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.

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Study IDST003566
Study TitleUnraveling cysteine deficiency-associated rapid weight loss
Study SummaryForty 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 NameVarghese
First NameAlan
Address550 First Avenue, NY, NY 10016
EmailAlan.Varghese@nyulangone.org
Phone212-263-7520
Submit Date2024-10-22
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2025-02-03
Release Version1
Alan Varghese Alan Varghese
https://dx.doi.org/10.21228/M87Z47
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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:SU003695
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Factor Sample source
SA389468SQ1615_Blank_0Blank blank
SA389469SQ1441_Blank_2Blank blank
SA389470SQ1441_Blank_3Blank blank
SA389471SQ1441_Blank_4Blank blank
SA389472SQ1441_Blank_5Blank blank
SA389473SQ1441_Blank_6Blank blank
SA389474SQ1441_Blank_7Blank blank
SA389475SQ1441_Blank_8Blank blank
SA389476SQ1441_Blank_9Blank blank
SA389477SQ1432_Blank_1Blank blank
SA389478SQ1432_Blank_2Blank blank
SA389479SQ1432_Blank_3Blank blank
SA389480SQ1432_Blank_4Blank blank
SA389481SQ1432_Blank_5Blank blank
SA389482SQ1615_Blank_1Blank blank
SA389483SQ1441_Blank_0Blank blank
SA389484SQ1615_Blank_2Blank blank
SA389485SQ1615_Blank_3Blank blank
SA389486SQ1615_Blank_4Blank blank
SA389487SQ1615_Blank_5Blank blank
SA389488SQ1615_Blank_6Blank blank
SA389489SQ1615_Blank_7Blank blank
SA389490SQ1616_Blank_0Blank blank
SA389491SQ1616_Blank_1Blank blank
SA389492SQ1616_Blank_2Blank blank
SA389493SQ1616_Blank_3Blank blank
SA389494SQ1616_Blank_4Blank blank
SA389495SQ1616_Blank_5Blank blank
SA389496SQ1616_Blank_6Blank blank
SA389497SQ1616_Blank_7Blank blank
SA389498SQ1441_Blank_1Blank blank
SA389499SQ1432_Blank_0Blank blank
SA389500SQ1403_Blank_2Blank blank
SA389501SQ1403_Blank_6Blank blank
SA389502SQ1403_Blank_5Blank blank
SA389503SQ1403_Blank_4Blank blank
SA389504SQ1403_Blank_3Blank blank
SA389505SQ1403_Blank_0Blank blank
SA389506SQ1403_Blank_7Blank blank
SA389507SQ1403_Blank_1Blank blank
SA389508F3Food food
SA389509F1Food food
SA389510F2Food food
SA389511SQ1616_U5Het 2 hr urine
SA389512SQ1616_U6Het 2 hr urine
SA389513SQ1616_U9Het 2 hr urine
SA389514SQ1616_U1Het 2 hr urine
SA389515SQ1616_U2Het 2 hr urine
SA389516SQ1616_U8Het 2 hr urine
SA389517SQ1615_L6Het 45 min liver
SA389518SQ1615_L5Het 45 min liver
SA389519SQ1615_L2Het 45 min liver
SA389520SQ1615_L1Het 45 min liver
SA389521SQ1615_ISTD_0ISTD standard
SA389522SQ1615_ISTD_1ISTD standard
SA389523SQ1615_ISTD_2ISTD standard
SA389524SQ1615_ISTD_3ISTD standard
SA389525SQ1403_ISTD_0ISTD standard
SA389526SQ1432_ISTD_1ISTD standard
SA389527SQ1432_ISTD_2ISTD standard
SA389528SQ1616_ISTD_3ISTD standard
SA389529SQ1616_ISTD_2ISTD standard
SA389530SQ1432_ISTD_0ISTD standard
SA389531SQ1403_ISTD_1ISTD standard
SA389532SQ1616_ISTD_1ISTD standard
SA389533SQ1616_ISTD_0ISTD standard
SA389534SQ1403_ISTD_2ISTD standard
SA389535SQ1403_ISTD_3ISTD standard
SA389544AMP KO3Knockout urine
SA389545AMP KO2Knockout urine
SA389546AMP KO1Knockout urine
SA389536SQ1616_U10KO 2 hr urine
SA389537SQ1616_U3KO 2 hr urine
SA389538SQ1616_U4KO 2 hr urine
SA389539SQ1616_U7KO 2 hr urine
SA389540SQ1615_L8KO 45 min liver
SA389541SQ1615_L7KO 45 min liver
SA389542SQ1615_L4KO 45 min liver
SA389543SQ1615_L3KO 45 min liver
SA389547SQ1432_L13No Cys KO liver
SA389548SQ1432_L12No Cys KO liver
SA389549SQ1432_L14No Cys KO liver
SA389550SQ1441_U23No Cys KO urine
SA389551SQ1441_U22No Cys KO urine
SA389552SQ1441_U8No Cys KO urine
SA389553SQ1441_U21No Cys KO urine
SA389554SQ1441_U20No Cys KO urine
SA389555SQ1441_U10No Cys KO urine
SA389556SQ1441_U9No Cys KO urine
SA389557SQ1432_L9No Cys WT liver
SA389558SQ1432_L8No Cys WT liver
SA389559SQ1432_L10No Cys WT liver
SA389560SQ1432_L11No Cys WT liver
SA389561SQ1441_U6No Cys WT urine
SA389562SQ1441_U16No Cys WT urine
SA389563SQ1441_U17No Cys WT urine
SA389564SQ1441_U18No Cys WT urine
SA389565SQ1441_U19No Cys WT urine
SA389566SQ1441_U7No Cys WT urine
SA389567AMP WT3Wild Type urine
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Collection:

Collection ID:CO003688
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

Treatment:

Treatment ID:TR003704
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:SP003702
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 AN005859
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:CH004450
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:MS005579
Analysis ID:AN005859
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
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