Pyruvate Dehydrogenase E1α Subunit Research Articles

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.

PDK inhibition promotes glucose utilization, reduces hepatic lipid deposition, and improves oxidative stress in largemouth bass (Micropterus salmoides) by increasing pyruvate oxidative phosphorylation.

In omnivorous fish, the pyruvate dehydrogenase kinases (PDKs)-pyruvate dehydrogenase E1α subunit (PDHE1α) axis is essential in the regulation of carbohydrate oxidative catabolism. Among the existing research, the role of the PDKs-PDHE1α axis in carnivorous fish with poor glucose utilization is unclear. In the present study, we determined the effects of PDK inhibition on the liver glycolipid metabolism of largemouth bass (Micropterus salmoides). DCA is a PDK-specific inhibitor that inhibits PDK by binding the allosteric sites. A total of 160 juvenile largemouth bass were randomly divided into two groups, with four replicates of 20 fish each, fed a control diet and a control diet supplemented with dichloroacetate (DCA) for 8 weeks. The present results showed that DCA supplementation significantly decreased the hepatosomatic index, triglycerides in liver and serum, and total liver lipids of largemouth bass compared with the control group. In addition, compared with the control group, DCA treatment significantly down-regulated gene expression associated with lipogenesis. Furthermore, DCA supplementation significantly decreased the mRNA expression of pdk3a and increased PDHE1α activity. In addition, DCA supplementation improved glucose oxidative catabolism and pyruvate oxidative phosphorylation (OXPHOS) in the liver, as evidenced by low pyruvate content in the liver and up-regulated expressions of glycolysis-related and TCA cycle/OXPHOS-related genes. Moreover, DCA consumption decreased hepatic malondialdehyde (MDA) content, enhanced the activities of superoxide dismutase (SOD), and increased transforming growth factor beta (tgf-β), glutathione S-transferase (gst), and superoxide dismutase 1 (sod1) gene expression compared with the control diet. This study demonstrated that inhibition of PDKs by DCA promoted glucose utilization, reduced hepatic lipid deposition, and improved oxidative stress in largemouth bass by increasing pyruvate OXPHOS. Our findings contribute to the understanding of the underlying mechanism of the PDKs-PDHE1α axis in glucose metabolism and improve the utilization of dietary carbohydrates in farmed carnivorous fish.

PDHA1 Alleviates Myocardial Ischemia-Reperfusion Injury by Improving Myocardial Insulin Resistance During Cardiopulmonary Bypass Surgery in Rats.

Cardiopulmonary bypass (CPB) is a requisite technique for thoracotomy in advanced cardiovascular surgery. However, the consequent myocardial ischemia-reperfusion injury (MIRI) is the primary culprit behind cardiac dysfunction and fatal consequences post-operation. Prior research has posited that myocardial insulin resistance (IR) plays a vital role in exacerbating the progression of MIRI. Nonetheless, the exact mechanisms underlying this phenomenon remain obscure. We constructed pyruvate dehydrogenase E1 α subunit (PDHA1) interference and overexpression rats and used ascending aorta occlusion in an in vivo model of CPB-MIRI. We devised an in vivo model of CPB-MIRI by constructing rat models with both pyruvate dehydrogenase E1α subunit (PDHA1) interference and overexpression through ascending aorta occlusion. We analyzed myocardial glucose metabolism and the degree of myocardial injury using functional monitoring, biochemical assays, and histological analysis. We discovered a clear downregulation of glucose transporter 4 (GLUT4) protein content expression in the CPB I/R model. In particular, cardiac-specific PDHA1 interference resulted in exacerbated cardiac dysfunction, significantly increased myocardial infarction area, more pronounced myocardial edema, and markedly increased cardiomyocyte apoptosis. Notably, the opposite effect was observed with PDHA1 overexpression, leading to a mitigated cardiac dysfunction and decreased incidence of myocardial infarction post-global ischemia. Mechanistically, PDHA1 plays a crucial role in regulating the protein content expression of GLUT4 on cardiomyocytes, thereby controlling the uptake and utilization of myocardial glucose, influencing the development of myocardial insulin resistance, and ultimately modulating MIRI. Overall, our study sheds new light on the pivotal role of PDHA1 in glucose metabolism and the development of myocardial insulin resistance. Our findings hold promising therapeutic potential for addressing the deleterious effects of MIRI in patients.

Inhibition of pyruvate dehydrogenase kinase improves carbohydrate utilization in Nile tilapia by regulating PDK2/4-PDHE1α axis and insulin sensitivity.

Pyruvate dehydrogenase kinases (PDKs)-pyruvate dehydrogenase E1α subunit (PDHE1α) axis plays an important role in regulating glucose metabolism in mammals. However, the regulatory function of PDKs-PDHE1α axis in the glucose metabolism of fish is not well known. This study determined whether PDKs inhibition could enhance PDHE1α activity, and improve glucose catabolism in fish. Nile tilapia fingerlings (1.90 ± 0.11 g) were randomly divided into 4 treatments in triplicate (30 fish each) and fed control diet without dichloroacetate (DCA) (38% protein, 7% lipid and 45% corn starch) and the control diet supplemented with DCA, which inhibits PDKs through binding the allosteric sites, at 3.75 (DCA3.75), 7.50 (DCA7.50) and 11.25 g/kg (DCA11.25), for 6 wk. The results showed that DCA3.75, DCA7.50 and DCA11.25 significantly increased weight gain, carcass ratio and protein efficiency ratio (P < 0.05) and reduced feed efficiency (P < 0.05) of Nile tilapia. To investigate the effects of DCA on growth performance of Nile tilapia, we selected the lowest dose DCA3.75 for subsequent analysis. Nile tilapia fed on DCA3.75 significantly reduced the mesenteric fat index, serum and liver triglyceride concentration and total lipid content in whole fish, and down-regulated the expressions of genes related to lipogenesis (P < 0.05) compared to the control. The DCA3.75 treatment significantly improved glucose oxidative catabolism and glycogen synthesis in the liver, but significantly reduced the conversion of glucose to lipid (P < 0.05). Furthermore, the DCA3.75 treatment significantly decreased the PDK2/4 gene and protein expressions (P < 0.05), accordingly stimulated PDHE1α activity by decreasing the phosphorylated PDHE1α protein level. In addition, DCA3.75 treatment significantly increased the phosphorylated levels of key proteins involved in insulin signaling pathway and glycogen synthase kinase 3β (P < 0.05). Taken together, the present study demonstrates that PDK2/4 inhibition by using DCA promotes glucose utilization in Nile tilapia by activating PDHE1α and improving insulin sensitivity. Our study helps to understand the regulatory mechanism of glucose metabolism for improving dietary carbohydrate utilization in farmed fish.

Rheb mediates neuronal-activity-induced mitochondrial energetics through mTORC1-independent PDH activation.

Neuronal activity increases energy consumption and requires balanced production to maintain neuronal function. How activity is coupled to energy production remains incompletely understood. Here, we report that Rheb regulates mitochondrial tricarboxylic acid cycle flux of acetyl-CoA by activating pyruvate dehydrogenase (PDH) to increase ATP production. Rheb is induced by synaptic activity and lactate and dynamically trafficked to the mitochondrial matrix through its interaction with Tom20. Mitochondria-localized Rheb protein is required for activity-induced PDH activation and ATP production. Cell-type-specific gain- and loss-of-function genetic models for Rheb reveal reciprocal changes in PDH phosphorylation/activity, acetyl-CoA, and ATP that are not evident with genetic or pharmacological manipulations of mTORC1. Mechanistically, Rheb physically associates with PDH phosphatase (PDP), enhancing its activity and association with the catalytic E1α-subunit of PDH to reduce PDH phosphorylation and increase its activity. Findings identify Rheb as a nodal point that balances neuronal activity and neuroenergetics via Rheb-PDH axis.

The neglected foodborne mycotoxin Fusaric acid induces bioenergetic adaptations by switching energy metabolism from mitochondrial processes to glycolysis in a human liver (HepG2) cell line

Metabolic flexibility defines the capacity of cells to respond to changes in nutrient status. Mitochondria are important mediators of metabolic flexibility and dysfunction is associated with metabolic inflexibility and pathology. Foodborne toxins are often overlooked as potential factors contributing to metabolic toxicity. Fusaric acid (FA), a neglected mycotoxin, is known to disrupt mitochondrial function. The aim of this study was to investigate the molecular mechanisms underlying a metabolic switch in response to FA. This study investigated the effects of FA on energy homeostasis in cultured human liver (HepG2) cells. HepG2 cells poised to undergo oxidative and glycolytic metabolism were exposed to a range of FA concentrations (4, 63 and 250 μg/mL) for 6 h. We determined mitochondrial toxicity, acetyl CoA levels and cell viability using luminometric, fluorometric and spectrophotometric methods. Expression of metabolic proteins (PDK1, PKM2, phosphorylated-PDH E1α and HIF-1α) and mRNAs (HIF-1α, PKM2, LDHa and PDK1) were determined using western blot and qPCR respectively. Our data connects a constitutive expression of HIF-1α in response to FA, to the inhibition of pyruvate decarboxylation through up-regulation of PDK-1 and phosphorylation of Pyruvate Dehydrogenase E1α subunit. Moreover, we highlight the potential of FA to induce a glucose “addiction” and phenotype reminiscent of the Warburg effect. The findings provide novel insights into the impact of this neglected foodborne mycotoxin in the dysregulation of energy metabolism.

Huzhangoside A Suppresses Tumor Growth through Inhibition of Pyruvate Dehydrogenase Kinase Activity.

Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1α subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for cancer treatment. Here, we report that huzhangoside A (Hu.A), a triterpenoid glycoside compound isolated from several plants of the Anemone genus, acts as a novel PDHK inhibitor. Hu.A was found to decrease the cell viability of human breast cancer MDA-MB-231, hepatocellular carcinoma Hep3B, colon cancer HT-29, DLD-1, and murine lewis lung carcinoma LLC cell lines. The activity of PDHK1 was decreased by Hu.A in both in vitro assays and in vivo assays in DLD-1 cells. Hu.A significantly increased the oxygen consumption and decreased the secretory lactate levels in DLD-1 cells. In addition, Hu.A interacted with the ATP-binding pocket of PDHK1 without affecting the interaction of PDHK1 and pyruvate dehydrogenase complex (PDC) subunits. Furthermore, Hu.A significantly induced mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in DLD-1 cells. Consistently, when Hu.A was intraperitoneally injected into LLC allograft mice, the tumor growth was significantly decreased. In conclusion, Hu.A suppressed the growth of tumors in both in vitro and in vivo models via inhibition of PDHK activity.

Overexpression of Pyruvate Dehydrogenase E1α Subunit Inhibits Warburg Effect and Induces Cell Apoptosis Through Mitochondria-Mediated Pathway in Hepatocellular Carcinoma.

Most cancers rely disproportionately on glycolysis for energy even in the presence of an adequate oxygen supply, a condition known as “aerobic glycolysis,” or the “Warburg effect.” Pyruvate dehydrogenase E1α subunit (PDHA1) is one of the main factors for the metabolic switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and has been suggested to be closely associated with tumorigenesis. Here we observed that the PDHA1 protein was reduced in hepatocellular carcinoma (HCC) specimens by immunohistochemistry and Western blot, which was significantly associated with poor overall survival. To further analyze the function of PDHA1 in cancer cells, PDHA1 was upregulated in the HCC cell lines SMMC-7721 and HepG2. The results demonstrated that overexpression of the PDHA1 gene inhibited aerobic glycolysis with lower lactate via increased PDH activity; meanwhile, mitochondrial OXPHOS was enhanced accompanied with higher ATP and lower glucose consumption. We also found that apoptosis was promoted and intrinsic pathway proteins were increased in PDHA1-overexpressing cells. Collectively, our data indicate that reduced PDHA1 protein expression is associated with the poor clinical outcome of HCC. Upregulated PDHA1 gene expression can inhibit the Warburg effect and enhance the mitochondria-mediated apoptosis pathway.

Calcium-sensitive pyruvate dehydrogenase phosphatase is required for energy metabolism, growth, differentiation, and infectivity of Trypanosoma cruzi

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work, we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and T. brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in T. cruzi Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.

Bi-allelic mutations ofLONP1encoding the mitochondrial LonP1 protease cause pyruvate dehydrogenase deficiency and profound neurodegeneration with progressive cerebellar atrophy

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C>T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings' fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings' fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings' fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

Insulin is a critical signaling molecule in reducing blood glucose levels, and pyruvate dehydrogenase (PDH) is an essential enzyme in regulating glucose metabolism. However, the insulin effect on PDH function has not been well established. We observed that insulin attenuated the phosphorylation (p) of Ser264 (p-Ser264) in the PDH E1α subunit (PDHA1) in normal rat hepatocyte. In contrast, insulin induced an increase of p-Ser264 PDHA1 levels in hepatocellular carcinoma HepG2 and Huh7 cells. Insulin activated RhoA and Rho-dependent coiled coil kinase, an effector protein of active RhoA, which regulated p-Ser264 PDHA1 levels, along with both p-Ser9 and p-Tyr216 forms of glycogen synthase kinase-3β (GSK-3β) in HepG2 cells. Only p-Tyr216 GSK-3β, the active form was involved in an increase of p-Ser264 PDHA1. Akt was also engaged in p-Ser9 of GSK-3β, but neither in p-Tyr216 of GSK-3β nor p-Ser264 of PDHA1 upon insulin. Reconstituted dephospho-mimic forms PDHA1 S264A and GSK-3β Y216F impaired, but wild-types PDHA1 and GSK-3β and phospho-mimic forms PDHA1 S264D and GSK-3β Y216E increased cell proliferation upon insulin through expression of c-Myc and cyclin D1. Therefore, we propose that insulin-mediated p-PDHA1 is involved in the regulation of HepG2 cell proliferation through RhoA signaling pathway.-Islam, R., Kim, J.-G., Park, Y., Cho, J.-Y., Cap, K.-C., Kho, A.-R., Chung, W.-S., Suh, S.-W., Park, J.-B. Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

The effect of dichloroacetate in mouse models of epilepsy

A recent report has found that glucose oxidation and the activity of pyruvate dehydrogenase (PDH) are reduced in the chronic stage of the pilocarpine mouse epilepsy model. This is likely caused by increased phosphorylation by PDH kinase of the E1α subunit of PDH, downregulating its activity. Inhibition of this phosphorylation has not yet been explored as a possible approach to treat epilepsy. Chronic dichloroacetate (DCA, 50 and 100 mg/kg/day) treatment was tested in acute seizure and the chronic pilocarpine models. We also determined the effects on phosphorylation state, activity and protein levels of PDH in the chronic stage of the pilocarpine model. DCA treatment did not increase latencies to seizures in the acute flurothyl seizure test and was slightly proconvulsant in the 6 Hz test. The latencies to seizures in a second-hit flurothyl test were decreased in SE vs. No SE mice in the chronic stage, but were not restored by DCA. In mice that had experienced pilocarpine-induced SE and were in the chronic “epileptic” stage of the model, PDH activity was reduced by 65% compared to “healthy” No SE mice. This was partially alleviated with DCA treatment. Also, PDH protein levels were decreased by 37% and phosphorylation at Ser300 of PDH was increased by 52% in SE mice, but were not significantly changed with DCA. Moreover DCA treatment decreased the amounts of total PDH by 23% in No SE mice, which may explain the proconvulsant effects in the 6 Hz test. The reduction in PDH protein levels during the chronic epileptic stage suggests increased degradation of the protein, which may contribute to the deficient glucose oxidation found in epilepsy. Taken together, DCA did not have any anti-convulsant effects in the tested models. Future studies utilising other PDH kinase inhibitors are required to determine whether this treatment approach is viable.

Role of the Pyruvate Dehydrogenase Complex in Metabolic Remodeling: Differential Pyruvate Dehydrogenase Complex Functions in Metabolism

Mitochondrial dysfunction is a hallmark of metabolic diseases such as obesity, type 2 diabetes mellitus, neurodegenerative diseases, and cancers. Dysfunction occurs in part because of altered regulation of the mitochondrial pyruvate dehydrogenase complex (PDC), which acts as a central metabolic node that mediates pyruvate oxidation after glycolysis and fuels the Krebs cycle to meet energy demands. Fine-tuning of PDC activity has been mainly attributed to post-translational modifications of its subunits, including the extensively studied phosphorylation and de-phosphorylation of the E1α subunit of pyruvate dehydrogenase (PDH), modulated by kinases (pyruvate dehydrogenase kinase [PDK] 1-4) and phosphatases (pyruvate dehydrogenase phosphatase [PDP] 1-2), respectively. In addition to phosphorylation, other covalent modifications, including acetylation and succinylation, and changes in metabolite levels via metabolic pathways linked to utilization of glucose, fatty acids, and amino acids, have been identified. In this review, we will summarize the roles of PDC in diverse tissues and how regulation of its activity is affected in various metabolic disorders.

Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours.

Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs.

Abstract LB-314: Oncogenic Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine phosphorylation

Abstract The Warburg effect, which reflects cancer cells’ preference for aerobic glycolysis over glucose oxidation, contributes to tumor growth, progression and therapy resistance. Cancer cells restrain pyruvate flux into mitochondrial oxidative metabolism in large part by inhibiting the pyruvate dehydrogenase (PDH) complex. Src is a prominent oncogenic non-receptor tyrosine kinase that promotes cancer cell proliferation, invasion, metastasis and resistance to conventional and targeted therapies. However, the potential implication of Src in tumor metabolism remained largely unclear. Here we report that active Src enzyme directly tyrosine-phosphorylates the PDH E1α subunit (PDHA1). Conversely, pharmacological Src inhibitors or depletion of Src by shRNA abolish PDHA1 tyrosine phosphorylation in cancer cells, suggesting that Src is the main kinase responsible for PDHA1 tyrosine phosphorylation. This Src-mediated modification inactivates PDH. Consistently, activation of Src attenuates PDH activity and generation of reactive oxygen species (ROS), which are byproducts of mitochondrial respiration. Src inhibitors activate PDH and increase cellular ROS levels. Importantly, expression of a tyrosine non-phosphorable PDHA1 mutant in Src-activated cancer cells restores PDH activity, increases mitochondrial respiration and oxidative stress, decreases experimental metastasis, and sensitizes cancer cells to pro-oxidant treatment. The results suggest that Src contributes to the Warburg metabolic phenotype by inactivating PDH through tyrosine phosphorylation, and this metabolic effect of Src is essential for Src-driven malignancy and therapy resistance. As inhibition of Src sensitizes cancer cells to pro-oxidants, combination therapies consisting of both Src inhibitors and pro-oxidants may improve anticancer efficacy. Citation Format: Jianrong Lu. Oncogenic Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine phosphorylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-314.

Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine-289 phosphorylation.

The Warburg effect, which reflects cancer cells' preference for aerobic glycolysis over glucose oxidation, contributes to tumor growth, progression and therapy resistance. The restraint on pyruvate flux into mitochondrial oxidative metabolism in cancer cells is in part attributed to the inhibition of pyruvate dehydrogenase (PDH) complex. Src is a prominent oncogenic non-receptor tyrosine kinase that promotes cancer cell proliferation, invasion, metastasis and resistance to conventional and targeted therapies. However, the potential role of Src in tumor metabolism remained unclear. Here we report that activation of Src attenuated PDH activity and generation of reactive oxygen species (ROS). Conversely, Src inhibitors activated PDH and increased cellular ROS levels. Src inactivated PDH through direct phosphorylation of tyrosine-289 of PDH E1α subunit (PDHA1). Indeed, Src was the main kinase responsible for PDHA1 tyrosine phosphorylation in cancer cells. Expression of a tyrosine-289 non-phosphorable PDHA1 mutant in Src-hyperactivated cancer cells restored PDH activity, increased mitochondrial respiration and oxidative stress, decreased experimental metastasis, and sensitized cancer cells to pro-oxidant treatment. The results suggest that Src contributes to the Warburg phenotype by inactivating PDH through tyrosine phosphorylation, and the metabolic effect of Src is essential for Src-driven malignancy and therapy resistance. Combination therapies consisting of both Src inhibitors and pro-oxidants may improve anticancer efficacy.

Effects of Taurine Administration on Carbohydrate Metabolism in Skeletal Muscle during the Post-Exercise Phase.

We previously reported that taurine (2-aminoethanesulfonic acid; dose: 0.5 mg/g body weight) administration after treadmill running at 25 m/min for 90 min increased the glycogen concentration in the skeletal muscle of ICR mice at 120 min after the exercise (Takahashi et al. 2014). In the current study, we further investigated the effects of taurine administration on glycogen repletion and carbohydrate metabolism in the tibialis anterior muscle after endurance exercise. The metabolomic profiles of the tibialis anterior muscle at 120 min after the exercise were analyzed by a capillary electrophoresis-time-of-flight mass spectrometry (n=6). Fructose-1,6-bisphosphate (F1,6P), a glycogenolytic/glycolytic intermediate produced by phosphofructokinase, was significantly lower in the taurine-treated group than that in the control group (p<0.01). Dihydroxyacetonephosphate (DHAP), a downstream product of F1,6P was lower (p=0.05) and glycerol 3-phosphate, a downstream product of F1,6P and DHAP, tended to be lower (p=0.09) in the taurine-treated group than in the controls. At that time, phosphorylated Ser293 on the E1α subunit of pyruvate dehydrogenase (PDH) tended to be higher in the taurine-treated mice than in the controls (p=0.09, n=5). There was a positive correlation between phosphorylation of the PDH E1α subunit at Ser293 and glycogen concentration (r=0.73, p<0.05). Our results showed that the enhanced glycogen repletion in skeletal muscle by taurine treatment during the post-exercise phase was accompanied by the lower levels of glycogenolytic/glycolytic intermediates.

Abstract 1140: PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit

Abstract The PI3K/mTOR pathway plays a central role in coupling metabolic processes to the cellular proliferative state. Pharmacologic inhibitors of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in transformed cell lines in vitro by 30-40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, an inhibitory modification of this critical gatekeeper of mitochondrial respiration. Expressing wild type PTEN in a doxycycline-inducible manner in a glioblastoma cell line with mutant PTEN led to an increase in PDH E1α phosphorylation and a decrease in OCR. The decrease in OCR with PI3K/Akt/mTOR inhibition was recapitulated by knocking down PDH E1α expression using siRNA. Pre-treatment with dichloroacetate (DCA), a known inhibitor of the pyruvate dehydrogenase kinases (PDKs) that phosphorylate PDH E1α, prevented the upregulation in PDH E1α phosphorylation, induced by inhibition of the pathway and also blunted the decrease in OCR. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. Citation Format: George John Cerniglia, Souvik Day, Shannon M. Gallagher-Colombo, Natalie Daurio, Stephen Tuttle, Theresa M. Busch, Theresa M. Busch, Alexander Lin, Tatiana V. Esipova, Sergei A. Vinogradov, Constantinos Koumenis, Amit Maity. PI3K/Akt inhibition decreases oxygen consumption In tumor cells by phosphorylating and inactivating pyruvate dehydrogenase PDH E1α subunit. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1140. doi:10.1158/1538-7445.AM2015-1140

Overexpression of Arabidopsis WRI1 enhanced seed mass and storage oil content in Camelina sativa

Vegetable oils are essential for human and animal diets and have been used for industrial applications such as cosmetics and lubricants. Camelina sativa, which contains 35–45 % storage oils of seed dry weight, is an emerging oilseed crop. In addition, Arabidopsis WRINKLED1 (AtWRI1) is known to be an AP2/EREBP-type transcription factor that regulates the expression of genes that encode enzymes involved in the glycolytic pathway and fatty acid synthesis. In this study, AtWRI1 was expressed in C. sativa under the control of the seed-specific SiW6 promoter. The introduction of the AtWRI1 gene was identified from polymerase chain reaction (PCR) analysis using genomic DNA, and its mRNA expression was analyzed by reverse transcription-PCR (RT-PCR) using gene-specific primers in transgenic C. sativa plants. The expression of AtWRI1 caused an increase of seed mass through an increase in size, but not through an increase in number of cells. Moreover, the total seed oil contents increased by approximately 14 % in the T3 transgenic C. sativa lines compared with the non-transgenic plants. It was revealed that the elevation in storage oil contents is caused by the upregulation of three isoforms encoding a pyruvate dehydrogenase E1α subunit and three isoforms encoding a biotin carboxyl carrier protein of acetyl-CoA carboxylase complex, which are involved in fatty acid biosynthesis. Finally, the increased expression levels of C. sativa expansin1, which may be involved in cell-wall loosening during cell expansion, was observed in transgenic C. sativa developing seeds. Transgenic C. sativa with enhanced seed oil contents will be useful for the production of non-petroleum-based biomaterials and biofuels.

Coordinating bacterial cell division with nutrient availability: a role for glycolysis.

ABSTRACTCell division in bacteria is driven by a cytoskeletal ring structure, the Z ring, composed of polymers of the tubulin-like protein FtsZ. Z-ring formation must be tightly regulated to ensure faithful cell division, and several mechanisms that influence the positioning and timing of Z-ring assembly have been described. Another important but as yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. In this study, we demonstrated for the first time that cell division is intimately linked to central carbon metabolism in the model Gram-positive bacterium Bacillus subtilis. We showed that a deletion of the gene encoding pyruvate kinase (pyk), which produces pyruvate in the final reaction of glycolysis, rescues the assembly defect of a temperature-sensitive ftsZ mutant and has significant effects on Z-ring formation in wild-type B. subtilis cells. Addition of exogenous pyruvate restores normal division in the absence of the pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the coordination of bacterial growth and division. Our results support a model in which pyruvate levels are coupled to Z-ring assembly via an enzyme that actually metabolizes pyruvate, the E1α subunit of pyruvate dehydrogenase. We have shown that this protein localizes over the nucleoid in a pyruvate-dependent manner and may stimulate more efficient Z-ring formation at the cell center under nutrient-rich conditions, when cells must divide more frequently.