#METABOLOMICS WORKBENCH Wu_Yandi_20230208_041417 DATATRACK_ID:3728 STUDY_ID:ST002474 ANALYSIS_ID:AN004041 PROJECT_ID:PR001597 VERSION 1 CREATED_ON February 10, 2023, 9:58 pm #PROJECT PR:PROJECT_TITLE Retinol Dehydrogenase 10 Reduction Mediated Retinol Metabolism Disorder Promotes PR:PROJECT_TITLE Diabetic Cardiomyopathy in Male Mice. PR:PROJECT_SUMMARY In this study, we identify disordered cardiac retinol metabolism in type 2 PR:PROJECT_SUMMARY diabetic male mice and patients characterized by retinol overload, all-trans PR:PROJECT_SUMMARY retinoic acid deficiency. By supplementing type 2 diabetic male mice with PR:PROJECT_SUMMARY retinol or all-trans retinoic acid, we demonstrate that both cardiac retinol PR:PROJECT_SUMMARY overload and all-trans retinoic acid deficiency promote diabetic cardiomyopathy. PR:PROJECT_SUMMARY Mechanistically, by constructing cardiomyocyte-specific conditional retinol PR:PROJECT_SUMMARY dehydrogenase 10-knockout male mice and overexpressing retinol dehydrogenase 10 PR:PROJECT_SUMMARY in male type 2 diabetic mice via adeno-associated virus, we verify that the PR:PROJECT_SUMMARY reduction in cardiac retinol dehydrogenase 10 is the initiating factor for PR:PROJECT_SUMMARY cardiac retinol metabolism disorder and results in diabetic cardiomyopathy. PR:PROJECT_SUMMARY Therefore, we suggest that the reduction of cardiac retinol dehydrogenase 10 and PR:PROJECT_SUMMARY its mediated disorder of cardiac retinol metabolism is a new mechanism PR:PROJECT_SUMMARY underlying diabetic cardiomyopathy. PR:INSTITUTE Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University PR:LAST_NAME Yandi PR:FIRST_NAME Wu PR:ADDRESS 74, Zhongshan second street PR:EMAIL wuyd3@mail2.sysu.edu.cn PR:PHONE 15622158754 PR:PUBLICATIONS Retinol Dehydrogenase 10 Reduction Mediated Retinol Metabolism Disorder Promotes PR:PUBLICATIONS Diabetic Cardiomyopathy in Male Mice. PR:CONTRIBUTORS Yandi Wu; Tongsheng Huang; Xinghui Li; Conghui Shen; Honglin Ren; Haiping Wang; PR:CONTRIBUTORS Teng Wu; Xinlu Fu; Shijie Deng; Ziqi Feng; Shijie Xiong; Hui Li; Saifei Gao; PR:CONTRIBUTORS Zhenyu Yang; Fei Gao; Lele Dong; Jianding Cheng & Weibin Cai #STUDY ST:STUDY_TITLE Retinol Dehydrogenase 10 Reduction Mediated Retinol Metabolism Disorder Promotes ST:STUDY_TITLE Diabetic Cardiomyopathy in Male Mice. ST:STUDY_SUMMARY In this study, we identify disordered cardiac retinol metabolism in type 2 ST:STUDY_SUMMARY diabetic male mice and patients characterized by retinol overload, all-trans ST:STUDY_SUMMARY retinoic acid deficiency. By supplementing type 2 diabetic male mice with ST:STUDY_SUMMARY retinol or all-trans retinoic acid, we demonstrate that both cardiac retinol ST:STUDY_SUMMARY overload and all-trans retinoic acid deficiency promote diabetic cardiomyopathy. ST:STUDY_SUMMARY Mechanistically, by constructing cardiomyocyte-specific conditional retinol ST:STUDY_SUMMARY dehydrogenase 10-knockout male mice and overexpressing retinol dehydrogenase 10 ST:STUDY_SUMMARY in male type 2 diabetic mice via adeno-associated virus, we verify that the ST:STUDY_SUMMARY reduction in cardiac retinol dehydrogenase 10 is the initiating factor for ST:STUDY_SUMMARY cardiac retinol metabolism disorder and results in diabetic cardiomyopathy. ST:STUDY_SUMMARY Therefore, we suggest that the reduction of cardiac retinol dehydrogenase 10 and ST:STUDY_SUMMARY its mediated disorder of cardiac retinol metabolism is a new mechanism ST:STUDY_SUMMARY underlying diabetic cardiomyopathy. ST:INSTITUTE Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University ST:LAST_NAME Yandi ST:FIRST_NAME Wu ST:ADDRESS 74, Zhongshan second street ST:EMAIL wuyd3@mail2.sysu.edu.cn ST:PHONE 15622158754 ST:PUBLICATIONS Retinol Dehydrogenase 10 Reduction Mediated Retinol Metabolism Disorder Promotes ST:PUBLICATIONS Diabetic Cardiomyopathy in Male Mice. #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN db/m; db/db; db/db+retinol; db/db+retinoic acid; db/db+AAV9-RDH10; RDH10-cKO; SU:GENOTYPE_STRAIN RDH10fl/fl SU:AGE_OR_AGE_RANGE 32-week; 36-week; 21-week 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 - 32-week db/m-1 Genotype:db/m+ | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-1 SUBJECT_SAMPLE_FACTORS - 32-week db/m-2 Genotype:db/m+ | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-2 SUBJECT_SAMPLE_FACTORS - 32-week db/m-3 Genotype:db/m+ | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-3 SUBJECT_SAMPLE_FACTORS - 32-week db/db-1 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-4 SUBJECT_SAMPLE_FACTORS - 32-week db/db-2 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-5 SUBJECT_SAMPLE_FACTORS - 32-week db/db-3 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-6 SUBJECT_SAMPLE_FACTORS - 32-week db/db-4 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-7 SUBJECT_SAMPLE_FACTORS - 32-week db/db-5 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-8 SUBJECT_SAMPLE_FACTORS - 32-week db/db-6 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-9 SUBJECT_SAMPLE_FACTORS - 32-week db/db+Rol-1 Genotype:db/db | Treatment:retinol RAW_FILE_NAME=20201203-Retinoic acid-Sample-10 SUBJECT_SAMPLE_FACTORS - 32-week db/db+Rol-2 Genotype:db/db | Treatment:retinol RAW_FILE_NAME=20201203-Retinoic acid-Sample-11 SUBJECT_SAMPLE_FACTORS - 32-week db/db+Rol-3 Genotype:db/db | Treatment:retinol RAW_FILE_NAME=20201203-Retinoic acid-Sample-12 SUBJECT_SAMPLE_FACTORS - RDH10-FL-1 Genotype:RDH10fl/fl | Treatment:tamoxifen RAW_FILE_NAME=20201203-Retinoic acid-Sample-13 SUBJECT_SAMPLE_FACTORS - RDH10-FL-2 Genotype:RDH10fl/fl | Treatment:tamoxifen RAW_FILE_NAME=20201203-Retinoic acid-Sample-14 SUBJECT_SAMPLE_FACTORS - RDH10-FL-3 Genotype:RDH10fl/fl | Treatment:tamoxifen RAW_FILE_NAME=20201203-Retinoic acid-Sample-15 SUBJECT_SAMPLE_FACTORS - RDH10-CKO-1 Genotype:"RDH10fl/fl, MYH6-iCre" | Treatment:tamoxifen RAW_FILE_NAME=20201203-Retinoic acid-Sample-16 SUBJECT_SAMPLE_FACTORS - RDH10-CKO-2 Genotype:"RDH10fl/fl, MYH6-iCre" | Treatment:tamoxifen RAW_FILE_NAME=20201203-Retinoic acid-Sample-17 SUBJECT_SAMPLE_FACTORS - RDH10-CKO-3 Genotype:"RDH10fl/fl, MYH6-iCre" | Treatment:tamoxifen RAW_FILE_NAME=20201203-Retinoic acid-Sample-18 SUBJECT_SAMPLE_FACTORS - 36-week db/m-1 Genotype:db/m+ | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-19 SUBJECT_SAMPLE_FACTORS - 36-week db/m-2 Genotype:db/m+ | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-20 SUBJECT_SAMPLE_FACTORS - 36-week db/m-3 Genotype:db/m+ | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-21 SUBJECT_SAMPLE_FACTORS - 36-week db/db-1 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-22 SUBJECT_SAMPLE_FACTORS - 36-week db/db-2 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-23 SUBJECT_SAMPLE_FACTORS - 36-week db/db-3 Genotype:db/db | Treatment:no RAW_FILE_NAME=20201203-Retinoic acid-Sample-24 SUBJECT_SAMPLE_FACTORS - 36-week db/db+atRA-1 Genotype:db/db | Treatment:all-trans retinoic acid RAW_FILE_NAME=20201203-Retinoic acid-Sample-25 SUBJECT_SAMPLE_FACTORS - 36-week db/db+atRA-2 Genotype:db/db | Treatment:all-trans retinoic acid RAW_FILE_NAME=20201203-Retinoic acid-Sample-26 SUBJECT_SAMPLE_FACTORS - 36-week db/db+atRA-3 Genotype:db/db | Treatment:all-trans retinoic acid RAW_FILE_NAME=20201203-Retinoic acid-Sample-27 SUBJECT_SAMPLE_FACTORS - 36-week db/db+aav9-RDH10-1 Genotype:db/db | Treatment:aav9-rdh10 RAW_FILE_NAME=20201203-Retinoic acid-Sample-28 SUBJECT_SAMPLE_FACTORS - 36-week db/db+aav9-RDH10-2 Genotype:db/db | Treatment:aav9-rdh11 RAW_FILE_NAME=20201203-Retinoic acid-Sample-29 SUBJECT_SAMPLE_FACTORS - 36-week db/db+aav9-RDH10-3 Genotype:db/db | Treatment:aav9-rdh12 RAW_FILE_NAME=20201203-Retinoic acid-Sample-30 #COLLECTION CO:COLLECTION_SUMMARY Mice were fasted for 12 hours before sampling and testing. All animal CO:COLLECTION_SUMMARY experiments were approved by the Animal Care and Ethics Committee of Zhongshan CO:COLLECTION_SUMMARY School of Medicine, Sun Yat-sen University, and followed the National Institutes CO:COLLECTION_SUMMARY of Health Guidelines on the Care and Use of Animals (the protocol number is CO:COLLECTION_SUMMARY SYSU-IACUC-2019-B027) CO:SAMPLE_TYPE Cardiac tissue #TREATMENT TR:TREATMENT_SUMMARY RDH10-cKO mice: RDH10-cKO mice, which contain both RDH10fl/fl and MYH6-iCre, TR:TREATMENT_SUMMARY were bred by RDH10fl/fl mice and MYH6-iCre mice and were injected tamoxifen TR:TREATMENT_SUMMARY intraperitoneally (50 mg/kg, T2859, Sigma -Aldrich, St. Louis, MO) for 5 TR:TREATMENT_SUMMARY consecutive days from 5 weeks of age. Age-matched male RDH10fl/fl mice that also TR:TREATMENT_SUMMARY received TMX injections served as normal controls for RDH10-cKO mice. Animal TR:TREATMENT_SUMMARY treatments: Mice in the Rol treatment group received Rol gavage (800 IU/each, TR:TREATMENT_SUMMARY 17772, Sigma-Aldrich, St. Louis, MO) every two days from 8 weeks of age. Mice in TR:TREATMENT_SUMMARY the atRA treatment group received atRA intraperitoneal injection (5 mg/kg body TR:TREATMENT_SUMMARY weight, R2625, Sigma-Aldrich, St. Louis, MO) daily from 8 weeks of age. Gene TR:TREATMENT_SUMMARY therapy: A recombinant AAV9 vector carrying the mouse RDH10 sequence TR:TREATMENT_SUMMARY (AAV9-RDH10, DZ-AAV-Rdh10-OE, Dongze, Hanbio Inc, Shanghai, China) was used to TR:TREATMENT_SUMMARY overexpress RDH10. 0.8*10^11 vg/per animal of AA9-RDH10 was transferred into TR:TREATMENT_SUMMARY T2DM mice, respectively, by tail vein injection at the age of 16 weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sample preparation Homogenized the heart tissue in 200μL of cold NaCl solution SP:SAMPLEPREP_SUMMARY (0.9%) for 5 seconds, add 100μL retinol-D4 (IR-23012, IsoSciences, Ambler, PA, SP:SAMPLEPREP_SUMMARY USA) as internal standard for Rol and atRA, and retinyl acetate (46958, SP:SAMPLEPREP_SUMMARY Sigma-Aldrich, St. Louis, MO) as internal standard for retinyl esters, then SP:SAMPLEPREP_SUMMARY homogenize for another 5 seconds. For Rol and at RA, 1mL formic acid n-hexane SP:SAMPLEPREP_SUMMARY solution (1%) was added to the homogenizing to facilitate phase separation by a SP:SAMPLEPREP_SUMMARY 5-minutes vortex and 5-min centrifugation (16,200 g). The supernatants were SP:SAMPLEPREP_SUMMARY collected in a new tube and blown dry with nitrogen followed by re-dilution with SP:SAMPLEPREP_SUMMARY 100μL 70% methanol. For retinyl esters, 24 μL 0.5 M NaOH solution was added to SP:SAMPLEPREP_SUMMARY tissue homogenate, followed by 1 mL n-hexane. The mixture was votexed 5 min and SP:SAMPLEPREP_SUMMARY centrifuged at 16,200 g for 5 min. The supernatants were collected in a new tube SP:SAMPLEPREP_SUMMARY and blown dry with nitrogen followed by re-dilution with 100μL 100% methanol. SP:SAMPLEPREP_SUMMARY The processes above should be protected from the light. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Sciex Triple Quad TM 4500MD MS CH:COLUMN_NAME Phenomenex Kinetex C18 (50 x 2.1mm,1.7um) CH:SOLVENT_A 40% acetonitrile/30% methanol/30% water; 0.1 % formic acid CH:SOLVENT_B 55% acetonitrile/30% methanol/15% water; 0.1 % formic acid CH:FLOW_GRADIENT initial conditions 75% B; from 0 to 3.5min linear increase to 95% B; between 3.5 CH:FLOW_GRADIENT and 4.5min 95% B was retained; at 4.6 min back to initial conditions with 75% B; CH:FLOW_GRADIENT finally, 75% B was held from 4.6 to 5.9min CH:FLOW_RATE 0.2ml/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5500 QTrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The MS conditions were as follows: electrospray ionization (ESI) under positive MS:MS_COMMENTS mode; nebulizer gas: nitrogen; curtain gas,30 psi; ion spray voltage, 4000V; MS:MS_COMMENTS temperature, 400ºC; gas 1 and gas 2, 35 and 40 psi, respectively; collision MS:MS_COMMENTS gas, 10 psi. The parameters of the mass spectrometer were optimized, and the MS:MS_COMMENTS multiple reaction monitoring (MRM) transitions of retinol, all-trans-retinoic MS:MS_COMMENTS acid, as well as D4-retinol were chosen as 269.2>93.1, 273.1>94.0, and MS:MS_COMMENTS 301.2>123.1, respectively. The MRM transitions for retinyl esters were chosen as MS:MS_COMMENTS 329.3>269.3 for retinyl acetate, 524.4>268.1 for retinyl palmitate (16:0), MS:MS_COMMENTS 552.5>268.2 for retinyl stearate (18:0), 522.4>268, retinyl palmitoleate (16:1), MS:MS_COMMENTS and retinyl oleate (18:1). The quantification was performed using the MS:MS_COMMENTS calibration curve. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ml MS_METABOLITE_DATA_START Samples 32-week db/m-1 32-week db/m-2 32-week db/m-3 32-week db/db-1 32-week db/db-2 32-week db/db-3 32-week db/db-4 32-week db/db-5 32-week db/db-6 32-week db/db+Rol-1 32-week db/db+Rol-2 32-week db/db+Rol-3 RDH10-FL-1 RDH10-FL-2 RDH10-FL-3 RDH10-CKO-1 RDH10-CKO-2 RDH10-CKO-3 36-week db/m-1 36-week db/m-2 36-week db/m-3 36-week db/db-1 36-week db/db-2 36-week db/db-3 36-week db/db+atRA-1 36-week db/db+atRA-2 36-week db/db+atRA-3 36-week db/db+aav9-RDH10-1 36-week db/db+aav9-RDH10-2 36-week db/db+aav9-RDH10-3 Factors Genotype:db/m+ | Treatment:no Genotype:db/m+ | Treatment:no Genotype:db/m+ | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:retinol Genotype:db/db | Treatment:retinol Genotype:db/db | Treatment:retinol Genotype:RDH10fl/fl | Treatment:tamoxifen Genotype:RDH10fl/fl | Treatment:tamoxifen Genotype:RDH10fl/fl | Treatment:tamoxifen Genotype:"RDH10fl/fl, MYH6-iCre" | Treatment:tamoxifen Genotype:"RDH10fl/fl, MYH6-iCre" | Treatment:tamoxifen Genotype:"RDH10fl/fl, MYH6-iCre" | Treatment:tamoxifen Genotype:db/m+ | Treatment:no Genotype:db/m+ | Treatment:no Genotype:db/m+ | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:no Genotype:db/db | Treatment:all-trans retinoic acid Genotype:db/db | Treatment:all-trans retinoic acid Genotype:db/db | Treatment:all-trans retinoic acid Genotype:db/db | Treatment:aav9-rdh10 Genotype:db/db | Treatment:aav9-rdh11 Genotype:db/db | Treatment:aav9-rdh12 all-trans retinoic acid 0.4225 0.2801 0.465 0.3109 0.2123 0.2443 0.2457 0.2077 0.2331 0.8069 0.1854 0.1568 0.3221 0.2427 0.738 0.1387 0.1652 0.2088 0.2639 0.2968 0.4983 0.1145 0.2287 0.1264 0.4869 0.3228 0.3495 0.2571 0.2941 0.2698 retinol 2.7287 3.9078 8.4067 11.4845 4.3457 11.8574 8.2282 7.8723 7.5237 1.7974 8.3747 13.6601 5.056 1.3794 8.2627 4.3456 6.1914 9.9269 5.4485 7.9932 8.1485 6.4338 12.4819 7.311 5.9866 4.9836 6.6919 7.2647 7.4051 9.6483 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID all-trans retinoic acid None 301.2>123.1 444795 C00777 retinol None 269.2>93.1 445354 C00473 METABOLITES_END #END