#METABOLOMICS WORKBENCH mwortham_20180427_120539 DATATRACK_ID:1390 STUDY_ID:ST000964 ANALYSIS_ID:AN001578 PROJECT_ID:PR000662
VERSION             	1
CREATED_ON             	April 30, 2018, 12:09 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                   	U13C Glucose Tracing of Young and Old Pancreatic Islets
ST:STUDY_SUMMARY                 	Pancreatic islets from young and old mice were traced with either 2.8mM or
ST:STUDY_SUMMARY                 	16.8mM U13C glucose to determine the effect of age upon glucose metabolism in
ST:STUDY_SUMMARY                 	basal and stimulated states.
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           	-	10_25_2016_islet_YA_1	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YB_2	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YC_3	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YD_4	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YE_5	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YHA_6	Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YHB_7	Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YHC_8	Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YHD_9	Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YHE_10	Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_YHF_11	Age:Young (4 weeks) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OA_12	Age:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OB_13	Age:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OC_14	Age:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OD_15	Age:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OHA_16	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OHB_17	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OHC_18	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OHD_19	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	
SUBJECT_SAMPLE_FACTORS           	-	10_25_2016_islet_OHE_20	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 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	10_25_2016_islet_YA_1	10_25_2016_islet_YB_2	10_25_2016_islet_YC_3	10_25_2016_islet_YD_4	10_25_2016_islet_YE_5	10_25_2016_islet_YHA_6	10_25_2016_islet_YHB_7	10_25_2016_islet_YHC_8	10_25_2016_islet_YHD_9	10_25_2016_islet_YHE_10	10_25_2016_islet_YHF_11	10_25_2016_islet_OA_12	10_25_2016_islet_OB_13	10_25_2016_islet_OC_14	10_25_2016_islet_OD_15	10_25_2016_islet_OHA_16	10_25_2016_islet_OHB_17	10_25_2016_islet_OHC_18	10_25_2016_islet_OHD_19	10_25_2016_islet_OHE_20	10_25_2016_islet_YHE_10	10_25_2016_islet_YHF_11
Factors	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	Age:Young (4 weeks) | Treatment:2.8mM Glucose (60 min)	Age:Young (4 weeks) | Treatment:2.8mM 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:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	Age:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	Age:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	Age:Old (10-15 months) | Treatment:2.8mM Glucose (60 min)	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	Age:Old (10-15 months) | Treatment:16.8mM Glucose (60 min)	Age:Old (10-15 months) | 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)
Pyruvic acid	127872	112204	144461	100184	127227	171618	125279	238191	147351	137885	273982	125525	125669	120114	138049	143772	263368	160353	165116	257911	137885	273982
Alanine	149331	176595	170978	92567	158648	225310	199434	297371	263588	216610	270899	129018	124160	157740	165844	166813	331445	188564	322864	276523	216610	270899
Glycine	1039556	1144609	1070311	626542	962526	906185	846861	1137254	1129803	906314	984481	1111236	933364	1404425	1202776	800997	1461625	965475	1538193	1330172	906314	984481
Norvaline	4871730	5862696	5271742	3623930	4816000	4481891	4621497	5607100	4805219	4890895	4986328	5060059	5192042	6270470	5029168	4209004	5193592	4220621	4848393	4889503	4890895	4986328
Fumaric acid	76644	89683	95824	70225	65259	204151	218227	164364	235746	223585	186575	88000	104780	63468	90678	268930	333936	222095	450449	313598	223585	186575
Malic acid	34985	47351	41842	145211	43924	409443	378689	597403	414248	430598	436624	50596	50206	56805	49845	443331	753516	488383	891031	754076	430598	436624
Aspartic acid	1258378	1631993	1709001	744237	1441617	1017961	949464	1745570	1419668	998841	1405252	991603	691648	1059798	1069099	558917	1480591	685144	1369715	905781	998841	1405252
Glutamic acid	1551470	2384163	2342152	1045410	2031316	1613981	1502895	2538828	2054041	1631004	2000736	1733091	1220167	1825296	1808941	828959	2399020	1142550	2499607	1707531	1631004	2000736
Citric acid	227796	403797	338957	133857	216051	515702	498069	761536	629775	495454	404636	146875	112900	137748	158354	294254	664080	361940	731834	528354	495454	404636
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	m/z
Pyruvic acid	174
Alanine	260
Glycine	246
Norvaline	260
Fumaric acid	287
Malic acid	419
Aspartic acid	418
Glutamic acid	432
Citric acid	459
METABOLITES_END
#END