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, 11 (4), e0152935
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Contribution of Drosophila TRPA1 to Metabolism

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Contribution of Drosophila TRPA1 to Metabolism

Jung-Eun Lee et al. PLoS One.

Abstract

Transient receptor potential (TRP) cation channels are highly conserved in humans and insects. Some of these channels are expressed in internal organs and their functions remain incompletely understood. By direct knock-in of the GAL4 gene into the trpA1 locus in Drosophila, we identified the expression of this gene in the subesophageal ganglion (SOGs) region. In addition, the neurites present in the dorsal posterior region as well as the drosophila insulin-like peptide 2 (dILP2)-positive neurons send signals to the SOGs. The signal is sent to the crop, which is an enlarged organ of the esophagus and functions as a storage place for food in the digestive system. To systematically investigate the role of TRPA1 in metabolism, we applied non-targeted metabolite profiling analysis together with gas-chromatography/time-of-flight mass spectrometry, with an aim to identify a wide range of primary metabolites. We effectively captured distinctive metabolomic phenotypes and identified specific metabolic dysregulation triggered by TRPA1 mutation based on reconstructed metabolic network analysis. Primarily, the network analysis pinpointed the simultaneous down-regulation of intermediates in the methionine salvation pathway, in contrast to the synchronized up-regulation of a range of free fatty acids. The gene dosage-dependent dynamics of metabolite levels among wild-type, hetero- and homozygous mutants, and their coordinated metabolic modulation under multiple gene settings across five different genotypes confirmed the direct linkages of TRPA1 to metabolism.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. A GAL4 knock-in to the trpA1 locus drives the expression in the digestive system.
(A-B) Brains dissected and stained with anti-GFP from UAS-mCD8::GFP;trpA1GAL4 flies at the adult stage. (C–E) Brains dissected and stained with anti-dILP2 (C) and anti-GFP (D) from UAS-mCD8::GFP;trpA1GAL4 flies in the adult stage. The merged image is shown (E). Broad expression of the trpA1 reporter is apparent in the subesophageal ganglions (SOG). Scale bars, 50 m.
Fig 2
Fig 2. Metabolomic phenotypes integrated with primary metabolic features.
(A) Principal component analysis of metabolomic profiles of 109 metabolites of the wild type (orange) and the homozygous mutant (purple). T1 indicates discriminating vector 1 that explained the largest degree of variation in the dataset. Likewise, T2 indicates principal component 2 with the second largest degree of variation. (B) Hierarchical clustering analysis. Clustering analysis was performed across the metabolites and samples by using Spearman rank correlation and average linkage methods. Each column and each row represent a fly sample and an individual metabolite, respectively.
Fig 3
Fig 3. Metabolic networks of biochemical reaction pairs and chemical similarity.
The regulation of all identified metabolites in files is depicted. Blue = down-regulated metabolites, red = up-regulated metabolites in the heterozygous mutants (trpA11) compared to the wild-type (Student’s t-test, P<0.05). Node sizes reflect the magnitude of differential metabolite expression. Metabolites that did not show significant difference in levels were left unnamed to maintain visual clarity. Dark blue edges represent connections determined via Kegg reaction pair information, and light blue edges represent assemblies as evaluated using Tanimoto scores (score > 0.7)
Fig 4
Fig 4. Gene-dosage effect of trpA1 on the primary metabolism.
Pavlidis template matching (PTM) analysis was performed to identify (A) the gradual increase and (B) decrease in metabolite abundances.
Fig 5
Fig 5. Metabolites showing consensus expression pattern across different genetic settings.
(A) Venn diagram showing allele-specific metabolite changes in trpA11 and trpA1GAL4 respectively, compared with wild-type (B) Venn diagram showing the chemical overlap between trpA1-expressed strains and trpA1-repressed strains along with two independent comparisons. (C) List of metabolites that are coordinately altered according to trpA1 gene expression.
Fig 6
Fig 6. Proposed functional linkage of trpA1 to central carbon metabolism and methionine salvage pathway in Drosophila.
Arrows with filled colors indicate significant changes (P<0.05) in trpA1 mutants compared to wild-type, and arrows without any colors presented the down-regulation pattern but without statistical significance. Data distributions were displayed by box-whisker plots, giving the mean value, the standard error as the box, and whiskers indicating 1.96 fold the standard.

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Grant support

This work was supported by a grant to Y.L. from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2012M3A9B2052525), and the Basic Science Research Program of the NRF of Korea funded by the Ministry of Education (2014R1A1A2058094), and a grant to D.Y.L. from the Bio & Medical Technology Development Program of the National Research Foundation (NRF) (grant numbers: NRF-2014M3A9B6069345 and NRF 2013M3A9B6046519). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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