Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC–MS/MS

AbstractIdentification of proteins that are synthesized de novo in response to specific microenvironmental cues is critical to understanding the molecular mechanisms that underpin key physiological processes and pathologies. Here we report that a brief period of pulsed SILAC diet (Stable Isotope Labelling by Amino acids in Cell culture) enables determination of biological functions corresponding to actively translating proteins in the mouse brain. Our data demonstrate that the hippocampus, cortex and cerebellum are highly active sites of protein synthesis, rapidly expressing key mediators of nutrient sensing and lipid metabolism, as well as critical regulators of synaptic function, axon guidance, and circadian entrainment. Together, these findings confirm that protein metabolic activity varies significantly between brain regions in vivo and indicate that pSILAC-based approaches can identify specific anatomical sites and biological pathways likely to be suitable for drug targeting in neurodegenerative disorders.AbbreviationsApoA1: Apolipoprotein A1, ApoA4: Apolipoprotein A4, ApoE: Apolipoprotein E, ApoJ/Clu: Apolipoprotein J/Clusterin, App: Amyloid-β precursor/A4 protein: App, HDL: high density lipoprotein, Lrp1: Low density lipoprotein receptor-related protein 1, pSILAC: pulsed SILAC, pSIVOM: pulsed-SILAC in vivo labelling in mouse, SILAC: Stable Isotope Labelling by Amino acids in Cell culture)

Download Full-text

Investigation into the flux distribution of central carbon metabolism in Corynebacterium glutamicum using principal component analysis

Archives of Biological Sciences ◽

10.2298/abs150107035s ◽

2015 ◽

Vol 67 (3) ◽

pp. 767-774

Author(s):

Chuanyu Shang ◽

Xiangfei Zhou ◽

Wenwei Zhou ◽

Xiaoyao Xie ◽

Yin Yi

Keyword(s):

Corynebacterium Glutamicum ◽

Carbon Metabolism ◽

Flux Distribution ◽

Environmental Changes ◽

Principal Component ◽

Central Carbon Metabolism ◽

Preliminary Examination ◽

Genetic Modifications ◽

Central Carbon

Central carbon metabolism is the main source of energy required by organisms and it provides precursors for other in vivo metabolic processes. The flux flowing through the pathways involved in central carbon metabolism characterizes its biological function and genetic readout between species or environments. In recent years, using a 13C tracer technique, researchers have measured the flux of central carbon metabolism in Corynebacterium glutamicum under a variety of nutritional and environmental changes or genetic modifications. However, there is no integrated and comparative analysis of these measured flux values. In this study, the flux values of central carbon metabolism in Corynebacterium glutamicum that were obtained in other recent studies were consolidated. A preliminary examination of the distribution characteristics of flux values in each metabolic pathway was conducted and the regression relationship between different fluxes was investigated. The principal components of the flux vector were further extracted and aggregated based on the components, and the general features of flux distribution of central carbon metabolism as well as the influence of environmental and genetic factors on the flux distribution were determined. This study provides a foundation for further investigation into the flux distribution and regulation characteristics of central carbon metabolism.

Download Full-text

Stable isotope labelling of phosphatidylcholine synthesis in vivo in children with acute lung injury

Archives of Disease in Childhood ◽

10.1136/adc.2010.186338.93 ◽

2010 ◽

Vol 95 (Suppl 1) ◽

pp. A43.1-A43

Author(s):

K Goss ◽

L Ward ◽

V Ledger ◽

G Koster ◽

P Townsend ◽

...

Keyword(s):

Acute Lung Injury ◽

Stable Isotope ◽

Lung Injury ◽

Stable Isotope Labelling ◽

Isotope Labelling ◽

Phosphatidylcholine Synthesis

Download Full-text

Using high-resolution Twin-Ion Metabolite Extraction (HiTIME) mass spectrometry with stable isotope labelling to investigate the metabolism of valproic acid in vivo

International Journal of Mass Spectrometry ◽

10.1016/j.ijms.2019.116187 ◽

2019 ◽

Vol 444 ◽

pp. 116187 ◽

Cited By ~ 2

Author(s):

Michael G. Leeming ◽

Noel Cranswick ◽

Christine E. Wright ◽

James Ziogas ◽

Terence J. O'Brien ◽

...

Keyword(s):

Mass Spectrometry ◽

Valproic Acid ◽

High Resolution ◽

Stable Isotope ◽

Stable Isotope Labelling ◽

Isotope Labelling ◽

Metabolite Extraction

Download Full-text

How the molecular weight affects the in vivo fate of exogenous hyaluronan delivered intravenously: A stable-isotope labelling strategy

Carbohydrate Polymers ◽

10.1016/j.carbpol.2021.117927 ◽

2021 ◽

Vol 263 ◽

pp. 117927

Author(s):

Matěj Šimek ◽

Kristina Nešporová ◽

Anna Kocurková ◽

Tereza Foglová ◽

Gabriela Ambrožová ◽

...

Keyword(s):

Molecular Weight ◽

Stable Isotope ◽

Stable Isotope Labelling ◽

Isotope Labelling

Download Full-text

Shifts in rotifer life history in response to stable isotope enrichment: testing theories of isotope effects on organismal growth

Royal Society Open Science ◽

10.1098/rsos.160810 ◽

2017 ◽

Vol 4 (3) ◽

pp. 160810 ◽

Cited By ~ 5

Author(s):

Elena Gorokhova

Keyword(s):

Stable Isotope ◽

Brachionus Plicatilis ◽

Isotope Effects ◽

Experimental Studies ◽

Isotope Enrichment ◽

Heavy Isotope ◽

Material Transfer ◽

Stable Isotope Labelling ◽

Isotope Labelling

In ecology, stable isotope labelling is commonly used for tracing material transfer in trophic interactions, nutrient budgets and biogeochemical processes. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism growth and metabolism. This assumption is, however, challenged by theoretical considerations and experimental studies on kinetic isotope effects in vivo . Here, I demonstrate profound changes in life histories of the rotifer Brachionus plicatilis fed 15 N-enriched algae (0.4–5.0 at%); i.e. at the enrichment levels commonly used in ecological studies. These findings support theoretically predicted effects of heavy isotope enrichment on growth, metabolism and ageing in biological systems and underline the importance of accounting for such effects when using stable isotope labelling in experimental studies.

Download Full-text

Central carbon metabolism of Leishmania parasites

Parasitology ◽

10.1017/s0031182010000077 ◽

2010 ◽

Vol 137 (9) ◽

pp. 1303-1313 ◽

Cited By ~ 49

Author(s):

ELEANOR C. SAUNDERS ◽

DAVID P. DE SOUZA ◽

THOMAS NADERER ◽

MARIJKE F. SERNEE ◽

JULIE E. RALTON ◽

...

Keyword(s):

Carbon Metabolism ◽

Drug Targets ◽

Carbon Sources ◽

Central Carbon Metabolism ◽

Mammalian Host ◽

Insect Vector ◽

Carbon Utilization ◽

Central Carbon ◽

Host Infection

SUMMARYLeishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.

Download Full-text

Lighting Up Live-Cell and In Vivo Central Carbon Metabolism with Genetically Encoded Fluorescent Sensors

Annual Review of Analytical Chemistry ◽

10.1146/annurev-anchem-091619-091306 ◽

2020 ◽

Vol 13 (1) ◽

pp. 293-314 ◽

Cited By ~ 1

Author(s):

Zhuo Zhang ◽

Xiawei Cheng ◽

Yuzheng Zhao ◽

Yi Yang

Keyword(s):

Carbon Metabolism ◽

Tricarboxylic Acid Cycle ◽

Cell Metabolism ◽

Central Carbon Metabolism ◽

Fluorescent Sensors ◽

Pentose Phosphate ◽

Spatiotemporal Information ◽

Extant Species ◽

Central Carbon

As the core component of cell metabolism, central carbon metabolism, consisting of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle converts nutrients into metabolic precursors for biomass and energy to sustain the life of virtually all extant species. The metabolite levels or distributions in central carbon metabolism often change dynamically with cell fates, development, and disease progression. However, traditional biochemical methods require cell lysis, making it challenging to obtain spatiotemporal information about metabolites in living cells and in vivo. Genetically encoded fluorescent sensors allow the rapid, sensitive, specific, and real-time readout of metabolite dynamics in living organisms, thereby offering the potential to fill the gap in current techniques. In this review, we introduce recent progress made in the development of genetically encoded fluorescent sensors for central carbon metabolism and discuss their advantages, disadvantages, and applications. Moreover, several future directions of metabolite sensors are also proposed.

Download Full-text