Showing papers on "Pyruvate dehydrogenase kinase published in 2018"
TL;DR: It is suggested that T cell function may be finely tuned through modulation of glycolytic activity, which is required acutely for cytokine synthesis but dispensable for cytotoxicity.
295 citations
TL;DR: The roles of PDC in diverse tissues and how regulation of its activity is affected in various metabolic disorders are summarized.
Abstract: 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.
105 citations
TL;DR: HFD mediated activation of PPAR-γ is associated with the related mitochondrial enzymes HMGCS2, BDH1 and PDK4 in HFD-induced diabetic hearts and in PA-treated NRCM, and demonstrates a more robust functional recovery after diabetes induction.
Abstract: Background/aims Systemic hyperlipidemia and intracellular lipid accumulation induced by chronic high fat diet (HFD) leads to enhanced fatty acid oxidation (FAO) and ketogenesis. The present study was aimed to determine whether activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) by surplus free fatty acids (FA) in hyperlipidemic condition, has a positive feedback regulation over FAO and ketogenic enzymes controlling lipotoxicity and cardiac apoptosis. Methods 8 weeks old C57BL/6 wild type (WT) or PPAR-γ-/- mice were challenged with 16 weeks 60% HFD to induce obesity mediated type 2 diabetes mellitus (T2DM) and diabetic cardiomyopathy. Treatment course was followed by echocardiographic measurements, glycemic and lipid profiling, immunoblot, qPCR and immunohistochemistry (IHC) analysis of PPAR-γ and following mitochondrial metabolic enzymes 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2), mitochondrial β- hydroxy butyrate dehydrogenase (BDH1) and pyruvate dehydrogenase kinase isoform 4 (PDK4). In vivo model was translated in vitro, with neonatal rat cardiomyocytes (NRCM) treated with PPAR-γ agonist/antagonist and PPAR-γ overexpression adenovirus in presence of palmitic acid (PA). Apoptosis was determined in vivo from left ventricular heart by TUNEL assay and immunoblot analysis. Results We found exaggerated circulating ketone bodies production and expressions of the related mitochondrial enzymes HMGCS2, BDH1 and PDK4 in HFD-induced diabetic hearts and in PA-treated NRCM. As a mechanistic approach we found HFD mediated activation of PPAR-γ is associated with the above-mentioned mitochondrial enzymes. HFD-fed PPAR-γ-/-mice display decreased hyperglycemia, hyperlipidemia associated with increased insulin responsiveness as compared to HFD-fed WT mice PPAR-γ-/-HFD mice demonstrated a more robust functional recovery after diabetes induction, as well as significantly reduced myocyte apoptosis and improved cardiac function. Conclusions PPAR-γ has been described previously to regulate lipid metabolism and adipogenesis. The present study suggests for the first time that increased PPAR-γ expression by HFD is responsible for cardiac dysfunction via upregulation of mitochondrial enzymes HMGCS2, BDH1 and PDK4. Targeting PPAR-γ and its downstream mitochondrial enzymes will provide novel strategies in preventing metabolic and myocardial dysfunction in diabetes mellitus.
83 citations
TL;DR: It is demonstrated that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution and offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.
Abstract: While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ2) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.
81 citations
TL;DR: The data show that NRF2 acting as a central node in the maintenance of low ROS levels and stemness associated properties of the CICs, which is significantly associated with the clinical outcome, but independent from ROS stress.
Abstract: Cancer-initiating cells (CICs) are responsible for tumor initiation, progression, and therapeutic resistance; moreover, redox homeostasis is important in regulating cancer stemness. Previously, we have identified that cancer cells containing low intracellular reactive oxygen species levels (ROSLow cells) display enhanced features of CICs. However, the specific metabolic signatures of CICs remain unclear and are required for further characterization by systemic screenings. Herein, we first showed CICs mainly relying on glycolysis that was important for the maintenance of stemness properties. Next, we revealed that NRF2, a master regulator of antioxidants, was able to maintain low intracellular ROS levels of CICs, even though in the absence of oxidative stress. We further characterized that NRF2 activation was required for the maintenance of CICs properties. Of ROSLow cells, NRF2 activation not only directly activates the transcription of genes encoding glycolytic enzymes but also inhibited the conversion of pyruvate to acetyl-CoA by directly activating pyruvate dehydrogenase kinase 1 (PDK1) to lead to inhibition of tricarboxylic acid (TCA) cycle; therefore, to promote Warburg effect. A positive regulatory ROS-independent ER stress pathway (GRP78/p-PERK/NRF2 signaling) was identified to mediate the metabolic shift (Warburg effect) and stemness of CICs. Lastly, co-expression of p-PERK and p-NRF2 was significantly associated with the clinical outcome. Our data show that NRF2 acting as a central node in the maintenance of low ROS levels and stemness associated properties of the CICs, which is significantly associated with the clinical outcome, but independent from ROS stress. Future treatments by inhibiting NRF2 activation may exhibit great potential in targeting CICs.
69 citations
TL;DR: Findings indicate inhibition of PDK4 may potentiate cisplatin-induced cell death and warrant further studies investigating the mechanism through which this occurs.
Abstract: Advanced bladder cancer (BCa) remains a major source of mortality, with poor treatment options. Cisplatin based chemotherapy is the standard treatment, however many patients are or become resistant. One potential cause of chemoresistance is the Warburg Effect, a metabolic switch to aerobic glycolysis that occurs in many cancers. Upregulation of the pyruvate dehydrogenase kinase family (PDK1-4) is associated with aerobic glycolysis and chemoresistance through inhibition of the pyruvate dehydrogenase complex (PDH). We have previously observed upregulation of PDK4 in high grade compared to low grade bladder cancers. We initiated this study to determine if inhibition of PDK4 could reduce tumor growth rates or sensitize BCa cells to cisplatin. Upregulation of PDK4 in malignant BCa cell lines as compared to benign transformed urothelial cells was confirmed using qPCR. Inhibition of PDK4 with dichloroacetate (DCA) resulted in increased PDH activity, reduced cell growth, and G0/G1 phase arrest in BCa cells. Similarly, siRNA knockdown of PDK4 inhibited BCa cell proliferation. Co-treatment of BCa cells with cisplatin and DCA did not increase caspase-3 activity but did enhance overall cell death in vitro. While daily treatment with 200mg/kg DCA alone did not reduce tumor volumes in a xenograft model, combination treatment with cisplatin resulted in dramatically reduced tumor volumes as compared to either DCA or cisplatin alone. This was attributed to substantial intra-tumoral necrosis. These findings indicate inhibition of PDK4 may potentiate cisplatin induced cell death and warrant further studies investigating the mechanism through which this occurs.
66 citations
TL;DR: It is reported here that mitochondrial sirtuin 4 (SIRT4) physiologically breaks tolerance and resolves acute inflammation in human monocytes by coordinately reprogramming of metabolism and bioenergetics.
Abstract: The goal of this investigation was to define the molecular mechanism underlying physiologic conversion of immune tolerance to resolution of the acute inflammatory response, which is unknown. An example of this knowledge gap and its clinical importance is the broad-based energy deficit and immunometabolic paralysis in blood monocytes from non-survivors of human and mouse sepsis that precludes sepsis resolution. This immunometabolic dysregulation is biomarked by ex vivo endotoxin tolerance to increased glycolysis and TNF-α expression. To investigate how tolerance switches to resolution, we adapted our previously documented models associated with acute inflammatory, immune, and metabolic reprogramming that induces endotoxin tolerance as a model of sepsis in human monocytes. We report here that mitochondrial sirtuin 4 (SIRT4) physiologically breaks tolerance and resolves acute inflammation in human monocytes by coordinately reprogramming of metabolism and bioenergetics. We find that increased SIRT4 mRNA and protein expression during immune tolerance counters the increase in pyruvate dehydrogenase kinase 1 (PDK1) and SIRT1 that promote tolerance by switching glucose-dependent support of immune resistance to fatty acid oxidation support of immune tolerance. By decreasing PDK1, pyruvate dehydrogenase complex reactivation rebalances mitochondrial respiration, and by decreasing SIRT1, SIRT4 represses fatty acid oxidation. The precise mechanism for the mitochondrial SIRT4 nuclear feedback is unclear. Our findings are consistent with a new concept in which mitochondrial SIRT4 directs the axis that controls anabolic and catabolic energy sources.
64 citations
TL;DR: This review focuses on the hypothesis of Warburg effect stimulated by the overexpression of the canonical WNT/β-catenin pathway in ASD, which could be an attractive target for developing therapeutic interventions in ASD.
Abstract: Autism spectrum disorder (ASD) is a neurodevelopmental disease which is characterized by a deficit in social interactions and communication with repetitive and restrictive behavior. In altered cells, metabolic enzymes are modified by the dysregulation of the canonical WNT/β-catenin pathway. In ASD, the canonical WNT/β-catenin pathway is upregulated. We focus this review on the hypothesis of Warburg effect stimulated by the overexpression of the canonical WNT/β-catenin pathway in ASD. Upregulation of WNT/β-catenin pathway induces aerobic glycolysis, named Warburg effect, through activation of glucose transporter (Glut), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase kinase 1(PDK1), monocarboxylate lactate transporter 1 (MCT-1), lactate dehydrogenase kinase-A (LDH-A) and inactivation of pyruvate dehydrogenase complex (PDH). The aerobic glycolysis consists to a supply of a large part of glucose into lactate regardless of oxygen. Aerobic glycolysis is less efficient in terms of ATP production than oxidative phosphorylation because of the shunt of the TCA cycle. Dysregulation of energetic metabolism might promote cell deregulation and progression of ASD. Warburg effect regulation could be an attractive target for developing therapeutic interventions in ASD.
61 citations
TL;DR: It is established that AMPK/GSK3b/ b‐catenin cascade‐triggered CEMIP overexpression might promote migration and invasion in anoikis‐resistant PCa cells by enhancing PDK4‐associated metabolic reprogramming, which may provide a novel, promising therapeutic target for the treatment of advanced PCa.
Abstract: Prostate cancer (PCa) represents one of the most common solid neoplasms, and metastasis is the second leading cause of death in adult males. Anoikis is a programmed cell death that is induced upon cell detachment from the extracellular matrix (ECM), which behaves as a critical protective mechanism for anchorage-independent cell growth and metastasis formation. However, in the absence of ECM attachment, shift of metabolic pattern and tolerance to anoikis facilitate the survival of aggressive cancer cells in the circulatory system as well as their metastasis to distant sites. Few molecular targets in PCa have thus far been reported to prevent anoikis resistance, metabolic reprogramming, and metastasis simultaneously. In the present study, elevated migration, invasion, pyruvate production, lactate generation, ATP level, and impaired detachment-induced apoptosis were found in anoikis-resistant PCa cells, and genome microarray analysis demonstrated that the cell migration-inducing protein (CEMIP) was a potential molecular target for the regulation of the aforementioned malignant behaviors. Additional investigation revealed that the AMPK/glycogen synthase kinase 3β (GSK3β)/β-catenin cascade-triggered CEMIP overexpression in anoikis-resistant PCa cells might be implicated in local progression, metabolic shift, and cellular migration and invasion, whereas knockout of CEMIP by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 in anoikis-resistant PCa cells reversed the described bioeffects by reducing expressions of matrix metalloproteinase 2 (MMP2), VEGF, pyruvate dehydrogenase kinase isoform 4 (PDK4), and lactate dehydrogenase A. In addition, inhibition of glycolysis by CEMIP-mediated PDK4 down-regulation impaired the migration and invasion of anoikis-resistant PCa cells by attenuating MMP2 and VEGF expressions. Our findings establish that AMPK/GSK3β/β-catenin cascade-triggered CEMIP overexpression might promote migration and invasion in anoikis-resistant PCa cells by enhancing PDK4-associated metabolic reprogramming, which may provide a novel, promising therapeutic target for the treatment of advanced PCa.-Zhang, P., Song, Y., Sun, Y., Li, X., Chen, L., Yang, L., Xing, Y. AMPK/GSK3β/β-catenin cascade-triggered overexpression of CEMIP promotes migration and invasion in anoikis-resistant prostate cancer cells by enhancing metabolic reprogramming.
58 citations
TL;DR: The findings show that the miR-422a–PDK2 axis is an important mediator in metabolic reprogramming and a promising therapeutic target for antitumor treatment.
Abstract: Increasing evidence indicates that dysregulation of microRNAs (miRNAs) plays a crucial role in human malignancies. Here, we showed that microRNA-422a (miR-422a) expression was dramatically downregulated in gastric cancer (GC) samples and cell lines compared with normal controls, and that its expression level was inversely related to tumor size and depth of infiltration. Functional studies revealed that the overexpression of miR-422a in GC tumor cells suppressed cell proliferation and migration, and drove a metabolic shift from aerobic glycolysis to oxidative phosphorylation. Mechanistic analysis suggested that miR-422a repressed pyruvate dehydrogenase kinase 2 (PDK2) to restore activity of the pyruvate dehydrogenase (PDH), the gatekeeping enzyme that catalyzes the decarboxylation of pyruvate to produce acetyl-CoA. Importantly, we further demonstrated that the mir-422a-PDK2 axis also influenced another metabolic pathway, de novo lipogenesis in cancer cells, and that it subsequently affected reactive oxygen species (ROS) and RB phosphorylation levels, ultimately resulting in cell cycle arrest in G1 phase. Our findings show that the miR-422a-PDK2 axis is an important mediator in metabolic reprogramming and a promising therapeutic target for antitumor treatment.
51 citations
TL;DR: The role of PDK in tumor progression, metabolic reprogramming in stem cells, and their regulation by miRNAs and lncRNAs, oncogenes and tumor suppressors are discussed.
Abstract: Cancer cells alter their metabolism to support the uninterrupted supply of biosynthetic molecules required for continuous proliferation. Glucose metabolism is frequently reprogrammed in several tumors in addition to fatty acid, amino acid and glutamine metabolism. Pyruvate Dehydrogenase Kinase (PDK) is a gatekeeper enzyme involved in altered glucose metabolism in tumors. There are four isoforms of PDK (1 to 4) in humans. PDK phosphorylates E1α subunit of pyruvate dehydrogenase complex (PDC) and inactivates it. PDC decarboxylates pyruvate to acetyl CoA, which is further metabolized in mitochondria. Overexpression of PDK was observed in several tumors and is frequently associated with chemotherapy related drug resistance, invasion and metastasis. Elevated expression of PDK leads to a shift in glucose metabolism towards glycolysis instead of oxidative phosphorylation. This review summarizes recent literature related to the role of PDKs in cancer and their inhibition as a strategy. In particular, we discuss the role of PDK in tumor progression, metabolic reprogramming in stem cells, and their regulation by miRNAs and lncRNAs, oncogenes and tumor suppressors. Further, we review strategies aimed at targeting PDK to halt tumor growth and progression.
TL;DR: The data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK 4 may be a new therapeutic strategy against NAFLD.
TL;DR: Results indicate that the PDC/PDK axis is a druggable mitochondrial target for promoting immunometabolic and organ homeostasis during sepsis.
Abstract: Limited understanding of the mechanisms responsible for life-threatening organ and immune failure hampers scientists' ability to design sepsis treatments. Pyruvate dehydrogenase kinase 1 (PDK1) is persistently expressed in immune-tolerant monocytes of septic mice and humans and deactivates mitochondrial pyruvate dehydrogenase complex (PDC), the gate-keeping enzyme for glucose oxidation. Here, we show that targeting PDK with its prototypic inhibitor dichloroacetate (DCA) reactivates PDC; increases mitochondrial oxidative bioenergetics in isolated hepatocytes and splenocytes; promotes vascular, immune, and organ homeostasis; accelerates bacterial clearance; and increases survival. These results indicate that the PDC/PDK axis is a druggable mitochondrial target for promoting immunometabolic and organ homeostasis during sepsis.
TL;DR: The addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell‐specific productivity or antibody quality.
Abstract: Lactate is one of the key waste metabolites of mammalian cell culture. High lactate levels are caused by high aerobic glycolysis, also known as the Warburg effect, and are usually associated with adverse culture performance. Therefore, reducing lactate accumulation has been an ongoing challenge in cell culture development in order to improve growth, productivity and process robustness. The pyruvate dehydrogenase complex (PDC) plays a crucial role for the fate of pyruvate, as it converts pyruvate to acetyl-CoA. PDC activity can be indirectly increased by inhibiting the PDC inhibitor, pyruvate dehydrogenase kinase, using dichloroacetate (DCA); resulting in less pyruvate being available for lactate formation. Here, Chinese hamster ovary cells were cultivated either with 5 mM DCA or without DCA in various batch and fed-batch bioreactor processes. In all cultures, DCA increased peak viable cell density (VCD), culture length and final antibody titer. The strongest effect was observed in a fed-batch with media and glucose feeding in which peak VCD was increased by more than 50%, culture length was extended by over three days and final antibody titer increased by more than 2-fold. In cultures with DCA, lactate production and glucose consumption during exponential growth were on average reduced by about 40% and 35%, respectively. Metabolic flux analysis showed reduced glycolytic fluxes while fluxes in TCA cycle were not affected suggesting that cultures with DCA use glucose more efficiently. In a proteomics analysis only few proteins were identified as being differentially expressed indicating that DCA acts on a post-translational level. Antibody quality in terms of aggregation, charge variant and glycosylation pattern was unaffected. Subsequent bioreactor experiments with sodium lactate and sodium chloride feeding indicated that lower osmolality, rather than lower lactate concentration itself, improved culture performance in DCA cultures. In conclusion, the addition of DCA to the cell culture improved culture performance and increased antibody titers without any disadvantages for cell specific productivity or antibody quality.
TL;DR: This review is focused on the opposing interactions observed in NDs between the canonical WNT/beta-catenin pathway and PPAR gamma and their metabolic and thermodynamic implications.
Abstract: Entropy production rate is increased by several metabolic and thermodynamics abnormalities in neurodegenerative diseases (NDs). Irreversible processes are quantified by changes in the entropy production rate. This review is focused on the opposing interactions observed in NDs between the canonical WNT/beta-catenin pathway and PPAR gamma and their metabolic and thermodynamic implications. In amyotrophic lateral sclerosis and Huntington's disease, WNT/beta-catenin pathway is upregulated, whereas PPAR gamma is downregulated. In Alzheimer's disease and Parkinson's disease, WNT/beta-catenin pathway is downregulated while PPAR gamma is upregulated. The dysregulation of the canonical WNT/beta-catenin pathway is responsible for the modification of thermodynamics behaviors of metabolic enzymes. Upregulation of WNT/beta-catenin pathway leads to aerobic glycolysis, named Warburg effect, through activated enzymes, such as glucose transporter (Glut), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase kinase 1(PDK1), monocarboxylate lactate transporter 1 (MCT-1), lactic dehydrogenase kinase-A (LDH-A) and inactivation of pyruvate dehydrogenase complex (PDH). Downregulation of WNT/beta-catenin pathway leads to oxidative stress and cell death through inactivation of Glut, PKM2, PDK1, MCT-1, LDH-A but activation of PDH. In addition, in NDs, PPAR gamma is dysregulated, whereas it contributes to the regulation of several key circadian genes. NDs show many dysregulation in the mediation of circadian clock genes and so of circadian rhythms. Thermodynamics rhythms operate far-from-equilibrium and partly regulate interactions between WNT/beta-catenin pathway and PPAR gamma. In NDs, metabolism, thermodynamics and circadian rhythms are tightly interrelated.
TL;DR: Metabolic flux analysis showed that the reduction in ATP was a consequence of a diminished rate of fatty acid oxidation (FAO), but overexpression of PDK4 increased FAO and increased ATP levels, which decreased p-AMPK and p-PDE4B and allowed greater accumulation of cAMP and p -CREB.
Abstract: In fasting or diabetes, gluconeogenic genes are transcriptionally activated by glucagon stimulation of the cAMP-protein kinase A (PKA)-CREB signaling pathway. Previous work showed pyruvate dehydrogenase kinase (PDK) inhibition in skeletal muscle increases pyruvate oxidation, which limits the availability of gluconeogenic substrates in the liver. However, this study found upregulation of hepatic PDK4 promoted glucagon-mediated expression of gluconeogenic genes, whereas knockdown or inhibition of hepatic PDK4 caused the opposite effect on gluconeogenic gene expression and decreased hepatic glucose production. Mechanistically, PDK4 deficiency decreased ATP levels, thus increasing phosphorylated AMPK (p-AMPK), which increased p-AMPK-sensitive phosphorylation of cyclic nucleotide phosphodiesterase 4B (p-PDE4B). This reduced cAMP levels and consequently p-CREB. Metabolic flux analysis showed that the reduction in ATP was a consequence of a diminished rate of fatty acid oxidation (FAO). However, overexpression of PDK4 increased FAO and increased ATP levels, which decreased p-AMPK and p-PDE4B and allowed greater accumulation of cAMP and p-CREB. The latter were abrogated by the FAO inhibitor etomoxir, suggesting a critical role for PDK4 in FAO stimulation and the regulation of cAMP levels. This finding strengthens the possibility of PDK4 as a target against diabetes.
TL;DR: This review focused on the hypothesis of aerobic glycolysis stimulated by the upregulation of WNT/β-catenin pathway in ALS and HD, an attractive mechanism for developing therapeutic interventions.
Abstract: Neurodegenerative cells are the sites of numerous metabolic and energetic abnormalities with abnormalities in energy production. Energy is the primary determinant of neuronal viability. In neurodegenerative cells, metabolic enzymes are modified by the dysregulation of the canonical WNT/β-catenin pathway. In amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD), WNT/β-catenin pathway is upregulated. We focused this review on the hypothesis of aerobic glycolysis stimulated by the upregulation of WNT/β-catenin pathway in ALS and HD. Upregulation of WNT/β-catenin pathway induces aerobic glycolysis, named Warburg effect, through activation of glucose transporter (Glut), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase kinase 1 (PDK1), monocarboxylate lactate transporter 1 (MCT-1), lactate dehydrogenase kinase-A (LDH-A), and inactivation of pyruvate dehydrogenase complex (PDH). Aerobic glycolysis consists of a supply of a large part of glucose into lactate regardless of oxygen. Aerobic glycolysis is less efficient in terms of ATP production compared with oxidative phosphorylation because of the shunt of the TCA cycle. Dysregulation of energetic metabolism promotes cell death and disease progression in ALD and HD. Aerobic glycolysis regulation is an attractive mechanism for developing therapeutic interventions.
TL;DR: The results suggest that the anabolic actions of r-irisin on the regulation of osteoblast lineage cells are preferentially through aerobic glycolysis, which may help to develop new irisin-based bone anabolic agents.
TL;DR: The present results suggest that PS10 is a more suitable PDK inhibitor for treatment of diabetic cardiomyopathy.
TL;DR: Results suggest that, although PDH activity controls the conversion of pyruvate to acetyl-CoA for oxidation, this has little effect on glucose uptake into muscle under insulin-stimulated conditions.
Abstract: Pyruvate dehydrogenase (PDH) activity is a key component of the glucose/fatty acid cycle hypothesis for the regulation of glucose uptake and metabolism We have investigated whether acute activation of PDH in muscle can alleviate the insulin resistance caused by feeding animals a high-fat diet (HFD) The importance of PDH activity in muscle glucose disposal under insulin-stimulated conditions was determined by infusing the PDH kinase inhibitor dichloroacetate (DCA) into HFD-fed Wistar rats during a hyperinsulinemic-euglycemic clamp Acute DCA infusion did not alter glucose infusion rate, glucose disappearance, or hepatic glucose production but did decrease plasma lactate levels DCA substantially increased muscle PDH activity; however, this did not improve insulin-stimulated glucose uptake in insulin-resistant muscle of HFD rats DCA infusion increased the flux of pyruvate to acetyl-CoA and reduced glucose incorporation into glycogen and alanine in muscle Similarly, in isolated muscle, DCA treatment increased glucose oxidation and decreased glycogen synthesis without changing glucose uptake These results suggest that, although PDH activity controls the conversion of pyruvate to acetyl-CoA for oxidation, this has little effect on glucose uptake into muscle under insulin-stimulated conditions
TL;DR: In conclusion, inhibition of the Akt/GSK-3β pathway improved the pro-apoptotic effect of DCA on human PASMCs, which may be attributed to a reversal of the Warburg effect by blocking the mutual interaction between HIF-1α and PDK-1, consequently downregulating HK-2.
Abstract: There is accumulating evidence indicating that the growth inhibitory effect of dichloroacetate (DCA) on pulmonary arterial smooth muscle cells (PASMCs) may be associated with the reversal of the Warburg effect and initiation of the mitochondria‑dependent apoptotic pathway. Previous studies indicated that platelet‑derived growth factor (PDGF) promoted the Warburg effect and resulted in apoptotic resistance of PASMCs, which was attributed to activation of the phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) signalling pathway. However, the mechanism underlying the pro‑apoptotic effect of DCA on PDGF‑treated PASMCs has not been thoroughly elucidated, and the effect of the Akt/glycogen synthase kinase‑3β (GSK‑3β) pathway inhibition concomitant with the effect of DCA on PASMC proliferation remains unclear. The growth of human PASMCs and the lactate concentration in extracellular medium of PASMCs were detected by Cell Counting Kit‑8 assays and a Lactate Colorimetric Assay kit, respectively. Cell apoptosis was evaluated by fluorescence activated cell sorting. The mitochondrial membrane potential (ΔΨm) was assessed with 5,5',6,6'‑tetrachloro‑1,1',3,3'‑tetraethylbenzimidazol‑carbocyanine iodide assays. The expression levels of phosphorylated Akt and GSK‑3β, pyruvate dehydrogenase, cleaved caspase‑3, pyruvate dehydrogenase kinase‑1 (PDK‑1), hypoxia inducible factor‑1α (HIF‑1α) and hexokinase‑2 (HK‑2) were measured with western blot analysis. Confocal analyses were employed to determine HK‑2 co‑localisation with the mitochondria. The results indicated that DCA inhibited human PASMC proliferation in a dose‑dependent manner. DCA at 10 mM promoted apoptosis and the upregulation of activated caspase‑3 in PASMCs pre‑treated with 20 ng/ml PDGF‑homeodimer BB (BB). Treatment with 5 µM LY294002 produced minimal anti‑proliferative effects on human PASMCs and barely induced cellular apoptosis and caspase‑3 activation. However, co‑administration of 10 mM DCA with LY294002 significantly decreased the cell proliferation index and induced cell apoptosis and caspase‑3 activation. The combined administration of LY294002 with DCA significantly decreased lactate concentration, promoted the depolarisation of the ΔΨm and repressed HIF‑1α upregulation and HK‑2 activation in PASMCs treated with PDGF, which was attributed to the potentiation of DCA‑induced PDK‑1 inhibition by LY294002 via blockade of the Akt/GSK‑3β/HIF‑1α signalling pathway. In conclusion, inhibition of the Akt/GSK‑3β pathway improved the pro‑apoptotic effect of DCA on human PASMCs, which may be attributed to a reversal of the Warburg effect by blocking the mutual interaction between HIF‑1α and PDK‑1, consequently downregulating HK‑2. Therefore, combinatory treatment with DCA and PI3K inhibitors may represent a novel therapeutic strategy for the reversal of apoptosis resistance exhibited by PASMCs as a result of mitochondrial bioenergetic abnormalities, as well as the treatment of pulmonary vascular remodelling in pulmonary arterial hypertension.
TL;DR: Lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms.
Abstract: Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate-limiting glycolytic enzymes. Moreover, pyruvate production of CGΔura3-Sp-MPC1, CGΔura3-Sp-MPC2, and CGΔura3-Sp-MPC1-Sp-MPC2 increased 134.4%, 120.3%, and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms.
TL;DR: Compound 1f could be a promising lead for the development of therapeutic PDK1 inhibitor in cancer treatment and reprogram the glucose metabolic pathways in cancer cells.
09 Feb 2018
TL;DR: Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells.
Abstract: Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo, hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo. Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia-inducible factors (HIF)-1/2α western blotting, and real-time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF-2α expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3-phosphate dehydrogenase [Gapdh], glucose transporter 1 [Glut1], pyruvate dehydrogenase kinase 1 [Pdk1], and lactate dehydrogenase A [Ldha]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (Cpt1), apolipoprotein B (Apob), and acetyl-coenzyme A carboxylase 1 (Acc1), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4α (Hnf4α) and farnesoid X receptor (Fxr) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF-2 signaling was involved in hypoxia-induced down-regulation of Fxr. Conclusion: Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. (Hepatology Communications 2018;2:299-312).
TL;DR: It is found that the introduction of Cpd64 in EGFR‐TKi therapy enhanced the anti‐proliferative effects in EGfr‐mutant NSCLC cells under hypoxia, and suggested that combined use of selective PDK inhibitor and EG FR‐Tki could be a potential strategy for NSCLc therapy.
TL;DR: Findings suggest that limitation of mitochondrial pyruvate import might be a therapeutic strategy to prevent excessive hepatic glucose production.
University of Alabama at Birmingham1, Northwestern University2, Sun Yat-sen University3, Huazhong University of Science and Technology4, South Central University for Nationalities5, Boston University6, Indiana University7, Indiana University – Purdue University Indianapolis8, Loma Linda University9, University of Chicago10
TL;DR: In this paper, the role of E2F1 in the regulation of BM PC function was defined, and strategies that inhibit or enhance oxidative metabolism in BM PC may improve the effectiveness of cell therapy.
Abstract: Rationale: The majority of current cardiovascular cell therapy trials use bone marrow progenitor cells (BM PCs) and achieve only modest efficacy; the limited potential of these cells to differentiate into endothelial-lineage cells is one of the major barriers to the success of this promising therapy. We have previously reported that the E2F transcription factor 1 (E2F1) is a repressor of revascularization after ischemic injury. Objective: We sought to define the role of E2F1 in the regulation of BM PC function. Methods and Results: Ablation of E2F1 (E2F1 deficient) in mouse BM PCs increases oxidative metabolism and reduces lactate production, resulting in enhanced endothelial differentiation. The metabolic switch in E2F1-deficient BM PCs is mediated by a reduction in the expression of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase kinase 2; overexpression of pyruvate dehydrogenase kinase 4 reverses the enhancement of oxidative metabolism and endothelial differentiation. Deletion of E2F1 in the BM increases the amount of PC-derived endothelial cells in the ischemic myocardium, enhances vascular growth, reduces infarct size, and improves cardiac function after myocardial infarction. Conclusion: Our results suggest a novel mechanism by which E2F1 mediates the metabolic control of BM PC differentiation, and strategies that inhibit E2F1 or enhance oxidative metabolism in BM PCs may improve the effectiveness of cell therapy.
TL;DR: 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.
Abstract: 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.
TL;DR: The results suggested that A549 cells exhibited distinct metabolite profiles following the treatment with DCA or AZD7545, which may reflect the different pharmacological indications of these two drugs.
Abstract: Pyruvate dehydrogenase kinases (PDKs) dominate the critical switch between mitochondria-based respiration and cytoplasm-based glycolysis by controlling pyruvate dehydrogenase (PDH) activity. Up-regulated PDKs play a great role in the Warburg effect in cancer cells and accordingly present a therapeutic target. Dichloroacetate (DCA) and AZD7545 are the two most-well-known PDK inhibitors exhibiting distinct pharmacological profiles. DCA showed anticancer effects in various preclinical models and clinical studies, while the primary preclinical indication of AZD7545 was on the improvement of glucose control in type II diabetes. Little, if any, study has been undertaken the elucidation of the effects of PDK inhibition on the metabolites in the tricarboxylic acid (TCA) cycle. Herein, the metabolite alterations of lung cancer cells (A549) upon the treatment with PDK inhibitors were studied using a reliable liquid-chromatography-based tandem mass spectrometry method. The developed method was validated for quantification of all common glycolysis and TCA cycle catabolites with good sensitivity and reproducibility, including glucose, pyruvate, lactate, acetyl coenzyme A, citrate, α-ketoglutarate, fumarate, succinate, malate, and oxaloacetate. Our results suggested that A549 cells exhibited distinct metabolite profiles following the treatment with DCA or AZD7545, which may reflect the different pharmacological indications of these two drugs.
TL;DR: It is suggested that betulin’s in vivo biological effectiveness is limited by its poor solubility, so polar glycosylated derivatives to increase its hydrosolubility and enhance its pharmacological properties are synthesized to induce apoptotic cell death via disturbance of mitochondrial electron transfer chain, reduced ROS production, and decreased membrane potential.
Abstract: Betulin has a wide range of biological and pharmacological properties with its anticancer activity attracting most of the attention as it offers a possible alternative treatment to chemotherapy. However, betulin’s in vivo biological effectiveness is limited by its poor solubility. As such, we synthesized polar glycosylated derivatives to increase its hydrosolubility and enhance its pharmacological properties. Among these synthesized compounds, 28-O-α-l-rhamnopyranosylbetulin 3β-O-α-l-rhamnopyranoside (Bi-L-RhamBet) was assessed for its cytotoxic effects against a suite of lung cancer cell lines. We also investigated its mechanism of action using an A549 lung cancer cell line. Our results showed that Bi-L-RhamBet exhibited potent cytotoxic activity toward lung cancer cell lines including A549, NCI-H2087, NCI-H522, NCI-H1993 NCI-H1755, and LLC1 having IC50 values ranging from 2.9 to 5.9 μM. Moreover, Bi-L-RhamBet (50 mg/kg) significantly inhibited tumor growth with a treatment-to-control ratio (T/C) of 0.54 and a tumor growth inhibition rate of 46% at day 18 (p < 0.05). Microscopic observations of A549 cells, double stained with acridine orange and ethidium bromide, showed apoptotic features. Bi-L-RhamBet induced activation of pro-apoptotic caspases 8, 9, and 3/7 as well as causing DNA fragmentation. Moreover, a marked increase in mitochondrial ROS (mROS) was coupled with a reduction of mitochondrial potential. Interestingly, the presence of mitochondrial electron transport chain (ETC) inhibitors, including rotenone, malonate, and antimycin A, reduced mROS production, and the activation of caspases suggesting that Bi-L-RhamBet disturbs the ETC. Finally, dichloroacetate, a pyruvate dehydrogenase kinase inhibitor potentiated the cytotoxicity of Bi-L-RhamBet against A549 cells. Taken together, these data suggest that Bi-L-RhamBet can induce apoptotic cell death via disturbance of mitochondrial electron transfer chain, reduced ROS production, and decreased membrane potential.