#METABOLOMICS WORKBENCH philmus_lab_20200728_134003 DATATRACK_ID:2105 STUDY_ID:ST001436 ANALYSIS_ID:AN002399 PROJECT_ID:PR000987 VERSION 1 CREATED_ON July 30, 2020, 5:12 pm #PROJECT PR:PROJECT_TITLE Gut microbiota mediates the interplay between immunity and glucose metabolism PR:PROJECT_TYPE preclinical studies PR:PROJECT_SUMMARY Diabetes, obesity and metabolic disease have reached epidemic proportions. Much PR:PROJECT_SUMMARY research on these diseases has focused on tissues such as adipose, liver, muscle PR:PROJECT_SUMMARY and pancreas. The role of the gut in glucose metabolism is relatively unstudied; PR:PROJECT_SUMMARY however, it can also have a significant effect on systemic glucose control. PR:PROJECT_SUMMARY Residing in the gut is a complex community of microbes, termed microbiota, which PR:PROJECT_SUMMARY are important contributors to immunity and metabolism, frequently mediating PR:PROJECT_SUMMARY cross-talks between these two functions. Changes in the gut microbiota of type 2 PR:PROJECT_SUMMARY diabetes (T2D) patients are directly linked to metabolic dysregulation in the PR:PROJECT_SUMMARY disease. Therefore, the goal of the project is to identify and test microbes and PR:PROJECT_SUMMARY microbial factors involved in regulation of glucose metabolism. For this, PR:PROJECT_SUMMARY microbiota perturbation by western diet followed by global analyses of gut PR:PROJECT_SUMMARY microbiome and host transcriptome combined with causal inference analysis is PR:PROJECT_SUMMARY employed. Newly inferred probiotics are tested in mice fed with western diet PR:PROJECT_SUMMARY followed by metabolomics analysis. This research provides a mechanistic PR:PROJECT_SUMMARY explanation of how probiotics helps disclose mechanisms of T2D as well as will PR:PROJECT_SUMMARY identify protective microbial factors for development of therapy of diabetes. PR:INSTITUTE Oregon State University PR:DEPARTMENT Pharmaceutical Sciences PR:LABORATORY Morgun and Shulzhenko PR:LAST_NAME Andriy PR:FIRST_NAME Morgun PR:ADDRESS 203 Pharmacy Bldg, Corvallis, OR 97331 PR:EMAIL andriy.morgun@oregonstate.edu PR:PHONE 1 541 737 8047 PR:FUNDING_SOURCE NIH R01 DK103761 #STUDY ST:STUDY_TITLE Transkingdom interactions between Lactobacilli and hepatic mitochondria ST:STUDY_TITLE attenuate western diet induced diabetes ST:STUDY_TYPE Supplementation of mice with probiotic bacteria ST:STUDY_SUMMARY For WD + Microbes 1 and WD + Microbes 3 experiments, C57BL/6 mice were fed ST:STUDY_SUMMARY western diet or western diet supplemented with Lactobacillus gasseri or ST:STUDY_SUMMARY Lactobacillus johnsonii for 8 weeks and serum was collected from each mice. For ST:STUDY_SUMMARY Pooled_1 (control group, western diet only), and Pooled_2 (western diet + ST:STUDY_SUMMARY Lactobacillus gasseri) groups, serum from 5 mice each was pooled. For Pooled_3 ST:STUDY_SUMMARY (western diet + Lactobacillus johnsonii) group, serum from 4 mice was pooled. ST:STUDY_SUMMARY For Pooled_7 (control group, western diet only), Pooled_8 (western diet + ST:STUDY_SUMMARY Lactobacillus gasseri) and Pooled_9 (western diet + Lactobacillus johnsonii) ST:STUDY_SUMMARY groups, serum from 6 mice each was pooled. For WD + Microbes 6 experiment, ST:STUDY_SUMMARY C57BL/6 mice were fed western diet for 8 weeks. Then, one group (n = 5) of mice ST:STUDY_SUMMARY was supplemented with Lactobacillus gasseri for 12 weeks along with continuation ST:STUDY_SUMMARY of western diet and serum was collected. For Pooled_10 (control group, western ST:STUDY_SUMMARY diet only) and Pooled_11 (western diet + Lactobacillus gasseri) groups, serum ST:STUDY_SUMMARY from 5 mice each was pooled. For GF + WD + LG experiment, C57BL/6 germ free mice ST:STUDY_SUMMARY were fed western diet or western diet supplemented with Lactobacillus gasseri ST:STUDY_SUMMARY for 2 weeks. For Pooled_12 (control, western diet only) and Pooled_13 (western ST:STUDY_SUMMARY diet + Lactobacillus gasseri) groups, serum from 2 mice each was pooled. ST:INSTITUTE Oregon State University ST:DEPARTMENT Pharmaceutical Sciences ST:LABORATORY Morgun and Shulzhenko ST:LAST_NAME Morgun ST:FIRST_NAME Andriy ST:ADDRESS 203 Pharmacy Bldg ST:EMAIL andriy.morgun@oregonstate.edu ST:PHONE 1 541 737 8047 ST:NUM_GROUPS 7 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57Bl/6 SU:AGE_OR_AGE_RANGE 10-20 weeks SU:GENDER Male #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 - Sample_01 Treatment:Western diet Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_01.d; RAW_FILE_NAME=Sample-neg_01.d SUBJECT_SAMPLE_FACTORS - Sample_02 Treatment:Western diet + Lactobacillus gasseri Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_02.d; RAW_FILE_NAME=Sample-neg_02.d SUBJECT_SAMPLE_FACTORS - Sample_03 Treatment:Western diet + Lactobacillus johnsonii Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_03.d; RAW_FILE_NAME=Sample-neg_03.d SUBJECT_SAMPLE_FACTORS - Sample_07 Treatment:Western diet Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_07.d; RAW_FILE_NAME=Sample-neg_07.d SUBJECT_SAMPLE_FACTORS - Sample_08 Treatment:Western diet + Lactobacillus gasseri Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_08.d; RAW_FILE_NAME=Sample-neg_08.d SUBJECT_SAMPLE_FACTORS - Sample_09 Treatment:Western diet + Lactobacillus johnsonii Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_09.d; RAW_FILE_NAME=Sample-neg_09.d SUBJECT_SAMPLE_FACTORS - Sample_10 Treatment:Western diet Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_10.d; RAW_FILE_NAME=Sample-neg_10.d SUBJECT_SAMPLE_FACTORS - Sample_11 Treatment:Western diet + Lactobacillus gasseri Genotype=Wild-type, Specific pathogen free; RAW_FILE_NAME=Sample-pos_11.d; RAW_FILE_NAME=Sample-neg_11.d SUBJECT_SAMPLE_FACTORS - Sample_12 Treatment:Western diet Genotype=Wild-type, Germ free; RAW_FILE_NAME=Sample-pos_12.d; RAW_FILE_NAME=Sample-neg_12.d SUBJECT_SAMPLE_FACTORS - Sample_13 Treatment:Western diet + Lactobacillus gasseri Genotype=Wild-type, Germ free; RAW_FILE_NAME=Sample-pos_13.d; RAW_FILE_NAME=Sample-neg_13.d #COLLECTION CO:COLLECTION_SUMMARY After the end of the experiment, whole blood was collected in BD Microtainer CO:COLLECTION_SUMMARY Serum Separator Tube and allowed to clot by leaving undistubred at room CO:COLLECTION_SUMMARY temperature for 30 minutes. Then the tubes were centrifuged at 2000 x g for 10 CO:COLLECTION_SUMMARY minutes in 4°C and the supernatant was immediately transferred into a CO:COLLECTION_SUMMARY polypropylene tube and stored at -80°C until further analysis. CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Western diet fed mice were supplemented with Lactobacillus gasseri or TR:TREATMENT_SUMMARY Lactobacillus johnsonii. For control groups, equal volume of vehicle (PBS) was TR:TREATMENT_SUMMARY gavaged. Supplementation was done for 2 to 8 weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted with 4 volumes of cold methanol/acetonitrile (1:1, SP:SAMPLEPREP_SUMMARY v/v). To precipitate proteins, the samples were incubated for 1h at -20°C. SP:SAMPLEPREP_SUMMARY After the samples were centrifuged at 4°C for 15 min at 13,000 rpm, the SP:SAMPLEPREP_SUMMARY supernatant was collected and evaporated to dryness in a vacuum concentrator. SP:SAMPLEPREP_SUMMARY The dry extracts were then reconstituted in 90 μL of acetonitrile/H2O (1:1, SP:SAMPLEPREP_SUMMARY v/v) containing 10 ng/mL CUDA (12-[[(cyclohexylamino)carbonyl] amino]-dodecanoic SP:SAMPLEPREP_SUMMARY acid). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Poroshell EC-C18 (100 x 3.0 mm, 2.7 um) CH:FLOW_RATE 0.4 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QToF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS None MS:MS_RESULTS_FILE ST001436_AN002399_Results.txt UNITS:AUC Has m/z:Yes Has RT:Yes RT units:Minutes #END