#METABOLOMICS WORKBENCH Asaelr_20201007_075605_mwtab.txt DATATRACK_ID:2207 STUDY_ID:ST001508 ANALYSIS_ID:AN002499 PROJECT_ID:000000 VERSION 1 CREATED_ON October 12, 2020, 12:13 pm #PROJECT PR:PROJECT_TITLE Hepatic [U-13C]Lactate tracing and metabolomics in young and old WT and SIRT6 PR:PROJECT_TITLE overexpressing mice PR:PROJECT_TYPE MS analysis PR:PROJECT_SUMMARY Our previous data had suggested that gluconeogenesis capacity from the PR:PROJECT_SUMMARY precursors lactate and glycerol declines in old C57BL/6 mice. This decline was PR:PROJECT_SUMMARY rescued by whole body SIRT6 overexpression, but not in liver-specific SIRT6 PR:PROJECT_SUMMARY overexpression. Here we preformed liver metabolomics in old liver-specific SIRT6 PR:PROJECT_SUMMARY overexpression and control mice. PR:INSTITUTE Bar Ilan University PR:DEPARTMENT The Mina & Everard Goodman Faculty of Life Sciences PR:LABORATORY Laboratory of Prof. Haim Cohen PR:LAST_NAME Cohen PR:FIRST_NAME Chaim PR:ADDRESS Ramat Gan, Ramat Gan, Israel, 5290002, Israel PR:EMAIL Haim.Cohen@biu.ac.il PR:PHONE +97235318383 PR:FUNDING_SOURCE Israel Science Foundation (621/13 and 777/16), I-Core foundation (1775/12) and PR:FUNDING_SOURCE (41/11) and Laura and Isaac Perlmutter Foundation PR:CONTRIBUTORS Asael Roichman, Ifat Abramovich, Eyal Gottlieb, Haim Yosef Cohen #STUDY ST:STUDY_TITLE Liver metabolomics in old liver-specific SIRT6 overexpressing mice ST:STUDY_SUMMARY Our previous data had suggested that gluconeogenesis capacity from the ST:STUDY_SUMMARY precursors lactate and glycerol declines in old C57BL/6 mice. This decline was ST:STUDY_SUMMARY rescued by whole body SIRT6 overexpression, but not in liver-specific SIRT6 ST:STUDY_SUMMARY overexpression. Here we preformed liver metabolomics in old liver-specific SIRT6 ST:STUDY_SUMMARY overexpression and control mice. ST:INSTITUTE Bar Ilan University ST:DEPARTMENT The Mina & Everard Goodman Faculty of Life Sciences ST:LABORATORY Laboratory of Prof. Haim Cohen ST:LAST_NAME Cohen ST:FIRST_NAME Chaim ST:ADDRESS Bar Ilan University, Ramat Gan, Israel, 5290002 ST:EMAIL Haim.Cohen@biu.ac.il ST:PHONE +97235318383 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 20 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6JOlaHsd SU:AGE_OR_AGE_RANGE 23-25 months SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Envigo SU:ANIMAL_HOUSING Animal rooms were maintained at 21°C-24°C and 35%-75% relative humidity SU:ANIMAL_LIGHT_CYCLE 12/12-h (7am to 7pm) dark-light cycle SU:ANIMAL_FEED Altromin 1324 (chow) SU:ANIMAL_WATER Ad lib #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 1 01_Liver_OE Genotype:Liver specific SIRT6 overexpression RAW_FILE_NAME=01_Liver_OE SUBJECT_SAMPLE_FACTORS 2 02_Liver_KISS Genotype:Flox control RAW_FILE_NAME=02_Liver_KISS SUBJECT_SAMPLE_FACTORS 3 03_Liver_OE Genotype:Liver specific SIRT6 overexpression RAW_FILE_NAME=03_Liver_OE SUBJECT_SAMPLE_FACTORS 4 04_Liver_CRE Genotype:Albumin Cre control RAW_FILE_NAME=04_Liver_CRE SUBJECT_SAMPLE_FACTORS 5 05_Liver_WT Genotype:Wild-type RAW_FILE_NAME=05_Liver_WT SUBJECT_SAMPLE_FACTORS 6 06_Liver_KISS Genotype:Flox control RAW_FILE_NAME=06_Liver_KISS SUBJECT_SAMPLE_FACTORS 7 07_Liver_CRE Genotype:Albumin Cre control RAW_FILE_NAME=07_Liver_CRE SUBJECT_SAMPLE_FACTORS 8 08_Liver_CRE Genotype:Albumin Cre control RAW_FILE_NAME=08_Liver_CRE SUBJECT_SAMPLE_FACTORS 9 09_Liver_WT Genotype:Wild-type RAW_FILE_NAME=09_Liver_WT SUBJECT_SAMPLE_FACTORS 10 10_Liver_OE Genotype:Liver specific SIRT6 overexpression RAW_FILE_NAME=10_Liver_OE SUBJECT_SAMPLE_FACTORS 11 11_Liver_KISS Genotype:Flox control RAW_FILE_NAME=11_Liver_KISS SUBJECT_SAMPLE_FACTORS 12 12_Liver_KISS Genotype:Flox control RAW_FILE_NAME=12_Liver_KISS SUBJECT_SAMPLE_FACTORS 13 13_Liver_WT Genotype:Wild-type RAW_FILE_NAME=13_Liver_WT SUBJECT_SAMPLE_FACTORS 14 14_Liver_KISS Genotype:Flox control RAW_FILE_NAME=14_Liver_KISS SUBJECT_SAMPLE_FACTORS 15 15_Liver_WT Genotype:Wild-type RAW_FILE_NAME=15_Liver_WT SUBJECT_SAMPLE_FACTORS 16 16_Liver_CRE Genotype:Albumin Cre control RAW_FILE_NAME=16_Liver_CRE SUBJECT_SAMPLE_FACTORS 17 17_Liver_OE Genotype:Liver specific SIRT6 overexpression RAW_FILE_NAME=17_Liver_OE SUBJECT_SAMPLE_FACTORS 18 18_Liver_OE Genotype:Liver specific SIRT6 overexpression RAW_FILE_NAME=18_Liver_OE SUBJECT_SAMPLE_FACTORS 19 19_Liver_CRE Genotype:Albumin Cre control RAW_FILE_NAME=19_Liver_CRE SUBJECT_SAMPLE_FACTORS 20 20_Liver_WT Genotype:Wild-type RAW_FILE_NAME=20_Liver_WT #COLLECTION CO:COLLECTION_SUMMARY Mice were euthanized by cervical dislocation, left liver lobe was collected as CO:COLLECTION_SUMMARY fast as possible (<30 sec) and immediately flash-frozen in liquid nitrogen. CO:COLLECTION_SUMMARY Tissues were stored at -80C until metabolite extraction. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY All mice were fasted for 6h (8AM-2PM). Tail blood was collected from all mice TR:TREATMENT_SUMMARY for less than 2 minutes, followed by mouse sacrifice. TR:ANIMAL_FASTING 6h morning fast TR:ANIMAL_ENDP_EUTHANASIA Cervical dislocation #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen liver pieces weighting ~30mg were transferred into soft tissue SP:SAMPLEPREP_SUMMARY homogenizing CK 14 tubes containing 1.4 mm ceramic beads (Bertin corp.) SP:SAMPLEPREP_SUMMARY prefilled with 1 ml of cold (-20°C) metabolites extraction solvent (Methanol: SP:SAMPLEPREP_SUMMARY Acetonitrile:H2O, 50:30:20) and kept on ice. Samples were homogenized using SP:SAMPLEPREP_SUMMARY precellys 24 tissue homogenizer (3X20sec at 6000 rpm, with a 30sec gap between SP:SAMPLEPREP_SUMMARY each of the three cycles, Bertin Technologies) cooled to 2°C. Homogenized SP:SAMPLEPREP_SUMMARY extracts were centrifuged in the Precellys tubes at 18,000g for 15 minutes at SP:SAMPLEPREP_SUMMARY 4°C, supernatants were collected in microcentrifuge tubes and centrifuged again SP:SAMPLEPREP_SUMMARY at 18,000g for 10 minutes at 4°C. The supernatants were transferred to glass SP:SAMPLEPREP_SUMMARY HPLC vials and kept at -75 °C prior to LC-MS analysis. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC-HILIC (150 x 2.1mm, 5um) CH:FLOW_RATE 0.2 ml/min CH:COLUMN_TEMPERATURE 45 CH:SOLVENT_A 20 mM ammonium carbonate adjusted to pH 9.2 with 0.1% of 25% ammonium hydroxide CH:SOLVENT_B acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Prof. Eyal Gottlieb AN:OPERATOR_NAME Ifat Abramovich #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS polarity switching mode to enable both positive and negative ions was used #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples 01_Liver_OE 02_Liver_KISS 03_Liver_OE 04_Liver_CRE 05_Liver_WT 06_Liver_KISS 07_Liver_CRE 08_Liver_CRE 09_Liver_WT 10_Liver_OE 11_Liver_KISS 12_Liver_KISS 13_Liver_WT 14_Liver_KISS 15_Liver_WT 16_Liver_CRE 17_Liver_OE 18_Liver_OE 19_Liver_CRE 20_Liver_WT Factors Genotype:Liver specific SIRT6 overexpression Genotype:Flox control Genotype:Liver specific SIRT6 overexpression Genotype:Albumin Cre control Genotype:Wild-type Genotype:Flox control Genotype:Albumin Cre control Genotype:Albumin Cre control Genotype:Wild-type Genotype:Liver specific SIRT6 overexpression Genotype:Flox control Genotype:Flox control Genotype:Wild-type Genotype:Flox control Genotype:Wild-type Genotype:Albumin Cre control Genotype:Liver specific SIRT6 overexpression Genotype:Liver specific SIRT6 overexpression Genotype:Albumin Cre control Genotype:Wild-type Acetoacetate 127084.9983 999443.0165 790517.8538 966428.1878 1033130.001 1095551.536 1544269.469 778113.5969 598420.7528 222553.0582 744297.8193 736593.3499 1020195.437 448078.7548 926218.0015 372857.2959 908632.7231 1780549.332 638207.2021 629998.0201 Acetyl CoA 3136063.986 5144632.104 8259400.965 4804478.916 5832341.046 5354130.482 6596955.092 4281381.318 5090962.285 4971963.593 6254648.927 5121737.919 5478406.382 9706382.253 8370840.465 5318609.185 7368794.456 6840295.482 7097032.155 6088965.848 aKG 5953099.565 10504805.42 12186579.92 19589224.94 14427985.21 16224614.48 16571371.37 10673567.41 12723022.45 12944186.63 16332652.53 9893577.402 16358531.22 11845380.28 11428423.23 14205399.64 11706920.51 11038294 8227429.528 13507015.75 Arginine 1615353.1 1257820.643 697882.9898 894402.748 1099746.198 696609.2792 680926.9012 1634123.698 704249.3457 786678.8479 689345.2992 1400634.394 646634.5882 624637.8868 509998.773 725835.1903 517275.7161 579202.1715 751040.8395 932120.3408 Asparagine 18569933.46 21966005.94 21216861.6 19985660.62 20521877.4 20287818.71 19215814.08 20694044.5 18857844.29 29132413.52 18903980.07 25430558.89 19312608.65 19067200.78 19378729.41 20864970.6 21420332.35 28064804.56 20687889.38 19628554.46 Aspartate 96252060.6 115712671.2 55945394.35 97369995.69 48821572.12 54784480.31 56013456.81 56157530.07 151487238.5 117727876.8 99692488.1 117383469.4 66739496.31 101346626.2 88980603.84 107295010.5 69293984.87 87702680.14 73099426.22 112396204.2 cis-aconitate 1927495.465 2932669.196 2498581.543 2847543.031 3268500.679 2221548.463 2749976.8 3469843.538 2047188.716 2368110.233 2769199.929 1770021.474 2224624.96 1887794.638 2048842.867 2821394.845 2360362.181 3068232.578 1261037.993 1838566.312 Citrate 68791194.15 97323618.78 90516770.97 108626234.5 110119094.1 83971012.84 105570532.8 87334274.83 85447609.45 89131701.19 95434245.06 64956210.93 86440975.48 81540239.6 82900010.17 99815017.15 94665657.51 106436105.6 60940751.27 80637463.59 Cysteine 27547.42073 5876.812933 0 5958.375454 8228.444015 13644.93549 12949.24819 7221.566738 6006.218893 12038.93103 5963.895244 12974.67255 0 0 5117.979916 0 6270.911411 6114.079005 0 0 Cystine 677484.358 282114.7135 121602.7692 24041.31086 233713.2709 178985.1157 149225.2787 290393.0943 36264.84814 321462.3923 126072.0436 370520.7397 178791.8653 66076.59719 251415.6784 143099.4733 70089.30595 150823.9924 92094.41073 12731.21529 dihydroxyacetone phosphate (DHAP) 132615.7487 32909.21939 73524.43529 19912.46628 44795.55564 71062.93668 22095.82155 67810.63929 16498.63063 103888.9685 27617.73869 114081.8454 74351.99353 126916.4368 125644.1753 71473.41741 19019.80611 56952.19314 134283.6509 54954.29245 Fructose 5728692.572 2916018.982 5347626.427 4471998.965 3188338.919 3907577.317 4985180.059 3990758.495 5050266.702 4520171.129 2596291.219 4651819.856 4678075.334 6581435.449 4237612.998 4769600.306 3377499.335 6501138.074 3868540.153 3534358.603 fructose 1,6-diphosphate 0 0 0 0 0 0 0 7234.600286 0 0 6094.020189 0 0 0 0 0 0 0 0 0 Fructose-6-phosphate 24903192.19 23689573.66 25591791.21 22080420.58 21834208.54 18885734.57 20225655.38 21986055.58 31525855.74 28534338.12 20794863.51 26341556.18 30354986.66 41413178.59 30389387.65 30330734.65 23748573.18 31133535.45 26511729.22 26917804.38 G6P 32702612.26 30112977.6 29061629.39 25605036.39 24481742.45 20871562.15 22286359.65 26871745.3 35862707.34 32045274.9 24426003.8 31384247.59 35054541.05 51605809.99 34568332.55 36662764.06 26879165.44 36474407.78 29498226.91 29616819.43 Glucose 304052367.1 470818579.6 429953162.8 537523436.9 445640012.4 477508597.2 467005127.6 483592541.1 390316388.2 467919187.6 376533477.2 530823236.2 460430567.8 377754145.7 518516728.5 528199005.7 440592438.7 520576060.6 536656105.4 523468367.5 Glutamate 479377037.4 990328375.1 737310601.5 860294796.6 733727610.5 683709115.1 553791931.8 706625822.2 1251392178 1176105863 1163460729 693304460.3 716533813.2 842836624.8 787592464.5 938498921.9 848645940.9 921025736.3 514700719.5 950035204.2 Glutamine 3658927651 2986232837 2961462566 2858919386 2602879934 2817759972 2565387172 2410257294 2851035890 2446061422 2674151842 3126225773 2353867380 2359377719 3227827074 2289038298 2938397208 3145624981 3128736259 3396241855 Glyceraldehyde 3-phosphate 2370697.203 2278023.065 2064118.206 1908502.475 2051002.459 1814347.454 2504593.93 2529006.799 2408665.047 2137114.998 2353164.945 2120484.709 1951189.971 1964441.563 2002882.206 2623303.235 2329221.148 2591662.699 2426777.924 2504960.615 Glycerol 3303836.45 5137205.174 4209027.606 4544160.063 4686353.12 4846295.889 6854151.892 4553437.27 3678617.713 3620945.719 3669056.574 5026152.118 4175673.925 3854411.28 4505891.325 4871139.285 3974263.032 7169584.972 5824681.862 5521546.369 Glycine 41358501.93 48195136.81 49852620.91 61479096.32 48781642.78 57667705.16 58344792.38 59324657.65 45911272.66 59012364.49 50131367.65 52627201.61 44993386.78 50070101.21 45549282.68 49576733.82 53308137.9 50830429.76 52937087.84 48806115.97 Histidine 7175102.257 13153186.88 4857611.714 9327461.777 5737061.731 6296123.084 9161084.474 6159171.971 10652244.89 9994269.072 9610978.773 4786468.601 532053.452 6714714.343 13411274.97 10124904.91 10939479.31 4747685.686 8785541.007 3800390.202 IsoLeucine 202159919.1 383119721.1 308592618.4 357378064.5 225631312 302441436.2 335624318.9 285296009.5 207074954.7 353976909.5 191762867.8 374887704.6 261201223.3 184566641.5 309496745.9 327936830.1 245824906 391613210.2 300504260.5 328555571.3 Lactate 297034465.5 296251559.8 313755799.8 382573669.7 328510901.8 361234761.1 443120665.8 389509447.3 220375696.7 474217266.1 328432729.7 452950370.2 384634051.5 279368052.5 402176527.8 406234104.3 270917686.9 366172501.1 460282348.9 410322887.8 Leucine 227271612.3 342257771.5 312785076.5 328912771 266426173.5 321410989.8 325365608 303158874.1 197454482.6 344560472.8 176263646.2 390658939.6 263940280.8 200865369.1 288596346.5 307281045.4 258258698.4 351016262 276561349 357300092.2 Lysine 101133899.2 65682927.19 43154387.41 50395119.38 41766704.79 37800710.99 43238054.81 65482546.86 72146048.31 74745770.97 36708142.45 116413818.8 64604645.52 52387861.51 57687464.13 87700385.59 44839474.29 53010109.04 57124912.59 63399307.84 Malate 295226882 373085000.9 242224798.7 498185654.8 311973967.9 260234911.7 349563578.3 310526707.3 439427779.8 368022758.2 382989672.1 361397555 316614637.2 362296495.2 219175176.1 418682831.6 282152788 364861963 287128013.6 478434803.9 Methionine 55506239.01 92879735.74 79442796.53 105719453 76310199.14 83812181.19 92138754.01 98304881.12 79404891.7 102107941.3 57494905.66 94502603.8 70316834.51 53517937.36 83123523.57 95420976.45 78318908.11 97559945.6 94015562.31 96065928.74 NAD+ 100503968.7 177088083.2 157948026.7 167820601.1 150200014.3 149524326.3 243999947.9 192970607.3 125656261 113186448.5 143127759.1 159327314.6 235034390.5 180294969.9 268166230.2 168175397 155437012.6 153548100.9 149011022.1 177955213.5 NADH 6303912.969 9613117.379 13786152.66 12861678.6 9216329.685 9178910.812 20076094.64 10018197.65 10770378.43 9451923.487 9872728.778 8351147.604 15948795.12 14753460.24 14975796.71 14173142.33 15694465.29 15183284.62 12454595.8 14536066.21 NADP+ 4701694.48 7954181.282 5996917.216 7449101.519 6916509.037 7873.501629 16012.78781 9198617.586 5857263.977 7707053.957 7937546.286 6533166.44 6839994.255 8120574.592 9505062.036 6524964.633 7151211.414 8102101.21 8065679.602 6617154.916 NADPH 3221636.925 4961216.344 8208775.54 7462638.037 5058321.773 4325487.155 7819411.265 3138189.803 3456602.788 5504045.626 3141497.821 6351260.334 8850366.512 9224493.504 8185721.81 11005724.35 9984784.101 10851849.08 6529851.92 9085121.907 PEP 1849892.544 874078.7565 907302.0353 1552951.585 928589.3696 1245798.233 1669865.433 1034536.936 1605342.786 1836818.713 2094205.651 1176858.993 1910534.844 2946896.281 1612069.838 1304537.797 1246059.544 1155552.442 1695539.679 1176063.937 Phenylalanine 158951707.2 181279616.5 141276517.3 160663982.5 142657252.4 147712032.5 167160930.9 170860263.3 121701045 192164328.4 110226766.6 193935828.6 152741609.4 121665230.8 168370382.4 166814064.6 143151905.2 186027965.3 157790959.8 173501935.3 Proline 465642751.8 353221393.5 318159262.9 373202261.9 276616336.7 354177135.1 335512732.8 360097363.9 324654673.5 578945898.6 295006178 576709023.3 344390886.2 396814171.7 339946335.7 341045857.9 256109248.9 464087412 519745101.9 420393336.5 Pyruvate 8367417.473 9180307.913 10744026.46 11897734.33 11246888.38 13973946.06 14268935.93 10462238.64 7388991.923 18111507.43 12290076.38 16681296.6 13521025.61 10471733.37 15313666.08 13794594.59 8303847.946 9910843.304 11998726.35 10010915.01 Serine 22921254.74 22316623.23 18017261.54 28110780.89 23626586.08 25828600.54 19962448.38 27164061.8 13560582 30239352.46 12823027 29713231.48 24961580.8 12021142.79 15048101.65 24084850.85 23359343.51 22993969.49 21386126.25 22220118.04 Succinic Acid 46807519.72 107161266.9 87622953.66 131173370.7 85023510.37 90712591.17 109134972.7 111032456.7 75701304.33 138482766.1 91652225.56 83320547.13 103906579.6 94069830.22 94365236.62 115082431.9 80865797.79 100327345 106723093.7 112033959 Threonine 83246796.22 91388273.79 63384983.57 95321666.2 84698459.11 95930587.63 75165751.06 96537839.57 52040811.67 110734208.9 61692518.53 128138767.2 76347844.43 42344397.66 75706162.37 74352353.28 60912128.88 98005868.73 69115972.94 85393920.77 Tryptophan 65582249.27 72246064.53 72813535.2 71920627.51 64261313.38 69851404.48 72084033.31 66869148.43 77566678.45 91782617.68 69699608.84 65483486.38 64376283.42 65101978.7 92535090.51 69785826 68035244 80981291.56 77761450.66 73266505.3 Tyrosine 184949408.7 205184876.6 194624503 247467759.7 199888404.7 212648937 231119208.5 247395114.5 215349571.2 305748119.6 164047204.8 267309668.2 203345992.2 164897280.4 181458719.6 246304280 211460754.4 278153321.5 205580526.4 239603965.8 Valine 97551003.43 196705293.2 177140583.3 198196539.7 121089407.9 182943137.7 225047090.2 188326819.6 106313966.6 186866308.5 124644170.4 217754636.8 195659433.2 118772857.1 162156768.3 208400827.3 130101208.4 203312391 132146578.8 203968564.6 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Acetoacetate Acetyl CoA aKG Arginine Asparagine Aspartate cis-aconitate Citrate Cysteine Cystine dihydroxyacetone phosphate (DHAP) Fructose fructose 1,6-diphosphate Fructose-6-phosphate G6P Glucose Glutamate Glutamine Glyceraldehyde 3-phosphate Glycerol Glycine Histidine IsoLeucine Lactate Leucine Lysine Malate Methionine NAD+ NADH NADP+ NADPH PEP Phenylalanine Proline Pyruvate Serine Succinic Acid Threonine Tryptophan Tyrosine Valine METABOLITES_END #END