Measurement Of Oxygen Consumption Rate Research Articles

OR18-02 Mammary Insulin Sensitivity Increases At The Onset Of Lactation And Promotes Metabolic Pathways Supporting Breast Milk Synthesis

Abstract Disclosure: X. Meng: None. H. Rostom: None. A. Fry: None. T. Elajnaf: None. F. Hannan: None. The initiation of lactation during postpartum days 1-4 is hormonally regulated and critical for successful breastfeeding. Insulin represents a key lactogenic hormone as evidenced by women with diabetes mellitus who have delayed lactation onset. However, the role of insulin in lactation and its influence on mammary cells are unclear. We utilised clinical and cellular approaches to investigate this, and recruited n=38 healthy pregnant women (age range 26-42 years) following informed consent and measured serum insulin at 36 weeks’ gestation and during post-partum days 1-4 (1). Serum insulin progressively decreased from 36 weeks’ gestation to day 4 post-partum (244±33pmol/L vs 37±4pmol/L, p<0.0001). Moreover, reverse transcription-quantitative PCR (RT-qPCR) of lactating human mammary epithelial cells (HMECs) isolated from breast milk showed a >20-fold increase in insulin receptor expression compared to non-lactating HMECs (p<0.01, n=4). These findings indicated an increase in both systemic and mammary insulin sensitivity at lactation onset. We hypothesised that insulin promotes lactation by influencing mammary metabolism and used cultured HMECs to evaluate this. Stimulation of HMECs with 10nM insulin caused a >7-fold increase (p=0.001, n=5) in the phosphorylation of Akt, a signalling protein required for initiating lactation. Akt influences oxidative phosphorylation and glycolysis, and we assessed these processes by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. Cultured HMECs treated with 10nM insulin for ≤18hrs showed increased OCR (22±0.9 vs. 17±0.4pmol O2/min/106 cells for control HMECs, p<0.05, n=4) and ECAR (0.23±0.003 vs. 0.18±0.002mpH/min/106 cells for control HMECs, p<0.001, n=4), consistent with increased oxidative phosphorylation and glycolysis. HMECs treated with 10nM insulin for 8hrs had significant upregulation of genes encoding glycolytic enzymes, namely hexokinase 2 (>4-fold increase, p<0.0001, n=4) and pyruvate kinase M1/2 (1.4-fold increase, p<0.05, n=4). However, RT-qPCR analysis of HMECs showed that insulin did not increase expression of genes mediating oxidative phosphorylation, suggesting an Akt-mediated post-transcriptional mechanism. In summary, increased mammary insulin sensitivity together with insulin-stimulated oxidative phosphorylation and glycolysis may support mammary function and milk component synthesis at the onset of lactation. Reference: (1) Rostom, H. et al. Protocol for an observational study investigating hormones triggering the onset of sustained lactation: the INSIGHT study. BMJ Open 12, e062478. Presentation: Saturday, June 17, 2023

SAT649 Org43553, The First Low-molecular-weight Agonist Of The Human Luteinizing Hormone Receptor Induces Leanness And Energy Expenditure In Mice

Abstract Disclosure: D. Lizneva: None. A. Gumerova: None. K. Ievleva: None. O. Barak: None. F. Korkmaz: None. J. Gimenez Roig: None. T. Frolinger: None. G. Pevnev: None. F. Sultana: None. N. Kramskiy: None. S. Wizman: None. M. Orloff: None. A.R. Pallapati: None. S. Rojekar: None. V. Ryu: None. O. Moldavski: None. T. Yuen: None. M. Zaidi: None. We provide compelling evidence that LH is a pro–lean hormone, and that ORG43553, the first low-molecular-weight agonist of the luteinizing hormone receptor (LHCGR) induces leanness and promotes energy expenditure in mice. We detected Lhcgr transcripts in white adipose tissue (WAT) depots in male and female C57BL/6 mice (qPCR, IHC, RNAscope, Sanger sequencing), but not in Lhcgr-/- mice. Intraperitoneal AlexaFluor-488–labeled hCG bound to gonadal and subcutaneous fat pads in wild type mice, but not in Lhcgr-/- mice. Following i.v. 89Zr–LH injection, radioactivity was detected in mesenteric, inguinal and gonadal WAT. LH, hCG and ORG43553 rapidly induced ERK1/2 phosphorylation in differentiated 3T3.L1 cells. Female and male Lhcgr+/- mice on normal chow and high–fat diet, respectively, became obese, importantly, without changes in sex steroids. The gene–dosage effect of Lhcgr depletion suggests a dominant physiologic action of LH on body composition. 14–week–old male C57BL/6 mice were fed on high–fat diet and injected, i.p., with LH, hCG or vehicle, twice–a–week, for 6 weeks. Both LH and hCG markedly reduced body fat accrual (qNMR) but triggered a rise in serum testosterone. The hCG–induced lean phenotype persisted despite androgen receptor blockade by flutamide—confirming that the pro–lean actions of LH and hCG were largely independent of testosterone. Furthermore that LH failed to inhibit fat accrual in Lhcgr-/- mice testified to its action via the LHCGR. Injection of ORG43553 into male C57BL/6 mice for 9 weeks caused a significant reduction in fat mass (qNMR), with reduced WAT weight in fat depots. Remarkably, serum testosterone remained unchanged confirming that the anti–adiposity effect of ORG43553 was independent of testosterone. To study the mechanism of LHCGR activation on body fat, 3T3.L1 adipocytes were treated with hCG or ORG43553 for 12 hours. This resulted in reduced oil droplet accumulation. 3T3-L1–derived organoids treated with LH, hCG or ORG43553 also displayed a substantial reduction in the differentiated layer compared to vehicle–treated organoids. To study whether, in addition to reducing adipocyte differentiation, LHCGR agonism also enhanced thermogenesis, 3T3-L1 adipocytes were pretreated with ORG43553 for 1 hour before measuring oxygen consumption rate (OCR) on a Seahorse Xf96 Analyzer. ORG43553 increased OCR at baseline and upon oligomycin exposure, indicating mitochondria proton leak (thermogenesis). To confirm increased energy expenditure in vivo, mice were injected, s.c., with ORG43553 or vehicle. Oxygen consumption (VO2) and energy expenditure (EE) increased acutely in ORG43553–treated mice, with no change in locomotor activity. The findings suggest that ORG45335, which has been tested in humans for infertility, can potentially become a candidate drug for obesity. Presentation: Saturday, June 17, 2023

Efficient single-cell oxygen consumption rate characterization based on frequency domain fluorescence lifetime imaging microscopy measurement and microfluidic platform.

Cell metabolism is critical in regulating normal cell functions to maintain energy homeostasis. In order to monitor cell metabolism, the oxygen consumption rate (OCR) of cells has been characterized as an important factor. In conventional cell analysis, the cells are characterized in bulk due to technical limitations. However, the heterogeneity between the cells cannot be identified. Therefore, single-cell analysis has been proposed to reveal cellular functions and their heterogeneity. In this research, an approach integrating a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell OCR characterization in an efficient manner is developed. The microfluidic device provides an efficient platform to trap and isolate single cells in microwells with the buffer saline containing an oxygen-sensitive phosphorescent dye. The oxygen tension variation within the microwells can be efficiently estimated by measuring the fluorescence lifetime change using the FD-FLIM, and the OCR values of the single cells can then be calculated. In the experiments, breast cancer (MCF-7) cells are exploited for the OCR measurement. The results demonstrate the functionality of the developed approach and show the heterogeneity among the cells. The developed approach possesses great potential to advance cellular metabolism studies with single-cell resolution.

Energy stress-induced circZFR enhances oxidative phosphorylation in lung adenocarcinoma via regulating alternative splicing

BackgroundCircular RNAs (circRNAs) contribute to multiple biological functions and are also involved in pathological conditions such as cancer. However, the role of circRNAs in metabolic reprogramming, especially upon energy stress in lung adenocarcinoma (LUAD), remains largely unknown.MethodsEnergy stress-induced circRNA was screened by circRNA profiling and glucose deprivation assays. RNA-seq, real-time cell analyzer system (RTCA) and measurement of oxygen consumption rate (OCR) were performed to explore the biological functions of circZFR in LUAD. The underlying mechanisms were investigated using circRNA pull-down, RNA immunoprecipitation, immunoprecipitation and bioinformatics analysis of alternative splicing. Clinical implications of circZFR were assessed in 92 pairs of LUAD tissues and adjacent non-tumor tissues, validated in established patient-derived tumor xenograft (PDTX) model.ResultsCircZFR is induced by glucose deprivation and is significantly upregulated in LUAD compared to adjacent non-tumor tissues, enhancing oxidative phosphorylation (OXPHOS) for adaptation to energy stress. CircZFR is strongly associated with higher T stage and poor prognosis in patients with LUAD. Mechanistically, circZFR protects heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL) from degradation by ubiquitination to regulate alternative splicing, such as myosin IB (MYO1B), and subsequently activates the AKT-mTOR pathway to facilitate OXPHOS.ConclusionOur study provides new insights into the role of circRNAs in anticancer metabolic therapies and expands our understanding of alternative splicing.

Early dynamic changes in iPSC oxygen consumption rate predict future cardiomyocyte differentiation.

Human induced pluripotent stem cells (iPSCs) hold great promise for reducing the mortality of cardiovascular disease by cellular replacement of infarcted cardiomyocytes (CMs). CM differentiation via iPSCs is a lengthy multiweek process and is highly subject to batch-to-batch variability, presenting challenges in current cell manufacturing contexts. Real-time, label-free control quality attributes (CQAs) are required to ensure efficient iPSC-derived CM manufacturing. In this work, we report that live oxygen consumption rate measurements are highly predictive CQAs of CM differentiation outcome as early as the first 72 h of the differentiation protocol with an accuracy of 93%. Oxygen probes are already incorporated in commercial bioreactors, thus methods presented in this work are easily translatable to the manufacturing setting. Detecting deviations in the CM differentiation trajectory early in the protocol will save time and money for both manufacturers and patients, bringing iPSC-derived CM one step closer to clinical use.

Can Single Cell Respiration be Measured by Scanning Electrochemical Microscopy (SECM)?

Ultramicroelectrode (UME), or, equivalently, microelectrode, probes are increasingly used for single-cell measurements of cellular properties and processes, including physiological activity, such as metabolic fluxes and respiration rates. Major challenges for the sensitivity of such measurements include: (i) the relative magnitude of cellular and UME fluxes (manifested in the current); and (ii) issues around the stability of the UME response over time. To explore the extent to which these factors impact the precision of electrochemical cellular measurements, we undertake a systematic analysis of measurement conditions and experimental parameters for determining single cell respiration rates via the oxygen consumption rate (OCR) in single HeLa cells. Using scanning electrochemical microscopy (SECM), with a platinum UME as the probe, we employ a self-referencing measurement protocol, rarely employed in SECM, whereby the UME is repeatedly approached from bulk solution to a cell, and a short pulse to oxygen reduction reaction (ORR) potential is performed near the cell and in bulk solution. This approach enables the periodic tracking of the bulk UME response to which the near-cell response is repeatedly compared (referenced) and also ensures that the ORR near the cell is performed only briefly, minimizing the effect of the electrochemical process on the cell. SECM experiments are combined with a finite element method (FEM) modeling framework to simulate oxygen diffusion and the UME response. Taking a realistic range of single cell OCR to be 1 × 10-18 to 1 × 10-16 mol s-1, results from the combination of FEM simulations and self-referencing SECM measurements show that these OCR values are at, or below, the present detection sensitivity of the technique. We provide a set of model-based suggestions for improving these measurements in the future but highlight that extraordinary improvements in the stability and precision of SECM measurements will be required if single cell OCR measurements are to be realized.

235-OR: Myc Activity Is Required for Mitochondrial Function and Glucose-Stimulated Insulin Secretion (GSIS) in Pancreatic ß-Cells

Glucose enhances mitochondrial function, insulin secretion and Myc expression in β-cells. β-cell-specific Myc knockout mice show glucose intolerance, hypoinsulinemia and lack of adaptive β-cell mass expansion following high-fat diet feeding. However, whether Myc regulates GSIS and mitochondrial function in β-cells is unknown. Here, we tested the effects of the Myc activity inhibitor 10058-F4 (1RH) in GSIS, mitochondrial function and metabolism in islets using islet perifusion, Seahorse and metabolomics/transcriptomics approaches. Mouse and human islets incubated 6h with 40µM 1RH displayed impaired phase 1 and phase 2 insulin secretion induced by 11mM glucose. Insulin secretion was not affected in the presence of 25mM KCl suggesting that Myc is required for glucose- but not membrane depolarization-induced insulin secretion. Since adequate mitochondrial function is essential for GSIS, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to analyze mitochondrial respiration and glycolytic flux in these islets. 1RH significantly reduced OCR, ECAR and glucose-induced ATP production. Conversely, the Myc inducer harmine (10µM), increased basal OCR, ATP production, and ECAR and partially reversed 1RH effects on mitochondrial function. RNAseq of mouse islets treated with 1RH in 11mM glucose revealed reduced expression of both oxidative phosphorylation and β-cell signature genes but enhanced gene expression of glycolysis, ROS, fatty acid metabolism and autophagy/mitophagy pathways. Metabolomics analysis of mouse islets treated with 1RH in 11mM glucose confirmed the reduction in ATP and highlighted the accumulation of L-palmitoylcarnitine and palmitic acid suggesting inefficient β-oxidation. In conclusion, Myc is required for GSIS and mitochondrial function in β-cells. Impaired Myc action can lead to unbalanced metabolism and autophagy/mytophagy leading to β-cell dysfunction. Disclosure G.Lu: None. R.Kang: None. P.Diaz-pozo: None. J.Lee: None. M.Li: None. V.M.Victor: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding National Institutes of Health (R01DK126450)

#5403 PTEN-INDUCED KINASE 1 HAS ASSOCIATION WITH RENAL AGING PROCESS THROUGH THE CGAS -STING PATHWAY

Abstract Background and Aims The global increase of an aging population has encouraged the research on renal aging, as the kidney is one of the organs that shows the greatest change as aging progresses. Although the mechanism of renal aging has not been clearly identified, dysfunctional mitochondria has been suggested as one of the factors that induce inflammation of the kidney, which is the major mediator of the pro-aging process of chronic kidney disease. PTEN-induced kinase 1(PINK1) is a protein involved in the quality control of mitochondria and regulates mitochondrial dysfunction. Although it is known that the mitochondrial DNA release promoted by PINK1 deficiency stimulates cyclic GMP–AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway eventually resulting in the inflammatory response, the role of PINK1 and cGAS-STING pathway in renal aging has not yet been clarified. This study aimed to investigate the relationship between PINK1 and renal aging, especially through the cGAS-STING pathway. Method To determine the role of PINK1 on the renal aging process, renal fibrosis, and tubular injury were compared in 4- and 24-month-old wild-type (Pink1+/+) and PINK1 knockout (Pink1-/-) mice. To establish in vitro senescence model, hydrogen peroxide (H2O2) treatment on human renal proximal cells (HKC-8) was used. The changes in gene expression levels related to PINK1 were analyzed by RNA sequencing, applying transcriptomic and metabolomic analyses. To validate the results of RNA sequencing, we measured mitochondrial oxygen consumption rate (OCR) by Seahorse Mito Stress Test. To investigate the relationship between PINK1 and renal aging through the cGAS-STING pathway, we explored the change of cGAS-STING expression on senescence-induced HKC-8 cells and additionally used H-151 treatment, a specific STING inhibitor. Results The renal fibrosis and tubular injury were significantly aggravated in 24-month-old Pink1-/- mice compared to 24-month-old Pink1+/+ mice. Western blot and RT-qPCR confirmed remarkably increased senescence markers and senescence-associated secretory phenotype (SASPs) in 24-month Pink1-/- mice and senescence-induced HKC-8 cells. The RNA sequencing of mice kidneys showed that inflammation-related pathways significantly increased in 24 months Pink1-/-mice, and transcriptomic and metabolomic analyses showed that PINK1 has an association with mitochondrial metabolism dysregulation. On OCR measurement, the basal respiration, maximal respiration, ATP production, and respiratory capacity significantly declined in H2O2-treated siPINK1 cells, suggesting that PINK1 deficiency might have effects on mitochondrial dysfunction. Finally, the STING pathway was significantly activated in 24-month Pink1-/- mice and senescence-induced HKC-8 cells, which was inhibited by a specific inhibitor of STING, H-151. Conclusion In conclusion, PINK1 is associated with renal aging, and the dysregulation of mitochondria caused by PINK1 deficiency might lead to aging-related inflammatory responses through the cGAS-STING pathway.

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

AimChemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches.MethodsGene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipitation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses.ResultsWe identified IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was significantly associated with resistance to selumetinib, gefitinib, and regorafenib in PDOs and to 5-fluorouracil and oxaliplatin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabilization of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confirmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells.ConclusionsIGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mitochondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance.

PO-01-124 THE CALCIUM HANDLING MACHINERY IS REMODELED IN FRIEDREICH’S ATAXIA

Friedreich's ataxia (FA) is an inherited neurodegenerative disorder that causes progressive nervous system damage. FA is caused by a deficiency in the frataxin protein due to an expansion of GAA repeats in the first intron of the frataxin gene. Reduced frataxin protein expression is thought to negatively affect mitochondrial function, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, it is not completely understood why most patients present with left ventricular systolic dysfunction and die from cardiac events. The objective of our study is to determine whether the abnormal calcium handling machinery is a molecular mechanism that perpetuates cardiac dysfunction in FA. We used the frataxin knock-out (KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. The expression of calcium handling machinery proteins was assessed with proteomics and western blot. In left ventricular myocytes from KO and WT mice, the IonOptix system was used for calcium imaging, the seahorse assay was utilized to measure oxygen consumption rate (OCR) and confocal imaging was used to quantify the mitochondrial membrane potential (Δψm), and reactive oxygen species (ROS). ECG and echocardiography were used to assess cardiac function in the mice. In the proteomic analysis and western blot, SERCA2, Ryr2 and CaMKII were significantly downregulated, and the mitochondrial calcium handling proteins voltage-dependent anion channel and mitochondrial calcium uniporter were significantly upregulated in left ventricular tissue from humans with FA and from KO mice. On the ECG, the RR, PR, QRS, and QTc intervals were significantly longer in the KO mice. Echocardiography showed that the ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the KO mice. Moreover, the calcium transient decay was significantly prolonged in ventricular myocytes from KO mice compared to WT. Additionally, the Δψm was significantly depolarized, the OCR was significantly decreased, and ROS levels were significantly elevated in left ventricular myocytes from KO compared to WT. The development of left ventricular contractile dysfunction in FA is associated with reduced expression of calcium handling proteins, abnormal calcium cycling, and mitochondrial dysfunction.

Sustained mitochondrial dysfunction in male, but not female Spontaneous Hypertensive Rats (SHR) corresponds with delayed renal recovery from ischemic injury

Acute kidney injury (AKI) due to ischemia-reperfusion (IR) is a serious and frequent complication in hospital settings with high mortality rates. The mechanisms mediating renal IR injury and leading to increased risk of later developing cardiovascular and renal diseases in either sex remain poorly understood. We previously reported that there is sex difference in renal recovery from IR where males have a delayed recovery vs females. Mitochondrial dysfunction plays a pivotal role in the pathogenesis of AKI in males, resulting in abnormal kidney repair and disease progression. However, the impact of IR on mitochondrial function in females has not been examined. The goal of the current study was to test the hypothesis that IR results in greater sustained mitochondrial dysfunction in males vs. females corresponds to delayed recovery of renal function in males. 13-week-old male and female SHR were subjected to sham or 30-minute warm bilateral IR. Subsets of rats were euthanized 1- or 7-days post-IR (n=6/group). Blood was collected to measure kidney function. The right kidney was dissected and processed for electron microscopy (EM) to assess mitochondrial structure, histological analysis of renal structure, and measurement of mitochondrial DNA (mtDNA) copy number via RT-PCR. Tubular cells were isolated from the left kidney to measure oxygen consumption rate (OCR) to assess mitochondrial respiration using Seahorse XFe24 Extracellular Flex Analyzer. IR increased plasma creatinine (Pcr) in male and female SHR vs. respective sham controls 1-day post-IR (PIR =0.0001; Psex✕IR=0.2). Pcr remained elevated in male, but not female, SHR 7 days post-IR (Pcr: PIR =0.03; Psex✕IR=0.04). OCR was reduced in male and female tubules 1 day post IR (PIR =0.001; Psex=0.94) and remained reduced only in tubules from males 7 days post-IR (PIR =0.0001; Psex✕IR=0.0001). Consistent with decreased OCR, EM revealed swollen round mitochondria, reduced mitochondria density with complete loss of cristae membranes vs. sham controls 1-day post-IR in both sexes, which was sustained 7 days post-IR only in males. Mitochondrial DNA copy number and peroxisome proliferator-activator receptor-y coactivator (PGC-1α) mRNA expression was also reduced 1-day post-IR in both sexes (PIR =0.05; Psex✕IR=0.59) and remained down only in males 7 days post-IR (PIR =0.02; Psex✕IR=0.001). In conclusion, our data demonstrate that there is sex difference in mitochondria after renal IR. Males have sustained mitochondrial dysfunction with delayed renal recovery compared to females. Further studies will be carried out to better understand the direct impact of mitochondria dysfunction on renal recovery post-IR in male and females. This work was supported by career development grant from AHA (20CDA35310362) to R Mohamed; National Institutes of Health (1P01HL134604-01 and R01 HL127091 to J. C. Sullivan. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase.

The emergence of drug-resistant variants of malaria-causing Plasmodium parasites is a life-threatening problem worldwide. Investigation of the physiological function of individual parasite proteins is a prerequisite for a deeper understanding of the metabolic pathways required for parasite survival and therefore a requirement for the development of novel antimalarials. A Plasmodium membrane protein, malate-quinone oxidoreductase (MQO), is thought to contribute to the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) and is an antimalarial drug target. However, there is little information on its expression and function. Here, we investigated the function of Plasmodium falciparum MQO (PfMQO) in mitochondria using a yeast heterologous expression system. Using a yeast deletion mutant of mitochondrial malate dehydrogenase (MDH1), which is expected to be functionally similar to MQO, as a background strain, we successfully constructed PfMQO-expressing yeast. We confirmed that expression of PfMQO complemented the growth defect of the MDH1 deletion, indicating that PfMQO can adopt the metabolic role of MDH1 in energy transduction for growth in the recombinant yeast. Analysis of cell fractions confirmed that PfMQO was expressed and enriched in yeast mitochondria. By measuring MQO activity, we also confirmed that PfMQO expressed in yeast mitochondria was active. Measurement of oxygen consumption rates showed that mitochondrial respiration was driven by the TCA cycle through PfMQO. In addition, we found that MQO activity was enhanced when intact mitochondria were sonicated, indicating that the malate binding site of PfMQO is located facing the mitochondrial matrix. IMPORTANCE We constructed a model organism to study the physiological role and function of P. falciparum malate-quinone oxidoreductase (PfMQO) in a yeast expression system. PfMQO is actively expressed in yeast mitochondria and functions in place of yeast mitochondrial malate dehydrogenase, which catalyzes the oxidation of malate to oxaloacetate in the TCA cycle. The catalytic site for the oxidation of malate in PfMQO, which is a membrane-bound protein, faces into the mitochondrial matrix, not the mitochondrial inner membrane space. Our findings clearly show that PfMQO is a TCA cycle enzyme and is coupled with the ETC via ubiquinone reduction.

Abstract 5341: Data mining of PubChem bioassay data reveals OXPHOS inhibitors with anti-cancer activity

Abstract High Grade Serous Ovarian Cancer (HGSOC) is a fatal disease that is typically detected at an advanced stage when there are limited modalities for effective treatment. Treatment efficacy against cancer is dependent on tumor metabolism and its adaptation for most therapies. Warburg effect is an important adaptation found in tumors where cells primarily catabolize glucose even in presence of sufficient oxygen and nutrients. Nevertheless, recent evidence suggests cells in tumor microenvironments remain dependent on mitochondrial ATP production via oxidative phosphorylation (OXPHOS). OXPHOS also represents a major metabolic pathway in cancer stem cells and tumor cells of relapse patients. Molecular signatures of glycolysis and OXPHOS are both detected in tumors. This points to the OXPHOS pathway as a potential therapeutic target in cancer. Here we apply a data mining approach to identify OXPHOS inhibitors to evaluate as candidates for therapies against HGSOC. High-Throughput Screening (HTS) on mechanistic targets like OXPHOS are cost prohibitive for academic laboratories. As an alternative, we deployed a data mining pipeline to find active chemical classes among 8415 available OXPHOS-related bioassays in the PubChem data repository. Active compound clusters frequently contain α,β-unsaturated carbonyl groups, which are also observed in both endogenous quinone electron carriers and atovaquone–a potent inhibitor of OXPHOS. Out of 312041 unique compounds tested in the assays, we selected 6 representative molecules from each of the 6 largest active chemical clusters to test for OXPHOS inhibition, ROS production, and reduced cancer cell proliferation. We assayed cell viability (MTT) to check for proliferation of human (OVCAR3, OVCAR5, OVISE) and mouse (ID8) ovarian cancer cells against 6 compounds, Oryzalin, Allylyestrenol, Esbiothrin, Lacidipine, Coumatetralyl, and CID929419 (MLS000713029), 72 h post treatment. ID8 and OVCAR5 significantly decreased cell proliferation after Lacidipine and Oryzalin treatment. Significant increase in intracellular ROS, a frequent consequence of OXPHOS inhibition, was observed in all cell lines after Lacidipine treatment. Elevated ROS was also observed in ID8 and OVISE cells after Esbiothrin treatment. To confirm OXPHOS inhibition as the mechanism of action, we performed Mito Stress Test on the Seahorse Analyzer and measured Oxygen Consumption Rate. Treatment with Oryzalin, Allylyestrenol, Esbiothrin, and Lacidipine significantly decreased basal respiration rate and ATP production in ID8 and OVCAR3 cells. This suggests that 4 of the 6 compound classes prioritized by the data mining pipeline inhibits OXPHOS in ovarian cancer cells. Thus, our pipeline provides an efficient alternative to HTS for identifying OXPHOS inhibitors. Next, we plan to test the in-vivo efficacy of these drugs against ovarian tumors in a mouse model and investigate their molecular mechanisms. Citation Format: Sejal T. Sharma, Liping Feng, Nicha Boonpattrawong, Lisa M. Barroilhet, Manish Patankar, Spencer S. Ericksen. Data mining of PubChem bioassay data reveals OXPHOS inhibitors with anti-cancer activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5341.

Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

ObjectiveMitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis. MethodsIn this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis. ResultsTranscriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy. ConclusionsAhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Abstract P6-11-15: Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which limited targeted therapies are available. Therefore, conventional chemotherapy remains the backbone of standard neoadjuvant treatment (NACT) for TNBC patients. Unfortunately, ~45% of patients will have substantial residual tumor burden post neoadjuvant chemotherapy, leading to poor prognoses (PMID: 28135148). Recently, it has been demonstrated that mitochondrial oxidative phosphorylation (oxphos) is upregulated and is a therapeutic vulnerability in chemoresistant TNBC (PMID: 30996079; Baek et al., BioRxiv doi.org/10.1101/2022.02.25.481996). However, mechanisms driving increased oxphos in chemoresistant TNBC are not understood. Upregulated fatty acid (FA) metabolism is a common adaptation in tumors, providing an energy source through fatty acid β-oxidation (FAO), and promoting lipid accumulation after fatty acid synthesis (FAS) when energy needs are met. Chemotherapy can induce oxidative stress through the generation of reactive oxygen species. Cancer cells adapt to these damaging molecules by increasing de novo lipogenesis, resulting in the accumulation of lipid droplets (LDs) in the cytosol (PMID: 32782526, 20876798). We hypothesize that TNBC cells metabolically adapt to the stress of NACT by upregulating lipid metabolic pathways, providing highly energetic molecules that can be utilized to drive oxphos in chemoresistant TNBC. Methods: Using orthotopic patient-derived xenograft (PDX) models of TNBC (PIM001-P, PMID: 30996079, HCI-010, PMID: 22019887; WHIM14, PMID:24055055), we are measuring protein levels of fatty acid synthase (FASN) in vehicle tumors vs residual tumors surviving treatment with the standard front-line neoadjuvant chemotherapy regimens (Adriamycin plus cyclophosphamide (AC), docetaxel, carboplatin, or docetaxel+carboplatin) using immunohistochemistry (IHC). Vectra 3 microscopy (Akoya) is being used to quantify tumor cell-specific staining. We complemented our IHC analysis with reverse-phase protein array (RPPA). To assess LD accumulation in residual PDX tumors, we conducted transmission electron microscopy (TEM). To complement these PDX studies, we modeled the residual tumor metabolic state in cultured human TNBC cells. Following treatment with the IC50 of standard chemotherapeutic agents (AC, carboplatin, paclitaxel, docetaxel), we assessed oxphos by measuring oxygen consumption rate (OCR) using a Seahorse Bioanalyzer (Agilent). Further, we tested LD accumulation using LipidTOX staining. In ongoing studies, we are measuring incorporation of 13C palmitate into the tricarboxylic acid cycle (TCA) prior to and following chemotherapy treatments to assess if lipids fuel mitochondrial metabolism in residual TNBC cells. Results/Discussion: IHC in the PIM001-P PDX model after in vivo AC treatment revealed increased levels of FASN in post-AC residual tumors compared to the treatment-naive tumors. Further, key proteins involved in fatty acid synthesis, FASN and Acetyl-CoA carboxylase, were significantly increased in residual PIM001-P cells that survived AC compared to vehicle by RPPA. TEM analysis of the HCI-010 PDX revealed significantly more LDs in carboplatin-treated tumors compared to vehicle. This finding was supported by increased LDs observed in TNBC cell lines treated with NACT compared to vehicle in our LipidTOX analyses. Taken together, these data indicate that NACT induces increased expression of key lipid metabolism proteins and accumulation of cytosolic LDs. Our future experiments will reveal if chemoresistant TNBC cells preferentially utilize and incorporate lipids into the tricarboxylic acid cycle, in turn driving oxphos. These data have the potential to provide rationale for the incorporation of FAO/LD inhibitors in sequential combinations with conventional chemotherapies to more effectively kill TNBC cells that are chemo-refractory. Citation Format: Katherine E. Pendleton, Mokryun L. Baek, Junegoo Lee, Lin Tan, Hannah L. Johnson, Lacey E. Dobrolecki, James P. Barrish, Michael T. Lewis, Philip L. Lorenzi, Fabio Stossi, Gloria V. Echeverria. Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-15.

Abstract TMP109: Labeling And Isolating Cell Specific Neuronal Mitochondria And Their Functional Analysis In Mice Post Stroke

Introduction: In vivo neuronal mitochondria dysfunction post stroke has not been well investigated. Methods: Using Mito-tag mice in combine with local infection of AAV9 produced from plasmids carrying Cre-recombinase controlled by neuronal promoters, we generated reporter mice expressing a hemagglutinin (HA)-tagged EGFP that localizes to mitochondrial outer membranes in synaptophysin (SYN) and calmodulin-kinase II (CaMKII) glutamatergic neurons localized to cerebral cortex. These mice were subjected to permanent middle cerebral artery occlusion and sacrificed 14 days post stroke. Fresh brain tissues were excised from the ischemic core (IC), ischemic boundary zone (IBZ) as well as from the homologous contralateral hemisphere, and the crude mitochondrial fractions (CMF) were extracted. Neuronal mitochondria were then isolated from the CMF by anti-HA magnetic beads (Thermo Scientific) and their specificity and oxidative phosphorylation (OXPHOS) complexes were confirmed by means of Western-blot. Seahorse assay was employed to measure oxygen consumption rate (OCR) of the isolated mitochondria as an indicator of mitochondrial function. Results: The bead pulled components were co-precipitated with GFP and enriched with mitochondrial markers, e.g. voltage-dependent anion channel, cytochrome C, and COX IV, but lacked the Golgi protein RCAS1 as well as endoplasmic reticulum markers: Heme-oxygenase 1 and Calnexin, indicating that specific neuronal mitochondria have been selectively isolated. Western-blot data showed that OXPHOS components in SYN + and CAMKII + neuronal mitochondria were significantly downregulated in the IBZ and further decreased in the IC compared to the contralateral tissue, which was associated with the significant reductions of mitochondrial function measured by OCR (p<0.05, respectively, for both neuron types). These data suggesting dysfunction of neuronal mitochondria post stroke. Conclusions: We, for the first time, measure mitochondrial function in 2 types of neurons post stroke. By introducing specific neuronal promoter controlled Cre recombinase to Mito-tag mouse, mitochondria from individual neuronal linage cells can be efficiently labeled and isolated to investigate functional change post stroke.

Immuno-metabolic effect of pancreastatin inhibitor PSTi8 in diet induced obese mice: In vitro and in vivo findings

AimsPancreastatin (PST), an anti-insulin peptide derived from chromogranin A. Its levels increase in cases of obesity, which contributes to adipose tissue inflammation and insulin resistance. This study aims to investigate the immunometabolic effect of PST inhibitor (PSTi8) against PST by using in vitro and in vivo finding. Main methods3T3-L1 cells were differentiated with or without PSTi8, and Oil Red O staining was performed. J774A.1 cells were used for macrophage polarization study. The diet-induced obesity and T2DM model was developed in C57BL/6 mice through high-fat diet for 8 weeks. Alzet osmotic pumps were filled with PSTi8 (release rate: 2 mg/kg/day) and implanted in mice for eight weeks. Further, insulin and glucose tolerance tests were performed. Liver and eWAT sections were stained with hematoxylin and eosin. FACS was used to measure mitochondrial ROS and membrane potential, while Oroboros O2k was used to measure oxygen consumption rate. Immunocytochemistry and qRT-PCR were done for protein and gene expression, respectively. Key findingsPSTi8 inhibited the expression of lipolytic genes and proteins in 3T3-L1 adipocytes. PSTi8 improved the inulin sensitivity, lipid profile, MMP, and OCR levels in the 3T3-L1 adipocyte and eWAT. It also increased the M1 to M2 macrophage polarization in J77A.1 cells and eWAT. Further, PSTi8 attenuated inflammatory CD4+ T, CD8+ T cells and increased the anti-inflammatory T-reg and eosinophil populations in the eWAT. It also reduced the expression of pro-inflammatory genes like Mcp1, Tnfα, and Il-6. SignificanceCollectively, PSTi8 exerted its beneficial effect on adipose tissue inflammation and restored energy expenditure against diet-induced obesity.

Dietary essential amino acids for the treatment of heart failure with reduced ejection fraction.

Heart failure with reduced ejection fraction (HFrEF) is a leading cause of mortality worldwide, requiring novel therapeutic and lifestyle interventions. Metabolic alterations and energy production deficit are hallmarks and thereby promising therapeutic targets for this complex clinical syndrome. We aim to study the molecular mechanisms and effects on cardiac function in rodents with HFrEF of a designer diet in which free essential amino acids-in specifically designed percentages-substituted for protein. Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricle (LV) pressure overload or sham surgery. Whole-body glucose homeostasis was studied with glucose tolerance test, while myocardial dysfunction and fibrosis were measured with echocardiogram and histological analysis. Mitochondrial bioenergetics and morphology were investigated with oxygen consumption rate measurement and electron microscopy evaluation. Circulating and cardiac non-targeted metabolite profiles were analyzed by ultrahigh performance liquid chromatography-tandem mass spectroscopy, while RNA-sequencing was used to identify signalling pathways mainly affected. The amino acid-substituted diet shows remarkable preventive and therapeutic effects. This dietary approach corrects the whole-body glucose metabolism and restores the unbalanced metabolic substrate usage-by improving mitochondrial fuel oxidation-in the failing heart. In particular, biochemical, molecular, and genetic approaches suggest that renormalization of branched-chain amino acid oxidation in cardiac tissue, which is suppressed in HFrEF, plays a relevant role. Beyond the changes of systemic metabolism, cell-autonomous processes may explain at least in part the diet's cardioprotective impact. Collectively, these results suggest that manipulation of dietary amino acids, and especially essential amino acids, is a potential adjuvant therapeutic strategy to treat systolic dysfunction and HFrEF in humans.

Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Muscle Stem Cells Using a Seahorse Analyzer: Applicability for Aging Studies.

In recent years, evidence showing metabolism as a fundamental regulator of stem cell functions has emerged. In skeletal muscle, its stem cells (satellite cells) sustain muscle regeneration, although they lose their regenerative potential with aging, and this has been attributed, at least in part, to changes in their metabolism. In this chapter, we describe a protocol to analyze the metabolism of satellite cells using the Seahorse technology, which can be applied to aging mice.