Mitochondrial Pyruvate Oxidation Research Articles

Disruption of mitochondrial pyruvate oxidation in dorsal root ganglia drives persistent nociceptive sensitization and causes pervasive transcriptomic alterations.

Metabolism is inextricably linked to every aspect of cellular function. In addition to energy production and biosynthesis, metabolism plays a crucial role in regulating signal transduction and gene expression. Altered metabolic states have been shown to maintain aberrant signaling and transcription, contributing to diseases like cancer, cardiovascular disease, and neurodegeneration. Metabolic gene polymorphisms and defects are also associated with chronic pain conditions, as are increased levels of nerve growth factor (NGF). However, the mechanisms by which NGF may modulate sensory neuron metabolism remain unclear. This study demonstrated that intraplantar NGF injection reprograms sensory neuron metabolism. Nerve growth factor suppressed mitochondrial pyruvate oxidation and enhanced lactate extrusion, requiring 24 hours to increase lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 (PDHK1) expression. Inhibiting these metabolic enzymes reversed NGF-mediated effects. Remarkably, directly disrupting mitochondrial pyruvate oxidation induced severe, persistent allodynia, implicating this metabolic dysfunction in chronic pain. Nanopore long-read sequencing of poly(A) mRNA uncovered extensive transcriptomic changes upon metabolic disruption, including altered gene expression, splicing, and poly(A) tail lengths. By linking metabolic disturbance of dorsal root ganglia to transcriptome reprogramming, this study enhances our understanding of the mechanisms underlying persistent nociceptive sensitization. These findings imply that impaired mitochondrial pyruvate oxidation may drive chronic pain, possibly by impacting transcriptomic regulation. Exploring these metabolite-driven mechanisms further might reveal novel therapeutic targets for intractable pain.

Artemether regulates liver glycogen and lipid utilization through mitochondrial pyruvate oxidation in db/db mice

Artemether regulates liver glycogen and lipid utilization through mitochondrial pyruvate oxidation in db/db mice

Decreased Pyruvate but Not Fatty Acid Driven Mitochondrial Respiration in Skeletal Muscle of Growth Restricted Fetal Sheep.

Fetuses with intrauterine growth restriction (FGR) have impaired oxidative and energy metabolism, with persistent consequences on their postnatal development. In this study, we test the hypothesis that FGR skeletal muscle has lower mitochondrial respiration rate and alters the transcriptomic profiles associated with energy metabolism in an ovine model. At late gestation, mitochondrial oxygen consumption rates (OCRs) and transcriptome profiles were evaluated in the skeletal muscle collected from FGR and control fetuses. The ex vivo mitochondrial OCRs were reduced (p < 0.01) in permeabilized FGR soleus muscle compared to the control muscle but only with pyruvate as the metabolic substrate. Mitochondrial OCRs were similar between the FGR and control groups for palmitoyl-carnitine (fatty acid-driven) or pyruvate plus palmitoyl-carnitine metabolic substrates. A total of 2284 genes were differentially expressed in the semitendinosus muscle from growth restricted fetuses (false discovery rate (FDR) ≤ 0.05). A pathway analysis showed that the upregulated genes (FGR compared to control) were overrepresented for autophagy, HIF-1, AMPK, and FOXO signaling pathways (all with an FDR < 0.05). In addition, the expression of genes modulating pyruvate's entry into the TCA cycle was downregulated, whereas the genes encoding key fatty acid oxidation enzymes were upregulated in the FGR muscle. These findings show that FGR skeletal muscle had attenuated mitochondrial pyruvate oxidation, possibly associated with the inability of pyruvate to enter into the TCA cycle, and that fatty acid oxidation might compensate for the attenuated energy metabolism. The current study provided phenotypic and molecular evidence for adaptive deficiencies in FGR skeletal muscle.

1510-P: Lipid-Induced Renal Cortical Insulin Resistance Perturbs Gluconeogenic and Oxidative Metabolism via an sn-1,2-diacylglycerol-PKCe-Insulin Receptor Kinase Axis In Vivo

Recently, our group demonstrated that 3-week high fat diet feeding (HFD) impairs insulin signaling in the renal cortex at the level of the insulin receptor kinase (IRK). Specifically, IRKY1162 and AKTS473 phosphorylation were reduced by ~50% during hyperinsulinemic-euglycemic clamp (HEC); however, it is unknown how renal cortical insulin resistance (IR) may affect gluconeogenic (GNG) and oxidative metabolism. To address this, here we measure renal cortical GNG fluxes in vivo using Q-Flux. In regular chow (RC) fed mice, hyperinsulinemia significantly suppressed mitochondrial pyruvate carboxylase (PC), as PC flux relative to α-ketoglutarate dehydrogenase flux (VPC/VOGDH) was 1.25 under basal conditions, but decreased by ~50%, to 0.57 during HEC. Similarly, PEPCK flux relative OGDH flux (VPEPCK/VOGDH) was reduced by ~60% during HEC. In contrast, insulin failed to suppress both PC and PEPCK fluxes with HFD. Using a [13C6]glucose tracer, we also find insulin-stimulated mitochondrial pyruvate oxidation to be impaired by ~30% with HFD. Mechanistically, we previously found HFD led to a 2-fold increase in plasma membrane (PM) sn-1,2-diacylglycerol (DAG) and a 4-fold increase in Protein Kinase Cε (PKCε) translocation, suggesting renal cortical IR may be mediated by an sn-1,2-DAG-PKCε-IRK axis. We further interrogated this possibility here by assessing inhibitory phosphorylation at IRKT1150, the key target of PKCε, finding a 42% increase with HFD. In addition, IRKT1150A knockin mice, whose IRK cannot be phosphorylated by PKCε, were protected from HFD-induced signaling and pyruvate oxidation defects. Conclusion: HFD inhibits renal cortical insulin signaling at the level of the IRK, which is associated with perturbed mitochondrial GNG and oxidative metabolism. IRKT1150A mice are protected from HFD-induced defects, indicating a PM sn-1,2-DAG-PKCε-pIRKT1150 mechanism for lipid-induced renal cortical IR. Disclosure B.T.Hubbard: None. R.C.Gaspar: None. D.Zhang: None. M.Kahn: None. A.Nasiri: Other Relationship; Medtronic. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck &amp; Co., Inc., Janssen Research &amp; Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.

Dichloroacetic acid and rapamycin synergistically inhibit tumor progression.

Mammalian target of rapamycin (mTOR) controls cellular anabolism, and mTOR signaling is hyperactive in most cancer cells. As a result, inhibition of mTOR signaling benefits cancer patients. Rapamycin is a US Food and Drug Administration (FDA)-approved drug, a specific mTOR complex 1 (mTORC1) inhibitor, for the treatment of several different types of cancer. However, rapamycin is reported to inhibit cancer growth rather than induce apoptosis. Pyruvate dehydrogenase complex (PDHc) is the gatekeeper for mitochondrial pyruvate oxidation. PDHc inactivation has been observed in a number of cancer cells, and this alteration protects cancer cells from senescence and nicotinamide adenine dinucleotide (NAD+‍) exhaustion. In this paper, we describe our finding that rapamycin treatment promotes pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) phosphorylation and leads to PDHc inactivation dependent on mTOR signaling inhibition in cells. This inactivation reduces the sensitivity of cancer cells' response to rapamycin. As a result, rebooting PDHc activity with dichloroacetic acid (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, promotes cancer cells' susceptibility to rapamycin treatment in vitro and in vivo.

Endogenous Energy Stores Maintain a High ATP Concentration for Hours in Glucose-Depleted Cultured Primary Rat Astrocytes

Adenosine triphosphate (ATP) is the central energy currency of all cells. Cultured primary rat astrocytes contain a specific cellular ATP content of 27.9 ± 4.7 nmol/mg. During incubation in a glucose- and amino acid-free incubation buffer, this high cellular ATP content was maintained for at least 6 h, while within 24 h the levels of ATP declined to around 30% of the initial value without compromising cell viability. In contrast, cells exposed to 1 mM and 5 mM glucose maintained the initial high cellular ATP content for 24 and 72 h, respectively. The loss in cellular ATP content observed during a 24 h glucose-deprivation was fully prevented by the presence of glucose, fructose or mannose as well as by the mitochondrial substrates lactate, pyruvate, β-hydroxybutyrate or acetate. The high initial specific ATP content in glucose-starved astrocytes, was almost completely abolished within 30 min after application of the respiratory chain inhibitor antimycin A or the mitochondrial uncoupler BAM-15, while these inhibitors lowered in glucose-fed cells the ATP content only to 60% (BAM-15) and 40% (antimycin A) within 5 h. Inhibition of the mitochondrial pyruvate carrier by UK5099 alone or of mitochondrial fatty acid uptake by etomoxir alone hardly affected the high ATP content of glucose-deprived astrocytes during an incubation for 8 h, while the co-application of both inhibitors depleted cellular ATP levels almost completely within 5 h. These data underline the importance of mitochondrial metabolism for the ATP regeneration of astrocytes and demonstrate that the mitochondrial oxidation of pyruvate and fatty acids strongly contributes to the maintenance of a high ATP concentration in glucose-deprived astrocytes.

Rg3 regulates myocardial pyruvate metabolism via P300-mediated dihydrolipoamide dehydrogenase 2-hydroxyisobutyrylation in TAC-induced cardiac hypertrophy

The failing heart is characterized by an increase in glucose uptake and glycolytic rates that is not accompanied by a concomitant increase in glucose oxidation. Lower coupling of glucose oxidation to glycolysis possibly owes to unchanged or reduced pyruvate oxidation in mitochondria. Therefore, increasing pyruvate oxidation may lead to new therapies for heart disease. Dihydrolipoamide dehydrogenase (DLD) is a component of the pyruvate dehydrogenase complex (PDH). DLD mutations or defects are closely associated with metabolic diseases. However, few studies explore the effects of DLD mutants or acylation status on PDH activity and pyruvate metabolism. P300 is protein 2-hydroxyisobutyryltransferases in cells, and P300-dependent lysine 2-hydroxyisobutyrylation of glycolytic enzymes affects glucose metabolism. However, there are no relevant reports on the effect of 2-hydroxyisobutyrylation on the energy metabolism of heart failure, and it is worth further in-depth study. In this study, we showed that 2-hydroxyisobutyrylation is an essential protein translational modification (PTM) that regulates the activity of pyruvate dehydrogenase complex (PDHc). In a mouse model of transverse aortic constriction (TAC)-induced cardiac hypertrophy, the 2-hydroxyisobutylation of DLD was significantly increased, related to the decrease in PDH activity. In addition, our data provide clear evidence that DLD is a direct substrate of P300. As one of the main active ingredients of ginseng, ginsenoside Rg3 (Rg3) can reduce the 2-hydroxyisobutylation levels of DLD and restore the PDH activity by inhibiting the acyltransferase activity of P300, thereby producing beneficial effects whenever the heart is injured. Therefore, this study suggests a novel strategy for reversing myocardial hypertrophy.

MPC2 variants disrupt mitochondrial pyruvate metabolism and cause an early-onset mitochondriopathy.

Pyruvate is an essential metabolite produced by glycolysis in the cytosol and must be transported across the inner mitochondrial membrane into the mitochondrial matrix, where it is oxidized to fuel mitochondrial respiration. Pyruvate import is performed by the mitochondrial pyruvate carrier (MPC), a hetero-oligomeric complex composed by interdependent subunits MPC1 and MPC2. Pathogenic variants in the MPC1 gene disrupt mitochondrial pyruvate uptake and oxidation and cause autosomal-recessive early-onset neurological dysfunction in humans. The present work describes the first pathogenic variants in MPC2 associated with human disease in four patients from two unrelated families. In the first family, patients presented with antenatal developmental abnormalities and harboured a homozygous c.148T>C (p.Trp50Arg) variant. In the second family, patients that presented with infantile encephalopathy carried a missense c.2T>G (p.Met1?) variant disrupting the initiation codon. Patient-derived skin fibroblasts exhibit decreased pyruvate-driven oxygen consumption rates with normal activities of the pyruvate dehydrogenase complex and mitochondrial respiratory chain and no defects in mitochondrial content or morphology. Re-expression of wild-type MPC2 restored pyruvate-dependent respiration rates in patient-derived fibroblasts. The discovery of pathogenic variants in MPC2 therefore broadens the clinical and genetic landscape associated with inborn errors in pyruvate metabolism.

Vitamin B3, nicotinamide, enhances mitochondrial metabolism to promote differentiation of the retinal pigment epithelium

In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception.

323-OR: SGLT2 Inhibition Promotes Myocardial Ketone Utilization in the Normal and Failing Heart

Recent clinical outcome studies demonstrate that SGLT2 inhibitors (i) significantly reduce major adverse cardiovascular events and heart failure in patients with and without type 2 diabetes; however, the mechanisms by which SGLT2i exert their cardiovascular benefits remain unclear. Here, we aimed to elucidate the acute effects of SGLT2i (dapagliflozin, dapa) on cardiac mitochondrial substrate oxidation. Using LC-MS/MS methodology combined with an infusion of [13C6]glucose and [13C4]βOHB, we assessed the ratio of cardiac mitochondrial pyruvate (VPDH) and βOHB (VβOHB) oxidation rates to rates of mitochondrial citrate synthase flux (VCS) in awake male Sprague Dawley (SD) rats. Metabolic studies were performed 4-6 h after oral dapa (1.5 mg/kg) or vehicle treatment in control rats and after induction of heart failure induced by permanent ligation of the left anterior descending coronary artery. Acute dapa treatment led to marked glycosuria with a 15% reduction in fasting plasma glucose concentrations and 50% increase in whole-body βOHB turnover and plasma βOHB concentrations (all P&amp;lt;0.05 vs. VEH). Dapa caused a 40-60% decrease in myocardial [14C]deoxyglucose uptake and pyruvate oxidation (VPDH/VCS) and a 60% increase in myocardial ketone oxidation (VβOHB/VCS) (all P&amp;lt;0.05 vs. VEH). Similar effects were observed in heart failure rats. Dapa treatment caused a 15% decrease in fasting plasma glucose concentrations, 50% increase in plasma βOHB levels and 60% increase in left ventricular VβOHB/VCS (all P&amp;lt;0.05 vs. VEH). Conclusion: Collectively, these studies demonstrate that dapa shifts both the normal and failing heart to increased mitochondrial ketone oxidation, which may be mediated by increased hepatic ketogenesis and βOHB availability. Disclosure L. Goedeke: None. X. Zhang: None. R. J. Perry: None. G. Cline: None. L. H. Young: None. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc., The Liver Company. J. Lee: None. Y. Ma: None. X. Hu: None. J. Zhang: n/a. J. Dong: None. K. D. Galsgaard: None. N. Guerrera: None. S. Haedersdal: None. Funding National Institutes of Health (R01DK113984, R01DK045735, K99HL150234); AstraZeneca

Arrangement and Symmetry of the Fungal E3BP-Containing Core of the Pyruvate Dehydrogenase Complex

The pyruvate dehydrogenase complex (PDC) is a multienzyme protein complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Its three main components (E1, E2, E3) exhibit a fascinating organization with multiple levels of symmetry. Despite its central metabolic role, important questions regarding its composition and regulation remain. Similar to the E3-binding protein (E3BP) of mammalian PDC, there is a “Protein X” (PX) that selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. To explain the variation in stoichiometry reported for PX in fungal PDC, we have used cryo-electron microscopy to reconstruct PDC densities from the filamentous fungus Neurospora crassa, and show how PX is present interior to the PDC core instead of substituting E2 subunits as in mammals. We show how steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, which explains the observations of a variety of stoichiometries in Saccharomyces cerevisiae. The PX-binding site is conserved in - and specific to - fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC, and suggests the PX oligomer stability and size are potential mechanisms that might adjust PDC composition in response to external cues.

The pyruvate-lactate axis modulates cardiac hypertrophy and heart failure

The metabolic rewiring of cardiomyocytes is a widely accepted hallmark of heart failure (HF). These metabolic changes include a decrease in mitochondrial pyruvate oxidation and an increased export of lactate. We identify the mitochondrial pyruvate carrier (MPC) and the cellular lactate exporter monocarboxylate transporter 4 (MCT4) as pivotal nodes in this metabolic axis. We observed that cardiac assist device-induced myocardial recovery in chronic HF patients was coincident with increased myocardial expression of the MPC. Moreover, the genetic ablation of the MPC in cultured cardiomyocytes and in adult murine hearts was sufficient to induce hypertrophy and HF. Conversely, MPC overexpression attenuated drug-induced hypertrophy in a cell-autonomous manner. We also introduced a novel, highly potent MCT4 inhibitor that mitigated hypertrophy in cultured cardiomyocytes and in mice. Together, we find that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure.

Mitochondrial Spare Reserve Capacity : A New Predictive Metabolic Biomarker for Aggressiveness of Acute Myeloid Leukemia

Introduction The persistence of leukemic cells after treatment limits the effectiveness of anticancer drugs and is the cause of relapse in patients with acute myeloid leukemia (AML). After exposure to chemotherapeutic drugs, the survival of leukemic cells is mainly supported by mitochondrial energy metabolism. Several preclinical studies have shown that the combination of mitochondrial oxidative phosphorylation inhibitors with various anticancer treatments constitutes an effective therapeutic combination in vitro to eradicate the surviving leukemic cells. Evaluating the mitochondrial bioenergetic activity of blasts from AML patients could therefore provide predictive information on treatment response. The basal oxygen consumption of cells varies according to hematopoietic differentiation and depends on the energy needs in the in vitro condition of measurement. But it is necessary to treat the cells with uncoupling agents (eg FCCP) to assess the maximum activity that the respiratory chain could reach to respond to energy stress. Then, the switch from a basal level of oxygen consumption to a maximum level defines the mitochondrial spare reserve capacity (SRC). In this study, we propose to determine whether spare reserve capacity of blasts is a potential biomarker of AML aggressiveness in patients and to characterize the biochemical processes involved in the control of SRC in leukemic cells. Results Using the XFe24 Seahorse fluorometric oximeter, we first determined the mitochondrial oxygen consumption and glycolytic activity in hematopoietic cells (monocytes, lymphocytes, dendritic cells) of healthy donors, in AML patient blasts at diagnosis or at relapse and in AML cell lines (HL-60, MOLM-13, THP-1, KG1, OCI-AML3, MV-4-11, U-937). All measures have been assessed from freshly collected samples of peripheral blood and of bone marrow. As expected, AMLs are characterized by low oxidative phosphorylation activity compared to normal hematopoietic cells. From all the OXPHOS values obtained we defined a SRC threshold above which the SRC is considered high. This threshold has been set at a capacity to increase basal respiration by 250%. From patients blasts, we have therefore defined two groups characterized by high (n=14) or low (n=21) mitochondrial spare reserve capacity. Blasts with high SRC exhibit high glycolytic activity suggesting a link between spare reserve capacity and glucose metabolism. Using U-13C6 glucose and pharmacological inhibitors, we have demonstrated that the utilization of the mitochondrial spare reserve capacity of leukemic cells is supported through glycolysis and that mitochondrial oxidation of pyruvate is a key element for SRC recruitment. Mitochondrial pyruvate carrier inhibitors (as UK-5099) or gene silencing of BRP44 abolish the SRC of leukemic cells highlighting the importance of pyruvate oxidation to increase oxygen consumption. Since high mutation rate is recognized as an unfavorable prognostic factor in AML, we have also sequenced 45 commonly genes mutated in AMLs characterized by high or low SRC blasts. Interestingly, DNA sequencing analysis showed that AML with low SRC blasts have a higher mutation rate than high SRC blasts and also exhibited exclusive mutations such as ASXL1 (25%), IDH2 (25%), NPM1 (25%), IDH1 (13%), JAK2 (13%) and SF3B1 (13%). Conclusion Currently, most of the clinical biomarkers used to predict AML aggressiveness are based on DNA analysis, but the emergence of mutations is not always associated with phenotypic changes. This study shows that the mitochondrial spare reserve capacity of blasts represents a new functional biomarker based on the assessment of the energetic phenotype and could help the clinicians to determine the prognosis of AML. Moreover we have showed that altering pyruvate metabolism highly decrease spare reserve capacity of blasts and then could be evaluated as metabolic strategies to improve the therapeutic response in patients with AML. Disclosures Kluza: Daiichi-Sankyo: Research Funding.

Chronic cold exposure enhances glucose oxidation in brown adipose tissue.

The cultured brown adipocytes can oxidize glucose in vitro, but it is still not fully clear whether brown adipose tissue (BAT) could completely oxidize glucose in vivo. Although positron emission tomography (PET) with 18F‐fluorodeoxyglucose (18F‐FDG) showed a high level of glucose uptake in the activated BAT, the non‐metabolizable 18F‐FDG cannot fully demonstrate intracellular glucose metabolism. Through in vivo [U‐13C]glucose tracing, here we show that chronic cold exposure dramatically activates glucose oxidation in BAT and the browning/beiging subcutaneous white adipose tissue (sWAT). Specifically, chronic cold exposure enhances glucose flux into the mitochondrial TCA cycle. Metabolic flux analysis models that β3‐adrenergic receptor (β3‐AR) agonist significantly enhances the flux of mitochondrial pyruvate uptake through mitochondrial pyruvate carrier (MPC) in the differentiated primary brown adipocytes. Furthermore, in vivo MPC inhibition blocks cold‐induced glucose oxidation and impairs body temperature maintenance in mice. Together, mitochondrial pyruvate uptake and oxidation serve an important energy source in the chronic cold exposure activated BAT and beige adipose tissue, which supports a role for glucose oxidation in brown fat thermogenesis.

Dissociation of Muscle Insulin Resistance from Alterations in Mitochondrial Substrate Preference

Alterations in muscle mitochondrial substrate preference have been postulated to play a major role in the pathogenesis of muscle insulin resistance. In order to examine this hypothesis, we assessed the ratio of mitochondrial pyruvate oxidation (VPDH) to rates of mitochondrial citrate synthase flux (VCS) in muscle. Contrary to this hypothesis, we found that high-fat-diet (HFD)-fed insulin-resistant rats did not manifest altered muscle substrate preference (VPDH/VCS) in soleus or quadriceps muscles in the fasting state. Furthermore, hyperinsulinemic-euglycemic (HE) clamps increased VPDH/VCS in both muscles in normal and insulin-resistant rats. We then examined the muscle VPDH/VCS flux in insulin-sensitive and insulin-resistant humans and found similar relative rates of VPDH/VCS, following an overnight fast (∼20%), and similar increases in VPDH/VCS fluxes during a HE clamp. Altogether, these findings demonstrate that alterations in mitochondrial substrate preference are not an essential step in the pathogenesis of muscle insulin resistance.

Arrangement and symmetry of the fungal E3BP-containing core of the pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Similar to the E3-binding protein (E3BP) of mammalian PDC, PX selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. Here, we report reconstructions of PDC from the filamentous fungus Neurospora crassa by cryo-electron microscopy, where we find protein X (PX) interior to the PDC core as opposed to substituting E2 core subunits as in mammals. Steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, explaining previous observations in Saccharomyces cerevisiae. The PX-binding site is conserved in (and specific to) fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC.

Abstract 2783: Monitoring of a novel LDH inhibitor and mitochondrial inhibitor efficacy in vivo in a glycolytic pancreatic cancer model using hyperpolarized13C MRI

Abstract Increased “glycolysis”, conversion of pyruvate(Pyr) to lactate by Lactate(Lac) Dehydrogenase (LDH), is a feature of many neoplasms. Therefore, LDH inhibition is considered a promising approach toward developing a new therapeutic strategy against glycolityic cancers. In tumor, however, it's still unclear that glycolysis and oxidative phosphorylation(OXPHOS) are functionally integrated in vivo and both are essential to maintain metabolic plasticity and tumor growth. To elucidate these points, a feasible noninvasive-imaging approach that can dynamically evaluate LDH activity in vivo would be highly beneficial. A new imaging thechonology, Hyperpolarized 13C-pyruvate(Pyr) magnetic resonance spectroscopic imaging (HP-MRSI), can permit real-time monitoring of pyruvate flux in vivo through dynamic observation of conversion of 13C-Pyr into its metabolite(s). This study aimed to apply HP-MRSI in support of a therapeutic strategy to explore efficacy of a newly developed and highly potent in vivo-active LDH inhibitor (NCI-006) and the mitochondrial inhibitor (Metformin) in a glycolytic tumor model using MiaPaCa-2(MP2) xenografts in mice. First, HP-MRSI was performed before and after a single NCI-006 IV injection to assess the inhibitor impact on intratumoral metabolic flux in vivo. HP-MRSI confirmed that NCI-006 suppressed intratumor LDH activity. In addition, HP-MRSI could visualize lactate production in the tumor was suppressed 30 minutes after IV administration of NCI-006, as was the 13C-lac to 13C-pyr (13C-L/P) ratio decreased by 83.3 ± 4.4 % compared to vehicle controls. Importantly, the close correlation of these data with the resuts of the ex vivo LDH activity assay, suggests that HP-MRSI can reliably monitor in vivo on target effects of NCI-006. Next, we investigated the impact of metformin on the 13C-L/P ratio in MP2 xenografts using HP-MRSI. HP-MRSI revealed a significant increase in tumor 13C-L/P ratio by 3 h after PO dosing (69.9±6.6% increase) and L/P ratio remained elevated at 5 h in MP2 xenografts. These results are consistent with inhibition of mitochondrial pyruvate oxidation by metformin.Further, to examine the anticipated combinatorial impact on in vivo metabolic flux, HP-MRSI was carried out using MP2 xenografts treated with NCI-006 (50 mg/kg, IV) and/or metformin (50 mg/kg, PO). This treatment resulted in a significantly decreased 13C-L/P ratio. In contrast to LDH inhibitor treatment alone, the 13C Bicarbonate/Pyr ratio did not changesignificantly while the 13C Alanin/Pyr ratio significantly increased. The enhanced transaminase-mediated conversion of Pyr to alanine with combination therapy is consistent with re-wiring of flux away from both Lac (through LDH) and bicarbonate (through the mitochondrion).Finally, we explored whether this combinatorial activity was reflected in greater growth inhibition of MP2 xenografts. We did find that combination treatment caused greater growth reduction in both tumor models compared to either treatment alone.In conclusion, intratumoral inhibition of glycolysis by NCI-006 and of OXPHOS by metformin in vivo was readily visualized by HP-MRSI, confirming the utility of this noninvasive method. This methodology can be of great value in developing new therapeutic strategies using glycolysis and OXPHOS inhibitors to treat cancers. Citation Format: Nobu Oshima, Ryo Ishida, Shun Kishimoto, Jeffrey R. Brender, Tomoaki Okada, Kazuataka Obama, Yoshiharu Sakai, Murali C. Krishna, Leonard M. Neckers. Monitoring of a novel LDH inhibitor and mitochondrial inhibitor efficacy in vivo in a glycolytic pancreatic cancer model using hyperpolarized13C MRI [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2783.

Bipotent Liver Progenitors Depend on Glycolysis and Mitochondrial Pyruvate Oxidation for Stem Cell Functions

Aerobic glycolysis, characterized by pyruvate reduction to lactate, serves proliferation in cancer cells and stem cells. To clarify whether this metabolic profile is universal to epithelial stem cells, despite vast differences in physiological turnover rates, we characterized the metabolic phenotype of bipotent liver progenitors (low turnover) relative to intestinal progenitors (high turnover). Using human liver and intestinal organoids, we show high glycolytic fluxes which provide substrates for cellular building blocks and reducing equivalents in proliferating progenitors, compared to their quiescent differentiating counterparts. Similar to cancer and pluripotent stem cells, intestinal progenitors display aerobic glycolysis with pyruvate reduction to lactate to serve their high proliferative demands. Strikingly, liver progenitors combine high glycolysis with substantial mitochondrial oxidation of pyruvate, which they require for both proliferation and maintenance of stemness. This concurs with the anabolic and epigenetic effects of mitochondrial pyruvate oxidation and the homeostatic liver function with low physiological turnover rates.

Inhibition of Anaplerosis Attenuated Vascular Proliferation in Pulmonary Arterial Hypertension

Vascular remodeling is considered a key event in the pathogenesis of pulmonary arterial hypertension (PAH). However, mechanisms of gaining the proliferative phenotype by pulmonary vascular cells are still unresolved. Due to well-established pyruvate dehydrogenase (PDH) deficiency in PAH pathogenesis, we hypothesized that the activation of another branch of pyruvate metabolism, anaplerosis, via pyruvate carboxylase (PC) could be a key contributor to the metabolic reprogramming of the vasculature. In sugen/hypoxic PAH rats, vascular proliferation was found to be accompanied by increased activation of Akt signaling, which upregulated membrane Glut4 translocation and caused upregulation of hexokinase and pyruvate kinase-2, and an overall increase in the glycolytic flux. Decreased PDH activity and upregulation of PC shuttled more pyruvate to oxaloacetate. This results in the anaplerotic reprogramming of lung vascular cells and their subsequent proliferation. Treatment of sugen/hypoxia rats with the PC inhibitor, phenylacetic acid 20 mg/kg, starting after one week from disease induction, significantly attenuated right ventricular systolic pressure, Fulton index, and pulmonary vascular cell proliferation. PC inhibition reduced the glycolytic shift by attenuating Akt-signaling, glycolysis, and restored mitochondrial pyruvate oxidation. Our findings suggest that targeting PC mediated anaplerosis is a potential therapeutic intervention for the resolution of vascular remodeling in PAH.

Defective Mitochondrial Pyruvate Flux Affects Cell Bioenergetics in Alzheimer’s Disease-Related Models

Mitochondria are key organelles for brain health. Mitochondrial alterations have been reported in several neurodegenerative disorders, including Alzheimer's disease (AD), and the comprehension of the underlying mechanisms appears crucial to understand their relationship with the pathology. Using multiple genetic, pharmacological, imaging, and biochemical approaches, we demonstrate that, in different familial AD cell models, mitochondrial ATP synthesis is affected. The defect depends on reduced mitochondrial pyruvate oxidation, due to both lower Ca2+-mediated stimulation of the Krebs cycle and dampened mitochondrial pyruvate uptake. Importantly, this latter event is linked to glycogen-synthase-kinase-3β (GSK-3β) hyper-activation, leading, in turn, to impaired recruitment of hexokinase 1 (HK1) to mitochondria, destabilization of mitochondrial-pyruvate-carrier (MPC) complexes, and decreased MPC2 protein levels. Remarkably, pharmacological GSK-3β inhibition in AD cells rescues MPC2 expression and improves mitochondrial ATP synthesis and respiration. The defective mitochondrial bioenergetics influences glutamate-induced neuronal excitotoxicity, thus representing a possible target for future therapeutic interventions.