{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000964","ANALYSIS_ID":"AN001578","VERSION":"1","CREATED_ON":"April 30, 2018, 12:09 pm"},

"PROJECT":{"PROJECT_TITLE":"Integrated in vivo quantitative proteomics and nutrient tracing reveals age-related metabolic rewiring of pancreatic beta cell function","PROJECT_SUMMARY":"Aging is associated with fundamental changes in pancreatic β-cell physiology; yet, the mechanisms that drive these age-related changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from adolescent and old mice. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in aged islets, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides the first in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in β-cell function.","INSTITUTE":"University of California, San Diego","LAST_NAME":"Wortham","FIRST_NAME":"Matthew","ADDRESS":"2880 Torrey Pines Scenic Drive, Sanford Consortium for Regenerative Medicine, Room 3102, La Jolla, California, 92037, USA","EMAIL":"mwortham@ucsd.edu","PHONE":"8582460588"},

"STUDY":{"STUDY_TITLE":"U13C Glucose Tracing of Young and Old Pancreatic Islets","STUDY_SUMMARY":"Pancreatic islets from young and old mice were traced with either 2.8mM or 16.8mM U13C glucose to determine the effect of age upon glucose metabolism in basal and stimulated states.","INSTITUTE":"University of California, San Diego","LAST_NAME":"Wortham","FIRST_NAME":"Matthew","ADDRESS":"2880 Torrey Pines Scenic Drive, Sanford Consortium for Regenerative Medicine, Room 3102","EMAIL":"mwortham@ucsd.edu","PHONE":"8582460588"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"C57BL/6N","AGE_OR_AGE_RANGE":"4 weeks old (young) or 10-15 months old (old)","GENDER":"Male and female","ANIMAL_ANIMAL_SUPPLIER":"Charles River Labs","ANIMAL_FEED":"standard chow diet (PicoLab Rodent Diet 20 pellets, #5053"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YA_1",
"Factors":{"Age":"Young (4 weeks)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YB_2",
"Factors":{"Age":"Young (4 weeks)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YC_3",
"Factors":{"Age":"Young (4 weeks)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YD_4",
"Factors":{"Age":"Young (4 weeks)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YE_5",
"Factors":{"Age":"Young (4 weeks)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YHA_6",
"Factors":{"Age":"Young (4 weeks)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YHB_7",
"Factors":{"Age":"Young (4 weeks)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YHC_8",
"Factors":{"Age":"Young (4 weeks)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YHD_9",
"Factors":{"Age":"Young (4 weeks)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YHE_10",
"Factors":{"Age":"Young (4 weeks)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_YHF_11",
"Factors":{"Age":"Young (4 weeks)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OA_12",
"Factors":{"Age":"Old (10-15 months)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OB_13",
"Factors":{"Age":"Old (10-15 months)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OC_14",
"Factors":{"Age":"Old (10-15 months)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OD_15",
"Factors":{"Age":"Old (10-15 months)","Treatment":"2.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OHA_16",
"Factors":{"Age":"Old (10-15 months)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OHB_17",
"Factors":{"Age":"Old (10-15 months)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OHC_18",
"Factors":{"Age":"Old (10-15 months)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OHD_19",
"Factors":{"Age":"Old (10-15 months)","Treatment":"16.8mM Glucose (60 min)"}
},
{
"Subject ID":"-",
"Sample ID":"10_25_2016_islet_OHE_20",
"Factors":{"Age":"Old (10-15 months)","Treatment":"16.8mM Glucose (60 min)"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Pancreata were digested using Liberase TL, and islets were purified using a Histopaque gradient followed by hand-picking under a dissecting microscope. Islets were allowed to recover overnight in RPMI 1640 media supplemented with 8 mM glucose, 10% FBS, 2 mM L-glutamine, 100 U/mL Pen/Strep, 1 mM sodium pyruvate, 10 mM HEPES, and 0.25 mg/mL amphoterecin B.","COLLECTION_PROTOCOL_FILENAME":"Methods.pdf","SAMPLE_TYPE":"Pancreatic Islets"},

"TREATMENT":{"TREATMENT_SUMMARY":"Islets were starved for 1 hour in KRBH supplemented with 2.8 mM glucose as for GSIS assays. Islets were then transferred to petri dishes containing U-13C-labeled glucose as indicated and immediately seeded into wells of non-treated 12-well plates at a density of 100 size-matched islets per replicate in a final volume of 500 μl KRBH containing basal (2.8 mM) or stimulatory (16.8 mM) concentrations of labeled glucose. Tracing was performed for the indicated durations at 37oC with 5% CO2. Following the trace, islets were rapidly chilled by swirling plate to resuspend islets then transferring entire contents of each well to an Eppendorf tube on ice. Islets were then pelleted by centrifugation at 500 x g for 2 min at 4oC, washed in 1 mL of ice cold 0.9% NaCl, centrifuged as before, then pellets were frozen in a dry ice/ethanol bath and stored at -80oC."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Metabolites were extracted from pellets using a bligh and dyer-based methanol/chloroform/water extraction with inclusion of norvaline as a polar internal standard. Briefly, 250 μl MeOH, 250 μl CHCL3, 100 μl water containing norvaline were added to pellets. This was vortexed for 10 minutes followed by centrifugation at 10,000 g for 5 minutes at 4°C. The upper phase was separated and dried under vacuum at 4°C until dry. Polar metabolites were derivatized in 2% (w/v) methoxyamine hydrochloride in pyridine and incubated at 45°C for 60 minutes. Samples were then silylated with N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) with 1% tert-butyldimethylchlorosilane (tBDMCS) at 45°C for 30 minutes. Polar derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm i.d. x 0.25 μm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with an Agilent 5977A mass spectrometer (MS) with an XTR EI source using the following temperature program: 100 °C initial, increase by 3.5 °C/min to 255 °C, increase by 15 °C/min to 320 °C and hold for 3 min."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Polar derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm i.d. x 0.25 μm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with an Agilent 5977A mass spectrometer (MS) with an XTR EI source using the following temperature program: 100 °C initial, increase by 3.5 °C/min to 255 °C, increase by 15 °C/min to 320 °C and hold for 3 min.","CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 7890B","COLUMN_NAME":"Agilent DB5-MS (15m × 0.25mm)"},

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

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 5977A","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","ION_MODE":"POSITIVE"},

"MS_METABOLITE_DATA":{
"Units":"Total ion counts",

"Data":[{"Metabolite":"Pyruvic acid","10_25_2016_islet_YA_1":"127872","10_25_2016_islet_YB_2":"112204","10_25_2016_islet_YC_3":"144461","10_25_2016_islet_YD_4":"100184","10_25_2016_islet_YE_5":"127227","10_25_2016_islet_YHA_6":"171618","10_25_2016_islet_YHB_7":"125279","10_25_2016_islet_YHC_8":"238191","10_25_2016_islet_YHD_9":"147351","10_25_2016_islet_YHE_10":"137885","10_25_2016_islet_YHF_11":"273982","10_25_2016_islet_OA_12":"125525","10_25_2016_islet_OB_13":"125669","10_25_2016_islet_OC_14":"120114","10_25_2016_islet_OD_15":"138049","10_25_2016_islet_OHA_16":"143772","10_25_2016_islet_OHB_17":"263368","10_25_2016_islet_OHC_18":"160353","10_25_2016_islet_OHD_19":"165116","10_25_2016_islet_OHE_20":"257911"},{"Metabolite":"Alanine","10_25_2016_islet_YA_1":"149331","10_25_2016_islet_YB_2":"176595","10_25_2016_islet_YC_3":"170978","10_25_2016_islet_YD_4":"92567","10_25_2016_islet_YE_5":"158648","10_25_2016_islet_YHA_6":"225310","10_25_2016_islet_YHB_7":"199434","10_25_2016_islet_YHC_8":"297371","10_25_2016_islet_YHD_9":"263588","10_25_2016_islet_YHE_10":"216610","10_25_2016_islet_YHF_11":"270899","10_25_2016_islet_OA_12":"129018","10_25_2016_islet_OB_13":"124160","10_25_2016_islet_OC_14":"157740","10_25_2016_islet_OD_15":"165844","10_25_2016_islet_OHA_16":"166813","10_25_2016_islet_OHB_17":"331445","10_25_2016_islet_OHC_18":"188564","10_25_2016_islet_OHD_19":"322864","10_25_2016_islet_OHE_20":"276523"},{"Metabolite":"Glycine","10_25_2016_islet_YA_1":"1039556","10_25_2016_islet_YB_2":"1144609","10_25_2016_islet_YC_3":"1070311","10_25_2016_islet_YD_4":"626542","10_25_2016_islet_YE_5":"962526","10_25_2016_islet_YHA_6":"906185","10_25_2016_islet_YHB_7":"846861","10_25_2016_islet_YHC_8":"1137254","10_25_2016_islet_YHD_9":"1129803","10_25_2016_islet_YHE_10":"906314","10_25_2016_islet_YHF_11":"984481","10_25_2016_islet_OA_12":"1111236","10_25_2016_islet_OB_13":"933364","10_25_2016_islet_OC_14":"1404425","10_25_2016_islet_OD_15":"1202776","10_25_2016_islet_OHA_16":"800997","10_25_2016_islet_OHB_17":"1461625","10_25_2016_islet_OHC_18":"965475","10_25_2016_islet_OHD_19":"1538193","10_25_2016_islet_OHE_20":"1330172"},{"Metabolite":"Norvaline","10_25_2016_islet_YA_1":"4871730","10_25_2016_islet_YB_2":"5862696","10_25_2016_islet_YC_3":"5271742","10_25_2016_islet_YD_4":"3623930","10_25_2016_islet_YE_5":"4816000","10_25_2016_islet_YHA_6":"4481891","10_25_2016_islet_YHB_7":"4621497","10_25_2016_islet_YHC_8":"5607100","10_25_2016_islet_YHD_9":"4805219","10_25_2016_islet_YHE_10":"4890895","10_25_2016_islet_YHF_11":"4986328","10_25_2016_islet_OA_12":"5060059","10_25_2016_islet_OB_13":"5192042","10_25_2016_islet_OC_14":"6270470","10_25_2016_islet_OD_15":"5029168","10_25_2016_islet_OHA_16":"4209004","10_25_2016_islet_OHB_17":"5193592","10_25_2016_islet_OHC_18":"4220621","10_25_2016_islet_OHD_19":"4848393","10_25_2016_islet_OHE_20":"4889503"},{"Metabolite":"Fumaric acid","10_25_2016_islet_YA_1":"76644","10_25_2016_islet_YB_2":"89683","10_25_2016_islet_YC_3":"95824","10_25_2016_islet_YD_4":"70225","10_25_2016_islet_YE_5":"65259","10_25_2016_islet_YHA_6":"204151","10_25_2016_islet_YHB_7":"218227","10_25_2016_islet_YHC_8":"164364","10_25_2016_islet_YHD_9":"235746","10_25_2016_islet_YHE_10":"223585","10_25_2016_islet_YHF_11":"186575","10_25_2016_islet_OA_12":"88000","10_25_2016_islet_OB_13":"104780","10_25_2016_islet_OC_14":"63468","10_25_2016_islet_OD_15":"90678","10_25_2016_islet_OHA_16":"268930","10_25_2016_islet_OHB_17":"333936","10_25_2016_islet_OHC_18":"222095","10_25_2016_islet_OHD_19":"450449","10_25_2016_islet_OHE_20":"313598"},{"Metabolite":"Malic acid","10_25_2016_islet_YA_1":"34985","10_25_2016_islet_YB_2":"47351","10_25_2016_islet_YC_3":"41842","10_25_2016_islet_YD_4":"145211","10_25_2016_islet_YE_5":"43924","10_25_2016_islet_YHA_6":"409443","10_25_2016_islet_YHB_7":"378689","10_25_2016_islet_YHC_8":"597403","10_25_2016_islet_YHD_9":"414248","10_25_2016_islet_YHE_10":"430598","10_25_2016_islet_YHF_11":"436624","10_25_2016_islet_OA_12":"50596","10_25_2016_islet_OB_13":"50206","10_25_2016_islet_OC_14":"56805","10_25_2016_islet_OD_15":"49845","10_25_2016_islet_OHA_16":"443331","10_25_2016_islet_OHB_17":"753516","10_25_2016_islet_OHC_18":"488383","10_25_2016_islet_OHD_19":"891031","10_25_2016_islet_OHE_20":"754076"},{"Metabolite":"Aspartic acid","10_25_2016_islet_YA_1":"1258378","10_25_2016_islet_YB_2":"1631993","10_25_2016_islet_YC_3":"1709001","10_25_2016_islet_YD_4":"744237","10_25_2016_islet_YE_5":"1441617","10_25_2016_islet_YHA_6":"1017961","10_25_2016_islet_YHB_7":"949464","10_25_2016_islet_YHC_8":"1745570","10_25_2016_islet_YHD_9":"1419668","10_25_2016_islet_YHE_10":"998841","10_25_2016_islet_YHF_11":"1405252","10_25_2016_islet_OA_12":"991603","10_25_2016_islet_OB_13":"691648","10_25_2016_islet_OC_14":"1059798","10_25_2016_islet_OD_15":"1069099","10_25_2016_islet_OHA_16":"558917","10_25_2016_islet_OHB_17":"1480591","10_25_2016_islet_OHC_18":"685144","10_25_2016_islet_OHD_19":"1369715","10_25_2016_islet_OHE_20":"905781"},{"Metabolite":"Glutamic acid","10_25_2016_islet_YA_1":"1551470","10_25_2016_islet_YB_2":"2384163","10_25_2016_islet_YC_3":"2342152","10_25_2016_islet_YD_4":"1045410","10_25_2016_islet_YE_5":"2031316","10_25_2016_islet_YHA_6":"1613981","10_25_2016_islet_YHB_7":"1502895","10_25_2016_islet_YHC_8":"2538828","10_25_2016_islet_YHD_9":"2054041","10_25_2016_islet_YHE_10":"1631004","10_25_2016_islet_YHF_11":"2000736","10_25_2016_islet_OA_12":"1733091","10_25_2016_islet_OB_13":"1220167","10_25_2016_islet_OC_14":"1825296","10_25_2016_islet_OD_15":"1808941","10_25_2016_islet_OHA_16":"828959","10_25_2016_islet_OHB_17":"2399020","10_25_2016_islet_OHC_18":"1142550","10_25_2016_islet_OHD_19":"2499607","10_25_2016_islet_OHE_20":"1707531"},{"Metabolite":"Citric acid","10_25_2016_islet_YA_1":"227796","10_25_2016_islet_YB_2":"403797","10_25_2016_islet_YC_3":"338957","10_25_2016_islet_YD_4":"133857","10_25_2016_islet_YE_5":"216051","10_25_2016_islet_YHA_6":"515702","10_25_2016_islet_YHB_7":"498069","10_25_2016_islet_YHC_8":"761536","10_25_2016_islet_YHD_9":"629775","10_25_2016_islet_YHE_10":"495454","10_25_2016_islet_YHF_11":"404636","10_25_2016_islet_OA_12":"146875","10_25_2016_islet_OB_13":"112900","10_25_2016_islet_OC_14":"137748","10_25_2016_islet_OD_15":"158354","10_25_2016_islet_OHA_16":"294254","10_25_2016_islet_OHB_17":"664080","10_25_2016_islet_OHC_18":"361940","10_25_2016_islet_OHD_19":"731834","10_25_2016_islet_OHE_20":"528354"}],

"Metabolites":[{"Metabolite":"Pyruvic acid","m/z":"174"},{"Metabolite":"Alanine","m/z":"260"},{"Metabolite":"Glycine","m/z":"246"},{"Metabolite":"Norvaline","m/z":"260"},{"Metabolite":"Fumaric acid","m/z":"287"},{"Metabolite":"Malic acid","m/z":"419"},{"Metabolite":"Aspartic acid","m/z":"418"},{"Metabolite":"Glutamic acid","m/z":"432"},{"Metabolite":"Citric acid","m/z":"459"}]
}

}