#METABOLOMICS WORKBENCH Liukan_20240513_021542 DATATRACK_ID:4836 STUDY_ID:ST003300 ANALYSIS_ID:AN005407 PROJECT_ID:PR002050 VERSION 1 CREATED_ON May 13, 2024, 9:17 pm #PROJECT PR:PROJECT_TITLE Hypothalamic SLC7A14 accounts for aging-reduced lipolysis in white adipose PR:PROJECT_TITLE tissue PR:PROJECT_TYPE Mass spectrometry PR:PROJECT_SUMMARY The central nervous system has been implicated in the age-induced reduction in PR:PROJECT_SUMMARY adipose tissue lipolysis. SLC7A14 is a lysosomal membrane protein highly PR:PROJECT_SUMMARY expressed in the brain. Herein, we investigated the possible role of PR:PROJECT_SUMMARY hypothalamic SLC7A14 in the age-induced lipolysis reduction. In this study, we PR:PROJECT_SUMMARY demonstrated the expression of SLC7A14 was reduced in proopiomelanocortin (POMC) PR:PROJECT_SUMMARY neurons of aged mice. Overexpression of SLC7A14 in POMC neurons alleviated the PR:PROJECT_SUMMARY age-induced reduction in white adipose tissue (WAT) lipolysis, whereas SLC7A14 PR:PROJECT_SUMMARY deletion mimicked the age-induced lipolysis impairment. Moreover, POMC SLC7A14 PR:PROJECT_SUMMARY regulated WAT lipolysis independently of sympathetic nerves in WAT. Metabolomics PR:PROJECT_SUMMARY analysis revealed that POMC SLC7A14 increased the primary bile acid PR:PROJECT_SUMMARY taurochenodeoxycholic acid (TCDCA) content, which mediated the SLC7A14 knockout- PR:PROJECT_SUMMARY or age-induced WAT lipolysis impairment. Furthermore, SLC7A14-increased TCDCA PR:PROJECT_SUMMARY content is dependent on intestinal apical sodium-dependent bile acid transporter PR:PROJECT_SUMMARY (ASBT), which is regulated by intestinal sympathetic afferent nerves. Finally, PR:PROJECT_SUMMARY SLC7A14 regulated the intestinal sympathetic afferent nerves by inhibiting PR:PROJECT_SUMMARY mTORC1 signaling through inhibiting TSC1 phosphorylation. Collectively, our PR:PROJECT_SUMMARY study suggests the function for central SLC7A14 and an upstream mechanism for PR:PROJECT_SUMMARY the mTORC1 signaling pathway. Moreover, our data provides insights into the PR:PROJECT_SUMMARY brain–gut–adipose tissue crosstalk in age-induced lipolysis impairment. PR:INSTITUTE Shanghai Institutes for Biological Sciences (SIBS) Chinese Academy of Sciences PR:INSTITUTE (CAS) PR:LAST_NAME Liu PR:FIRST_NAME Kan PR:ADDRESS No. 320, Yueyang Road, Shanghai, Shanghai, Shanghai/Shanghai/xuhui, 200000, PR:ADDRESS China PR:EMAIL liukan2019@sibs.ac.cn PR:PHONE 021-17718134725 #STUDY ST:STUDY_TITLE Hypothalamic SLC7A14 accounts for aging-reduced lipolysis in white adipose ST:STUDY_TITLE tissue ST:STUDY_SUMMARY The central nervous system has been implicated in the age-induced reduction in ST:STUDY_SUMMARY adipose tissue lipolysis. SLC7A14 is a lysosomal membrane protein highly ST:STUDY_SUMMARY expressed in the brain. Herein, we investigated the possible role of ST:STUDY_SUMMARY hypothalamic SLC7A14 in the age-induced lipolysis reduction. In this study, we ST:STUDY_SUMMARY demonstrated the expression of SLC7A14 was reduced in proopiomelanocortin (POMC) ST:STUDY_SUMMARY neurons of aged mice. Overexpression of SLC7A14 in POMC neurons alleviated the ST:STUDY_SUMMARY age-induced reduction in white adipose tissue (WAT) lipolysis, whereas SLC7A14 ST:STUDY_SUMMARY deletion mimicked the age-induced lipolysis impairment. Moreover, POMC SLC7A14 ST:STUDY_SUMMARY regulated WAT lipolysis independently of sympathetic nerves in WAT. Metabolomics ST:STUDY_SUMMARY analysis revealed that POMC SLC7A14 increased the primary bile acid ST:STUDY_SUMMARY taurochenodeoxycholic acid (TCDCA) content, which mediated the SLC7A14 knockout- ST:STUDY_SUMMARY or age-induced WAT lipolysis impairment. Furthermore, SLC7A14-increased TCDCA ST:STUDY_SUMMARY content is dependent on intestinal apical sodium-dependent bile acid transporter ST:STUDY_SUMMARY (ASBT), which is regulated by intestinal sympathetic afferent nerves. Finally, ST:STUDY_SUMMARY SLC7A14 regulated the intestinal sympathetic afferent nerves by inhibiting ST:STUDY_SUMMARY mTORC1 signaling through inhibiting TSC1 phosphorylation. Collectively, our ST:STUDY_SUMMARY study suggests the function for central SLC7A14 and an upstream mechanism for ST:STUDY_SUMMARY the mTORC1 signaling pathway. Moreover, our data provides insights into the ST:STUDY_SUMMARY brain–gut–adipose tissue crosstalk in age-induced lipolysis impairment. ST:INSTITUTE Shanghai Institutes for Biological Sciences (SIBS) Chinese Academy of Sciences ST:INSTITUTE (CAS) ST:LAST_NAME Liu ST:FIRST_NAME Kan ST:ADDRESS No. 320, Yueyang Road, Shanghai ST:EMAIL liukan2019@sibs.ac.cn ST:PHONE 021-17718134725 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 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 - control3 Sample source:Blood serum | Genotype:WT | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN1_FZTM210013175-1A.mzML SUBJECT_SAMPLE_FACTORS - control1 Sample source:Blood serum | Genotype:WT | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN1_FZTM210013176-1A.mzML SUBJECT_SAMPLE_FACTORS - control2 Sample source:Blood serum | Genotype:WT | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN1_FZTM210013174-1A.mzML SUBJECT_SAMPLE_FACTORS - control4 Sample source:Blood serum | Genotype:WT | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN1_FZTM210013177-1A.mzML SUBJECT_SAMPLE_FACTORS - control5 Sample source:Blood serum | Genotype:WT | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN1_FZTM210013178-1A.mzML SUBJECT_SAMPLE_FACTORS - oe1 Sample source:Blood serum | Genotype:SLC7A14_OE | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN2_FZTM210013180-1A.mzML SUBJECT_SAMPLE_FACTORS - oe2 Sample source:Blood serum | Genotype:SLC7A15_OE | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN2_FZTM210013181-1A.mzML SUBJECT_SAMPLE_FACTORS - oe3 Sample source:Blood serum | Genotype:SLC7A16_OE | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN2_FZTM210013182-1A.mzML SUBJECT_SAMPLE_FACTORS - oe4 Sample source:Blood serum | Genotype:SLC7A17_OE | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_CN2_FZTM210013183-1A.mzML SUBJECT_SAMPLE_FACTORS - QC1 Sample source:Blood serum | Genotype:QC | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_663032_CN_QC1.mzML SUBJECT_SAMPLE_FACTORS - QC2 Sample source:Blood serum | Genotype:QC | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_663032_CN_QC2.mzML SUBJECT_SAMPLE_FACTORS - QC3 Sample source:Blood serum | Genotype:QC | Treatment:control RAW_FILE_NAME(Raw file name)=HFX5_663032_CN_QC3.mzML #COLLECTION CO:COLLECTION_SUMMARY Serum was collected from control or overexpression of SLC7A14 in POMC neuron CO:COLLECTION_SUMMARY mice. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY To overexpression of SLC7A14 in ARC POMC neurons, POMC Cre mice were bilaterally TR:TREATMENT_SUMMARY injected either with a Cre-dependent AAV vector containing SLC7A14 in the TR:TREATMENT_SUMMARY opposite orientation flanked by two inverted loxP sites TR:TREATMENT_SUMMARY (AAV9-Syn-DIO-SLC7A14-mCherry, 1.5 × 1012 Pfu/mL, HANBIO) at a volume of 200 nL TR:TREATMENT_SUMMARY into the ARC or an AAV vector containing only mCherry in the opposite TR:TREATMENT_SUMMARY orientation flanked by two inverted loxP sites (AAV9-Syn-DIO-mCherry, 1.5 × TR:TREATMENT_SUMMARY 1012 Pfu/mL, HANBIO) as a control.Serum was collected from control or TR:TREATMENT_SUMMARY overexpression of SLC7A14 in POMC neuron mice. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The samples (100 μL) were placed in the EP tubes and resuspended with SP:SAMPLEPREP_SUMMARY prechilled 80% methanol and 0.1% formic acid by well vortex. Then the SP:SAMPLEPREP_SUMMARY sampleswere incubated on ice for 5 min and centrifuged at 15,000 g, 4°C for 20 SP:SAMPLEPREP_SUMMARY min. Some of supernatant was diluted to final concentration containing 53% SP:SAMPLEPREP_SUMMARY methanol by LC-MS grade water.The samples were subsequently transferred to a SP:SAMPLEPREP_SUMMARY fresh Eppendorf tube and then were centrifuged at 15000 g, 4°C for 20 min. SP:SAMPLEPREP_SUMMARY Finally, the supernatant was injected into the LC-MS/MS system analysis #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) CH:SOLVENT_A The eluents for the negative polarity mode were eluent A (5 mMammonium acetate, CH:SOLVENT_A pH 9.0 CH:SOLVENT_B Methanol CH:FLOW_GRADIENT 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min；100-2% B, 14.1 min；2% B, CH:FLOW_GRADIENT 17 min. CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The raw data files generated by UHPLC-MS/MS were processed using the Compound MS:MS_COMMENTS Discoverer 3.1 (CD3.1, ThermoFisher) to perform peak alignment, peak picking, MS:MS_COMMENTS and quantitation for each metabolite. The main parameterswere set as follows: MS:MS_COMMENTS retention time tolerance, 0.2 minutes; actual mass tolerance, 5ppm; signal MS:MS_COMMENTS intensity tolerance, 30%; signal/noise ratio, 3; and minimum intensity, et al. MS:MS_COMMENTS After that, peak intensities were normalized to the total spectral intensity.The MS:MS_COMMENTS normalized data was used to predict the molecular formula based on additive MS:MS_COMMENTS ions, molecular ion peaks and fragment ions. And then peaks were matched with MS:MS_COMMENTS the mzCloud (https://www.mzcloud.org/)，mzVault and MassList database to obtain MS:MS_COMMENTS the accurate qualitative and relative quantitative results.Statistical analyses MS:MS_COMMENTS were performed using the statistical software R (R version R-3.4.3),Python MS:MS_COMMENTS (Python 2.7.6 version) and CentOS (CentOS release 6.6),When data were not MS:MS_COMMENTS normally distributed, normal transformations were attempted using of area MS:MS_COMMENTS normalization method. MS:MS_RESULTS_FILE ST003300_AN005407_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END