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DOI: 10.1055/a-2192-3167
A Metabolomic Approach to Investigate the Effect of Phytonutrients on Proteostasis and Metabolic Pathways in Drosophila melanogaster [ # ]
This work was funded by the “NutraFood” project (T7ΔKI-00296), Bilateral and Multilateral S&T Cooperation Greece-China, 2018″ (EPAnEK 2014 – 2020).
Abstract
The use of Drosophila melanogaster as a biological platform to study the effect of diet and food bioactives on the metabolome remains a highly unexplored subject. Aiming to establish alternative solutions for the investigation of nutritional interventions with bioactive natural products by employing LC-MS-based metabolomics approaches, we assessed the effect of a phytonutrient-rich extract from the endemic Mediterranean plant Cichorium spinosum (stamnagkàthi) on a Drosophila population. The extractʼs modulating effect on the proteostasis network and metabolism of young D. melanogaster flies was evaluated. Furthermore, an untargeted metabolomics approach, employing a C18 UPLC-ESI-Orbitrap-HRMS/MS platform, permitted the detection of several biomarkers in the metabolic profile of Drosophila’s tissues; while targeted amino acid quantification in Drosophila tissue was simultaneously performed by employing aTRAQ labeling and an ion-pairing UPLC-ESI-SWATH-HRMS/MS platform. The detected metabolites belong to different chemical classes, and statistical analysis with chemometrics tools was utilized to reveal patterns and trends, as well as to uncover potential class-distinguishing features and possible biomarkers. Our findings suggest that Drosophila can serve as a valuable in vivo model for investigating the role of bioactive phytoconstituents, like those found in C. spinosum’s decoction, on diverse metabolic processes. Additionally, the fruit fly represents a highly effective platform to investigate the molecular mechanisms underlying sex differences in diverse aspects of nutrition and physiology in higher metazoans.
Key words
Cichorium spinosum - Asteraceae - Drosophila melanogaster - amino acids - metabolomics - Orbitrap - TripleTOF - proteostasis network# This work is dedicated to Professors Rudolf Bauer, Chlodwig Franz, Brigitte Kopp, and Hermann Stuppner for their invaluable contributions and commitment to Austrian Pharmacognosy.
Supporting Information
- Supporting Information
The following are available as Supporting Information: 1. Supplementary Materials and Methods: Cichorium spinosum extraction, analysis, and profile characterization; Proteasome peptidase activity in fly tissue extracts; Measurement of ARE-GFP; Total RNA extraction and Quantitative Real Time PCR (Q RT PCR) analysis; Tissue protein extract preparation, immunoblotting analysis, and detection of protein carbonyl groups; Statistical analysis for bioactivity assays; Preprocessing parameters for the untargeted metabolomics data; Metabolomics statistical analysis and metabolite annotation; 2. Supplementary Results and Discussion: Cichorium spinosum extract profiling; Drosophila untargeted metabolomics method development; 3. Supplementary Tables: Table 1S. UPLC-ESI(−)-HRMS data for the C. spinosum decoction; Table 2S. UPLC-ESI(−)-HRMS&HRMS/MS dereplication data for Drosophila QC pooled extract; Table 3S. UPLC-ESI(+)-HRMS&HRMS/MS dereplication data for Drosophila QC pooled extract; Table 4S. Untargeted metabolomics sample list; Table 5S. Targeted AA quantification sample list. 4. Supplementary Figures: Fig. 1S. UPLC-ESI(−)-HRMS chromatogram of C. spinosum’ s decoction.; Fig. 2S. UPLC-ESI-HRMS profiles of Drosophila pooled sample in negative (upper) and positive (lower) ionization modes.; Fig. 3S. PCA (a), PLS-DA (b) and OPLS-DA (c) scores plots of the control group in negative mode, after UV scaling. For a) R2X = 0.630, Q2 = − 0.171, b) R2X = 0.547, R2Y = 0.994, Q2 = 0.865, c) R2X = 0.547, R2Y = 0.994, Q2 = 0.805; Fig. 4S. PCA (a), PLS-DA (b) and OPLS-DA (c) scores plots of the control group in positive mode, after UV scaling. For a) R2X = 0.502, Q2 = − 0.21, b) R2X = 0.425, R2Y = 1, Q2 = 0.738, c) R2X = 0.425, R2Y = 1, Q2 = 0.746.; Fig. 5S. (a) PCA, (b) PLS-DA and (c) OPLS-DA scores plots of all Drosophila samples in negative mode, separated according to sex, after UV scaling. (a) R2X = 0.444, Q2 = 0.153, (b) R2X = 0.316, R2Y = 0.897, Q2 = 0.631, (c) R2X = 0.316, R2Y = 0.897, Q2 = 0.628. (d, e) Permutation tests (n = 200) referring to the female (d) and male (e) classes of the OPLS-DA model. (f) Loadings plot referring to (c).; Fig. 6S. (a) PCA, (b) PLS-DA and (c) OPLS-DA scores plots of all Drosophila samples in positive mode, separated according to sex, after UV scaling. (a) R2X = 0.300, Q2 = 0.0578, (b) R2X = 0.189, R2Y = 0.926, Q2 = 0.434, (c) R2X = 0.189, R2Y = 0.926, Q2 = 0.419. (d,e) Permutation tests (n = 200) referring to the female (d) and male (e) classes of the OPLS-DA model. (f) Loadings plot referring to (c).; Fig. 7S. Network of likely affected metabolic pathways according to our metabolomics study.; Fig. 8S. Biplot of the respective OPLS-DA model for the different AA concentrations in the Drosophila samples, separated according to sex, after UV scaling. R2X = 0.704, R2Y = 0.939, Q2 = 0.789.
Publication History
Received: 13 June 2023
Accepted after revision: 06 October 2023
Article published online:
06 June 2024
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