Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA025099

Local Sample ID:	MS5040_9_180
Subject ID:	SU000506
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000506
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
MS5040_9_180	SA025099	FL006151	2	visit
MS5040_9_180	SA025099	FL006151	180	time (mins)
MS5040_9_180	SA025099	FL006151	Control	Category

Collection:

Collection ID:	CO000500
Collection Summary:	Blood samples were collected in a heated hotbox (131°F) through a retrograde intravenous catheter at baseline for glucose and hormone levels, and every 10 min during the clamp to maintain euglycemia. In addition, blood samples were collected every 20 min from 0600 to 0700, 0900 to 1000, and 1200 to 1300 to measure plasma [6,62H2]glucose. At 1330 h, a percutaneous needle muscle biopsy specimen (350–400 mg) was obtained from the vastus lateralis muscle under local anesthesia, immediately frozen in liquid nitrogen, and stored at −80°F for future analysis (27). This biopsy sample was used for analysis of TXNIP mRNA and protein content. The participant remained in the CRU through the remainder of the day and was given a weight-maintenance diet until 2200 h. At 0700 h the following morning, a second muscle biopsy specimen was obtained under local anesthesia, and ∼100 mg was used immediately for mitochondrial function measurements of isolated mitochondria and mtH2O2 emissions (28). The remainder was immediately frozen in liquid nitrogen and stored at −80°F for future analysis, including DAG, ceramide, and amino acid measurements (Fig. 1).
Sample Type:	Blood

Treatment:

Treatment ID:	TR000520
Treatment Summary:	Before and after 16 weeks of CR or CON, two outpatient visits and one inpatient visit were scheduled. Before the outpatient visits, participants were instructed to fast overnight from 10:00 p.m. the evening before and to avoid strenuous exercise for 24 h preceding the visits. One outpatient visit consisted of an MRI to measure subcutaneous and visceral fat distribution and magnetic resonance spectroscopy to measure skeletal muscle oxidative capacity (25). The second outpatient visit was for measurements of resting energy expenditure (REE) for the calculation of a weight-maintenance diet (Parvo Medics TrueOne 2400 Canopy system), DEXA scan (Lunar DPX-L; Lunar Radiation, Madison, WI), and VO2peak test on a bicycle ergometer (Fig. 1). Participants were admitted to the Clinical Research Unit (CRU) on the evening of the fifth day of the weight-maintaining diet provided by the CRU metabolic kitchen (Supplementary Fig. 1). The weight-maintenance meals (diet composition: 20% protein, 30% fat, 50% carbohydrate) were monitored daily to ensure that the correct calorie level was achieved. Upon admission to the CRU, no calories were consumed after 2100 h to achieve a 10-h fast before the two-stage insulin euglycemic pancreatic clamp the following morning, as previously published (26), with modifications as follows: the following morning at 0400 h, a primed [6,62H2]glucose bolus (6 mg ⋅ kg fat-free mass[FFM]−1) was administered, followed by a 9-h continuous infusion of [6,62H2]glucose (started at 4 mg ⋅ kgFFM−1 ⋅ h−1 then titrated downward over the infusion time period to match anticipated changes in endogenous glucose production [EGP]). At 0600 h, gas exchange was measured by indirect calorimetry for 30 min for REE determination. Then at 0700 h, glucagon (0.001 μg ⋅ kgFFM−1 ⋅ min−1), somatostatin (0.093 μg ⋅ kgFFM−1 ⋅ min−1), and growth hormone (0.0047 μg ⋅ kgFFM−1 ⋅ min−1) were infused for 6 h. Insulin was infused from 0700 to 1000 h at 0.62 mU ⋅ kgFFM−1 ⋅ min−1 and then from 1000 to 1300 h at 2.3 mU ⋅ kgFFM−1 ⋅ min−1. A 40% dextrose with 2% enrichment of [6,62H2]glucose was infused as needed to maintain blood glucose above 4.7 mmol/L from 0700 to 1000 h and then between 4.7 and 5.3 mmol/L from 1000 to 1300 h.

Treatment ID:

TR000520

Treatment Summary:

Before and after 16 weeks of CR or CON, two outpatient visits and one inpatient visit were scheduled. Before the outpatient visits, participants were instructed to fast overnight from 10:00 p.m. the evening before and to avoid strenuous exercise for 24 h preceding the visits. One outpatient visit consisted of an MRI to measure subcutaneous and visceral fat distribution and magnetic resonance spectroscopy to measure skeletal muscle oxidative capacity (25). The second outpatient visit was for measurements of resting energy expenditure (REE) for the calculation of a weight-maintenance diet (Parvo Medics TrueOne 2400 Canopy system), DEXA scan (Lunar DPX-L; Lunar Radiation, Madison, WI), and VO2peak test on a bicycle ergometer (Fig. 1). Participants were admitted to the Clinical Research Unit (CRU) on the evening of the fifth day of the weight-maintaining diet provided by the CRU metabolic kitchen (Supplementary Fig. 1). The weight-maintenance meals (diet composition: 20% protein, 30% fat, 50% carbohydrate) were monitored daily to ensure that the correct calorie level was achieved. Upon admission to the CRU, no calories were consumed after 2100 h to achieve a 10-h fast before the two-stage insulin euglycemic pancreatic clamp the following morning, as previously published (26), with modifications as follows: the following morning at 0400 h, a primed [6,62H2]glucose bolus (6 mg ⋅ kg fat-free mass[FFM]−1) was administered, followed by a 9-h continuous infusion of [6,62H2]glucose (started at 4 mg ⋅ kgFFM−1 ⋅ h−1 then titrated downward over the infusion time period to match anticipated changes in endogenous glucose production [EGP]). At 0600 h, gas exchange was measured by indirect calorimetry for 30 min for REE determination. Then at 0700 h, glucagon (0.001 μg ⋅ kgFFM−1 ⋅ min−1), somatostatin (0.093 μg ⋅ kgFFM−1 ⋅ min−1), and growth hormone (0.0047 μg ⋅ kgFFM−1 ⋅ min−1) were infused for 6 h. Insulin was infused from 0700 to 1000 h at 0.62 mU ⋅ kgFFM−1 ⋅ min−1 and then from 1000 to 1300 h at 2.3 mU ⋅ kgFFM−1 ⋅ min−1. A 40% dextrose with 2% enrichment of [6,62H2]glucose was infused as needed to maintain blood glucose above 4.7 mmol/L from 0700 to 1000 h and then between 4.7 and 5.3 mmol/L from 1000 to 1300 h.

Sample Preparation:

Sampleprep ID:	SP000513
Sampleprep Summary:	Glucose concentration was measured every 10 min during the insulin clamp with an Analox glucose analyzer (Analox Instruments, London, U.K.). [6,6-2H2]-d-glucose enrichment in the plasma and infusate was measured using gas chromatography–mass spectrometry. As described previously, the steady-state equations of Steele et al. (29) were used to calculate the rate of glucose appearance (Ra) and disappearance (Rd). EGP was calculated as the difference between total glucose Ra and the exogenous glucose infusion rate, peripheral insulin sensitivity was assessed from the rate of glucose infusion required to maintain euglycemia during the high-dose insulin clamp, and hepatic insulin sensitivity was assessed by the extent to which EGP was suppressed from baseline to low-dose hyperinsulinemia (26).

Sampleprep ID:

SP000513

Sampleprep Summary:

Glucose concentration was measured every 10 min during the insulin clamp with an Analox glucose analyzer (Analox Instruments, London, U.K.). [6,6-2H2]-d-glucose enrichment in the plasma and infusate was measured using gas chromatography–mass spectrometry. As described previously, the steady-state equations of Steele et al. (29) were used to calculate the rate of glucose appearance (Ra) and disappearance (Rd). EGP was calculated as the difference between total glucose Ra and the exogenous glucose infusion rate, peripheral insulin sensitivity was assessed from the rate of glucose infusion required to maintain euglycemia during the high-dose insulin clamp, and hepatic insulin sensitivity was assessed by the extent to which EGP was suppressed from baseline to low-dose hyperinsulinemia (26).

Combined analysis:

Analysis ID	AN000751
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent
Column	Agilent DB5-MS (30m × 0.25mm, 0.25um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5973
Ion Mode	POSITIVE
Units	mole percent enrichment

Chromatography:

Chromatography ID:	CH000539
Chromatography Summary:	Glucose concentration was measured every 10 min during the insulin clamp with an Analox glucose analyzer (Analox Instruments, London, U.K.). [6,6-2H2]-d-glucose enrichment in the plasma and infusate was measured using gas chromatography–mass spectrometry. As described previously, the steady-state equations of Steele et al. (29) were used to calculate the rate of glucose appearance (Ra) and disappearance (Rd). EGP was calculated as the difference between total glucose Ra and the exogenous glucose infusion rate, peripheral insulin sensitivity was assessed from the rate of glucose infusion required to maintain euglycemia during the high-dose insulin clamp, and hepatic insulin sensitivity was assessed by the extent to which EGP was suppressed from baseline to low-dose hyperinsulinemia (26).
Instrument Name:	Agilent
Column Name:	Agilent DB5-MS (30m × 0.25mm, 0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS000665
Analysis ID:	AN000751
Instrument Name:	Agilent 5973
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE