#METABOLOMICS WORKBENCH kameiyasutomi_20190422_012754 DATATRACK_ID:1708 STUDY_ID:ST001178 ANALYSIS_ID:AN001954 PROJECT_ID:PR000790 VERSION 1 CREATED_ON May 1, 2019, 2:29 pm #PROJECT PR:PROJECT_TITLE Metabolomic analysis of C2C12 myoblasts induced by the transcriptional factor PR:PROJECT_TITLE FOXO1 PR:PROJECT_SUMMARY To determine the metabolic changes occurring due to the activation of PR:PROJECT_SUMMARY transcriptional factor FOXO1, we analyzed the metabolic profile of C2C12 PR:PROJECT_SUMMARY myoblasts expressing FOXO1-estrogen receptor fusion protein using CE-TOFMS. PR:INSTITUTE Kyoto Prefectural University PR:LAST_NAME Kamei PR:FIRST_NAME Yasutomi PR:ADDRESS 1-5 Hangi-cho, Shimogamo, Sakyo-ku Kyoto, Kyoto, Kyoto, 606-8522, Japan PR:EMAIL cnqwb974@yahoo.co.jp PR:PHONE +81-75-703-5661 #STUDY ST:STUDY_TITLE Metabolomic analysis of C2C12 myoblasts induced by the transcriptional factor ST:STUDY_TITLE FOXO1 ST:STUDY_SUMMARY The transcriptional factor FOXO1 is considered to play roles in the regulation ST:STUDY_SUMMARY of energy metabolism in various tissues. To determine the metabolic changes ST:STUDY_SUMMARY occurring due to the activation of FOXO1, we analyzed the metabolic profile of ST:STUDY_SUMMARY C2C12 myoblasts expressing FOXO1-estrogen receptor fusion protein using ST:STUDY_SUMMARY CE-TOFMS. In the FOXO1-activated cells, the metabolite levels during glycolysis ST:STUDY_SUMMARY were higher. In addition, the gene expression of pyruvate dehydrogenase kinase, ST:STUDY_SUMMARY an enzyme that inhibits glucose utilization, increased. In the FOXO1-activated ST:STUDY_SUMMARY cells, the metabolite levels of numerous amino acids decreased, with increased ST:STUDY_SUMMARY gene expression of branched chain amino acid metabolism enzymes. Our results ST:STUDY_SUMMARY suggest that FOXO1 suppresses glucose utilization and promotes the use of ST:STUDY_SUMMARY proteins/amino acids as energy sources in muscle cells, potentially during ST:STUDY_SUMMARY starvation. ST:INSTITUTE Kyoto Prefectural University ST:LAST_NAME Kamei ST:FIRST_NAME Yasutomi ST:ADDRESS 1-5 Hangi-cho, Shimogamo, Sakyo-ku Kyoto, Kyoto, Kyoto, 606-8522, Japan ST:EMAIL cnqwb974@yahoo.co.jp ST:PHONE +81-75-703-5661 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Control 1 treatment:control Hours=24; tamoxifen(µM）=-; Cells(×106cells)=3.9 SUBJECT_SAMPLE_FACTORS - Control 2 treatment:control Hours=24; tamoxifen(µM）=-; Cells(×106cells)=3.9 SUBJECT_SAMPLE_FACTORS - Control 3 treatment:control Hours=24; tamoxifen(µM）=-; Cells(×106cells)=3.9 SUBJECT_SAMPLE_FACTORS - FOXO1-ER1 treatment:tamoxifen Hours=24; tamoxifen(µM）=2; Cells(×106cells)=3.7 SUBJECT_SAMPLE_FACTORS - FOXO1-ER2 treatment:tamoxifen Hours=24; tamoxifen(µM）=2; Cells(×106cells)=3.7 SUBJECT_SAMPLE_FACTORS - FOXO1-ER3 treatment:tamoxifen Hours=24; tamoxifen(µM）=2; Cells(×106cells)=3.7 #COLLECTION CO:COLLECTION_SUMMARY Cells were washed with cold PBS and then flash-frozen in liquid N2. CO:SAMPLE_TYPE Muscle #TREATMENT TR:TREATMENT_SUMMARY Two days after confluence, cells were treated with tamoxifen for 24 h and used TR:TREATMENT_SUMMARY for the metabolomic analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cells treated with vehicle (control, N = 3) or tamoxifen (FOXO1-ER, N = 3) SP:SAMPLEPREP_SUMMARY were used for the metabolomic analysis (Human Metabolome Technologies Inc., SP:SAMPLEPREP_SUMMARY Tsuruoka, Japan). 3.9 x 106 cells were transferred into 500 μL of methanol SP:SAMPLEPREP_SUMMARY containing 50 mM external standard. After homogenization by BMSM10N21 (BMS, SP:SAMPLEPREP_SUMMARY Tokyo) at 1,500 rpm for 120 s performed 5 times, 500 μL of chloroform and 200 SP:SAMPLEPREP_SUMMARY μL of ultrapure water were added to the homogenate. The solution was mixed well SP:SAMPLEPREP_SUMMARY and centrifuged at 2,300 × g for 5 min at 4 °C. The resultant water phase was SP:SAMPLEPREP_SUMMARY ultrafiltered using a Millipore Ultrafree-MC PLHCC HMT Centrifugal Filter SP:SAMPLEPREP_SUMMARY Device, 5 kDa (Millipore, Billerica, MA). The filtrate was dried and then SP:SAMPLEPREP_SUMMARY dissolved in 50 μL of ultrapure water. The samples obtained were then subjected SP:SAMPLEPREP_SUMMARY to capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) SP:SAMPLEPREP_SUMMARY analysis using the Agilent CE-TOFMS system (Agilent Technologies, Santa Clara, SP:SAMPLEPREP_SUMMARY CA) at 4 °C. The detected peaks were aligned according to their m/z values and SP:SAMPLEPREP_SUMMARY normalized migration times. The peaks were mean-centered and scaled using their SP:SAMPLEPREP_SUMMARY standard deviations on a per peak basis as a pretreatment. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent CE-TOFMS MS:INSTRUMENT_TYPE Other MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS - #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Relative Area MS_METABOLITE_DATA_START Samples Control 1 Control 2 Control 3 FOXO1-ER1 FOXO1-ER2 FOXO1-ER3 Factors treatment:control treatment:control treatment:control treatment:tamoxifen treatment:tamoxifen treatment:tamoxifen Urea 0.25 0.18 0.15 0.20 0.20 0.15 Ethanolamine 0.02 0.02 0.03 0.03 0.03 0.03 Aminoacetone 0.04 0.03 0.03 Gly 5.40 5.30 5.30 4.50 4.40 4.70 Putrescine 0.01 0.00 0.00 Ala 4.30 4.20 4.30 3.10 3.30 3.10 β-Ala 1.40 1.40 1.40 1.10 1.20 1.20 Glycerol 1.20 1.00 0.96 1.20 1.30 1.10 Cyclohexylamine 0.03 0.01 0.05 1-Aminocyclopropane-1-carboxylic acid 0.00 Homoserinelactone 0.00 2-Aminoisobutyric acid 0.03 0.03 0.03 0.02 0.02 0.02 2-Aminobutyric acid 0.03 0.03 0.03 0.02 0.02 0.02 GABA 0.10 0.11 0.11 0.07 0.08 0.07 3-Aminoisobutyric acid 0.01 0.01 0.01 Choline 0.57 0.51 0.59 0.71 0.79 0.77 Ser 0.69 0.64 0.62 0.60 0.62 0.58 Diethanolamine 0.01 0.01 0.01 0.01 0.02 Hypotaurine 0.02 0.02 0.02 0.01 0.01 0.01 Creatinine 0.05 0.04 0.04 0.05 0.04 0.04 Pro 0.87 0.88 0.86 0.90 0.92 0.89 Guanidoacetic acid 0.53 0.51 0.56 0.22 0.23 0.22 Val 1.60 1.30 1.20 1.00 1.00 0.77 Betaine 0.22 0.20 0.20 0.21 0.22 0.21 5-Aminovaleric acid 0.03 0.04 0.03 0.02 Thr 3.70 3.60 3.60 2.70 2.80 2.70 Cys 0.00 0.00 0.00 0.00 0.01 0.01 Nicotinamide 0.02 0.01 0.01 0.01 0.01 0.01 Taurine 0.22 0.26 0.26 0.25 0.24 0.26 XC0016 0.13 0.12 0.12 0.07 0.07 0.07 Hydroxyproline 0.10 0.10 0.09 0.07 0.06 0.06 Creatine 2.90 3.10 3.10 3.30 2.70 2.80 3-Guanidinopropionic acid 0.02 0.01 0.02 0.01 0.01 0.01 Ile 1.70 1.40 1.20 0.97 0.92 0.68 Leu 1.80 1.40 1.20 1.10 1.00 0.68 Asn 0.13 0.12 0.15 0.09 0.09 0.08 Ornithine 0.05 0.04 0.04 0.02 0.02 0.02 Asp 0.79 0.85 0.93 0.46 0.55 0.52 Hypoxanthine 0.01 1-Methylnicotinamide 0.07 0.07 0.07 0.04 0.04 0.04 4-Guanidinobutyric acid 0.01 0.01 0.01 0.00 0.00 γ-Butyrobetaine 0.06 0.05 0.06 0.06 0.06 0.06 Spermidine 0.02 0.02 0.02 0.02 0.02 0.01 Gln 5.80 5.70 5.60 3.00 3.00 2.90 Lys 2.10 2.10 2.00 1.10 1.10 1.00 Glu 7.80 8.30 8.20 7.40 7.60 7.50 N-Acetylserine 0.01 0.01 0.01 0.02 0.02 0.02 Met 0.19 0.14 0.14 0.11 0.11 0.08 Octopamine 0.00 0.00 0.00 0.00 Dopamine 0.00 0.00 0.00 0.00 His 0.34 0.28 0.27 0.21 0.19 0.17 N6-Methyllysine 0.01 0.01 0.01 0.00 0.00 O-Acetylhomoserine 0.02 0.03 0.03 0.02 0.03 0.02 2-Aminoadipic acid 0.02 0.03 0.03 0.02 0.03 0.02 Carnitine 0.27 0.25 0.27 0.30 0.33 0.33 Methionine sulfoxide 0.05 0.05 0.04 0.03 0.03 0.03 Phe 0.77 0.61 0.53 0.37 0.36 0.24 Pyridoxine 0.03 0.02 0.02 0.03 0.03 0.02 1-Methylhistidine 0.01 0.01 0.01 0.01 0.01 0.01 3-Methylhistidine 0.01 0.01 0.01 0.01 0.01 0.01 N-Acetylornithine 0.00 0.00 N5-Ethylglutamine 0.00 Arg 1.20 1.10 1.10 0.56 0.59 0.52 Citrulline 0.01 0.00 0.00 0.00 0.00 0.00 Tyr 0.54 0.47 0.43 0.25 0.23 0.17 Phosphorylcholine 1.10 1.10 1.10 0.86 0.87 0.90 N8-Acetylspermidine 0.00 0.00 0.00 N-Acetyllysine 0.00 0.01 0.01 0.01 Gly-Asp 0.02 0.02 0.02 0.02 0.02 0.02 SDMA 0.00 0.00 0.00 0.00 Spermine 0.01 0.01 0.01 0.01 0.01 0.01 O-Acetylcarnitine 0.16 0.21 0.21 0.21 0.17 0.20 Trp 0.14 0.12 0.12 0.07 0.06 0.05 Lipoamide 0.01 0.01 0.01 0.01 0.01 0.01 XC0061 0.00 0.01 0.01 0.01 Cystathionine 0.02 0.02 0.02 0.01 0.01 0.01 Carnosine 0.01 0.01 0.01 0.01 0.02 0.01 Butyrylcarnitine 0.02 0.02 0.01 0.02 0.02 0.01 Pyridoxamine 5'-phosphate 0.00 0.00 0.00 0.00 0.00 0.00 γ-Glu-Cys 0.00 0.01 0.00 0.01 0.01 0.00 XC0089 0.00 0.00 0.00 0.00 0.00 0.00 Glycerophosphocholine 1.20 1.30 1.30 0.93 0.94 0.92 Thiamine 0.01 0.00 0.00 0.01 0.01 0.00 Adenosine 0.01 0.00 0.00 0.01 0.01 0.01 Inosine 0.00 0.01 0.01 0.01 Glu-Glu 0.00 0.00 0.00 0.00 0.00 Guanosine 0.00 0.00 0.00 0.00 0.00 Ophthalmic acid 0.18 0.18 0.18 0.11 0.12 0.11 Argininosuccinic acid 0.01 0.01 0.01 0.00 0.01 0.00 γ-Glu-Val-Gly 0.00 0.00 0.00 Glutathione (GSSG)_divalent 0.35 0.31 0.27 0.26 0.21 0.23 Glutathione (GSH) 1.80 2.10 2.00 1.50 1.60 1.50 XC0132 0.07 0.06 0.07 0.06 0.07 0.07 XC0137 0.01 0.01 0.01 0.01 0.01 0.01 S-Lactoylglutathione 0.02 0.00 S-Adenosylmethionine 0.03 0.03 0.03 0.03 0.02 0.03 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID HMDB ID quantified m/z MT/RT Urea C00086 HMDB00294 61.04 20.95 Ethanolamine C00189 HMDB00149 62.061 6.4 Aminoacetone C01888 HMDB02134 74.059 6.93 Gly C00037 HMDB00123 76.039 8.34 Putrescine C00134 HMDB01414 89.107 4.77 Ala C00041,C00133,C01401 HMDB00161,HMDB01310 90.055 9.06 β-Ala C00099 HMDB00056 90.055 7.39 Glycerol C00116 HMDB00131 93.054 21.84 Cyclohexylamine C00571 100.112 7.74 1-Aminocyclopropane-1-carboxylic acid C01234 102.056 7.16 Homoserinelactone 102.056 7.16 2-Aminoisobutyric acid C03665 HMDB01906 104.07 9.66 2-Aminobutyric acid C02261,C02356 HMDB00452 104.07 9.66 GABA C00334 HMDB00112 104.07 7.74 3-Aminoisobutyric acid C03284,C05145 HMDB03911 104.07 7.89 Choline C00114 HMDB00097 104.106 6.91 Ser C00065,C00716,C00740 HMDB00187,HMDB03406 106.049 10.01 Diethanolamine C06772 HMDB04437 106.086 7.7 Hypotaurine C00519 HMDB00965 110.027 17.97 Creatinine C00791 HMDB00562 114.066 7.35 Pro C00148,C00763,C16435 HMDB00162,HMDB03411 116.07 10.81 Guanidoacetic acid C00581 HMDB00128 118.061 8.27 Val C00183,C06417,C16436 HMDB00883 118.085 10.04 Betaine C00719 HMDB00043 118.086 11.26 5-Aminovaleric acid C00431 HMDB03355 118.086 8.09 Thr C00188,C00820 HMDB00167 120.065 10.52 Cys C00097,C00736,C00793 HMDB00574,HMDB03417 122.026 11.3 Nicotinamide C00153 HMDB01406 123.054 7.45 Taurine C00245 HMDB00251 126.021 21.81 XC0016 129.065 8.81 Hydroxyproline C01157 HMDB00725 132.065 12.01 Creatine C00300 HMDB00064 132.076 8.89 3-Guanidinopropionic acid C03065 132.077 8.05 Ile C00407,C06418,C16434 HMDB00172 132.101 10.23 Leu C00123,C01570,C16439 HMDB00687 132.101 10.34 Asn C00152,C01905,C16438 HMDB00168 133.06 10.49 Ornithine C00077,C00515,C01602 HMDB00214,HMDB03374 133.097 6.87 Asp C00049,C00402,C16433 HMDB00191,HMDB06483 134.044 11.55 Hypoxanthine C00262 HMDB00157 137.046 11.1 1-Methylnicotinamide C02918 HMDB00699 137.07 7.4 4-Guanidinobutyric acid C01035 HMDB03464 146.092 8.31 γ-Butyrobetaine C01181 HMDB01161 146.116 8.14 Spermidine C00315 HMDB01257 146.164 4.59 Gln C00064,C00303,C00819 HMDB00641,HMDB03423 147.075 10.76 Lys C00047,C00739,C16440 HMDB00182,HMDB03405 147.111 6.93 Glu C00025,C00217,C00302 HMDB00148,HMDB03339 148.059 10.94 N-Acetylserine HMDB02931 148.059 22.88 Met C00073,C00855,C01733 HMDB00696 150.057 10.73 Octopamine C04227 HMDB04825 154.087 8.68 Dopamine C03758 HMDB00073 154.087 8.68 His C00135,C00768,C06419 HMDB00177 156.076 7.35 N6-Methyllysine C02728 HMDB02038 161.13 7.15 O-Acetylhomoserine C01077 162.076 10.95 2-Aminoadipic acid C00956 HMDB00510 162.076 10.95 Carnitine C00318,C00487,C15025 HMDB00062 162.111 8.55 Methionine sulfoxide C02989 HMDB02005 166.052 11.8 Phe C00079,C02057,C02265 HMDB00159 166.085 11.08 Pyridoxine C00314 HMDB00239 170.08 8.67 1-Methylhistidine HMDB00001 170.091 7.53 3-Methylhistidine C01152 HMDB00479 170.091 7.53 N-Acetylornithine C00437 HMDB03357 175.107 9.56 N5-Ethylglutamine C01047 175.107 11.36 Arg C00062,C00792 HMDB00517,HMDB03416 175.118 7.16 Citrulline C00327 HMDB00904 176.102 11.06 Tyr C00082,C01536,C06420 HMDB00158 182.08 11.35 Phosphorylcholine C00588 HMDB01565 184.072 20.34 N8-Acetylspermidine C01029 HMDB02189 188.175 6.39 N-Acetyllysine C12989 HMDB00446 189.122 9.81 Gly-Asp 191.065 9.9 SDMA HMDB03334 203.149 7.84 Spermine C00750 HMDB01256 203.222 4.54 O-Acetylcarnitine C02571 HMDB00201 204.122 9.07 Trp C00078,C00525,C00806 HMDB00929 205.096 11.02 Lipoamide C00248 HMDB00962 206.069 21.63 XC0061 218.138 9.35 Cystathionine C00542,C02291 HMDB00099 223.074 9.92 Carnosine C00386 HMDB00033 227.112 6.82 Butyrylcarnitine C02862 HMDB02013 232.152 9.6 Pyridoxamine 5'-phosphate C00647 HMDB01555 249.063 10.48 γ-Glu-Cys C00669 HMDB01049 251.068 12.76 XC0089 255.097 9.55 Glycerophosphocholine C00670 HMDB00086 258.109 21.43 Thiamine C00378 HMDB00235 265.111 6.64 Adenosine C00212 HMDB00050 268.104 9.89 Inosine C00294 HMDB00195 269.088 19.37 Glu-Glu C01425 277.102 10.89 Guanosine C00387 HMDB00133 284.095 12.5 Ophthalmic acid HMDB05765 290.133 13.16 Argininosuccinic acid C03406 HMDB00052 291.128 9.45 γ-Glu-Val-Gly 304.149 13.24 Glutathione (GSSG)_divalent C00127 HMDB03337 307.081 12.16 Glutathione (GSH) C00051 HMDB00125 308.089 13.18 XC0132 325.159 8.72 XC0137 350.1 13.44 S-Lactoylglutathione C03451 HMDB01066 380.112 13.7 S-Adenosylmethionine C00019 HMDB01185 399.143 7.16 METABOLITES_END #END