Summary of Study ST000483

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 PR000367. The data can be accessed directly via it's Project DOI: 10.21228/M8MC7X This work is supported by NIH grant, U2C- DK119886.

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

Study ID	ST000483
Study Title	Amino Acid Quantifcation of obese patients on a 16 week caloric restriction from Plasma
Study Type	timecourse, quantitative measurements of amino acid
Study Summary	Caloric restriction (CR) improves insulin sensitivity and reduces the incidence of diabetes in obese individuals. The underlying mechanisms whereby CR improves insulin sensitivity are not clear. We evaluated the effect of 16 weeks of CR on whole-body insulin sensitivity by pancreatic clamp before and after CR in 11 obese participants (BMI = 35 kg/m2) compared with 9 matched control subjects (BMI = 34 kg/m2). Compared with the control subjects, CR increased the glucose infusion rate needed to maintain euglycemia during hyperinsulinemia, indicating enhancement of peripheral insulin sensitivity. This improvement in insulin sensitivity was not accompanied by changes in skeletal muscle mitochondrial oxidative capacity or oxidant emissions, nor were there changes in skeletal muscle ceramide, diacylglycerol, or amino acid metabolite levels. However, CR lowered insulin-stimulated thioredoxin-interacting protein (TXNIP) levels and enhanced nonoxidative glucose disposal. These results support a role for TXNIP in mediating the improvement in peripheral insulin sensitivity after CR.
Institute	Mayo Clinic
Department	Endocrinology
Laboratory	Mayo Clinic Metabolomics Resource Core
Last Name	Nair
First Name	Sreekumaran
Address	200 First Street SW, Rochester, MN 55905
Email	Nair.K@mayo.edu
Phone	507-285-2415
Submit Date	2016-09-23
Publications	Mechanism by Which Caloric Restriction Improves Insulin Sensitivity in Sedentary Obese Adults. DOI: 10.2337/db15-0675
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2016-12-22
Release Version	1

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Project:

Project ID:	PR000367
Project DOI:	doi: 10.21228/M8MC7X
Project Title:	Mechanism by Which Caloric Restriction Improves Insulin Sensitivity in Sedentary Obese Adults
Project Type:	skeletal muscle ceramide, diacylglycerol, or amino acid metabolite levels
Project Summary:	effect of caloric restriction on insulin sensitivity through skeletal muscle ceramide, diacylglycerol, or amino acid metabolite levels
Institute:	Mayo Clinic
Department:	Endocrinology
Laboratory:	Mayo Clinic Metabolomics Resource Core
Last Name:	Nair
First Name:	Sreekumaran
Address:	200 First Street SW, Rochester, MN 55905
Email:	Nair.K@mayo.edu
Phone:	507-285-2415

Subject:

Subject ID:	SU000504
Subject Type:	Animal
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Subject type: Animal; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	time (mins)	Category
SA024582	ms5361-11	1	Caloric Restriction
SA024583	ms5361-33	1	Caloric Restriction
SA024584	ms5361-31	1	Caloric Restriction
SA024585	ms5361-23	1	Caloric Restriction
SA024586	ms5361-1	1	Caloric Restriction
SA024587	ms5361-19	1	Caloric Restriction
SA024588	ms5361-13	1	Caloric Restriction
SA024589	ms5361-9	1	Caloric Restriction
SA024590	ms5361-5	1	Caloric Restriction
SA024591	ms5361-3	1	Caloric Restriction
SA024592	ms5361-7	1	Caloric Restriction
SA024593	ms5361-39	1	Control
SA024594	ms5361-25	1	Control
SA024595	ms5361-29	1	Control
SA024596	ms5361-37	1	Control
SA024597	ms5361-35	1	Control
SA024598	ms5361-27	1	Control
SA024599	ms5361-21	1	Control
SA024600	ms5361-17	1	Control
SA024601	ms5361-15	1	Control
SA024602	ms5361-12	2	Caloric Restriction
SA024603	ms5361-20	2	Caloric Restriction
SA024604	ms5361-32	2	Caloric Restriction
SA024605	ms5361-10	2	Caloric Restriction
SA024606	ms5361-8	2	Caloric Restriction
SA024607	ms5361-34	2	Caloric Restriction
SA024608	ms5361-14	2	Caloric Restriction
SA024609	ms5361-6	2	Caloric Restriction
SA024610	ms5361-24	2	Caloric Restriction
SA024611	ms5361-2	2	Caloric Restriction
SA024612	ms5361-4	2	Caloric Restriction
SA024613	ms5361-38	2	Control
SA024614	ms5361-36	2	Control
SA024615	ms5361-40	2	Control
SA024616	ms5361-30	2	Control
SA024617	ms5361-18	2	Control
SA024618	ms5361-22	2	Control
SA024619	ms5361-16	2	Control
SA024620	ms5361-26	2	Control
SA024621	ms5361-28	2	Control

Showing results 1 to 40 of 40

Showing results 1 to 40 of 40

Collection:

Collection ID:	CO000498
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. Plasma was isolated for MS analysis.

Treatment:

Treatment ID:	TR000518
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:

TR000518

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:	SP000511
Sampleprep Summary:	Plasma samples and amino acid calibration standards were prepared with MassTrak Amino Acid Analysis Solution (AAA) kit from Waters according to instructions with slight modifications for detection on a mass spectrometer. A 10 point standard concentration curve was made from the calibration standard solution to calculate amino acid concentrations in plasma samples. A solution containing U-13C4-L-aspartic acid, U-13C3-L-alanine, U-13C4-L-threonine, U-13C5-L-proline, U-13C5-L-valine, U-13C6-leucine, U-13C6-phenylalanine all from Cambridge Isotope Laboratories, 13C6-tyrosine from Isotec, L-arginine (15N2, 2H2) from MassTrace, norvaline from Sigma dissolved in 0.01N HCl was used as the internal standard solution. Frozen plasma samples were thawed, spiked with internal standard then deproteinized with cold MeOH followed by centrifugation at 10,000 g for 5 minutes prior to derivatization according to MassTrak instructions. The amino acid derivatizing reagent used was 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

Sampleprep ID:

SP000511

Sampleprep Summary:

Plasma samples and amino acid calibration standards were prepared with MassTrak Amino Acid Analysis Solution (AAA) kit from Waters according to instructions with slight modifications for detection on a mass spectrometer. A 10 point standard concentration curve was made from the calibration standard solution to calculate amino acid concentrations in plasma samples. A solution containing U-13C4-L-aspartic acid, U-13C3-L-alanine, U-13C4-L-threonine, U-13C5-L-proline, U-13C5-L-valine, U-13C6-leucine, U-13C6-phenylalanine all from Cambridge Isotope Laboratories, 13C6-tyrosine from Isotec, L-arginine (15N2, 2H2) from MassTrace, norvaline from Sigma dissolved in 0.01N HCl was used as the internal standard solution. Frozen plasma samples were thawed, spiked with internal standard then deproteinized with cold MeOH followed by centrifugation at 10,000 g for 5 minutes prior to derivatization according to MassTrak instructions. The amino acid derivatizing reagent used was 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

Combined analysis:

Analysis ID	AN000749
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	C18
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Thermo Quantum Ultra
Ion Mode	POSITIVE
Units	micromolar

Chromatography:

Chromatography ID:	CH000537
Chromatography Summary:	High resolution separation was done using an Acquity UPLC system, injecting 1 µl of derviatized solution, with a UPLC BEH C18 1.7 micron 2.1×150 mm column from Waters. Column flow was set to 400 µl/min with a gradient from 99.9%A to 98%B where buffer A is 1% acetonitrile in 0.1% formic acid and buffer B is 100% acetonitrile. A column temp of 43 degrees Celsius and a sample tray temp of 6% Celsius. Mass detection was completed on a TSQ Ultra Quantum from Thermo Finnigan running in ESI positive mode. A scan width of 0.002, scan time of 0.04 seconds per transition mass, collision energy of 25, collision gas pressure of 1.5 mTorr, tube lens value set to 90, monitoring a signature ion of the derivitized amines at m/z 171.04 by selected reaction monitoring.
Instrument Name:	Waters Acquity
Column Name:	C18
Column Temperature:	43
Flow Gradient:	99.9% A to 98% B
Flow Rate:	400 µl/min
Solvent A:	99% water/1% acetonitrile; 0.1% formic acid
Solvent B:	100% acetonitrile
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000663
Analysis ID:	AN000749
Instrument Name:	Thermo Quantum Ultra
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE