Summary of Study ST000115

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 PR000104. The data can be accessed directly via it's Project DOI: 10.21228/M80W2M This work is supported by NIH grant, U2C- DK119886.

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Study IDST000115
Study TitleImpact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice
Study TypeInsulin depravation
Study SummaryExperiments were conducted using 13-wk-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME). Mice were housed individually with free access to water and chow (TD.10112; Harlan Laboratories, Indianapolis, IN), with a 12:12-h light-dark cycle and temperature and humidity control. Mice were acclimated for 1 wk prior to the beginning of the experiment. The protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Following a 6-h fast, mice were given intraperitoneal injections of STZ (125 mg/kg; in sodium acetate buffer, pH = 4.5) (67). Injections were repeated on the following day. Control animals received intraperitoneal injection of vehicle. Only mice that displayed blood glucose ?300 mg/dl and an increase in blood ketones (both values by Precision Xtra glucometer; Abbott Laboratories, Abbott Park, IL), hyperphagia, and polyuria and were positive for urine glucose presence via dipstick (Uristix, Bayer, Pittsburgh, PA) on day 7 after the first STZ dose were included in the experiment. Animals that were positive for STZ diabetes received LinBit subcutaneous insulin implant (LinShin Canada, Toronto, ON, Canada) (79) under pentobarbital sodium anesthesia (Nebutal, 40 mg/kg of body wt) according to the manufacturer's protocol. Each animal received two subcutaneous implants (total dose: 0.2 U/24 h for >30 days, 10 U/kg for 20-g mice). Insulin treatment was continued for 3 wk. Control animals (C; n = 13) received blank implants. Diabetic control was confirmed by biweekly measurements of blood and urinary glucose. In some cases, when urine glucose was present and blood glucose was >288 mg/dl, the animal received a third implant. The insulin treatment was continued until initially lower plasma glucose content in diabetic animals reached control values. Three weeks following implantation, diabetic mice were divided randomly into diabetic-treated (D + I; n = 13) and diabetic-deprived (D ? I; n = 13) groups. Insulin implants were removed from the D ? I group under pentobarbital anesthesia, which led to the return of the diabetic phenotype within 24 h. Animals from the D + I group continued on insulin treatment (Fig. 1). At the age of 18 wk, animals from all groups were analyzed for body composition by an Echo-MRI Body Composition Analyzer (EchoMRI, Houston, TX) and euthanized by decapitation 5 wk after the initial STZ or vehicle dose. Figure 1 depicts the timeline of the experiment and blood glucose profiles for each experimental group. Additional animals were used for estimation of skeletal muscle insulin sensitivity by acute insulin stimulation. The mice were divided into the C (n = 6), D ? I (n = 7), and D + I (n = 7) groups and followed appropriate experimental treatment, except for acute insulin stimulation 10 min prior to euthanization by pentobarbital overdose. Figure 1 of the attached PDF of the article summarizes the study design
Institute
Mayo Clinic
DepartmentEndocrinology
Last NameNair
First NameSreekumaran
EmailDasari.Surendra@mayo.edu
Submit Date2014-09-30
Num Groups3
Total Subjects39
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Uploaded File Size24 M
Analysis Type DetailLC-MS
Release Date2015-02-03
Release Version1
Sreekumaran Nair Sreekumaran Nair
https://dx.doi.org/10.21228/M80W2M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000104
Project DOI:doi: 10.21228/M80W2M
Project Title:Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice.
Project Type:Targeted metabolomics
Project Summary:Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Akt? and GYS3? phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3? when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.
Institute:Mayo Clinic
Department:Endocrinology
Laboratory:Dr. Sreekumaran Nair's lab
Last Name:Nair
First Name:Sreekumaran
Email:Dasari.Surendra@mayo.edu
Funding Source:R01-DK-41973, UL1 TR000135, the David Murdock Dole Professorship (K. S. Nair), and the Stephenson Fellowship (P. Zabielski).
Project Comments:24368672

Subject:

Subject ID:SU000134
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:13-wk-old
Gender:Male
Species Group:Mammals

Factors:

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

mb_sample_id local_sample_id Treatment
SA006096C3control
SA006097C51control
SA006098C4control
SA006099C6control
SA006100C38control
SA006101C35control
SA006102C50control
SA006103C2control
SA006104C44control
SA006105C49control
SA006106C45control
SA006107C5control
SA006108C47control
SA006109C48control
SA006110D+32diabetic treated
SA006111D+52diabetic treated
SA006112D+24diabetic treated
SA006113D+21diabetic treated
SA006114D+14diabetic treated
SA006115D+23diabetic treated
SA006116D+70diabetic treated
SA006117D+57diabetic treated
SA006118D+58diabetic treated
SA006119D+53diabetic treated
SA006120D+56diabetic treated
SA006121D+55diabetic treated
SA006122D+54diabetic treated
SA006123D-69diabetic untreated
SA006124D-68diabetic untreated
SA006125D-41diabetic untreated
SA006126D-36diabetic untreated
SA006127D-39diabetic untreated
SA006128D-33diabetic untreated
SA006129D-31diabetic untreated
SA006130D-25diabetic untreated
SA006131D-28diabetic untreated
SA006132D-40diabetic untreated
SA006133D-42diabetic untreated
SA006134D-63diabetic untreated
SA006135D-64diabetic untreated
SA006136D-61diabetic untreated
SA006137D-60diabetic untreated
SA006138D-43diabetic untreated
SA006139D-67diabetic untreated
Showing results 1 to 44 of 44

Collection:

Collection ID:CO000118
Collection Summary:Mitochondria were isolated from quadriceps muscle by differential centrifugation, as described previously (38). Briefly, quadriceps muscle samples were homogenized on ice using a motor-driven Potter-Elvehjem tissue grinder. After initial centrifugation, the supernatant containing the mitochondrial and sarcoplasmic fraction was transferred to a chilled microcentrifuge tube and centrifuged at 10,000 g for 2 min to pellet mitochondria. The supernatant containing sarcoplasmic fraction was frozen for further analysis. Mitochondrial pellet was washed twice by resuspending/centrifugation and finally suspended in a mitochondrial storage buffer. The levels of both the LCFa-CoA and sphingolipids in homogenates and various muscle fractions were normalized to total protein content, as measured by 660 nm Protein Assay (Thermo Scientific; Pierce Protein Biology Products, Rockford, IL).
Sample Type:Blood

Treatment:

Treatment ID:TR000136
Treatment Summary:Control/Diabetic; insulin treated/Diabetic; insulin deprived
Treatment Compound:blank/Insulin/Insulin
Treatment Route:Skin implants
Animal Anesthesia:phenobarbital
Animal Endp Euthanasia:5 weeks after treatment
Animal Endp Tissue Coll List:plasma, muscle, liver and skin
Animal Endp Tissue Proc Method:see attached reference publication PDF

Sample Preparation:

Sampleprep ID:SP000131
Sampleprep Summary:Mitochondria were isolated from quadriceps muscle by differential centrifugation, as described previously (38). Briefly, quadriceps muscle samples were homogenized on ice using a motor-driven Potter-Elvehjem tissue grinder. After initial centrifugation, the supernatant containing the mitochondrial and sarcoplasmic fraction was transferred to a chilled microcentrifuge tube and centrifuged at 10,000 g for 2 min to pellet mitochondria. The supernatant containing sarcoplasmic fraction was frozen for further analysis. Mitochondrial pellet was washed twice by resuspending/centrifugation and finally suspended in a mitochondrial storage buffer. The levels of both the LCFa-CoA and sphingolipids in homogenates and various muscle fractions were normalized to total protein content, as measured by 660 nm Protein Assay (Thermo Scientific; Pierce Protein Biology Products, Rockford, IL). / Plasma free fatty acid concentrations were measured by liquid chromatography/mass spectrometry (LC/MS), as described previously (51). Briefly, 50 ?l of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M ? H]? ions. plasma LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 × 150 mm, 1.7 ?m (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids.
Sampleprep Protocol Filename:PMID-24368672-Zabielski-Nair-AJPEM-2014.pdf
Sampleprep Protocol Comments:Pubmed ID: 24368672

Combined analysis:

Analysis ID AN000195 AN000196
Analysis type MS MS
Chromatography type
Chromatography system
Column
MS Type ESI ESI
MS instrument type Triple quadrupole Triple quadrupole
MS instrument name ABI Sciex API 5000 QQQ Thermo TSQ Quantum Ultra
Ion Mode NEGATIVE POSITIVE
Units uM uM

Chromatography:

Chromatography ID:CH000129
Chromatography Summary:Plasma free fatty acid concentrations were measured by liquid chromatography/mass spectrometry (LC/MS), as described previously (51). Briefly, 50 ?l of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M ? H]? ions. LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 150 mm, 1.7 ?m (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids.
Methods Filename:PMID-24368672-Zabielski-Nair-AJPEM-2014.pdf
Chromatography Comments:Pubmed ID: 24368672

MS:

MS ID:MS000158
Analysis ID:AN000195
Instrument Name:ABI Sciex API 5000 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Briefly, 50 ul of plasma was spiked with heptadecanoate internal standard (ISTD) and analyzed with Applied Biosystems (Foster City, CA) API5000 mass spectrometer coupled with a Cohesive (Franklin, MA) TX2 liquid chromatography system. Concentration of individual FFA was measured against a six-point standard curve prepared for each analyte. Both the ISTD and individual FFA standard curves were prepared in 2% fatty acid-free human albumin solution. All analytes were monitored as their [M - H]- ions.
Ion Mode:NEGATIVE
  
MS ID:MS000159
Analysis ID:AN000196
Instrument Name:Thermo TSQ Quantum Ultra
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:LCFa-CoA esters were estimated using the LC-MS/MS method (9). After extraction in the presence of internal standard (20 ng of heptadecanoyl-CoA), samples were analyzed by UHPLC-ESI-MS/MS operating in multiple reaction monitoring mode [Waters Acquity UHPLC, C8 UPLC BEH column 2.1 × 150 mm, 1.7 um (Waters, Milford, MA) and TSQ Quantum Ultra triple-quad mass spectrometer (Thermo Fisher Scientific, Waltham, MA)]. All standard curves were prepared using chemicals from Avanti Polar Lipids.
Ion Mode:POSITIVE
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