Summary of Study ST004490
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 | ST004490 |
| Study Title | Comparative Untargeted Metabolomics of TIGAR-Knockout and Control HIEC-6 cells |
| Study Summary | In a previous untargeted metabolomics study ID6876, we observed significant accumulation of 6-phosphogluconate (6-PG) and elevated lactate levels in TIGAR-knockout HT-29 cells. To determine whether this metabolic phenotype is conserved across other cell types, we generated stable TIGAR-knockout HIEC-6 cells using CRISPR/Cas9 technology. A double-stranded oligonucleotide targeting the TIGAR gene was cloned into the lentiCRISPRv2 vector and co-transfected with packaging plasmids into HEK293T cells. Viral supernatant collected at 48 hours post-transfection was used to infect HIEC-6 cells in the presence of polybrene. Following selection, wild-type (WT) and TIGAR-knockout (sgTIGAR) HIEC-6 cells (100,000 cells per sample; n = 6 biological replicates per group) were cultured in sodium bicarbonate–free DMEM supplemented with 10% dialyzed FBS, 2 mM L-glutamine, and 5 mM glucose in 6-cm plates. Metabolite analysis at 24 hours post-treatment confirmed that both 6-PG and lactate levels were markedly increased in TIGAR-deficient HIEC-6 cells, recapitulating the metabolic alterations observed in HT-29 cells. These findings suggest that TIGAR plays a conserved role in regulating glycolytic and pentose phosphate pathway flux across distinct intestinal epithelial cell models. |
| Institute | Southwest Hospital, Third Military Medical University |
| Last Name | Su |
| First Name | Sen |
| Address | Gaotanyan Street 30, Shapingba District, Chongqing, Chongqing, 400038, China |
| 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 |
Select appropriate tab below to view additional metadata details:
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: | SU004667 |
| 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 | Sample source |
|---|---|---|---|
| SA534251 | KO1 | TIGAR-knockout | HIEC-6 cells |
| SA534252 | KO2 | TIGAR-knockout | HIEC-6 cells |
| SA534253 | KO3 | TIGAR-knockout | HIEC-6 cells |
| SA534254 | KO4 | TIGAR-knockout | HIEC-6 cells |
| SA534255 | KO5 | TIGAR-knockout | HIEC-6 cells |
| SA534256 | KO6 | TIGAR-knockout | HIEC-6 cells |
| SA534257 | Cont1 | Wild-type | HIEC-6 cells |
| SA534258 | Cont2 | Wild-type | HIEC-6 cells |
| SA534259 | Cont3 | Wild-type | HIEC-6 cells |
| SA534260 | Cont4 | Wild-type | HIEC-6 cells |
| SA534261 | Cont5 | Wild-type | HIEC-6 cells |
| SA534262 | Cont6 | Wild-type | HIEC-6 cells |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO004660 |
| 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. The shRNA sequence was cloned into the pLKO.1-Puro vector and co-transfected with packaging plasmids to construct stable knockdown cell lines. The viral supernatant was collected 48 hours post-transfection to infect cells, and infected cells were selected using puromycin for 7 days. |
| Collection Protocol Filename: | collection_protocol_-HIEC6.txt |
| Sample Type: | Intestine |
Treatment:
| Treatment ID: | TR004676 |
| Treatment Summary: | No treatment |
Sample Preparation:
| Sampleprep ID: | SP004673 |
| 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 | AN007532 |
|---|---|
| Chromatography ID | CH005714 |
| MS ID | MS007229 |
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | Waters BEH Amide HILIC column (2.1 × 150 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: | CH005714 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters BEH Amide HILIC column (2.1 × 150 mm, 1.7 μm) |
| Column Temperature: | 45°C |
| Flow Gradient: | 0 -0.1 min: 99% B; 0.1-10 min: 99% B-30% B; 10-10.5 min: 30% B-99% B; 10.5-15 min: 99% B |
| Flow Rate: | 0.4 mL/min |
| Solvent A: | 5% Acetonitrile/95% water; 10 mM ammonium bicarbonate (pH 9) |
| Solvent B: | 95% Acetonitrile/5% water; 10 mM ammonium bicarbonate (pH 9) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS007229 |
| Analysis ID: | AN007532 |
| 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 six 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 |
| Analysis Protocol File: | MS-Mehods-20240608.txt methods2-20240608_.txt |
