Autocrine phosphatase PDP2 inhibits ferroptosis by dephosphorylating ACSL4 in the Luminal A Breast Cancer.

Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p < 0.001), as indicated by data from flow cytometry detection of membrane-permeability cell death stained with 7-aminoactinomycin, lipid peroxidation, and Fe2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1).

Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. Recent studies have identified the inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS model systems. However, the molecular mechanisms linking aberrant CL remodeling, particularly the primary, direct consequence of reduced tetralinoleoyl-CL (TLCL) levels, to PDH activity deficiency are not yet understood. In the current study, we found that remodeled TLCL promotes PDH function by directly binding to and enhancing the activity of PDH phosphatase 1 (PDP1). This is supported by our findings that TLCL uniquely activates PDH in a dose-dependent manner, TLCL binds to PDP1 in vitro, TLCL-mediated PDH activation is attenuated in the presence of phosphatase inhibitor, and PDP1 activity is decreased in Tafazzin-knockout (TAZ-KO) C2C12 myoblasts. Additionally, we observed decreased mitochondrial calcium levels in TAZ-KO cells and treating TAZ-KO cells with calcium lactate (CaLac) increases mitochondrial calcium and restores PDH activity and mitochondrial oxygen consumption rate. Based on our findings, we conclude that reduced mitochondrial calcium levels and decreased binding of PDP1 to TLCL contribute to decreased PDP1 activity in TAZ-KO cells.

Pyruvate dehydrogenase phosphatase 1 (PDP1) stimulates HIF activity by supporting histone acetylation under hypoxia.

Cancer cells, when exposed to the hypoxic tumour microenvironment, respond by activating hypoxia-inducible factors (HIFs). HIF-1 mediates extensive metabolic re-programming, and expression of HIF-1α, its oxygen-regulated subunit, is associated with poor prognosis in cancer. Here we analyse the role of pyruvate dehydrogenase phosphatase 1 (PDP1) in the regulation of HIF-1 activity. PDP1 is a key hormone-regulated metabolic enzyme that dephosphorylates and activates pyruvate dehydrogenase (PDH), thereby stimulating the conversion of pyruvate into acetyl-CoA. Silencing of PDP1 down-regulated HIF transcriptional activity and the expression of HIF-dependent genes, including that of PDK1, the kinase that phosphorylates and inactivates PDH, opposing the effects of PDP1. Inversely, PDP1 stimulation enhanced HIF activity under hypoxia. Alteration of PDP1 levels or activity did not have an effect on HIF-1α protein levels, nuclear accumulation or interaction with its partners ARNT and NPM1. However, depletion of PDP-1 decreased histone H3 acetylation of HIF-1 target gene promoters and inhibited binding of HIF-1 to the respective hypoxia-response elements (HREs) under hypoxia. Furthermore, the decrease of HIF transcriptional activity upon PDP1 depletion could be reversed by treating the cells with acetate, as an exogenous source of acetyl-CoA, or the histone deacetylase (HDAC) inhibitor trichostatin A. These data suggest that the PDP1/PDH/HIF-1/PDK1 axis is part of a homeostatic loop which, under hypoxia, preserves cellular acetyl-CoA production to a level sufficient to sustain chromatin acetylation and transcription of hypoxia-inducible genes.

Association of structural variation with cardiometabolic traits in Finns.

The contribution of genome structural variation (SV) to quantitative traits associated with cardiometabolic diseases remains largely unknown. Here, we present the results of a study examining genetic association between SVs and cardiometabolic traits in the Finnish population. We used sensitive methods to identify and genotype 129,166 high-confidence SVs from deep whole-genome sequencing (WGS) data of 4,848 individuals. We tested the 64,572 common and low-frequency SVs for association with 116 quantitative traits and tested candidate associations using exome sequencing and array genotype data from an additional 15,205 individuals. We discovered 31 genome-wide significant associations at 15 loci, including 2 loci at which SVs have strong phenotypic effects: (1) a deletion of the ALB promoter that is greatly enriched in the Finnish population and causes decreased serum albumin level in carriers (p = 1.47 × 10-54) and is also associated with increased levels of total cholesterol (p = 1.22 × 10-28) and 14 additional cholesterol-related traits, and (2) a multi-allelic copy number variant (CNV) at PDPR that is strongly associated with pyruvate (p = 4.81 × 10-21) and alanine (p = 6.14 × 10-12) levels and resides within a structurally complex genomic region that has accumulated many rearrangements over evolutionary time. We also confirmed six previously reported associations, including five led by stronger signals in single nucleotide variants (SNVs) and one linking recurrent HP gene deletion and cholesterol levels (p = 6.24 × 10-10), which was also found to be strongly associated with increased glycoprotein level (p = 3.53 × 10-35). Our study confirms that integrating SVs in trait-mapping studies will expand our knowledge of genetic factors underlying disease risk.

Detailed evaluation of pyruvate dehydrogenase complex inhibition in simulated exercise conditions.

The mammalian pyruvate dehydrogenase complex (PDC) is a mitochondrial multienzyme complex that connects glycolysis to the tricarboxylic acid cycle by catalyzing pyruvate oxidation to produce acetyl-CoA, NADH, and CO2. This reaction is required to aerobically utilize glucose, a preferred metabolic fuel, and is composed of three core enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). The pyruvate-dehydrogenase-specific kinase (PDK) and pyruvate-dehydrogenase-specific phosphatase (PDP) are considered the main control mechanism of mammalian PDC activity. However, PDK and PDP activity are allosterically regulated by several effectors fully overlapping PDC substrates and products. This collection of positive and negative feedback mechanisms confounds simple predictions of relative PDC flux, especially when all effectors are dynamically modulated during metabolic states that exist in physiologically realistic conditions, such as exercise. Here, we provide, to our knowledge, the first globally fitted, pH-dependent kinetic model of the PDC accounting for the PDC core reaction because it is regulated by PDK, PDP, metal binding equilibria, and numerous allosteric effectors. The model was used to compute PDH regulatory complex flux as a function of previously determined metabolic conditions used to simulate exercise and demonstrates increased flux with exercise. Our model reveals that PDC flux in physiological conditions is primarily inhibited by product inhibition (∼60%), mostly NADH inhibition (∼30-50%), rather than phosphorylation cycle inhibition (∼40%), but the degree to which depends on the metabolic state and PDC tissue source.

Crystal structure of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase.

Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Šresolution is reported. The crystals belonged to space group P3221, with unit-cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (Rfree = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.

Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts.

A splice site mutation in the canine pyruvate dehydrogenase kinase 4 (PDK4) gene has been shown to be associated with the development of dilated cardiomyopathy (DCM) in Doberman Pinchers (DPs). Subsequent studies have successfully demonstrated the use of dermal fibroblasts isolated from DPs as models for PDK4 deficiency and have shown activation of the intrinsic (mitochondrial mediated) apoptosis pathway in these cells under starvation conditions. For this study, we sought to further explore the functional consequences of PDK4 deficiency in DP fibroblasts representing PDK4wt/wt, PDK4wt/del, and PDK4del/del genotypes. Our results show that starvation conditions cause increased perinuclear localization of mitochondria and decreased cell proliferation, altered expression levels of pyruvate dehydrogenase phosphatase (PDP) and pyruvate dehydrogenase (PDH), dramatically increased PDH activity, and an impaired response to mitochondrial stress in affected cells. In sum, these results show the broad impact of PDK4 deficiency and reveal mechanistic pathways used by these cells in an attempt to compensate for the condition. Our data help to elucidate the mechanisms at play in this extremely prevalent DP disorder and provide further support demonstrating the general importance of metabolic flexibility in cell health.

The mitochondrial PKCδ/retinol signal complex exerts real-time control on energy homeostasis.

The review focuses on the role of vitamin A (retinol) in the control of energy homeostasis, and on the manner in which certain retinoids subvert this process, leading potentially to disease. In eukaryotic cells, the pyruvate dehydrogenase complex (PDHC) is negatively regulated by four pyruvate dehydrogenase kinases (PDKs) and two antagonistically acting pyruvate dehydrogenase phosphatases (PDPs). The second isoform, PDK2, is regulated by an autonomous mitochondrial signal cascade that is anchored on protein kinase Cδ (PKCδ), where retinoids play an indispensible co-factor role. Along with its companion proteins p66Shc, cytochrome c, and vitamin A, the PKCδ/retinol complex is located in the intermembrane space of mitochondria. At this site, and in contrast to cytosolic locations, PKCδ is activated by the site-specific oxidation of its cysteine-rich activation domain (CRD) that is configured into a complex RING-finger. Oxidation involves the transfer of electrons from cysteine moieties to oxidized cytochrome c, a step catalyzed by vitamin A. The PKCδ/retinol signalosome monitors the internal cytochrome c redox state that reflects the workload of the respiratory chain. Upon sensing demands for energy PKCδ signals the PDHC to increase glucose-derived fuel flux entering the KREBS cycle. Conversely, if excessive fuel flux surpasses the capacity of the respiratory chain, threatening the release of damaging reactive oxygen species (ROS), the polarity of the cytochrome c redox system is reversed, resulting in the chemical reduction of the PKCδ CRD, restoration of the RING-finger, refolding of PKCδ into the inactive, globular form, and curtailment of PDHC output, thereby constraining the respiratory capacity within safe margins. Several retinoids, notably anhydroretinol and fenretinide, capable of displacing retinol from binding sites on PKCδ, can co-activate PKCδ signaling but, owing to their extended system of conjugated double bonds, are unable to silence PKCδ in a timely manner. Left in the ON position, PKCδ causes chronic overload of the respiratory chain leading to mitochondrial dysfunction. This review explores how defects in the PKCδ signal machinery potentially contribute to metabolic and degenerative diseases.

FOXK1 and FOXK2 regulate aerobic glycolysis.

Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others-including humans and several other mammals-have developed a high degree of flexibility1. A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients2. Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria-instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis.

Identification of hepatic thyroid hormone-responsive genes in neonatal rats: Potential targets for thyroid hormone-disrupting chemicals.

There have been many concerns about the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. Because thyroid hormones are essential for regulating the growth and differentiation of many tissues, disruption of thyroid hormones during the neonatal period of an organism might lead to permanent effects on that organism. We postulated that there are target genes that are sensitive to thyroid hormones particularly during the neonatal period and that would thus be susceptible to thyroid hormone-disrupting chemicals. Global gene expression analysis was used to identify these genes in the liver of rat neonates. The changes in hepatic gene expression were examined 24 h after administering 1.0, 10, and 100 ng/g body weight (bw) triiodothyronine (T3) to male rats on postnatal day 3. Thirteen upregulated and four downregulated genes were identified in the neonatal liver. Among these, Pdp2 and Slc25a25 were found to be upregulated and more sensitive to T3 than the others, whereas Cyp7b1 and Hdc were found to be downregulated even at the lowest dose of 1.0 ng/g bw T3. Interestingly, when the responses of gene expression to T3 were examined in adult rats (8-week old), one-third of them did not respond to T3. The environmental chemicals with thyroid hormone-like activity, hydroxylated polybrominated diphenyl ethers, were then administered to neonatal rats to examine the effects on expression of the identified genes. The results showed that these chemicals were indeed capable of changing the expression of Slc25a25 and Hdc. Our results demonstrated a series of hepatic T3-responsive genes that are more sensitive to hormones during the neonatal period than during adulthood. These genes might be the potential targets of thyroid hormone-disrupting chemicals in newborns.

MiR-195 modulates oxidative stress-induced apoptosis and mitochondrial energy production in human trophoblasts via flavin adenine dinucleotide-dependent oxidoreductase domain-containing protein 1 and pyruvate dehydrogenase phosphatase regulatory subunit.

OBJECTIVE: Preeclampsia is a severe pregnancy-specific syndrome defined as newly onset hypertension and proteinuria. Abnormal placental development has been generally accepted as the initial cause of the disorder. Recently, miR-195 was identified as one of the downregulated small RNAs in preeclamptic placentas. METHODS: The potential targets of miR-195 in human trophoblast cells were screened by isobaric tags for relative and absolute quantification-based mass spectrum analysis. Localization of miR-195 and its targets was examined by in-situ hybridization and immunohistochemistry in human placenta. Real-time PCR, western blotting and luciferase assay were used for target validation. Apoptosis was accessed by Annexin V/PI costaining, whereas mitochondrial function by ATP measurement and tetramethylrhodamine ethyl ester fluorescence. RESULTS: Two mitochondria-associated proteins, flavin adenine dinucleotide-dependent oxidoreductase domain-containing protein 1 (FOXRED1) and pyruvate dehydrogenase phosphatase regulatory subunit (PDPR), were identified as targets of miR-195. Overexpression of miR-195 in HTR8/SVneo cells resulted in enhanced apoptosis, decreased mitochondrial membrane potential and cellular ATP content upon hydrogen peroxide stimulation. The effects could be partially rescued by FOXRED1 or PDPR. In preeclamptic patients, lowered circulating level of miR-195 were found at early-to-mid gestation and term pregnancy, and marked increase in FOXRED1 and PDPR expression were observed in the placenta when compared with gestational week-matched controls. In addition, chronic hydrogen peroxide stimuli suppressed miR-195 expression in trophoblast cells. CONCLUSION: MiR-195 could suppress mitochondrial energy production via targeting FOXRED1 and PDPR, and lead to trophoblast cell apoptosis under oxidative stress. In preeclamptic placenta, lowered level of miR-195 might be induced by chorionic oxidative stress and subsequently form a compensation mechanism to defend the disturbed energy production and cell apoptosis upon oxidative stress.

Targeted knockdown of polo-like kinase 1 alters metabolic regulation in melanoma.

A limited number of studies have indicated an association of the mitotic kinase polo-like kinase 1 (PLK1) and cellular metabolism. Here, employing an inducible RNA interference approach in A375 melanoma cells coupled with a PCR array and multiple validation approaches, we demonstrated that PLK1 alters a number of genes associated with cellular metabolism. PLK1 knockdown resulted in a significant downregulation of IDH1, PDP2 and PCK1 and upregulation of FBP1. Ingenuity Pathway Analysis (IPA) identified that 1) glycolysis and the pentose phosphate pathway are major canonical pathways associated with PLK1, and 2) PLK1 inhibition-modulated genes were largely associated with cellular proliferation, with FBP1 being the key modulator. Further, BI 6727-mediated inhibition of PLK1 caused a decrease in PCK1 and increase in FBP1 in A375 melanoma cell implanted xenografts in vivo. Furthermore, an inverse correlation between PLK1 and FBP1 was found in melanoma cells, with FBP1 expression significantly downregulated in a panel of melanoma cells. In addition, BI 6727 treatment resulted in an upregulation in FBP1 in A375, Hs294T and G361 melanoma cells. Overall, our study suggests that PLK1 may be an important regulator of metabolism maintenance in melanoma cells.

Pyruvate Dehydrogenase Phosphatase Regulatory Gene Expression Correlates with Exercise Training Insulin Sensitivity Changes.

PURPOSE: Whole body insulin sensitivity (Si) typically improves after aerobic exercise training; however, individual responses can be highly variable. The purpose of this study was to use global gene expression to identify skeletal muscle genes that correlate with exercise-induced Si changes. METHODS: Longitudinal cohorts from the Studies of Targeted Risk Reduction Intervention through Defined Exercise were used as Discovery (Affymetrix) and Confirmation (Illumina) of vastus lateralis gene expression profiles. Discovery (n = 39; 21 men) and Confirmation (n = 42; 19 men) cohorts were matched for age (52 ± 8 vs 51 ± 10 yr), body mass index (30.4 ± 2.8 vs 29.7 ± 2.8 kg·m), and V˙O2max (30.4 ± 2.8 vs 29.7 ± 2.8 mL·kg·min). Si was determined via intravenous glucose tolerance test pretraining and posttraining. Pearson product-moment correlation coefficients determined relationships between a) baseline and b) training-induced changes in gene expression and %ΔSi after training. RESULTS: Expression of 2454 (Discovery) and 1778 genes (Confirmation) at baseline were significantly (P < 0.05) correlated to %ΔSi; 112 genes overlapped. Pathway analyses identified Ca signaling-related transcripts in this 112-gene list. Expression changes of 1384 (Discovery) and 1288 genes (Confirmation) after training were significantly (P < 0.05) correlated to %ΔSi; 33 genes overlapped, representing contractile apparatus of skeletal and smooth muscle genes. Pyruvate dehydrogenase phosphatase regulatory subunit expression at baseline (P = 0.01, r = 0.41) and posttraining (P = 0.01, r = 0.43) were both correlated with %ΔSi. CONCLUSIONS: Exercise-induced adaptations in skeletal muscle Si are related to baseline levels of Ca-regulating transcripts, which may prime the muscle for adaptation. Relationships between %ΔSi and pyruvate dehydrogenase phosphatase regulatory, a regulatory subunit of the pyruvate dehydrogenase complex, indicate that the Si response is strongly related to key steps in metabolic regulation.

1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells.

Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength.

NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation.

Metabolic reprogramming is implicated in macrophage activation, but the underlying mechanisms are poorly understood. Here, we demonstrate that the NOTCH1 pathway dictates activation of M1 phenotypes in isolated mouse hepatic macrophages (HMacs) and in a murine macrophage cell line by coupling transcriptional upregulation of M1 genes with metabolic upregulation of mitochondrial oxidative phosphorylation and ROS (mtROS) to augment induction of M1 genes. Enhanced mitochondrial glucose oxidation was achieved by increased recruitment of the NOTCH1 intracellular domain (NICD1) to nuclear and mitochondrial genes that encode respiratory chain components and by NOTCH-dependent induction of pyruvate dehydrogenase phosphatase 1 (Pdp1) expression, pyruvate dehydrogenase activity, and glucose flux to the TCA cycle. As such, inhibition of the NOTCH pathway or Pdp1 knockdown abrogated glucose oxidation, mtROS, and M1 gene expression. Conditional NOTCH1 deficiency in the myeloid lineage attenuated HMac M1 activation and inflammation in a murine model of alcoholic steatohepatitis and markedly reduced lethality following endotoxin-mediated fulminant hepatitis in mice. In vivo monocyte tracking further demonstrated the requirement of NOTCH1 for the migration of blood monocytes into the liver and subsequent M1 differentiation. Together, these results reveal that NOTCH1 promotes reprogramming of mitochondrial metabolism for M1 macrophage activation.

Ras-mediated modulation of pyruvate dehydrogenase activity regulates mitochondrial reserve capacity and contributes to glioblastoma tumorigenesis.

BACKGROUND: Even though altered metabolism representing a hallmark of cancer was proposed nearly a century ago, recent technological advances have allowed investigators to continue uncovering a previously unrecognized complexity of metabolic programs that drive tumorigenesis beyond that of aerobic glycolysis. METHODS: The bioenergetic state of a diverse panel of glioblastoma models, including isogenic lines derived from a genetically engineered adult astrocytic mouse model and patient-derived glioblastoma stem cells, was determined at baseline and in stressed conditions. Mechanisms contributing to the discovered metabolic phenotypes were determined through molecular and chemical perturbation, and their biological consequences were evaluated in vivo and in patient samples. RESULTS: Attenuated mitochondrial reserve capacity was identified as a common metabolic phenotype in glioblastoma lines. This phenotype was linked mechanistically with the capacity of Ras-mediated signaling to inhibit pyruvate dehydrogenase (PDH) activity through downregulation of PDH phosphatase (PDP) expression. PDP1 repression was validated clinically in patient-derived samples, suggesting that aberrant cellular signaling typical of glioblastoma actively modulates PDH activity. This phenotype was reversed through both chemical and molecular perturbation. Restoration of PDH activity through stable expression of PDP1-impaired tumorigenic potential. CONCLUSIONS: These findings support the central role that PDH regulation plays as a downstream consequence of aberrant signaling associated with gliomagenesis and the scientific rationale to continue to develop and test clinical strategies designed to activate PDH as a form of anticancer therapy in glioblastoma.

Tyr-94 phosphorylation inhibits pyruvate dehydrogenase phosphatase 1 and promotes tumor growth.

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.

Tyr phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate the pyruvate dehydrogenase complex.

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here, we report that lysine acetylation of PDHA1 and PDP1 is common in epidermal growth factor (EGF)-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1, and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important in promoting glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct posttranslational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.

A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence.

In response to tenacious stress signals, such as the unscheduled activation of oncogenes, cells can mobilize tumour suppressor networks to avert the hazard of malignant transformation. A large body of evidence indicates that oncogene-induced senescence (OIS) acts as such a break, withdrawing cells from the proliferative pool almost irreversibly, thus crafting a vital pathophysiological mechanism that protects against cancer. Despite the widespread contribution of OIS to the cessation of tumorigenic expansion in animal models and humans, we have only just begun to define the underlying mechanism and identify key players. Although deregulation of metabolism is intimately linked to the proliferative capacity of cells, and senescent cells are thought to remain metabolically active, little has been investigated in detail about the role of cellular metabolism in OIS. Here we show, by metabolic profiling and functional perturbations, that the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) is a crucial mediator of senescence induced by BRAF(V600E), an oncogene commonly mutated in melanoma and other cancers. BRAF(V600E)-induced senescence was accompanied by simultaneous suppression of the PDH-inhibitory enzyme pyruvate dehydrogenase kinase 1 (PDK1) and induction of the PDH-activating enzyme pyruvate dehydrogenase phosphatase 2 (PDP2). The resulting combined activation of PDH enhanced the use of pyruvate in the tricarboxylic acid cycle, causing increased respiration and redox stress. Abrogation of OIS, a rate-limiting step towards oncogenic transformation, coincided with reversion of these processes. Further supporting a crucial role of PDH in OIS, enforced normalization of either PDK1 or PDP2 expression levels inhibited PDH and abrogated OIS, thereby licensing BRAF(V600E)-driven melanoma development. Finally, depletion of PDK1 eradicated melanoma subpopulations resistant to targeted BRAF inhibition, and caused regression of established melanomas. These results reveal a mechanistic relationship between OIS and a key metabolic signalling axis, which may be exploited therapeutically.