#METABOLOMICS WORKBENCH mwortham_20180427_190224_mwtab.txt DATATRACK_ID:1392 STUDY_ID:ST000965 ANALYSIS_ID:AN001579 PROJECT_ID:PR000662 VERSION 1 CREATED_ON April 30, 2018, 12:21 pm #PROJECT PR:PROJECT_TITLE Integrated in vivo quantitative proteomics and nutrient tracing reveals PR:PROJECT_TITLE age-related metabolic rewiring of pancreatic beta cell function PR:PROJECT_SUMMARY Aging is associated with fundamental changes in pancreatic β-cell physiology; PR:PROJECT_SUMMARY yet, the mechanisms that drive these age-related changes are poorly understood. PR:PROJECT_SUMMARY Here, we performed comprehensive in vivo quantitative proteomic profiling of PR:PROJECT_SUMMARY pancreatic islets from adolescent and old mice. Nutrient tracing and targeted PR:PROJECT_SUMMARY metabolomics demonstrated accelerated accumulation of glucose-derived PR:PROJECT_SUMMARY metabolites and coupling factors in aged islets, indicating that age-related PR:PROJECT_SUMMARY changes in glucose metabolism contribute to improved glucose-stimulated insulin PR:PROJECT_SUMMARY secretion with age. Together, our study provides the first in-depth PR:PROJECT_SUMMARY characterization of age-related changes in the islet proteome and establishes PR:PROJECT_SUMMARY metabolic rewiring as an important mechanism for age-associated changes in PR:PROJECT_SUMMARY β-cell function. PR:INSTITUTE University of California, San Diego PR:LAST_NAME Wortham PR:FIRST_NAME Matthew PR:ADDRESS 2880 Torrey Pines Scenic Drive, Sanford Consortium for Regenerative Medicine, PR:ADDRESS Room 3102, La Jolla, California, 92037, USA PR:EMAIL mwortham@ucsd.edu PR:PHONE 8582460588 #STUDY ST:STUDY_TITLE Timecourse of U13C glucose labeling of pancreatic islets in young mice ST:STUDY_SUMMARY Pancreatic islets from young mice were traced with 16.8mM U13C glucose over a 90 ST:STUDY_SUMMARY minute timecourse to determine the rate of metabolite labeling during glucose ST:STUDY_SUMMARY stimulation. ST:INSTITUTE University of California, San Diego ST:LAST_NAME Wortham ST:FIRST_NAME Matthew ST:ADDRESS 2880 Torrey Pines Scenic Drive, Sanford Consortium for Regenerative Medicine, ST:ADDRESS Room 3102 ST:EMAIL mwortham@ucsd.edu ST:PHONE 8582460588 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6N SU:AGE_OR_AGE_RANGE 4 weeks old (young) or 10-15 months old (old) SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Charles River Labs SU:ANIMAL_FEED standard chow diet (PicoLab Rodent Diet 20 pellets, #5053 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_0_1 Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_0_2 Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_0_3 Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_0_5 Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_0_6 Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_0_7 Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_30mins_10 Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_30mins_11 Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_30mins_12 Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_30mins_13 Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_30mins_14 Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_30mins_8 Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_30mins_9 Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_60mins_15 Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_60mins_16 Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_60mins_17 Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_60mins_18 Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_60mins_19 Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_60mins_20 Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_60mins_21 Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_90mins_22 Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_90mins_23 Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_90mins_24 Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_90mins_25 Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_90mins_26 Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_90mins_27 Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) SUBJECT_SAMPLE_FACTORS - 1_9_2018_islet_ugluc_timecourse_90mins_28 Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) #COLLECTION CO:COLLECTION_SUMMARY Pancreata were digested using Liberase TL, and islets were purified using a CO:COLLECTION_SUMMARY Histopaque gradient followed by hand-picking under a dissecting microscope. CO:COLLECTION_SUMMARY Islets were allowed to recover overnight in RPMI 1640 media supplemented with 8 CO:COLLECTION_SUMMARY mM glucose, 10% FBS, 2 mM L-glutamine, 100 U/mL Pen/Strep, 1 mM sodium pyruvate, CO:COLLECTION_SUMMARY 10 mM HEPES, and 0.25 mg/mL amphoterecin B. CO:COLLECTION_PROTOCOL_FILENAME Methods.pdf CO:SAMPLE_TYPE Pancreatic Islets #TREATMENT TR:TREATMENT_SUMMARY Islets were starved for 1 hour in KRBH supplemented with 2.8 mM glucose as for TR:TREATMENT_SUMMARY GSIS assays. Islets were then transferred to petri dishes containing TR:TREATMENT_SUMMARY U-13C-labeled glucose as indicated and immediately seeded into wells of TR:TREATMENT_SUMMARY non-treated 12-well plates at a density of 100 size-matched islets per replicate TR:TREATMENT_SUMMARY in a final volume of 500 μl KRBH containing basal (2.8 mM) or stimulatory (16.8 TR:TREATMENT_SUMMARY mM) concentrations of labeled glucose. Tracing was performed for the indicated TR:TREATMENT_SUMMARY durations at 37oC with 5% CO2. Following the trace, islets were rapidly chilled TR:TREATMENT_SUMMARY by swirling plate to resuspend islets then transferring entire contents of each TR:TREATMENT_SUMMARY well to an Eppendorf tube on ice. Islets were then pelleted by centrifugation at TR:TREATMENT_SUMMARY 500 x g for 2 min at 4oC, washed in 1 mL of ice cold 0.9% NaCl, centrifuged as TR:TREATMENT_SUMMARY before, then pellets were frozen in a dry ice/ethanol bath and stored at -80oC. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from pellets using a bligh and dyer-based SP:SAMPLEPREP_SUMMARY methanol/chloroform/water extraction with inclusion of norvaline as a polar SP:SAMPLEPREP_SUMMARY internal standard. Briefly, 250 μl MeOH, 250 μl CHCL3, 100 μl water SP:SAMPLEPREP_SUMMARY containing norvaline were added to pellets. This was vortexed for 10 minutes SP:SAMPLEPREP_SUMMARY followed by centrifugation at 10,000 g for 5 minutes at 4°C. The upper phase SP:SAMPLEPREP_SUMMARY was separated and dried under vacuum at 4°C until dry. Polar metabolites were SP:SAMPLEPREP_SUMMARY derivatized in 2% (w/v) methoxyamine hydrochloride in pyridine and incubated at SP:SAMPLEPREP_SUMMARY 45°C for 60 minutes. Samples were then silylated with SP:SAMPLEPREP_SUMMARY N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) with 1% SP:SAMPLEPREP_SUMMARY tert-butyldimethylchlorosilane (tBDMCS) at 45°C for 30 minutes. Polar SP:SAMPLEPREP_SUMMARY derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm i.d. x SP:SAMPLEPREP_SUMMARY 0.25 μm) installed in an Agilent 7890B gas chromatograph (GC) interfaced with SP:SAMPLEPREP_SUMMARY an Agilent 5977A mass spectrometer (MS) with an XTR EI source using the SP:SAMPLEPREP_SUMMARY following temperature program: 100 °C initial, increase by 3.5 °C/min to 255 SP:SAMPLEPREP_SUMMARY °C, increase by 15 °C/min to 320 °C and hold for 3 min. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Polar derivatives were analyzed by GC-MS using a DB-35MS column (30m x 0.25 mm CH:CHROMATOGRAPHY_SUMMARY i.d. x 0.25 μm) installed in an Agilent 7890B gas chromatograph (GC) interfaced CH:CHROMATOGRAPHY_SUMMARY with an Agilent 5977A mass spectrometer (MS) with an XTR EI source using the CH:CHROMATOGRAPHY_SUMMARY following temperature program: 100 °C initial, increase by 3.5 °C/min to 255 CH:CHROMATOGRAPHY_SUMMARY °C, increase by 15 °C/min to 320 °C and hold for 3 min. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent DB5-MS (15m × 0.25mm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 5977A MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Total ion counts MS_METABOLITE_DATA_START Samples 1_9_2018_islet_ugluc_timecourse_0_1 1_9_2018_islet_ugluc_timecourse_0_2 1_9_2018_islet_ugluc_timecourse_0_3 1_9_2018_islet_ugluc_timecourse_0_5 1_9_2018_islet_ugluc_timecourse_0_6 1_9_2018_islet_ugluc_timecourse_0_7 1_9_2018_islet_ugluc_timecourse_30mins_10 1_9_2018_islet_ugluc_timecourse_30mins_11 1_9_2018_islet_ugluc_timecourse_30mins_12 1_9_2018_islet_ugluc_timecourse_30mins_13 1_9_2018_islet_ugluc_timecourse_30mins_14 1_9_2018_islet_ugluc_timecourse_30mins_8 1_9_2018_islet_ugluc_timecourse_30mins_9 1_9_2018_islet_ugluc_timecourse_60mins_15 1_9_2018_islet_ugluc_timecourse_60mins_16 1_9_2018_islet_ugluc_timecourse_60mins_17 1_9_2018_islet_ugluc_timecourse_60mins_18 1_9_2018_islet_ugluc_timecourse_60mins_19 1_9_2018_islet_ugluc_timecourse_60mins_20 1_9_2018_islet_ugluc_timecourse_60mins_21 1_9_2018_islet_ugluc_timecourse_90mins_22 1_9_2018_islet_ugluc_timecourse_90mins_23 1_9_2018_islet_ugluc_timecourse_90mins_24 1_9_2018_islet_ugluc_timecourse_90mins_25 1_9_2018_islet_ugluc_timecourse_90mins_26 1_9_2018_islet_ugluc_timecourse_90mins_27 1_9_2018_islet_ugluc_timecourse_90mins_28 Factors Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (0 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (30 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) Age:Young (4 weeks) | Treatment:16.8mM Glucose (90 min) Pyruvic acid 105678 118276 103753 912684 793655 115132 151525 1114846 163697 223372 130901 152476 144068 105596 104228 157458 150224 162775 165366 138384 134363 1303553 126347 155266 171205 150289 142408 Glycine 1610906 3902266 6340278 3207792 4277278 12109448 3243655 1281232 3299245 15100260 2896121 1101077 1380239 3218487 2498200 1827924 850694 1658494 4834282 4996556 2087014 2124704 891144 2140573 2913820 3333413 2077356 Norvaline 63213622 67311591 67727917 62135261 59277912 59141049 64265368 58493183 59935350 71137201 65196161 45122279 53213176 65172806 64432296 62050601 44219693 63251476 69884373 69248549 65287849 59414906 44588154 57155996 69952630 58696768 63034328 Fumaric acid 316220 311338 478746 251572 129810 141974 161411 176206 143084 372259 271617 121710 136647 203096 258367 196043 90131 168080 419420 421876 347800 249689 159034 320178 517622 468193 302344 Malic acid 38899 49616 82238 38810 51113 88342 182595 155581 155829 368742 230372 115466 121625 238932 324502 300304 147395 271204 486228 483453 475237 430227 276978 439641 661849 743674 428451 Aspartic acid 3763174 4068933 7238059 3015331 4199882 2683545 3313734 2418784 2258601 3729468 4181958 1986551 1939683 2195052 2443394 2632088 1056271 2254619 3934926 4127907 2850077 2206535 1385623 2531118 4152871 4476148 2256039 Glutamic acid 4175172 4691716 7902542 3257584 4227381 2963733 4966154 3746117 3470247 6155259 6290759 2877306 2963780 3724927 4051439 4469036 1790789 3754654 6492208 7104865 4438011 3617421 2177253 4222967 6624137 7124972 3488410 Citric acid 193335 228847 480151 181374 249386 63229 381429 292802 204671 142040 158133 77569 74062 100437 140423 181955 87747 107874 363790 537502 456037 422550 299545 367599 499290 642121 343076 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z Pyruvic acid 174 Glycine 246 Norvaline 260 Fumaric acid 287 Malic acid 419 Aspartic acid 418 Glutamic acid 432 Citric acid 459 METABOLITES_END #END