Emetine inhibits glycolysis in isolated, perfused rat hearts

This work was designed to test whether phosphofructokinase is a target for emetine action on the heart. The effects of 37, μM emetine on the activities of phosphofructokinase and hexokinase were measured in homogenates from perfused hearts. The action of increasing concentrations of emetine was determined in nonperfused heart homogenates. The effect of 37 μM emetine or control solutions on the concentration of fructose-6-phosphate and fructose-1,6-phosphate was measured. The effect of 37 μM emetine or control perfusion on the utilization of fructose-6-phosphate by phosphofructokinase in centrifugation supernatants of homogenates and in reconstituted 27,000g pellets was measured. Double-reciprocal plots of fructose-6-phosphate concentrations vs phosphofructokinase activities were plotted. Emetine decreased phosphofructokinase activity in homogenates from both perfused and nonperfused hearts. Emetine did not inhibit cardiac hexokinase activity. In homogenates from nonperfused hearts, the maximal inhibition with high concentrations of emetine was approx 50%. Emetine perfusion caused a simultaneous increase in the phosphofructokinase substrate fructose-6-phosphate and a decrease in the phosphofructokinase product fructose-1,6-bisphosphate. Phosphofructokinase and, consequently, glycolytic flux appear to be subcellular targets for emetine in the heart. Homogenate centrifugation studies indicate that emetine acts on bound rather than unbound phosphofructokinase. The inhibition may be uncompetitive in nature.     

A mutation in Drosophila Aldolase Causes Temperature-Sensitive Paralysis,Shortened Lifespan,and Neurodegeneration

We describe the characterization of m4, an autosomal recessive, temperature-sensitive paralytic mutant in Drosophila that is associated with shortened lifespan and neurodegeneration. Deletion mapping places the mutation in the gene encoding the glycolytic enzyme, Aldolase. The mutant enzyme contains a single amino acid substitution, which results in decreased steady-state levels of Aldolase with a consequent reduction in adenosine triphosphate (ATP) levels. Transgenic-rescue experiments with a genomic construct containing the entire Aldolase gene confirm that paralysis, reduced lifespan, and neurodegeneration all result from the same mutation. Tissue-specific rescue and RNA interference (RNAi) knockdown experiments indicate that Aldolase function (and presumably glycolysis) is important both in neurons and in glia for normal lifespan and neuronal maintenance over time. Impaired glycolysis in neurons can apparently be rescued in part by glycolytically active glia. However, this rescue may depend on the exact physiological state of the neurons and may also vary in different subsets of neurons. Further studies of m4 and related mutants in Drosophila should help elucidate the connections between energy production and utilization in glia and neurons and lead to better understanding of how metabolic defects impair neuronal function and maintenance.     

Cancer‐generated lactic acid: a regulatory,immunosuppressive metabolite?

The common preference of cancers for lactic acid‐generating metabolic energy pathways has led to proposals that their reprogrammed metabolism confers growth advantages such as decreased susceptibility to hypoxic stress. Recent observations, however, suggest that it generates a novel way for cancer survival. There is increasing evidence that cancers can escape immune destruction by suppressing the anti‐cancer immune response through maintaining a relatively low pH in their micro‐environment. Tumours achieve this by regulating lactic acid secretion via modification of glucose/glutamine metabolisms. We propose that the maintenance by cancers of a relatively low pH in their micro‐environment, via regulation of their lactic acid secretion through selective modification of their energy metabolism, is another major mechanism by which cancers can suppress the anti‐cancer immune response. Cancer‐generated lactic acid could thus be viewed as a critical, immunosuppressive metabolite in the tumour micro-environment rather than a ‘waste product’. This paradigm shift can have major impact on therapeutic strategy development. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pim-2 Modulates Aerobic Glycolysis and Energy Production during the Development of Colorectal Tumors

Tumor cells have higher rates of glucose uptake and aerobic glycolysis to meet energy demands for proliferation and metastasis. The characteristics of increased glucose uptake, accompanied with aerobic glycolysis, has been exploited for the diagnosis of cancers. Although much progress has been made, the mechanisms regulating tumor aerobic glycolysis and energy production are still not fully understood. Here, we demonstrate that Pim-2 is required for glycolysis and energy production in colorectal tumor cells. Our results show that Pim-2 is highly expressed in colorectal tumor cells, and may be induced by nutrient stimulation. Activation of Pim-2 in colorectal cells led to increase glucose utilization and aerobic glycolysis, as well as energy production. While knockdown of Pim-2 decreased energy production in colorectal tumor cells and increased their susceptibility to apoptosis. Moreover, the effects of Pim-2 kinase on aerobic glycolysis seem to be partly dependent on mTORC1 signaling, because inhibition of mTORC1 activity reversed the aerobic glycolysis mediated by Pim-2. Our findings suggest that Pim-2-mediated aerobic glycolysis is critical for monitoring Warburg effect in colorectal tumor cells, highlighting Pim-2 as a potential metabolic target for colorectal tumor therapy.     

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation.Altered metabolism is a hallmark of malignantly transformed cells. Cancer risk is linked to metabolic syndrome, a disease state that includes obesity, type 2 diabetes, high cholesterol, and atherosclerosis. Retrospective studies of type 2 diabetes patients treated with metformin, the most widely prescribed antidiabetic drug, show a strong correlation between drug intake and reduced tumor incidence or reduced cancer-related deaths (1–4).In the breast lineage, metformin inhibits growth of cancer cell lines (5–7), blocks transformation in a Src-inducible cell system (8, 9), and selectively inhibits the growth of cancer stem cells (CSCs) (8). As a consequence of its selective effects on CSCs, combinatorial therapy of metformin and standard chemotherapeutic drugs (doxorubicin, paclitaxel, and cisplatin) increases tumor regression and prolongs remission in mouse xenografts (8, 10). In addition, metformin can decrease the chemotherapeutic dose for prolonging tumor remission in xenografts involving multiple cancer types (10).Phenformin, a related biguanide and formerly used diabetes drug, acts as an anticancer agent in tumors including lung, lymphoma, and breast cancer with a greater potency than metformin. Phenformin mediates antineoplastic effects at a lower concentration than metformin in cell lines, a PTEN-deficient mouse model, breast cancer xenografts, and drug-induced mitochondrial impairment (11–14). The chemical similarities of these biguanides, as well as their similar effects in diabetes and cancer, have led to the untested assumption that phenformin is essentially a stronger version of metformin.In a Src-inducible model of cellular transformation and CSC formation, multiple lines of evidence suggest that metformin inhibits a signal transduction pathway that results in an inflammatory response (15). In the context of atherosclerosis, metformin inhibits NF-κB activation and the inflammatory response via a pathway involving AMP kinase (AMPK) and the tumor suppressor PTEN (16, 17). As metformin alters energy metabolism in diabetics, we speculated that metformin might block a metabolic stress response that stimulates the inflammatory pathway (15). However, very little is known about the metabolic changes that inhibit the inflammatory pathway.Previous studies on metformin-induced metabolic effects in cancer have focused on single metabolic alterations or pathways in already established cancer cell lines. Metformin leads to activation of AMPK, which plays a key role in insulin signaling and energy sensing (18). Metformin can reduce protein synthesis via mTOR inhibition (19). In addition, metformin may directly impair mitochondrial respiration through complex I inhibition and has been described to boost glycolysis as a compensation mechanism (14, 20). In this regard, lactic acidosis can be a side effect of metformin and phenformin treatment of diabetic patients, presumably because inhibition of complex I prevents NADH oxidation, thereby leading to a requirement for cytosolic NADH to be oxidized by the conversion of pyruvate to lactate. There is some knowledge about the metabolic effects of metformin (21, 22), but very little is known about the specific metabolic alterations linking biguanides to inhibition of neoplastic transformation.Here, we perform a metabolomic analysis on the effects of metformin and phenformin in a Src-inducible model of transformation and in CSCs. This inducible model permits an analysis of the transition from nontransformed to transformed cells in an isogenic cell system and hence differs from analyses of already established cancer cell lines. We studied CSCs to address why this population, which is resistant to standard chemotherapeutics and hypothesized to be a major reason for tumor recurrence, is selectively inhibited by metformin. Our results indicate the metabolic effects of metformin and phenformin are remarkably similar to each other, with only a few differences. Both biguanides dramatically decrease tricarboxylic acid (TCA) cycle intermediates in the early stages of transformation, and they inhibit the boost in select glycolytic intermediates that normally occurs with transformation along with increases in glycerol 3-phosphate and lactate, which are metabolites branching from glycolysis. Unexpectedly, in CSCs, biguanides have only marginal effects on glycolytic and TCA cycle metabolites, but they severely decrease nucleotide triphosphates. These detailed metabolic analyses provide independent support for the idea that metformin inhibits mitochondrial complex 1 (14, 20), and they indicate that the metabolic effects of biguanides depend on the stage of the cellular transformation.     

IGFBP-3 Mediates Metabolic Homeostasis During Hyperosmolar Stress in the Corneal Epithelium

PurposeThe insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional secretory protein with well-known roles in cell growth and survival. Data in our laboratory suggest that IGFBP-3 may be functioning as a stress response protein in the corneal epithelium. The purpose of this study is to determine the role of IGFBP-3 in mediating the corneal epithelial cell stress response to hyperosmolarity, a well-known pathophysiological event in the development of dry eye disease.MethodsTelomerase-immortalized human corneal epithelial (hTCEpi) cells were used in this study. Cells were cultured in serum-free media with (growth) or without (basal) supplements. Hyperosmolarity was achieved by increasing salt concentrations to 450 and 500 mOsM. Metabolic and mitochondrial changes were assessed using Seahorse metabolic flux analysis and assays for mitochondrial calcium, polarization and mtDNA. Levels of IGFBP-3 and inflammatory mediators were quantified using ELISA. Cytotoxicity was evaluated using a lactate dehydrogenase assay. In select experiments, cells were cotreated with 500 ng/mL recombinant human (rh)IGFBP-3.ResultsHyperosmolar stress altered metabolic activity, shifting cells towards a respiratory phenotype. Hyperosmolar stress further altered mitochondrial calcium levels, depolarized mitochondria, decreased levels of ATP, mtDNA, and expression of IGFBP-3. In contrast, hyperosmolar stress increased production of the proinflammatory cytokines IL-6 and IL-8. Supplementation with rhIGFBP-3 abrogated metabolic and mitochondrial changes with only marginal effects on IL-8.ConclusionsThese findings indicate that IGFBP-3 is a critical protein involved in hyperosmolar stress responses in the corneal epithelium. These data further support a new role for IGFBP-3 in the control of cellular metabolism.     

Glucose Content and Efficiency of Glycolysis in Protected Ischemic Kidneys of Different Species

In ischemic canine kidneys protected by Bretschneider's HTK solution the glycolytic lactate production is limited by a low renal substrate content. However, for anaerobic energy supply ischemic organs depend on glycolysis. To evaluate the role of glycolysis in renal protection, the relationship between lactate production and anaerobic energy supply was examined in protected kidneys of dogs, sheep, and swine. Additionally, in canine kidneys an attempt was made to improve anaerobic energy provision by adding glucose to the protective solution. The results were as follows: (1) According to increasing lactate production from swine to dog to sheep, intraischemic ATP decay was delayed least in swine and most in sheep. (2) Glucose addition (10 mM) to the HTK solution roughly doubled the time for ATP to fall to 1 μmollg dry wt (t_Atp) in dogs. (3) The greater the lactate production in all three species, the lower the decrease in SAN (ATP + ADP + AMP) from 5 to 120 min of ischemia. (4) A glucose additive in the protective solution led to a significant (p >. 005) increase of SAN in dogs at 120 min of ischemia. A sufficient substrate supply seems to be an essential component of a reliable renal protection.     

Proteomic and metabolomic profiling reveals time-dependent changes in hippocampal metabolism upon paroxetine treatment and biomarker candidates

Most of the commonly used antidepressants block monoamine reuptake transporters to enhance serotonergic or noradrenergic neurotransmission. Effects besides or downstream of monoamine reuptake inhibition are poorly understood and yet presumably important for the drugs' mode of action. In the present study we aimed at identifying hippocampal cellular pathway alterations in DBA/2 mice using paroxetine as a representative Selective Serotonin Reuptake Inhibitor (SSRI). Furthermore we identified biomarker candidates for the assessment of antidepressant treatment effects in plasma. Hippocampal protein levels were compared between chronic paroxetine- and vehicle-treated animals using in vivo¹⁵N metabolic labeling combined with mass spectrometry. We also studied the time course of metabolite level changes in hippocampus and plasma using a targeted polar metabolomics profiling platform. In silico pathway analyses revealed profound alterations related to hippocampal energy metabolism. Glycolytic metabolite levels acutely increased while Krebs cycle metabolite levels decreased upon chronic treatment. Changes in energy metabolism were influenced by altered glycogen metabolism rather than by altered glycolytic or Krebs cycle enzyme levels. Increased energy levels were reflected by an increased ATP/ADP ratio and by increased ratios of high-to-low energy purines and pyrimidines. In the course of our analyses we also identified myo-inositol as a biomarker candidate for the assessment of antidepressant treatment effects in the periphery. This study defines the cellular response to paroxetine treatment at the proteome and metabolome levels in the hippocampus of DBA/2 mice and suggests novel SSRI modes of action that warrant consideration in antidepressant development efforts.