Kinetic flux profiling for quantitation of cellular metabolic fluxes

Yuan, Jie; Bennett, Bryson D.; Rabinowitz, Joshua D.

doi:10.1038/nprot.2008.131

Cited by 237 publications

(238 citation statements)

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“…2), we attempted to identify a defect in metabolism using [U- 13 C]glucose and mass spectrometry-based kinetic flux profiling (30). Using this technique, we were able to quantify 118 metabolites in each glucose treatment.…”

Section: Resultsmentioning

confidence: 99%

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Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet β-Cell Function

Burke

May

Noland³

et al. 2015

Journal of Biological Chemistry

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Background: Glucocorticoids impair islet ␤-cell function via glucocorticoid receptor (GR) activation. Results: Thiobenzothiazole-modified hydrocortisone compounds exhibit anti-inflammatory properties with reduced impact on insulin secretion. Conclusion: Novel glucocorticoids can be engineered to reduce impact on ␤-cell mass and function. Significance: Improved GR agonists will be beneficial in a variety of clinical settings.

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Section: Resultsmentioning

confidence: 99%

“…3. For harvesting, cells were washed with cold PBS, pelleted by centrifugation at 250 ϫ g for 5 min, and metabolome-extracted as described (30). Sample analysis was accomplished with an ultraperformance liquid chromatography-mass spectrometric (UPLC-MS) untargeted metabolomics and flux method adapted from a previous report (31).…”

Section: Methodsmentioning

confidence: 99%

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet β-Cell Function

Burke

May

Noland³

et al. 2015

Journal of Biological Chemistry

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“…The targeted metabolomic methods employed to measure relative intracellular metabolite concentrations and turnover rates used slight modifications of a known metabolite extraction procedure (Rabinowitz and Kimball, 2007;Bennett et al, 2008;Yuan et al, 2008). Briefly, 10 ml of cells were rapidly collected on Magna nylon filters (Millipore, Billerica, MA, USA) via vacuum filtration and then extracted by placing the filter directly in a petri dish containing extraction solvent at 4 1C as previously described.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting liquids were lyophilized and then resuspended in 300 ml 40:40:20 acetonitrile/methanol/water with 0.1 M formic acid for MS analysis. Two LC-MS/MS analyses, one in each of positive and negative ion modes, were performed for each sample, and relative metabolite levels and fluxes were analyzed as previously described (Rabinowitz and Kimball, 2007;Bennett et al, 2008;Yuan et al, 2008).…”

Section: Methodsmentioning

confidence: 99%

Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition

Ankrah

May²,

Middleton³

et al. 2013

The ISME Journal

130

126

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Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects B75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that 470% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry.

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“…At the start of the experiment, cells received fresh medium containing U-13 C-glucose. After labeling, metabolites were extracted, dried, and resuspended as previously described (26,49). Metabolites were separated by liquid chromatography and analyzed using liquid chromatography coupled to an Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) according to established parameters (50).…”

Section: Methodsmentioning

confidence: 99%

Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection

Terry¹,

Vastag

Rabinowitz

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Human cytomegalovirus (HCMV) modulates numerous cellular signaling pathways. Alterations in signaling are evident from the broad changes in cellular phosphorylation that occur during HCMV infection and from the altered activity of multiple kinases. Here we report a comprehensive RNAi screen, which predicts that 106 cellular kinases influence growth of the virus, most of which were not previously linked to HCMV replication. Multiple elements of the AMP-activated protein kinase (AMPK) pathway scored in the screen. As a regulator of carbon and nucleotide metabolism, AMPK is poised to activate many of the metabolic pathways induced by HCMV infection. An AMPK inhibitor, compound C, blocked a substantial portion of HCMV-induced metabolic changes, inhibited the accumulation of all HCMV proteins tested, and markedly reduced the production of infectious progeny. We propose that HCMV requires AMPK or related activity for viral replication and reprogramming of cellular metabolism.herpesvirus | siRNA V iruses are dependent on host cell signaling pathways for replication and spread. Infection with the prevalent β-herpes virus human cytomegalovirus (HCMV) induces increased levels of protein phosphorylation and markedly alters host cell signal transduction pathways (1). A portion of the phosphorylation changes induced by HCMV infection are attributed to the Ser/ Thr kinase encoded by the viral genome, pUL97 (2), and others may derive from cellular kinase(s) packaged into virions (3) or from cellular kinases known to be activated by HCMV (4).Here we sought to more completely delineate the effects of HCMV infection on kinase signaling by performing an siRNA screen of the entire cellular kinome. The screen identified 106 kinases predicted to influence the production of virus. The hits included the 5′-AMP-activated protein kinase (AMPK), a sensor of cellular energy homeostasis. AMPK is composed of three subunits: a catalytic subunit, AMPKα, and two regulatory subunits, AMPKβ and AMPKγ. Activation of the kinase requires cooperative AMP binding to AMPKγ, which occurs stochastically with shifts in the AMP:ATP ratio, and phosphorylation of AMPKα at Thr172 (5, 6). At least three different kinases are reported to phosphorylate Thr172 of AMPKα: Ca 2+ /calmodulindependent kinase kinase (CaMKK), TGF-β-activated kinase 1 (TAK1), and liver kinase B1 (LKB1) (7). Activated AMPK phosphorylates a number of substrates to effect changes in central carbon metabolism, lipid metabolism, physiological homeostasis, cell growth, apoptosis, and gene expression (5).HCMV induces glycolysis (8-10) and also causes increased levels of the glucose transporter GLUT4 at the plasma membrane increasing glucose uptake (11).AMPK controls GLUT4 relocalization to the plasma membrane (5), and this regulation likely links the kinase to altered metabolism in HCMV-infected cells. However, previous work indicates that pharmacological activation of AMPK during the early phase of HCMV infection can be deleterious to viral replication (12), yet CaMKK activity is required for ...

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Kinetic flux profiling for quantitation of cellular metabolic fluxes

Cited by 237 publications

References 40 publications

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet β-Cell Function

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet β-Cell Function

Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition

Human kinome profiling identifies a requirement for AMP-activated protein kinase during human cytomegalovirus infection

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