Mitochondrial Pyruvate Transport Research Articles

Loss of mitochondrial pyruvate transport initiates cardiac glycogen accumulation and heart failure.

Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of U-13C-glucose. Cardiac glycogen was assessed biochemically and by transmission electron microscopy. Cardiac uptake of 2-deoxyglucose was measured and western blotting performed to analyze insulin signaling and enzymatic regulators of glycogen synthesis and degradation. Isotope tracing and glycogen analysis was also performed in hearts from mice fed either low-fat diet or a ketogenic diet previously shown to reverse the CS-Mpc2-/- heart failure. Cardiac glycogen was also assessed in mice infused with angiotensin-II that were fed low-fat or ketogenic diet. Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, yet these hearts displayed increased enrichment from U-13C-glucose and increased glycolytic metabolite pool sizes. 13C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. This increased glycogen synthesis occurred despite enhanced inhibitory phosphorylation of glycogen synthase and reduced expression of glycogenin-1. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic 13C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin-II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Our results indicate that loss of MPC in the heart causes glycogen accumulation and heart failure, while a ketogenic diet can reverse both the glycogen accumulation and heart failure. We conclude that maintaining mitochondrial pyruvate import and metabolism is critical for the heart, unless cardiac pyruvate metabolism is reduced by consumption of a ketogenic diet.

Meteorin-like regulates cardiac hypertrophy and associated metabolic plasticity during gestation and lactation

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de investigación y ciencia de España Introduction Pregnancy-induced cardiac hypertrophy is considered a physiological, reversible process (1, 2). However, cardiac remodeling after delivery and through lactation, as well as mechanisms driving hypertrophy reversion, remain undefined. In recent studies, we described the cardioprotective role of Meteorin-like (Metrnl) during pathological remodeling of the myocardium (3). Its role during gestation and lactation in the maternal heart is completely unknown. Purpose To characterize pregnancy-induced cardiac hypertrophy and its underlying mechanisms, and to determine the role of Metrnl in the cardiac remodeling associated to pregnancy and lactation. Methods C57BL/6J wild-type (WT) and Metrnl knockout (Metrnl-KO) female mice were studied during gestation, lactation and weaning. Echocardiographic measurements and cardiac samples were obtained from virgin control animals (V), pregnant mice at term (E18), lactating females at days 14 and 21 (L14 and L21), and mice 7 and 21 days after L21 weaning (W7 and W21). Results In WT females, pregnancy induces eccentric cardiac hypertrophy characterized by a major dilation of the left ventricle, a phenomenon that is maintained and further increased during lactation, reaching the highest degree of eccentric hypertrophy by L14. After weaning, hypertrophy is totally reversed (Figure 1A). By contrast, Metrnl-KO mice show a more pathological cardiac phenotype characterised by an exacerbated eccentric hypertrophy development accompanied by reduced ejection fraction. Post-weaning resolution is also delayed compared to WT. Next, we analysed cardiac metabolism since during the latest stages of gestation, the heart relies more on fatty acid utilization due to the increased glucose demands of the foetus (Figure 1B). We found that in WT mice, cardiac expression of genes linked to fatty acid oxidation (FAO; Acadm, Pdk4 and Cpt1b) is increased during gestation and repressed during lactation in parallel with cardiac Metrnl expression, recovering basal levels by W7. By contrast, in Metrnl-KO mice mitochondrial pyruvate transporter Mpc1 expression is increased during the whole process, while Pdk4 and Cpt1b protein levels are lower, suggesting a reliance of these hearts on glucose metabolism during pregnancy and post-partum. Conclusions The maternal heart develops an adaptive pregnancy-induced cardiac hypertrophy, a condition that initiates at gestation, peaks by day 14 of lactation and starts reverting upon weaning. Metrnl is necessary for the correct development of cardiac adaptations associated with pregnancy/lactation.Figure 1

Abstract 4426: Targeting MCT1 and PFKFB3 provokes synthetic lethality in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is the third most common lung cancer and is extremely aggressive and highly metastatic. SCLC responds to radiation and standard platinum-based chemotherapy, yet this is invariably followed by relapse and the rapid emergence of lethal chemoresistant disease. Hence, SCLC has been declared a recalcitrant malignancy by NCI, and there is an urgent need to identify new and actionable therapeutic vulnerabilities. Unbiased metabolomic and shotgun and activity-based proteomic analyses of NSCLC and SCLC patient derived tumor tissues, PDX and a panel of cell lines revealed marked upregulation of glycolytic and nucleotide metabolic pathways in SCLC. Further, unbiased screens with metabolic inhibitors revealed selective sensitivity of SCLC cells to the MCT1 lactate transporter inhibitor SR13800 and to the PFKFB3 inhibitor PFK15, and that combined treatment provokes rapid apoptosis. Metabolic, flux and molecular analyses revealed that MCT1 inhibition (MCT1i) or loss in SCLC cells impairs glycolysis and triggers rapid increases in intracellular lactate that then diminish, and this is driven by the induction of lactate dehydrogenase-B (LDH-B) that oxidizes lactate into pyruvate. This is accompanied by a marked shift in the NAD+ to NADH ratio towards NADH, and increased pyruvate dehydrogenase (PDH) activity and OXPHOS. Further, MCT1 inhibition provoked increases in the Aspartate-Malate and DHAP-SnG3P shuttles, which restored the NAD+/NADH balance in the cytosol. In contrast, treatment of SCLC cells with PFKFB3 inhibitors (PFKFB3i) leads to inhibition of PDH activity, a collapse in OXPHOS and increases in lactate efflux that are due to flux of glucose through pentose phosphate pathway and re-entry into glycolysis via 3-phosphoglygerate (3-PGA). Combined MCT1/PFKFB3 inhibition amplifies metabolic alterations provoked by both MCT1i and PFKFB3i, triggering a collapse in both glycolysis and OXPHOS. Similarly, inhibition or silencing of enzymes that drive the Aspartate-Malate and DHAP-SnG3P shuttles, or inhibition of the mitochondrial pyruvate transporter, provoked metabolic collapse and cell death following inhibition or loss of MCT1. Finally, SCLC xenografts and primary SCLC CDX treated with a combination of SR13800 and PFK15 showed improved survival compared to vehicle or monotherapy treatments. Thus, co-targeting adaptive metabolic circuits provoked by inhibition of MCT1 and PFKFB3 provokes synthetic lethality in SCLC, supporting the notion that inhibition of these metabolic chokepoints will be an effective strategy for treatment of this lethal malignancy. Citation Format: Antony Herold Prabhu, Kristen Scott, Paul Stewart, Daniel Grass, Mario Fernandez, Dalia Ercan, John Koomen, Thomas Bannister, Charles Rudin, Gina DeNicola, Eric Haura, John Cleveland. Targeting MCT1 and PFKFB3 provokes synthetic lethality in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4426.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

Glucose-derived glutamate drives neuronal terminal differentiation in vitro

Neuronal maturation is the phase during which neurons acquire their final characteristics in terms of morphology, electrical activity, and metabolism. However, little is known about the metabolic pathways governing neuronal maturation. Here, we investigate the contribution of the main metabolic pathways, namely glucose, glutamine, and fatty acid oxidation, during the maturation of primary rat hippocampal neurons. Blunting glucose oxidation through the genetic and chemical inhibition of the mitochondrial pyruvate transporter reveals that this protein is critical for the production of glutamate, which is required for neuronal arborization, proper dendritic elongation, and spine formation. Glutamate supplementation in the early phase of differentiation restores morphological defects and synaptic function in mitochondrial pyruvate transporter-inhibited cells. Furthermore, the selective activation of metabotropic glutamate receptors restores the impairment of neuronal differentiation due to the reduced generation of glucose-derived glutamate and rescues synaptic local translation. Fatty acid oxidation does not impact neuronal maturation. Whereas glutamine metabolism is important for mitochondria, it is not for endogenous glutamate production. Our results provide insights into the role of glucose-derived glutamate as a key player in neuronal terminal differentiation.

TRAF3-Inactivated Chronic Lymphocytic Leukemia Cells Show an Enhanced Metabolic Plasticity That Can be Attenuated By Glutaminolysis and Mitochondrial Pyruvate Import Inhibition

I ntroduction: TRAF3 gene is located in 14q32.32 and has been identified as a tumor suppressor gene involved in B-cell survival, immune response, and cellular metabolism. TRAF3 is frequently encompassed within 14q deletion and affected by inactivating mutations in 4% of chronic lymphocytic leukemia (CLL) patients. Recently, our group found that CLL patients harboring biallelic TRAF3 inactivation (deletion and mutation) showed a dismal prognosis, with a short time to first treatment (Pérez Carretero, AJH 2022). However, the biological role of TRAF3 alterations in CLL pathogenesis remains to be elucidated. Thus, the aims of this study wereto assess the functional consequences of these alterations and to determine their impact on therapy response. Methods: Previously reported TRAF3 mutations were mimicked in a CRISPR/Cas9-edited CLL model by using the PGA1 cell line (PGA1- TRAF3 mut). Metabolomic (liquid chromatography-mass spectrometry), transcriptomic (RNAseq) and functional analyses were carried out. To evaluate the metabolic specificities of PGA1- TRAF3 mut cells, oxidative phosphorylation (OXPHOS) and glycolytic capacity were studied by assessing the mitochondrial oxygen consumption rate (OCR) and extracellular acidification (ECAR), respectively. Additionally, we assessed the metabolite profile of 25 primary CLL cells (n=3 TRAF3 mut cells, n=22 TRAF3 wt cells) and their response to a library of drugs (CLL drugs and metabolic inhibitors), in the presence and absence of microenvironment (with and without HS-5 co-culture and IL-2 and CpG stimulation). Results: Metabolomic analysis of CRISPR/Cas9-edited CLL models showed an enrichment of metabolites involved in Warburg effect (pyruvate, phosphoenolpyruvate, and lactate), citric acid cycle (TCA- acetyl-CoA, α-ketoglutarate, succinate) and glutamate metabolism (α-ketoglutarate) in PGA1- TRAF3 mut cells. Moreover, PGA1- TRAF3 mut cells exhibited higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis was also slightly enhanced in the TRAF3 mut cells. Remarkably, primary CLL cells with TRAF3 mutations presented a higher concentration of phosphoenolpyruvate, glutamate and the TCA intermediates fumarate and malate, as well as higher levels of NADH and ATP, proving an increased energy production in these cells. Altogether, these results reveal that TRAF3 mut cells showed an enhanced mitochondrial glycolytic metabolism. Given the metabolic changes identified in TRAF3 mut cells, we subsequently assessed their OCR and ECAR after treatment with metabolic inhibitors. Unlike in WT cells, UK5099 administration (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) induced an increase in the maximal respiration of TRAF3 mut cells, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. Therefore, we tested the effect of C968 (glutaminolysis inhibitor), finding the same effect on TRAF3 mut cells, which reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis). Notably, the transcriptomic analysis revealed an upregulation of the glutamine transporter SLC1A4 (p-adj=0.007), what could be favoring glutamine uptake for an enhanced glutaminolysis. Interestingly, by blocking pyruvate import and glutaminolysis simultaneously, a decrease in TRAF3 mut CLL cells proliferation was observed (p=0.03), with equal levels of the previously dysregulated metabolites to those of the WT, which may indicate that this treatment sensitizes TRAF3 mut cells through its relieving effect on metabolic reprogramming. Moreover, primary CLL cells with TRAF3 mutations were also sensitive to the combination, in the presence and absence of microenvironment (p=0.01, p=0.04 respectively), demonstrating the potential of the combination of metabolic inhibitors as a new therapeutic approach in TRAF3 mut patients. Conclusion: Our work identifies a novel role of TRAF3 alterations in the CLL cellular metabolism. Specifically, TRAF3 inactivation induced an increased mitochondrial glycolytic activity and distinct metabolic dependencies in CLL cells, contributing to an enhanced metabolic plasticity (mitochondrial glycolysis/glutaminolysis) potentially targetable in CLL. Funding: PI21/00983 FEHH-GELLC(CPC), JYCL(ARS)

Lysine acetylation regulates mitochondrial pyruvate carrier activity and cardiac pyruvate oxidation

Background/Objective: The normal heart can acutely switch fuel sources based on delivery. While this is beneficial in physiological scenarios, this flexibility is often lost in cardiac pathologies. During fasting, blood glucose levels drop and increased free fatty acid delivery causes the heart to shut off glucose/pyruvate oxidation and increase fat oxidation. This balance, known as the Randle cycle, has mainly been attributed to regulation of pyruvate dehydrogenase (PDH) activity via PDH phosphorylation. Hypothesis: Decreased pyruvate transport into the mitochondria via posttranslational modifications of the mitochondrial pyruvate carrier (MPC) plays a role in regulating cardiac pyruvate oxidation. Methods: Wildtype mice were allowed to consume normal chow ad libitum or were fasted for 24 hours. After euthanasia, plasma and hearts were collected and left-ventricular muscle fibers were saponin permeabilized and mitochondrial respiration measured in an Oroboros Oxygraph O2k. Cardiac protein lysates were immunoprecipitated with anti-acetyl-lysine beads, and western blotted using standard procedures. A bioluminescent resonance transfer (BRET) assay to detect conformational changes caused by pyruvate/inhibitor binding the MPC was used, and lysine mutant MPC2 constructs were generated by site-directed mutagenesis. Lastly, MPC2-/- H9C2 cardiomyocytes were generated by CRISPR, and respiration measured by Seahorse bioanalyzer in these cells after transfection with WT or lysine mutant MPC2 constructs. Results: 24h fasting reduced blood glucose and insulin concentrations, and increased circulating free fatty acids. Cardiac mitochondrial oxidation of pyruvate/malate was decreased, while oxidation of fatty acids (palmitoyl-CoA+carnitine) was increased in fasted hearts. Western blotting of cardiac lysates showed the expected increase in phosphorylation of PDH-E1α in fasted hearts, known to decrease PDH activity. Immunoprecipitating cardiac lysates with anti-acetyl-lysine beads and blotting for MPC2 suggested increased acetylation of MPC2 in fasted hearts. Using a model structure of the MPC, we have recently proposed and validated lysine 49 (K49) of MPC2 as a critical pyruvate binding site within the MPC. To assess the possible importance of K49 acetylation we mutated K49 to glutamine to mimic acetylation and observed that this K49Q-MPC2 mutant was no longer able to bind pyruvate or competitive inhibitors in a MPC BRET assay. We next created MPC2 knockout H9C2 cardiomyocytes by CRISPR-Cas9 and observed complete loss of MPC2 and MPC1 expression in these cells which decreased pyruvate respiration compared to wildtype H9C2s. Overexpression of wildtype MPC1 and MPC2 constructs increased pyruvate respiration in these MPC-/- cells, while overexpression of the MPC2-K49Q mutant was unable to improve pyruvate respiration. Conclusion: Fasting results in decreased cardiac oxidation of pyruvate in part by acetylation of the MPC and decreased mitochondrial pyruvate transport. National Institutes of Health and SLU institutional funding. 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.

The Mitochondrial Pyruvate Carrier Coupling Glycolysis and the Tricarboxylic Acid Cycle Is Required for the Asexual Reproduction of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite capable of infecting humans and animals. The organism has extraordinary metabolic resilience that allows it to establish parasitism in varied nutritional milieus of diverse host cells. Our earlier work has shown that, despite flexibility in the usage of glucose and glutamine as the major carbon precursors, the production of pyruvate by glycolytic enzymes is central to the parasite's growth. Pyruvate is metabolized in a number of subcellular compartments, including the mitochondrion, apicoplast, and cytosol. With the objective of examining the mechanism and importance of the mitochondrial pool of pyruvate imported from the cytosol, we identified the conserved mitochondrial pyruvate carrier (MPC) complex, consisting of two subunits, MPC1 and MPC2, in T. gondii. The two parasite proteins could complement a yeast mutant deficient in growth on leucine and valine. Genetic ablation of either one or both subunits reduced the parasite's growth, mimicking the deletion of branched-chain ketoacid dehydrogenase (BCKDH), which has been reported to convert pyruvate into acetyl-coenzyme A (CoA) in the mitochondrion. Metabolic labeling of the MPC mutants by isotopic glucose revealed impaired synthesis of acetyl-CoA, correlating with a global decrease in carbon flux through glycolysis and the tricarboxylic acid (TCA) cycle. Disruption of MPC proteins exerted only a modest effect on the parasite's virulence in mice, further highlighting its metabolic flexibility. In brief, our work reveals the modus operandi of pyruvate transport from the cytosol to the mitochondrion in the parasite, providing the missing link between glycolysis and the TCA cycle in T. gondii. IMPORTANCE T. gondii is a zoonotic parasite capable of infecting many warm-blooded organisms, including humans. Among others, a feature that allows it to parasitize multiple hosts is its exceptional metabolic plasticity. Although T. gondii can utilize different carbon sources, pyruvate homeostasis is critical for parasite growth. Pyruvate is produced primarily in the cytosol but metabolized in other organelles, such as the mitochondrion and apicoplast. The mechanism of import and physiological significance of pyruvate in these organelles remains unclear. Here, we identified the transporter of cytosol-derived pyruvate into the mitochondrion and studied its constituent subunits and their relevance. Our results show that cytosolic pyruvate is a major source of acetyl-CoA in the mitochondrion and that the mitochondrial pyruvate transporter is needed for optimal parasite growth. The mutants lacking the transporter are viable and virulent in a mouse model, underscoring the metabolic plasticity in the parasite's mitochondrion.

Islet-on-a-chip device reveals first phase glucose-stimulated respiration is substrate limited by glycolysis independent of Ca2+ activity.

Pancreatic islets respond metabolically to glucose by closing KATP channels resulting in Ca2+-influx and insulin secretion. Previous work has revealed the importance of glycolytic flux in triggering insulin secretion. However, it is unclear whether the triggered (‘first phase’) secretion is further amplified by Ca2+-stimulation of mitochondrial NADH production and/or oxidative phosphorylation (OxPhos). Although commercially available tools have been developed to explore islet metabolism, these methods often overlook islet variability and have poor spatiotemporal resolution. To tease apart first phase glucose-stimulated respiration, we designed an islet-on-a-chip microfluidic device to simultaneously measure O2-consumption rate (OCR) and Ca2+-activity of individual islets with high temporal resolution. We used finite element analysis to optimize placement of sensor in optically clear microwells on a thin glass coverslip. The microfluidic channels were subsequently fabricated using O2-impermeable plastic to limit outside-in diffusion and push islets against the microsensor. We validated our device using living mouse islets and well-established modulators of respiration. By inhibiting glycolysis and mitochondrial pyruvate transport, we show that islet OxPhos is limited by NADH-substrate rather than ADP in low and high glucose. We subsequently imaged glucose-stimulated OCR and Ca2+-influx simultaneously to reveal a biphasic respiratory response that is determined by glycolytic flux through pyruvate kinase (PKM2) and independent of Ca2+. These data demonstrate the unique utility of our modular and optically clear O2-sensor to simultaneously measure glucose-stimulated OCR and Ca2+ activity of multiple individual islets.

TRAF3 Alterations Identify a Subgroup of Patients with Adverse Clinical Outcome and Induce Metabolic Reprogramming in Chronic Lymphocytic Leukemia

The TNF receptor-associated factor3 (TRAF3) gene located in 14q32 is frequently altered by deletion and/or mutation in multiple myeloma, lymphoma and other B-cell malignancies such as chronic lymphocytic leukemia (CLL). TRAF3 is a negative regulator of the non-canonical NF-KB signaling, and recent data uncovered additional functions of this gene in inflammation, antiviral immunity or cellular metabolism. Nevertheless, the biological and clinical significance of TRAF3 alterations in CLL is unknown. For that, the aim of this study was to assess the clinical impact of TRAF3 alterations in a CLL cohort as well as its biological role in CLL pathogenesis through CRISPR/Cas9-generated models. TRAF3 alterations (mutations and copy number variations-CNVs) were assessed by NGS in our previously published cohort of 340 CLLs (Pérez Carretero, AJH 2022). A total of 14 patients exhibited a TRAF3 CNV loss, and 10 of them showed additional TRAF3 loss-of-function mutations, indicating a biallelic inactivation of this gene in CLL. Interestingly, the presence of TRAF3 alterations was significantly associated with shorter time to first treatment (6 vs 75 months, p<0.001), being an independent risk factor in the multivariate analysis (p=0.029). To further investigate the biological role of TRAF3, we used the CRISPR/Cas9 system to model TRAF3 loss-of-function mutations in the CLL-derived cell line PGA1, and characterized these cells by transcriptomics, metabolomics and functional studies. RNA-seq analyses identified 54 dysregulated genes enriched in pathways such as NIK/NF-kB signaling (FDR=0.029). Concretely, NIK (a direct target of TRAF3-mediated degradation) was significantly upregulated (Q<0.001) in TRAF3mut clones, which correlated with a higher nuclear activity of the non-canonical NF-kB transcription factors p52 (p=0.05) and RelB (p=0.02). Moreover, we validated these results in primary CLL cells (n=15; 3 TRAF3mut cells). Besides, PGA1-TRAF3mut cells showed increased levels of BAX (p=0.05) and more sensitivity to venetoclax (IC50 3.1 vs 13.8μM, p=0.003). In the metabolomic study, we found an enrichment of metabolites involved in Warburg effect, citric acid cycle (TCA) and glutamate metabolism (FDR<0.001, 0.003, 0.1 resp.), with significant higher levels of α-ketoglutarate (TCA intermediate synthesized from glutamate), pyruvate and lactate (products of glycolysis) (p<0.001, p<0.001, p=0.002) in PGA1-TRAF3mut cells. To better analyze their metabolic specificities, we evaluated the mitochondrial oxygen consumption rate (OCR) extracellular acidification (ECAR) and cell viability at basal state and in response to metabolic inhibitors. Of note, PGA1-TRAF3mut cells exhibited distinct mitochondrial characteristics: higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis and glycolytic reserve were also enhanced in the TRAF3mut cells. In this way, they were more sensitive to 2-deoxy-D-glucose (glucose uptake inhibitor), and more resistant to oxamate (lactate-dehydrogenase inhibitor, marker of anaerobic glycolysis). Overall, these results indicate, unlike WT, TRAF3mut cells instead utilize a mitochondrial glycolytic metabolism. Surprisingly, by using UK5099 (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) mutant cells showed higher maximal respiration levels than WT, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. We therefore tested the effect of C968 (glutaminolysis inhibitor) and we found that TRAF3mut cells were also more resistant to this drug, what reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis) in the PGA1-TRAF3mut cells that does not exist in the WT. Interestingly, the combined treatment (UK5099 and C968) induced more cell death in the TRAF3mut than in the WT cells (p=0.03), suggesting a new potential opportunity to treat patients with TRAF3 alterations. In conclusion, TRAF3 deficiency appeared to be an independent prognostic factor of time to first treatment in CLL. This alteration results in a non-canonical NF-KB activation and in a metabolic reprogramming towards an enhanced mitochondrial glycolysis and glutaminolysis, which could be potentially targetable in CLL patients. Fund.: PI21/00983 FI19/00191 FEHH

Abstract 12: Social isolation promotes mammary cancer recurrence, increases IL6/JAK/STAT3 signaling and causes mitochondrial dysfunction in tamoxifen treated rats, and a traditional herbal mixture reverses all these changes

Abstract Social isolation is associated with increased risk and mortality from many diseases, such as breast cancer. Socially isolated breast cancer survivors have a 43% higher risk of recurrence and a 64% higher risk of breast cancer-specific mortality than socially integrated survivors. Since Covid-19 has dramatically increased the incidence of social isolation, it is important to determine if social isolation affects the response to endocrine therapy and/or recurrence after the therapy is completed. Since previous studies indicate that social isolation increases circulating inflammatory cytokines, we investigated if an anti-inflammatory herbal mixture Jaeumkanghwa-tang (JGT) prevents the adverse effects of social isolation on breast cancer mortality. Estrogen receptor positive mammary tumors were initiated with 7,12-dimethylbenz[a]anthracene. When a rat developed a palpable mammary tumor, it was either socially isolated (SI) by housing it singly or a rat was allowed to remain group-housed (GH). Tamoxifen (340ppm via diet) or tamoxifen + JGT (500ppm via drinking water) started when the first mammary tumor reached a size of 11 mm in diameter. Tamoxifen administration ended when a complete response to this therapy had lasted for 9 weeks (corresponds to 5 years in women). During tamoxifen therapy, social isolation non-significantly reduced the rate of complete responses to 21%, from 31% in GH group (p&amp;gt;0.05). After the therapy was completed, SI significantly increased local mammary tumor recurrence (p&amp;lt;0.001; 45% GH vs 75% SI). RNAseq analysis was performed in the mammary glands. Gene set enrichment analysis (GSEA) of transcriptome showed that the increased recurrence risk in socially isolated rats was associated with an enrichment of IL6/JAK/STAT3 signaling: this result was confirmed in the tumors. In addition, oxidative phosphorylation (OXPHOS) pathway was suppressed: the suppressed genes included those involved in mitochondrial pyruvate transport and conversion of pyruvate to acetyl CoA as well as genes in the TCA cycle and mediating electron transport in mitochondrial complexes I-IV. Social isolation also increased the expression of inflammatory receptor for advanced glycation end-products (RAGE) (p≤0.05). Consumption of an anti-inflammatory JGT inhibited IL6/JAK/STAT3 signaling, upregulated OXPHOS signaling and prevented the increased risk of mammary cancer recurrence in socially isolated animals. The percentage of recurrences in the SI rats dropped from 75% without JGT to 22% with JGT (p&amp;lt;0.001). Breast cancer mortality among socially isolated survivors may be most effectively prevented by focusing on the period following endocrine therapy using tools that inhibit IL6/JAK/STAT3 inflammatory cytokine signaling and correct disrupted OXPHOS and mitochondrial dysfunction. Citation Format: Fabia de Oliveira Andrade, Lu Jin, Robert Clarke, Imani Wood, MaryAnn Dutton, Chezaray Anjorin, Grace Rubin, Audrey Gao, Surojeet Sengupta, Kevin FitzGerald, Leena Hilakivi-Clarke. Social isolation promotes mammary cancer recurrence, increases IL6/JAK/STAT3 signaling and causes mitochondrial dysfunction in tamoxifen treated rats, and a traditional herbal mixture reverses all these changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 12.

Phenylketonuria oxidative stress and energy dysregulation: Emerging pathophysiological elements provide interventional opportunity.

Phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is rightfully considered the paradigm treatable metabolic disease. Dietary substrate restriction (i.e. phenylalanine (Phe) restriction) was applied >60 years ago and remains the primary PKU management means. The traditional model of PKU neuropathophysiology dictates blood Phe over-representation directs asymmetric blood:brain barrier amino acid transport through the LAT1 transporter with subsequent increased cerebral Phe concentration and low concentrations of tyrosine (Tyr), tryptophan (Trp), leucine (Leu), valine (Val), and isoleucine (Ile). Low Tyr and Trp concentrations generate secondary serotonergic and dopaminergic neurotransmitter paucities, widely attributed as drivers of PKU neurologic phenotypes. White matter disease, a central PKU characteristic, is ascribed to Phe-mediated tissue toxicity. Impaired cerebral protein synthesis, by reduced concentrations of non-Phe large neutral amino acids, is another cited pathological mechanism. The PKU amino acid transport model suggests Phe management should be more efficacious than is realized, as even early identified, continuously treated patients that retain therapy compliance into adulthood, demonstrate neurologic disease elements. Reduced cerebral metabolism was an early-recognized element of PKU pathology. Legacy data (late 1960's to mid-1970's) determined the Phe catabolite phenylpyruvate inhibits mitochondrial pyruvate transport. Respirometry of Pahenu2 cerebral mitochondria have attenuated respiratory chain complex 1 induction in response to pyruvate substrate, indicating reduced energy metabolism. Oxidative stress is intrinsic to PKU and Pahenu2 brain tissue presents increased reactive oxygen species. Phenylpyruvate inhibits glucose-6-phosphate dehydrogenase that generates reduced niacinamide adenine dinucleotide phosphate the obligatory cofactor of glutathione reductase. Pahenu2 brain tissue metabolomics identified increased oxidized glutathione and glutathione disulfide. Over-represented glutathione disulfide argues for reduced glutathione reductase activity secondary to reduced NADPH. Herein, we review evidence of energy and oxidative stress involvement in PKU pathology. Data suggests energy deficit and oxidative stress are features of PKU pathophysiology, providing intervention-amenable therapeutic targets to ameliorate disease elements refractory to standard of care.

Targeting temporal metabolic needs of T follicular helper cells to treat cGVHD utilizing a mitochondrial pyruvate carrier inhibitor

Abstract The curative potential of allogeneic hematopoietic stem cell transplantation is limited by development of chronic graft versus host disease (cGVHD) and metabolic regulation of immune cells is a promising therapeutic target. During cGVHD, interactions between T follicular helper (TFH) and germinal center B (GCB) cells result in production of alloreactive class-switched antibodies that attack host tissues, causing multi-organ damage and tissue fibrosis. We show the metabolic dependencies of TFH change throughout cGVHD progression and targeting mitochondrial pyruvate transport is an effective strategy to mitigate disease. Pyruvate, the end product of glycolysis, is transported by the mitochondrial pyruvate carrier (MPC) for use in the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS). TFH require glycolysis in order to induce a GC reaction, both in an immunization model and in an in vivo systemic cGVHD model with bronchiolitis obliterans (BO). Furthermore, Seahorse data from TFH extracted early in disease development in the BO model demonstrate increased dependency on glycolysis. But Seahorse data also indicates TFH undergo a shift away from glycolysis and toward OXPHOS as cGVHD continues. Based on the central role of MPC in tying pyruvate to multiple metabolic pathways, we examined an inhibitor of MPC (7ACC1) in reducing TFH activity. Administration of 7ACC1 resulted in inhibition of GCB production of class switched antibody in an in vitro assay. These findings were additionally supported in vivo, whereby daily treatment after establishment of cGVHD resulted in a decrease in disease severity. These data provide evidence that inhibiting mitochondrial pyruvate transport is a viable therapeutic strategy for cGVHD. This work was supported by grants from National Institutes of Health, National Institute of Allergy, and Infectious Diseases R37 AI34495, T32 AI007313; National Heart, Lung, and Blood Institute R01 HL56067, R01 HL11879, R01 HL115114 ; National Cancer Institute P01 CA065493

Abstract P4-02-13: Social isolation promotes mammary cancer recurrence, increases IL6/JAK/STAT3 signaling and causes mitochondrial dysfunction in tamoxifen treated rats, and a traditional herbal mixture reverses all these changes

Abstract BACKGROUND: Social isolation is associated with increased risk and mortality from many diseases, such as breast cancer. Socially isolated breast cancer survivors have a 43% higher risk of recurrence and a 64% higher risk of breast cancer-specific mortality than socially integrated survivors. Since Covid-19 has dramatically increased the incidence of social isolation, it is important to determine if social isolation affects the response to endocrine therapy and/or recurrence after the therapy is completed. Further, since previous studies indicate that social isolation increases circulating inflammatory cytokines, we investigated if an anti-inflammatory herbal mixture Jaeumkanghwa-tang (JEKHT) prevents the adverse effects of social isolation on breast cancer mortality. METHODS: Estrogen receptor positive mammary tumors were initiated with 7,12-dimethylbenz[a]anthracene. When a rat developed a palpable mammary tumor, it was either socially isolated (SI) by housing it singly or a rat was allowed to remain group-housed (GH). Tamoxifen (340ppm via diet) or tamoxifen + JEKHT (500ppm via drinking water) started when the first mammary tumor was detected. Tamoxifen administration ended when a complete response to this therapy had lasted for 9 weeks (corresponds to 5 years in women). Rats which received JEKHT during tamoxifen therapy continued to take it also after tamoxifen therapy ended. RESULTS: During tamoxifen therapy, social isolation non-significantly reduced the rate of complete responses to 21%, from 31% in GH group (p&amp;gt;0.05). After the therapy was completed, SI significantly increased local mammary tumor recurrence (p&amp;lt;0.001; 45% GH vs 75% SI). RNAseq analysis was performed in the mammary glands, since only tamoxifen resistant or recurring mammary tumors were available for analysis. Gene set enrichment analysis (GSEA) of transcriptome showed that the increased recurrence risk in socially isolated rats was associated with an enrichment of IL6/JAK/STAT3 signaling: this result was confirmed in the tumors. In addition, oxidative phosphorylation (OXPHOS) pathway was suppressed: the suppressed genes included those involved in mitochondrial pyruvate transport and conversion of pyruvate to acetyl CoA as well as genes in the TCA cycle and mediating electron transport in mitochondrial complexes I-IV. Despite the reduction in pyruvate transporting and converting genes, lactate levels or lactate dehydrogenase activity were not increased in socially isolated rats, indicating no changes in Warburg effect. Social isolation also increased the expression of inflammatory receptor for advanced glycation end-products (RAGE) (p≤0.05). Consumption of an anti-inflammatory JEKHT inhibited IL6/JAK/STAT3 signaling, upregulated OXPHOS signaling and prevented the increased risk of mammary cancer recurrence in socially isolated animals. The percentage of recurrences in the SI rats dropped from 75% without JEKHT to 22% with JEKHT (p&amp;lt;0.001). CONCLUSION: Breast cancer mortality among socially isolated survivors may be most effectively prevented by focusing on the period following endocrine therapy using tools that inhibit IL6/JAK/STAT3 inflammatory cytokine signaling and correct disrupted OXPHOS and mitochondrial dysfunction. Citation Format: Fabia Andrade, Lu Jin, Robert Clarke, Imani Wood, MaryAnn Dutton, Chezaray Anjorin, Grace Rubin, Audrey Gao, Surojeet Sengupta, Kevin FitzGerald, Leena Hilakivi-Clarke. Social isolation promotes mammary cancer recurrence, increases IL6/JAK/STAT3 signaling and causes mitochondrial dysfunction in tamoxifen treated rats, and a traditional herbal mixture reverses all these changes [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 P4-02-13.

Branched-chain amino acid metabolism regulates gluconeogenesis via mitochondrial pyruvate transport

Branched-chain amino acid metabolism regulates gluconeogenesis via mitochondrial pyruvate transport

Thyroid hormones and the potential for regulating glucose metabolism in cardiomyocytes during insulin resistance and T2DM.

In order for the heart to maintain its continuous mechanical work and provide the systolic movement to uphold coronary blood flow, substantial synthesis of adenosine triphosphate (ATP) is required. Under normal conditions cardiac tissue utilizes roughly 70% fatty acids (FA), and 30% glucose for the production of ATP; however, during impaired metabolic conditions like insulin resistance and diabetes glucose metabolism is dysregulated and FA account for 99% of energy production. One of the major consequences of a shift in FA metabolism in cardiac tissue is an increase in reactive oxygen species (ROS) and lipotoxicity, which ultimately lead to mitochondrial dysfunction. Thyroid hormones (TH) have direct effects on cardiac function and glucose metabolism during impaired metabolic conditions suggesting that TH may improve glucose metabolism in an insulin resistant condition. None‐classical TH signaling in the heart has shown to phosphorylate protein kinase B (Akt) and increase activity of phosphoinositide‐3‐kinase (PI3K), which are critical mediators in the insulin‐stimulated glucose uptake pathway. Studies on peripheral tissues such as skeletal muscle and adipocytes have demonstrated TH treatment improved glucose intolerance in a diabetic model and increased insulin‐regulated glucose transporter (GLUT4) mRNA levels. GLUT4 is a downstream target of thyroid response element (TRE), which demonstrates that THs regulate glucose via GLUT4. Elevated 3,5,3′‐triiodothyronine (T3) increased glucose oxidation rate and decreased the glycolytic intermediate, fructose 6‐phosphate (F6P) in cardiomyocytes, in addition to increasing mitochondrial biogenesis and pyruvate transport across the mitochondrial membrane. These findings along with a few other studies on T3 treatment in cardiac tissue suggest TH may improve glucose metabolism in an insulin resistant model and ameliorate the effects of diabetes and metabolic syndrome. This review highlights the potential benefits of exogenous TH on ameliorating metabolic dysfunction in the heart.

Lactic Acid Fermentation Is Required for NLRP3 Inflammasome Activation.

Activation of the Nod-like receptor 3 (NLRP3) inflammasome is important for activation of innate immune responses, but improper and excessive activation can cause inflammatory disease. We previously showed that glycolysis, a metabolic pathway that converts glucose into pyruvate, is essential for NLRP3 inflammasome activation in macrophages. Here, we investigated the role of metabolic pathways downstream glycolysis – lactic acid fermentation and pyruvate oxidation—in activation of the NLRP3 inflammasome. Using pharmacological or genetic approaches, we show that decreasing lactic acid fermentation by inhibiting lactate dehydrogenase reduced caspase-1 activation and IL-1β maturation in response to various NLRP3 inflammasome agonists such as nigericin, ATP, monosodium urate (MSU) crystals, or alum, indicating that lactic acid fermentation is required for NLRP3 inflammasome activation. Inhibition of lactate dehydrogenase with GSK2837808A reduced lactate production and activity of the NLRP3 inflammasome regulator, phosphorylated protein kinase R (PKR), but did not reduce the common trigger of NLRP3 inflammasome, potassium efflux, or reactive oxygen species (ROS) production. By contrast, decreasing the activity of pyruvate oxidation by depletion of either mitochondrial pyruvate carrier 2 (MPC2) or pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) enhanced NLRP3 inflammasome activation, suggesting that inhibition of mitochondrial pyruvate transport enhanced lactic acid fermentation. Moreover, treatment with GSK2837808A reduced MSU-mediated peritonitis in mice, a disease model used for studying the consequences of NLRP3 inflammasome activation. Our results suggest that lactic acid fermentation is important for NLRP3 inflammasome activation, while pyruvate oxidation is not. Thus, reprograming pyruvate metabolism in mitochondria and in the cytoplasm should be considered as a novel strategy for the treatment of NLRP3 inflammasome-associated diseases.

Metabolic Engineering of Saccharomyces cerevisiae for Ethyl Acetate Biosynthesis

Ethyl acetate can be synthesized from acetyl-CoA and ethanol via a reaction by alcohol acetyltransferases (AATase) in yeast. In order to increase the yield of acetyl-CoA, different terminators were used to optimize the expressions of acetyl-CoA synthetase (ACS1/2) and aldehyde dehydrogenase (ALD6) to increase the contents of acetyl-CoA in Saccharomyces cerevisiae. ATF1 coding AATase was coexpressed in expression cassettes of ACS1/ACS2 and ALD6 to promote the carbon flux toward ethyl acetate from acetyl-CoA. Further to improve ethyl acetate production, four heterologous AATase including HuvEAT1 (Hanseniaspora uvarum), KamEAT1 (Kluyveromyces marxianus), VAAT (wild strawberry), and AeAT9 (kiwifruit) were introduced. Subsequently mitochondrial transport and utilization of pyruvate and acetyl-CoA were impeded to increase the ethyl acetate accumulation in cytoplasm. Under the optimal fermentation conditions, the engineered strain of PGAeΔPOR2 produced 1.69 g/L ethyl acetate, which was the highest value reported to date by metabolic engineering methods.

TAMI-38. CYSTEINE-PROMOTING COMPOUNDS INDUCE MITOCHONDRIAL TOXICITY IN GLIOBLASTOMA THROUGH ALTERED PYRUVATE AND SERINE METABOLISM

Abstract Glioblastoma (GBM) remains a poorly treatable disease with high mortality. Tumor metabolism in GBM is a critical mechanism responsible for accelerated growth because of upregulation of glucose, amino acid, and fatty acid utilization. However, little is known about the metabolic alterations that are specific to GBM and that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated the TCGA and a cancer metabolite database for alterations in glucose and amino acid signatures in GBM relative to other human cancers and relative to low-grade glioma. From these analyses, we found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers and that GBM exhibits high levels of cysteine-related metabolites compared to low-grade gliomas. To study the role of cysteine in GBM pathogenesis, we treated patient-derived GBM cells with a variety of FDA-approved cyst(e)ine-promoting compounds in vitro, including N-acetylcysteine (NAC) and the cephalosporin antibiotic, Ceftriaxone (CTX), which induces cystine import through System Xc transporter upregulation. Cysteine-promoting compounds, including NAC and CTX, inhibit growth of GBM cells, which is exacerbated by glucose deprivation. This growth inhibition is associated with reduced mitochondrial metabolism, manifest by reduction in ATP, NADPH/NADP+ ratio, mitochondrial membrane potential, and oxygen consumption rate. Metabolic tracing experiments with 13C6-glucose demonstrate that L-serine is rapidly depleted in GBM cells upon treatment with NAC and CTX, and exogenous serine rescues NAC- and CTX-mediated cell growth inhibition. In addition, these compounds reduce GBM mitochondrial pyruvate transport. We show that cysteine-promoting compounds reduce cell growth and induce mitochondrial toxicity in GBM, which may be due to rapid serine depletion and reduced mitochondrial pyruvate transport. This metabolic phenotype is exacerbated by glucose deprivation. This pathway is targetable with FDA-approved cysteine-promoting compounds and could synergize with glucose-lowering treatments, including the ketogenic diet, for GBM.

Enhanced Production of Ethyl Lactate in Saccharomyces cerevisiae by Genetic Modification.

Ethyl lactate is an important flavor substance in baijiu, and it is also one of the common raw materials in the production of flavors and spices. In this study, we first established the ethyl lactate biosynthesis pathway in Saccharomyces cerevisiae α(L) by introducing propionyl coenzyme A transferase (Pct) and alcohol acyltransferase (AAT), and the results showed that strain α(L)-CP-Ae produced the most ethyl lactate 239.53 ± 5.45 mg/L. Subsequently, the copy number of the Pctcp gene and AeAT9 gene was increased, and the modified strain α(L)-tCP-tAe produced 346.39 ± 3.99 mg/L ethyl lactate. Finally, the porin gene (por2) and the mitochondrial pyruvate carrier gene (MPC2) were knocked to impede mitochondrial transport of pyruvate, and the final modified strain α(L)-tCP-tAeΔpor2 produced ethyl lactate 420.48 ± 6.03 mg/L.