Summary of Study ST003698
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002295. The data can be accessed directly via it's Project DOI: 10.21228/M8ZZ54 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
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| Study ID | ST003698 |
| Study Title | Impact of High Fat diet-induced metabolic dysfunction-associated steatotic liver disease (MASLD) on Heart, Kidney and Skeletal Muscle Metabolomes in Wild-type Mice |
| Study Type | 1H NMR metabolomics to study the effects high-fat diet-induced MASLD on extrahepatic tissues metabolomes, namely the heart, kidney and skeletal muscle, in C57BL6J wild-type mice. |
| Study Summary | Excessive caloric intake is a primary driver of metabolic dysfunction-associated steatotic liver disease (MASLD), and this has been recapitulated in mice fed a high-fat diet. In 2023, the global prevalence of MASLD was estimated at 30%, with high incidences affecting wealthy urbanised countries. This implication of hypercaloric diets can also perturb metabolism and function of extrahepatic tissues such as heart, kidney and skeletal muscle. These effects that can take place in extrahepatic tissues are still poorly understood in terms of metabolic alterations and physiology, and represent an important point of improvement in the knowledge gap that connects early stage MASLD with other obesity related comorbidities, such as type 2 diabetes, insulin resistance, cardiovascular and renal complications, and overall, with the so known metabolic syndrome. In this study, we aimed to evaluate the potential of using metabolomics to unravel the effects and interactions taking place in a diet-induced MASLD model related to the development of the disorder. Black-6 mice were subjected to either a control diet or a high-fat diet for 18 weeks, from which at the end their heart, kidney and skeletal muscle metabolites were extracted. The metabolites, divided into aqueous and lipophilic fractions, were acquired by 1H-NMR, and then processed using a untargeted Metabolomics and Lipidomics analysis approach, to identify key changes occurring between control and high-fat diet in these models. These results added important information to better understand the link between early onset MASLD and the Metabolic Syndrome and its comorbidities, though several metabolic changes in the extrahepatic tissues, namely in ectopic fat deposition and alterations to Randle cycle and gut microbiota activity. |
| Institute | Center for Innovative Biomedicine and Biotechnology (CIBB UC) |
| Department | Institute of Interdisciplinary Research |
| Laboratory | Metabolic Modelling and Systems Biology |
| Last Name | Silva |
| First Name | João |
| Address | Rua Larga - Faculdade de Medicina, 1ºandar - POLO I Universidade de Coimbra |
| jgsilva@cnc.uc.pt | |
| Phone | (+351) 239 820 190 |
| Submit Date | 2025-01-24 |
| Num Groups | 2 |
| Total Subjects | 23 |
| Num Males | 23 |
| Study Comments | Full NMR sample preparation and analysis procedures are available in the accompanying document entitled 1. MASLD Extrahepatic Metabolomics experimental procedure. The normalized data that was used in uni- and multivariate analysis is available in the accompanying files: 4. MASLD Extrahepatic Metabolomics results data.txt The raw fid as well as 1r file can be found in 5. MASLD Extrahepatic Metabolomics 1H NMR Raw Data.zip |
| Raw Data Available | Yes |
| Raw Data File Type(s) | fid |
| Analysis Type Detail | NMR |
| Release Date | 2025-02-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002295 |
| Project DOI: | doi: 10.21228/M8ZZ54 |
| Project Title: | Impact of High Fat diet-induced MASLD on Heart, Kidney and Skeletal Muscle Metabolomes in Wild-type Mice |
| Project Type: | 1H NMR metabolomics to study the effects high-fat diet-induced MASLD on extrahepatic tissues metabolomes, namely the heart, kidney and skeletal muscle, in C57BL6J wild-type mice. |
| Project Summary: | Excessive caloric intake is a primary driver of metabolic dysfunction-associated steatotic liver disease (MASLD), and this has been recapitulated in mice fed a high-fat diet. In 2023, the global prevalence of MASLD was estimated at 30%, with high incidences affecting wealthy urbanised countries. This implication of hypercaloric diets can also perturb metabolism and function of extrahepatic tissues such as heart, kidney and skeletal muscle. These effects that can take place in extrahepatic tissues are still poorly understood in terms of metabolic alterations and physiology, and represent an important point of improvement in the knowledge gap that connects early stage MASLD with other obesity related comorbidities, such as type 2 diabetes, insulin resistance, cardiovascular and renal complications, and overall, with the so known metabolic syndrome. In this study, we aimed to evaluate the potential of using metabolomics to unravel the effects and interactions taking place in a diet-induced MASLD model related to the development of the disorder. Black-6 mice were subjected to either a control diet or a high-fat diet for 18 weeks, from which at the end their heart, kidney and skeletal muscle metabolites were extracted. The metabolites, divided into aqueous and lipophilic fractions, were acquired by 1H-NMR, and then processed using a untargeted Metabolomics and Lipidomics analysis approach, to identify key changes occurring between control and high-fat diet in these models. These results added important information to better understand the link between early onset MASLD and the Metabolic Syndrome and its comorbidities, though several metabolic changes in the extrahepatic tissues, namely in ectopic fat deposition and alterations to Randle cycle and gut microbiota activity. |
| Institute: | Center for Innovative Biomedicine and Biotechnology (CIBB UC) |
| Department: | Institute of Interdisciplinary Research |
| Last Name: | Silva |
| First Name: | João |
| Address: | Rua Larga - Faculdade de Medicina, 1ºandar - POLO I Universidade de Coimbra |
| Email: | jgsilva@cnc.uc.pt |
| Phone: | (+351) 239 820 190 |
| Project Comments: | Full NMR sample preparation and analysis procedures are available in the accompanying document entitled 1. MASLD Extrahepatic Metabolomics experimental procedure. The normalized data that was used in uni- and multivariate analysis is available in the accompanying files: 4. MASLD Extrahepatic Metabolomics results data.txt The raw fid as well as 1r file can be found in 5. MASLD Extrahepatic Metabolomics 1H NMR Raw Data.zip |
Subject:
| Subject ID: | SU003830 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL6J |
| Age Or Age Range: | 10 weeks |
| Weight Or Weight Range: | Average 25g |
| Gender: | Male |
| Animal Animal Supplier: | Charles River Labs (Barcelona, Spain. RRID: IMSR_JAX:000664) |
| Animal Housing: | Twenty-four adult male C57BL6J mice obtained from Charles River Labs (Barcelona, Spain. RRID: IMSR_JAX:000664) at 8 weeks of age were housed at the University of Coimbra UC Biotech Bioterium. The mice were kept in a well-ventilated environment with a 12-hour light/dark cycle. Upon arrival to the Bioterium, mice were randomly assigned into cages with four mice per cage and given a two-week adaptation period with free access to water and standard chow. |
| Animal Light Cycle: | 12-hour light/dark cycle |
| Animal Feed: | After acclimatisation, twelve of the mice were provided with high-fat chow (41% carbohydrate, 30% fat, 25% protein and 4% ash) (HF group), while the remaining mice were kept on standard chow (73% carbohydrate, 4% fat, 19% protein and 4% ash) (SC group) during the following 18 weeks. |
| Animal Water: | Normal water |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Diet Group | Sample source | Tissue |
|---|---|---|---|---|
| SA404575 | AQ_H_HF_A1 | HF | Heart | Heart |
| SA404576 | AQ_H_HF_C3 | HF | Heart | Heart |
| SA404577 | AQ_H_HF_C2 | HF | Heart | Heart |
| SA404578 | AQ_H_HF_C1 | HF | Heart | Heart |
| SA404579 | AQ_H_HF_B4 | HF | Heart | Heart |
| SA404580 | AQ_H_HF_B3 | HF | Heart | Heart |
| SA404581 | AQ_H_HF_B2 | HF | Heart | Heart |
| SA404582 | LE_H_HF_C3 | HF | Heart | Heart |
| SA404583 | AQ_H_HF_A3 | HF | Heart | Heart |
| SA404584 | AQ_H_HF_A2 | HF | Heart | Heart |
| SA404585 | AQ_H_HF_B1 | HF | Heart | Heart |
| SA404586 | LE_H_HF_A1 | HF | Heart | Heart |
| SA404587 | LE_H_HF_C1 | HF | Heart | Heart |
| SA404588 | LE_H_HF_B4 | HF | Heart | Heart |
| SA404589 | LE_H_HF_B3 | HF | Heart | Heart |
| SA404590 | LE_H_HF_B2 | HF | Heart | Heart |
| SA404591 | LE_H_HF_B1 | HF | Heart | Heart |
| SA404592 | LE_H_HF_A3 | HF | Heart | Heart |
| SA404593 | LE_H_HF_A2 | HF | Heart | Heart |
| SA404594 | LE_H_HF_C2 | HF | Heart | Heart |
| SA404595 | AQ_K_HF_B2 | HF | Kidney | Kidney |
| SA404596 | AQ_K_HF_A3 | HF | Kidney | Kidney |
| SA404597 | AQ_K_HF_A4 | HF | Kidney | Kidney |
| SA404598 | AQ_K_HF_B1 | HF | Kidney | Kidney |
| SA404599 | AQ_K_HF_C3 | HF | Kidney | Kidney |
| SA404600 | AQ_K_HF_B3 | HF | Kidney | Kidney |
| SA404601 | AQ_K_HF_B4 | HF | Kidney | Kidney |
| SA404602 | AQ_K_HF_C1 | HF | Kidney | Kidney |
| SA404603 | AQ_K_HF_C2 | HF | Kidney | Kidney |
| SA404604 | AQ_K_HF_A2 | HF | Kidney | Kidney |
| SA404605 | AQ_K_HF_A1 | HF | Kidney | Kidney |
| SA404606 | LE_K_HF_A1 | HF | Kidney | Kidney |
| SA404607 | LE_K_HF_B3 | HF | Kidney | Kidney |
| SA404608 | LE_K_HF_A2 | HF | Kidney | Kidney |
| SA404609 | LE_K_HF_C3 | HF | Kidney | Kidney |
| SA404610 | LE_K_HF_C1 | HF | Kidney | Kidney |
| SA404611 | LE_K_HF_B4 | HF | Kidney | Kidney |
| SA404612 | LE_K_HF_C2 | HF | Kidney | Kidney |
| SA404613 | LE_K_HF_B2 | HF | Kidney | Kidney |
| SA404614 | LE_K_HF_B1 | HF | Kidney | Kidney |
| SA404615 | LE_K_HF_A4 | HF | Kidney | Kidney |
| SA404616 | LE_K_HF_A3 | HF | Kidney | Kidney |
| SA404617 | AQ_M_HF_A1 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404618 | AQ_M_HF_C2 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404619 | AQ_M_HF_B4 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404620 | AQ_M_HF_B3 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404621 | AQ_M_HF_B2 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404622 | AQ_M_HF_B1 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404623 | AQ_M_HF_A4 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404624 | AQ_M_HF_C3 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404625 | AQ_M_HF_A3 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404626 | AQ_M_HF_A2 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404627 | LE_M_HF_B3 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404628 | LE_M_HF_C2 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404629 | LE_M_HF_C1 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404630 | LE_M_HF_B4 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404631 | LE_M_HF_B2 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404632 | LE_M_HF_B1 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404633 | LE_M_HF_A4 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404634 | LE_M_HF_A3 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404635 | LE_M_HF_A2 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404636 | LE_M_HF_A1 | HF | Skeletal Muscle | Skeletal Muscle |
| SA404637 | LE_H_SC_B3 | SC | Heart | Heart |
| SA404638 | LE_H_SC_C4 | SC | Heart | Heart |
| SA404639 | LE_H_SC_C3 | SC | Heart | Heart |
| SA404640 | LE_H_SC_C2 | SC | Heart | Heart |
| SA404641 | LE_H_SC_C1 | SC | Heart | Heart |
| SA404642 | LE_H_SC_B4 | SC | Heart | Heart |
| SA404643 | AQ_H_SC_A1 | SC | Heart | Heart |
| SA404644 | LE_H_SC_B2 | SC | Heart | Heart |
| SA404645 | LE_H_SC_B1 | SC | Heart | Heart |
| SA404646 | AQ_H_SC_A3 | SC | Heart | Heart |
| SA404647 | AQ_H_SC_A4 | SC | Heart | Heart |
| SA404648 | AQ_H_SC_B1 | SC | Heart | Heart |
| SA404649 | AQ_H_SC_B2 | SC | Heart | Heart |
| SA404650 | AQ_H_SC_B3 | SC | Heart | Heart |
| SA404651 | AQ_H_SC_B4 | SC | Heart | Heart |
| SA404652 | AQ_H_SC_C1 | SC | Heart | Heart |
| SA404653 | AQ_H_SC_C2 | SC | Heart | Heart |
| SA404654 | AQ_H_SC_C3 | SC | Heart | Heart |
| SA404655 | AQ_H_SC_C4 | SC | Heart | Heart |
| SA404656 | LE_H_SC_A3 | SC | Heart | Heart |
| SA404657 | LE_H_SC_A4 | SC | Heart | Heart |
| SA404658 | AQ_H_SC_A2 | SC | Heart | Heart |
| SA404659 | LE_H_SC_A2 | SC | Heart | Heart |
| SA404660 | LE_H_SC_A1 | SC | Heart | Heart |
| SA404661 | AQ_K_SC_B4 | SC | Kidney | Kidney |
| SA404662 | AQ_K_SC_C3 | SC | Kidney | Kidney |
| SA404663 | AQ_K_SC_A1 | SC | Kidney | Kidney |
| SA404664 | AQ_K_SC_A3 | SC | Kidney | Kidney |
| SA404665 | AQ_K_SC_A4 | SC | Kidney | Kidney |
| SA404666 | AQ_K_SC_B1 | SC | Kidney | Kidney |
| SA404667 | AQ_K_SC_B2 | SC | Kidney | Kidney |
| SA404668 | AQ_K_SC_B3 | SC | Kidney | Kidney |
| SA404669 | AQ_K_SC_C1 | SC | Kidney | Kidney |
| SA404670 | AQ_K_SC_C2 | SC | Kidney | Kidney |
| SA404671 | AQ_K_SC_A2 | SC | Kidney | Kidney |
| SA404672 | LE_K_SC_C4 | SC | Kidney | Kidney |
| SA404673 | LE_K_SC_B1 | SC | Kidney | Kidney |
| SA404674 | LE_K_SC_C3 | SC | Kidney | Kidney |
Collection:
| Collection ID: | CO003823 |
| Collection Summary: | All mice were deeply anesthetized with ketamine/xylazine and sacrificed by cardiac puncture. Whole livers, hearts, kidneys, and hind limb skeletal muscles were freeze-clamped and stored at -80ºC until further analysis. The MASLD profile was characterized by liver histology as well as measurements of hepatic triglyceride content, and whole body adiposity. |
| Collection Protocol Filename: | MASLD_Extrahepatic_Metabolomics_Experimental_Procedure.pdf |
| Sample Type: | Tissues: Heart, Kidney, and Skeletal Muscle |
| Volumeoramount Collected: | Whole heart, Kidney and pieces of Hind limb skeletal muscle |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003839 |
| Treatment Summary: | After acclimatisation, twelve of the mice were provided with high-fat chow (41% carbohydrate, 30% fat, 25% protein and 4% ash) (HF group), while the remaining mice were kept on standard chow (73% carbohydrate, 4% fat, 19% protein and 4% ash) (SC group) during the following 18 weeks. |
| Treatment Protocol Filename: | MASLD_Extrahepatic_Metabolomics_Experimental_Procedure.pdf |
| Treatment Compound: | Diet - Standard Chow vs High-fat Chow |
| Treatment Route: | Feed |
| Treatment Dose: | High-fat chow (41% carbohydrate, 30% fat, 25% protein and 4% ash) vs Standard chow (73% carbohydrate, 4% fat, 19% protein and 4% ash) |
| Treatment Doseduration: | 18 weeks |
| Treatment Vehicle: | Feed |
| Animal Anesthesia: | Ketamine/xylazine |
| Animal Acclimation Duration: | 2 Weeks |
| Animal Endp Euthanasia: | Cardiac Puncture |
| Animal Endp Tissue Coll List: | Heart, Kidney, Skeletal Muscle |
Sample Preparation:
| Sampleprep ID: | SP003837 |
| Sampleprep Summary: | Whole hearts, whole kidneys, and sections of skeletal muscle, all maintained in dry ice, were submerged in 500 µL of ice-cold methanol, pulverized using a tissue homogenizer (IKA ULTRA-TURRAX), then kept on ice. Polar and non-polar metabolites were obtained using the methyl tert-butyl ether (MTBE) extraction protocol.To the homogenized tissue, 4.6 mL of ice-cold methanol/g wet weight was added followed by rigorous mixing by a vortex mixer. To this, 15.4 mL of MTBE/g wet weight was added and then vigorously mixed at room temperature. The mixture was then centrifuged for 10 min at 13000 g at room temperature followed by the addition of 4 mL of water/g wet weight to the supernatant. After resting for 10 min, the mixture was centrifuged at 1000 g for 10 minutes at room temperature and the lipophilic and aqueous layers were collected into different vials. The aqueous fractions were then lyophilized and stored at -80 ºC until NMR analysis, while the lipophilic fractions were kept from the light and dried in room air for 24 hours or until fully dried, followed by storage at -20 ºC until NMR analysis. |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Water/methanol/methyl tert-butyl ether (MTBE) extraction protocol as described in (V. Matyash et al., 2007) and (G. D. Belew et al 2019) |
| Extract Storage: | Described in summary |
| Sample Resuspension: | For aqueous extracts, each sample was resuspended in 600 µL of 2H2O phosphate buffer (0.1 M Na2HPO4/NaH2PO4) with 0.1 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) and the pH adjusted to 7.4, using deuterated hydrochloric acid and deuterated potassium hydroxide. For lipophilic extracts, each sample was resuspended in 600 µL of 99.98% deuterated chloroform containing 0.24 mM of pyrazine. From each sample, a 500 µL aliquot was transferred to a 5 mm diameter NMR sample tube for analysis. |
| Sample Spiking: | 0.1 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) for aqueous samples, and 0.24 mM of pyrazine for lipophilic samples, as a chemical shift references. |
Analysis:
| Analysis ID: | AN006068 |
| Analysis Type: | NMR |
| Analysis Protocol File: | MASLD_Extrahepatic_Metabolomics_Experimental_Procedure.pdf |
| Data Format: | fid, 1r |
NMR:
| NMR ID: | NM000306 |
| Analysis ID: | AN006068 |
| Instrument Name: | Bruker AVANCE III 500 spectrometer |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Spectrometer Frequency: | 500 MHz |
| NMR Probe: | TXI, BBI (only heart aqueous samples) |
| NMR Solvent: | Sodium phosphate buffer (0.1 M in D2O, 99.96% D, pH 7.4, containing 0.1 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP) chemical shift referencing) for aqueous samples. 99.98% deuterated chloroform containing 0.24 mM of pyrazine for lipophilic samples. |
| NMR Tube Size: | 5 mm NMR tubes |
| Shimming Method: | Topshim |
| Pulse Sequence: | noesypr1d (aqueous samples), and zg (lipophilic samples) |
| Water Suppression: | presat |
| Pulse Width: | 90-degree |
| Receiver Gain: | 203 |
| Offset Frequency: | 2352 Hz |
| Chemical Shift Ref Cpd: | 0.1 mM sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP)/ pyrazine |
| Temperature: | 298 K |
| Number Of Scans: | 256 scans |
| Dummy Scans: | 8 |
| Acquisition Time: | 2.33 s |
| Relaxation Delay: | 4 s |
| Spectral Width: | 7,002.8 Hz |
| Num Data Points Acquired: | 32 k points |
| Line Broadening: | 0.3 Hz |
| Zero Filling: | 64 k points |
| Apodization: | Exponential |
| Baseline Correction Method: | Manual |
| Chemical Shift Ref Std: | 0 ppm for TSP, 8.6 ppm for Pyrazine |
| NMR Results File: | MASLD_Extrahepatic_Metabolomics_results_data.txt UNITS:PPM |
