Article

Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotechnol

Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544, USA.
Nature Biotechnology (Impact Factor: 41.51). 10/2008; 26(10):1179-86. DOI: 10.1038/nbt.1500
Source: PubMed

ABSTRACT

Viruses rely on the metabolic network of their cellular hosts to provide energy and building blocks for viral replication. We developed a flux measurement approach based on liquid chromatography-tandem mass spectrometry to quantify changes in metabolic activity induced by human cytomegalovirus (HCMV). This approach reliably elucidated fluxes in cultured mammalian cells by monitoring metabolome labeling kinetics after feeding cells (13)C-labeled forms of glucose and glutamine. Infection with HCMV markedly upregulated flux through much of the central carbon metabolism, including glycolysis. Particularly notable increases occurred in flux through the tricarboxylic acid cycle and its efflux to the fatty acid biosynthesis pathway. Pharmacological inhibition of fatty acid biosynthesis suppressed the replication of both HCMV and influenza A, another enveloped virus. These results show that fatty acid synthesis is essential for the replication of two divergent enveloped viruses and that systems-level metabolic flux profiling can identify metabolic targets for antiviral therapy.

Download full-text

Full-text

Available from: Thomas Shenk, Oct 24, 2014
    • "Acid hydrolysis is commonly used in metabolic flux analysis (MFA) applications, but the extent of labeling in glycans is not commonly measured [34] . This is particularly true in MFA applied to mammalian cells353637. To validate our approach we analyzed standards for specific glycan residues and compared the mass isotopomer distributions (MIDs) of specific fragments to those measured in cells cultured in the presence of [U-13 C 6 ] glucose. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging; however, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    No preview · Article · Aug 2015 · Biotechnology Journal
  • Source
    • "The fatty acids and other lipid materials that are generated by these processes are essential substrates for energy metabolism (Menendez and Lupu, 2007; Mashima et al., 2009) as well as the major components of all biological lipid membranes (Menendez and Lupu, 2007; Vander Heiden et al., 2009; Mashima et al., 2009). The Warburg Effect is also seen in vertebrate cells infected by oncogenic viruses such as KSHV (Kaposi's sarcoma-associated herpesvirus) (Delgado et al., 2010) and HCMV (Human cytomegalovirus ) (Munger et al., 2008), and a recent paper (Chen et al., 2011) further showed that it occurs in shrimp hemocytes that are infected by the White Spot Syndrome Virus (WSSV). WSSV, which is a large enveloped (approximately 300 kbp) dsDNA invertebrate virus with an in vivo replication cycle of 22e24 h, was found to trigger the Warburg effect at the WSSV genome replication stage (12 hpi [hours post infection]) via the activation of the PI3K-Akt-mTOR pathway (Su et al., 2014). "
    [Show abstract] [Hide abstract]
    ABSTRACT: White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD), is a serious and aggressive shrimp viral pathogen with a worldwide distribution. At the genome replication stage (12 hpi), WSSV induces a metabolic rerouting known as the invertebrate Warburg effect, which boosts the availability of energy and biosynthetic building blocks in the host cell. Here we show that unlike the lipogenesis that is seen in cancer cells that are undergoing the Warburg effect, at 12 hpi, all of the long chain fatty acids (LCFAs) were significantly decreased in the stomach cells of WSSV-infected shrimp. By means of this non-selective WSSV-induced lipolysis, the LCFAs were apparently diverted into β-oxidation and used to replenish the TCA cycle. Conversely, at 24 hpi, when the Warburg effect had ceased, most of the LCFAs were significantly up-regulated and the composition was also significantly altered. In crayfish these changes were in a direction that appeared to favor the formation of WSSV virion particles. We also found that, at 24 hpi, but not at 12 hpi, the PI3K-Akt-mTOR-HIF1α pathway induced the expression of fatty acid synthase (FAS), an enzyme which catalyzes the conversion of acetyl-CoA into LCFAs. WSSV virion formation was impaired in the presence of the FAS inhibitor C75, although viral gene and viral DNA levels were unaffected. WSSV therefore appears to use the PI3K-Akt-mTOR pathway to induce lipid biosynthesis at 24 hpi in order to support viral morphogenesis. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Jun 2015 · Developmental and comparative immunology
    • "ethanol (70 C). Supernatant of extracted samples were dried under vacuum and resuspended in LC-MS grade water for analysis of the relative abundance of 13 C and 15 N metabolites (Munger et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Macrophage polarization involves a coordinated metabolic and transcriptional rewiring that is only partially understood. By using an integrated high-throughput transcriptional-metabolic profiling and analysis pipeline, we characterized systemic changes during murine macrophage M1 and M2 polarization. M2 polarization was found to activate glutamine catabolism and UDP-GlcNAc-associated modules. Correspondingly, glutamine deprivation or inhibition of N-glycosylation decreased M2 polarization and production of chemokine CCL22. In M1 macrophages, we identified a metabolic break at Idh, the enzyme that converts isocitrate to alpha-ketoglutarate, providing mechanistic explanation for TCA cycle fragmentation. (13)C-tracer studies suggested the presence of an active variant of the aspartate-arginosuccinate shunt that compensated for this break. Consistently, inhibition of aspartate-aminotransferase, a key enzyme of the shunt, inhibited nitric oxide and interleukin-6 production in M1 macrophages, while promoting mitochondrial respiration. This systems approach provides a highly integrated picture of the physiological modules supporting macrophage polarization, identifying potential pharmacologic control points for both macrophage phenotypes. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Mar 2015 · Immunity
Show more