Summary of Study ST002079

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 PR001319. The data can be accessed directly via it's Project DOI: 10.21228/M87125 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	ST002079
Study Title	Defining the mammalian coactivation of hepatic 12-hour clock and lipid metabolism
Study Summary	The 12-hour clock coordinates lipid homeostasis, energy metabolism and stress rhythms via the transcriptional regulator XBP1. However, the biochemical and physiological basis for integrated control of the 12-hour clock and diverse metabolic pathways remains unclear. Here, we show that steroid receptor coactivator SRC-3 coactivates XBP1 transcription and regulates hepatic 12-hour cistrome and gene rhythmicity. Mice lacking SRC-3 show abnormal 12-hour rhythms in hepatic transcription, metabolic functions, systemic energetics, and rate-limiting lipid metabolic processes including triglyceride, phospholipid and cardiolipin pathways. Notably, 12-hour clock coactivation is not only preserved, with its cistromic activation priming ahead of the zeitgeber cue of light, but concomitant with rhythmic remodeling in the absence of food. These findings reveal that SRC-3 integrates the mammalian 12-hour clock, energy metabolism, and membrane and lipid homeostasis, and demonstrates a role for the 12-hour clock machinery as an active transcriptional mechanism in anticipating physiological and metabolic energy needs and stresses.
Institute	Baylor College of Medicine
Last Name	Meng
First Name	Huan
Address	One Baylor Plaza BCM 130 Houston, TX 77030
Email	huanm@bcm.edu
Phone	5127729532
Submit Date	2022-02-04
Num Groups	12
Total Subjects	24
Num Males	24
Raw Data Available	Yes
Raw Data File Type(s)	wiff
Analysis Type Detail	Other
Release Date	2022-02-22
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002168
Sampleprep Summary:	Before MS analysis, the dried extract was resuspended in 100 ?L of Buffer B (10:5:85 Acetonitrile/water/Isopropyl alcohol) containing 10mM NH4OAc and subjected to LC/MS. The lipidome was separated using reverse-phase chromatography. High-performance Liquid Chromatography (LC) grade water, methanol, acetonitrile, dichloromethane, isopropanol from Fisher scientific were used per the manufacturer’s instructions. Mass spectrometry (MS) grade lipid standards from Avanti Polar Lipids (Alabaster, AL) and MS grade ammonium acetate from sigma Aldrich (St. Louis, MO) were used. For internal standards and quality controls, the lipid stock solution was prepared by weighing an exact amount of the lipid internal standards in Chloroform/Methanol/H2O, resulting in sample aliquots at concentration of 1mg/mL as stock solutions. The stock solutions were diluted to 100 pmol/?L by mixing an appropriate volume of the internal standards LPC 17:0/0:0, PG 17:0/17:0, PE 17:0/17:0, PC 17:0/17:0, TAG 17:0/17:0/17:0, SM 18:1/17:0, MAG 17:0, DAG 16:0/18:1, CE 17:0, ceramide d 18:1/17:0, PA 17:0, PI 17:0/20:4, and PS 17:0/17:0. To monitor instrument performance, 10 ?L of a dried matrix-free mixture of the internal standards was used, reconstituted in 100 ?L of buffer B (5% water, 85%Isopropanolol: 10%Acetonitrile in 10mM NH4OAc). To monitor the lipid extraction process, a standard pool representing the tissue sample aliquots was used.Shimadzu CTO-20A Nexera X2 UHPLC systems was used for data acquisition, equipped with a degasser, binary pump, thermostat-regulated auto sampler, and a column oven for chromatographic separation. For lipid separation, 5 uL of the lipid extract was injected to a 1.8 ?m particle 50 ? 2.1 mm Acquity HSS UPLC T3 column (Waters, Milford, MA). The column heater temperature was set at 55° C. For chromatographic elution, a linear gradient was used over a 20 min total run time, with 60% Solvent A (acetonitrile/water (40:60, v/v) with 10 mM ammonium acetate) and 40% Solvent B (acetonitrile/water/isopropanol (10:5:85 v/v) with 10 mM ammonium acetate) gradient in the first 10 minutes. The gradient was ramped in a linear fashion to 100% Solvent B for 7 minutes. Then the system was switched back to 60% Solvent B and 40% Solvent A for 3 minutes. A flow rate of 0.4 mL/min was used at an injection volume of 5?L. The column was equilibrated for 3 min and run at a flow rate of 0.4 mL/min for a total run time of 20 min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada).

Sampleprep ID:

SP002168

Sampleprep Summary:

Before MS analysis, the dried extract was resuspended in 100 ?L of Buffer B (10:5:85 Acetonitrile/water/Isopropyl alcohol) containing 10mM NH4OAc and subjected to LC/MS. The lipidome was separated using reverse-phase chromatography. High-performance Liquid Chromatography (LC) grade water, methanol, acetonitrile, dichloromethane, isopropanol from Fisher scientific were used per the manufacturer’s instructions. Mass spectrometry (MS) grade lipid standards from Avanti Polar Lipids (Alabaster, AL) and MS grade ammonium acetate from sigma Aldrich (St. Louis, MO) were used. For internal standards and quality controls, the lipid stock solution was prepared by weighing an exact amount of the lipid internal standards in Chloroform/Methanol/H2O, resulting in sample aliquots at concentration of 1mg/mL as stock solutions. The stock solutions were diluted to 100 pmol/?L by mixing an appropriate volume of the internal standards LPC 17:0/0:0, PG 17:0/17:0, PE 17:0/17:0, PC 17:0/17:0, TAG 17:0/17:0/17:0, SM 18:1/17:0, MAG 17:0, DAG 16:0/18:1, CE 17:0, ceramide d 18:1/17:0, PA 17:0, PI 17:0/20:4, and PS 17:0/17:0. To monitor instrument performance, 10 ?L of a dried matrix-free mixture of the internal standards was used, reconstituted in 100 ?L of buffer B (5% water, 85%Isopropanolol: 10%Acetonitrile in 10mM NH4OAc). To monitor the lipid extraction process, a standard pool representing the tissue sample aliquots was used.Shimadzu CTO-20A Nexera X2 UHPLC systems was used for data acquisition, equipped with a degasser, binary pump, thermostat-regulated auto sampler, and a column oven for chromatographic separation. For lipid separation, 5 uL of the lipid extract was injected to a 1.8 ?m particle 50 ? 2.1 mm Acquity HSS UPLC T3 column (Waters, Milford, MA). The column heater temperature was set at 55° C. For chromatographic elution, a linear gradient was used over a 20 min total run time, with 60% Solvent A (acetonitrile/water (40:60, v/v) with 10 mM ammonium acetate) and 40% Solvent B (acetonitrile/water/isopropanol (10:5:85 v/v) with 10 mM ammonium acetate) gradient in the first 10 minutes. The gradient was ramped in a linear fashion to 100% Solvent B for 7 minutes. Then the system was switched back to 60% Solvent B and 40% Solvent A for 3 minutes. A flow rate of 0.4 mL/min was used at an injection volume of 5?L. The column was equilibrated for 3 min and run at a flow rate of 0.4 mL/min for a total run time of 20 min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada).