#METABOLOMICS WORKBENCH Mahamogren_20230529_060250 DATATRACK_ID:4052 STUDY_ID:ST002735 ANALYSIS_ID:AN004434 PROJECT_ID:PR001699 VERSION 1 CREATED_ON June 12, 2023, 10:41 am #PROJECT PR:PROJECT_TITLE Untargeted metabolomics revealed multiple metabolic perturbations in plasma of PR:PROJECT_TITLE T2D patients in response to Liraglutide PR:PROJECT_SUMMARY Despite the global efforts put into the clinical research and studies in order PR:PROJECT_SUMMARY to protect against Type-2 diabetes mellitus (T2DM), the incidence of T2DM PR:PROJECT_SUMMARY remains high causing a major health problem and impacting the health and care PR:PROJECT_SUMMARY systems. Therefore, T2DM-related treatments and therapies are continuously PR:PROJECT_SUMMARY invented for the clinical use, including Liraglutide. The last is a GLP-1 PR:PROJECT_SUMMARY analogue and shows its beneficial health outcomes e.g., improved glycemic PR:PROJECT_SUMMARY control, lower body weight, and reduced cardiovascular disease risks. The PR:PROJECT_SUMMARY intrinsic mechanisms of these beneficial effects are not fully understood; PR:PROJECT_SUMMARY however, our research group has previously published proteomics work PR:PROJECT_SUMMARY demonstrating the involvement of certain important proteins in part in the PR:PROJECT_SUMMARY beneficial health outcomes of Liraglutide. Since proteomics and metabolomics are PR:PROJECT_SUMMARY complementary to each other in the context of the biological pathways, studying PR:PROJECT_SUMMARY the metabolic impacts of Liraglutide on T2DM patients would add further PR:PROJECT_SUMMARY information about the beneficial health outcomes of Liraglutide. Thus, herein, PR:PROJECT_SUMMARY we performed an untargeted metabolomics approach for identifying metabolic PR:PROJECT_SUMMARY pathways impacted by the treatment of Liraglutide in T2DM patients. Methods: PR:PROJECT_SUMMARY Untargeted liquid chromatography coupled with mass spectrometry was used for PR:PROJECT_SUMMARY metabolomics analysis of plasma samples collected from T2DM patients (n=20) PR:PROJECT_SUMMARY before and after receiving Liraglutide treatment. Metabolic profiling and PR:PROJECT_SUMMARY related pathway and network analyses were conducted. Results: The metabolic PR:PROJECT_SUMMARY profiling analyses identified 93 endogenous metabolites were significantly PR:PROJECT_SUMMARY affected by the Liraglutide treatments, which 49 metabolites up-regulated and 44 PR:PROJECT_SUMMARY metabolites down-regulated. Moreover, the metabolic pathway analyses revealed PR:PROJECT_SUMMARY that the most pronounced metabolite and metabolic pathways that were affected by PR:PROJECT_SUMMARY the Liraglutide treatment was Pentose and glucuronate interconversion, PR:PROJECT_SUMMARY suggesting the last may be a potential target of the Liraglutide treatment could PR:PROJECT_SUMMARY be involved in part in the beneficial effects seen in T2DM patients, specially, PR:PROJECT_SUMMARY we found that glucuronate interconversion pathway which is known by its role in PR:PROJECT_SUMMARY eliminating toxic and undesirable substances from the human body, impacted in PR:PROJECT_SUMMARY Liraglutide treated patients. The last findings ar consistence with our previous PR:PROJECT_SUMMARY proteomics findings. Conclusion: These findings, taken together with our PR:PROJECT_SUMMARY previous results, provide a deeper understanding of the underlying mechanisms PR:PROJECT_SUMMARY involved in the beneficial effects of Liraglutide at the proteomic and metabolic PR:PROJECT_SUMMARY levels in T2DM patients. PR:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) PR:LAST_NAME Al Mogren PR:FIRST_NAME Maha PR:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia PR:EMAIL malmogren@alfaisal.edu PR:PHONE 966541205332 #STUDY ST:STUDY_TITLE Untargeted metabolomics revealed multiple metabolic perturbations in plasma of ST:STUDY_TITLE T2D patients in response to Liraglutide ST:STUDY_SUMMARY Despite the global efforts put into the clinical research and studies in order ST:STUDY_SUMMARY to protect against Type-2 diabetes mellitus (T2DM), the incidence of T2DM ST:STUDY_SUMMARY remains high causing a major health problem and impacting the health and care ST:STUDY_SUMMARY systems. Therefore, T2DM-related treatments and therapies are continuously ST:STUDY_SUMMARY invented for the clinical use, including Liraglutide. The last is a GLP-1 ST:STUDY_SUMMARY analogue and shows its beneficial health outcomes e.g., improved glycemic ST:STUDY_SUMMARY control, lower body weight, and reduced cardiovascular disease risks. The ST:STUDY_SUMMARY intrinsic mechanisms of these beneficial effects are not fully understood; ST:STUDY_SUMMARY however, our research group has previously published proteomics work ST:STUDY_SUMMARY demonstrating the involvement of certain important proteins in part in the ST:STUDY_SUMMARY beneficial health outcomes of Liraglutide. Since proteomics and metabolomics are ST:STUDY_SUMMARY complementary to each other in the context of the biological pathways, studying ST:STUDY_SUMMARY the metabolic impacts of Liraglutide on T2DM patients would add further ST:STUDY_SUMMARY information about the beneficial health outcomes of Liraglutide. Thus, herein, ST:STUDY_SUMMARY we performed an untargeted metabolomics approach for identifying metabolic ST:STUDY_SUMMARY pathways impacted by the treatment of Liraglutide in T2DM patients. Methods: ST:STUDY_SUMMARY Untargeted liquid chromatography coupled with mass spectrometry was used for ST:STUDY_SUMMARY metabolomics analysis of plasma samples collected from T2DM patients (n=20) ST:STUDY_SUMMARY before and after receiving Liraglutide treatment. Metabolic profiling and ST:STUDY_SUMMARY related pathway and network analyses were conducted. Results: The metabolic ST:STUDY_SUMMARY profiling analyses identified 93 endogenous metabolites were significantly ST:STUDY_SUMMARY affected by the Liraglutide treatments, which 49 metabolites up-regulated and 44 ST:STUDY_SUMMARY metabolites down-regulated. Moreover, the metabolic pathway analyses revealed ST:STUDY_SUMMARY that the most pronounced metabolite and metabolic pathways that were affected by ST:STUDY_SUMMARY the Liraglutide treatment was Pentose and glucuronate interconversion, ST:STUDY_SUMMARY suggesting the last may be a potential target of the Liraglutide treatment could ST:STUDY_SUMMARY be involved in part in the beneficial effects seen in T2DM patients, specially, ST:STUDY_SUMMARY we found that glucuronate interconversion pathway which is known by its role in ST:STUDY_SUMMARY eliminating toxic and undesirable substances from the human body, impacted in ST:STUDY_SUMMARY Liraglutide treated patients. The last findings ar consistence with our previous ST:STUDY_SUMMARY proteomics findings. Conclusion: These findings, taken together with our ST:STUDY_SUMMARY previous results, provide a deeper understanding of the underlying mechanisms ST:STUDY_SUMMARY involved in the beneficial effects of Liraglutide at the proteomic and metabolic ST:STUDY_SUMMARY levels in T2DM patients. ST:INSTITUTE King Faisal Specialist Hospital and Research Centre (KFSHRC) ST:LAST_NAME Al Mogren ST:FIRST_NAME Maha ST:ADDRESS Zahrawi Street, Al Maather, Riyadh 11211, Saudi Arabia ST:EMAIL malmogren@alfaisal.edu ST:PHONE 966541205332 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #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 - RS_1 Factor:Pre-treatment RAW_FILE_NAME=RS_1 SUBJECT_SAMPLE_FACTORS - RS_2 Factor:Pre-treatment RAW_FILE_NAME=RS_2 SUBJECT_SAMPLE_FACTORS - RS_3 Factor:Pre-treatment RAW_FILE_NAME=RS_3 SUBJECT_SAMPLE_FACTORS - RS_4 Factor:Pre-treatment RAW_FILE_NAME=RS_4 SUBJECT_SAMPLE_FACTORS - RS_5 Factor:Pre-treatment RAW_FILE_NAME=RS_5 SUBJECT_SAMPLE_FACTORS - RS_6 Factor:Pre-treatment RAW_FILE_NAME=RS_6 SUBJECT_SAMPLE_FACTORS - RS_7 Factor:Pre-treatment RAW_FILE_NAME=RS_7 SUBJECT_SAMPLE_FACTORS - RS_8 Factor:Pre-treatment RAW_FILE_NAME=RS_8 SUBJECT_SAMPLE_FACTORS - RS_9 Factor:Pre-treatment RAW_FILE_NAME=RS_9 SUBJECT_SAMPLE_FACTORS - RS_10 Factor:Pre-treatment RAW_FILE_NAME=RS_10 SUBJECT_SAMPLE_FACTORS - RS_11 Factor:Pre-treatment RAW_FILE_NAME=RS_11 SUBJECT_SAMPLE_FACTORS - RS_12 Factor:Pre-treatment RAW_FILE_NAME=RS_12 SUBJECT_SAMPLE_FACTORS - RS_13 Factor:Pre-treatment RAW_FILE_NAME=RS_13 SUBJECT_SAMPLE_FACTORS - RS_14 Factor:Pre-treatment RAW_FILE_NAME=RS_14 SUBJECT_SAMPLE_FACTORS - RS_15 Factor:Pre-treatment RAW_FILE_NAME=RS_15 SUBJECT_SAMPLE_FACTORS - RS_16 Factor:Pre-treatment RAW_FILE_NAME=RS_16 SUBJECT_SAMPLE_FACTORS - RS_17 Factor:Pre-treatment RAW_FILE_NAME=RS_17 SUBJECT_SAMPLE_FACTORS - RS_18 Factor:Pre-treatment RAW_FILE_NAME=RS_18 SUBJECT_SAMPLE_FACTORS - RS_19 Factor:Pre-treatment RAW_FILE_NAME=RS_19 SUBJECT_SAMPLE_FACTORS - RS_20 Factor:Pre-treatment RAW_FILE_NAME=RS_20 SUBJECT_SAMPLE_FACTORS - RS_P1 Factor:Post-treatment RAW_FILE_NAME=RS_P1 SUBJECT_SAMPLE_FACTORS - RS_P2 Factor:Post-treatment RAW_FILE_NAME=RS_P2 SUBJECT_SAMPLE_FACTORS - RS_P3 Factor:Post-treatment RAW_FILE_NAME=RS_P3 SUBJECT_SAMPLE_FACTORS - RS_P4 Factor:Post-treatment RAW_FILE_NAME=RS_P4 SUBJECT_SAMPLE_FACTORS - RS_P5 Factor:Post-treatment RAW_FILE_NAME=RS_P5 SUBJECT_SAMPLE_FACTORS - RS_P6 Factor:Post-treatment RAW_FILE_NAME=RS_P6 SUBJECT_SAMPLE_FACTORS - RS_P7 Factor:Post-treatment RAW_FILE_NAME=RS_P7 SUBJECT_SAMPLE_FACTORS - RS_P8 Factor:Post-treatment RAW_FILE_NAME=RS_P8 SUBJECT_SAMPLE_FACTORS - RS_P9 Factor:Post-treatment RAW_FILE_NAME=RS_P9 SUBJECT_SAMPLE_FACTORS - RS_P10 Factor:Post-treatment RAW_FILE_NAME=RS_P10 SUBJECT_SAMPLE_FACTORS - RS_P11 Factor:Post-treatment RAW_FILE_NAME=RS_P11 SUBJECT_SAMPLE_FACTORS - RS_P12 Factor:Post-treatment RAW_FILE_NAME=RS_P12 SUBJECT_SAMPLE_FACTORS - RS_P13 Factor:Post-treatment RAW_FILE_NAME=RS_P13 SUBJECT_SAMPLE_FACTORS - RS_P14 Factor:Post-treatment RAW_FILE_NAME=RS_P14 SUBJECT_SAMPLE_FACTORS - RS_P15 Factor:Post-treatment RAW_FILE_NAME=RS_P15 SUBJECT_SAMPLE_FACTORS - RS_P16 Factor:Post-treatment RAW_FILE_NAME=RS_P16 SUBJECT_SAMPLE_FACTORS - RS_P17 Factor:Post-treatment RAW_FILE_NAME=RS_P17 SUBJECT_SAMPLE_FACTORS - RS_P18 Factor:Post-treatment RAW_FILE_NAME=RS_P18 SUBJECT_SAMPLE_FACTORS - RS_P19 Factor:Post-treatment RAW_FILE_NAME=RS_P19 SUBJECT_SAMPLE_FACTORS - RS_P20 Factor:Post-treatment RAW_FILE_NAME=RS_P20 #COLLECTION CO:COLLECTION_SUMMARY The study was approved by the Institutional Review Board of the College of CO:COLLECTION_SUMMARY Medicine, King Saud University, Riyadh, Saudi Arabia (registration no. CO:COLLECTION_SUMMARY E-18-3075). Recruited patients were asked to sign a written informed consent CO:COLLECTION_SUMMARY form before enrolling. Twenty patients who were diagnosed with T2DM were CO:COLLECTION_SUMMARY referred to the King Khaled University Hospital's (KKUH), Obesity Research CO:COLLECTION_SUMMARY Center, where this study took place. Patients were treated with an appropriate CO:COLLECTION_SUMMARY dose of Liraglutide for a three months as described previously (8). Samples were CO:COLLECTION_SUMMARY taken pre-treatment and post-treatment. Note: the T2DM participants were on CO:COLLECTION_SUMMARY other medications including insulin and metformin beside the Liraglutide CO:COLLECTION_SUMMARY treatment. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Patients with indications of add-on liraglutide were started on treatment by TR:TREATMENT_SUMMARY their physician in a scaled-up dose from 0.6 mg to 1.8 mg of a once-daily TR:TREATMENT_SUMMARY subcutaneous injection over a period of three weeks. The follow-up visit was TR:TREATMENT_SUMMARY scheduled 3 months after receiving the full dose (1.8 mg) of liraglutide. Urine TR:TREATMENT_SUMMARY samples were collected at two time points: one sample before and another sample TR:TREATMENT_SUMMARY after treatment with liraglutide. Blood samples were collected by venipuncture TR:TREATMENT_SUMMARY into plain tubes (Vacutainer, BD Biosciences, San Jose, CA, USA) from each TR:TREATMENT_SUMMARY patient after a 10 h fast. The plasma was separated by centrifugation (15 min, TR:TREATMENT_SUMMARY 3000× g), divided into several aliquots, and stored at −80 °C for further TR:TREATMENT_SUMMARY analysis. TR:TREATMENT_COMPOUND Liraglutide #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted from plasma were collected from 20 type2 diabetic SP:SAMPLEPREP_SUMMARY patients, pre-and post-treatment with liraglutide (n=40 samples) (10). Briefly, SP:SAMPLEPREP_SUMMARY 100 μL plasma sample were mixed with 900 μL of extraction solvent 50% SP:SAMPLEPREP_SUMMARY acetonitrile (ACN) in methanol (MeOH). Meanwhile, QC samples were prepared with SP:SAMPLEPREP_SUMMARY aliquots from all samples to check for system stability. The mixtures were mixed SP:SAMPLEPREP_SUMMARY on thermomixer at 600 rpm at room temperature for one hour (Eppendorf, CITY, SP:SAMPLEPREP_SUMMARY Germany). Afterward, the samples were centrifuged at 16000 rpm at 4ºC for 10 SP:SAMPLEPREP_SUMMARY min. The supernatant was transferred into new Eppendrof tube, and then SP:SAMPLEPREP_SUMMARY evaporated completely in a SpeedVac (Christ, Germany). The dried samples were SP:SAMPLEPREP_SUMMARY reconstituted with100 μl of 50% mobile phase A: B (A: 0.1% Formic acid in dH2O, SP:SAMPLEPREP_SUMMARY B: 0.1% Formic acid in 50% ACN: MeOH). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity UPLC CH:COLUMN_NAME Waters XSelect HSS C18 (100 × 2.1mm,2.5um) CH:SOLVENT_A 0.1% formic acid in dH2O CH:SOLVENT_B 0.1% formic acid in 50% MeOH and ACN CH:FLOW_GRADIENT 0-16 min 95- 5% A, 16-19 min 5% A, 19-20 min 5-95% A, 20-22 min 95- 95% A CH:FLOW_RATE 300 µL/min CH:COLUMN_TEMPERATURE 55 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-S MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The DIA data were collected with a Masslynx™ V4.1 workstation in continuum MS:MS_COMMENTS mode (Waters Inc., Milford, MA, USA). The raw MS data were processed following a MS:MS_COMMENTS standard pipeline using the Progenesis QI v.3.0 software. MS:MS_RESULTS_FILE ST002735_AN004434_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END