#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