AMPK’s double-faced role in advanced stages of prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer deaths in men. Unfortunately, a very limited number of drugs are available for the relapsed and advanced stages of PCa, adding only a few months to survival; therefore, it is vital to develop new drugs. 5´ AMP-activated protein kinase (AMPK) is a master regulator of cell metabolism. It plays a significant role in the metabolism of PCa; hence, it can serve well as a treatment option for the advanced stages of PCa. However, whether this pathway contributes to cancer cell survival or death remains unknown. The present study reviews the possible pathways by which AMPK plays role in the advanced stages of PCa, drug resistance, and metastasis: (1) AMPK has a contradictory role in promoting glycolysis and the Warburg effect which are correlated with cancer stem cells (CSCs) survival and advanced PCa. It exerts its effect by interacting with hypoxia-induced factor 1 (HIF1) α, pyruvate kinase 2 (PKM2), glucose transporter (GLUT) 1 and pyruvate dehydrogenase complex (PDHC), which are key regulators of glycolysis; however, whether it promotes or discourage glycolysis is not conclusive. It can also exert an anti-CSC effect by negative regulation of NANOG and epithelial–mesenchymal transition (EMT) transcription factors, which are the major drivers of CSC maintenance; (2) the regulatory effect of AMPK on autophagy is also noticeable. Androgen receptors’ expression increases AMPK activation through Calcium/calmodulin-dependent protein kinase 2 (CaMKK2) and induces autophagy. (AU)

AMPK's double-faced role in advanced stages of prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer deaths in men. Unfortunately, a very limited number of drugs are available for the relapsed and advanced stages of PCa, adding only a few months to survival; therefore, it is vital to develop new drugs. 5´ AMP-activated protein kinase (AMPK) is a master regulator of cell metabolism. It plays a significant role in the metabolism of PCa; hence, it can serve well as a treatment option for the advanced stages of PCa. However, whether this pathway contributes to cancer cell survival or death remains unknown. The present study reviews the possible pathways by which AMPK plays role in the advanced stages of PCa, drug resistance, and metastasis: (1) AMPK has a contradictory role in promoting glycolysis and the Warburg effect which are correlated with cancer stem cells (CSCs) survival and advanced PCa. It exerts its effect by interacting with hypoxia-induced factor 1 (HIF1) α, pyruvate kinase 2 (PKM2), glucose transporter (GLUT) 1 and pyruvate dehydrogenase complex (PDHC), which are key regulators of glycolysis; however, whether it promotes or discourage glycolysis is not conclusive. It can also exert an anti-CSC effect by negative regulation of NANOG and epithelial-mesenchymal transition (EMT) transcription factors, which are the major drivers of CSC maintenance; (2) the regulatory effect of AMPK on autophagy is also noticeable. Androgen receptors' expression increases AMPK activation through Calcium/calmodulin-dependent protein kinase 2 (CaMKK2) and induces autophagy. In addition, AMPK itself increases autophagy by downregulating the mammalian target of rapamycin complex (mTORC). However, whether increased autophagy inhibits or promotes cell death and drug resistance is contradictory. This study reveals that there are numerous pathways other than cell metabolism by which AMPK exerts its effects in the advanced stages of PCa, making it a priceless treatment target. Finally, we mention some drugs developed to treat the advanced stages of PCa by acting on AMPK.

Dichloroacetate reveals the presence of metabolic inertia at the start of exercise in rainbow trout (Oncorhynchus mykiss, Walbaum 1792).

A lag in the increase in oxygen consumption (MO2 ) occurs at the start of sustainable exercise in trout. Waterborne dichloroacetate (0.58 and 3.49 mmol l-1 ), a compound which activates pyruvate dehydrogenase (PDH) by inhibiting PDH kinase in muscle, accelerates the increase in MO2 during the first 10 min of sustainable exercise when velocity is elevated to 75% critical swimming speed in a swim tunnel. There are no effects on MO2 thereafter or at rest. This indicates that a delay in PDH activation ("metabolic inertia") contributes to the lag phenomenon.

Adaptations in Protein Expression and Regulated Activity of Pyruvate Dehydrogenase Multienzyme Complex in Human Systolic Heart Failure.

Pyruvate dehydrogenase (PDH) complex, a multienzyme complex at the nexus of glycolytic and Krebs cycles, provides acetyl-CoA to the Krebs cycle and NADH to complex I thus supporting a critical role in mitochondrial energy production and cellular survival. PDH activity is regulated by pyruvate dehydrogenase phosphatases (PDP1, PDP2), pyruvate dehydrogenase kinases (PDK 1-4), and mitochondrial pyruvate carriers (MPC1, MPC2). As NADH-dependent oxidative phosphorylation is diminished in systolic heart failure, we tested whether the left ventricular myocardium (LV) from end-stage systolic adult heart failure patients (n = 26) exhibits altered expression of PDH complex subunits, PDK, MPC, PDP, and PDH complex activity, compared to LV from nonfailing donor hearts (n = 21). Compared to nonfailing LV, PDH activity and relative expression levels of E2, E3bp, E1α, and E1ß subunits were greater in LV failure. PDK4, MPC1, and MPC2 expressions were decreased in failing LV, whereas PDP1, PDP2, PDK1, and PDK2 expressions did not differ between nonfailing and failing LV. In order to examine PDK4 further, donor human LV cardiomyocytes were induced in culture to hypertrophy with 0.1 µM angiotensin II and treated with PDK inhibitors (0.2 mM dichloroacetate, or 5 mM pyruvate) or activators (0.6 mM NADH plus 50 µM acetyl CoA). In isolated hypertrophic cardiomyocytes in vitro, PDK activators and inhibitors increased and decreased PDK4, respectively. In conclusion, in end-stage failing hearts, greater expression of PDH proteins and decreased expression of PDK4, MPC1, and MPC2 were evident with higher rates of PDH activity. These adaptations support sustained capacity for PDH to facilitate glucose metabolism in the face of other failing bioenergetic pathways.

Effects of altered pyruvate dehydrogenase activity on contracting skeletal muscle bioenergetics.

During aerobic exercise (>65% of maximum oxygen consumption), the primary source of acetyl-CoA to fuel oxidative ATP synthesis in muscle is the pyruvate dehydrogenase (PDH) reaction. This study investigated how regulation of PDH activity affects muscle energetics by determining whether activation of PDH with dichloroacetate (DCA) alters the dynamics of the phosphate potential of rat gastrocnemius muscle during contraction. Twitch contractions were induced in vivo over a broad range of intensities to sample submaximal and maximal aerobic workloads. Muscle phosphorus metabolites were measured in vivo before and after DCA treatment by phosphorus nuclear magnetic resonance spectroscopy. At rest, DCA increased PDH activation compared with control (90 ± 12% vs. 23 ± 3%, P < 0.05), with parallel decreases in inorganic phosphate (Pi) of 17% (1.4 ± 0.2 vs. 1.7 ± 0.1 mM, P < 0.05) and an increase in the free energy of ATP hydrolysis (ΔGATP) (-66.2 ± 0.3 vs. -65.6 ± 0.2 kJ/mol, P < 0.05). During stimulation DCA increased steady-state phosphocreatine (PCr) and the magnitude of ΔGATP, with concomitant reduction in Pi and ADP concentrations. These effects were not due to kinetic alterations in PCr hydrolysis, resynthesis, or glycolytic ATP production and altered the flow-force relationship between mitochondrial ATP synthesis rate and ΔGATP. DCA had no significant effect at 1.0- to 2.0-Hz stimulation because physiological mechanisms at these high stimulation levels cause maximal activation of PDH. These data support a role of PDH activation in the regulation of the energetic steady state by altering the phosphate potential (ΔGATP) at rest and during contraction.

Exogenous Catalase and Pyruvate Dehydrogenase Improve Survival and Regeneration and Affect Oxidative Stress in Cryopreserved Dendrobium nobile Protocorm-like Bodies.

BACKGROUND: Reactive oxygen species (ROS)-induced oxidative damage is responsible for viability loss in plant tissues following cryopreservation. Antioxidants may improve viability by preventing or repairing the injury. OBJECTIVE: This work aimed at studying the effect of catalase (CAT) and pyruvate dehydrogenase (PDH), which are involved in ROS metabolism and are differentially expressed during pollen cryopreservation, for cryopreservation of Dendrobium nobile Lindl. 'Hamana Lake Dream' protocorm-like bodies (PLBs). MATERIALS AND METHODS: Different concentrations of exogenous CAT or PDH were added at the loading, PVS2 treatment, unloading steps during vitrification-cryopreservation of PLBs. Their survival and regeneration were evaluated and correlated with physiological oxidative indexes. RESULTS: PLB survival increased significantly when CAT and PDH were added separately to the unloading solution at a suitable concentration. CAT at 400 U·ml-1 increased PLB survival and regeneration by 33.5 and 14.6 percent respectively. It had no impact on the production of superoxide anion radical (·O2-) and on superoxide dismutase (SOD) activity, but it reduced the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and enhanced ascorbic acid (AsA) and endogenous CAT levels compared to PLBs cryopreserved using the standard vitrification protocol (CK1). PDH at 0.1 U·ml-1 significantly improved PLB survival (by 2.5 percent), but it had no marked effect on regeneration compared to the CK1 group. It induced the same variations in ·O2-, AsA and endogenous CAT levels that were observed following CAT addition. However, PDH did not affect the H2O2 and MDA content but significantly increased SOD activity. CONCLUSION: These results indicate that the addition of 400 U·ml-1 CAT and 0.1 U·ml-1 PDH at the unloading step increased survival of cryopreserved PLBs and that this improvement was associated with scavenging of H2O2 and the repair of oxidative damage. Exogenous CAT also significantly improved PLB regeneration after cryopreservation, while PDH had no obvious effect. The effect of exogenous CAT on PLB survival and regeneration was stronger than that of PDH, which may be due to the increased SOD activity by PDH addition.

Activation of pyruvate dehydrogenase by dichloroacetate has the potential to induce epigenetic remodeling in the heart.

Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. (13)C6-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 2.3% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart.

Design, synthesis and molecular modeling of novel N-acylhydrazone derivatives as pyruvate dehydrogenase complex E1 inhibitors.

As potential inhibitors of pyruvate dehydrogenase complex E1 (PDHc-E1), a series of 19 1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-methyl-N'-(substituent)benzylidene-1H-1,2,3-triazole-4-carbohydrazide 4 has been synthesized and tested for their PDHc-E1 inhibitory activity in vitro. Some of these compounds such as 4a, 4g, 4l, 4o, 4p, and 4q were demonstrated to be effective inhibitors by the bioassay of Escherichia coli PDHc-E1. SAR analysis indicated that the PDHc-E1 inhibitory activity could be further enhanced by optimizing the substituted groups in the parent compound. Molecular modeling study with compound 4o as a model was performed to evaluate docking. The results of modeling study suggested a probable inhibition mechanism.

Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.

BACKGROUND & AIMS: Previous work has suggested that CD4+ CD28- or costimulation-independent T cells are increased in autoimmune diseases. In this study, we compared frequency and qualitative characteristics of autoreactive costimulation-independent or CD4+ CD28- T cells in primary biliary cirrhosis (PBC) by taking advantage of the well-defined immunodominant autoepitope of the E2 component of pyruvate dehydrogenase (PDC-E2). METHODS: We determined the frequency of costimulation-independent autoreactive T cells that respond to PDC-E2 163-176 and the frequency of CD4+ CD28- T cells. Finally, we determined the role of biliary epithelial cells (BEC) as both an antigen-presenting cell or, alternatively, as a target cell for T-cell-mediated cytotoxicity. RESULTS: The precursor frequency of costimulation-independent CD4+ T cells that respond to PDC-E2 163-176 and the frequency of CD4+ CD28- T cells were dramatically elevated in PBC. Furthermore, 2 types of T-cell clones that respond to PDC-E2 163-176 emerged from this study. One type was costimulation dependent and the other costimulation independent. Both types of clones lyse BEC in a similar effector target (E/T) ratio distribution. However, BEC did not help the proliferation of any T-cell clones. Furthermore, costimulation-independent T-cell clones do not become anergic by BEC. CONCLUSIONS: In PBC, costimulation-independent autoreactive T cells, which do not become anergic, increase and maintain the autoimmune response. In controls, although autoantigens are expressed on BEC and autoantigen-reactive T cells exist around BEC, autoantigen-reactive T cells are costimulation dependent and will become anergic and maintain peripheral tolerance.

Inhibition of the alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes by a putative aberrant metabolite of serotonin, tryptamine-4,5-dione.

A transient energy impairment with resultant release and subsequent reuptake of 5-hydroxytryptamine (5-HT) and NMDA receptor activation with consequent cytoplasmic superoxide (O(2)(-)(*)), nitric oxide (NO(*)), and peroxynitrite (ONOO(-)) generation have all been implicated in a neurotoxic cascade which ultimately leads to the degeneration of serotonergic neurons evoked by methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). Such observations raise the possibility that the O(2)(-)(*)/NO(*)/ONOO(-)-mediated oxidation of 5-HT, as it returns via the plasma membrane transporter to the cytoplasm of serotonergic neurons when the MA/MDMA-induced energy impairment begins to subside, may generate an endogenous neurotoxin. In vitro the O(2)(-)(*)/NO(*)/ONOO(-)-mediated oxidation of 5-HT forms tryptamine-4,5-dione (T-4,5-D). When incubated with intact rat brain mitochondria, T-4,5-D strongly inhibits state 3 respiration with pyruvate or alpha-ketoglutarate as substrates at concentrations which do not affect succinate-supported (complex II) respiration. Experiments with freeze-thawed rat brain mitochondria reveal that T-4,5-D inhibits the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. These and other properties of T-4,5-D raise the possibility that it may be an endogenously formed intraneuronal metabolite of 5-HT that contributes to the serotonergic neurotoxicity of MA and MDMA.

Oral tolerisation to pyruvate dehydrogenase complex as a potential therapy for primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterised by immune-mediated damage to the intra-hepatic biliary epithelial cells (BEC). Immuno-modulatory/suppressive therapy represents, therefore, alogical approach to treatment in this disease. Conventional immuno-suppressive and immuno-modulatory agents suffer from the breadth of their action and/or excessive side effects. Autoreactive responses to pyruvate dehydrogenase complex (PDC) have been extensively characterised in PBC, and implicated in target cell damage. The aim of the current study was to study the potential efficacy of an antigen specific approach (oral tolerisation with autoantigen) to modulation of anti-PDC immune responses characteristic of PBC, utilising a mouse model of PDC immuno-reactivity. Groups of SJL/J mice were orally dosed with PDC alone, dosed with carrier only (saline) but systemically sensitised with PDC in adjuvant, or orally dosed with PDC at high (5 mg) or low (0.01 mg) dose and systemically sensitised with PDC. Oral dosing with PDC in isolation had no adverse effects on the animals and did not prime anti-PDC responses at doses below 1 mg. Pre-dosing with PDC at both high and low doses was effective at skewing the phenotype of the T-cell response to PDC induced by subsequent sensitisation away from the disease associated Th-1 phenotype (IL-2 and IFN-gamma secreting) and towards a theoretically protective Th-2 phenotype (IL-4 secreting) in a majority of, but not all, animals. No augmentation of Th-1 response was seen in any animal. Although the effects on liver histology remain to established oral tolerisation with PDC holds promise as a novel, antigen specific approach to therapy in PBC.

Mimicry peptides of human PDC-E2 163-176 peptide, the immunodominant T-cell epitope of primary biliary cirrhosis.

The human PDC-E2 163-176 peptide (GDLLAEIETDKATI) is an immunodominant autoreactive T-cell epitope in patients with primary biliary cirrhosis (PBC), restricted by HLA DRB4*0101. We have previously reported that the ExDK sequence is essential for recognition of this epitope and identified 1 mimicry peptide, Escherichia coli PDC-E2 peptide (EQSLITVEGDKASM), which can activate human PDC-E2 163-176 peptide-reactive T-cell clones. In the present study, to further investigate mimicry peptides possibly involved in PBC, we generated 13 different T-cell clones reactive to the human PDC-E2 163-176 peptide following repeated in vitro stimulation of peripheral T lymphocytes with the human PDC-E2 163-176 peptide (native peptide) and tested for the reactivity of these T-cell clones to 30 different mimicry peptides derived from various self- and nonself proteins that have an ExDK-sequence. We found 7 mimicry peptides derived from microbial proteins that can activate at least 1 of these T-cell clones; 7 of 7 T-cell clones from patients with PBC and 2 of 6 T-cell clones from healthy subjects were activated by at least 1 to 6 different mimicry peptides. Two of 6 T-cell clones from healthy subjects were activated by specific mimicry peptides more strongly than by the native peptide, and 2 of 6 T-cell clones from healthy subjects were not activated by any mimicry peptides tested. Thus, the pattern and degree of activation by mimicry peptides differed in each T-cell clone, indicating the presence of a diverse spectrum of autoreactive T cells that are reactive to a single minimal epitope of the human PDC-E2 163-176 peptide.

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.

[Effect of pyruvate dehydrogenase coenzymes and mitochondrial proteins on the accumulation of [35S]lipoic acid]. / Vliianie kofermentov piruvatdegidrogenazy i belkov mitokhondrii na nakoplenie v nikh [35S]lipoevoi kisloty.

Coenzymes introduced in the ratio, peculiar for pyruvate dehydrogenase complex into the medium containing fresh-isolated mitochondria and oxidation substrate--pyruvate increase accumulation of [35S] lipoate by these organelles. This process is highly stimulated by introducing either the only CoA or a coenzyme mixture (CoA, thiamine pyrophosphate, FAD, NAD). Addition of phosphate-extracted components of mitochondria and their protein fraction with coenzymes in the ratio indicated above provides maximum accumulation of [35S] lipoate by liver mitochondria. An equimolar mixture of coenzymes as well as protein components evoke no reliable variations in [35S] lipoate accumulation by albino rat liver mitochondria, while addition of the only thiamine pyrophosphate decreases this accumulation. Reconstruction of multienzyme complexes of coenzymes and apoenzymes on mitochondrion membranes accounts for the results obtained.