Summary of Study ST004492

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 PR002830. The data can be accessed directly via it's Project DOI: 10.21228/M8VG27 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004492
Study Title	Metabolic Flux Analysis of Glycolysis and the Pentose Phosphate Pathway Using U-¹³C-Glucose Stable Isotope Tracing in WT type and TIGAR-knockout HIEC-6 cells
Study Summary	To investigate the mechanisms underlying glucose metabolism reprogramming upon TIGAR depletion, we performed stable isotope tracing using U ¹³C glucose. In HIEC-6 cells incubated with U ¹³C glucose, TIGAR knockout resulted in reduced carbon flux through the pentose phosphate pathway (PPP) and enhanced flux through glycolysis.
Institute	Southwest Hospital, Third Military Medical University
Last Name	Su
First Name	Sen
Address	Gaotanyan Street 30, Shapingba District, Chongqing, Chongqing, 400038, China
Email	1441suse@163.com
Phone	0086015023351789
Submit Date	2025-12-08
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2026-01-02
Release Version	1

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Project:

Project ID:	PR002830
Project DOI:	doi: 10.21228/M8VG27
Project Title:	TIGAR Regulates Intestinal Mucus Barrier Integrity by Inhibiting Lactylation of G6PD/6PGD in Ulcerative Colitis
Project Summary:	Oxidative stress and metabolic dysregulation in goblet cells represent significant contributors to the pathogenesis of ulcerative colitis (UC). TIGAR (TP53-induced glycolysis and apoptosis regulator) plays a critical role as a metabolic regulatory enzyme by promoting NADPH synthesis, thereby counteracting oxidative stress. However, the precise mechanisms through which TIGAR regulates NADPH synthesis and its impact on UC remain incompletely understood. Here, we demonstrate that TIGAR inhibits the lactylation of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), both pivotal enzymes in the NADPH biosynthesis pathway, hence preserving their enzymatic activities. We further identify specific lactylation sites at lysine 432 (K432) in G6PD and lysine 38 (K38) in 6PGD. Lactylation modifications impact the formation of G6PD homodimers and the binding of 6PGD with NADP+. In male UC mice, persistently low TIGAR expression results in elevated lactic acid levels, which enhance the lactylation of G6PD and 6PGD, inhibit NADPH synthesis, and exacerbate oxidative stress in goblet cells. Consequently, these alterations lead to a reduction in thioredoxin 1 (Trx1) reductase activity, inducing S-nitrosylation of anterior gradient homolog 2 (AGR2), a key enzyme involved in MUC2 modification, thus impeding mature MUC2 production and compromising the integrity of the intestinal mucus barrier. Overall, our study elucidates the critical mechanisms by which TIGAR regulates NADPH synthesis, provides novel insights into how TIGAR maintains cellular redox homeostasis, and offers experimental evidence for considering TIGAR as a potential target for UC therapy.
Institute:	Southwest Hospital, Third Military Medical University
Department:	Clinical Medical Research Center
Laboratory:	Clinical Medical Research Center
Last Name:	Su
First Name:	Sen
Address:	Gaotanyan Street 30, Shapingba District, Chongqing, Chongqing, 400038, China
Email:	1441suse@163.com
Phone:	0086015023551789

Subject:

Subject ID:	SU004669
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype	Treatment	Time	Sample source
SA534279	C13-KO-1H-1	TIGAR-knockout	13C Glc	1H	HIEC6 cells
SA534280	C13-KO-1H-2	TIGAR-knockout	13C Glc	1H	HIEC6 cells
SA534281	C13-KO-1H-3	TIGAR-knockout	13C Glc	1H	HIEC6 cells
SA534282	C13-KO-1H-4	TIGAR-knockout	13C Glc	1H	HIEC6 cells
SA534283	C13-KO-24H-4	TIGAR-knockout	13C Glc	24H	HIEC6 cells
SA534284	C13-KO-24H-3	TIGAR-knockout	13C Glc	24H	HIEC6 cells
SA534285	C13-KO-24H-2	TIGAR-knockout	13C Glc	24H	HIEC6 cells
SA534286	C13-KO-24H-1	TIGAR-knockout	13C Glc	24H	HIEC6 cells
SA534287	C13-KO-2H-1	TIGAR-knockout	13C Glc	2H	HIEC6 cells
SA534288	C13-KO-2H-2	TIGAR-knockout	13C Glc	2H	HIEC6 cells
SA534289	C13-KO-2H-3	TIGAR-knockout	13C Glc	2H	HIEC6 cells
SA534290	C13-KO-2H-4	TIGAR-knockout	13C Glc	2H	HIEC6 cells
SA534291	C13-KO-4H-2	TIGAR-knockout	13C Glc	4H	HIEC6 cells
SA534292	C13-KO-4H-4	TIGAR-knockout	13C Glc	4H	HIEC6 cells
SA534293	C13-KO-4H-3	TIGAR-knockout	13C Glc	4H	HIEC6 cells
SA534294	C13-KO-4H-1	TIGAR-knockout	13C Glc	4H	HIEC6 cells
SA534295	C13-KO-8H-2	TIGAR-knockout	13C Glc	8H	HIEC6 cells
SA534296	C13-KO-8H-1	TIGAR-knockout	13C Glc	8H	HIEC6 cells
SA534297	C13-KO-8H-4	TIGAR-knockout	13C Glc	8H	HIEC6 cells
SA534298	C13-KO-8H-3	TIGAR-knockout	13C Glc	8H	HIEC6 cells
SA534299	C13-Cont-1H-3	Wild-type	13C Glc	1H	HIEC6 cells
SA534300	C13-Cont-1H-4	Wild-type	13C Glc	1H	HIEC6 cells
SA534301	C13-Cont-1H-1	Wild-type	13C Glc	1H	HIEC6 cells
SA534302	C13-Cont-1H-2	Wild-type	13C Glc	1H	HIEC6 cells
SA534303	C13-Cont-24H-1	Wild-type	13C Glc	24H	HIEC6 cells
SA534304	C13-Cont-24H-3	Wild-type	13C Glc	24H	HIEC6 cells
SA534305	C13-Cont-24H-4	Wild-type	13C Glc	24H	HIEC6 cells
SA534306	C13-Cont-24H-2	Wild-type	13C Glc	24H	HIEC6 cells
SA534307	C13-Cont-2H-3	Wild-type	13C Glc	2H	HIEC6 cells
SA534308	C13-Cont-2H-1	Wild-type	13C Glc	2H	HIEC6 cells
SA534309	C13-Cont-2H-4	Wild-type	13C Glc	2H	HIEC6 cells
SA534310	C13-Cont-2H-2	Wild-type	13C Glc	2H	HIEC6 cells
SA534311	C13-Cont-4H-4	Wild-type	13C Glc	4H	HIEC6 cells
SA534312	C13-Cont-4H-2	Wild-type	13C Glc	4H	HIEC6 cells
SA534313	C13-Cont-4H-1	Wild-type	13C Glc	4H	HIEC6 cells
SA534314	C13-Cont-4H-3	Wild-type	13C Glc	4H	HIEC6 cells
SA534315	C13-Cont-8H-2	Wild-type	13C Glc	8H	HIEC6 cells
SA534316	C13-Cont-8H-4	Wild-type	13C Glc	8H	HIEC6 cells
SA534317	C13-Cont-8H-3	Wild-type	13C Glc	8H	HIEC6 cells
SA534318	C13-Cont-8H-1	Wild-type	13C Glc	8H	HIEC6 cells

Showing results 1 to 40 of 40

Showing results 1 to 40 of 40

Collection:

Collection ID:	CO004662
Collection Summary:	The HIEC-6 cell line was purchased from ATCC and cultured in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 5% FBS (Gibco, Carlsbad, CA, USA), 10 mM HEPES, 5 mg/ml EGF, 100 U/ml penicillin/streptomycin, 4 mM Glutamax at 37℃ in a 5% CO2 incubator. Stable knockout cell lines of TIGAR were generated utilizing CRISPR/Cas9 technology. The double-stranded oligonucleotide complementary to the target sequence was cloned into the lentiCRISPRv2 vector and co-transfected with the packaging plasmid into HEK293 cells. Subsequently, a 48h viral supernatant was collected to infect HIEC-6 cells with polybrene. For functional assays, 1000 000 cells (WT or sgTIGAR HIEC-6 cells; n = 4 biological replicates per group) were seeded in 6-cm culture plates in sodium bicarbonate-free DMEM medium supplemented with 10% dialyzed FBS, 2 mM L‐glutamine and 10 mM U‐13C‐Glucose. Samples were collected at 1, 2, 4, 8, and 24 hours post-treatment.
Sample Type:	Colon

Treatment:

Treatment ID:	TR004678
Treatment Summary:	1000 000 cells (WT or sgTIGAR HIEC-6 cells; n = 4 biological replicates per group) were seeded in 6-cm culture plates in sodium bicarbonate-free DMEM medium supplemented with 10% dialyzed FBS, 2 mM L‐glutamine and 10 mM U‐13C‐Glucose. Samples were collected at 1, 2, 4, 8, and 24 hours post-treatment.

Sample Preparation:

Sampleprep ID:	SP004675
Sampleprep Summary:	For cellular samples, 1 mL of pre-chilled (at -80°C) 80% (v/v) methanol of MS grade was added to the culture dish, and cell debris was scraped off using a spatula. The cells were disrupted using a low-temperature ultrasonic disrupter for 60 seconds at -20°C, followed by a pause of 30 seconds, with this cycle repeated three times. The resulting homogenates from cells and colons were transferred and incubated at -20°C for 60 minutes. Subsequently, samples were centrifuged at 18,410 x g for 10 minutes at 4°C. The supernatant was collected after centrifugation, dried under a nitrogen gas, reconstituted, and centrifuged again to obtain the supernatant for subsequent analysis. A 20 μL aliquot from each sample was combined to prepare a quality control (QC) mixture for mass spectrometry analyses.

Sampleprep ID:

SP004675

Sampleprep Summary:

For cellular samples, 1 mL of pre-chilled (at -80°C) 80% (v/v) methanol of MS grade was added to the culture dish, and cell debris was scraped off using a spatula. The cells were disrupted using a low-temperature ultrasonic disrupter for 60 seconds at -20°C, followed by a pause of 30 seconds, with this cycle repeated three times. The resulting homogenates from cells and colons were transferred and incubated at -20°C for 60 minutes. Subsequently, samples were centrifuged at 18,410 x g for 10 minutes at 4°C. The supernatant was collected after centrifugation, dried under a nitrogen gas, reconstituted, and centrifuged again to obtain the supernatant for subsequent analysis. A 20 μL aliquot from each sample was combined to prepare a quality control (QC) mixture for mass spectrometry analyses.

Combined analysis:

Analysis ID	AN007534
Chromatography ID	CH005716
MS ID	MS007231
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Waters Atlantis Premier BEH Z-HILIC Column (2.1 × 100 mm, 1.7 μm)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Orbitrap Exploris 120
Ion Mode	NEGATIVE
Units	peak area

Chromatography:

Chromatography ID:	CH005716
Instrument Name:	Thermo Vanquish
Column Name:	Waters Atlantis Premier BEH Z-HILIC Column (2.1 × 100 mm, 1.7 μm)
Column Temperature:	30°C
Flow Gradient:	0-5 min: 90% B-65% B; 5-6 min: 65% B-65% B; 6 -8 min: 65% B-90% B; 8-10 min: 90% B
Flow Rate:	0.5 mL/min
Solvent A:	5% ACN/95% water; 15 mM ammonium acetate (pH 9)
Solvent B:	95% ACN/5% water; 15 mM ammonium acetate (pH 9)
Chromatography Type:	HILIC

MS:

MS ID:	MS007231
Analysis ID:	AN007534
Instrument Name:	Thermo Orbitrap Exploris 120
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The MS parameters for detection were: ESI source voltage 3.0 kV in negative ion scanning mode with scan range of m/z 70-1050 for mass spectrometry scanning, full scanning resolution of 120000, and MS2 scanning resolution of 60000, the top four precursor ions were broken up with the higher energy collisional dissociation cell in MS2 set to 30% normalized collision energy. the sheath gas was set at 60 arbitrary units, the auxiliary gas was set at 20 arbitrary units, and the blow air flow rate of 2Arb; the ion transport tube was set to 380°C; the vaporizer temperature of the ion source was set to 350°C. Software tools Xcalibur 4.3 (Thermo Fisher Scientific) and Compound Discover 3.3 (Thermo Fisher Scientific) were used for data processing and analyzing. Retention time alignment across all samples was achieved using the ChromeAlign node, with a pooled quality control (QC) sample serving as the reference. The pooled QC sample was prepared by combining equal aliquots from all biological samples. It was injected repeatedly—once every 10 analytical runs—to monitor instrument stability, enable batch effect correction, and support data normalization. Putative metabolite features were detected and grouped across all samples using the following key parameters (all other settings remained at default values): minimum peak intensity threshold of 1 × 10⁵ (area under the curve); mass tolerance of 5 ppm; retention time tolerance of 0.25 min; ionization modes limited to [M – H]⁻; and peak rating filter set to 4. Missing values were imputed using the built-in “Fill Gap” function, configured with a mass tolerance of 5 ppm and a signal-to-noise ratio threshold of 1.5. Metabolite identification was carried out through a tiered annotation strategy. Matching of both accurate mass (±5 ppm) and retention time (±0.5 min) to an in-house spectral library generated from authentic commercial standards, or spectral matching against the mzCloud database (https://www.mzcloud.org/) and mzVault database using MS/MS fragmentation data, with precursor and fragment mass tolerances of 10 ppm and a minimum match factor of 50.
Ion Mode:	NEGATIVE