{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002219","ANALYSIS_ID":"AN003628","VERSION":"1","CREATED_ON":"July 14, 2022, 3:06 pm"},

"PROJECT":{"PROJECT_TITLE":"Spatially resolved characterization of tissue metabolic compartments in fasted and high-fat diet livers","PROJECT_SUMMARY":"Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI that can be implemented to achieve broad coverage of central carbon, nucleotide, and lipid metabolism pathways. Herein, we used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic tissue compartmentalization in livers, which becomes disrupted under high fat diet. Fasting caused changes in the abundance of several metabolites, including increased levels of fatty acids and TCA intermediates while fatty livers had higher levels of purine and pentose phosphate-related metabolites, which generate reducing equivalents to counteract oxidative stress. This spatially conserved approach allowed the visualization of liver metabolic compartmentalization at 30 µm pixel resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo.","INSTITUTE":"Brigham and Women's Hospital","LAST_NAME":"Stopka","FIRST_NAME":"Sylwia","ADDRESS":"60 Fenway Rd","EMAIL":"sstopka@bwh.harvard.edu","PHONE":"617-525-9746"},

"STUDY":{"STUDY_TITLE":"Spatially resolved characterization of tissue metabolic compartments in fasted and high-fat diet livers","STUDY_SUMMARY":"Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI that can be implemented to achieve broad coverage of central carbon, nucleotide, and lipid metabolism pathways. Herein, we used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic tissue compartmentalization in livers, which becomes disrupted under high fat diet. Fasting caused changes in the abundance of several metabolites, including increased levels of fatty acids and TCA intermediates while fatty livers had higher levels of purine and pentose phosphate-related metabolites, which generate reducing equivalents to counteract oxidative stress. This spatially conserved approach allowed the visualization of liver metabolic compartmentalization at 30 µm pixel resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo.","INSTITUTE":"Brigham and Women's Hospital","DEPARTMENT":"Brigham and Women's Hospital","LAST_NAME":"Stopka","FIRST_NAME":"Sylwia","ADDRESS":"60 Fenway Rd","EMAIL":"sstopka@bwh.harvard.edu","PHONE":"617-525-9746"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"fed_fasted",
"Factors":{"Treatment":"fed_fasted"},
"Additional sample data":{"RAW_FILE_NAME":"fed_fasted.zip"}
},
{
"Subject ID":"-",
"Sample ID":"heat-freezing_treatments_liver",
"Factors":{"Treatment":"heat-freezing_treatments_liver"},
"Additional sample data":{"RAW_FILE_NAME":"heat-freezing_treatments_liver.zip"}
},
{
"Subject ID":"-",
"Sample ID":"High_fat_diet",
"Factors":{"Treatment":"High_fat_diet"},
"Additional sample data":{"RAW_FILE_NAME":"High_fat_diet.zip"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"C57BL/6J (000664) and BALB/cJ (000651) mice were obtained from The Jackson Laboratory. Mice were housed at 20-22°C on a 12 h light/dark cycle with ad libitum access to food (PicoLab Rodent Diet 5053) and water. All animal studies were performed in accordance with Haigis lab protocols approved by the Standing Committee on Animals, the Institutional Animal Care and Use Committee at Harvard Medical School. For heat inactivation studies, 3 mice were used (C57BL/6J, female, 7 weeks old) and kidneys, brain halves, and liver lobes from the same individual animal were subjected to the different heat inactivation treatments (overview in Supplementary Fig. 1A, E). For desiccation experiments, 2 mice were used (C57BL/6J, male, 7 weeks old). For fasting experiments, two independent cohorts of 5 mice were used per treatment group (BALB/cJ, female, 10-11 weeks old) and mice were subjected to a 16 hour overnight fast. For HFD experiments, two independent cohorts of 4 mice were used per treatment group (C57BL/6J, female). Mice were assigned at 5 weeks old to the control diet (PicoLab Rodent Diet 5053) or HFD (Research Diets, Inc. #12492) and maintained on this diet for 4.5 months. The control diet is 4.07 Gross Energy Kcal/g. The HFD is 5.21 Kcal/g. for 8-10 weeks. Comparative MALDI MSI and LC-MS analyses of tissues were always performed on the same tissue specimens.","SAMPLE_TYPE":"Liver_Brain_Kidney"},

"TREATMENT":{"TREATMENT_SUMMARY":"N/A"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Tissue preparation for MALDI MSI Frozen tissues were placed at -20 °C before sectioning in a Microm HM550 cryostat (Thermo Scientific™). Tissues were sectioned at 10 µm thickness and thaw mounted onto indium-tin-oxide (ITO)-coated slides (Bruker Daltonics) for MALDI MSI analysis with serial sections mounted onto glass slides for histological analyses. The microtome chamber and specimen holder were maintained between -15 °C and -20 °C. Slides were stored at -80 °C until further processing. For desiccation experiments, slides were subjected to desiccation in a tabletop vacuum desiccator before freezing. Matrix deposition A 1,5-Diaminonaphthalene(DAN)-HCl matrix solution was used for all experiments. To generate the hydrochloride derivative of 1,5-DAN, 39.5 mg of 1,5-DAN was dissolved in 500 µL of 1 mol/L hydrochloride solution with 4 mL HPLC-grade water. The solution was sonicated for 20 minutes to dissolve 1,5-DAN, after which 4.5 mL ethanol was added to yield the matrix solution. Matrices were deposited on slides and tissues using a TM-sprayer (HTX imaging, Carrboro, NC). DAN-HCl matrix spray conditions used where: a flow rate of 0.09 mL/min, spray nozzle temperature of 75 °C, and spray nozzle velocity of 1200 mm/min. A four-pass cycle was used with 2 mm track spacing and the nitrogen gas pressure was maintained at 10 psi."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"None (Direct infusion)","INSTRUMENT_NAME":"timsTOF fleX","COLUMN_NAME":"none"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Bruker timsTOF fleX","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"MALDI","ION_MODE":"NEGATIVE","MS_COMMENTS":"SCilS 2022b pro","MS_RESULTS_FILE":"ST002219_AN003628_Results.txt UNITS:Da Has m/z:Yes Has RT:No RT units:No RT data"}

}