Summary of Study ST003507

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002153. The data can be accessed directly via it's Project DOI: 10.21228/M89Z57 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST003507
Study Title	A NRF2/β3-adrenoreceptor axis drives a sustained antioxidant and metabolic rewiring through the pentose-phosphate pathway to alleviate cardiac stress
Study Summary	WT TAC vs TG TAC vs WT baseline vs TG baseline Background: Cardiac β3-adrenergic receptors (β3AR) are upregulated in diseased hearts and mediate antithetic effects to those of β1AR and β2AR. β3AR agonists were recently shown to protect from myocardial remodeling in preclinical studies and to improve systolic function in patients with severe heart failure. The underlying mechanisms, however, remain elusive. Methods: To dissect functional, transcriptional and metabolic effects, hearts and isolated ventricular myocytes from mice harboring a moderate, cardiac-specific expression of a human ADRB3 transgene (β3AR-Tg) and subjected to transverse aortic constriction (TAC) were assessed using echocardiography, RNAseq, PET scan, metabolomics, seahorse and metabolic flux analysis. Subsequently, signaling and metabolic pathways were investigated further in vivo in β3AR-Tg and in vitro in neonatal rat ventricular myocytes adenovirally infected to express β3AR and subjected to neurohormonal stress. These results were completed with an analysis of single nucleus RNAseq data from human cardiac myocytes from heart failure patients. Results: Compared with WT littermate, β3AR-Tg mice were protected from hypertrophy after transaortic constriction (TAC), while systolic function was preserved. β3AR-expressing hearts displayed enhanced myocardial glucose uptake under stress in absence of increased lactate levels. Instead, metabolomic and metabolic flux analyses in stressed hearts revealed an increase in intermediates of the Pentose-Phosphate Pathway (PPP) in β3AR-Tg, an alternative route of glucose utilization, paralleled with increased transcript levels of NADPH-producing and rate-limiting enzymes of the PPP, without fueling the hexosamine metabolism. The ensuing increased content of NADPH and of reduced glutathione decreased myocyte oxidant stress, while downstream oxidative metabolism assessed by oxygen consumption was preserved with higher glucose oxidation in β3ARTg post-TAC compared to WT, together with increased mitochondrial biogenesis. Unbiased transcriptomics and pathway analysis identified NRF2 (NFE2L2) as upstream transcription factor which was functionally verified in β3AR- expressing cardiac myocytes where its translocation and nuclear activity was dependent on β3AR activation of nitric-oxide synthase (NOS) NO production. Conclusion: Moderate expression of cardiac β3AR, at levels observed in human cardiac myocardium, exerts antioxidant effects through activation of the PPP and NRF2 pathway, thereby preserving myocardial oxidative metabolism, function and integrity under pathophysiological stress.
Institute	UCLouvain
Last Name	Dewulf
First Name	Joseph
Address	Avenue Hippocrate 10
Email	joseph.dewulf@uclouvain.be
Phone	027646727
Submit Date	2024-08-30
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-11-01
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002153
Project DOI:	doi: 10.21228/M89Z57
Project Title:	A NRF2/β3-adrenoreceptor axis drives a sustained antioxidant and metabolic rewiring through the pentose-phosphate pathway to alleviate cardiac stress
Project Summary:	Background Cardiac β3-adrenergic receptors (β3AR) are upregulated in diseased hearts and mediate antithetic effects to those of β1AR and β2AR. β3AR agonists were recently shown to protect from myocardial remodeling in preclinical studies and to improve systolic function in patients with severe heart failure. The underlying mechanisms, however, remain elusive. Methods To dissect functional, transcriptional and metabolic effects, hearts and isolated ventricular myocytes from mice harboring a moderate, cardiac-specific expression of a human ADRB3 transgene (β3AR-Tg) and subjected to transverse aortic constriction (TAC) were assessed using echocardiography, RNAseq, PET scan, metabolomics, seahorse and metabolic flux analysis. Subsequently, signaling and metabolic pathways were investigated further in vivo in β3AR-Tg and in vitro in neonatal rat ventricular myocytes adenovirally infected to express β3AR and subjected to neurohormonal stress. These results were completed with an analysis of single nucleus RNAseq data from human cardiac myocytes from heart failure patients. Results Compared with WT littermate, β3AR-Tg mice were protected from hypertrophy after transaortic constriction (TAC), while systolic function was preserved. β3AR-expressing hearts displayed enhanced myocardial glucose uptake under stress in absence of increased lactate levels. Instead, metabolomic and metabolic flux analyses in stressed hearts revealed an increase in intermediates of the Pentose-Phosphate Pathway (PPP) in β3AR-Tg, an alternative route of glucose utilization, paralleled with increased transcript levels of NADPH-producing and rate-limiting enzymes of the PPP, without fueling the hexosamine metabolism. The ensuing increased content of NADPH and of reduced glutathione decreased myocyte oxidant stress, while downstream oxidative metabolism assessed by oxygen consumption was preserved with higher glucose oxidation in β3AR-Tg post-TAC compared to WT, together with increased mitochondrial biogenesis. Unbiased transcriptomics and pathway analysis identified NRF2 (NFE2L2) as upstream transcription factor which was functionally verified in β3AR-expressing cardiac myocytes where its translocation and nuclear activity was dependent on β3AR activation of nitric-oxide synthase (NOS) NO production. Conclusion Moderate expression of cardiac β3AR, at levels observed in human cardiac myocardium, exerts antioxidant effects through activation of the PPP and NRF2 pathway, thereby preserving myocardial oxidative metabolism, function and integrity under pathophysiological stress.
Institute:	UCLouvain
Last Name:	Dewulf
First Name:	Joseph
Address:	Avenue Hippocrate 10, Brussels, Brussels, 1200, Belgium
Email:	joseph.dewulf@uclouvain.be
Phone:	027646727

Subject:

Subject ID:	SU003636
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Condition
SA385864	027 sample 15 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385865	023 sample 11 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385866	022 sample 10 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385867	015 sample 3 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385868	016 sample 4 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385869	019 sample 7 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385870	020 sample 8 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385871	020 sample 8 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385872	019 sample 7 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385873	027 sample 15 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385874	016 sample 4 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385875	015 sample 3 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385876	027 sample 15 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385877	015 sample 3 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385878	016 sample 4 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385879	022 sample 10 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385880	019 sample 7 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385881	019 sample 7 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385882	020 sample 8 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385883	023 sample 11 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385884	027 sample 15 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385885	022 sample 10 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385886	023 sample 11 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG Baseline
SA385887	015 sample 3 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385888	023 sample 11 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385889	022 sample 10 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385890	016 sample 4 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385891	020 sample 8 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG Baseline
SA385892	043 sample 31 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385893	038 sample 26 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385894	042 sample 30 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385895	039 sample 27 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385896	032 sample 20 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385897	044 sample 32 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385898	033 sample 21 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385899	044 sample 32 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385900	043 sample 31 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385901	036 sample 24 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385902	042 sample 30 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385903	036 sample 24 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385904	036 sample 24 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385905	033 sample 21 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385906	044 sample 32 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385907	032 sample 20 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385908	033 sample 21 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385909	036 sample 24 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385910	038 sample 26 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385911	039 sample 27 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385912	044 sample 32 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385913	043 sample 31 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385914	043 sample 31 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385915	042 sample 30 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385916	042 sample 30 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385917	033 sample 21 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385918	032 sample 20 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385919	032 sample 20 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385920	038 sample 26 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385921	039 sample 27 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385922	038 sample 26 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385923	039 sample 27 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	TG TAC 9W
SA385924	026 sample 14 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385925	024 sample 12 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385926	013 sample 1 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385927	021 sample 9 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385928	014 sample 2 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385929	013 sample 1 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385930	014 sample 2 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385931	025 sample 13 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385932	021 sample 9 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385933	024 sample 12 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385934	025 sample 13 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385935	028 sample 16 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385936	013 sample 1 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385937	026 sample 14 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385938	028 sample 16 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385939	025 sample 13 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385940	028 sample 16 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385941	013 sample 1 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385942	014 sample 2 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385943	021 sample 9 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385944	014 sample 2 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385945	026 sample 14 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385946	028 sample 16 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385947	026 sample 14 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385948	025 sample 13 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385949	024 sample 12 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385950	021 sample 9 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT Baseline
SA385951	024 sample 12 ACNH2O 1_1 AF M1_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT Baseline
SA385952	041 sample 29 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385953	040 sample 28 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385954	041 sample 29 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385955	030 sample 18 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385956	040 sample 28 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385957	037 sample 25 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385958	035 sample 23 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385959	034 sample 22 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385960	031 sample 19 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385961	029 sample 17 ACNH2O 1_1 AF M1_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385962	037 sample 25 M2_ MSE_res_neg IDC10_ 3ul inj	mouse serum	WT TAC 9W
SA385963	040 sample 28 PREM M2_ MSE_res_pos IDC10_ 3ul inj	mouse serum	WT TAC 9W

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Collection:

Collection ID:	CO003629
Collection Summary:	Metabolites from serum samples were extracted in microcentrifuge tubes after the addition of 3 volumes of 100% acetonitrile containing 3 internal standards [caffeine-(trimethyl-d9), succinic acid-2,2,3,3-d4 and N-acetyl-aspartic acid-2,3,3-d3] on one volume of serum. The samples were thoroughly mixed, sonicated and incubated at -20°C for 30 min, then centrifuged at 10,000g for 10 minutes at 4°C. The upper-phase was collected into a new microcentrifuge tube and 3 volumes of pure acetonitrile were added to the initial pellet for a second similar extraction. The combined upper-phases resulting from the two extractions were mixed and divided into four equal parts. Each part was dried down under a gentle stream of nitrogen at 30°C and kept frozen until analysis. Prior to analysis, the samples were reconstituted in [water:acetonitrile, 50:50 (v:v), 0,1% formic acid], [water:acetonitrile, 50:50 (v:v)], [water:acetonitrile, 25:75 (v:v), 0,1% formic acid] and [water:acetonitrile, 25:75 (v:v)] depending of the method used ,“M1 POS”, “M1 NEG”, “M2 POS” and “M2 NEG”, respectively. The tubes were centrifuged at 10 000g for 5 minutes at 4°C to remove any particulates before being transferred into polypropylene autosampler vials.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR003645
Treatment Summary:	Metabolites from serum samples were extracted in microcentrifuge tubes after the addition of 3 volumes of 100% acetonitrile containing 3 internal standards [caffeine-(trimethyl-d9), succinic acid-2,2,3,3-d4 and N-acetyl-aspartic acid-2,3,3-d3] on one volume of serum. The samples were thoroughly mixed, sonicated and incubated at -20°C for 30 min, then centrifuged at 10,000g for 10 minutes at 4°C. The upper-phase was collected into a new microcentrifuge tube and 3 volumes of pure acetonitrile were added to the initial pellet for a second similar extraction. The combined upper-phases resulting from the two extractions were mixed and divided into four equal parts. Each part was dried down under a gentle stream of nitrogen at 30°C and kept frozen until analysis. Prior to analysis, the samples were reconstituted in [water:acetonitrile, 50:50 (v:v), 0,1% formic acid], [water:acetonitrile, 50:50 (v:v)], [water:acetonitrile, 25:75 (v:v), 0,1% formic acid] and [water:acetonitrile, 25:75 (v:v)] depending of the method used ,“M1 POS”, “M1 NEG”, “M2 POS” and “M2 NEG”, respectively. The tubes were centrifuged at 10 000g for 5 minutes at 4°C to remove any particulates before being transferred into polypropylene autosampler vials. L

Treatment ID:

TR003645

Treatment Summary:

Metabolites from serum samples were extracted in microcentrifuge tubes after the addition of 3 volumes of 100% acetonitrile containing 3 internal standards [caffeine-(trimethyl-d9), succinic acid-2,2,3,3-d4 and N-acetyl-aspartic acid-2,3,3-d3] on one volume of serum. The samples were thoroughly mixed, sonicated and incubated at -20°C for 30 min, then centrifuged at 10,000g for 10 minutes at 4°C. The upper-phase was collected into a new microcentrifuge tube and 3 volumes of pure acetonitrile were added to the initial pellet for a second similar extraction. The combined upper-phases resulting from the two extractions were mixed and divided into four equal parts. Each part was dried down under a gentle stream of nitrogen at 30°C and kept frozen until analysis. Prior to analysis, the samples were reconstituted in [water:acetonitrile, 50:50 (v:v), 0,1% formic acid], [water:acetonitrile, 50:50 (v:v)], [water:acetonitrile, 25:75 (v:v), 0,1% formic acid] and [water:acetonitrile, 25:75 (v:v)] depending of the method used ,“M1 POS”, “M1 NEG”, “M2 POS” and “M2 NEG”, respectively. The tubes were centrifuged at 10 000g for 5 minutes at 4°C to remove any particulates before being transferred into polypropylene autosampler vials. L

Sample Preparation:

Sampleprep ID:	SP003643
Sampleprep Summary:	Metabolites from serum samples were extracted in microcentrifuge tubes after the addition of 3 volumes of 100% acetonitrile containing 3 internal standards [caffeine-(trimethyl-d9), succinic acid-2,2,3,3-d4 and N-acetyl-aspartic acid-2,3,3-d3] on one volume of serum. The samples were thoroughly mixed, sonicated and incubated at -20°C for 30 min, then centrifuged at 10,000g for 10 minutes at 4°C. The upper-phase was collected into a new microcentrifuge tube and 3 volumes of pure acetonitrile were added to the initial pellet for a second similar extraction. The combined upper-phases resulting from the two extractions were mixed and divided into four equal parts. Each part was dried down under a gentle stream of nitrogen at 30°C and kept frozen until analysis. Prior to analysis, the samples were reconstituted in [water:acetonitrile, 50:50 (v:v), 0,1% formic acid], [water:acetonitrile, 50:50 (v:v)], [water:acetonitrile, 25:75 (v:v), 0,1% formic acid] and [water:acetonitrile, 25:75 (v:v)] depending of the method used ,“M1 POS”, “M1 NEG”, “M2 POS” and “M2 NEG”, respectively. The tubes were centrifuged at 10 000g for 5 minutes at 4°C to remove any particulates before being transferred into polypropylene autosampler vials. L

Sampleprep ID:

SP003643

Sampleprep Summary:

Combined analysis:

Analysis ID	AN005757	AN005758	AN005759	AN005760
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Waters Acquity Premier	Waters Acquity Premier	Waters Acquity Premier	Waters Acquity Premier
Column	Waters Acquity Premier HSS T3 (100 x 2.1 mm, 1.8um)	Waters Acquity Premier HSS T3 (100 x 2.1 mm, 1.8um)	Waters Acquity Premier BEH amide (100 x 2.1 mm, 1.7um)	Waters Acquity Premier BEH amide (100 x 2.1 mm, 1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF	QTOF
MS instrument name	Waters Synapt-XS	Waters Synapt-XS	Waters Synapt-XS	Waters Synapt-XS
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	arbitrary unit	arbitrary unit	arbitrary unit	arbitrary unit

Chromatography:

Chromatography ID:	CH004369
Chromatography Summary:	M1POS
Instrument Name:	Waters Acquity Premier
Column Name:	Waters Acquity Premier HSS T3 (100 x 2.1 mm, 1.8um)
Column Temperature:	40°C
Flow Gradient:	1 min 99%A, 85% over 2 min, 50% over 3min, 5% over 3 min
Flow Rate:	0.5 mL/min
Solvent A:	100% Water; 0.1% Formic acid
Solvent B:	100% Acetonitrile; 0.1% Formic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH004370
Chromatography Summary:	M1NEG
Instrument Name:	Waters Acquity Premier
Column Name:	Waters Acquity Premier HSS T3 (100 x 2.1 mm, 1.8um)
Column Temperature:	40°C
Flow Gradient:	1 min 99%A, 85% over 2 min, 50% over 3min, 5% over 3 min
Flow Rate:	0.5 mL/min
Solvent A:	100% Water; 0.1% Acetic acid
Solvent B:	100% Acetonitrile; 0.1% Acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH004371
Chromatography Summary:	M2POS
Instrument Name:	Waters Acquity Premier
Column Name:	Waters Acquity Premier BEH amide (100 x 2.1 mm, 1.7um)
Column Temperature:	40°C
Flow Gradient:	0.2 min 0%A, 20% over 8.3 min, 40% over 1 min
Flow Rate:	0.7 mL/min
Solvent A:	93% Water/7% Acetonitrile; 10mM Ammonium formate pH3
Solvent B:	7% Water/93% Acetonitrile; 10mM Ammonium formate pH3
Chromatography Type:	HILIC

Chromatography ID:	CH004372
Chromatography Summary:	M2NEG
Instrument Name:	Waters Acquity Premier
Column Name:	Waters Acquity Premier BEH amide (100 x 2.1 mm, 1.7um)
Column Temperature:	40°C
Flow Gradient:	0.2 min 0%A, 20% over 8.3 min, 40% over 1 min
Flow Rate:	0.7 mL/min
Solvent A:	93% Water/7% Acetonitrile; 10mM Ammonium formate pH9
Solvent B:	7% Water/93% Acetonitrile; 10mM Ammonium formate pH9
Chromatography Type:	HILIC

MS:

MS ID:	MS005479
Analysis ID:	AN005757
Instrument Name:	Waters Synapt-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Acquisition: Masslynx The separation columns, operated at 40°C, were an Acquity Premier HSS T3 column 1,8 µm, 2,1 x 100 mm (Waters) and an Acquity Premier BEH Amide 1,8 µm, 2,1 x 100 mm (Waters), for reverse phase and HILIC chromatography, respectively. Mass spectrometry data was acquired in profile (continuum) format over the mass range of m/z 50-1200 using a Waters Synapt XS high resolution Q-TOF mass spectrometer set in MSE resolution mode (scan time 0,1 sec, the low and high trap collision energy were 4V and a ramp between 20 and 50 V, respectively). A dual electrospray ionization (ESI) source was used in positive or negative mode. Capillary and sampling cone, voltages were set to 1 kV and 30 V or 1 kV and 25 V in positive and negative mode, respectively. Source temperature was set to 150 °C and desolvation temperature to 600 °C (550°C in negative ESI). Gas flow rates were set at 1200 L/h for the desolvation gas (1100 L/h in negative ESI) and 50 L/h for the cone gas. Acquisition of leucine enkephalin infused at 10ul/min through a lockspray probe allowed a real time mass correction (30 sec scan intervals). Data processing: Raw LC-MS data were imported and processed in Progenesis QI software (Nonlinear Dynamics), which performed chromatogram alignment, peak picking, ion deconvolution, normalization, peak annotation and statistical analysis. Thresholds of area under the curve > 1000 were set and yielded the following number of detected metabolites: for Control WT vs Control TG (M1 neg : 1886 ; M1 pos : 3881 ; M2 neg : 785 ; M2 pos : 2411) TAC 9W WT vs Control TG (M1 neg : 2022 ; M1 pos : 3690 ; M2 neg : 905 ; M2 pos : 2405) and Control WT vs TAC WT (M1 neg : 2165 ; M1 pos : 4068 ; M2 neg : 944 ; M2 pos : 2448). Volcano plot and FDR threshold of significance were performed on Graphpad Prism.
Ion Mode:	POSITIVE

MS ID:	MS005480
Analysis ID:	AN005758
Instrument Name:	Waters Synapt-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Acquisition: Masslynx The separation columns, operated at 40°C, were an Acquity Premier HSS T3 column 1,8 µm, 2,1 x 100 mm (Waters) and an Acquity Premier BEH Amide 1,8 µm, 2,1 x 100 mm (Waters), for reverse phase and HILIC chromatography, respectively. Mass spectrometry data was acquired in profile (continuum) format over the mass range of m/z 50-1200 using a Waters Synapt XS high resolution Q-TOF mass spectrometer set in MSE resolution mode (scan time 0,1 sec, the low and high trap collision energy were 4V and a ramp between 20 and 50 V, respectively). A dual electrospray ionization (ESI) source was used in positive or negative mode. Capillary and sampling cone, voltages were set to 1 kV and 30 V or 1 kV and 25 V in positive and negative mode, respectively. Source temperature was set to 150 °C and desolvation temperature to 600 °C (550°C in negative ESI). Gas flow rates were set at 1200 L/h for the desolvation gas (1100 L/h in negative ESI) and 50 L/h for the cone gas. Acquisition of leucine enkephalin infused at 10ul/min through a lockspray probe allowed a real time mass correction (30 sec scan intervals). Data processing: Raw LC-MS data were imported and processed in Progenesis QI software (Nonlinear Dynamics), which performed chromatogram alignment, peak picking, ion deconvolution, normalization, peak annotation and statistical analysis. Thresholds of area under the curve > 1000 were set and yielded the following number of detected metabolites: for Control WT vs Control TG (M1 neg : 1886 ; M1 pos : 3881 ; M2 neg : 785 ; M2 pos : 2411) TAC 9W WT vs Control TG (M1 neg : 2022 ; M1 pos : 3690 ; M2 neg : 905 ; M2 pos : 2405) and Control WT vs TAC WT (M1 neg : 2165 ; M1 pos : 4068 ; M2 neg : 944 ; M2 pos : 2448). Volcano plot and FDR threshold of significance were performed on Graphpad Prism.
Ion Mode:	NEGATIVE

MS ID:	MS005481
Analysis ID:	AN005759
Instrument Name:	Waters Synapt-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Acquisition: Masslynx The separation columns, operated at 40°C, were an Acquity Premier HSS T3 column 1,8 µm, 2,1 x 100 mm (Waters) and an Acquity Premier BEH Amide 1,8 µm, 2,1 x 100 mm (Waters), for reverse phase and HILIC chromatography, respectively. Mass spectrometry data was acquired in profile (continuum) format over the mass range of m/z 50-1200 using a Waters Synapt XS high resolution Q-TOF mass spectrometer set in MSE resolution mode (scan time 0,1 sec, the low and high trap collision energy were 4V and a ramp between 20 and 50 V, respectively). A dual electrospray ionization (ESI) source was used in positive or negative mode. Capillary and sampling cone, voltages were set to 1 kV and 30 V or 1 kV and 25 V in positive and negative mode, respectively. Source temperature was set to 150 °C and desolvation temperature to 600 °C (550°C in negative ESI). Gas flow rates were set at 1200 L/h for the desolvation gas (1100 L/h in negative ESI) and 50 L/h for the cone gas. Acquisition of leucine enkephalin infused at 10ul/min through a lockspray probe allowed a real time mass correction (30 sec scan intervals). Data processing: Raw LC-MS data were imported and processed in Progenesis QI software (Nonlinear Dynamics), which performed chromatogram alignment, peak picking, ion deconvolution, normalization, peak annotation and statistical analysis. Thresholds of area under the curve > 1000 were set and yielded the following number of detected metabolites: for Control WT vs Control TG (M1 neg : 1886 ; M1 pos : 3881 ; M2 neg : 785 ; M2 pos : 2411) TAC 9W WT vs Control TG (M1 neg : 2022 ; M1 pos : 3690 ; M2 neg : 905 ; M2 pos : 2405) and Control WT vs TAC WT (M1 neg : 2165 ; M1 pos : 4068 ; M2 neg : 944 ; M2 pos : 2448). Volcano plot and FDR threshold of significance were performed on Graphpad Prism.
Ion Mode:	POSITIVE

MS ID:	MS005482
Analysis ID:	AN005760
Instrument Name:	Waters Synapt-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Acquisition: Masslynx The separation columns, operated at 40°C, were an Acquity Premier HSS T3 column 1,8 µm, 2,1 x 100 mm (Waters) and an Acquity Premier BEH Amide 1,8 µm, 2,1 x 100 mm (Waters), for reverse phase and HILIC chromatography, respectively. Mass spectrometry data was acquired in profile (continuum) format over the mass range of m/z 50-1200 using a Waters Synapt XS high resolution Q-TOF mass spectrometer set in MSE resolution mode (scan time 0,1 sec, the low and high trap collision energy were 4V and a ramp between 20 and 50 V, respectively). A dual electrospray ionization (ESI) source was used in positive or negative mode. Capillary and sampling cone, voltages were set to 1 kV and 30 V or 1 kV and 25 V in positive and negative mode, respectively. Source temperature was set to 150 °C and desolvation temperature to 600 °C (550°C in negative ESI). Gas flow rates were set at 1200 L/h for the desolvation gas (1100 L/h in negative ESI) and 50 L/h for the cone gas. Acquisition of leucine enkephalin infused at 10ul/min through a lockspray probe allowed a real time mass correction (30 sec scan intervals). Data processing: Raw LC-MS data were imported and processed in Progenesis QI software (Nonlinear Dynamics), which performed chromatogram alignment, peak picking, ion deconvolution, normalization, peak annotation and statistical analysis. Thresholds of area under the curve > 1000 were set and yielded the following number of detected metabolites: for Control WT vs Control TG (M1 neg : 1886 ; M1 pos : 3881 ; M2 neg : 785 ; M2 pos : 2411) TAC 9W WT vs Control TG (M1 neg : 2022 ; M1 pos : 3690 ; M2 neg : 905 ; M2 pos : 2405) and Control WT vs TAC WT (M1 neg : 2165 ; M1 pos : 4068 ; M2 neg : 944 ; M2 pos : 2448). Volcano plot and FDR threshold of significance were performed on Graphpad Prism.
Ion Mode:	NEGATIVE