Summary of Study ST002564
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 PR001653. The data can be accessed directly via it's Project DOI: 10.21228/M81Q6Q 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 | ST002564 |
| Study Title | Metabolomic profiling of PMM2-CDG after siRNA mediated KD of AKR1b1 and neuraminidase treatment |
| Study Summary | Abnormal polyol metabolism has been predominantly associated with diabetes, where excess glucose is converted to sorbitol by aldose reductase (AR). Recently, abnormal polyol metabolism has also been implicated in phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG), and epalrestat, an AR inhibitor, proposed as a potential therapy for this disorder. Given that the PMM enzyme is not closely connected to polyol metabolism, and, unlike in diabetes, PMM2-CDG does not present with hyperglycemia in blood, the increased polyol production, and the therapeutic mechanism of epalrestat in PMM2-CDG remained largely elusive. PMM2-CDG is caused by deficiency of the PMM enzyme and results in a depletion of mannose-1-P and guanosine diphosphate mannose (GDP-mannose), which is essential for glycosylation. Here, we show that apart from glycosylation abnormalities, PMM2 deficiency also leads to changes in intracellular glucose flux, which results in an increase in intracellular polyols. Ssing tracer glucose studies, we demonstrate that AR inhibition diverts glucose flux away from polyol production towards the synthesis of sugar nucleotides, which results in increase in glucose flux towards glycans. |
| Institute | Mayo Clinic |
| Last Name | Radenkovic |
| First Name | Silvia |
| Address | 200 2nd Ave SW Rochester MN, USA |
| radenkovic.silvia@mayo.edu | |
| Phone | 507(77) 6-6107 |
| Submit Date | 2023-04-18 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-08-18 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001653 |
| Project DOI: | doi: 10.21228/M81Q6Q |
| Project Title: | Metabolomic profiling of PMM2-CDG zebrafish in presence and absence of epalrestat |
| Project Summary: | Abnormal polyol metabolism has been predominantly associated with diabetes, where excess glucose is converted to sorbitol by aldose reductase (AR). Recently, abnormal polyol metabolism has also been implicated in phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG), and epalrestat, an AR inhibitor, proposed as a potential therapy for this disorder. Given that the PMM enzyme is not closely connected to polyol metabolism, and, unlike in diabetes, PMM2-CDG does not present with hyperglycemia in blood, the increased polyol production, and the therapeutic mechanism of epalrestat in PMM2-CDG remained largely elusive. PMM2-CDG is caused by deficiency of the PMM enzyme and results in a depletion of mannose-1-P and guanosine diphosphate mannose (GDP-mannose), which is essential for glycosylation. Here, we show that apart from glycosylation abnormalities, PMM2 deficiency also leads to changes in intracellular glucose flux, which results in an increase in intracellular polyols. Targeting AR with epalrestat decreases polyol levels and increases GDP-mannose in vivo in pmm2 mutant zebrafish. |
| Institute: | Mayo Clinic |
| Last Name: | Radenkovic |
| First Name: | Silvia |
| Address: | 200 2nd Ave SW Rochester MN, USA |
| Email: | radenkovic.silvia@mayo.edu |
| Phone: | 507(77) 6-6107 |
| Funding Source: | NIH, KU Leuven |
| Publications: | Tracer metabolomics reveals the role of aldose reductase in glycosylation |
| Contributors: | Silvia Radenkovic, Anna N. Ligezka, Sneha S. Mokashi, Karen Driesen, Lynn Dukes-Rimsky, Graeme Preston, Luckio F. Owuocha, Leila Sabbagh, Jehan Mousa, Christina Lam, Andrew Edmondson, Austin Larson, Matthew Schultz, Pieter Vermeersch, David Cassiman, Peter Witters, Lesa J. Beamer, Tamas Kozicz, Heather Flanagan-Steet, Bart Ghesquière, Eva Morava |
Subject:
| Subject ID: | SU002665 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | WT/PMM2-CDG |
| Age Or Age Range: | 5-45 |
| Gender: | Male and female |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Treatment |
|---|---|---|---|
| SA257911 | SR02 | PMM2-CDG | 5.5mM 12C GLU negative siRNA |
| SA257912 | SR06 | PMM2-CDG | 5.5mM 12C GLU negative siRNA |
| SA257913 | SR04 | PMM2-CDG | 5.5mM 12C GLU siRNA |
| SA257914 | SR08 | PMM2-CDG | 5.5mM 12C GLU siRNA |
| SA257915 | SR01 | PMM2-CDG | 5.5mM 13C GLU negative siRNA |
| SA257916 | SR05 | PMM2-CDG | 5.5mM 13C GLU negative siRNA |
| SA257917 | SR07 | PMM2-CDG | 5.5mM 13C GLU siRNA |
| SA257918 | SR03 | PMM2-CDG | 5.5mM 13C GLU siRNA |
| SA257919 | SR10 | WT | 5.5mM 12C GLU negative siRNA |
| SA257920 | SR14 | WT | 5.5mM 12C GLU negative siRNA |
| SA257921 | SR16 | WT | 5.5mM 12C GLU siRNA |
| SA257922 | SR12 | WT | 5.5mM 12C GLU siRNA |
| SA257923 | SR09 | WT | 5.5mM 13C GLU negative siRNA |
| SA257924 | SR13 | WT | 5.5mM 13C GLU negative siRNA |
| SA257925 | SR11 | WT | 5.5mM 13C GLU siRNA |
| SA257926 | SR15 | WT | 5.5mM 13C GLU siRNA |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO002658 |
| Collection Summary: | Briefly, medium was removed from the cells and the cells were washed 3 times with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 (Invitrogen) was added to each sample and the samples were washed twice with 500 µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each sample, and samples were incubated, shaking overnight at 4 °C. Samples were put on a dynabead rack (Invitrogen) and the supernatant was transferred to a new Eppendorf tube and used for protein concentration assay. Beads containing membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each sample and samples were incubated overnight at 37 °C, shaking. Next, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS). Sialic acid was measured by LC/MS as described below. El Maven Polly software was used to annotate sialic acid based on m/z ratio and elution time, and determine fractional contribution of glucose in sialic acid. |
| Sample Type: | Fibroblasts |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002677 |
| Treatment Summary: | Cells were treated with vehicle or 5nM siRNA targeting AKR1B1, Before collection, medium was removed from the cells and the cells were washed 3 times with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 (Invitrogen) was added to each sample and the samples were washed twice with 500 µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each sample, and samples were incubated, shaking overnight at 4 °C. Samples were put on a dynabead rack (Invitrogen) and the supernatant was transferred to a new Eppendorf tube and used for protein concentration assay. Beads containing membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each sample and samples were incubated overnight at 37 °C, shaking. Next, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS). Sialic acid was measured by LC/MS as described below. El Maven Polly software was used to annotate sialic acid based on m/z ratio and elution time, and determine fractional contribution of glucose in sialic acid. |
Sample Preparation:
| Sampleprep ID: | SP002671 |
| Sampleprep Summary: | After treatment with neuraminidase, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, lyophilized pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS). |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN004225 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE |
| Units | AUC |
Chromatography:
| Chromatography ID: | CH003134 |
| Chromatography Summary: | C18 iP REVERSE PHASE |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) |
| Column Temperature: | 40 |
| Flow Gradient: | The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min. |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 100% water; 10mM tributylamine; 15mM acetic acid |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003972 |
| Analysis ID: | AN004225 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | El-Maven polly, ThermoFisher Xcalibur; Sialic acid was annotated using the inhouse standard metabolite library- elution time and m/z values. |
| Ion Mode: | NEGATIVE |
