#METABOLOMICS WORKBENCH jgsilva_20250124_090213 DATATRACK_ID:5566 STUDY_ID:ST003698 ANALYSIS_ID:AN006068 PROJECT_ID:PR002295 VERSION 1 CREATED_ON January 30, 2025, 8:51 am #PROJECT PR:PROJECT_TITLE Impact of High Fat diet-induced MASLD on Heart, Kidney and Skeletal Muscle PR:PROJECT_TITLE Metabolomes in Wild-type Mice PR:PROJECT_TYPE 1H NMR metabolomics to study the effects high-fat diet-induced MASLD on PR:PROJECT_TYPE extrahepatic tissues metabolomes, namely the heart, kidney and skeletal muscle, PR:PROJECT_TYPE in C57BL6J wild-type mice. PR:PROJECT_SUMMARY Excessive caloric intake is a primary driver of metabolic dysfunction-associated PR:PROJECT_SUMMARY steatotic liver disease (MASLD), and this has been recapitulated in mice fed a PR:PROJECT_SUMMARY high-fat diet. In 2023, the global prevalence of MASLD was estimated at 30%, PR:PROJECT_SUMMARY with high incidences affecting wealthy urbanised countries. This implication of PR:PROJECT_SUMMARY hypercaloric diets can also perturb metabolism and function of extrahepatic PR:PROJECT_SUMMARY tissues such as heart, kidney and skeletal muscle. These effects that can take PR:PROJECT_SUMMARY place in extrahepatic tissues are still poorly understood in terms of metabolic PR:PROJECT_SUMMARY alterations and physiology, and represent an important point of improvement in PR:PROJECT_SUMMARY the knowledge gap that connects early stage MASLD with other obesity related PR:PROJECT_SUMMARY comorbidities, such as type 2 diabetes, insulin resistance, cardiovascular and PR:PROJECT_SUMMARY renal complications, and overall, with the so known metabolic syndrome. In this PR:PROJECT_SUMMARY study, we aimed to evaluate the potential of using metabolomics to unravel the PR:PROJECT_SUMMARY effects and interactions taking place in a diet-induced MASLD model related to PR:PROJECT_SUMMARY the development of the disorder. Black-6 mice were subjected to either a control PR:PROJECT_SUMMARY diet or a high-fat diet for 18 weeks, from which at the end their heart, kidney PR:PROJECT_SUMMARY and skeletal muscle metabolites were extracted. The metabolites, divided into PR:PROJECT_SUMMARY aqueous and lipophilic fractions, were acquired by 1H-NMR, and then processed PR:PROJECT_SUMMARY using a untargeted Metabolomics and Lipidomics analysis approach, to identify PR:PROJECT_SUMMARY key changes occurring between control and high-fat diet in these models. These PR:PROJECT_SUMMARY results added important information to better understand the link between early PR:PROJECT_SUMMARY onset MASLD and the Metabolic Syndrome and its comorbidities, though several PR:PROJECT_SUMMARY metabolic changes in the extrahepatic tissues, namely in ectopic fat deposition PR:PROJECT_SUMMARY and alterations to Randle cycle and gut microbiota activity. PR:INSTITUTE Center for Innovative Biomedicine and Biotechnology (CIBB UC) PR:DEPARTMENT Institute of Interdisciplinary Research PR:LAST_NAME Silva PR:FIRST_NAME João PR:ADDRESS Rua Larga - Faculdade de Medicina, 1ºandar - POLO I Universidade de Coimbra PR:EMAIL jgsilva@cnc.uc.pt PR:PHONE (+351) 239 820 190 PR:PROJECT_COMMENTS Full NMR sample preparation and analysis procedures are available in the PR:PROJECT_COMMENTS accompanying document entitled 1. MASLD Extrahepatic Metabolomics experimental PR:PROJECT_COMMENTS procedure. The normalized data that was used in uni- and multivariate analysis PR:PROJECT_COMMENTS is available in the accompanying files: 4. MASLD Extrahepatic Metabolomics PR:PROJECT_COMMENTS results data.txt The raw fid as well as 1r file can be found in 5. MASLD PR:PROJECT_COMMENTS Extrahepatic Metabolomics 1H NMR Raw Data.zip #STUDY ST:STUDY_TITLE Impact of High Fat diet-induced metabolic dysfunction-associated steatotic liver ST:STUDY_TITLE disease (MASLD) on Heart, Kidney and Skeletal Muscle Metabolomes in Wild-type ST:STUDY_TITLE Mice ST:STUDY_TYPE 1H NMR metabolomics to study the effects high-fat diet-induced MASLD on ST:STUDY_TYPE extrahepatic tissues metabolomes, namely the heart, kidney and skeletal muscle, ST:STUDY_TYPE in C57BL6J wild-type mice. ST:STUDY_SUMMARY Excessive caloric intake is a primary driver of metabolic dysfunction-associated ST:STUDY_SUMMARY steatotic liver disease (MASLD), and this has been recapitulated in mice fed a ST:STUDY_SUMMARY high-fat diet. In 2023, the global prevalence of MASLD was estimated at 30%, ST:STUDY_SUMMARY with high incidences affecting wealthy urbanised countries. This implication of ST:STUDY_SUMMARY hypercaloric diets can also perturb metabolism and function of extrahepatic ST:STUDY_SUMMARY tissues such as heart, kidney and skeletal muscle. These effects that can take ST:STUDY_SUMMARY place in extrahepatic tissues are still poorly understood in terms of metabolic ST:STUDY_SUMMARY alterations and physiology, and represent an important point of improvement in ST:STUDY_SUMMARY the knowledge gap that connects early stage MASLD with other obesity related ST:STUDY_SUMMARY comorbidities, such as type 2 diabetes, insulin resistance, cardiovascular and ST:STUDY_SUMMARY renal complications, and overall, with the so known metabolic syndrome. In this ST:STUDY_SUMMARY study, we aimed to evaluate the potential of using metabolomics to unravel the ST:STUDY_SUMMARY effects and interactions taking place in a diet-induced MASLD model related to ST:STUDY_SUMMARY the development of the disorder. Black-6 mice were subjected to either a control ST:STUDY_SUMMARY diet or a high-fat diet for 18 weeks, from which at the end their heart, kidney ST:STUDY_SUMMARY and skeletal muscle metabolites were extracted. The metabolites, divided into ST:STUDY_SUMMARY aqueous and lipophilic fractions, were acquired by 1H-NMR, and then processed ST:STUDY_SUMMARY using a untargeted Metabolomics and Lipidomics analysis approach, to identify ST:STUDY_SUMMARY key changes occurring between control and high-fat diet in these models. These ST:STUDY_SUMMARY results added important information to better understand the link between early ST:STUDY_SUMMARY onset MASLD and the Metabolic Syndrome and its comorbidities, though several ST:STUDY_SUMMARY metabolic changes in the extrahepatic tissues, namely in ectopic fat deposition ST:STUDY_SUMMARY and alterations to Randle cycle and gut microbiota activity. ST:INSTITUTE Center for Innovative Biomedicine and Biotechnology (CIBB UC) ST:DEPARTMENT Institute of Interdisciplinary Research ST:LABORATORY Metabolic Modelling and Systems Biology ST:LAST_NAME Silva ST:FIRST_NAME João ST:ADDRESS Rua Larga - Faculdade de Medicina, 1ºandar - POLO I Universidade de Coimbra ST:EMAIL jgsilva@cnc.uc.pt ST:PHONE (+351) 239 820 190 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 23 ST:NUM_MALES 23 ST:STUDY_COMMENTS Full NMR sample preparation and analysis procedures are available in the ST:STUDY_COMMENTS accompanying document entitled 1. MASLD Extrahepatic Metabolomics experimental ST:STUDY_COMMENTS procedure. The normalized data that was used in uni- and multivariate analysis ST:STUDY_COMMENTS is available in the accompanying files: 4. MASLD Extrahepatic Metabolomics ST:STUDY_COMMENTS results data.txt The raw fid as well as 1r file can be found in 5. MASLD ST:STUDY_COMMENTS Extrahepatic Metabolomics 1H NMR Raw Data.zip #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL6J SU:AGE_OR_AGE_RANGE 10 weeks SU:WEIGHT_OR_WEIGHT_RANGE Average 25g SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Charles River Labs (Barcelona, Spain. RRID: IMSR_JAX:000664) SU:ANIMAL_HOUSING Twenty-four adult male C57BL6J mice obtained from Charles River Labs (Barcelona, SU:ANIMAL_HOUSING Spain. RRID: IMSR_JAX:000664) at 8 weeks of age were housed at the University of SU:ANIMAL_HOUSING Coimbra UC Biotech Bioterium. The mice were kept in a well-ventilated SU:ANIMAL_HOUSING environment with a 12-hour light/dark cycle. Upon arrival to the Bioterium, mice SU:ANIMAL_HOUSING were randomly assigned into cages with four mice per cage and given a two-week SU:ANIMAL_HOUSING adaptation period with free access to water and standard chow. SU:ANIMAL_LIGHT_CYCLE 12-hour light/dark cycle SU:ANIMAL_FEED After acclimatisation, twelve of the mice were provided with high-fat chow (41% SU:ANIMAL_FEED carbohydrate, 30% fat, 25% protein and 4% ash) (HF group), while the remaining SU:ANIMAL_FEED mice were kept on standard chow (73% carbohydrate, 4% fat, 19% protein and 4% SU:ANIMAL_FEED ash) (SC group) during the following 18 weeks. SU:ANIMAL_WATER Normal water #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 - AQ_H_SC_A1 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_H_SC_A1 SUBJECT_SAMPLE_FACTORS - AQ_H_SC_A2 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_H_SC_A2 SUBJECT_SAMPLE_FACTORS - AQ_H_SC_A3 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_H_SC_A3 SUBJECT_SAMPLE_FACTORS - AQ_H_SC_A4 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_H_SC_A4 SUBJECT_SAMPLE_FACTORS - AQ_H_SC_B1 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_H_SC_B1 SUBJECT_SAMPLE_FACTORS - AQ_H_SC_B2 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Aqueous; 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RAW_FILE_NAME(Raw file name)=JGS_AQ_M_SC_C4 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_A1 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_A1 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_A2 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_A2 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_A3 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_A3 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_A4 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_A4 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_B1 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_B1 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_B2 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_B2 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_B3 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_B3 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_B4 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_B4 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_C2 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_C2 SUBJECT_SAMPLE_FACTORS - AQ_M_HF_C3 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Aqueous; RAW_FILE_NAME(Raw file name)=JGS_AQ_M_HF_C3 SUBJECT_SAMPLE_FACTORS - LE_H_SC_A1 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_H_SC_A1 SUBJECT_SAMPLE_FACTORS - LE_H_SC_A2 Diet Group:SC | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; 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RAW_FILE_NAME(Raw file name)=JGS_LE_H_HF_B1 SUBJECT_SAMPLE_FACTORS - LE_H_HF_B2 Diet Group:HF | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_H_HF_B2 SUBJECT_SAMPLE_FACTORS - LE_H_HF_B3 Diet Group:HF | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_H_HF_B3 SUBJECT_SAMPLE_FACTORS - LE_H_HF_B4 Diet Group:HF | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_H_HF_B4 SUBJECT_SAMPLE_FACTORS - LE_H_HF_C1 Diet Group:HF | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_H_HF_C1 SUBJECT_SAMPLE_FACTORS - LE_H_HF_C2 Diet Group:HF | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_H_HF_C2 SUBJECT_SAMPLE_FACTORS - LE_H_HF_C3 Diet Group:HF | Sample source:Heart | Tissue:Heart Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_H_HF_C3 SUBJECT_SAMPLE_FACTORS - LE_K_SC_A1 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_A1 SUBJECT_SAMPLE_FACTORS - LE_K_SC_A2 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_A2 SUBJECT_SAMPLE_FACTORS - LE_K_SC_A3 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_A3 SUBJECT_SAMPLE_FACTORS - LE_K_SC_A4 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_A4 SUBJECT_SAMPLE_FACTORS - LE_K_SC_B1 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_B1 SUBJECT_SAMPLE_FACTORS - LE_K_SC_B2 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_B2 SUBJECT_SAMPLE_FACTORS - LE_K_SC_B3 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_B3 SUBJECT_SAMPLE_FACTORS - LE_K_SC_B4 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_B4 SUBJECT_SAMPLE_FACTORS - LE_K_SC_C1 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_C1 SUBJECT_SAMPLE_FACTORS - LE_K_SC_C2 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_C2 SUBJECT_SAMPLE_FACTORS - LE_K_SC_C3 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_C3 SUBJECT_SAMPLE_FACTORS - LE_K_SC_C4 Diet Group:SC | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_SC_C4 SUBJECT_SAMPLE_FACTORS - LE_K_HF_A1 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_A1 SUBJECT_SAMPLE_FACTORS - LE_K_HF_A2 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_A2 SUBJECT_SAMPLE_FACTORS - LE_K_HF_A3 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_A3 SUBJECT_SAMPLE_FACTORS - LE_K_HF_A4 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_A4 SUBJECT_SAMPLE_FACTORS - LE_K_HF_B1 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_B1 SUBJECT_SAMPLE_FACTORS - LE_K_HF_B2 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_B2 SUBJECT_SAMPLE_FACTORS - LE_K_HF_B3 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_B3 SUBJECT_SAMPLE_FACTORS - LE_K_HF_B4 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_B4 SUBJECT_SAMPLE_FACTORS - LE_K_HF_C1 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_C1 SUBJECT_SAMPLE_FACTORS - LE_K_HF_C2 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_C2 SUBJECT_SAMPLE_FACTORS - LE_K_HF_C3 Diet Group:HF | Sample source:Kidney | Tissue:Kidney Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_K_HF_C3 SUBJECT_SAMPLE_FACTORS - LE_M_SC_A1 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_A1 SUBJECT_SAMPLE_FACTORS - LE_M_SC_A2 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_A2 SUBJECT_SAMPLE_FACTORS - LE_M_SC_A3 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_A3 SUBJECT_SAMPLE_FACTORS - LE_M_SC_A4 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_A4 SUBJECT_SAMPLE_FACTORS - LE_M_SC_B1 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_B1 SUBJECT_SAMPLE_FACTORS - LE_M_SC_B2 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_B2 SUBJECT_SAMPLE_FACTORS - LE_M_SC_B3 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_B3 SUBJECT_SAMPLE_FACTORS - LE_M_SC_B4 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_B4 SUBJECT_SAMPLE_FACTORS - LE_M_SC_C1 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_C1 SUBJECT_SAMPLE_FACTORS - LE_M_SC_C2 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_C2 SUBJECT_SAMPLE_FACTORS - LE_M_SC_C3 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_C3 SUBJECT_SAMPLE_FACTORS - LE_M_SC_C4 Diet Group:SC | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_SC_C4 SUBJECT_SAMPLE_FACTORS - LE_M_HF_A1 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_A1 SUBJECT_SAMPLE_FACTORS - LE_M_HF_A2 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_A2 SUBJECT_SAMPLE_FACTORS - LE_M_HF_A3 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_A3 SUBJECT_SAMPLE_FACTORS - LE_M_HF_A4 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_A4 SUBJECT_SAMPLE_FACTORS - LE_M_HF_B1 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_B1 SUBJECT_SAMPLE_FACTORS - LE_M_HF_B2 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_B2 SUBJECT_SAMPLE_FACTORS - LE_M_HF_B3 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_B3 SUBJECT_SAMPLE_FACTORS - LE_M_HF_B4 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_B4 SUBJECT_SAMPLE_FACTORS - LE_M_HF_C1 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_C1 SUBJECT_SAMPLE_FACTORS - LE_M_HF_C2 Diet Group:HF | Sample source:Skeletal Muscle | Tissue:Skeletal Muscle Extract type=Lipophilic; RAW_FILE_NAME(Raw file name)=JGS_LE_M_HF_C2 #COLLECTION CO:COLLECTION_SUMMARY All mice were deeply anesthetized with ketamine/xylazine and sacrificed by CO:COLLECTION_SUMMARY cardiac puncture. Whole livers, hearts, kidneys, and hind limb skeletal muscles CO:COLLECTION_SUMMARY were freeze-clamped and stored at -80ºC until further analysis. The MASLD CO:COLLECTION_SUMMARY profile was characterized by liver histology as well as measurements of hepatic CO:COLLECTION_SUMMARY triglyceride content, and whole body adiposity. CO:COLLECTION_PROTOCOL_FILENAME MASLD_Extrahepatic_Metabolomics_Experimental_Procedure.pdf CO:SAMPLE_TYPE Tissues: Heart, Kidney, and Skeletal Muscle CO:VOLUMEORAMOUNT_COLLECTED Whole heart, Kidney and pieces of Hind limb skeletal muscle CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY After acclimatisation, twelve of the mice were provided with high-fat chow (41% TR:TREATMENT_SUMMARY carbohydrate, 30% fat, 25% protein and 4% ash) (HF group), while the remaining TR:TREATMENT_SUMMARY mice were kept on standard chow (73% carbohydrate, 4% fat, 19% protein and 4% TR:TREATMENT_SUMMARY ash) (SC group) during the following 18 weeks. TR:TREATMENT_PROTOCOL_FILENAME MASLD_Extrahepatic_Metabolomics_Experimental_Procedure.pdf TR:TREATMENT_COMPOUND Diet - Standard Chow vs High-fat Chow TR:TREATMENT_ROUTE Feed TR:TREATMENT_DOSE High-fat chow (41% carbohydrate, 30% fat, 25% protein and 4% ash) vs Standard TR:TREATMENT_DOSE chow (73% carbohydrate, 4% fat, 19% protein and 4% ash) TR:TREATMENT_DOSEDURATION 18 weeks TR:TREATMENT_VEHICLE Feed TR:ANIMAL_ANESTHESIA Ketamine/xylazine TR:ANIMAL_ACCLIMATION_DURATION 2 Weeks TR:ANIMAL_ENDP_EUTHANASIA Cardiac Puncture TR:ANIMAL_ENDP_TISSUE_COLL_LIST Heart, Kidney, Skeletal Muscle #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Whole hearts, whole kidneys, and sections of skeletal muscle, all maintained in SP:SAMPLEPREP_SUMMARY dry ice, were submerged in 500 µL of ice-cold methanol, pulverized using a SP:SAMPLEPREP_SUMMARY tissue homogenizer (IKA ULTRA-TURRAX), then kept on ice. Polar and non-polar SP:SAMPLEPREP_SUMMARY metabolites were obtained using the methyl tert-butyl ether (MTBE) extraction SP:SAMPLEPREP_SUMMARY protocol.To the homogenized tissue, 4.6 mL of ice-cold methanol/g wet weight was SP:SAMPLEPREP_SUMMARY added followed by rigorous mixing by a vortex mixer. To this, 15.4 mL of MTBE/g SP:SAMPLEPREP_SUMMARY wet weight was added and then vigorously mixed at room temperature. The mixture SP:SAMPLEPREP_SUMMARY was then centrifuged for 10 min at 13000 g at room temperature followed by the SP:SAMPLEPREP_SUMMARY addition of 4 mL of water/g wet weight to the supernatant. After resting for 10 SP:SAMPLEPREP_SUMMARY min, the mixture was centrifuged at 1000 g for 10 minutes at room temperature SP:SAMPLEPREP_SUMMARY and the lipophilic and aqueous layers were collected into different vials. The SP:SAMPLEPREP_SUMMARY aqueous fractions were then lyophilized and stored at -80 ºC until NMR SP:SAMPLEPREP_SUMMARY analysis, while the lipophilic fractions were kept from the light and dried in SP:SAMPLEPREP_SUMMARY room air for 24 hours or until fully dried, followed by storage at -20 ºC until SP:SAMPLEPREP_SUMMARY NMR analysis. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Water/methanol/methyl tert-butyl ether (MTBE) extraction protocol as described SP:EXTRACTION_METHOD in (V. Matyash et al., 2007) and (G. D. Belew et al 2019) SP:EXTRACT_STORAGE Described in summary SP:SAMPLE_RESUSPENSION For aqueous extracts, each sample was resuspended in 600 µL of 2H2O phosphate SP:SAMPLE_RESUSPENSION buffer (0.1 M Na2HPO4/NaH2PO4) with 0.1 mM sodium SP:SAMPLE_RESUSPENSION 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) and the pH adjusted to 7.4, using SP:SAMPLE_RESUSPENSION deuterated hydrochloric acid and deuterated potassium hydroxide. For lipophilic SP:SAMPLE_RESUSPENSION extracts, each sample was resuspended in 600 µL of 99.98% deuterated chloroform SP:SAMPLE_RESUSPENSION containing 0.24 mM of pyrazine. From each sample, a 500 µL aliquot was SP:SAMPLE_RESUSPENSION transferred to a 5 mm diameter NMR sample tube for analysis. SP:SAMPLE_SPIKING 0.1 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) for aqueous samples, SP:SAMPLE_SPIKING and 0.24 mM of pyrazine for lipophilic samples, as a chemical shift references. #ANALYSIS AN:ANALYSIS_TYPE NMR AN:ANALYSIS_PROTOCOL_FILE MASLD_Extrahepatic_Metabolomics_Experimental_Procedure.pdf AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Bruker AVANCE III 500 spectrometer NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500 MHz NM:NMR_PROBE TXI, BBI (only heart aqueous samples) NM:NMR_SOLVENT Sodium phosphate buffer (0.1 M in D2O, 99.96% D, pH 7.4, containing 0.1 mM NM:NMR_SOLVENT sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) chemical shift referencing) NM:NMR_SOLVENT for aqueous samples. 99.98% deuterated chloroform containing 0.24 mM of pyrazine NM:NMR_SOLVENT for lipophilic samples. NM:NMR_TUBE_SIZE 5 mm NMR tubes NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d (aqueous samples), and zg (lipophilic samples) NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 203 NM:OFFSET_FREQUENCY 2352 Hz NM:CHEMICAL_SHIFT_REF_CPD 0.1 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP)/ pyrazine NM:TEMPERATURE 298 K NM:NUMBER_OF_SCANS 256 scans NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 2.33 s NM:RELAXATION_DELAY 4 s NM:SPECTRAL_WIDTH 7,002.8 Hz NM:NUM_DATA_POINTS_ACQUIRED 32 k points NM:LINE_BROADENING 0.3 Hz NM:ZERO_FILLING 64 k points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Manual NM:CHEMICAL_SHIFT_REF_STD 0 ppm for TSP, 8.6 ppm for Pyrazine NM:NMR_RESULTS_FILE MASLD_Extrahepatic_Metabolomics_results_data.txt UNITS:PPM #END