Summary of Study ST003920

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 PR002453. The data can be accessed directly via it's Project DOI: 10.21228/M8JZ5D 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.

Study ID	ST003920
Study Title	Mitochondrial control of fuel switching through de novo carnitine synthesis
Study Summary	Metabolic adaptation to fasting and cold exposure involves a dynamic fuel switch from glucose to fatty acid oxidation in peripheral organs, a process that depends on carnitine-mediated fatty acid oxidation in mitochondria. While dietary sources of animal origin (e.g., red meat) contribute to the carnitine pool, de novo synthesis from trimethyllysine (TML) is essential, particularly for those whose dietary sources are vegetables and fruits that contain negligible amounts of carnitine. However, the molecular pathway of de novo carnitine synthesis, in particular the mitochondrial transport step of TML, remains undefined. MBP-SLC25A45 in E. coli is used as a non-mammalian system to study trimethyllysine uptake. Here, we identify SLC25A45 as a previously uncharacterized mitochondrial TML carrier that is required for de novo carnitine biosynthesis. Genetic loss of SLC25A45 results in systemic carnitine and acylcarnitine deficiency, leading to impaired fatty acid oxidation and thermogenesis during cold adaptation. Unexpectedly, SLC25A45-deficient mice were somewhat resistant to the effects of a GLP1 receptor agonist (GLP-1RA) to lower food intake and maintained a high respiratory exchange ratio and low lipolysis following treatment with a GLP1 receptor agonist (GLP-1RA), rendering them resistant to GLP-1RA-induced adipose tissue loss. We further demonstrate a decrease in acylcarnitines via our lipidomic analysis, leading to decreased cold tolerance in vivo. Together, the present study uncovers mitochondrial metabolite transport as a key regulatory checkpoint in fuel switching during adaptation, with implications for systemic energy balance and response to GLP-1RA-mediated anti-obesity therapy.
Institute	BIDMC
Last Name	Christopher
First Name	Auger
Address	3 Blackfan Street, 02115, Boston, MA
Email	cauger1@bidmc.harvard.edu
Phone	8579282065
Submit Date	2025-05-03
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-12-15
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002453
Project DOI:	doi: 10.21228/M8JZ5D
Project Title:	Mitochondrial control of fuel switching through de novo carnitine synthesis
Project Summary:	Mitochondrial transport of trimethyllysine is the key first step in de novo biosynthesis of carnitine. In this study, we show that the uncharacterized transporter SLC25A45 mediates this step. Knockout of SLC25A45 leads to impaired fatty acid oxidation due to a decrease in carnitine. To demonstrate this, deuterated trimethyllysine (d9-TML) is traced in vitro (293 cells and primary hepatocytes) as well as subjected to mitochondrial uptake assays. MBP-SLC25A45 in E. coli is used as a non-mammalian system to study trimethyllysine uptake. We further demonstrate a decrease in acylcarnitines via our lipidomic analysis, leading to decreased cold tolerance in vivo.
Institute:	BIDMC
Last Name:	Auger
First Name:	Christopher
Address:	3 Blackfan Street, Boston, MA, 02115, USA
Email:	cauger1@bidmc.harvard.edu
Phone:	8579282065

Subject:

Subject ID:	SU004055
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	Sample type	Genotype	Treatment
SA443881	S60	293 cells	293 cells	Control	10 uM D9-TML
SA443882	S61	293 cells	293 cells	Control	10 uM D9-TML
SA443883	S62	293 cells	293 cells	Control	10 uM D9-TML
SA443884	S63	293 cells	293 cells	Control	10 uM D9-TML
SA443885	S64	293 cells	293 cells	Control	10 uM D9-TML
SA443886	S66	293 cells	293 cells	Overexpression	10 uM D9-TML
SA443887	S67	293 cells	293 cells	Overexpression	10 uM D9-TML
SA443888	S68	293 cells	293 cells	Overexpression	10 uM D9-TML
SA443889	S69	293 cells	293 cells	Overexpression	10 uM D9-TML
SA443890	S65	293 cells	293 cells	Overexpression	10 uM D9-TML
SA443873	2K1	293 Mito	293 Mito	KO	10 uM D9-TML
SA443874	2K2	293 Mito	293 Mito	KO	10 uM D9-TML
SA443875	2K3	293 Mito	293 Mito	KO	10 uM D9-TML
SA443876	2K4	293 Mito	293 Mito	KO	10 uM D9-TML
SA443877	2R1	293 Mito	293 Mito	Rescue	10 uM D9-TML
SA443878	2R4	293 Mito	293 Mito	Rescue	10 uM D9-TML
SA443879	2R3	293 Mito	293 Mito	Rescue	10 uM D9-TML
SA443880	2R2	293 Mito	293 Mito	Rescue	10 uM D9-TML
SA443891	pSamp52	Ecoli	Ecoli	Control	10 uM D9-TML
SA443892	pSamp43	Ecoli	Ecoli	Control	10 uM D9-TML
SA443893	pSamp44	Ecoli	Ecoli	Control	10 uM D9-TML
SA443894	pSamp49	Ecoli	Ecoli	Control	10 uM D9-TML
SA443895	pSamp50	Ecoli	Ecoli	Control	10 uM D9-TML
SA443896	pSamp51	Ecoli	Ecoli	Control	10 uM D9-TML
SA443897	pSamp59	Ecoli	Ecoli	Control	10 uM D9-TML
SA443898	pSamp57	Ecoli	Ecoli	Control	10 uM D9-TML
SA443899	pSamp58	Ecoli	Ecoli	Control	10 uM D9-TML
SA443900	pSamp60	Ecoli	Ecoli	Control	10 uM D9-TML
SA443901	pSamp65	Ecoli	Ecoli	Control	10 uM D9-TML
SA443902	pSamp66	Ecoli	Ecoli	Control	10 uM D9-TML
SA443903	pSamp41	Ecoli	Ecoli	Control	10 uM D9-TML
SA443904	pSamp42	Ecoli	Ecoli	Control	10 uM D9-TML
SA443905	pSamp67	Ecoli	Ecoli	Control	10 uM D9-TML
SA443906	pSamp73	Ecoli	Ecoli	Control	10 uM D9-TML
SA443907	pSamp74	Ecoli	Ecoli	Control	10 uM D9-TML
SA443908	pSamp75	Ecoli	Ecoli	Control	10 uM D9-TML
SA443909	pSamp76	Ecoli	Ecoli	Control	10 uM D9-TML
SA443910	S40	Ecoli	Ecoli	Control	10 uM D9-TML
SA443911	S41	Ecoli	Ecoli	Control	10 uM D9-TML
SA443912	S42	Ecoli	Ecoli	Control	10 uM D9-TML
SA443913	S43	Ecoli	Ecoli	Control	10 uM D9-TML
SA443914	S44	Ecoli	Ecoli	Control	10 uM D9-TML
SA443915	pSamp68	Ecoli	Ecoli	Control	10 uM D9-TML
SA443916	pSamp70	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443917	pSamp69	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443918	pSamp64	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443919	pSamp63	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443920	pSamp71	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443921	pSamp80	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443922	pSamp72	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443923	pSamp77	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443924	pSamp78	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443925	pSamp61	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443926	S45	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443927	S46	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443928	S47	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443929	S48	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443930	S49	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443931	pSamp62	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443932	pSamp79	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443933	pSamp56	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443934	pSamp54	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443935	pSamp53	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443936	pSamp48	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443937	pSamp47	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443938	pSamp46	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443939	pSamp45	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443940	pSamp55	Ecoli	Ecoli	Overexpression	10 uM D9-TML
SA443941	S18	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443942	S13	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443943	S10	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443944	S14	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443945	S17	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443946	S12	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443947	S15	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443948	S16	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443949	S11	Hepatocytes	Hepatocytes	Control	10 uM D9-TML
SA443950	S31	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443951	S28	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443952	S29	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443953	S32	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443954	S35	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443955	S30	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443956	S33	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443957	S36	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443958	S34	Hepatocytes	Hepatocytes	KO	10 uM D9-TML
SA443959	C1P	Ing Mito	Ing Mito	Cre	10 uM D9-TML
SA443960	C2P	Ing Mito	Ing Mito	Cre	10 uM D9-TML
SA443961	C3P	Ing Mito	Ing Mito	Cre	10 uM D9-TML
SA443962	C4P	Ing Mito	Ing Mito	Cre	10 uM D9-TML
SA443963	E4P	Ing Mito	Ing Mito	Ctl	10 uM D9-TML
SA443964	E3P	Ing Mito	Ing Mito	Ctl	10 uM D9-TML
SA443965	E2P	Ing Mito	Ing Mito	Ctl	10 uM D9-TML
SA443966	E1P	Ing Mito	Ing Mito	Ctl	10 uM D9-TML
SA443967	plasma_ctl7	Serum	Serum	Control	72h 6C
SA443968	C6	Serum	Serum	Control	72h 6C
SA443969	C5	Serum	Serum	Control	72h 6C
SA443970	C4	Serum	Serum	Control	72h 6C
SA443971	C3	Serum	Serum	Control	72h 6C
SA443972	C2	Serum	Serum	Control	72h 6C

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Collection:

Collection ID:	CO004048
Collection Summary:	Serum was taken at termination of study. 10 uL was mixed with metabolomics extraction solvent (40% MeOH, 40% acetonitrile). Cells were counted for whole cell-tracing studies and normalized to protein prior to extraction with metabolomics extraction solvent. Mitochondria were normalized to protein before extraction with metabolomics extraction solvent
Sample Type:	Mouse blood serum, 293 cells, primary hepatocytes, e.coli, inguinal cell mitochondria and 293 cell mitochondria
Collection Method:	10 uL of mouse serum mixed 90 uL extraction solvent - 23000g spin for 15 min; Whole cells on ice with extraction solvent for 10 minutes - 23000g spin for 15 min; 100 ug of isolated mitochondria mixed with 80 uL of extraction solvent - 23000g spin for 15 min; For E. coli, twenty OD600*mL units of bacteria transformed with empty vector or overexpressing MBP-hSLC25A45 were resuspended in 1 mL of potassium HEPES buffer. For timecourse assays, 10 µM D9-TML was added to the sample with shaking at room temperature and aliquots taken at 1, 5, 10, 20, and 60 minutes. Metabolites were extracted with ice-cold solvent

Treatment:

Treatment ID:	TR004064
Treatment Summary:	Human embryonic kidney 293 cells (45 KO, WT or 45 Rescue) were treated with 10 uM D9-TML for 4 h prior to metabolite extraction. Primary hepatocytes from SLC25A45 KO mice or littermate controls were also used for D9-TML tracing for various timepoints. Serum from SLC25A45 whole body KO, liver-specific KO (Slc25a45AlbKO) or littermate controls was used at study termination. Select mice were exposed to cold for 72h (at 6 degrees C) prior to termination. Isolated mitochondria from inguinal adipocytes (empty vector or pMSCV-cre to induce slc25a45 ko)or 293 cells were treated with 10 uM D9-TML for five minutes at room temperature, then washed prior to extraction for metabolomics

Treatment ID:

TR004064

Treatment Summary:

Human embryonic kidney 293 cells (45 KO, WT or 45 Rescue) were treated with 10 uM D9-TML for 4 h prior to metabolite extraction. Primary hepatocytes from SLC25A45 KO mice or littermate controls were also used for D9-TML tracing for various timepoints. Serum from SLC25A45 whole body KO, liver-specific KO (Slc25a45AlbKO) or littermate controls was used at study termination. Select mice were exposed to cold for 72h (at 6 degrees C) prior to termination. Isolated mitochondria from inguinal adipocytes (empty vector or pMSCV-cre to induce slc25a45 ko)or 293 cells were treated with 10 uM D9-TML for five minutes at room temperature, then washed prior to extraction for metabolomics

Sample Preparation:

Sampleprep ID:	SP004061
Sampleprep Summary:	For metabolomics, samples were extracted with 40% MeOH, 40% acetonitrile. For lipidomics, samples were extracted in butanol/methanol (1:1) with 5 mM ammonium formate.

Combined analysis:

Analysis ID	AN006436	AN006437
Chromatography ID	CH004884	CH004885
MS ID	MS006137	MS006138
Analysis type	MS	MS
Chromatography type	HILIC	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Waters XBridge BEH Amide XP (150 x 2.1mm, 2.5um)	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF-X Orbitrap	Thermo Orbitrap Exploris 480
Ion Mode	POSITIVE	POSITIVE
Units	Peak area	Peak area

Chromatography:

Chromatography ID:	CH004884
Chromatography Summary:	Metabolomics
Instrument Name:	Thermo Vanquish
Column Name:	Waters XBridge BEH Amide XP (150 x 2.1mm, 2.5um)
Column Temperature:	4
Flow Gradient:	0 - 3 min, 100% B; 3.2 - 6.2 min, 90% B; 6.5 - 10.5 min, 80% B; 10.7 13.5 min, 70% B; 13.7 - 16 min, 45% B; and 16.5 - 22 min, 100% B
Flow Rate:	0.3 mL/min
Solvent A:	95% Water:5% acetonitrile; 10 mM ammonium acetate; 10 mM ammonium hydroxide
Solvent B:	20% Water/80% acetonitrile; 10 mM ammonium acetate; 10 mM ammonium hydroxide
Chromatography Type:	HILIC

Chromatography ID:	CH004885
Chromatography Summary:	Lipidomics
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	4
Flow Gradient:	20% B from 0 to 3 min, 55% B at 7 min, 65% B at 15 min, 70% B at 21 min, 88% B at 23 min, 100% B at 24 min held until 26 min, and 20% B at 28 min and held until 30 min
Flow Rate:	0.35 mL/min
Solvent A:	Water:acetonitrile 40:60; 10 mM ammonium formate and 0.1% formic acid
Solvent B:	Isopropranol:acetonitrile 90:10; 10 mM ammonium formate and 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS006137
Analysis ID:	AN006436
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The mass spectrometry used was Q Exactive HF (Thermo Fisher Scientific, San Jose, CA), and scanned from 70 to 1000 m/z with switching polarity. The resolution was 120,000. Metabolites were identified based on accurate mass and retention time using an in-house library, and the isotopic labeling was analyzed by El-Maven.
Ion Mode:	POSITIVE

MS ID:	MS006138
Analysis ID:	AN006437
Instrument Name:	Thermo Orbitrap Exploris 480
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The ion source was H-ESI. Spray voltage was 3500 V for positive ions, and 2500 V for negative ions. Sheath gas was set at 50 arbitrary unit (Arb), auxiliary gas 15 Arb, sweep gas 1 Arb, ion transfer tube at 325 °C, and vaporizer 350 °C. The scan mode was data dependent (dd)-MS2, covering 150-1600 m/z in both positive and negative polarities. Precursor ion scan had resolution 60,000. For product ion scan, the resolution was 15,000, isolation width 1.0 m/z, and collision energy 25%. Thermo Scientific LipidSearch software version 5.0 was used for lipid identification and quantitation. First, the product search mode was used to identify lipids based on the exact mass of the precursor ions and the MS2 mass spectra of product ion scan. The precursor and product tolerance was 10 ppm. The absolute intensity threshold of precursor ions and the relative intensity threshold of product ions were set to 30000 and 1%, respectively. Next, the search results from all samples were aligned within a retention time tolerance of 0.25 min. The annotated lipids were then filtered to reduce false positives by only including the lipids with a total grade of A and B.
Ion Mode:	POSITIVE