#METABOLOMICS WORKBENCH leaptrkl_20220826_093946 DATATRACK_ID:3434 STUDY_ID:ST002268 ANALYSIS_ID:AN003705 PROJECT_ID:PR001450 VERSION 1 CREATED_ON August 26, 2022, 11:32 am #PROJECT PR:PROJECT_TITLE Autophagy-related protein PIK3C3 maintains healthy brown and white adipose PR:PROJECT_TITLE tissues to prevent metabolic diseases PR:PROJECT_TYPE Lipidomics PR:PROJECT_SUMMARY Adequate mass and function of adipose tissues (ATs) play an essential role in PR:PROJECT_SUMMARY preventing metabolic perturbations. Pathological reduction of ATs in PR:PROJECT_SUMMARY lipodystrophy leads to an array of metabolic diseases. Understanding the PR:PROJECT_SUMMARY underlying mechanisms may benefit the development of effective therapies. PR:PROJECT_SUMMARY Several cellular processes, including autophagy, function collectively to PR:PROJECT_SUMMARY maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific PR:PROJECT_SUMMARY deletion of the autophagy-related lipid kinase PIK3C3 on AT homeostasis and PR:PROJECT_SUMMARY systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that PR:PROJECT_SUMMARY its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, PR:PROJECT_SUMMARY survival, and function with differential effects on brown and white ATs. These PR:PROJECT_SUMMARY abnormalities caused loss of white ATs, whitening followed by loss of brown ATs, PR:PROJECT_SUMMARY and impaired browning of white ATs. Consequently, mice exhibited compromised PR:PROJECT_SUMMARY thermogenic capacity and developed dyslipidemia, hepatic steatosis, insulin PR:PROJECT_SUMMARY resistance and type 2 diabetes. While these effects of PIK3C3 contrast previous PR:PROJECT_SUMMARY findings with the autophagy-related protein ATG7 in adipocytes, mice with a PR:PROJECT_SUMMARY combined deficiency in both factors revealed a dominant role of the PR:PROJECT_SUMMARY PIK3C3-deficient phenotype. We also found that dietary lipid excess exacerbates PR:PROJECT_SUMMARY AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance was PR:PROJECT_SUMMARY spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that was more PR:PROJECT_SUMMARY evident during dietary lipid excess. These findings reveal a crucial yet complex PR:PROJECT_SUMMARY role for PIK3C3 in ATs and suggest the potential of targeting this factor for PR:PROJECT_SUMMARY therapeutic intervention in metabolic diseases. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME Leaptrot PR:FIRST_NAME Katrina PR:ADDRESS 1234 Stevenson Center Ln PR:EMAIL katrina.l.leaptrot@vanderbilt.edu PR:PHONE 6158758422 #STUDY ST:STUDY_TITLE Autophagy-related protein PIK3C3 maintains healthy brown and white adipose ST:STUDY_TITLE tissues to prevent metabolic diseases ST:STUDY_TYPE Lipidomics ST:STUDY_SUMMARY Adequate mass and function of adipose tissues (ATs) play an essential role in ST:STUDY_SUMMARY preventing metabolic perturbations. Pathological reduction of ATs in ST:STUDY_SUMMARY lipodystrophy leads to an array of metabolic diseases. Understanding the ST:STUDY_SUMMARY underlying mechanisms may benefit the development of effective therapies. ST:STUDY_SUMMARY Several cellular processes, including autophagy, function collectively to ST:STUDY_SUMMARY maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific ST:STUDY_SUMMARY deletion of the autophagy-related lipid kinase PIK3C3 on AT homeostasis and ST:STUDY_SUMMARY systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that ST:STUDY_SUMMARY its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, ST:STUDY_SUMMARY survival, and function with differential effects on brown and white ATs. These ST:STUDY_SUMMARY abnormalities caused loss of white ATs, whitening followed by loss of brown ATs, ST:STUDY_SUMMARY and impaired browning of white ATs. Consequently, mice exhibited compromised ST:STUDY_SUMMARY thermogenic capacity and developed dyslipidemia, hepatic steatosis, insulin ST:STUDY_SUMMARY resistance and type 2 diabetes. While these effects of PIK3C3 contrast previous ST:STUDY_SUMMARY findings with the autophagy-related protein ATG7 in adipocytes, mice with a ST:STUDY_SUMMARY combined deficiency in both factors revealed a dominant role of the ST:STUDY_SUMMARY PIK3C3-deficient phenotype. We also found that dietary lipid excess exacerbates ST:STUDY_SUMMARY AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance was ST:STUDY_SUMMARY spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that was more ST:STUDY_SUMMARY evident during dietary lipid excess. These findings reveal a crucial yet complex ST:STUDY_SUMMARY role for PIK3C3 in ATs and suggest the potential of targeting this factor for ST:STUDY_SUMMARY therapeutic intervention in metabolic diseases. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME Leaptrot ST:FIRST_NAME Katrina ST:ADDRESS 1234 Stevenson Center Ln ST:EMAIL katrina.l.leaptrot@vanderbilt.edu ST:PHONE 6158758422 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 16 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN Pik3c3f/f mice SU:AGE_OR_AGE_RANGE W24 SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratory SU:ANIMAL_HOUSING pathogen-free conditions at a controlled room temperature SU:ANIMAL_LIGHT_CYCLE 12-hour light/dark cycle SU:ANIMAL_FEED regular chow diet (5LOD, LabDiet) SU:ANIMAL_WATER ad lib #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 S1 BAT-WT-S1 Genotype:WT | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_13_FS_S1_pos.d SUBJECT_SAMPLE_FACTORS S2 BAT-WT-S2 Genotype:WT | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_11_FS_S2_pos.d SUBJECT_SAMPLE_FACTORS S3 BAT-WT-S3 Genotype:WT | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_16_FS_S3_pos.d SUBJECT_SAMPLE_FACTORS S4 BAT-WT-S4 Genotype:WT | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_15_FS_S4_pos.d SUBJECT_SAMPLE_FACTORS S5 BAT-KO-S5 Genotype:KO | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_25_FS_S5_pos.d SUBJECT_SAMPLE_FACTORS S6 BAT-KO-S6 Genotype:KO | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_08_FS_S6_pos.d SUBJECT_SAMPLE_FACTORS S7 BAT-KO-S7 Genotype:KO | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_18_FS_S7_pos.d SUBJECT_SAMPLE_FACTORS S8 BAT-KO-S8 Genotype:KO | Lipid Type:Brown adipose tissue RAW_FILE_NAME=20220131_KLL_23_FS_S8_pos.d SUBJECT_SAMPLE_FACTORS S9 VAT-WT-S9 Genotype:WT | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_27_FS_S9_pos.d SUBJECT_SAMPLE_FACTORS S10 VAT-WT-S10 Genotype:WT | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_20_FS_S10_pos.d SUBJECT_SAMPLE_FACTORS S11 VAT-WT-S11 Genotype:WT | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_09_FS_S11_pos.d SUBJECT_SAMPLE_FACTORS S12 VAT-WT-S12 Genotype:WT | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_24_FS_S12_pos.d SUBJECT_SAMPLE_FACTORS S13 VAT-KO-S13 Genotype:KO | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_26_FS_S13_pos.d SUBJECT_SAMPLE_FACTORS S14 VAT-KO-S14 Genotype:KO | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_14_FS_S14_pos.d SUBJECT_SAMPLE_FACTORS S15 VAT-KO-S15 Genotype:KO | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_10_FS_S15_pos.d SUBJECT_SAMPLE_FACTORS S16 VAT-KO-S16 Genotype:KO | Lipid Type:White adipose tissue RAW_FILE_NAME=20220131_KLL_21_FS_S16_pos.d #COLLECTION CO:COLLECTION_SUMMARY Mice were housed under specific pathogen-free conditions, fed with a regular CO:COLLECTION_SUMMARY chow diet (5LOD, LabDiet), provided food and water ad lib unless otherwise CO:COLLECTION_SUMMARY specified, and maintained on a 12-hour light/dark cycle at a controlled room CO:COLLECTION_SUMMARY temperature of 22°C, except for the cold treatment studies. Mice were CO:COLLECTION_SUMMARY sacrificed at non-fasting state for further analysis unless otherwise specified. CO:COLLECTION_PROTOCOL_FILENAME Materials and Methods.pdf CO:SAMPLE_TYPE Adipose tissue CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY We generated Adipoq-Cre;Pik3c3f/f (cKO) and Pik3c3f/f (WT) mice and analyzed TR:TREATMENT_SUMMARY interscapular BAT (iBAT), inguinal subcutaneous WAT (iWAT), and perigonadal TR:TREATMENT_SUMMARY visceral WAT (pWAT). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The iBAT and pWAT was harvestedat sacrifice from mice at W24. Samples were SP:SAMPLEPREP_SUMMARY immediately frozen in liquid nitrogen followed by -80°C storage before SP:SAMPLEPREP_SUMMARY analysis. Both WT (n=4) and cKO (n=4) mice were analyzed. Adipose tissue SP:SAMPLEPREP_SUMMARY samples, ranging from 10-30 mg, were thawed on ice and mixed with 1 mL of cold SP:SAMPLEPREP_SUMMARY 1:1:2 (v:v:v) methanol MeOH:ACN:H2O with 50 mM ammonium bicarbonate lysis SP:SAMPLEPREP_SUMMARY buffer. Samples were homogenized using a tissue homogenizer operated at 20,000 SP:SAMPLEPREP_SUMMARY rpm for 10 seconds to break the tissue, then vortex mixed for 10 seconds. An SP:SAMPLEPREP_SUMMARY appropriate volume of lysate was transferred from each sample such that SP:SAMPLEPREP_SUMMARY individual samples were normalized based on tissue amount. Following volume SP:SAMPLEPREP_SUMMARY adjustment to 200 L, 800 L of cold MeOH was added to the samples. SP:SAMPLEPREP_SUMMARY Individual samples were vortexed for 30 seconds and incubated overnight at SP:SAMPLEPREP_SUMMARY -80°C for protein precipitation. Following incubation, samples were centrifuged SP:SAMPLEPREP_SUMMARY for 15 min at 15,000 rpm at 4°C and the supernatant was transferred to a new SP:SAMPLEPREP_SUMMARY labeled tube and dried down using a cold vacuum centrifuge. Samples were SP:SAMPLEPREP_SUMMARY reconstituted in 100 L H2O, 100 L MeOH, and 10 μL of SPLASH LIPIDOMIX SP:SAMPLEPREP_SUMMARY with vortex mixing after each addition. Samples were incubated at room SP:SAMPLEPREP_SUMMARY temperature for 10 min followed SP:SAMPLEPREP_PROTOCOL_FILENAME Global untargeted lipidomics.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6560 CH:COLUMN_NAME Thermo Hypersil Gold column (1.9 M, 2.1 mm x 100 mm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6560 Ion Mobility MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data analysis was performed using Progenesis QI software (version 3.0, Nonlinear MS:MS_COMMENTS Dynamics, Newcastle, UK). Retention time alignment, peak picking, and peak MS:MS_COMMENTS deconvolution used default parameters. Spectra were normalized to all compounds, MS:MS_COMMENTS and data were filtered for coefficients of variance < 25% in QC technical MS:MS_COMMENTS replicate injections. A prioritized compound list was generated via a one-factor MS:MS_COMMENTS ANOVA, with four experimental groups for comparison including wild type and MS:MS_COMMENTS Vps34 knockout for both brown and visceral adipose tissue. Lipids were MS:MS_COMMENTS considered to be differentially altered if the p-value < 0.05 and the fold MS:MS_COMMENTS change was greater than Ι2Ι. Significantly changed compounds were selected for MS:MS_COMMENTS annotation. Lipidomic annotations were performed using a previously described MS:MS_COMMENTS classification system with compounds being assigned a confidence level of 1 to 5 MS:MS_COMMENTS (1 being the highest confidence) with improved confidence requiring more MS:MS_COMMENTS supporting evidence such as accurate mass, MS/MS fragmentation, and retention MS:MS_COMMENTS time matching to standards. Lipid annotated were performed with reference to MS:MS_COMMENTS in-house and online databases (MS-DIAL, LipidMatch, and Lipid Annotator). MS:MS_COMMENTS Differentially abundant lipids (DALs) were uploaded into the LIPEA algorithm for MS:MS_COMMENTS pathway enrichment analysis. Corrected p-values were calculated using Benjamini MS:MS_COMMENTS correction and a p-value <0.05 was used to determine significantly affected MS:MS_COMMENTS pathways. MS:MS_RESULTS_FILE ST002268_AN003705_Results.txt UNITS:retention time underscore m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END