#METABOLOMICS WORKBENCH Jyoti_123_20240821_051157 DATATRACK_ID:5135 STUDY_ID:ST003475 ANALYSIS_ID:AN005710 PROJECT_ID:PR002134 VERSION 1 CREATED_ON September 16, 2024, 10:11 am #PROJECT PR:PROJECT_TITLE Differentially Altered Hepatocellular Global Metabolome in Response to PR:PROJECT_TITLE Endotoxemia and Lipotoxicity PR:PROJECT_SUMMARY The primary focus of the paper revolves around the exploration of the distinct PR:PROJECT_SUMMARY impacts of endotoxemia, induced by gut-derived endotoxin, and lipotoxicity, PR:PROJECT_SUMMARY resulting from adipose-derived free non-esterified fatty acids, on the PR:PROJECT_SUMMARY untargeted global metabolome of hepatocytes. The study elucidates the pivotal PR:PROJECT_SUMMARY significance of these two parameters on hepatotoxicity associated with the PR:PROJECT_SUMMARY pathogenesis of non-alcoholic fatty liver disease (NAFLD). The study highlights PR:PROJECT_SUMMARY the liver as a principal site for metabolic damage, which occurs primarily via PR:PROJECT_SUMMARY two main pathways. One of the pathways includes the transportation of microbial PR:PROJECT_SUMMARY products from the gut, namely endotoxin, which triggers the activation of PR:PROJECT_SUMMARY hepatic Kupffer cells and subsequently induces inflammation in the liver. The PR:PROJECT_SUMMARY second pathway encompasses the consequences of lipotoxicity, which arises from PR:PROJECT_SUMMARY the relocation of fatty acids from adipose tissue to the liver, ultimately PR:PROJECT_SUMMARY leading to the development of hepatic steatosis. The study used HepG2 cells as a PR:PROJECT_SUMMARY model system to assess the individual impacts of endotoxin (LPS) and fatty acids PR:PROJECT_SUMMARY on the intracellular global metabolome. This study elucidates the distinct PR:PROJECT_SUMMARY effects of these two parameters on the overall metabolic balance of hepatocytes. PR:PROJECT_SUMMARY The manuscript highlights the significance of comprehending the distinct impacts PR:PROJECT_SUMMARY of endotoxemia and lipotoxicity on hepatocellular metabolism, particularly in PR:PROJECT_SUMMARY relation to the growing incidence of lean nonalcoholic steatohepatitis (NASH). PR:PROJECT_SUMMARY This form of NASH is not linked to obesity or lipotoxicity originating from PR:PROJECT_SUMMARY adipose tissue. Moreover, it underscores the importance of metabolomics in the PR:PROJECT_SUMMARY detection of biomarkers, comprehension of disease processes, and formulation of PR:PROJECT_SUMMARY diagnostic and therapeutic approaches for liver illnesses and metabolic PR:PROJECT_SUMMARY disorders. PR:INSTITUTE THAPAR INSTITUTE OF ENGINEERING AND TECHNOLOGY PR:LAST_NAME Sharma PR:FIRST_NAME Jyoti PR:ADDRESS Bhadson Rd, Adarsh Nagar, Prem Nagar, Patiala, PATIALA, PUNJAB, 147004, India PR:EMAIL jjyoti_phd22@thapar.edu PR:PHONE +91-9064275660 #STUDY ST:STUDY_TITLE Intracellular metabolome and Intracellular metabolic pathway prediction ST:STUDY_SUMMARY The study used HepG2 cells as a model system to assess the individual impacts of ST:STUDY_SUMMARY endotoxin (LPS) and fatty acids on the intracellular global metabolome. This ST:STUDY_SUMMARY study elucidates the distinct effects of these two parameters on the overall ST:STUDY_SUMMARY metabolic balance of hepatocytes. ST:INSTITUTE THAPAR INSTITUTE OF ENGINEERING AND TECHNOLOGY ST:LAST_NAME Sharma ST:FIRST_NAME Jyoti ST:ADDRESS Bhadson Rd, Adarsh Nagar, Prem Nagar, Patiala, PATIALA, PUNJAB, 147004, India ST:EMAIL jjyoti_phd22@thapar.edu ST:PHONE +91-9064275660 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 Wild-type A_Cntrl Sample source:Human liver cancer cells | Treatment:Control Cell line=HepG2 Cells; RAW_FILE_NAME(Raw file name)=PD_GCMS_Cntrl SUBJECT_SAMPLE_FACTORS Wild-type B_LPS Sample source:Human liver cancer cells | Treatment:LPS Cell line=HepG2 Cells; RAW_FILE_NAME(Raw file name)=PD_GCMS_LPS SUBJECT_SAMPLE_FACTORS Wild-type C_FFA Sample source:Human liver cancer cells | Treatment:FFA Cell line=HepG2 Cells; RAW_FILE_NAME(Raw file name)=PD_GCMS_FFA #COLLECTION CO:COLLECTION_SUMMARY HepG2 cells were procured from National Institute of Cell Science (Pune, India) CO:COLLECTION_SUMMARY and cells were cultures as per our previously standardized method (Rezgui et CO:COLLECTION_SUMMARY al., 2023). In brief, cells were cultured (37°C and 5% CO2) in low-glucose CO:COLLECTION_SUMMARY Dulbecco's Modified Eagle Medium (HiMedia,India) supplemented with 10% fetal CO:COLLECTION_SUMMARY bovine serum, 100 UI/mL penicillin, 100 ug/mL streptomycin,and 25 ug/mL CO:COLLECTION_SUMMARY amphotericin B. An Olympus inverted microscope (model) was used to track cell CO:COLLECTION_SUMMARY development during sub-culturing, which was carried out at intervals of 45–50 CO:COLLECTION_SUMMARY h. A cell counting equipment (Far-scope B, Curiosis) was used to do the trypan CO:COLLECTION_SUMMARY blue cell count. CO:SAMPLE_TYPE HepG2 cells #TREATMENT TR:TREATMENT_SUMMARY HepG2 cells were seeded in 6 well-plates (2X106 119 cells/mL) in DMEM without TR:TREATMENT_SUMMARY serum for 24-h to achieve 85-90% confluency. Next, cells were either treated TR:TREATMENT_SUMMARY with FAA-albumin conjugate at 400µM concentration or with 200 ng/mL TR:TREATMENT_SUMMARY lipopolysaccharide (LPS; E. coli O55.B5, Sigma, USA) for 24 h under standard TR:TREATMENT_SUMMARY conditions. Untreated cells were considered as control. After 24-h of treatment, TR:TREATMENT_SUMMARY cells were separated from the culture supernatant and cell extracts were TR:TREATMENT_SUMMARY prepared as described before in Rezgui et al., Antioxidants 2023, 12(4), 930. TR:TREATMENT_SUMMARY https://doi.org/10.3390/antiox12040930. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Preparation of fatty acid-albumin conjugate preparation- Palmitic-acid albumin SP:SAMPLEPREP_SUMMARY conjugate was prepared based on a previously standardized method (Cousin et al., SP:SAMPLEPREP_SUMMARY 2001). In brief, palmitic acid (Sigma #P0500) was heated at 70°C with 0.1M NaOH SP:SAMPLEPREP_SUMMARY under constant shaking in a Thermo mixer to prepare a stock solution (0.1 M, SP:SAMPLEPREP_SUMMARY solution A). In parallel, 5% (w/v) FAA and endotoxin-free bovine serum albumin SP:SAMPLEPREP_SUMMARY (Sigma #126579) was mixed with water at 55°C under constant shaking (solution SP:SAMPLEPREP_SUMMARY B). Both the solutions were mixed at desired proportions at 55°C under constant SP:SAMPLEPREP_SUMMARY shaking to obtain 10 mM PA-BSA conjugate. The resultant was filtered through SP:SAMPLEPREP_SUMMARY 0.45 uM hydrophilic membrane syringe filter, cooled down at room temperature, SP:SAMPLEPREP_SUMMARY and stored at -20°C until further use (stability 3-4 wk). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Pooled metabolite extract (n = 3) was analyzed using a Shimadzu QP 2010 Ultra CH:CHROMATOGRAPHY_SUMMARY GC-MS instrument equipped with a TG‑5MS column (30 m X 0.25 mm X 0.25 µm). CH:CHROMATOGRAPHY_SUMMARY The injector temperature was set at 250 °C and the initial temperature of the CH:CHROMATOGRAPHY_SUMMARY program was set at 60 °C (solvent delay 4 min) with a hold of 4 min, followed CH:CHROMATOGRAPHY_SUMMARY by a ramp of 10 °C to 300 °C with a hold of 10 min. Derivatized samples (1 CH:CHROMATOGRAPHY_SUMMARY µL) were injected in a split mode (split ratio 20:1) with a splitless time of CH:CHROMATOGRAPHY_SUMMARY 0.80 min, with a constant flow of helium gas (1 mL/min). CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Shimadzu GCMS-QP2010 ultra CH:COLUMN_NAME ThermoFisher TG‑5MS (30 m x 0.25 mm, 0.25um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE - CH:COLUMN_TEMPERATURE Temperature-programmed GC: the initial temperature of the program was set at 60 CH:COLUMN_TEMPERATURE °C (solvent delay 4 min) with a hold of 4 min, followed by a ramp of 10 °C to CH:COLUMN_TEMPERATURE 300 °C with a hold of 10 min #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Shimadzu QP2010 Ultra MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS transfer line temperature was set at 290 °C with an ion source temperature MS:MS_COMMENTS of 200 °C (electron ionization). The samples were analyzed at electron energy MS:MS_COMMENTS 70 eV (vacuum pressure: 2.21e‑0.5 Torr), and the mass analyzer range was set MS:MS_COMMENTS to 50-650 amu. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS % Abundance MS_METABOLITE_DATA_START Samples A_Cntrl B_LPS C_FFA Factors Sample source:Human liver cancer cells | Treatment:Control Sample source:Human liver cancer cells | Treatment:LPS Sample source:Human liver cancer cells | Treatment:FFA 2,4-Pentanediol 0.02 N,N-Dimethylacetamide 0.17 Propanoic acid 0.02 1,3-Pentadiene 0.27 Benzo[b]thiophene, 2-ethyl- 0.28 3-Methyl-3-pentanol 0.22 2-Amino-4-tertbutylthiazole 0.1 4-Methoxy-2-allylphenol 0.02 2-Propanone 0.03 Sarcosylserine 0.13 2,6-Dimethylbenzenethiol, S-acetyl- 0.43 ?-D-Glucopyranoside 0 ?-Hydroxypyruvic acid 0.47 N-ethyl-butanamide 0.19 Thiazole, 5-methyl-2-phenyl 0.02 D-(+)-Arabitol 0.04 Acetamide 0.63 Benzo[b]thiophene, 2-ethyl 0.24 Pyrazine 0.03 Butanoic acid 0.63 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name MOL. WEIGHT RETENTION TIME 2,4-Pentanediol 104 7.335 N,N-Dimethylacetamide 87 7.3605 Propanoic acid 74 7.3715 1,3-Pentadiene 68 7.501 Benzo[b]thiophene, 2-ethyl- 162 7.5375 3-Methyl-3-pentanol 102 7.5525 2-Amino-4-tertbutylthiazole 156 7.565 4-Methoxy-2-allylphenol 164 7.63 2-Propanone 58 7.7235 Sarcosylserine †89 7.678 2,6-Dimethylbenzenethiol, S-acetyl- 210.41 7.9135 ?-D-Glucopyranoside 194 8.3015 ?-Hydroxypyruvic acid 104 8.3305 N-ethyl-butanamide 115 8.3325 Thiazole, 5-methyl-2-phenyl 175 8.089 D-(+)-Arabitol 152 7.409 Acetamide 59 7.49 Benzo[b]thiophene, 2-ethyl 162 7.495 Pyrazine 80 7.5895 Butanoic acid 88 7.6155 METABOLITES_END #END