{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002564","ANALYSIS_ID":"AN004225","VERSION":"1","CREATED_ON":"April 18, 2023, 4:51 pm"}, "PROJECT":{"PROJECT_TITLE":"Metabolomic profiling of PMM2-CDG after siRNA mediated KD of AKR1b1 and neuraminidase treatment","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. 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","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 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","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":"5-45","GENDER":"Male and female"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"P2", "Sample ID":"SR01", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 13C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR01"} }, { "Subject ID":"P2", "Sample ID":"SR02", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 12C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR02"} }, { "Subject ID":"P2", "Sample ID":"SR03", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 13C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR03"} }, { "Subject ID":"P2", "Sample ID":"SR04", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 12C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR04"} }, { "Subject ID":"P5", "Sample ID":"SR05", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 13C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR05"} }, { "Subject ID":"P5", "Sample ID":"SR06", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 12C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR06"} }, { "Subject ID":"P5", "Sample ID":"SR07", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 13C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR07"} }, { "Subject ID":"P5", "Sample ID":"SR08", "Factors":{"Genotype":"PMM2-CDG","Treatment":"5.5mM 12C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR08"} }, { "Subject ID":"C3", "Sample ID":"SR09", "Factors":{"Genotype":"WT","Treatment":"5.5mM 13C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR09"} }, { "Subject ID":"C3", "Sample ID":"SR10", "Factors":{"Genotype":"WT","Treatment":"5.5mM 12C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR10"} }, { "Subject ID":"C3", "Sample ID":"SR11", "Factors":{"Genotype":"WT","Treatment":"5.5mM 13C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR11"} }, { "Subject ID":"C3", "Sample ID":"SR12", "Factors":{"Genotype":"WT","Treatment":"5.5mM 12C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR12"} }, { "Subject ID":"C2", "Sample ID":"SR13", "Factors":{"Genotype":"WT","Treatment":"5.5mM 13C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR13"} }, { "Subject ID":"C2", "Sample ID":"SR14", "Factors":{"Genotype":"WT","Treatment":"5.5mM 12C GLU negative siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR14"} }, { "Subject ID":"C2", "Sample ID":"SR15", "Factors":{"Genotype":"WT","Treatment":"5.5mM 13C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR15"} }, { "Subject ID":"C2", "Sample ID":"SR16", "Factors":{"Genotype":"WT","Treatment":"5.5mM 12C GLU siRNA"}, "Additional sample data":{"Organism":"Homo sapiens","Tissue type":"Fibroblasts","RAW_FILE_NAME":"SR16"} } ], "COLLECTION":{"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_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."}, "SAMPLEPREP":{"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℃"}, "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; Sialic acid was annotated using the inhouse standard metabolite library- elution time and m/z values."}, "MS_METABOLITE_DATA":{ "Units":"AUC", "Data":[{"Metabolite":"Sialic acid","SR01":"1.68E+08","SR02":"1.57E+08","SR03":"1.40E+08","SR04":"1.38E+08","SR05":"1.65E+08","SR06":"1.94E+08","SR07":"2.42E+08","SR08":"2.88E+08","SR09":"2.28E+08","SR10":"1.68E+08","SR11":"2.65E+08","SR12":"3.21E+08","SR13":"1.48E+08","SR14":"1.73E+08","SR15":"1.24E+08","SR16":"1.71E+08"}], "Metabolites":[{"Metabolite":"Sialic acid","parent formula":"C11H19NO9","M/Z":"308.09845","HMDB ID":"HMDB00230","isotopeLabel":"C12 PARENT","isotope count":"m0-m11"}] } }