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Additional file 1: SupplementaryFigure 1. The comparison ofexperimental and theoretical values of M+1 relative abundance of representativemetabolites analyzed by orbitrap-based mass spectrometer. (A) The experimental and theoretical values of M+1 abundance ofrepresentative metabolites. (B) The abundance of adenosine M+1 (relative toM+0) in cells treated with or without [U-13C]-glucose. The relativeratio of M+0 is set as 1 and M+1 abundance is normalized to M+0 abundance. Thedata presented in this figure was without natural abundance correction. SupplementaryFigure 2. The mass isotopologue distribution of [U-13C]-glutaminedetected in glutamine free RPMI 1640 medium supplemented with [U-13C]-glutamine. The data presented in this figure waswithout natural abundance correction. SupplementaryFigure 3. The effect of exogenous CO2 on M+1 labeling in sarcomacells. (A) Schematic of in vitrotracing analysis. (B) 13C labeling patterns of metabolites fromsarcoma cells (in vitro) incubated in [U-13C]-glutamine medium inthe presence or absence of CO2 for 3 hrs. M+1/M+4 ratio (in theinsert) was used to account for the overall TCA activity perturbations due tomedium changes. Data are presented as mean ± SD of three replicates and afternatural abundance correction. Supplementary Figure 4. MS2 analysis of adenosine M+1. (A) TheMS2 spectrum of adenosine fragments. (B) the relative ratio of daughter ionadenine M+0 versus adenine M+1 in the absence of tracer (labeled as “12C-glucose”)or [U-13C]-glucose. Data in (B) are presented as mean ± SD of threereplicates. P value was calculated based on Student’s ttest, and “ns” denotes p value larger than 0.05. The data presented in thisfigure was without natural abundance correction. Supplementary Figure 5. The mass isotopologue distribution of succinyl-CoA in sarcomaof mice receiving [U-13C]-glucose tracing. Data are presented asmean ± SD of n=5 mice. Data with and without natural abundance (NA) correctionwas presented. Supplementary Figure 6. GlucoseM+1 in the plasma of mice receiving [U-13C]-glucose for 3 hrs. Datapresented here was after natural abundance correction. Supplementary Figure 7. Pyruvate labeling patterns in sarcoma of micereceiving [U-13C]-glucose or [U-13C]-glutamine tracingfor 3 hrs. Data are presented as mean ± SD of three replicates and afternatural abundance correction.

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: Lipids were extracted from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days and analyzed using 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 using MS/MS combining BRI-DIA and DDA 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 lysophosphorylcholine 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., 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)., Competing Interests: Competing interest Authors declare that they have no conflicts of interest with the contents of this article.