{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002565","ANALYSIS_ID":"AN004226","VERSION":"1","CREATED_ON":"April 18, 2023, 5:08 pm"},

"PROJECT":{"PROJECT_TITLE":"Metabolomic profiling of PMM2-CDG after fructose tracer experiments","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. To investigate the influence of fructose on polyol abundance, and glucose flux we performed 13C6 glucose and fructose study in patient and healthy control fibroblasts.","INSTITUTE":"Mayo Clinic","LAST_NAME":"Radenkovic","FIRST_NAME":"Silvia","ADDRESS":"200 2nd Ave SW Rochester MN","EMAIL":"radenkovic.silvia@mayo.edu","PHONE":"507(77) 6-6107","FUNDING_SOURCE":"NIH, KU Leuven"},

"STUDY":{"STUDY_TITLE":"Metabolomic profiling of PMM2-CDG after siRNA mediated KD of AKR1b1 - 13C6 glucose and fructose study","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.","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"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","GENOTYPE_STRAIN":"WT/PMM2-CDG","AGE_OR_AGE_RANGE":"40-45","GENDER":"Female"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"P2",
"Sample ID":"SR01",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"13C glu + 12C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR01"}
},
{
"Subject ID":"P2",
"Sample ID":"SR02",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"13C glu + 12C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR02"}
},
{
"Subject ID":"P2",
"Sample ID":"SR03",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR03"}
},
{
"Subject ID":"P2",
"Sample ID":"SR04",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR04"}
},
{
"Subject ID":"P2",
"Sample ID":"SR05",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"12C fructose+13C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR05"}
},
{
"Subject ID":"P2",
"Sample ID":"SR06",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"12C fructose+13C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR06"}
},
{
"Subject ID":"P2",
"Sample ID":"SR07",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR07"}
},
{
"Subject ID":"P2",
"Sample ID":"SR08",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR08"}
},
{
"Subject ID":"P2",
"Sample ID":"SR09",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 13C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR09"}
},
{
"Subject ID":"P2",
"Sample ID":"SR10",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 13C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR10"}
},
{
"Subject ID":"P2",
"Sample ID":"SR11",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM glucose +"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR11"}
},
{
"Subject ID":"P2",
"Sample ID":"SR12",
"Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM glucose +"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR12"}
},
{
"Subject ID":"C3",
"Sample ID":"SR13",
"Factors":{"Genotype":"WT","Treatment":"13C glu + 12C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR13"}
},
{
"Subject ID":"C3",
"Sample ID":"SR14",
"Factors":{"Genotype":"WT","Treatment":"13C glu + 12C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR14"}
},
{
"Subject ID":"C3",
"Sample ID":"SR15",
"Factors":{"Genotype":"WT","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR15"}
},
{
"Subject ID":"C3",
"Sample ID":"SR16",
"Factors":{"Genotype":"WT","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR16"}
},
{
"Subject ID":"C3",
"Sample ID":"SR17",
"Factors":{"Genotype":"WT","Treatment":"12C fructose+13C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR17"}
},
{
"Subject ID":"C3",
"Sample ID":"SR18",
"Factors":{"Genotype":"WT","Treatment":"12C fructose+13C fructose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR18"}
},
{
"Subject ID":"C3",
"Sample ID":"SR19",
"Factors":{"Genotype":"WT","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR19"}
},
{
"Subject ID":"C3",
"Sample ID":"SR20",
"Factors":{"Genotype":"WT","Treatment":"12C fructose + 12C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR20"}
},
{
"Subject ID":"C3",
"Sample ID":"SR21",
"Factors":{"Genotype":"WT","Treatment":"5.5mM glucose +"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR21"}
},
{
"Subject ID":"C3",
"Sample ID":"SR22",
"Factors":{"Genotype":"WT","Treatment":"5.5mM glucose +"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR22"}
},
{
"Subject ID":"C3",
"Sample ID":"SR23",
"Factors":{"Genotype":"WT","Treatment":"5.5mM 13C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR23"}
},
{
"Subject ID":"C3",
"Sample ID":"SR24",
"Factors":{"Genotype":"WT","Treatment":"5.5mM 13C glucose"},
"Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR24"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cells were washed with PBS, cells were incubated with extraction buffer for 2min before scraping and transferring to a fresh eppendorf. Samples were precipitated overnight at -80, then they were centrifuged at max rpm, 20min, 4 degrees C and supernatant transferred to an M/S vial.","SAMPLE_TYPE":"Fibroblasts","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cells were treated with 12C/13C glucose +/- 12C/13C fructose"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Metabolites were extracted with 80% Methanol and IS (d27 myristic acid)","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"C18 iP REVERSE PHASE","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um)","SOLVENT_A":"100% water; 10mM tributylamine; 15mM acetic acid","SOLVENT_B":"100% methanol","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","COLUMN_TEMPERATURE":"40"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"El-Maven polly, ThermoFisher Xcalibur; Metabolites were annotated using the inhouse standard metabolite library- elution time and m/z values."},

"MS_METABOLITE_DATA":{
"Units":"AUC",

"Data":[{"Metabolite":"Sorbitol","SR01":"9.11E+06","SR02":"6.44E+06","SR03":"1.53E+07","SR04":"1.26E+07","SR05":"9.77E+06","SR06":"9.28E+06","SR07":"1.62E+07","SR08":"1.50E+07","SR09":"1.12E+07","SR10":"1.02E+07","SR11":"1.03E+07","SR12":"9.66E+06","SR13":"6.51E+06","SR14":"6.59E+06","SR15":"9.50E+06","SR16":"9.70E+06","SR17":"8.00E+06","SR18":"6.70E+06","SR19":"7.77E+06","SR20":"6.93E+06","SR21":"9.42E+06","SR22":"9.56E+06","SR23":"7.11E+06","SR24":"6.48E+06"},{"Metabolite":"D-Glucose","SR01":"9.32E+08","SR02":"1.10E+09","SR03":"1.34E+09","SR04":"1.49E+09","SR05":"8.00E+08","SR06":"9.57E+08","SR07":"1.40E+09","SR08":"1.23E+09","SR09":"9.30E+08","SR10":"8.82E+08","SR11":"1.08E+09","SR12":"1.26E+09","SR13":"8.12E+08","SR14":"8.71E+08","SR15":"1.01E+09","SR16":"1.00E+09","SR17":"9.78E+08","SR18":"9.53E+08","SR19":"1.36E+09","SR20":"1.19E+09","SR21":"9.87E+08","SR22":"1.09E+09","SR23":"9.68E+08","SR24":"1.16E+09"}],

"Metabolites":[{"Metabolite":"Sorbitol","parent formula":"C6H14O6","M/Z":"181.07164","HMDB ID":"HMDB00247","isotopeLabel":"C12 PARENT","isotope count":"m0-6"},{"Metabolite":"D-Glucose","parent formula":"C6H12O6","M/Z":"179.056061","HMDB ID":"HMDB00122","isotopeLabel":"C12 PARENT","isotope count":"m0-6"}]
}

}