MTBLS3: A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans    

Cecilia Castro , Jules Griffin

Study Submission Date: 14-Feb-2012   Study public release date: 14-Feb-2012

Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the delta-9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. Gas chromatography mass spectrometry (GC-MS) was used to define the metabolome of all the possible knock-outs for the delta 9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence delta 9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. The propagation of metabolic changes across the network of metabolism demonstrates that modification of the delta 9 desaturases places C.elegans into a catabolic state compared with wildtype controls.

Organism(s):
Caenorhabditis elegans

Study Design Description:
  • metabolomic profiling
  • Free amino acid, target analysis
  • Caenorhabditis elegans
  • gas chromatography mass spectrometry

Publications
A metabolomic strategy defines the regulation of lipid content and global metabolism by Delta-9 desaturases in Caenorhabditis elegans.

Experimental Factors
  • gene knockout: [ Delta 9 fat 5 fat 7 , wild-type generic , Delta 9 fat 7 , Delta 9 fat 5 , Delta 9 fat 6 , Delta 9 fat 5 fat 6 ]
Protocol Description
Sample collection C. elegans were grown under standard conditions on Nematode Growth Media (NGM) with HB101 as food source, at 20ºC. The wild-type strain used was Bristol N2. Mutant strains were obtained from the Caenorhabditis Genetics Center, University of Minnesota, Twin Cities, MN and included: BX107 fat 5(tm420) V; BX106 fat 6(tm331) IV; BX153 fat 7(wa36) V; BX110 fat 7(wa36) V fat 5(tm420) V (referred to as fat 5;fat 7 in the text); BX160 fat 6(tm331) IV;fat-5(tm420) V refered to as fat 5;fat 6); and BX156 fat 6(tm331) IV;fat 7(wa36) V. Mutations were verified using PCR and DNA sequencing prior to starting experiments. For each experiment, early embryos were isolated by alkaline hypochlorite treatment and plated after synchronisation on NGM plates seeded with bacteria. For each biological replicate, ~8000 animals were harvested, washed and stored at -80ºC until extraction. Eight samples were grown for each strain. C. elegans were grown under standard conditions on Nematode Growth Media (NGM) with HB101 as food source, at 20ºC. The wild-type strain used was Bristol N2. Mutant strains were obtained from the Caenorhabditis Genetics Center, University of Minnesota, Twin Cities, MN and included: BX107 fat 5(tm420) V; BX106 fat 6(tm331) IV; BX153 fat 7(wa36) V; BX110 fat 7(wa36) V fat 5(tm420) V (referred to as fat 5;fat 7 in the text); BX160 fat 6(tm331) IV;fat-5(tm420) V refered to as fat 5;fat 6); and BX156 fat 6(tm331) IV;fat 7(wa36) V. Mutations were verified using PCR and DNA sequencing prior to starting experiments. For each experiment, early embryos were isolated by alkaline hypochlorite treatment and plated after synchronisation on NGM plates seeded with bacteria. For each biological replicate, ~8000 animals were harvested, washed and stored at -80ºC until extraction. Eight samples were grown for each strain.
Extraction Metabolites from whole nematodes were extracted using a methanol–chloroform procedure. 600 µl of methanol–chloroform (2:1 v:v) was added to the frozen nematodes and samples were sonicated for 15 min. 200 µl each of chloroform and water were added, the samples centrifuged and the aqueous layer separated from the lipid one. The procedure was repeated twice. The aqueous layer was dried overnight in an evacuated centrifuge, while the lipid fraction was dried overnight in the fumehood.
Chromatography EZ:faast Free (Physiological) Amino Acid provided column (Zebron™ ZB-AAA GC Column)
Mass spectrometry The derivatized sample was injected (3µ) split ratio of 10 into a Thermo Electron Trace GC Ultra equipped with amino acid column (Phenomenex; injector temperature = 220°C, helium carrier gas flow rate = 1.1 ml/min). The initial column temperature was 110°C; this was held for 2 min and then increased by 30°C/min to 310°C. The column effluent was introduced into a Trace DSQ quadrupole mass spectrometer (Thermo Electron; transfer line temperature = 320°C, ion source temperature = 240°C, electron beam = 70 eV). The detector was turned on after a solvent delay of 120 s, and data were collected in full scan mode using 3 scans/s across a mass range of 50–450 m/z.
Data transformation GC-MS chromatograms were analyzed using Xcalibur, version 2.0 (Thermo Electron), integrating each peak individually. Each integrated peak was normalized so that the total sum of peaks was set to 10,000. Deconvolution of overlapping peaks was achieved by traces of single ions. A 0.1 min threshold window was used for the deviation of peaks away from the predicted retention time across the data set.
Metabolite identification Amino acid identification was based on the information, retention time and fragmentation, provided by the EZ:faast Free (Physiological) Amino Acid GC kit

Technology - Platform: mass spectrometry - metabolite profiling - DSQ II Single Quadrupole GC/MS (Thermo Scientific)
Source gene knockout
Cecilia_AA_rerun05 Delta 9 fat 5 fat 7
Cecilia_AA_rerun05 Delta 9 fat 5 fat 7
Cecilia_AA_rerun05 wild-type generic
Cecilia_AA_rerun05 Delta 9 fat 7
Cecilia_AA_rerun05 wild-type generic
Cecilia_AA_rerun05 Delta 9 fat 7
Cecilia_AA_rerun05 Delta 9 fat 5
Cecilia_AA_rerun05 wild-type generic
Cecilia_AA_rerun05 Delta 9 fat 5
Cecilia_AA_rerun05 Delta 9 fat 6
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DL-Tyrosine (CHEBI:18186) C9H11NO3 5.06 C1=CC(=CC=C1CC(C(=O)O)N)O InChI=1S/C9H11NO3/c10-8(9(12)13)5-6-1-3-7(11)4-2-6/h1-4,8,11H,5,10H2,(H,12,13) 108.0947955 96.43555879 99.06564944 93.76657098 71.78632903 98.31552644 93.89727531 133.6661856 74.26765399 60.26177679 62.38683263 44.80764872 51.7141739 79.71905855 191.3932271 87.85839661 158.2912271 76.82843671 69.94565411 59.20712427 67.04090106 66.36607446 106.5004712 120.0241952 81.83932206 80.55735039 71.70080346 76.80197487 71.1865377 82.35763558 88.2685519 67.63092466 73.45617628 68.76356406 85.46517271 80.19190304 72.67426867 86.72225196 82.98795024 83.30994836 100.5819455 137.0329911 97.65600454 97.42362278 97.20796836 111.3195482 194.8823257 193.7758693 63.46408927 59.13474224 60.34961184 60.89487619 58.53272318 63.65928745 59.0289247 56.52582307 58.39558635 104.6045726 58.29291931 54.79329743 147.1788319 165.6146322 64.67248059
Description Formula m/z Retention time Smiles Inchi Cecilia_AA_rerun05 Cecilia_AA_rerun06 Cecilia_AA_rerun07 Cecilia_AA_rerun08 Cecilia_AA_rerun10 Cecilia_AA_rerun11 Cecilia_AA_rerun12 Cecilia_AA_rerun13 Cecilia_AA_rerun15 Cecilia_AA_rerun16 Cecilia_AA_rerun17 Cecilia_AA_rerun18 Cecilia_AA_rerun19 Cecilia_AA_rerun21 Cecilia_AA_rerun22 Cecilia_AA_rerun23 Cecilia_AA_rerun24 Cecilia_AA_rerun27 Cecilia_AA_rerun28 Cecilia_AA_rerun29 Cecilia_AA_rerun30 Cecilia_AA_rerun31 Cecilia_AA_rerun33 Cecilia_AA_rerun34 Cecilia_AA_rerun35 Cecilia_AA_rerun36 Cecilia_AA_rerun37 Cecilia_AA_rerun39 Cecilia_AA_rerun40 Cecilia_AA_rerun41 Cecilia_AA_rerun42 Cecilia_AA_rerun43 Cecilia_AA_rerun45 Cecilia_AA_batch23_05 Cecilia_AA_batch23_06 Cecilia_AA_batch23_07 Cecilia_AA_batch23_08 Cecilia_AA_batch23_10 Cecilia_AA_batch23_11 Cecilia_AA_batch23_12 Cecilia_AA_batch23_15 Cecilia_AA_batch23_16 Cecilia_AA_batch23_17 Cecilia_AA_batch23_18 Cecilia_AA_batch23_19 Cecilia_AA_batch23_21 Cecilia_AA_batch23_22 Cecilia_AA_batch23_23 Cecilia_AA_batch23_28 Cecilia_AA_batch23_29 Cecilia_AA_batch23_30 Cecilia_AA_batch23_31 Cecilia_AA_batch23_32 Cecilia_AA_batch23_33 Cecilia_AA_batch23_35 Cecilia_AA_batch23_36 Cecilia_AA_batch23_39 Cecilia_AA_batch23_40 Cecilia_AA_batch23_41 Cecilia_AA_batch23_42 Cecilia_AA_batch23_43 Cecilia_AA_batch23_45 Cecilia_AA_batch23_46
DL-Tryptophan (CHEBI:27897) C11H12N2O2 5.35 C1=CC=C2C(=C1)C(=CN2)CC(C(=O)O)N InChI=1S/C11H12N2O2/c12-9(11(14)15)5-7-6-13-10-4-2-1-3-8(7)10/h1-4,6,9,13H,5,12H2,(H,14,15) 19.83113537 6.322898364 7.385922132 12.10092028 4.543213897 12.98307779 14.61054969 15.23290468 14.29135796 11.62586778 12.59804526 5.830801786 6.210341047 10.24952292 30.38460202 14.18494686 26.76691607 10.43737176 12.26474678 12.4534862 10.92089819 11.35595462 1.905415802 10.31391435 8.854775118 10.7177827 12.87468755 10.95708442 10.08695289 13.54803544 10.8083365 12.05652643 12.51923852 11.64763955 17.40357411 18.66621928 13.00831109 17.5604242 18.8385324 16.84507784 15.23350041 19.16925017 15.75671698 19.22273667 15.05933305 17.20414067 20.92391827 19.61217376 18.37429711 17.98335974 17.98889837 16.88507037 18.45462579 17.37495593 15.86045744 15.80662544 10.31042566 21.8684016 13.66620294 16.36705864 21.65887827 29.43777094 22.65015084
DL-Cystathionine (CHEBI:17755) C7H14N2O4S C(CSCC(C(=O)O)N)C(C(=O)O)N InChI=1S/C7H14N2O4S/c8-4(6(10)11)1-2-14-3-5(9)7(12)13/h4-5H,1-3,8-9H2,(H,10,11)(H,12,13) 47.4363578 43.09356196 32.77395814 27.64814151 2.635777454 30.01043219 29.67616598 38.19488581 38.81908999 21.38112919 29.32836535 9.905220485 18.63454804 14.81816573 15.27768831 15.73453755 18.41213375 35.08116693 38.10014367 29.98185235 35.02136356 27.08160333 10.90487562 74.17110051 51.476666 46.46133263 56.3330843 57.44646398 41.22113974 51.11973307 49.03165873 50.39215032 43.93260163 5.99182726 14.26300985 19.05984976 10.7084473 5.117237817 8.062789461 9.5641002 6.1162625 9.031874511 7.647971167 8.219395651 4.203912242 8.048737006 10.5796651 19.87359632 25.53215091 31.25354208 31.234469 30.4043435 35.72272061 31.54504287 20.99839337 31.36519526 40.72804684 55.80506712 34.57782971 29.95715803 12.20245409 10.26465986 52.58630079
histidine (CHEBI:57595) C6H9N3O2 4.77 C1=C(NC=N1)CC(C(=O)[O-])[NH3+] InChI=1S/C6H9N3O2/c7-5(6(10)11)1-4-2-8-3-9-4/h2-3,5H,1,7H2,(H,8,9)(H,10,11)/t5-/m0/s1 27.53981437 13.36236975 7.651698011 9.694335327 9.416320795 20.70732416 11.62436436 1.233986769 15.15062291 5.114316908 17.02116798 6.121355863 4.601713204 7.659752035 31.53928841 64.63281297 52.5322162 10.57555188 15.94157007 14.5611543 17.65134994 26.76960786 51.60681674 38.23166858 18.33842599 18.02536288 19.22704248 16.23202731 21.53917319 25.15284208 25.18285797 19.75465528 14.34307096 11.62259443 17.75809254 22.07951236 15.77089972 2.852277464 10.36904937 15.34623586 13.92361659 17.66441058 7.061664948 7.505825172 11.09804825 12.2473197 24.59940444 29.44362004 19.93029246 21.57186928 24.42993718 21.47149409 24.17251291 20.17781921 14.34511026 23.51850885 17.87791113 33.12544522 21.96344696 23.09042007 49.54179818 43.8154193 28.02855098
DL-ALANINE (CHEBI:16449) C3H7NO2 1.29 CC(C(=O)O)N InChI=1S/C3H7NO2/c1-2(4)3(5)6/h2H,4H2,1H3,(H,5,6) 133.8929083 167.5201039 155.1238427 144.8666656 200.5420424 159.1994674 150.212371 176.6219909 217.1062906 233.5551411 219.1074799 247.4326908 239.894212 223.0036401 43.06870772 77.46659712 79.01989018 143.3414825 213.8605502 237.1337641 231.8380845 159.9097397 93.93089407 159.8983763 134.9615169 167.6428675 192.5067258 183.9130675 200.4523376 178.6083901 136.631747 173.3415144 185.8995693 163.4815053 151.1343187 147.4076571 149.2690829 173.4350251 140.9890206 136.9435485 94.27355959 39.56461971 114.4263984 103.2153103 149.7770186 131.2849043 13.7642026 47.35644396 155.2979545 153.7480489 146.8226996 146.1135373 148.5135503 143.4364078 157.8829262 152.073262 186.1619168 171.8050368 173.2703927 166.564495 108.4282699 84.50321181 159.231466
Glycine (CHEBI:15428) C2H5NO2 1.39 C(C(=O)O)N InChI=1S/C2H5NO2/c3-1-2(4)5/h1,3H2,(H,4,5) 16.3995923 18.76254188 17.60344711 16.96854877 28.66139462 14.86884931 16.20307964 31.84807726 26.64894852 27.66101884 27.52877272 29.97378149 34.80953979 20.1710859 9.496629839 14.67726241 22.71778214 22.48446821 26.44180579 25.96422024 24.20836363 52.04728477 14.36302083 33.10107571 24.25455899 22.40678627 24.27378475 26.66265909 27.19113513 26.15349742 22.62153238 21.91349277 24.45273431 25.9907992 22.92614508 23.16441103 47.71087008 44.85107312 22.59594727 24.39472099 21.9648098 20.50165408 28.56080092 23.10075027 44.23697125 43.8255013 19.24634875 65.29043492 21.52634986 21.68758382 23.89693649 22.24125269 25.37403865 22.58875955 29.22332634 25.87469939 29.53526122 18.98500798 29.50491365 36.85836529 13.40130702 15.13166789 33.23683831
DL-2-Aminobutyric acid (CHEBI:35621) C4H9NO2 1.49 CCC(C(=O)O)N InChI=1S/C4H9NO2/c1-2-3(5)4(6)7/h3H,2,5H2,1H3,(H,6,7) 3.8740694 4.513149447 4.663629821 4.062584578 8.460839022 4.789517633 4.458753663 17.82150081 8.264401596 12.3497396 10.39741795 12.6466602 11.26036662 7.693675065 7.300516433 8.955990349 10.82439024 5.515688904 6.521976041 7.664840477 8.152624339 5.654238498 7.177473355 7.899554559 5.787600612 5.534647264 5.652965656 5.415354625 6.614230225 5.47116826 6.487237781 5.496865481 6.629023099 6.636605154 5.79550144 5.75003779 4.563418528 7.009246476 5.293698793 5.284397969 4.332009506 3.952053467 5.928695762 4.528961062 6.194847554 5.975991382 5.121891113 14.17937919 9.775024474 10.11867238 9.947042435 9.412407308 9.673463786 8.060323088 8.361618947 10.0051261 11.24597528 9.297012981 11.3976353 10.36391381 6.494474799 4.089923826 11.34639702
valine (CHEBI:57762) C5H11NO2 1.6 CC(C)C(C(=O)[O-])[NH3+] InChI=1S/C5H11NO2/c1-3(2)4(6)5(7)8/h3-4H,6H2,1-2H3,(H,7,8)/t4-/m0/s1 72.93943407 92.1116529 85.49794556 86.56089356 87.93848558 76.36258645 86.54479459 40.25448215 63.67414699 99.99000183 88.53311767 107.762385 99.73711111 78.78288272 18.84737647 193.3243933 42.41503676 54.50175198 67.36865196 92.09774674 81.98777428 73.79177761 108.9167332 25.20629267 60.9579215 57.47467112 72.25312789 62.3176478 72.8811694 80.29391906 49.46319415 65.11021126 81.78195376 245.3836328 105.4873387 105.1638782 131.4463359 117.8266713 106.6863788 120.7987412 74.27297542 47.11915589 89.42449901 85.0819512 108.4091247 99.336369 13.25262842 24.16144072 88.4672174 88.84525071 83.94052208 84.35107912 85.64783301 85.04194001 83.64479267 80.59118607 77.17516855 32.38016432 77.38902543 86.92483886 28.56584991 34.32825676 68.52899021
DL-Leucine (CHEBI:25017) C6H13NO2 1.82 CC(C)CC(C(=O)O)N InChI=1S/C6H13NO2/c1-4(2)3-5(7)6(8)9/h4-5H,3,7H2,1-2H3,(H,8,9) 107.0526235 94.13920558 95.1766566 95.5405415 119.6954004 108.4529303 106.6114956 107.2578643 96.41392324 111.3709591 106.3232711 108.9657059 97.41046704 94.27697272 116.9845039 62.5676947 78.99176154 80.18855581 76.84330261 77.45154017 79.89617208 77.58448847 86.56888735 83.24548323 90.41190216 89.35419226 80.49026532 82.98482686 82.35068695 87.56423525 89.87497313 79.04990322 90.99414267 109.7625853 111.8581042 109.0602913 126.5282088 129.6835312 117.6831507 115.554136 119.6843209 132.6813524 107.4590257 107.3323618 115.1566794 122.8445578 87.20335726 95.72145601 95.77174744 93.79831835 92.4998347 93.24055426 89.75633019 94.87037084 108.60734 91.98169933 85.17727682 91.87998775 80.49832469 88.09236232 103.0124921 124.4715574 85.24530798
Isoleucine (CHEBI:24898) C6H13NO2 1.88 CCC(C)C(C(=O)O)N InChI=1S/C6H13NO2/c1-3-4(2)5(7)6(8)9/h4-5H,3,7H2,1-2H3,(H,8,9) 66.45993738 84.58053329 81.04339932 85.20892719 81.42918712 80.04834049 74.91125015 41.1894027 59.65274965 76.99585511 68.33510425 90.92001173 87.61418504 69.55466413 24.04794943 13.13391185 36.2623924 54.34510217 53.77988703 60.24518172 69.33564465 61.91835335 19.23077307 16.86830013 62.54183679 57.38724767 54.15650794 61.29014125 69.45067313 68.85997187 55.01940953 63.57774482 71.96646647 83.01205432 98.227633 100.3371882 95.4804885 103.1704342 103.6721011 110.4087787 91.66597057 72.8624692 101.3089813 89.57608325 99.53160489 93.71033192 23.30127257 57.71148383 82.07028075 79.31045057 77.28032601 77.13478547 75.88188504 78.83082835 76.16956417 71.92015448 64.07705641 19.76717716 65.99605835 74.50481465 24.21666004 29.13102199 58.09418996
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