breast cancer (BC), an aggressive subtype of BC, has high dependency for mitochondrial fatty acid oxidation (FAO). We have also reported that FAO activates c-Src, one of the frequently upregulated oncopathways in TNBC. Our mechanism-based mathematical model of cancer metabolism by coupling the master gene regulators (AMPK, HIF-1) suggested that the combination of mitochondrial electron transport chain (ETC) and FAO repressions acquire the highest efficacy in suppressing the bioenergetics (i.e., cellular ATP production) of the TNBC cells. Biguanides such as metformin are one of the most widely administered anti-diabetic agents. Phenformin, a biguanide derivative similar to metformin, has greater potency than metformin. Both these drugs have been widely studied for their antitumor activities but showed only limited clinical advantages in BC. Biguanides can suppress oxidative phosphorylation (OXPHOS) by inhibiting the complex-I activity of mitochondrial ETC. However, biguanides also activate the energy sensor AMPK and phosphorylate its downstream protein ACC. Since ACC inhibits the transport of fatty acids to mitochondria, its phosphorylation can lead to FAO activation. Thus, biguanides can play opposing roles in mitochondrial metabolism (suppression of ETC and activation of FAO). Here we have analyzed the significance of these opposing roles of biguanides in TNBC metabolism and oncopathway activation. Molecular biology and RNASeq analyses suggest that while higher concentrations of biguanides suppress ETC activity and Src pathway despite the activation of AMPK, lower concentrations of biguanides activate FAO-driven ETC and Src pathway in TNBC cells. To understand the translational significance of this observation, we have performed preclinical studies using TNBC organoids and patient-derived xenografts (PDX) models. Combination therapy with metformin and FAO inhibitor, etomoxir significantly improved the antitumor activity compared to the single drug therapy. For a better clinically relevant approach, we did preclinical studies using the combination of metformin and Src inhibitor, dasatinib in TNBC organoids and PDX models. As expected, the combination of metformin and dasatinib synergistically inhibited tumor growth and enhanced the overall survival of the mice. Our observations suggested that the tumor bioavailability of biguanides is critical for its anti-tumor activity, especially in FAO-dependent tumors like TNBC. Using metabolomic, genomic and preclinical experiments, we are currently evaluating the translational potential of this findings in FAO-dependent TNBCs. Citation Format: Jun Hyoung Park, Dongya Jia, Sukjin Yang, Kwang Hwa Jung, Abha Tiwari, Debasmita Dutta, Meron Ghidey, Nagireddy Putluri, Cristian Coarfa, Chad Creighton, José N. Onuchic, Benny A Kaipparettu. Metformin concentration is a deciding factor of its pro- or anti-tumor function in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-06.

Fisetin attenuates renal ischemia/reperfusion injury by improving mitochondrial quality, reducing apoptosis and oxidative stress.

Renal ischemic reperfusion (IR) injury is one of the major source of mortality and morbidity associated with acute kidney injury (AKI). Several flavonoids have shown to be renal protective against many nephrotoxic agents causing AKI. Fisetin, a promising flavonoid, is effective in the management of septic AKI, expected to ameliorate renal IR injury. The present study aimed to generate evidence for fisetin-mediated renal protection against IR injury. Male Wistar rats of 200-250g were subjected to IR protocol by performing bilateral clamping for 45min and reperfusion for 24h. Fisetin was administrated 30min (20mg/kg b.wt, ip) before the surgery. Renal injury was evaluated by measuring the biomarkers in plasma, examining the ultra-structure of the kidney, and analyzing the apoptotic changes. Oxidative stress, antioxidant levels, and mitochondrial function were analyzed in the renal tissue. Fisetin administration significantly reduced the renal damages associated with IR by improving estimated glomerular filtration rate (eGFR: IR-0.35ml/min, F_IR-9.03ml/min), reducing plasma creatinine level (IR-2.2mg/dl, F_IR-0.92mg/dl), and lowering urinary albumin/creatinine ratio (IR-6.09 F_IR-2.16), caspase activity, decreased DNA fragmentation and reduced tubular injury score (IR- 11 F_IR-6.5). At the cellular level, fisetin significantly reduced renal oxidative stress and augmented the antioxidant levels. Fisetin was found to preserve mitochondrial electron transport chain activities and improved the ATP producing capacity in the renal tissue upon IR injury. Fisetin pretreatment attenuates renal IR injury by improving renal function, reducing the renal injury mediated by apoptosis, reducing free radical release, and augmenting mitochondrial function.

Low intensity magnetic fields stimulate the electron transport chain in heart mitochondria

Recent studies suggested that low intensity magnetic fields (MF) exerted a stimulatory effect on the mitochondrial metabolic activity, when applied to the whole animals, organs, or single cells. However, the mechanisms and the ranges of MF strength for this stimulation are still unclear. Here we hypothesize that static low intensity MF can regulate mitochondrial electron transport chain activity as such enhance mitochondrial respiration. Nearly uniform low-strength magnetic fields were generated by Helmholtz coils, which were mounted into the water jacket of the measure chamber of an oxygraph.

The cardiac-enriched microprotein mitolamban regulates mitochondrial respiratory complex assembly and function in mice

Emerging evidence indicates that a subset of RNA molecules annotated as noncoding contain short open reading frames that code for small functional proteins called microproteins, which have largely been overlooked due to their small size. To search for cardiac-expressed microproteins, we used a comparative genomics approach and identified mitolamban (Mtlbn) as a highly conserved 47-amino acid transmembrane protein that is abundantly expressed in the heart. Mtlbn localizes specifically to the inner mitochondrial membrane where it interacts with subunits of complex III of the electron transport chain and with mitochondrial respiratory supercomplexes. Genetic deletion of Mtlbn in mice altered complex III assembly dynamics and reduced complex III activity. Unbiased metabolomic analysis of heart tissue from Mtlbn knockout mice further revealed an altered metabolite profile consistent with deficiencies in complex III activity. Cardiac-specific Mtlbn overexpression in transgenic (TG) mice induced cardiomyopathy with histological, biochemical, and ultrastructural pathologic features that contributed to premature death. Metabolomic analysis and biochemical studies indicated that hearts from Mtlbn TG mice exhibited increased oxidative stress and mitochondrial dysfunction. These findings reveal Mtlbn as a cardiac-expressed inner mitochondrial membrane microprotein that contributes to mitochondrial electron transport chain activity through direct association with complex III and the regulation of its assembly and function.