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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000115 |
| Study Title | Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice |
| Study Type | Insulin depravation |
| Study Summary | Experiments 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 |
| Department | Endocrinology |
| Last Name | Nair |
| First Name | Sreekumaran |
| Dasari.Surendra@mayo.edu | |
| Submit Date | 2014-09-30 |
| Num Groups | 3 |
| Total Subjects | 39 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Uploaded File Size | 24 M |
| Analysis Type Detail | LC-MS |
| Release Date | 2015-02-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | Mammal |
Factors:
Subject type: Animal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA006096 | C3 | control |
| SA006097 | C51 | control |
| SA006098 | C4 | control |
| SA006099 | C6 | control |
| SA006100 | C38 | control |
| SA006101 | C35 | control |
| SA006102 | C50 | control |
| SA006103 | C2 | control |
| SA006104 | C44 | control |
| SA006105 | C49 | control |
| SA006106 | C45 | control |
| SA006107 | C5 | control |
| SA006108 | C47 | control |
| SA006109 | C48 | control |
| SA006110 | D+32 | diabetic treated |
| SA006111 | D+52 | diabetic treated |
| SA006112 | D+24 | diabetic treated |
| SA006113 | D+21 | diabetic treated |
| SA006114 | D+14 | diabetic treated |
| SA006115 | D+23 | diabetic treated |
| SA006116 | D+70 | diabetic treated |
| SA006117 | D+57 | diabetic treated |
| SA006118 | D+58 | diabetic treated |
| SA006119 | D+53 | diabetic treated |
| SA006120 | D+56 | diabetic treated |
| SA006121 | D+55 | diabetic treated |
| SA006122 | D+54 | diabetic treated |
| SA006123 | D-69 | diabetic untreated |
| SA006124 | D-68 | diabetic untreated |
| SA006125 | D-41 | diabetic untreated |
| SA006126 | D-36 | diabetic untreated |
| SA006127 | D-39 | diabetic untreated |
| SA006128 | D-33 | diabetic untreated |
| SA006129 | D-31 | diabetic untreated |
| SA006130 | D-25 | diabetic untreated |
| SA006131 | D-28 | diabetic untreated |
| SA006132 | D-40 | diabetic untreated |
| SA006133 | D-42 | diabetic untreated |
| SA006134 | D-63 | diabetic untreated |
| SA006135 | D-64 | diabetic untreated |
| SA006136 | D-61 | diabetic untreated |
| SA006137 | D-60 | diabetic untreated |
| SA006138 | D-43 | diabetic untreated |
| SA006139 | D-67 | diabetic 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 |
