#METABOLOMICS WORKBENCH MTFoster_20190118_092702 DATATRACK_ID:1608 STUDY_ID:ST001145 ANALYSIS_ID:AN001890 PROJECT_ID:PR000765 VERSION 1 CREATED_ON March 4, 2019, 5:55 pm #PROJECT PR:PROJECT_TITLE UPLC-MS Analysis of Lipids From Insulin Resistant Femoral Muscles of PR:PROJECT_TITLE Diet-induced Obese Mice PR:PROJECT_TYPE Lipidomics PR:PROJECT_SUMMARY Muscle insulin resistance is a fundamental contributor in the pathogenesis of PR:PROJECT_SUMMARY obesity-related diseases like type 2 diabetes. Increased triglyceride PR:PROJECT_SUMMARY concentration in muscle tissue, as seen with obesity, is associated with PR:PROJECT_SUMMARY inhibition of insulin action and decreased glucose uptake. Here we use liquid PR:PROJECT_SUMMARY chromatography paired with mass spectrometry (LCMS) to identify patterns of PR:PROJECT_SUMMARY lipid species in femoral muscle of mice associated with diet-induced insulin PR:PROJECT_SUMMARY resistance. Mice were fed a standard CHOW diet for 5 weeks or HFD for 5 or 13 PR:PROJECT_SUMMARY weeks. 806 lipids were significantly different (p ≤ 0.05) between HFD-induced PR:PROJECT_SUMMARY insulin resistant muscle and CHOW insulin sensitive. Of these 217 lipid species PR:PROJECT_SUMMARY were quantified and annotated based on principle components analysis, PR:PROJECT_SUMMARY significance (p ≤ 0.01) and fold change of relative abundance values. CHOW PR:PROJECT_SUMMARY insulin sensitive muscle was associated with triglycerides and phospholipids PR:PROJECT_SUMMARY that contained higher abundance of long-chain highly unsaturated fatty acids. PR:PROJECT_SUMMARY Serine and inositol phospholipids favored insulin sensitive femoral muscle, yet PR:PROJECT_SUMMARY higher abundance also occurred in 13 week HFD mice compared with 5 week. PR:PROJECT_SUMMARY Consequently, phospholipid imbalance may be indicative of cell membrane PR:PROJECT_SUMMARY dysfunction. HFD insulin resistant femoral muscle contained triglycerides with PR:PROJECT_SUMMARY less carbons, compared with CHOW, which were predominantly saturated. In PR:PROJECT_SUMMARY addition, there was greater abundance of diacylglycerides and sphingomyelin, but PR:PROJECT_SUMMARY not ceramides. Extending HFD intake to 13 weeks did not cause increased PR:PROJECT_SUMMARY abundance of deleterious lipids with the exception of sphingomyelin. Overall, PR:PROJECT_SUMMARY distinct lipid combinations, perhaps even ratios, should be characterized when PR:PROJECT_SUMMARY identifying what contributes to the maintenance or dysregulation of muscle PR:PROJECT_SUMMARY insulin sensitivity. PR:INSTITUTE Colorado State University PR:DEPARTMENT Food Science and Human Nutrition PR:LABORATORY Adipose Tissue PR:LAST_NAME Foster PR:FIRST_NAME Michelle PR:ADDRESS 1571 Campus Delivery, Fort Collins Colorado PR:EMAIL Michelle.Foster@colostate.edu PR:PHONE 970-491-6189 PR:FUNDING_SOURCE NIH NIDDK #STUDY ST:STUDY_TITLE UPLC-MS Analysis of Lipids From Insulin Resistant Femoral Muscles of ST:STUDY_TITLE Diet-induced Obese Mice ST:STUDY_TYPE Lipidomics, Basic Research ST:STUDY_SUMMARY Muscle insulin resistance is a fundamental contributor in the pathogenesis of ST:STUDY_SUMMARY obesity-related diseases like type 2 diabetes. Increased triglyceride ST:STUDY_SUMMARY concentration in muscle tissue, as seen with obesity, is associated with ST:STUDY_SUMMARY inhibition of insulin action and decreased glucose uptake. Here we use liquid ST:STUDY_SUMMARY chromatography paired with mass spectrometry (LCMS) to identify patterns of ST:STUDY_SUMMARY lipid species in femoral muscle of mice associated with diet-induced insulin ST:STUDY_SUMMARY resistance. Mice were fed a standard CHOW diet for 5 weeks or HFD for 5 or 13 ST:STUDY_SUMMARY weeks. 806 lipids were significantly different (p ≤ 0.05) between HFD-induced ST:STUDY_SUMMARY insulin resistant muscle and CHOW insulin sensitive. Of these 217 lipid species ST:STUDY_SUMMARY were quantified and annotated based on principle components analysis, ST:STUDY_SUMMARY significance (p ≤ 0.01) and fold change of relative abundance values. CHOW ST:STUDY_SUMMARY insulin sensitive muscle was associated with triglycerides and phospholipids ST:STUDY_SUMMARY that contained higher abundance of long-chain highly unsaturated fatty acids. ST:STUDY_SUMMARY Serine and inositol phospholipids favored insulin sensitive femoral muscle, yet ST:STUDY_SUMMARY higher abundance also occurred in 13 week HFD mice compared with 5 week. ST:STUDY_SUMMARY Consequently, phospholipid imbalance may be indicative of cell membrane ST:STUDY_SUMMARY dysfunction. HFD insulin resistant femoral muscle contained triglycerides with ST:STUDY_SUMMARY less carbons, compared with CHOW, which were predominantly saturated. In ST:STUDY_SUMMARY addition, there was greater abundance of diacylglycerides and sphingomyelin, but ST:STUDY_SUMMARY not ceramides. Extending HFD intake to 13 weeks did not cause increased ST:STUDY_SUMMARY abundance of deleterious lipids with the exception of sphingomyelin. Overall, ST:STUDY_SUMMARY distinct lipid combinations, perhaps even ratios, should be characterized when ST:STUDY_SUMMARY identifying what contributes to the maintenance or dysregulation of muscle ST:STUDY_SUMMARY insulin sensitivity. ST:INSTITUTE Colorado State University ST:DEPARTMENT Food Science and Human Nutrition ST:LABORATORY Adipose Tissue ST:LAST_NAME Foster ST:FIRST_NAME Michelle ST:ADDRESS 1571 Campus Delivery, Fort Collins, Colorado 80523 ST:EMAIL Michelle.Foster@colostate.edu ST:PHONE 9704916189 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 21 ST:NUM_MALES 21 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL6 SU:AGE_OR_AGE_RANGE 2-3 months SU:WEIGHT_OR_WEIGHT_RANGE 28-46 SU:GENDER Female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratory SU:ANIMAL_HOUSING Single House SU:ANIMAL_LIGHT_CYCLE 12:12 SU:ANIMAL_FEED CHOW diet (Envigo Teklad 6% fat 7002, Madison, WI) and Western (H.D high-fat, SU:ANIMAL_FEED high-sugar; 21% milk fat and 34% sucrose (Envigo TD.08811) #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S1-D1-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 2 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S1-D2-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 3 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S1-D2-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 4 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S13-D1-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 5 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S13-D2-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 6 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S19-D1-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 7 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S19-D1-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 8 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S19-D2-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 9 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S19-D2-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 10 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S25-D1-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 11 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S25-D1-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 12 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S25-D2-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 13 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S31-D1-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 14 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S37-D1-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 15 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S37-D2-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 16 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S37-D2-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 17 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S43-D1-W2-T1-M1 SUBJECT_SAMPLE_FACTORS - 18 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S49-D1-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 19 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S55-D2-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 20 Diet:HFD | Time:5week Tissue=Control; Fat Muscle=Top; Label=S7-D1-W1-T1-M1 SUBJECT_SAMPLE_FACTORS - 21 Diet:CHOW | Time:13week Tissue=Control; Fat Muscle=Top; Label=S7-D2-W1-T1-M1 #COLLECTION CO:COLLECTION_SUMMARY Approximately 20 mg of muscle tissue was homogenized in a glass homogenizer with CO:COLLECTION_SUMMARY 1.5 ml of 2:1 chloroform:methanol and then brought to 4 ml using the same ratio. CO:COLLECTION_SUMMARY The mixture was poured through a 2V grade qualitative 12.5 cm Whatman filter CO:COLLECTION_SUMMARY into a clean 10 ml glass tube. The volume in the tube was again brought up to 4 CO:COLLECTION_SUMMARY ml with the same 2:1 solution as above. One ml of water was added to the tube, CO:COLLECTION_SUMMARY vortexed for 20 seconds, and then centrifuged for 10 minutes at 2500 rpm. The CO:COLLECTION_SUMMARY top non-lipid portion was removed and the lower lipid-containing layer was dried CO:COLLECTION_SUMMARY under nitrogen. CO:SAMPLE_TYPE Muscle CO:COLLECTION_METHOD excision CO:COLLECTION_LOCATION femoral muscle CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Male C57BL/6 mice, 3 months of age, (Jackson Laboratory, Bar Harbor, Maine) were TR:TREATMENT_SUMMARY allowed to acclimate for one week before experiment start. Mice were TR:TREATMENT_SUMMARY individually housed under controlled conditions (12:12 light-dark cycle, TR:TREATMENT_SUMMARY 50–60% humidity, and 25° C) and had ad libitum access to standard CHOW diet TR:TREATMENT_SUMMARY (Envigo Teklad 6% fat 7002, Madison, WI). Lipids in the CHOW diet consisted of TR:TREATMENT_SUMMARY an assortment of fatty acids where linoleic > oleic > palmitic > linolenic > TR:TREATMENT_SUMMARY stearic. Following a baseline glucose tolerance test (GTT), mice were grouped TR:TREATMENT_SUMMARY according to mean GTT and body mass into a standard 5 week CHOW (n = 10) or TR:TREATMENT_SUMMARY Western (H.D high-fat, high-sugar; 21% milk fat and 34% sucrose (Envigo TR:TREATMENT_SUMMARY TD.08811); 5 (n = 5) and 13 week (n = 6)) diet group. The saturated fatty acids TR:TREATMENT_SUMMARY in HFD ranged from 4:0 to 18:0, however, palmitate (16:0) followed by steric TR:TREATMENT_SUMMARY (18:0) and myristic (14;0) where highest in quantity. TR:TREATMENT Diet TR:TREATMENT_COMPOUND Envigo TD.08811 TR:TREATMENT_ROUTE oral #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Approximately 20 mg of muscle tissue was homogenized in a glass homogenizer with SP:SAMPLEPREP_SUMMARY 1.5 ml of 2:1 chloroform:methanol and then brought to 4 ml using the same ratio. SP:SAMPLEPREP_SUMMARY The mixture was poured through a 2V grade qualitative 12.5 cm Whatman filter SP:SAMPLEPREP_SUMMARY into a clean 10 ml glass tube. The volume in the tube was again brought up to 4 SP:SAMPLEPREP_SUMMARY ml with the same 2:1 solution as above. One ml of water was added to the tube, SP:SAMPLEPREP_SUMMARY vortexed for 20 seconds, and then centrifuged for 10 minutes at 2500 rpm. The SP:SAMPLEPREP_SUMMARY top non-lipid portion was removed and the lower lipid-containing layer was dried SP:SAMPLEPREP_SUMMARY under nitrogen.Lipid extracts were suspended in 100 uL of 2:1 SP:SAMPLEPREP_SUMMARY Chloroform:Methanol. Injections were normalized such that equal amounts of lipid SP:SAMPLEPREP_SUMMARY were analyzed for each sample, regardless of total lipid content of diet. 3 μL SP:SAMPLEPREP_SUMMARY of extract was injected twice (n=2 replicates) onto a Waters Acquity UPLC system SP:SAMPLEPREP_SUMMARY in discrete, randomized blocks. Next samples were separated using a Waters SP:SAMPLEPREP_SUMMARY Acquity UPLC CSH Phenyl Hexyl column (1.7 µM, 1.0 x 100 mm), using a gradient SP:SAMPLEPREP_SUMMARY from solvent A (water, 0.1% formic acid) to solvent B (Acetonitrile, 0.1% formic SP:SAMPLEPREP_SUMMARY acid). Injections were made in 100% A, held at 100% A for 1 min, ramped to 98% B SP:SAMPLEPREP_SUMMARY over 12 minutes, held at 98% B for 3 minutes, and then returned to starting SP:SAMPLEPREP_SUMMARY conditions over 0.05 minutes and allowed to re-equilibrate for 3.95 minutes, SP:SAMPLEPREP_SUMMARY with a 200 µL/min constant flow rate. The column and samples were held at 65 SP:SAMPLEPREP_SUMMARY °C and 6 °C, respectively. The column eluent was infused into a Waters Xevo G2 SP:SAMPLEPREP_SUMMARY TOF-MS with an electrospray source in positive mode, scanning 50-2000 m/z at 0.2 SP:SAMPLEPREP_SUMMARY seconds per scan, alternating between MS (6 V collision energy) and MSE mode SP:SAMPLEPREP_SUMMARY (15-30 V ramp). Calibration was performed using sodium iodide with 1 ppm mass SP:SAMPLEPREP_SUMMARY accuracy. The capillary voltage was held at 2200 V, source temp at 150 °C, and SP:SAMPLEPREP_SUMMARY nitrogen desolvation temp at 350 °C with a flow rate of 800 L/hr. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity UPLC CH:COLUMN_NAME Acquity CSH PhenylHexyl CH:FLOW_GRADIENT water + 0.1% Formic + 2 mM AmOH / Acetonitrile CH:FLOW_RATE 200 uL/min CH:SOLVENT_A water + 0.1% Formic + 2 mM AmOH CH:SOLVENT_B Acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 XS QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Binary Data Format: .cdf #_1 MS:MS_RESULTS_FILE ST001145_AN001890_Results.txt UNITS:Relative Abundance Has m/z:No Has RT:No RT units:No RT data #END