#METABOLOMICS WORKBENCH mlhomme_20221213_043543 DATATRACK_ID:3642 STUDY_ID:ST002400 ANALYSIS_ID:AN003907
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                   	Phenomenex Kinetex HILIC (150 x 3mm,2.6um)
CH:COLUMN_TEMPERATURE            	45
CH:FLOW_GRADIENT                 	-
CH:FLOW_RATE                     	300ul/min
CH:SOLVENT_A                     	100% water; 0.2% acetic acid; 30mM ammonium acetate
CH:SOLVENT_B                     	100% acetonitrile; 0.2% acetic acid
CH:CHROMATOGRAPHY_TYPE           	HILIC
#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 broad chromatographic peaks of low abundant phospho- and
MS:MS_COMMENTS                   	sphingolipid classes: PS, PA, LPC, LPE and some ceramides this acquisition is
MS:MS_COMMENTS                   	called "long_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	
LPC(16:0)	0.4605	0.4862	0.3549	0.3597	0.3847	0.3008	0.2893	0.2965	0.2718	0.2784
LPC(16:1)	0.0523	0.0613	0.0417	0.0340	0.0414	0.0307	0.0316	0.0366	0.0324	0.0323
LPC(18:0)	0.5466	0.5367	0.4829	0.5498	0.5094	0.2644	0.2558	0.2523	0.2401	0.2615
LPC(18:1)	0.2032	0.2137	0.1667	0.1261	0.1629	0.1160	0.1266	0.1406	0.1213	0.1269
LPC(18:2)				0.0088	0.0135	0.0104	0.0102	0.0109	0.0091	0.0096
LPC(20:3)	0.0182	0.0196	0.0131	0.0087	0.0132	0.0062	0.0059		0.0059	0.0059
LPC(20:4)	0.1111	0.1522	0.1006	0.0587	0.0958	0.0246	0.0299	0.0307	0.0282	0.0235
LPC(22:4)			0.0125	0.0081	0.0154	0.0059	0.0072		0.0066	0.0053
LPC(22:5)	0.0336	0.0386	0.0288	0.0160	0.0249	0.0115	0.0135	0.0170	0.0138	0.0119
LPC(22:6)	0.0274	0.0369	0.0265	0.0152	0.0223	0.0116	0.0135	0.0152	0.0133	0.0109
LPE(16:0)	0.0479	0.0551	0.0548	0.0398	0.0566	0.0642	0.0648	0.0627	0.0655	0.0514
LPE(16:1)	0.0218		0.0246	0.0163	0.0218	0.0155	0.0197	0.0166	0.0169	0.0144
LPE(18:0)	0.0696	0.0703	0.0705	0.0726	0.0648	0.0549	0.0569	0.0511	0.0530	0.0492
LPE(18:1)	0.3614	0.4264	0.4250	0.3154	0.3997	0.1835	0.2691	0.2068	0.2091	0.1971
LPE(18:2)	0.0326	0.0363	0.0384	0.0265	0.0335	0.0171	0.0238	0.0198	0.0179	0.0175
LPE(20:3)	0.0411	0.0533	0.0409	0.0353	0.0403	0.0171	0.0225	0.0187	0.0184	0.0170
LPE(20:4)	0.6046	0.7390	0.6820	0.4806	0.6349	0.2111	0.2959	0.2456	0.2331	0.1844
LPE(20:5)	0.0582	0.0656	0.0578	0.0480	0.0542	0.0261	0.0363	0.0297	0.0311	0.0245
LPE(22:3)			0.0093	0.0068	0.0103	0.0130	0.0187	0.0143	0.0127	0.0126
LPE(22:4)	0.1129	0.1428	0.1413	0.0928	0.1295	0.0884	0.1306	0.0959	0.1019	0.0871
LPE(22:5)	0.2082	0.2791	0.2624	0.1785	0.2413	0.1829	0.2549	0.2182	0.2112	0.1943
LPE(22:6)	0.2367	0.2800	0.2670	0.1801	0.2239	0.1700	0.2327	0.2041	0.1926	0.1771
PA(34:2)	0.0523	0.0979	0.0597		0.0818	0.0187	0.0584	0.0908	0.0580	0.0285
PA(36:1)	0.2963	0.6465	0.3532		0.3956		0.1670	0.1613	0.1597	
PA(36:2)	0.1266	0.3772	0.2196		0.1597	0.0008	0.1341	0.1763	0.1188	0.0200
PA(36:3)	0.0524	0.1199	0.0834		0.0741	0.0149	0.0602	0.0794	0.0406	0.0256
PA(38:2)	0.0087	0.0585	0.0035		0.0186		0.0231	0.0149	0.0139	
PA(38:3)	0.1052	0.2211	0.1379		0.1378		0.0632	0.0691	0.0667	0.0094
PA(38:4)	0.5620	0.9387	0.7992	0.2182	0.7288	0.1545	0.3355	0.3697	0.2673	0.1891
PA(38:5)	0.1179	0.2090	0.1476	0.0297	0.1375	0.0461	0.1021	0.1067	0.0748	0.0531
PA(38:6)	0.0522	0.0876	0.0648	0.0232	0.0512	0.0148	0.0342	0.0302	0.0264	0.0177
PA(40:5)	0.1262	0.1727	0.1837	0.0285	0.1309	0.0139	0.0689	0.0654	0.0650	0.0034
PS(34:0)	0.0119	0.0098	0.0129	0.0060	0.0097	0.0293	0.0483	0.0455	0.0397	0.0078
PS(34:1)	0.2952	0.4146	0.3405	0.1697	0.3077	0.1910	0.2946	0.2700	0.2303	0.0581
PS(34:2)	0.0241	0.0229	0.0193	0.0171	0.0194	0.0184	0.0228	0.0249	0.0231	0.0083
PS(34:3)	0.0028	0.0060	0.0022		0.0036	0.0006	0.0017	0.0029	0.0012	
PS(36:0)	0.0424	0.0621	0.0488	0.0207	0.0427	0.0296	0.0455	0.0379	0.0427	0.0099
PS(36:1)	0.7754	1.0710	0.8474	0.4447	0.8769	0.5126	0.7803	0.6767	0.6433	0.1126
PS(36:2)	0.1876	0.2392	0.1864	0.1055	0.1879	0.1653	0.2498	0.2016	0.2104	0.0530
PS(36:3)	0.0288	0.0390	0.0340	0.0180	0.0327	0.0211	0.0279	0.0246	0.0242	0.0090
PS(36:4)	0.0192	0.0338	0.0221	0.0108	0.0216	0.0081	0.0132	0.0133	0.0108	0.0045
PS(38:2)	0.0260	0.0437	0.0320	0.0169	0.0292	0.0271	0.0438	0.0388	0.0332	0.0082
PS(38:3)	0.2968	0.3971	0.3022	0.1701	0.2954	0.1384	0.2259	0.1983	0.1823	0.0422
PS(38:4)	0.2596	0.3466	0.2743	0.1479	0.2644	0.1129	0.1694	0.1583	0.1418	0.0381
PS(38:5)	0.0457	0.0558	0.0462	0.0246	0.0452	0.0274	0.0361	0.0391	0.0361	0.0147
PS(38:6)	0.0590	0.0752	0.0515	0.0338	0.0525	0.0258	0.0375	0.0404	0.0367	0.0139
PS(40:4)	0.1210	0.1641	0.1228	0.0648	0.1362	0.0718	0.1154	0.0985	0.0866	0.0230
PS(40:5)	0.3066	0.4090	0.3204	0.1587	0.3515	0.1820	0.2775	0.2646	0.2289	0.0726
PS(40:6)	0.3459	0.4590	0.3500	0.1798	0.3410	0.1645	0.2368	0.2314	0.2084	0.0573
PS(40:7)	0.0248	0.0308	0.0231	0.0116	0.0212	0.0144	0.0211	0.0184	0.0186	0.0060
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	
LPC(16:0)						10.1			
LPC(16:1)						9.93			
LPC(18:0)						9.91			
LPC(18:1)						9.72			
LPC(18:2)						9.84			
LPC(20:3)						9.66			
LPC(20:4)						9.63			
LPC(22:4)						9.55			
LPC(22:5)						9.57			
LPC(22:6)						9.56			
LPE(16:0)						7.85			
LPE(16:1)						7.88			
LPE(18:0)						7.38			
LPE(18:1)						7.76			
LPE(18:2)						7.81			
LPE(20:3)						7.72			
LPE(20:4)						7.69			
LPE(20:5)						7.76			
LPE(22:3)						7.63			
LPE(22:4)						7.65			
LPE(22:5)						7.67			
LPE(22:6)						7.65			
PA(34:2)						6.12			
PA(36:1)						6			
PA(36:2)						6			
PA(36:3)						6.02			
PA(38:2)						5.95			
PA(38:3)						5.81			
PA(38:4)						6			
PA(38:5)						5.81			
PA(38:6)						6			
PA(40:5)						5.79			
PS(34:0)						7.8			
PS(34:1)						7.7			
PS(34:2)						7.22			
PS(34:3)						7.69			
PS(36:0)						7.11			
PS(36:1)						7.6			
PS(36:2)						7.6			
PS(36:3)						7.6			
PS(36:4)						7.6			
PS(38:2)						7.6			
PS(38:3)						7.4			
PS(38:4)						7.28			
PS(38:5)						7.09			
PS(38:6)						7.4			
PS(40:4)						7.4			
PS(40:5)						7.4			
PS(40:6)						7.4			
PS(40:7)						7.4			
METABOLITES_END
#END