Summary of Study ST003566

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 PR002200. The data can be accessed directly via it's Project DOI: 10.21228/M87Z47 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	ST003566
Study Title	Unraveling cysteine deficiency-associated rapid weight loss
Study Summary	Forty percent of the US population and 1 in 6 individuals worldwide are obese, with the incidence surging globally. Various dietary interventions, including carbohydrate, fat and more recently amino acid restriction, have been explored to combat this epidemic. We investigated the impact of removing individual amino acids on the weight profiles of mice. Here, we show that conditional cysteine restriction resulted in the most dramatic weight loss when compared to essential amino acid restriction, amounting to 30% within one week, which was readily reversed. We found that cysteine deficiency activated the integrated stress response and oxidative stress response, which amplify each other, leading to the induction of GDF15 and FGF21, which partly explained the phenotype. Surprisingly, we observed lower tissue coenzyme A (CoA), which has been considered to be extremely stable10, resulting in reduced mitochondrial functionality and metabolic rewiring. This results in energetically inefficient anaerobic glycolysis and defective TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds, and amino acids In summary, our investigation reveals that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings suggest novel strategies for addressing a range of metabolic diseases and the growing obesity crisis.
Institute	NYU Grossman School of Medicine
Last Name	Varghese
First Name	Alan
Address	550 First Avenue, NY, NY 10016
Email	Alan.Varghese@nyulangone.org
Phone	212-263-7520
Submit Date	2024-10-22
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-03-05
Release Version	1

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Project:

Project ID:	PR002200
Project DOI:	doi: 10.21228/M87Z47
Project Title:	Unraveling cysteine deficiency-associated rapid weight loss
Project Summary:	Forty percent of the US population and 1 in 6 individuals worldwide are obese, with the incidence surging globally. Various dietary interventions, including carbohydrate, fat and more recently amino acid restriction, have been explored to combat this epidemic. We investigated the impact of removing individual amino acids on the weight profiles of mice. Here, we show that conditional cysteine restriction resulted in the most dramatic weight loss when compared to essential amino acid restriction, amounting to 30% within one week, which was readily reversed. We found that cysteine deficiency activated the integrated stress response and oxidative stress response, which amplify each other, leading to the induction of GDF15 and FGF21, which partly explained the phenotype. Surprisingly, we observed lower tissue coenzyme A (CoA), which has been considered to be extremely stable10, resulting in reduced mitochondrial functionality and metabolic rewiring. This results in energetically inefficient anaerobic glycolysis and defective TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds, and amino acids In summary, our investigation reveals that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings suggest novel strategies for addressing a range of metabolic diseases and the growing obesity crisis.
Institute:	NYU Grossman School of Medicine
Last Name:	Varghese
First Name:	Alan
Address:	550 First Avenue, NY, NY 10016
Email:	Alan.Varghese@nyulangone.org
Phone:	212-263-7520

Subject:

Subject ID:	SU003903
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	Factor	Sample source
SA409642	SQ1615_Blank_0	Blank	blank
SA409643	SQ1441_Blank_2	Blank	blank
SA409644	SQ1441_Blank_3	Blank	blank
SA409645	SQ1441_Blank_4	Blank	blank
SA409646	SQ1441_Blank_5	Blank	blank
SA409647	SQ1441_Blank_6	Blank	blank
SA409648	SQ1441_Blank_7	Blank	blank
SA409649	SQ1441_Blank_8	Blank	blank
SA409650	SQ1441_Blank_9	Blank	blank
SA409651	SQ1432_Blank_1	Blank	blank
SA409652	SQ1432_Blank_2	Blank	blank
SA409653	SQ1432_Blank_3	Blank	blank
SA409654	SQ1432_Blank_4	Blank	blank
SA409655	SQ1432_Blank_5	Blank	blank
SA409656	SQ1615_Blank_1	Blank	blank
SA409657	SQ1441_Blank_0	Blank	blank
SA409658	SQ1615_Blank_2	Blank	blank
SA409659	SQ1615_Blank_3	Blank	blank
SA409660	SQ1615_Blank_4	Blank	blank
SA409661	SQ1615_Blank_5	Blank	blank
SA409662	SQ1615_Blank_6	Blank	blank
SA409663	SQ1615_Blank_7	Blank	blank
SA409664	SQ1616_Blank_0	Blank	blank
SA409665	SQ1616_Blank_1	Blank	blank
SA409666	SQ1616_Blank_2	Blank	blank
SA409667	SQ1616_Blank_3	Blank	blank
SA409668	SQ1616_Blank_4	Blank	blank
SA409669	SQ1616_Blank_5	Blank	blank
SA409670	SQ1616_Blank_6	Blank	blank
SA409671	SQ1616_Blank_7	Blank	blank
SA409672	SQ1441_Blank_1	Blank	blank
SA409673	SQ1432_Blank_0	Blank	blank
SA409674	SQ1403_Blank_2	Blank	blank
SA409675	SQ1403_Blank_6	Blank	blank
SA409676	SQ1403_Blank_5	Blank	blank
SA409677	SQ1403_Blank_4	Blank	blank
SA409678	SQ1403_Blank_3	Blank	blank
SA409679	SQ1403_Blank_0	Blank	blank
SA409680	SQ1403_Blank_7	Blank	blank
SA409681	SQ1403_Blank_1	Blank	blank
SA409682	F3	Food	food
SA409683	F1	Food	food
SA409684	F2	Food	food
SA409685	SQ1616_U5	Het 2 hr	urine
SA409686	SQ1616_U6	Het 2 hr	urine
SA409687	SQ1616_U9	Het 2 hr	urine
SA409688	SQ1616_U1	Het 2 hr	urine
SA409689	SQ1616_U2	Het 2 hr	urine
SA409690	SQ1616_U8	Het 2 hr	urine
SA409691	SQ1615_L6	Het 45 min	liver
SA409692	SQ1615_L5	Het 45 min	liver
SA409693	SQ1615_L2	Het 45 min	liver
SA409694	SQ1615_L1	Het 45 min	liver
SA409695	SQ1615_ISTD_0	ISTD	standard
SA409696	SQ1615_ISTD_1	ISTD	standard
SA409697	SQ1615_ISTD_2	ISTD	standard
SA409698	SQ1615_ISTD_3	ISTD	standard
SA409699	SQ1403_ISTD_0	ISTD	standard
SA409700	SQ1432_ISTD_1	ISTD	standard
SA409701	SQ1432_ISTD_2	ISTD	standard
SA409702	SQ1616_ISTD_3	ISTD	standard
SA409703	SQ1616_ISTD_2	ISTD	standard
SA409704	SQ1432_ISTD_0	ISTD	standard
SA409705	SQ1403_ISTD_1	ISTD	standard
SA409706	SQ1616_ISTD_1	ISTD	standard
SA409707	SQ1616_ISTD_0	ISTD	standard
SA409708	SQ1403_ISTD_2	ISTD	standard
SA409709	SQ1403_ISTD_3	ISTD	standard
SA409718	AMP KO3	Knockout	Stool
SA409719	AMP KO2	Knockout	Stool
SA409720	AMP KO1	Knockout	Stool
SA409710	SQ1616_U10	KO 2 hr	urine
SA409711	SQ1616_U3	KO 2 hr	urine
SA409712	SQ1616_U4	KO 2 hr	urine
SA409713	SQ1616_U7	KO 2 hr	urine
SA409714	SQ1615_L8	KO 45 min	liver
SA409715	SQ1615_L7	KO 45 min	liver
SA409716	SQ1615_L4	KO 45 min	liver
SA409717	SQ1615_L3	KO 45 min	liver
SA409721	SQ1432_L13	No Cys KO	liver
SA409722	SQ1432_L12	No Cys KO	liver
SA409723	SQ1432_L14	No Cys KO	liver
SA409724	SQ1441_U23	No Cys KO	urine
SA409725	SQ1441_U22	No Cys KO	urine
SA409726	SQ1441_U8	No Cys KO	urine
SA409727	SQ1441_U21	No Cys KO	urine
SA409728	SQ1441_U20	No Cys KO	urine
SA409729	SQ1441_U10	No Cys KO	urine
SA409730	SQ1441_U9	No Cys KO	urine
SA409731	SQ1432_L9	No Cys WT	liver
SA409732	SQ1432_L8	No Cys WT	liver
SA409733	SQ1432_L10	No Cys WT	liver
SA409734	SQ1432_L11	No Cys WT	liver
SA409735	SQ1441_U6	No Cys WT	urine
SA409736	SQ1441_U16	No Cys WT	urine
SA409737	SQ1441_U17	No Cys WT	urine
SA409738	SQ1441_U18	No Cys WT	urine
SA409739	SQ1441_U19	No Cys WT	urine
SA409740	SQ1441_U7	No Cys WT	urine
SA409741	AMP WT3	Wild Type	Stool

Showing page 1 of 2 Results: 1 2 Next Showing results 1 to 100 of 102

Collection:

Collection ID:	CO003896
Collection Summary:	Food pellets were weighed into bead blaster tubes containing zircon beads. Extraction buffer containing 80% methanol with 500nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA) was added to each to reach a final concentration of 10 mg/mL. Samples were homogenized using D2400 BeadBlaster homogenizer (Benchmark Scientific, NJ) then spun at 21 kg for 3min. Then, 450uL of metabolite extract was transferred to a new 1.5mL Eppendorf tube and dried using Speedvac. Samples were reconstituted in 50uL of MS grade water and sonicated for 2 mins. Then, samples were spun at 21,000 g for 3 mins. Samples were transferred to glass LC vials for analysis by LCMS. Liver Samples Approximately 300 mg of liver was homogenized in 1 mL of PBS and then subjected to three freeze-thaw cycles. Liver samples were filtered using a 10kDa filter. Protein concentrations prior to deproteinization were measured using BSA standard curve and found to be between 12.8 – 42.0 mg/mL. For metabolomics extracts, on average protein concentration was determined to be 28.58 mg/mL per 300 mg of tissue. This value was used to scale all liver extracts to each other. Scaled liver extracts were transferred to bead blaster tubes with zircon beads and homogenized using D2400 BeadBlaster homogenizer (Benchmark Scientific, NJ) in cold 80% methanol spiked with 500 nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA). Samples were centrifuged at 21,000 g for 3 min to pellet any insoluble materials. Then, 450 µL of metabolite extract was transferred to a new 1.5mL Eppendorf tube and dried down using Speedvac. Samples were reconstituted in 50uL of MS grade water and sonicated for 2 mins. Then, samples were spun at 21,000 g for 3 min. Samples were transferred to glass LC vials for analysis by LCMS. Urine Samples Urine samples were collected and stored as frozen aliquots between ~5-7 µL. For metabolite extraction, 5µL of urine was transferred to a glass insert and extracted using 195 µL of cold 80% methanol spiked with 500nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA). Glass inserts were transferred into 1.5 mL Eppendorf tubes and spun at 3 kg for 10 min to pellet insoluble material. Then, 180 µL of extract was transferred to a 1.5 mL Eppendorf tube and dried down completely using Speedvac. To each, 20 µL of MS grade water was added to reconstitute metabolites. Samples were sonicated for 2 mins and then centrifuged for 3 min at 21 kg. Then 15 µL of samples was transferred to a glass LCMS vial for analysis.
Sample Type:	urine, liver, food, stool

Treatment:

Treatment ID:	TR003912
Treatment Summary:	All custom diets were procured from TestDiet. All diets were based on the defined amino acid diet 5CC7. For individual or dual amino acid depleted diets, the specific amino acid(s) was completely removed, and all other amino acids were increased in proportion. For pantothenic acid-deficient diet, it was removed from the same 5CC7 defined diet. Unless specified control diet refers to the 5CC7 diet. D-Galactose (Sigma G0750) and D-Glucose (Sigma G8270) were purchased from Sigma and were dissolved in water and filter sterilized. A list of diet names is available on request.

Treatment ID:

TR003912

Treatment Summary:

All custom diets were procured from TestDiet. All diets were based on the defined amino acid diet 5CC7. For individual or dual amino acid depleted diets, the specific amino acid(s) was completely removed, and all other amino acids were increased in proportion. For pantothenic acid-deficient diet, it was removed from the same 5CC7 defined diet. Unless specified control diet refers to the 5CC7 diet. D-Galactose (Sigma G0750) and D-Glucose (Sigma G8270) were purchased from Sigma and were dissolved in water and filter sterilized. A list of diet names is available on request.

Sample Preparation:

Sampleprep ID:	SP003909
Sampleprep Summary:	Food pellets were weighed into bead blaster tubes containing zircon beads. Extraction buffer containing 80% methanol with 500nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA) was added to each to reach a final concentration of 10 mg/mL. Samples were homogenized using D2400 BeadBlaster homogenizer (Benchmark Scientific, NJ) then spun at 21 kg for 3min. Then, 450 µL of metabolite extract was transferred to a new 1.5 mL Eppendorf tube and dried using Speedvac. Samples were reconstituted in 50 µL of MS grade water and sonicated for 2 mins. Then, samples were spun at 21,000 g for 3 mins. Samples were transferred to glass LC vials for analysis by LCMS. Liver Samples Approximately 300 mg of liver was homogenized in 1 mL of PBS and then subjected to three freeze-thaw cycles. Liver samples were filtered using a 10 kDa filter. Protein concentrations prior to deproteinization were measured using BSA standard curve and found to be between 12.8 – 42.0 mg/mL. For metabolomics extracts, on average protein concentration was determined to be 28.58 mg/mL per 300 mg of tissue. This value was used to scale all liver extracts to each other. Scaled liver extracts were transferred to bead blaster tubes with zircon beads and homogenized using D2400 BeadBlaster homogenizer (Benchmark Scientific, NJ) in cold 80% methanol spiked with 500 nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA). Samples were centrifuged at 21,000 g for 3 min to pellet any insoluble materials. Then, 450 µL of metabolite extract was transferred to a new 1.5mL Eppendorf tube and dried down using Speedvac. Samples were reconstituted in 50uL of MS grade water and sonicated for 2 mins. Then, samples were spun at 21,000 g for 3 min. Samples were transferred to glass LC vials for analysis by LCMS. Urine Samples Urine samples were collected and stored as frozen aliquots between ~5-7 µL. For metabolite extraction, 5µL of urine was transferred to a glass insert and extracted using 195µL of cold 80% methanol spiked with 500nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA). Glass inserts were transferred into 1.5mL Eppendorf tubes and spun at 3 kg for 10 min to pellet insoluble material. Then, 180 µL of extract was transferred to a 1.5 mL Eppendorf tube and dried down completely using Speedvac. To each, 20 µL of MS grade water was added to reconstitute metabolites. Samples were sonicated for 2 mins and then centrifuged for 3 min at 21 kg. Then 15 µL of samples was transferred to a glass LCMS vial for analysis.

Sampleprep ID:

SP003909

Sampleprep Summary:

Food pellets were weighed into bead blaster tubes containing zircon beads. Extraction buffer containing 80% methanol with 500nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA) was added to each to reach a final concentration of 10 mg/mL. Samples were homogenized using D2400 BeadBlaster homogenizer (Benchmark Scientific, NJ) then spun at 21 kg for 3min. Then, 450 µL of metabolite extract was transferred to a new 1.5 mL Eppendorf tube and dried using Speedvac. Samples were reconstituted in 50 µL of MS grade water and sonicated for 2 mins. Then, samples were spun at 21,000 g for 3 mins. Samples were transferred to glass LC vials for analysis by LCMS. Liver Samples Approximately 300 mg of liver was homogenized in 1 mL of PBS and then subjected to three freeze-thaw cycles. Liver samples were filtered using a 10 kDa filter. Protein concentrations prior to deproteinization were measured using BSA standard curve and found to be between 12.8 – 42.0 mg/mL. For metabolomics extracts, on average protein concentration was determined to be 28.58 mg/mL per 300 mg of tissue. This value was used to scale all liver extracts to each other. Scaled liver extracts were transferred to bead blaster tubes with zircon beads and homogenized using D2400 BeadBlaster homogenizer (Benchmark Scientific, NJ) in cold 80% methanol spiked with 500 nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA). Samples were centrifuged at 21,000 g for 3 min to pellet any insoluble materials. Then, 450 µL of metabolite extract was transferred to a new 1.5mL Eppendorf tube and dried down using Speedvac. Samples were reconstituted in 50uL of MS grade water and sonicated for 2 mins. Then, samples were spun at 21,000 g for 3 min. Samples were transferred to glass LC vials for analysis by LCMS. Urine Samples Urine samples were collected and stored as frozen aliquots between ~5-7 µL. For metabolite extraction, 5µL of urine was transferred to a glass insert and extracted using 195µL of cold 80% methanol spiked with 500nM Metabolomics amino acid standard mix (Cambridge Isotopes Laboratory, MA). Glass inserts were transferred into 1.5mL Eppendorf tubes and spun at 3 kg for 10 min to pellet insoluble material. Then, 180 µL of extract was transferred to a 1.5 mL Eppendorf tube and dried down completely using Speedvac. To each, 20 µL of MS grade water was added to reconstitute metabolites. Samples were sonicated for 2 mins and then centrifuged for 3 min at 21 kg. Then 15 µL of samples was transferred to a glass LCMS vial for analysis.

Combined analysis:

Analysis ID	AN006190
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Ultimate 3000 RS
Column	SeQuant ZIC-HILIC (150 x 2.1mm,5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	absolute intensity

Chromatography:

Chromatography ID:	CH004697
Instrument Name:	Thermo Ultimate 3000 RS
Column Name:	SeQuant ZIC-HILIC (150 x 2.1mm,5um)
Column Temperature:	25
Flow Gradient:	80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min)
Flow Rate:	0.1 mL/min
Solvent A:	100% Water; 10 mM ammonium carbonate in water, pH 9.0
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS005894
Analysis ID:	AN006190
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	both positive and negative polarities were collected in the same acquisition during polarity switching mechanism
Ion Mode:	UNSPECIFIED