#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