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Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism

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Abstract

Introduction

Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects.

Objectives

To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations.

Methods

A dual extraction method involving 80% methanol followed by MTBE (methyl tert-butyl ether) extraction enables the analysis of free fatty acids, polar metabolites, and lipids. Extracts from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days were analyzed using HILIC chromatography coupled to Q Exactive Plus mass spectrometer or reversed-phase liquid chromatography coupled to MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility.

Results

Lipidomics analysis of 6 mouse tissues and plasma allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that (1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; (2) the impact on lysophosphatidylcholine (lysoPC) and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations; (3) increase of intestinal tricarboxylic acid (TCA) cycle intermediates after metformin treatment.

Conclusion

The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

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Data availability

Lipidomics and metabolomics data have been uploaded to Metabolomics Workbench. The lipidomics dataset of tissue and plasma samples can be found under DataTrackID 4628, the lipid serial dilution analysis under DataTrack ID 4632, and the metabolomics dataset under DataTrack ID 4633. Other data is available upon request from the corresponding author.

Abbreviations

LC:

Liquid chromatography

HRMS:

High-resolution mass spectrometer

DDA:

Data-dependent acquisition

DIA:

Data-independent acquisition

BRI:

Biologically relevant ions

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Acknowledgements

We thank support from Molecular Education, Technology and Research Innovation Center (METRIC) at North Carolina State University.

Funding

This work was supported by Chemistry of Life pilot funds and startup funds (L.D., G.S., M.L., X.L., A.K.) at North Carolina State University (NCSU), National Institutes of Health 1R21DK128678-01A1 (A.K.), National Institutes of Health 1R35GM150985-01 (X.L.), Center for Human Health and the Environment Pilot Project Program Award (ES025128 to X.W.) at NCSU and Animal Reproduction Program (No. 2022-67015-36491 to X.W.) from the USDA National Institute of Food and Agriculture.

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Author notes

  1. Grace Scheidemantle and Likun Duan have equal contributions to this work.

Authors and Affiliations

  1. Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA

    Grace Scheidemantle, Likun Duan, Mareca Lodge, Dalton Hilovsky, Eva Pham, Arion Kennedy & Xiaojing Liu

  2. Department of Animal Science, North Carolina State University, Raleigh, NC, 27695, USA

    Magdalina J. Cummings & Xiaoqiu Wang

  3. The Comparative Medicine Institute, North Carolina State University, Raleigh, NC, 27695, USA

    Magdalina J. Cummings & Xiaoqiu Wang

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  1. Grace Scheidemantle
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Contributions

A.K., X.W., M.L, and M.C. performed all animal experiments. G.S., L.D., and X.L. prepared samples and performed LC–MS analysis. X.L., L.D., G.S., A.K., and X.W. participated in experimental design. X.L, L.D., and G.S. interpreted results and wrote the manuscript. E.P. helped with text editing. All authors provided input on the manuscript.

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Correspondence to Xiaojing Liu.

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All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at North Carolina State University.

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Scheidemantle, G., Duan, L., Lodge, M. et al. Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism. Metabolomics 20, 53 (2024). https://doi.org/10.1007/s11306-024-02113-2

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