#METABOLOMICS WORKBENCH chrisbarlow_20200403_172959 DATATRACK_ID:1981 STUDY_ID:ST001348 ANALYSIS_ID:AN002242 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Restriction of essential amino acids dictates the systemic metabolic response to PR:PROJECT_TITLE dietary protein dilution PR:PROJECT_SUMMARY The data provided here are in support of the publication “Restriction of PR:PROJECT_SUMMARY essential amino acids dictates the systemic metabolic response to dietary PR:PROJECT_SUMMARY protein dilution” Yann W. Yap et al. Nature Communications 2020 (accepted for PR:PROJECT_SUMMARY publication). Here we provide untargeted metabolomics LC-MS data from liver and PR:PROJECT_SUMMARY plasma from mice fed a diet in which dietary protein was restricted and PR:PROJECT_SUMMARY corresponding unrestricted controls. Specifically, part 1 and 2 correspond to PR:PROJECT_SUMMARY liver and plasma from the hepatic portal vein respectively from animals on a PR:PROJECT_SUMMARY low-protein diet following a week of diet adaptation and correspond controls PR:PROJECT_SUMMARY with n = 5 for each group. Part 3 of the data presented here corresponds to PR:PROJECT_SUMMARY liver from mice subject to a 3wk treatment with diets either containing 18% PR:PROJECT_SUMMARY digestible energy from amino acids or a diet with restricted amounts of PR:PROJECT_SUMMARY threonine and following prior treatments with adeno-associated viruses to PR:PROJECT_SUMMARY transduce the liver to express yeast threonine biosynthetic enzymes PR:PROJECT_SUMMARY (AAV-yTHR1+THR4) or a negative control (AAV-GFP). n= 6 individual mice per PR:PROJECT_SUMMARY group. PR:INSTITUTE Monash University PR:DEPARTMENT Department of Biochemistry and Molecular Biology PR:LABORATORY Nutrient Metabolism and Signalling Laboratory PR:LAST_NAME Rose PR:FIRST_NAME Adam PR:ADDRESS Monash University, Clayton Campus, 23 Innovation Walk, Clayton, Victoria, 3800, PR:ADDRESS Australia PR:EMAIL adam.rose@monash.edu PR:PHONE +61 3 99029340 PR:DOI http://dx.doi.org/10.21228/M8568J #STUDY ST:STUDY_TITLE Metabolomic changes in mouse liver on a threonine restricted diet (part-III) ST:STUDY_SUMMARY The data provided here are in support of the publication “Restriction of ST:STUDY_SUMMARY essential amino acids dictates the systemic metabolic response to dietary ST:STUDY_SUMMARY protein dilution” Yann W. Yap et al. Nature Communications 2020 (accepted for ST:STUDY_SUMMARY publication). The data in this study corresponds to liver from C57Bl/6JMarp mice ST:STUDY_SUMMARY subject to a 3wk treatment with diets either containing 18% digestible energy ST:STUDY_SUMMARY from amino acids with either a normal distribution of amino acids or with ST:STUDY_SUMMARY restricted amounts of threonine. This followed prior treatment with ST:STUDY_SUMMARY adeno-associated viruses to transduce the liver to express yeast threonine ST:STUDY_SUMMARY biosynthetic enzymes (AAV-yTHR1+THR4) or a negative control (AAV-GFP). n= 6 ST:STUDY_SUMMARY individual mice per group. ST:INSTITUTE Monash University ST:DEPARTMENT Department of Biochemistry and Molecular Biology ST:LABORATORY Nutrient Metabolism and Signalling Laboratory ST:LAST_NAME Rose ST:FIRST_NAME Adam ST:ADDRESS Monash University, Clayton Campus, 23 Innovation Walk, Clayton, Victoria, 3800, ST:ADDRESS Australia ST:EMAIL adam.rose@monash.edu ST:PHONE +61 3 99029340 ST:SUBMIT_DATE 2020-04-03 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57Bl/6JMarp SU:AGE_OR_AGE_RANGE 7 to 12 weeks SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Monash University Animal Research Platform, Clayton, AUS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - AR401_control_AA_GFP_1pt1 Diet:Control diet SF17-177 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR401_control_AA_GFP_1pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR402_control_AA_GFP_1pt2 Diet:Control diet SF17-177 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR402_control_AA_GFP_1pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR403_control_AA_GFP_1pt3 Diet:Control diet SF17-177 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR403_control_AA_GFP_1pt3_1.raw SUBJECT_SAMPLE_FACTORS - AR404_control_AA_GFP_2pt1 Diet:Control diet SF17-177 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR404_control_AA_GFP_2pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR405_control_AA_GFP_2pt2 Diet:Control diet SF17-177 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR405_control_AA_GFP_2pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR406_control_AA_GFP_2pt3 Diet:Control diet SF17-177 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR406_control_AA_GFP_2pt3_1.raw SUBJECT_SAMPLE_FACTORS - AR407_control_AA_THR1plus4_3pt1 Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR407_control_AA_THR1plus4_3pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR408_control_AA_THR1plus4_3pt2 Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR408_control_AA_THR1plus4_3pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR409_control_AA_THR1plus4_3pt3 Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR409_control_AA_THR1plus4_3pt3_1.raw SUBJECT_SAMPLE_FACTORS - AR410_control_AA_THR1plus4_4pt1 Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR410_control_AA_THR1plus4_4pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR411_control_AA_THR1plus4_4pt2 Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR411_control_AA_THR1plus4_4pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR412_control_AA_THR1plus4_4pt3 Diet:Control diet SF17-177 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR412_control_AA_THR1plus4_4pt3_1.raw SUBJECT_SAMPLE_FACTORS - AR413_Low_T_GFP_5pt1 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR413_Low_T_GFP_5pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR414_Low_T_GFP_5pt2 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR414_Low_T_GFP_5pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR415_Low_T_GFP_5pt3 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR415_Low_T_GFP_5pt3_1.raw SUBJECT_SAMPLE_FACTORS - AR416_Low_T_GFP_6pt1 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR416_Low_T_GFP_6pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR417_Low_T_GFP_6pt2 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR417_Low_T_GFP_6pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR418_Low_T_GFP_6pt3 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-GFP RAW_FILE_NAME=C120181105_AR418_Low_T_GFP_6pt3_1.raw SUBJECT_SAMPLE_FACTORS - AR419_Low_T_THR1plus4_7pt1 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR419_Low_T_THR1plus4_7pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR420_Low_T_THR1plus4_7pt2 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR420_Low_T_THR1plus4_7pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR421_Low_T_THR1plus4_7pt3 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR421_Low_T_THR1plus4_7pt3_1.raw SUBJECT_SAMPLE_FACTORS - AR422_Low_T_THR1plus4_8pt1 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR422_Low_T_THR1plus4_8pt1_1.raw SUBJECT_SAMPLE_FACTORS - AR423_Low_T_THR1plus4_8pt2 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR423_Low_T_THR1plus4_8pt2_1.raw SUBJECT_SAMPLE_FACTORS - AR424_Low_T_THR1plus4_8pt3 Diet:Low Threonine diet SF18-066 | Virus treatment:AAV-yTHR1+THR4 RAW_FILE_NAME=C120181105_AR424_Low_T_THR1plus4_8pt3_1.raw #COLLECTION CO:COLLECTION_SUMMARY Mice were humanely euthanised for tissue collection CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Male mice aged 7 weeks upon arrival, were acclimated to the local housing TR:TREATMENT_SUMMARY facility (12-12h light-dark cycle, 22-24°C) for one week prior to TR:TREATMENT_SUMMARY experimentation and were fed standard rodent chow (8720610, Barastoc, AUS). TR:TREATMENT_SUMMARY Following acclimation, mice were administered a total of 5E+11 virus particles TR:TREATMENT_SUMMARY per mouse via the tail vein. For the negative control (NC): 5E+11 GFP-AAV; and TR:TREATMENT_SUMMARY for the THR1/4 overexpression studies mice were administered 2.5E+11 virus TR:TREATMENT_SUMMARY particles each of yTHR1-AAV and yTHR4-AAV. One week following this time, the TR:TREATMENT_SUMMARY dietary intervention was initiated. In these studies, the low threonine diet TR:TREATMENT_SUMMARY (SF18-066, Specialty Feeds AUS) contained homoserine, the substrate of THR1. The TR:TREATMENT_SUMMARY corresponding control diet was SF17-177 (Specialty Feeds AUS). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Livers were cryogenically pulverized (cryopulverization) using a 12-well SP:SAMPLEPREP_SUMMARY biopulverizer (BioSpec Products, OK USA Part number 59012MS) according to the SP:SAMPLEPREP_SUMMARY manufacturer’s instructions. The frozen tissue powder was then weighed and SP:SAMPLEPREP_SUMMARY extracted in 20 µL of extraction solvent (0°C) per mg of tissue. The mixture SP:SAMPLEPREP_SUMMARY was then briefly vortexed before sonication in an ice-water bath for 10 minutes SP:SAMPLEPREP_SUMMARY followed by centrifugation (20,000 rcf, 4℃, 10 minutes). The supernatant was SP:SAMPLEPREP_SUMMARY then transferred to a mass spectrometry vial for LC-MS analysis. The extraction SP:SAMPLEPREP_SUMMARY solvent consisted of 2:6:1 CHCl3:MeOH:H2O v/v/v with 2 µM CHAPS, CAPS, PIPES SP:SAMPLEPREP_SUMMARY and TRIS as internal standards. Additionally where quantitative amino acid SP:SAMPLEPREP_SUMMARY analysis was performed, a mixture of 17 stable isotope labelled amino acids SP:SAMPLEPREP_SUMMARY (Gln, Asn and Trp were absent) were added at a concentration of 500 pmol of each SP:SAMPLEPREP_SUMMARY amino acid per mg liver (Cambridge Isotope Laboratories PN MSK-A2-1.2). SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatography utilized a ZIC-pHILIC column (column temperature 25 °C) with a CH:CHROMATOGRAPHY_SUMMARY gradient elution of 20 mM ammonium carbonate (A) and acetonitrile (B) (linear CH:CHROMATOGRAPHY_SUMMARY gradient time-%B as follows: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%, 24 CH:CHROMATOGRAPHY_SUMMARY min-80%, 32 min-80%) on a Dionex RSLC3000 UHPLC (Thermo). The flow rate was CH:CHROMATOGRAPHY_SUMMARY maintained at 300 μL/min. Samples were kept at 4 °C in the autosampler and 10 CH:CHROMATOGRAPHY_SUMMARY μL injected for analysis. CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME SeQuant ZIC-pHILIC (150 x 4.6mm,5um) CH:COLUMN_TEMPERATURE 25 CH:FLOW_GRADIENT linear gradient time-%B as follows: 0 min-80%, 15 min-50%, 18 min-5%, 21 min-5%, CH:FLOW_GRADIENT 24 min-80%, 32 min-80% CH:FLOW_RATE 300 µL/min CH:SOLVENT_A 100% water; 20 mM ammonium carbonate CH:SOLVENT_B 100% water; acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Mass spectrometry was performed at 35 000 resolution (accuracy calibrated to MS:MS_COMMENTS <1 ppm) on a Q-Exactive Orbitrap MS (Thermo) operating in rapid switching MS:MS_COMMENTS positive (4 kV) and negative (−3.5 kV) mode electrospray ionization (capillary MS:MS_COMMENTS temperature 300 °C; sheath gas 50; Aux gas 20; sweep gas 2; probe temp 120 MS:MS_COMMENTS °C). MS:ION_MODE UNSPECIFIED MS:MS_RESULTS_FILE ST001348_AN002242_Results.txt UNITS:Peak Area Has RT:Yes RT units:Minutes #END