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Planta Med 2024; 90(07/08): 512-522
DOI: 10.1055/a-2192-3167
Biological and Pharmacological Activity
Original Papers

A Metabolomic Approach to Investigate the Effect of Phytonutrients on Proteostasis and Metabolic Pathways in Drosophila melanogaster [ # ]

Eleni V. Mikropoulou
1   Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens, Greece
,
Zoi Evangelakou
2   Section of Cell Biology and Biophysics, Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens, Greece
,
Ioannis P. Trougakos
2   Section of Cell Biology and Biophysics, Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens, Greece
,
Maria Halabalaki
1   Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, Athens, Greece
› Author AffiliationsThis work was funded by the “NutraFood” project (T7ΔKI-00296), Bilateral and Multilateral S&T Cooperation Greece-China, 2018″ (EPAnEK 2014 – 2020).
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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

    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.

  • Supporting Information


Publication History

Received: 13 June 2023

Accepted after revision: 06 October 2023

Article published online:
06 June 2024

© 2024. Thieme. All rights reserved.

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