Summary of Study ST000965

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000662. The data can be accessed directly via it's Project DOI: 10.21228/M83Q2T This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST000965
Study Title	Timecourse of U13C glucose labeling of pancreatic islets in young mice
Study Summary	Pancreatic islets from young mice were traced with 16.8mM U13C glucose over a 90 minute timecourse to determine the rate of metabolite labeling during glucose stimulation.
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
Submit Date	2018-04-27
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	GC-MS
Release Date	2019-04-27
Release Version	1

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Project:

Project ID:	PR000662
Project DOI:	doi: 10.21228/M83Q2T
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

Subject:

Subject ID:	SU001004
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

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Age	Treatment
SA057712	1_9_2018_islet_ugluc_timecourse_0_1	Young (4 weeks)	16.8mM Glucose (0 min)
SA057713	1_9_2018_islet_ugluc_timecourse_0_7	Young (4 weeks)	16.8mM Glucose (0 min)
SA057714	1_9_2018_islet_ugluc_timecourse_0_6	Young (4 weeks)	16.8mM Glucose (0 min)
SA057715	1_9_2018_islet_ugluc_timecourse_0_2	Young (4 weeks)	16.8mM Glucose (0 min)
SA057716	1_9_2018_islet_ugluc_timecourse_0_3	Young (4 weeks)	16.8mM Glucose (0 min)
SA057717	1_9_2018_islet_ugluc_timecourse_0_5	Young (4 weeks)	16.8mM Glucose (0 min)
SA057718	1_9_2018_islet_ugluc_timecourse_30mins_8	Young (4 weeks)	16.8mM Glucose (30 min)
SA057719	1_9_2018_islet_ugluc_timecourse_30mins_9	Young (4 weeks)	16.8mM Glucose (30 min)
SA057720	1_9_2018_islet_ugluc_timecourse_30mins_14	Young (4 weeks)	16.8mM Glucose (30 min)
SA057721	1_9_2018_islet_ugluc_timecourse_30mins_12	Young (4 weeks)	16.8mM Glucose (30 min)
SA057722	1_9_2018_islet_ugluc_timecourse_30mins_10	Young (4 weeks)	16.8mM Glucose (30 min)
SA057723	1_9_2018_islet_ugluc_timecourse_30mins_13	Young (4 weeks)	16.8mM Glucose (30 min)
SA057724	1_9_2018_islet_ugluc_timecourse_30mins_11	Young (4 weeks)	16.8mM Glucose (30 min)
SA057725	1_9_2018_islet_ugluc_timecourse_60mins_20	Young (4 weeks)	16.8mM Glucose (60 min)
SA057726	1_9_2018_islet_ugluc_timecourse_60mins_21	Young (4 weeks)	16.8mM Glucose (60 min)
SA057727	1_9_2018_islet_ugluc_timecourse_60mins_19	Young (4 weeks)	16.8mM Glucose (60 min)
SA057728	1_9_2018_islet_ugluc_timecourse_60mins_15	Young (4 weeks)	16.8mM Glucose (60 min)
SA057729	1_9_2018_islet_ugluc_timecourse_60mins_18	Young (4 weeks)	16.8mM Glucose (60 min)
SA057730	1_9_2018_islet_ugluc_timecourse_60mins_16	Young (4 weeks)	16.8mM Glucose (60 min)
SA057731	1_9_2018_islet_ugluc_timecourse_60mins_17	Young (4 weeks)	16.8mM Glucose (60 min)
SA057732	1_9_2018_islet_ugluc_timecourse_90mins_27	Young (4 weeks)	16.8mM Glucose (90 min)
SA057733	1_9_2018_islet_ugluc_timecourse_90mins_28	Young (4 weeks)	16.8mM Glucose (90 min)
SA057734	1_9_2018_islet_ugluc_timecourse_90mins_26	Young (4 weeks)	16.8mM Glucose (90 min)
SA057735	1_9_2018_islet_ugluc_timecourse_90mins_24	Young (4 weeks)	16.8mM Glucose (90 min)
SA057736	1_9_2018_islet_ugluc_timecourse_90mins_22	Young (4 weeks)	16.8mM Glucose (90 min)
SA057737	1_9_2018_islet_ugluc_timecourse_90mins_23	Young (4 weeks)	16.8mM Glucose (90 min)
SA057738	1_9_2018_islet_ugluc_timecourse_90mins_25	Young (4 weeks)	16.8mM Glucose (90 min)

Showing results 1 to 27 of 27

Showing results 1 to 27 of 27

Collection:

Collection ID:	CO000998
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 ID:	TR001018
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.

Treatment ID:

TR001018

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.

Sample Preparation:

Sampleprep ID:	SP001011
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.

Sampleprep ID:

SP001011

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.

Combined analysis:

Analysis ID	AN001579
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890B
Column	Agilent DB5-MS (15m × 0.25mm)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5977A
Ion Mode	POSITIVE
Units	Total ion counts

Chromatography:

Chromatography ID:	CH001108
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.
Instrument Name:	Agilent 7890B
Column Name:	Agilent DB5-MS (15m × 0.25mm)
Chromatography Type:	GC

MS:

MS ID:	MS001457
Analysis ID:	AN001579
Instrument Name:	Agilent 5977A
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE