#METABOLOMICS WORKBENCH mlhomme_20221213_043543 DATATRACK_ID:3642 STUDY_ID:ST002400 ANALYSIS_ID:AN003910
VERSION                          	1
CREATED_ON                       	12-16-2022
#PROJECT
PR:PROJECT_TITLE                 	Alcohol dehydrogenase 1B is crucial for adipocyte homeostasis.
PR:PROJECT_SUMMARY               	Background. Alcohol dehydrogenase (ADH1B), encoded by the ADH1B gene, is a
PR:PROJECT_SUMMARY               	cytosolic enzyme mainly known for its role in ethanol catabolism in the liver. A
PR:PROJECT_SUMMARY               	few studies have paved the way to show an equally important role of ADH1B in
PR:PROJECT_SUMMARY               	adipocytes. This study aimed to better identify the cellular mechanisms and
PR:PROJECT_SUMMARY               	signaling pathways involving ADH1B in adipose tissue and to determine if ADH1B
PR:PROJECT_SUMMARY               	variants might contribute to adipose tissue dysfunction. Results. We showed that
PR:PROJECT_SUMMARY               	CRISPR-Cas9-mediated ADH1B knockout (KO) in human adipose stem cells (ASC)
PR:PROJECT_SUMMARY               	abolished adipocyte differentiation and decreased insulin response. This was
PR:PROJECT_SUMMARY               	accompanied by oxidative stress, altered mitochondrial functions, and cellular
PR:PROJECT_SUMMARY               	senescence. Lipidomic analysis revealed that ADH1B deficiency results in a major
PR:PROJECT_SUMMARY               	remodeling of lipid composition in ASC. An ADH1B homozygous loss-of-function
PR:PROJECT_SUMMARY               	variant was also identified in a patient presenting with a lipodystrophic and
PR:PROJECT_SUMMARY               	insulin resistant syndrome associated with major liver dysfunction, leading to
PR:PROJECT_SUMMARY               	early death. Discussion. This translational study underlines the crucial role of
PR:PROJECT_SUMMARY               	ADH1B in adipose tissue. It unveils cellular mechanisms accounting for its key
PR:PROJECT_SUMMARY               	role in adipogenesis, and adipocyte homeostasis. This study also identifies
PR:PROJECT_SUMMARY               	ADH1B as a candidate gene in monogenic forms of lipodystrophic and insulin
PR:PROJECT_SUMMARY               	resistant syndromes.
PR:INSTITUTE                     	INSERM
PR:LAST_NAME                     	Gautheron
PR:FIRST_NAME                    	Jérémie
PR:ADDRESS                       	27 rue Chaligny, 75012 Paris France
PR:EMAIL                         	jeremie.gautheron@gmail.com
PR:PHONE                         	+33623398373
PR:DOI                           	http://dx.doi.org/10.21228/M8VM64
#STUDY
ST:STUDY_TITLE                   	Alcohol dehydrogenase 1B is crucial for adipocyte homeostasis
ST:STUDY_SUMMARY                 	Background. Alcohol dehydrogenase (ADH1B), encoded by the ADH1B gene, is a
ST:STUDY_SUMMARY                 	cytosolic enzyme mainly known for its role in ethanol catabolism in the liver. A
ST:STUDY_SUMMARY                 	few studies have paved the way to show an equally important role of ADH1B in
ST:STUDY_SUMMARY                 	adipocytes. This study aimed to better identify the cellular mechanisms and
ST:STUDY_SUMMARY                 	signaling pathways involving ADH1B in adipose tissue and to determine if ADH1B
ST:STUDY_SUMMARY                 	variants might contribute to adipose tissue dysfunction. Results. We showed that
ST:STUDY_SUMMARY                 	CRISPR-Cas9-mediated ADH1B knockout (KO) in human adipose stem cells (ASC)
ST:STUDY_SUMMARY                 	abolished adipocyte differentiation and decreased insulin response. This was
ST:STUDY_SUMMARY                 	accompanied by oxidative stress, altered mitochondrial functions, and cellular
ST:STUDY_SUMMARY                 	senescence. Lipidomic analysis revealed that ADH1B deficiency results in a major
ST:STUDY_SUMMARY                 	remodeling of lipid composition in ASC. An ADH1B homozygous loss-of-function
ST:STUDY_SUMMARY                 	variant was also identified in a patient presenting with a lipodystrophic and
ST:STUDY_SUMMARY                 	insulin resistant syndrome associated with major liver dysfunction, leading to
ST:STUDY_SUMMARY                 	early death. Discussion. This translational study underlines the crucial role of
ST:STUDY_SUMMARY                 	ADH1B in adipose tissue. It unveils cellular mechanisms accounting for its key
ST:STUDY_SUMMARY                 	role in adipogenesis, and adipocyte homeostasis. This study also identifies
ST:STUDY_SUMMARY                 	ADH1B as a candidate gene in monogenic forms of lipodystrophic and insulin
ST:STUDY_SUMMARY                 	resistant syndromes.
ST:INSTITUTE                     	INSERM
ST:LAST_NAME                     	Gautheron
ST:FIRST_NAME                    	Jérémie
ST:ADDRESS                       	27 rue Chaligny
ST:EMAIL                         	jeremie.gautheron@gmail.com
ST:PHONE                         	+33623398373
ST:SUBMIT_DATE                   	2022-12-13
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	ADH1B_KO_01_LIP329	ADH1B_KO_01_LIP329	Genotype:ADH1B_KO	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_KO_01; RAW_FILE_NAME=LIP329_C18_10x_KO_01; RAW_FILE_NAME=LIP329_long_1x_KO_01; RAW_FILE_NAME=LIP329_short_1x_KO_01; RAW_FILE_NAME=LIP329_short_20x_KO_01
SUBJECT_SAMPLE_FACTORS           	ADH1B_KO_02_LIP329	ADH1B_KO_02_LIP329	Genotype:ADH1B_KO	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_KO_02; RAW_FILE_NAME=LIP329_C18_10x_KO_02; RAW_FILE_NAME=LIP329_long_1x_KO_02; RAW_FILE_NAME=LIP329_short_1x_KO_02; RAW_FILE_NAME=LIP329_short_20x_KO_02
SUBJECT_SAMPLE_FACTORS           	ADH1B_KO_03_LIP329	ADH1B_KO_03_LIP329	Genotype:ADH1B_KO	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_KO_03; RAW_FILE_NAME=LIP329_C18_10x_KO_03; RAW_FILE_NAME=LIP329_long_1x_KO_03; RAW_FILE_NAME=LIP329_short_1x_KO_03; RAW_FILE_NAME=LIP329_short_20x_KO_03
SUBJECT_SAMPLE_FACTORS           	ADH1B_KO_04_LIP329	ADH1B_KO_04_LIP329	Genotype:ADH1B_KO	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_KO_04; RAW_FILE_NAME=LIP329_C18_10x_KO_04; RAW_FILE_NAME=LIP329_long_1x_KO_04; RAW_FILE_NAME=LIP329_short_1x_KO_04; RAW_FILE_NAME=LIP329_short_20x_KO_04
SUBJECT_SAMPLE_FACTORS           	ADH1B_KO_05_LIP329	ADH1B_KO_05_LIP329	Genotype:ADH1B_KO	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_KO_05; RAW_FILE_NAME=LIP329_C18_10x_KO_05; RAW_FILE_NAME=LIP329_long_1x_KO_05; RAW_FILE_NAME=LIP329_short_1x_KO_05; RAW_FILE_NAME=LIP329_short_20x_KO_05
SUBJECT_SAMPLE_FACTORS           	CTL_01_LIP329	CTL_01_LIP329	Genotype:CTL	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_CTL_01; RAW_FILE_NAME=LIP329_C18_10x_CTL_01; RAW_FILE_NAME=LIP329_long_1x_CTL_01; RAW_FILE_NAME=LIP329_short_1x_CTL_01; RAW_FILE_NAME=LIP329_short_20x_CTL_01
SUBJECT_SAMPLE_FACTORS           	CTL_02_LIP329	CTL_02_LIP329	Genotype:CTL	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_CTL_02; RAW_FILE_NAME=LIP329_C18_10x_CTL_02; RAW_FILE_NAME=LIP329_long_1x_CTL_02; RAW_FILE_NAME=LIP329_short_1x_CTL_02; RAW_FILE_NAME=LIP329_short_20x_CTL_02
SUBJECT_SAMPLE_FACTORS           	CTL_03_LIP329	CTL_03_LIP329	Genotype:CTL	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_CTL_03; RAW_FILE_NAME=LIP329_C18_10x_CTL_03; RAW_FILE_NAME=LIP329_long_1x_CTL_03; RAW_FILE_NAME=LIP329_short_1x_CTL_03; RAW_FILE_NAME=LIP329_short_20x_CTL_03
SUBJECT_SAMPLE_FACTORS           	CTL_04_LIP329	CTL_04_LIP329	Genotype:CTL	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_CTL_04; RAW_FILE_NAME=LIP329_C18_10x_CTL_04; RAW_FILE_NAME=LIP329_long_1x_CTL_04; RAW_FILE_NAME=LIP329_short_1x_CTL_04; RAW_FILE_NAME=LIP329_short_20x_CTL_04
SUBJECT_SAMPLE_FACTORS           	CTL_05_LIP329	CTL_05_LIP329	Genotype:CTL	Batch=1; RAW_FILE_NAME=LIP329_C18_1x_CTL_05; RAW_FILE_NAME=LIP329_C18_10x_CTL_05; RAW_FILE_NAME=LIP329_long_1x_CTL_05; RAW_FILE_NAME=LIP329_short_1x_CTL_05; RAW_FILE_NAME=LIP329_short_20x_CTL_05
#COLLECTION
CO:COLLECTION_SUMMARY            	ASC were isolated from surgical samples of subcutaneous abdominal adipose tissue
CO:COLLECTION_SUMMARY            	from a 25-year-old healthy woman with a normal body mass index (BMI). Adipose
CO:COLLECTION_SUMMARY            	tissue was enzymatically digested with collagenase B (0.2%).
CO:SAMPLE_TYPE                   	Adipose tissue
#TREATMENT
TR:TREATMENT_SUMMARY             	After centrifugation, stromal vascular fraction was filtered, rinsed, plated and
TR:TREATMENT_SUMMARY             	cultured in α-MEM with 10% Fetal Calf Serum (FCS), 1% GlutaMAX (#35050061,
TR:TREATMENT_SUMMARY             	Thermo Fisher Scientific), 1% Penicillin/streptomycin (PS - 10,000 UI/mL), 1%
TR:TREATMENT_SUMMARY             	HEPES and Fibroblast Growth Factor-2 (FGF-2 -145 nmol/L). After 24 h, only ASC
TR:TREATMENT_SUMMARY             	adhered to plastic surfaces, while other cells were removed after culture medium
TR:TREATMENT_SUMMARY             	replacement. ASC were maintained in an undifferentiated state in α-MEM
TR:TREATMENT_SUMMARY             	supplemented with 10 % newborn calf serum (#CA-1151500; Biosera, MI, USA), 1%
TR:TREATMENT_SUMMARY             	GlutaMAX, HEPES and P/S, and FGF-2 (145 nmol/L). Adipocyte differentiation was
TR:TREATMENT_SUMMARY             	induced by treating 2-day post-confluent cultures with high-glucose (25 mmol/L)
TR:TREATMENT_SUMMARY             	DMEM supplemented with 10 % FCS, 1 % PS, 1 µmol/L dexamethasone (#D4902;
TR:TREATMENT_SUMMARY             	Sigma-Aldrich, MI, USA), 1 µM rosiglitazone (#D4902; Sigma-Aldrich), 250 µM
TR:TREATMENT_SUMMARY             	3-isobutyl-1-methyl xanthine (IBMX) (#I7018; Sigma-Aldrich) and 0.17 µmol/L
TR:TREATMENT_SUMMARY             	insulin (#I0516; Sigma-Aldrich) for ten days. The medium was then replaced with
TR:TREATMENT_SUMMARY             	high-glucose DMEM supplemented with 10% FCS, 1 % PS, 1 µmol/L rosiglitazone and
TR:TREATMENT_SUMMARY             	0.17 µM insulin, and changed to fresh medium every 2 days until the 20th day.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Lipid extraction. Lipids were extracted from ASC cells according to a modified
SP:SAMPLEPREP_SUMMARY            	Folch method. Cell pellets were resuspended in 100µl methanol and supplemented
SP:SAMPLEPREP_SUMMARY            	with deuterated internal standards. Lipids were extracted with 1.5 mL chloroform
SP:SAMPLEPREP_SUMMARY            	and 650 µL methanol, sonicated for 15 min. Phase separation was triggered by
SP:SAMPLEPREP_SUMMARY            	addition of 450 µL of ammonium carbonate (250 mM). Lower organic phase was
SP:SAMPLEPREP_SUMMARY            	dried and resuspended in 200 µL of liquid chromatography – mass spectrometry
SP:SAMPLEPREP_SUMMARY            	(LC-MS) solvent.
#CHROMATOGRAPHY
CH:INSTRUMENT_NAME               	Shimadzu 20AD
CH:COLUMN_NAME                   	Merck Supelco Ascentis Express C18 (150 x 2.1mm,2.7um)
CH:COLUMN_TEMPERATURE            	43
CH:FLOW_GRADIENT                 	-
CH:FLOW_RATE                     	300ul/min
CH:SOLVENT_A                     	60% acetonitrile/40% water; 0.1% formic acid; 10mM ammonium formate
CH:SOLVENT_B                     	10% acetonitrile/90% isopropanol; 0.1% formic acid; 10mM ammonium formate
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	ABI Sciex 4000 QTrap
MS:INSTRUMENT_TYPE               	QTRAP
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	MRM acquisition of low abundant neutral lipids: DG This acquisition is referred
MS:MS_COMMENTS                   	to as "C18_1x"
MS:ION_MODE                      	POSITIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	mol% of total lipids
MS_METABOLITE_DATA_START
Samples	ADH1B_KO_01_LIP329	ADH1B_KO_02_LIP329	ADH1B_KO_03_LIP329	ADH1B_KO_04_LIP329	ADH1B_KO_05_LIP329	CTL_01_LIP329	CTL_02_LIP329	CTL_03_LIP329	CTL_04_LIP329	CTL_05_LIP329
Factors	Genotype:ADH1B_KO	Genotype:ADH1B_KO	Genotype:ADH1B_KO	Genotype:ADH1B_KO	Genotype:ADH1B_KO	Genotype:CTL	Genotype:CTL	Genotype:CTL	Genotype:CTL	Genotype:CTL	
CE(16:0)	0.0418	0.0316	0.0274	0.0309	0.0412	0.1083	0.0897	0.1080	0.0701	0.0955
CE(16:1)	0.0281	0.0302	0.0291	0.0223	0.0350	0.0759	0.0666	0.0774	0.0590	0.0583
CE(18:0)	0.0172	0.0147	0.0126	0.0180	0.0180	0.0808	0.0569	0.0685	0.0396	0.0944
CE(18:1)	0.1733	0.1569	0.1580	0.1581	0.2004	0.9055	0.8795	0.8279	0.6789	0.8097
CE(18:2)	0.1375	0.1341	0.1242	0.1086	0.1625	0.4013	0.3628	0.3414	0.2812	0.3714
CE(18:3)	0.0398	0.0378	0.0376	0.0389	0.0426	0.1600	0.1207	0.1178	0.0988	0.1233
CE(20:3)	0.0142	0.0115	0.0115	0.0141	0.0155	0.0442	0.0385	0.0349	0.0271	0.0371
CE(20:4)	0.0232	0.0174	0.0192	0.0293	0.0334	0.0913	0.0618	0.0504	0.0474	0.0636
CE(22:5)	0.0598	0.0512	0.0570	0.0770	0.0793	0.2634	0.2069	0.1600	0.1514	0.1905
CE(22:6)	0.1103	0.0914	0.1105	0.1354	0.1462	0.3102	0.2719	0.2161	0.1895	0.2514
DG(30:1_14:0_16:1)	0.0065	0.0065	0.0051	0.0041	0.0055	0.0042	0.0042	0.0063	0.0047	0.0038
DG(32:0_16:0_16:0)	0.1645	0.1569	0.1751	0.2063	0.2357	0.1766	0.1806	0.1588	0.1423	0.1753
DG(32:1_14:0_18:1)	0.0207	0.0222	0.0212	0.0225	0.0215	0.0197	0.0222	0.0206	0.0219	0.0219
DG(32:2_14:0_18:2)	0.0077	0.0087	0.0073	0.0059	0.0078	0.0035	0.0035	0.0069	0.0040	0.0047
DG(34:0_16:0_18:0)	0.0629	0.0632	0.0535	0.0631	0.0834	0.0495	0.0520	0.0435	0.0377	0.0498
DG(34:1_16:0_18:1)	0.3331	0.3580	0.3775	0.4282	0.3995	0.3934	0.3760	0.3554	0.3332	0.3977
DG(34:2_16:0_18:2)	0.0957	0.1072	0.1107	0.1133	0.1047	0.0595	0.0605	0.1137	0.0527	0.0945
DG(34:2_16:1_18:1)	0.0696	0.0754	0.0786	0.0740	0.0763	0.0725	0.0754	0.0769	0.0750	0.0784
DG(34:3_16:0_18:3)	0.0110	0.0145	0.0131	0.0122	0.0136	0.0061	0.0081	0.0163	0.0055	0.0126
DG(34:3_16:1_18:2)	0.0147	0.0173	0.0188	0.0146	0.0157	0.0090	0.0092	0.0130	0.0097	0.0105
DG(34:4_16:1_18:3)	0.0036	0.0040	0.0029	0.0025	0.0036	0.0014	0.0019	0.0024	0.0017	0.0016
DG(36:0_18:0_18:0)	0.0352	0.0373	0.0107	0.0216	0.0341	0.0062	0.0195	0.0148	0.0082	0.0083
DG(36:1_18:0_18:1)	0.1158	0.1314	0.1454	0.1457	0.1617	0.0999	0.0894	0.0964	0.0792	0.1043
DG(36:2_18:0_18:2)	0.0441	0.0508	0.0552	0.0550	0.0569	0.0332	0.0308	0.1017	0.0281	0.0777
DG(36:2_18:1_18:1)	0.3437	0.3916	0.4077	0.4595	0.4194	0.3806	0.3544	0.3463	0.3310	0.3851
DG(36:3_16:0_20:3)	0.1093	0.1195	0.1244	0.1340	0.1230	0.0358	0.0344	0.0298	0.0282	0.0360
DG(36:3_18:1_18:2)	0.0877	0.0997	0.1015	0.1034	0.0966	0.0517	0.0545	0.0669	0.0454	0.0628
DG(36:4_16:0_20:4)	0.1607	0.1927	0.1882	0.1951	0.1860	0.1172	0.1013	0.1012	0.1027	0.1102
DG(36:4_18:1_18:3)	0.0089	0.0088	0.0086	0.0086	0.0089	0.0044	0.0050	0.0077	0.0045	0.0068
DG(36:4_18:2_18:2)	0.0146	0.0150	0.0152	0.0132	0.0131	0.0050	0.0056	0.0297	0.0039	0.0197
DG(36:5_16:0_20:5)	0.0276	0.0308	0.0333	0.0293	0.0325	0.0161	0.0174	0.0168	0.0163	0.0156
DG(36:5_16:1_20:4)	0.0273	0.0320	0.0270	0.0212	0.0308	0.0188	0.0208	0.0197	0.0205	0.0209
DG(36:5_18:2_18:3)	0.0027		0.0027	0.0022	0.0031	0.0009	0.0015	0.0050		0.0028
DG(38:3_18:0_20:3)	0.0580	0.0698	0.0746	0.0704	0.0733	0.0443	0.0423	0.0409	0.0404	0.0484
DG(38:3_18:1_20:2)	0.0231	0.0280	0.0285	0.0267	0.0281	0.0134	0.0128	0.0123	0.0116	0.0132
DG(38:4_18:0_20:4)	0.2429	0.2815	0.3048	0.3353	0.3105	0.3276	0.2889	0.2694	0.2888	0.3432
DG(38:4_18:1_20:3)	0.0966	0.1053	0.1111	0.1253	0.1071	0.0348	0.0317	0.0269	0.0269	0.0322
DG(38:5_16:0_22:5)	0.1494	0.1658	0.1771	0.1575	0.1664	0.0627	0.0646	0.0593	0.0601	0.0621
DG(38:5_18:1_20:4)	0.1347	0.1572	0.1564	0.1598	0.1505	0.1179	0.1089	0.1093	0.1137	0.1191
DG(38:6_16:0_22:6)	0.1187	0.1400	0.1385	0.1394	0.1359	0.0886	0.0842	0.0782	0.0798	0.0843
DG(38:6_18:2_20:4)	0.0199	0.0239	0.0190	0.0171	0.0204	0.0073	0.0069	0.0073	0.0069	0.0069
DG(40:7_18:1_22:6)	0.0959	0.1072	0.1091	0.1097	0.1103	0.0708	0.0676	0.0666	0.0636	0.0673
FC	20.2911	21.2272	21.9566	20.0937	22.5802	21.4107	18.6599	19.8820	25.5547	18.9283
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	pubchem_id	inchi_key	kegg_id	other_id	other_id_type	ri	ri_type	moverz_quant	
CE(16:0)						12.4			
CE(16:1)						11.6			
CE(18:0)						13.2			
CE(18:1)						12.4			
CE(18:2)						11.7			
CE(18:3)						11			
CE(20:3)						11.8			
CE(20:4)						11.3			
CE(22:5)						11.3			
CE(22:6)						10.8			
DG(30:1_14:0_16:1)						3.92			
DG(32:0_16:0_16:0)						5.61			
DG(32:1_14:0_18:1)						4.7			
DG(32:2_14:0_18:2)						4.01			
DG(34:0_16:0_18:0)						6.55			
DG(34:1_16:0_18:1)						5.63			
DG(34:2_16:0_18:2)						4.9			
DG(34:2_16:1_18:1)						4.79			
DG(34:3_16:0_18:3)						4.27			
DG(34:3_16:1_18:2)						4.16			
DG(34:4_16:1_18:3)						3.7			
DG(36:0_18:0_18:0)						7.49			
DG(36:1_18:0_18:1)						6.58			
DG(36:2_18:0_18:2)						5.84			
DG(36:2_18:1_18:1)						5.65			
DG(36:3_16:0_20:3)						5.07			
DG(36:3_18:1_18:2)						4.92			
DG(36:4_16:0_20:4)						4.67			
DG(36:4_18:1_18:3)						4.25			
DG(36:4_18:2_18:2)						4.2			
DG(36:5_16:0_20:5)						4.09			
DG(36:5_16:1_20:4)						3.91			
DG(36:5_18:2_18:3)						3.64			
DG(38:3_18:0_20:3)						6.03			
DG(38:3_18:1_20:2)						5.77			
DG(38:4_18:0_20:4)						5.59			
DG(38:4_18:1_20:3)						5.14			
DG(38:5_16:0_22:5)						4.67			
DG(38:5_18:1_20:4)						4.69			
DG(38:6_16:0_22:6)						4.38			
DG(38:6_18:2_20:4)						3.99			
DG(40:7_18:1_22:6)						4.38			
FC						3.72			
METABOLITES_END
#END