Mitochondrial Pyruvate Dehydrogenase Research Articles

Reciprocal regulation of cardiac β-oxidation and pyruvate dehydrogenase by insulin

The heart alters the rate and relative oxidation of fatty acids and glucose based on availability and energetic demand. Insulin plays a crucial role in this process diminishing fatty acid and increasing glucose oxidation when glucose availability increases. Loss of insulin sensitivity and metabolic flexibility can result in cardiovascular disease. It is therefore important to identify mechanisms by which insulin regulates substrate utilization in the heart. Mitochondrial pyruvate dehydrogenase (PDH) is the key regulatory site for the oxidation of glucose for ATP production. Nevertheless, the impact of insulin on PDH activity has not been fully delineated, particularly in the heart. We sought in vivo evidence that insulin stimulates cardiac PDH and that this process is driven by inhibition of fatty acid oxidation. Mice injected with insulin exhibited dephosphorylation and activation of cardiac PDH. This was accompanied by an increase in the content of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT1) and, thus, mitochondrial import of fatty acids. Administration of the CPT1 inhibitor oxfenicine was sufficient to activate PDH. Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC). Pharmacologic inhibition or knockout of cardiac ACC diminished insulin-dependent production of malonyl-CoA and activation of PDH. Finally, circulating insulin and cardiac glucose utilization exhibit daily rhythms reflective of nutritional status. We demonstrate that time of day-dependent changes in PDH activity are mediated, in part, by ACC-dependent production of malonyl-CoA. Thus, by inhibiting fatty acid oxidation, insulin reciprocally activates PDH. These studies identify potential molecular targets to promote cardiac glucose oxidation and treat heart disease.

The OGT-c-Myc-PDK2 axis rewires the TCA cycle and promotes colorectal tumor growth.

Deregulated glucose metabolism termed the "Warburg effect" is a fundamental feature of cancers, including the colorectal cancer. This is typically characterized with an increased rate of glycolysis, and a concomitant reduced rate of the tricarboxylic acid (TCA) cycle metabolism as compared to the normal cells. How the TCA cycle is manipulated in cancer cells remains unknown. Here, we show that O-linked N-acetylglucosamine (O-GlcNAc) regulates the TCA cycle in colorectal cancer cells. Depletion of OGT, the sole transferase of O-GlcNAc, significantly increases the TCA cycle metabolism in colorectal cancer cells. Mechanistically, OGT-catalyzed O-GlcNAc modification of c-Myc at serine 415 (S415) increases c-Myc stability, which transcriptionally upregulates the expression of pyruvate dehydrogenase kinase 2 (PDK2). PDK2 phosphorylates pyruvate dehydrogenase (PDH) to inhibit the activity of mitochondrial pyruvate dehydrogenase complex, which reduces mitochondrial pyruvate metabolism, suppresses reactive oxygen species production, and promotes xenograft tumor growth. Furthermore, c-Myc S415 glycosylation levels positively correlate with PDK2 expression levels in clinical colorectal tumor tissues. This study highlights the OGT-c-Myc-PDK2 axis as a key mechanism linking oncoprotein activation with deregulated glucose metabolism in colorectal cancer.

Pyruvate dehydrogenase kinase regulates macrophage polarization in metabolic and inflammatory diseases.

Macrophages are highly heterogeneous and plastic, and have two main polarized phenotypes that are determined by their microenvironment, namely pro- and anti-inflammatory macrophages. Activation of pro-inflammatory macrophages is closely associated with metabolic reprogramming, especially that of aerobic glycolysis. Mitochondrial pyruvate dehydrogenase kinase (PDK) negatively regulates pyruvate dehydrogenase complex activity through reversible phosphorylation and further links glycolysis to the tricarboxylic acid cycle and ATP production. PDK is commonly associated with the metabolism and polarization of macrophages in metabolic and inflammatory diseases. This review examines the relationship between PDK and macrophage metabolism and discusses the mechanisms by which PDK regulates macrophage polarization, migration, and inflammatory cytokine secretion in metabolic and inflammatory diseases. Elucidating the relationships between the metabolism and polarization of macrophages under physiological and pathological conditions, as well as the regulatory pathways involved, may provide valuable insights into the etiology and treatment of macrophage-mediated inflammatory diseases.

IAR4 mutation enhances cadmium toxicity by disturbing auxin homeostasis in Arabidopsis thaliana.

Cadmium (Cd) is highly toxic to plants, but the targets and modes of toxicity remain unclear. We isolated a Cd-hypersensitive mutant of Arabidopsis thaliana, Cd-induced short root 2 (cdsr2), in the background of the phytochelatin synthase-defective mutant cad1-3. Both cdsr2 and cdsr2 cad1-3 displayed shorter roots and were more sensitive to Cd than their respective wild type. Using genomic resequencing and complementation, IAR4 was identified as the causal gene, which encodes a putative mitochondrial pyruvate dehydrogenase E1α subunit. cdsr2 showed decreased pyruvate dehydrogenase activity and NADH content, but markedly increased concentrations of pyruvate and alanine in roots. Both Cd stress and IAR4 mutation decreased auxin level in the root tips, and the effect was additive. A higher growth temperature rescued the phenotypes in cdsr2. Exogenous alanine inhibited root growth and decreased auxin level in the wild type. Cadmium stress suppressed the expression of genes involved in auxin biosynthesis, hydrolysis of auxin-conjugates and auxin polar transport. Our results suggest that auxin homeostasis is a key target of Cd toxicity, which is aggravated by IAR4 mutation due to decreased pyruvate dehydrogenase activity. Decreased auxin level in cdsr2 is likely caused by increased auxin-alanine conjugation and decreased energy status in roots.

β-hydroxybutyrate impairs bovine oocyte maturation via pyruvate dehydrogenase (PDH) associated energy metabolism abnormality.

Background: Ketosis is one of the most frequent and costly metabolic disorders in high-producing dairy cows, and negatively associated with the health and reproductive performance of bovine. Ketosis is mainly caused by the accumulation of ketone body β-hydroxybutyric acid and its diagnosis is based on β-hydroxybutyrate (βHB) concentration in blood. Methods: In this study, we investigated the effects of βHB on bovine oocyte maturation in the concentration of subclinical (1.2mM) βHB and clinical (3.6mM). Results: The results showed βHB disrupted bovine oocyte maturation and development capacity. Further analysis showed that βHB induced oxidative stress and mitochondrial dysfunction, as indicated by the increased level of reactive oxygen species (ROS), disrupted mitochondrial structure and distribution, and depolarized membrane potential. Furthermore, oxidative stress triggered early apoptosis, as shown by the enhanced levels of Caspase-3 and Annexin-V. Moreover, 3.6mM βHB induced the disruption of the pyruvate dehydrogenase (PDH) activity, showing with the decrease of the global acetylation modification and the increase of the abnormal spindle rate. Conclusion: Our study showed that βHB in subclinical/clinical concentration had toxic effects on mitochondrial function and PDH activity, which might affect energy metabolism and epigenetic modification of bovine oocytes and embryos.

Glutathionylation of pyruvate dehydrogenase complex E2 and inflammatory cytokine production during acute inflammation are magnified by mitochondrial oxidative stress

Lipopolysaccharide (LPS) is a known inducer of inflammatory signaling which triggers generation of reactive oxygen species (ROS) and cell death in responsive cells like THP-1 promonocytes and freshly isolated human monocytes. A key LPS-responsive metabolic pivot point is the 9 MDa mitochondrial pyruvate dehydrogenase complex (PDC), which provides pyruvate dehydrogenase (E1), lipoamide-linked transacetylase (E2) and lipoamide dehydrogenase (E3) activities to produce acetyl-CoA from pyruvate. While phosphorylation-dependent decreases in PDC activity following LPS treatment or sepsis have been deeply investigated, redox-linked processes have received less attention. Data presented here demonstrate that LPS-induced reversible oxidation within PDC occurs in PDCE2 in both THP-1 cells and primary human monocytes. Knockout of PDCE2 by CRISPR and expression of FLAG-tagged PDCE2 in THP-1 cells demonstrated that LPS-induced glutathionylation is associated with wild type PDCE2 but not mutant protein lacking the lipoamide-linking lysine residues. Moreover, the mitochondrially-targeted electrophile MitoCDNB, which impairs both glutathione- and thioredoxin-based reductase systems, elevates ROS similar to LPS but does not cause PDCE2 glutathionylation. However, LPS and MitoCDNB together are highly synergistic for PDCE2 glutathionylation, ROS production, and cell death. Surprisingly, the two treatments together had differential effects on cytokine production; pro-inflammatory IL-1β production was enhanced by the co-treatment, while IL-10, an important anti-inflammatory cytokine, dropped precipitously compared to LPS treatment alone. This new information may expand opportunities to understand and modulate PDC redox status and activity and improve the outcomes of pathological inflammation.

Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Mitochondrial pyruvate dehydrogenase kinases contribute to platelet function and thrombosis in mice by regulating aerobic glycolysis.

Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) for their energy requirements. In contrast, platelet activation exhibits an increased rate of aerobic glycolysis relative to OXPHOS. Mitochondrial enzymes pyruvate dehydrogenase kinases (PDKs) phosphorylate the pyruvate dehydrogenase (PDH) complex to inhibit its activity, thereby diverting the pyruvate flux from OXPHOS to aerobic glycolysis upon platelet activation. Of four PDK isoforms, PDK2 and PDK4 (PDK2/4) are predominantly associated with metabolic diseases. Herein, we report that the combined deletion of PDK2/4 inhibits agonist-induced platelet functions, including aggregation, integrin αIIbβ3 activation, degranulation, spreading, and clot retraction. Additionally, collagen-mediated PLCγ2 phosphorylation and calcium mobilization were significantly reduced in PDK2/4-/- platelets, suggesting impaired GPVI signaling. The PDK2/4-/- mice were less susceptible to FeCl3-induced carotid and laser-induced mesenteric artery thrombosis without any effect on hemostasis. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with PDK2/4-/- platelets exhibited less susceptibility to FeCl3 injury-induced carotid thrombosis compared to hIL-4Rα/GPIbα-Tg mice transfused with WT platelets, suggesting a platelet-specific role of PDK2/4 in thrombosis. Mechanistically, the inhibitory effects of PDK2/4 deletion on platelet function were associated with reduced PDH phosphorylation and glycoPER in activated platelets, suggesting that PDK2/4 regulates aerobic glycolysis. Finally, using PDK2 or PDK4 single KO mice, we identified that PDK4 plays a more prominent role in regulating platelet secretion and thrombosis compared to PDK2. This study identifies the fundamental role of PDK2/4 in regulating platelet functions and identifies PDK/PDH axis as a potentially novel antithrombotic target.

Machine-learning Assisted Differentiation Between Physiological Oscillation Epochs and Muscle or Epileptiform activity in EEGs (S30.005)

<h3>Objective:</h3> Machine-learning assisted separation of spontaneous cognition-relevant EEG oscillations from EEG perturbation induced by epileptiform events and muscle artifacts. <h3>Background:</h3> Transient band-limited alpha and gamma frequency oscillations in EEGs of awake subjects reflect cognitive processes and integrity of excitation-inhibition balance. These oscillations are diminished in disorders such as mitochondrial encephalopathies as we have previously shown. Automated capture of such oscillation epochs in prolonged EEGs can prove valuable for numerous clinical and research studies. However, EEG recordings can be degraded by superimposed high frequency activity inherent in movement and epileptiform discharges. <h3>Design/Methods:</h3> Forty band-limited alpha and gamma spontaneous oscillation epochs were recorded from electrode C3 in a standard 20-lead EEG configuration. The gaussian distribution of non-event wavelet scalogram allowed automated identification of outlier putative events, which were then confirmed or rejected by a human observer. An instantaneous wavelet scalogram “cross-section” of the event was used to generate a representative frequency waveform for that event and analyzed using action potential waveform sorting Wave clus (<i>Neural Computation</i> 16, 1661–1687; 2004). This protocol was applied to EEGs containing muscle (27 events) and to EEGs with epileptiform activity (36 events) due to mitochondrial pyruvate dehydrogenase deficiency. <h3>Results:</h3> Oscillation epoch waveforms exhibited distinct band-limited peaks which were absent for muscle or epileptiform activity. Muscle events uniformly displayed broad-band high-frequencies, whereas epileptiform events were more variable. Oscillation epoch wavelet coefficients were reliably differentiated from other events (MANOVA p&lt;10⁻⁴). A support vector machine-learning protocol examining the difference between epochs and muscle and epileptiform events demonstrated a misclassification rate of zero and 1.3%, respectively, and an area under the curve for a receiver operating characteristic curve of 1 for both. <h3>Conclusions:</h3> Instantaneous wavelet scalogram profiles provide high precision in differentiating oscillation epochs from muscle or epileptiform activity, allowing for automated oscillation epoch detection in subjects with epilepsy. <b>Disclosure:</b> Dr. Jakkamsetti has nothing to disclose. Miss Kathote has nothing to disclose. Mr. Avila has nothing to disclose. Sharon Primeaux has nothing to disclose. Mr. Dobariya has nothing to disclose. Dr. Malaga Dieguez has nothing to disclose. Dr. Pascual has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Elsevir. The institution of Dr. Pascual has received research support from NIH.

Systemic sclerosis and primary biliary cholangitis share an antibody population with identical specificity.

Anti-centromere (ACA) and antimitochondrial antibodies (AMA) are specific for limited-cutaneous systemic sclerosis (lcSSc) and primary biliary cholangitis (PBC), respectively, and can coexist in up to 25 and 30% of SSc and PBC patients. Here, we evaluated whether anti-centromeric protein A (CENP-A) antibodies cross-react with mitochondrial antigens. To this end, sera from two lcSSc patients (pt1 and pt4), one of them (pt4) also affected by PBC, were used as the source of ACA, previously shown to recognize different groups of amino acids (motifs) in the CENP-A region spanning amino acids 1-17 (Ap1-17). Pt1 and pt4 Ap1-17-specific IgG were purified by affinity-chromatography on insolubilized Ap1-17-peptide column and tested by western blotting with nuclear and cytoplasmic protein extract from HeLa cells. Immunoreactive proteins were identified by mass spectrometry and validated by immunodot. The results showed that affinity-purified SSc/PBC pt4 anti-Ap1-17 and not SSc pt1 anti-Ap1-17 Ab, specifically cross-reacted with the E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen in PBC. Sequence homology analysis indicated that the motif A-x-x-P-x-A-P recognized by pt4 anti-Ap1-17 IgG and shared by CENP-A and PDC-E2, is also expressed by some members of the Human Herpesvirus family, suggesting that they may trigger the production of these cross-reacting antibodies.

Changes in the Expression of Pyruvate Dehydrogenase Kinase Isoenzymes During Early Differentiation of Mouse Embryonic Stem Cells

The embryonic stem cells (ESCs) are pluripotent, self-renewing cells that able to differentiate into any of the germ layers involved in embryogenesis. However, the molecular mechanisms that control ESC pluripotency and differentiation remain poorly understood. The family of Pyruvate dehydrogenase kinase (PDK1-4), inactivates the mitochondrial pyruvate dehydrogenase complex via phosphorylation, plays a crucial role in the control of glucose homeostasis. In the current study, gene expression levels of PDK isoenzymes were analyzed on undifferentiated mouse embryonic stem cells (mESCs) and compared to mESCs induced to differentiate by removal of leukemia inhibitory factor (LIF) for 1, 3 and 5 days. Besides, we performed gene expression analysis of several genes related to pluripotency and differentiation. In addition, we also determined glucose uptake rates by a colorimetric assay kit in early differentiated and undifferentiated mESCs. Differently expression level of PDK isoenzymes in pluripotent and LIF-depleted mESCs suggest that they may have roles in differentiation and pluripotency of ESCs. Furthermore, this study lays the foundation for detailed investigation of molecular mechanisms underlying the roles of PDKs in the pluripotency and transition to differentiated state of ESCs.

Phosphonate Inhibitors of Pyruvate Dehydrogenase Perturb Homeostasis of Amino Acids and Protein Succinylation in the Brain.

Mitochondrial pyruvate dehydrogenase complex (PDHC) is essential for brain glucose and neurotransmitter metabolism, which is dysregulated in many pathologies. Using specific inhibitors of PDHC in vivo, we determine biochemical and physiological responses to PDHC dysfunction. Dose dependence of the responses to membrane-permeable dimethyl acetylphosphonate (AcPMe2) is non-monotonous. Primary decreases in glutathione and its redox potential, methionine, and ethanolamine are alleviated with increasing PDHC inhibition, the alleviation accompanied by physiological changes. A comparison of 39 brain biochemical parameters after administration of four phosphinate and phosphonate analogs of pyruvate at a fixed dose of 0.1 mmol/kg reveals no primary, but secondary changes, such as activation of 2-oxoglutarate dehydrogenase complex (OGDHC) and decreased levels of glutamate, isoleucine and leucine. The accompanying decreases in freezing time are most pronounced after administration of methyl acetylphosphinate and dimethyl acetylphosphonate. The PDHC inhibitors do not significantly change the levels of PDHA1 expression and phosphorylation, sirtuin 3 and total protein acetylation, but increase total protein succinylation and glutarylation, affecting sirtuin 5 expression. Thus, decreased production of the tricarboxylic acid cycle substrate acetyl-CoA by inhibited PDHC is compensated by increased degradation of amino acids through the activated OGDHC, increasing total protein succinylation/glutarylation. Simultaneously, parasympathetic activity and anxiety indicators decrease.

Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.

Abstract P2083: Erk-mediated Phosphorylation Of Grk2 Leads To Reduced Pyruvate Dehydrogenase Activity And Alters Pyroptotic Signaling

G protein-coupled receptors (GPCRs) are important regulators of cellular functions where agonist binding leads to receptor conformational changes and downstream activation of g-protein-mediated signaling cascades. Signal transduction is terminated by receptor phosphorylation mediated by G-protein coupled receptor kinases (GRKs). GPCR kinase 2 (GRK2) is a main GRK in the heart and is upregulated in heart failure (HF) patients. Recently, we and others have shown that GRK2 can translocate to cardiac mitochondria where it regulates metabolism. Notably, we found that phosphorylated GRK2 at S670 post- ischemia/reperfusion (IR) decreases glucose mitochondrial utilization and pyruvate dehydrogenase (PDH) activity. Furthermore, 2D-SDS PAGE followed by LC/MS/MS revealed a potential link between mitochondrial GRK2 and PDH phosphorylation. Using a novel PDHα KI CRISPR Cas HEK cell line, we tested the hypothesize that PDH phosphorylation of this novel site was important for PDH activity. Utilizing cytosolic and mitochondrially-targeted pyruvate indicators we measured pyruvate levels in WT and KI HEK cells. In parallel, using a novel mouse model GRK2-S670A, we investigated the role of ERK in mediating GRK2 mitochondrial translocation and pyroptotic signaling. Further experiments will determine long-term impact of mitochondrial GRK2 in cell survival in cardiac pathological conditions.

Sepsis, pyruvate, and mitochondria energy supply chain shortage.

Balancing high energy-consuming danger resistance and low energy supply of disease tolerance is a universal survival principle that often fails during sepsis. Our research supports the concept that sepsis phosphorylates and deactivates mitochondrial pyruvate dehydrogenase complex control over the tricarboxylic cycle and the electron transport chain. StimulatIng mitochondrial energetics in septic mice and human sepsis cell models can be achieved by inhibiting pyruvate dehydrogenase kinases with the pyruvate structural analog dichloroacetate. Stimulating the pyruvate dehydrogenase complex by dichloroacetate reverses a disruption in the tricarboxylic cycle that induces itaconate, a key mediator of the disease tolerance pathway. Dichloroacetate treatment increases mitochondrial respiration and ATP synthesis, decreases oxidant stress, overcomes metabolic paralysis, regenerates tissue, organ, and innate and adaptive immune cells, and doubles the survival rate in a murine model of sepsis.

Abstract 6163: Targeting glucose metabolism through inhibition of pyruvate dehydrogenase kinase to improve response to immune-checkpoint inhibition in melanoma

Abstract Immune-checkpoint inhibitors have improved survival of patients with advanced melanoma, however, 40-50% of patients do not benefit sufficiently. Melanoma tumor lesions have high glucose uptake due to metabolic reprogramming, which supports tumor cell growth and is partially mediated through elevated expression of mitochondrial pyruvate dehydrogenase kinases (PDK1-4). Resultant decreased glucose levels and low pH in the tumor microenvironment are detrimental to antitumor immune cell function. Here, we aimed to reverse metabolic reprogramming in melanoma through PDK inhibition, thereby inhibiting tumor cell growth while maintaining or potentially enhancing antitumor immunity. We used a panel of four melanoma cell lines with genetic backgrounds similar to those most frequently found in patients, namely the BRAF-mutant A375 and S-MEL-28 cell lines, the NRAS-mutant SK-MEL-2 cell line and the BRAF/NRAS wild-type MeWo cell line. These were then treated with PDK inhibitor dichloroacetate (DCA) to determine effects on viability and metabolic phenotype. Furthermore, we investigated whether DCA synergized with treatment with the glutaminase inhibitor CB-839. Finally, we determined the effect of DCA on viability, interferon-γ (IFN-γ) secretion and antitumor activity of CD8+ T cells. MeWo cells were most sensitive to DCA, while SK-MEL-2 was the least sensitive, with IC50 values ranging from 13.3 to 27.0 mM. DCA led to an up to 6-fold increase in oxygen consumption rate:extracellular acidification rate (OCR:ECAR) ratio in all cell lines. SK-MEL-28 cells were not sensitive to CB-839, while the IC50 values in the other cell lines ranged from 7.9 nM in MeWo to 139.2 nM in SK-MEL-2 cells. DCA synergized with CB-839 in 2D and 3D culture, ranging from 2-fold sensitization compared to either drug alone in MeWo up to 5-fold sensitization in SK-MEL-2. In activated CD8+ T cells, viability was not affected by DCA treatment of up to 21 mM, whereas proliferation was only mildly inhibited. These cells also showed a 2.5-fold increase in OCR:ECAR ratio after DCA treatment. Interestingly, IFN-γ secretion by CD8+ T cells was increased 2.8-fold after DCA treatment and tumor cell killing by CD8+ T cells in a coculture with A375 cells was not impaired by DCA or CB-839 treatment. We conclude that DCA can indeed reprogram cellular metabolism in melanoma and synergizes with other metabolically targeted drugs in antitumor activity, while keeping the antitumor reactivity of CD8+ T cells intact. Supported by the Dutch Cancer Foundation (10913/2017-1). Citation Format: Jiske F. Tiersma, Bernardus Evers, Barbara M. Bakker, Steven de Jong, Mathilde Jalving. Targeting glucose metabolism through inhibition of pyruvate dehydrogenase kinase to improve response to immune-checkpoint inhibition in melanoma [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 6163.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

P2X7 inhibition prevents mitochondrial stress caused by alcohol and e‐cigarette exposure in primary vascular endothelial cells and restores barrier function

Studies in both human and animal models demonstrated that chronic alcohol/e‐cigarette (e‐Cig) exposure affects mitochondrial function and impairs barrier function in brain microvascular endothelial cells (BMVEC). Identification of the signaling pathways by which chronic alcohol/e‐Cig exposure induces mitochondrial damage in BMVEC is vital for protection of the blood‐brain barrier. We treated primary human BMVEC with ethanol (100 mM) or e‐Cig conditioned medium and showed reduced mitochondrial oxidative phosphorylation (OXPHOS) analyzed by a Seahorse analyzer. Chronic alcohol/e‐Cig treatment significantly reduced the expression of mitochondrial OXPHOS proteins and pyruvate dehydrogenase (a vital enzyme in the TCA cycle) detected by western blot. We observed the cytosolic escape of ATP and its extracellular release due to the disruption of mitochondrial membrane potential after alcohol or e‐Cig treatment of BMVEC. Further, alcohol or e‐Cig treatment resulted in an elevated purinergic receptor (P2X7) and TRPV1 channel expression by qPCR in primary BMVEC. We showed a protective role of the P2X7r antagonist which restored the mitochondrial oxidative phosphorylation levels and prevented ATP release. In summary, we identified potential common pathways of mitochondrial injury caused by alcohol and e‐Cig which allow new protective interventions. We are further investigating the potential link between P2X7 regulatory pathways and mitochondrial health.

Metabolomic Effects of the Dietary Inclusion of Hermetia illucens Larva Meal in Tilapia.

Black soldier fly (Hermetia illucens) larvae meal have been used as feed protein supplements in fish feed, but few researches have investigated the metabolomic effects of Hermetia illucens larvae meal supplements. Therefore, the metabolic effects on Nile tilapia were investigated by replacing 5%, 10%, and 20% of the dietary soybean meal in the basal diet with Hermetia illucens larvae meal, respectively. This study shows that 20% H. illucens larvae meal feed could promote tilapia average daily gain of upto 5.03 ± 0.18 g (mean ± SEM). It was found that the tricarboxylic acid cycle efficiency was improved by activating the enzymes of mitochondrial isocitrate dehydrogenase, NAD-malate dehydrogenase, succinate dehydrogenase, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase, which then increased the output of ATP and NADH. Furthermore, amino acid and protein biosynthesis was boosted by enhanced glutamine synthetase and glutamate synthase. In particular, GSH increased with increased H. illucens larvae meal. Unsaturated fatty acid biosynthesis was stimulated by higher levels of fatty acid synthase and acetyl CoA carboxylase. Additionally, there was no significant change in lipase levels. Thus, the higher acetyl Co-A content was primarily involved in fatty acid biosynthesis and energy metabolism. Flavor substances, such as nonanal and 2-methyl-3-furanthiol, also accumulated with the addition of H. illucens larvae meal, which increased the umami taste and meat flavor. Additionally, the flavor of tilapia was improved owing to a decrease in trimethylamine content, which causes an earthy and fishy taste. This study uncovers a previously unknown metabolic effect of dietary H. illucens larvae meal on Nile tilapia.

MFN2-driven mitochondria-to-nucleus tethering allows a non-canonical nuclear entry pathway of the mitochondrial pyruvate dehydrogenase complex

The mitochondrial pyruvate dehydrogenase complex (PDC) translocates into the nucleus, facilitating histone acetylation by producing acetyl-CoA. We describe a noncanonical pathway for nuclear PDC(nPDC) import that does not involve nuclear pore complexes (NPCs). Mitochondria cluster around the nucleus in response to proliferative stimuli and tether onto the nuclear envelope (NE) via mitofusin-2 (MFN2)-enriched contact points. A decrease in nuclear MFN2 levels decreases mitochondria tethering and nPDC levels. Mitochondrial PDC crosses the NE and interacts with lamin A, forming a ring below the NE before crossing through the lamin layer into the nucleoplasm, in areas away from NPCs. Effective blockage of NPC trafficking does not decrease nPDC levels. The PDC-lamin interaction is maintained during cell division, when lamin depolymerizes and disassembles before reforming daughter nuclear envelopes, providing another pathway for nPDC entry during mitosis. Our work provides a different angle to understanding mitochondria-to-nucleus communication and nuclear metabolism.