{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001145","ANALYSIS_ID":"AN001890","VERSION":"1","CREATED_ON":"March 4, 2019, 5:55 pm"},

"PROJECT":{"PROJECT_TITLE":"UPLC-MS Analysis of Lipids From Insulin Resistant Femoral Muscles of Diet-induced Obese Mice","PROJECT_TYPE":"Lipidomics","PROJECT_SUMMARY":"Muscle insulin resistance is a fundamental contributor in the pathogenesis of obesity-related diseases like type 2 diabetes. Increased triglyceride concentration in muscle tissue, as seen with obesity, is associated with inhibition of insulin action and decreased glucose uptake. Here we use liquid chromatography paired with mass spectrometry (LCMS) to identify patterns of lipid species in femoral muscle of mice associated with diet-induced insulin resistance. Mice were fed a standard CHOW diet for 5 weeks or HFD for 5 or 13 weeks. 806 lipids were significantly different (p ≤ 0.05) between HFD-induced insulin resistant muscle and CHOW insulin sensitive. Of these 217 lipid species were quantified and annotated based on principle components analysis, significance (p ≤ 0.01) and fold change of relative abundance values. CHOW insulin sensitive muscle was associated with triglycerides and phospholipids that contained higher abundance of long-chain highly unsaturated fatty acids. Serine and inositol phospholipids favored insulin sensitive femoral muscle, yet higher abundance also occurred in 13 week HFD mice compared with 5 week. Consequently, phospholipid imbalance may be indicative of cell membrane dysfunction. HFD insulin resistant femoral muscle contained triglycerides with less carbons, compared with CHOW, which were predominantly saturated. In addition, there was greater abundance of diacylglycerides and sphingomyelin, but not ceramides. Extending HFD intake to 13 weeks did not cause increased abundance of deleterious lipids with the exception of sphingomyelin. Overall, distinct lipid combinations, perhaps even ratios, should be characterized when identifying what contributes to the maintenance or dysregulation of muscle insulin sensitivity.","INSTITUTE":"Colorado State University","DEPARTMENT":"Food Science and Human Nutrition","LABORATORY":"Adipose Tissue","LAST_NAME":"Foster","FIRST_NAME":"Michelle","ADDRESS":"1571 Campus Delivery, Fort Collins Colorado","EMAIL":"Michelle.Foster@colostate.edu","PHONE":"970-491-6189","FUNDING_SOURCE":"NIH NIDDK"},

"STUDY":{"STUDY_TITLE":"UPLC-MS Analysis of Lipids From Insulin Resistant Femoral Muscles of Diet-induced Obese Mice","STUDY_TYPE":"Lipidomics, Basic Research","STUDY_SUMMARY":"Muscle insulin resistance is a fundamental contributor in the pathogenesis of obesity-related diseases like type 2 diabetes. Increased triglyceride concentration in muscle tissue, as seen with obesity, is associated with inhibition of insulin action and decreased glucose uptake. Here we use liquid chromatography paired with mass spectrometry (LCMS) to identify patterns of lipid species in femoral muscle of mice associated with diet-induced insulin resistance. Mice were fed a standard CHOW diet for 5 weeks or HFD for 5 or 13 weeks. 806 lipids were significantly different (p ≤ 0.05) between HFD-induced insulin resistant muscle and CHOW insulin sensitive. Of these 217 lipid species were quantified and annotated based on principle components analysis, significance (p ≤ 0.01) and fold change of relative abundance values. CHOW insulin sensitive muscle was associated with triglycerides and phospholipids that contained higher abundance of long-chain highly unsaturated fatty acids. Serine and inositol phospholipids favored insulin sensitive femoral muscle, yet higher abundance also occurred in 13 week HFD mice compared with 5 week. Consequently, phospholipid imbalance may be indicative of cell membrane dysfunction. HFD insulin resistant femoral muscle contained triglycerides with less carbons, compared with CHOW, which were predominantly saturated. In addition, there was greater abundance of diacylglycerides and sphingomyelin, but not ceramides. Extending HFD intake to 13 weeks did not cause increased abundance of deleterious lipids with the exception of sphingomyelin. Overall, distinct lipid combinations, perhaps even ratios, should be characterized when identifying what contributes to the maintenance or dysregulation of muscle insulin sensitivity.","INSTITUTE":"Colorado State University","DEPARTMENT":"Food Science and Human Nutrition","LABORATORY":"Adipose Tissue","LAST_NAME":"Foster","FIRST_NAME":"Michelle","ADDRESS":"1571 Campus Delivery, Fort Collins, Colorado 80523","EMAIL":"Michelle.Foster@colostate.edu","PHONE":"9704916189","NUM_GROUPS":"3","TOTAL_SUBJECTS":"21","NUM_MALES":"21"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"C57BL6","AGE_OR_AGE_RANGE":"2-3 months","WEIGHT_OR_WEIGHT_RANGE":"28-46","GENDER":"Female","ANIMAL_ANIMAL_SUPPLIER":"Jackson Laboratory","ANIMAL_HOUSING":"Single House","ANIMAL_LIGHT_CYCLE":"12:12","ANIMAL_FEED":"CHOW diet (Envigo Teklad 6% fat 7002, Madison, WI) and Western (H.D  high-fat, high-sugar; 21% milk fat and 34% sucrose (Envigo TD.08811)"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S1-D1-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"2",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S1-D2-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"3",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S1-D2-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"4",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S13-D1-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"5",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S13-D2-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"6",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S19-D1-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"7",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S19-D1-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"8",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S19-D2-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"9",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S19-D2-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"10",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S25-D1-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"11",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S25-D1-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"12",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S25-D2-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"13",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S31-D1-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"14",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S37-D1-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"15",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S37-D2-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"16",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S37-D2-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"17",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S43-D1-W2-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"18",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S49-D1-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"19",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S55-D2-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"20",
"Factors":{"Diet":"HFD","Time":"5week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S7-D1-W1-T1-M1"}
},
{
"Subject ID":"-",
"Sample ID":"21",
"Factors":{"Diet":"CHOW","Time":"13week"},
"Additional sample data":{"Tissue":"Control","Fat Muscle":"Top","Label":"S7-D2-W1-T1-M1"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Approximately 20 mg of muscle tissue was homogenized in a glass homogenizer with 1.5 ml of 2:1 chloroform:methanol and then brought to 4 ml using the same ratio. The mixture was poured through a 2V grade qualitative 12.5 cm Whatman filter into a clean 10 ml glass tube. The volume in the tube was again brought up to 4 ml with the same 2:1 solution as above. One ml of water was added to the tube, vortexed for 20 seconds, and then centrifuged for 10 minutes at 2500 rpm. The top non-lipid portion was removed and the lower lipid-containing layer was dried under nitrogen.","SAMPLE_TYPE":"Muscle","COLLECTION_METHOD":"excision","COLLECTION_LOCATION":"femoral muscle","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Male C57BL/6 mice, 3 months of age, (Jackson Laboratory, Bar Harbor, Maine) were allowed to acclimate for one week before experiment start. Mice were individually housed under controlled conditions (12:12 light-dark cycle, 50–60% humidity, and 25° C) and had ad libitum access to standard CHOW diet (Envigo Teklad 6% fat 7002, Madison, WI). Lipids in the CHOW diet consisted of an assortment of fatty acids where linoleic > oleic > palmitic > linolenic > stearic. Following a baseline glucose tolerance test (GTT), mice were grouped according to mean GTT and body mass into a standard 5 week CHOW (n = 10) or Western (H.D  high-fat, high-sugar; 21% milk fat and 34% sucrose (Envigo TD.08811); 5 (n = 5) and 13 week (n = 6)) diet group. The saturated fatty acids in HFD ranged from 4:0 to 18:0, however, palmitate (16:0) followed by steric (18:0) and myristic (14;0) where highest in quantity.","TREATMENT":"Diet","TREATMENT_COMPOUND":"Envigo TD.08811","TREATMENT_ROUTE":"oral"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Approximately 20 mg of muscle tissue was homogenized in a glass homogenizer with 1.5 ml of 2:1 chloroform:methanol and then brought to 4 ml using the same ratio. The mixture was poured through a 2V grade qualitative 12.5 cm Whatman filter into a clean 10 ml glass tube. The volume in the tube was again brought up to 4 ml with the same 2:1 solution as above. One ml of water was added to the tube, vortexed for 20 seconds, and then centrifuged for 10 minutes at 2500 rpm. The top non-lipid portion was removed and the lower lipid-containing layer was dried under nitrogen.Lipid extracts were suspended in 100 uL of 2:1 Chloroform:Methanol. Injections were normalized such that equal amounts of lipid were analyzed for each sample, regardless of total lipid content of diet. 3 μL of extract was injected twice (n=2 replicates) onto a Waters Acquity UPLC system in discrete, randomized blocks. Next samples were separated using a Waters Acquity UPLC CSH Phenyl Hexyl column (1.7 µM, 1.0 x 100 mm), using a gradient from solvent A (water, 0.1% formic acid) to solvent B (Acetonitrile, 0.1% formic acid). Injections were made in 100% A, held at 100% A for 1 min, ramped to 98% B over 12 minutes, held at 98% B for 3 minutes, and then returned to starting conditions over 0.05 minutes and allowed to re-equilibrate for 3.95 minutes, with a 200 µL/min constant flow rate. The column and samples were held at 65 °C and 6 °C, respectively. The column eluent was infused into a Waters Xevo G2 TOF-MS with an electrospray source in positive mode, scanning 50-2000 m/z at 0.2 seconds per scan, alternating between MS (6 V collision energy) and MSE mode (15-30 V ramp). Calibration was performed using sodium iodide with 1 ppm mass accuracy. The capillary voltage was held at 2200 V, source temp at 150 °C, and nitrogen desolvation temp at 350 °C with a flow rate of 800 L/hr.","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACT_STORAGE":"On ice"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity UPLC","COLUMN_NAME":"Acquity CSH PhenylHexyl","FLOW_GRADIENT":"water + 0.1% Formic + 2 mM AmOH / Acetonitrile","FLOW_RATE":"200 uL/min","SOLVENT_A":"water + 0.1% Formic + 2 mM AmOH","SOLVENT_B":"Acetonitrile"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Waters Synapt G2 XS QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Binary Data Format: .cdf"}

}