Summary of Study ST004494

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	ST004494
Study Title	Metabolic Flux Analysis of Glycolysis and the Pentose Phosphate Pathway Using U-¹³C-Glucose Stable Isotope Tracing in Wild-type and sgTIGAR HT-29 Cl.16E Cells
Study Summary	To investigate the mechanisms underlying glucose metabolism reprogramming upon TIGAR depletion, we performed stable isotope tracing using U ¹³C glucose. In HT29 CL.16E cells incubated with U ¹³C glucose, TIGAR knockout resulted in reduced carbon flux through the pentose phosphate pathway (PPP) and enhanced flux through glycolysis. Specifically, TIGAR-deficient cells exhibited increased fractional enrichment of ¹³C-labeled intermediates in glycolysis—including glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), 2-phosphoglycerate (2-PG), pyruvate (PYR), and lactate—compared to controls. Conversely, the fractional enrichment of ¹³C-labeled 6-phosphogluconate (6-PG) and ribose-5-phosphate (R5P), key metabolites in the PPP, was markedly decreased. Consistent with this, the isotope exchange rate for 6-PG was significantly lower in TIGAR-deficient cells than in control cells. Although total lactate levels were elevated in TIGAR-deficient cells, the isotope exchange rate of lactate was also increased, further supporting enhanced glycolytic activity in the absence of TIGAR.
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	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:	SU004671
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
SA534325	C13-1H-KO-1	TIGAR-knockout	13C Glc	1H	HT29 CL.16E cells
SA534326	C13-1H-KO-2	TIGAR-knockout	13C Glc	1H	HT29 CL.16E cells
SA534327	C13-1H-KO-3	TIGAR-knockout	13C Glc	1H	HT29 CL.16E cells
SA534328	C13-1H-KO-4	TIGAR-knockout	13C Glc	1H	HT29 CL.16E cells
SA534329	C13-24H-KO-4	TIGAR-knockout	13C Glc	24H	HT29 CL.16E cells
SA534330	C13-24H-KO-3	TIGAR-knockout	13C Glc	24H	HT29 CL.16E cells
SA534331	C13-24H-KO-2	TIGAR-knockout	13C Glc	24H	HT29 CL.16E cells
SA534332	C13-24H-KO-1	TIGAR-knockout	13C Glc	24H	HT29 CL.16E cells
SA534333	C13-2H-KO-1	TIGAR-knockout	13C Glc	2H	HT29 CL.16E cells
SA534334	C13-2H-KO-2	TIGAR-knockout	13C Glc	2H	HT29 CL.16E cells
SA534335	C13-2H-KO-3	TIGAR-knockout	13C Glc	2H	HT29 CL.16E cells
SA534336	C13-2H-KO-4	TIGAR-knockout	13C Glc	2H	HT29 CL.16E cells
SA534337	C13-4H-KO-2	TIGAR-knockout	13C Glc	4H	HT29 CL.16E cells
SA534338	C13-4H-KO-4	TIGAR-knockout	13C Glc	4H	HT29 CL.16E cells
SA534339	C13-4H-KO-3	TIGAR-knockout	13C Glc	4H	HT29 CL.16E cells
SA534340	C13-4H-KO-1	TIGAR-knockout	13C Glc	4H	HT29 CL.16E cells
SA534341	C13-8H-KO-2	TIGAR-knockout	13C Glc	8H	HT29 CL.16E cells
SA534342	C13-8H-KO-1	TIGAR-knockout	13C Glc	8H	HT29 CL.16E cells
SA534343	C13-8H-KO-4	TIGAR-knockout	13C Glc	8H	HT29 CL.16E cells
SA534344	C13-8H-KO-3	TIGAR-knockout	13C Glc	8H	HT29 CL.16E cells
SA534345	C13-1H-Cont-3	Wild-type	13C Glc	1H	HT29 CL.16E cells
SA534346	C13-1H-Cont-4	Wild-type	13C Glc	1H	HT29 CL.16E cells
SA534347	C13-1H-Cont-1	Wild-type	13C Glc	1H	HT29 CL.16E cells
SA534348	C13-1H-Cont-2	Wild-type	13C Glc	1H	HT29 CL.16E cells
SA534349	C13-24H-Cont-1	Wild-type	13C Glc	24H	HT29 CL.16E cells
SA534350	C13-24H-Cont-3	Wild-type	13C Glc	24H	HT29 CL.16E cells
SA534351	C13-24H-Cont-4	Wild-type	13C Glc	24H	HT29 CL.16E cells
SA534352	C13-24H-Cont-2	Wild-type	13C Glc	24H	HT29 CL.16E cells
SA534353	C13-2H-Cont-3	Wild-type	13C Glc	2H	HT29 CL.16E cells
SA534354	C13-2H-Cont-1	Wild-type	13C Glc	2H	HT29 CL.16E cells
SA534355	C13-2H-Cont-4	Wild-type	13C Glc	2H	HT29 CL.16E cells
SA534356	C13-2H-Cont-2	Wild-type	13C Glc	2H	HT29 CL.16E cells
SA534357	C13-4H-Cont-4	Wild-type	13C Glc	4H	HT29 CL.16E cells
SA534358	C13-4H-Cont-2	Wild-type	13C Glc	4H	HT29 CL.16E cells
SA534359	C13-4H-Cont-1	Wild-type	13C Glc	4H	HT29 CL.16E cells
SA534360	C13-4H-Cont-3	Wild-type	13C Glc	4H	HT29 CL.16E cells
SA534361	C13-8H-Cont-2	Wild-type	13C Glc	8H	HT29 CL.16E cells
SA534362	C13-8H-Cont-4	Wild-type	13C Glc	8H	HT29 CL.16E cells
SA534363	C13-8H-Cont-3	Wild-type	13C Glc	8H	HT29 CL.16E cells
SA534364	C13-8H-Cont-1	Wild-type	13C Glc	8H	HT29 CL.16E cells

Showing results 1 to 40 of 40

Showing results 1 to 40 of 40

Collection:

Collection ID:	CO004664
Collection Summary:	Sodium butyrate was utilized to differentiate HT29 cells into mature goblet cells, thereby obtaining mucus-secreting HT29 CL.16E cells. Briefly, Human colon cancer cell line HT29 (ATCC, HTB-38) was cultured in a 5 mmol/L sodium butyrate solution for 9 days. Subsequently, the cells were passaged and maintained in a medium supplemented with sodium butyrate for 14 days to generate the HT-29 Cl.16E cell line. Over time, these cells differentiate into goblet cell phenotypes characterized by their ability to produce substantial amounts of mucus. The HT-29 Cl.16E cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. The culture medium consisted of DMEM supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, and 1 mM sodium pyruvate.
Sample Type:	Tumor cells

Treatment:

Treatment ID:	TR004680
Treatment Summary:	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 HT-29 Cl.16E cells with polybrene. sample (wild-type or sgTIGAR HT-29 Cl.16E; n = 3 biological replicates per group) were seeded in 6-cm plates and cultured in sodium bicarbonate–free DMEM supplemented with 10% dialyzed FBS, 2 mM L-glutamine, and 5 mM glucose. For functional assays, 2000 000 cells (WT or sgTIGAR HT-29 Cl.16E 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.

Treatment ID:

TR004680

Treatment Summary:

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 HT-29 Cl.16E cells with polybrene. sample (wild-type or sgTIGAR HT-29 Cl.16E; n = 3 biological replicates per group) were seeded in 6-cm plates and cultured in sodium bicarbonate–free DMEM supplemented with 10% dialyzed FBS, 2 mM L-glutamine, and 5 mM glucose. For functional assays, 2000 000 cells (WT or sgTIGAR HT-29 Cl.16E 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:	SP004677
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 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:

SP004677

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 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	AN007536
Chromatography ID	CH005718
MS ID	MS007233
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:	CH005718
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:	MS007233
Analysis ID:	AN007536
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