#METABOLOMICS WORKBENCH Kaori_20250611_192128 DATATRACK_ID:6029 STUDY_ID:ST003985 ANALYSIS_ID:AN006564 PROJECT_ID:PR002491
VERSION             	1
CREATED_ON             	June 16, 2025, 11:23 pm
#PROJECT
PR:PROJECT_TITLE                 	Glucose-activated JMJD1A drives visceral adipogenesis via
PR:PROJECT_TITLE                 	α-ketoglutarate-dependent chromatin remodeling
PR:PROJECT_SUMMARY               	Understanding how extracellular glucose regulates adipose tissue remodeling is
PR:PROJECT_SUMMARY               	key to decoding metabolic health. Here, we show that the histone demethylase
PR:PROJECT_SUMMARY               	JMJD1A senses glucose availability via α-ketoglutarate (α-KG), a TCA cycle
PR:PROJECT_SUMMARY               	metabolite derived from glycolysis. Upon glucose stimulation, α-KG accumulates
PR:PROJECT_SUMMARY               	in the nucleus and activates JMJD1A to remove repressive H3K9me2 marks at
PR:PROJECT_SUMMARY               	glycolytic and adipogenic gene loci, including Pparg. This initiates a
PR:PROJECT_SUMMARY               	transcriptional feedforward loop that amplifies glycolysis and de novo
PR:PROJECT_SUMMARY               	adipogenesis. Mechanistically, JMJD1A is pre-recruited to chromatin by NFIC, and
PR:PROJECT_SUMMARY               	glucose-induced demethylation enables subsequent ChREBP binding. In vivo, mice
PR:PROJECT_SUMMARY               	lacking JMJD1A in adipocyte precursors exhibit impaired adipose tissue
PR:PROJECT_SUMMARY               	hyperplasia and compensatory hypertrophic expansion selectively in visceral fat,
PR:PROJECT_SUMMARY               	resulting in metabolically unfavorable remodeling. These findings uncover a
PR:PROJECT_SUMMARY               	glucose-sensing α-KG-JMJD1A pathway that regulates histone demethylation and de
PR:PROJECT_SUMMARY               	novo adipogenesis, enabling adaptive expansion of visceral adipose tissue under
PR:PROJECT_SUMMARY               	nutrient excess conditions.
PR:INSTITUTE                     	Tohoku University
PR:LAST_NAME                     	Sakai
PR:FIRST_NAME                    	Juro
PR:ADDRESS                       	2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
PR:EMAIL                         	juro.sakai.b6@tohoku.ac.jp
PR:PHONE                         	+81-22-717-8117
#STUDY
ST:STUDY_TITLE                   	Glucose-activated JMJD1A drives visceral adipogenesis via
ST:STUDY_TITLE                   	α-ketoglutarate-dependent chromatin remodeling (Study part 6 of 5)
ST:STUDY_SUMMARY                 	Understanding how extracellular glucose regulates adipose tissue remodeling is
ST:STUDY_SUMMARY                 	key to decoding metabolic health. Here, we show that the histone demethylase
ST:STUDY_SUMMARY                 	JMJD1A senses glucose availability via α-ketoglutarate (α-KG), a TCA cycle
ST:STUDY_SUMMARY                 	metabolite derived from glycolysis. Upon glucose stimulation, α-KG accumulates
ST:STUDY_SUMMARY                 	in the nucleus and activates JMJD1A to remove repressive H3K9me2 marks at
ST:STUDY_SUMMARY                 	glycolytic and adipogenic gene loci, including Pparg. This initiates a
ST:STUDY_SUMMARY                 	transcriptional feedforward loop that amplifies glycolysis and de novo
ST:STUDY_SUMMARY                 	adipogenesis. Mechanistically, JMJD1A is pre-recruited to chromatin by NFIC, and
ST:STUDY_SUMMARY                 	glucose-induced demethylation enables subsequent ChREBP binding. In vivo, mice
ST:STUDY_SUMMARY                 	lacking JMJD1A in adipocyte precursors exhibit impaired adipose tissue
ST:STUDY_SUMMARY                 	hyperplasia and compensatory hypertrophic expansion selectively in visceral fat,
ST:STUDY_SUMMARY                 	resulting in metabolically unfavorable remodeling. These findings uncover a
ST:STUDY_SUMMARY                 	glucose-sensing α-KG-JMJD1A pathway that regulates histone demethylation and de
ST:STUDY_SUMMARY                 	novo adipogenesis, enabling adaptive expansion of visceral adipose tissue under
ST:STUDY_SUMMARY                 	nutrient excess conditions.
ST:INSTITUTE                     	Tohoku University
ST:LAST_NAME                     	Sakai
ST:FIRST_NAME                    	Juro
ST:ADDRESS                       	2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
ST:EMAIL                         	juro.sakai.b6@tohoku.ac.jp
ST:PHONE                         	+81-22-717-8117
ST:STUDY_COMMENTS                	Figure S1H
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#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           	6	3T3-L1_0h	Sample source:3T3-L1 | Treatment:13C6-Glucose	RAW_FILE_NAME(Anion)=6_d2.mzML; RAW_FILE_NAME(Cation)=6_d5.mzML; RAW_FILE_NAME(Glucose)=g6_d2-3.mzML
SUBJECT_SAMPLE_FACTORS           	7	3T3-L1_1h	Sample source:3T3-L1 | Treatment:13C6-Glucose	RAW_FILE_NAME(Anion)=7_d2.mzML; RAW_FILE_NAME(Cation)=7_d5.mzML; RAW_FILE_NAME(Glucose)=g7_d2-2.mzML
SUBJECT_SAMPLE_FACTORS           	8	3T3-L1_2h	Sample source:3T3-L1 | Treatment:13C6-Glucose	RAW_FILE_NAME(Anion)=8_d2.mzML; RAW_FILE_NAME(Cation)=8_d5.mzML; RAW_FILE_NAME(Glucose)=g8_d2.mzML
SUBJECT_SAMPLE_FACTORS           	9	3T3-L1_4h	Sample source:3T3-L1 | Treatment:13C6-Glucose	RAW_FILE_NAME(Anion)=9_d2.mzML; RAW_FILE_NAME(Cation)=9_d5.mzML; RAW_FILE_NAME(Glucose)=g9_d2.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	3T3-L1 cells were washed twice with 5% mannitol before metabolite extraction.
CO:COLLECTION_SUMMARY            	Aqueous metabolites were extracted with 400 µL of methanol containing three
CO:COLLECTION_SUMMARY            	internal standards (3ISs; methionine sulfone, 2-(N-morpholino)ethanesulfonic
CO:COLLECTION_SUMMARY            	acid [MES] and D-camphol-10-sulfonic acid [CSA]; 25 µM each) at room
CO:COLLECTION_SUMMARY            	temperature for 10 min. Then 400 µL chloroform and 200 µL of water were added,
CO:COLLECTION_SUMMARY            	and the solution was centrifuged at 10,000 × g for 3 min at 4ºC. The upper
CO:COLLECTION_SUMMARY            	aqueous layer (400 µL) was filtered through a 5 kDa cutoff filter (Millipore),
CO:COLLECTION_SUMMARY            	and the filtrate was stored at -80ºC until analysis. At the time of analysis,
CO:COLLECTION_SUMMARY            	the filtrate was thawed and centrifugally concentrated and dissolved in 25 µL
CO:COLLECTION_SUMMARY            	of water containing reference compounds (3-aminopyrrolidine and
CO:COLLECTION_SUMMARY            	1,3,5-benzenetricarboxylic acid; 200 µM each).
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	3T3-L1 preadipocytes were differentiated with MDI mixture until day 4 and
TR:TREATMENT_SUMMARY             	treated with 25 mM 13C glucose.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	For metabolite extraction, the 3T3-L1 cells were washed twice with ice-cold 5%
SP:SAMPLEPREP_SUMMARY            	mannitol solution and covered with 1 mL of methanol containing 25 µM internal
SP:SAMPLEPREP_SUMMARY            	standards for 10 min. 400 µL of the resulting extracts were mixed with 200 µL
SP:SAMPLEPREP_SUMMARY            	of Milli-Q water and 400 µL of chloroform. 400 µL of the aqueous solution was
SP:SAMPLEPREP_SUMMARY            	centrifugally filtered through a 5-kDa cut-off filter (Human Metabolome
SP:SAMPLEPREP_SUMMARY            	Technologies, Tsuruoka, Japan) to remove proteins. The filtrate was
SP:SAMPLEPREP_SUMMARY            	centrifugally concentrated and dissolved in 50 µL of Milli-Q water that
SP:SAMPLEPREP_SUMMARY            	contained reference compounds (200 µM each of 3-aminopyrrolidine and trimesate)
SP:SAMPLEPREP_SUMMARY            	immediately prior to metabolome analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	CE
CH:INSTRUMENT_NAME               	Agilent 7100 CE
CH:COLUMN_NAME                   	Fused silica capillary i. d. 50 µm x 100 cm
CH:SOLVENT_A                     	100% Water; 1 M formic acid
CH:SOLVENT_B                     	N/A
CH:FLOW_GRADIENT                 	N/A
CH:FLOW_RATE                     	N/A
CH:COLUMN_TEMPERATURE            	N/A
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6230 TOF
MS:INSTRUMENT_TYPE               	TOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	ESI-TOFMS was performed in the positive ion mode. The capillary voltage was set
MS:MS_COMMENTS                   	at 4 kV, and a flow rate of nitrogen gas (heater temperature 300°C) was set at
MS:MS_COMMENTS                   	10 L/min. The spectrometer was scanned from m/z 50 to 1,000. Peaks were
MS:MS_COMMENTS                   	extracted using the automatic integration software MasterHands (Keio University,
MS:MS_COMMENTS                   	Tsuruoka, Japan)(Sugimoto et al., Metabolomics, 2009). Based on the in-house
MS:MS_COMMENTS                   	library, metabolites were identified from migration time and m/z. Quantification
MS:MS_COMMENTS                   	was performed using the Standard mixture measured in the same run.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	fmol / cell
MS_METABOLITE_DATA_START
Samples	3T3-L1_0h	3T3-L1_1h	3T3-L1_2h	3T3-L1_4h
Factors	Sample source:3T3-L1 | Treatment:13C6-Glucose	Sample source:3T3-L1 | Treatment:13C6-Glucose	Sample source:3T3-L1 | Treatment:13C6-Glucose	Sample source:3T3-L1 | Treatment:13C6-Glucose
Urea	7.2	N.D.	N.D.	N.D.
Gly	41	40	40	42
Putrescine(1,4-Butanediamine)	0.067	0.036	0.019	0.019
beta-Ala	9.2	8.8	8.0	9.1
Ala	20	5.7	6.5	7.8
GABA	1.0	0.81	0.74	0.64
2AB	0.18	0.17	0.18	0.16
Choline	0.067	0.12	0.29	0.40
Ser	1.1	8.0	7.1	6.1
Diethanolamine	0.34	0.71	0.32	0.78
Hypotaurine	8.6	8.9	8.3	9.3
Carnosine	0.031	0.024	0.032	0.032
Creatinine	0.18	0.11	0.093	0.10
Pro	1.3	4.7	5.1	4.7
5-Aminovalerate	0.26	0.17	0.21	0.15
Val	4.2	4.2	3.9	3.6
Betaine	0.30	0.30	0.30	0.33
Thr	21	20	19	20
Nicotinamide	N.D.	N.D.	N.D.	0.042
Taurine	3.9	3.8	4.4	4.6
Hydroxyproline	0.71	0.48	0.46	0.51
5-Aminolevulinate	0.024	0.016	N.D.	N.D.
Creatine	10	9.1	9.1	9.2
Ile	2.4	3.3	2.9	2.4
Leu	2.5	4.0	3.8	3.1
Gly-Gly	0.038	0.075	0.088	0.063
Asn	4.0	2.9	3.0	3.5
Ornithine	0.047	0.027	0.021	0.030
Asp	2.3	17	20	23
Adenine	N.D.	N.D.	N.D.	0.038
1-Methylnicotinamide	2.0	1.8	1.9	1.9
gamma-Butyrobetaine	0.071	0.080	0.094	0.12
Spermidine	0.031	0.029	0.016	0.025
Gln	67	79	75	73
Lys	1.7	1.7	1.5	1.5
Glu	10	18	15	14
Met	1.8	N.D.	N.D.	N.D.
His	2.0	1.4	1.5	1.7
alpha-Aminoadipate	0.075	0.11	0.15	0.14
Carnitine	0.39	0.31	0.32	0.33
Phe	5.2	3.3	3.4	4.0
Pyridoxal	N.D.	0.097	0.087	0.081
Pyridoxine	0.11	N.D.	N.D.	N.D.
3-Methylhistidine	0.11	0.055	0.070	0.076
Arg	0.64	0.89	0.76	0.76
Citrulline	0.098	0.046	0.047	0.057
Tyr	5.5	3.5	3.7	4.3
Phosphorylcholine	2.3	3.9	4.6	6.1
N1-Acetylspermidine	0.037	0.0065	N.D.	0.0040
N6,N6,N6-Trimethyllysine	0.017	0.024	0.026	0.024
SAMm+	0.49	0.45	0.40	0.49
o-Acetylcarnitine	0.080	0.040	0.047	0.040
Trp	1.0	0.78	0.78	0.93
Cysteine-glutathione disulphide	0.12	N.D.	N.D.	N.D.
Thymidine	3.7	3.8	3.0	3.2
Pyridoxamine 5'-phosphate	0.032	N.D.	N.D.	N.D.
Glycerophosphorylcholine	0.62	0.67	0.92	1.1
Thiamine	N.D.	N.D.	N.D.	0.0077
1-Methyladenosine	N.D.	N.D.	N.D.	0.0096
Guanosine	N.D.	0.067	0.030	0.011
Ophthalmate	0.029	0.052	0.036	0.026
Argininosuccinate	0.16	0.25	0.20	0.19
5-Methylthioadenosine	0.021	0.026	0.031	0.029
Glutathione(ox)	0.39	0.36	0.34	0.45
Glutathione(red)	10	11	10	11
S-Lactoylglutathione	N.D.	N.D.	N.D.	N.D.
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	PubChem
Urea	C00086	1176
Gly	C00037	5257127
Putrescine(1,4-Butanediamine)	C00134	1045
beta-Ala	C00099	239
Ala	C00041	5950
GABA	C00334	119
2AB	C02356	6971252
Choline	C00114	305
Ser	C00065	5951
Diethanolamine	C06772	8113
Hypotaurine	C00519	107812
Carnosine	C00386	439224
Creatinine	C00791	588
Pro	C00148	145742
5-Aminovalerate	C00431	138
Val	C00183	6287
Betaine	C00719	247
Thr	C00188	6288
Nicotinamide	C00153	936
Taurine	C00245	1123
Hydroxyproline	C01157	5810
5-Aminolevulinate	C00430	137
Creatine	C00300	586
Ile	C00407	6306
Leu	C00123	6106
Gly-Gly	C02037	11163
Asn	C00152	6267
Ornithine	C00077	6262
Asp	C00049	44367445
Adenine	C00147	190
1-Methylnicotinamide	C02918	457
gamma-Butyrobetaine	C01181	134
Spermidine	C00315	1102
Gln	C00064	5961
Lys	C00047	5962
Glu	C00025	33032
Met	C00073	6137
His	C00135	6274
alpha-Aminoadipate	C00956	59066632
Carnitine	C00318	10917
Phe	C00079	6140
Pyridoxal	C00250	1050
Pyridoxine	C00314	1054
3-Methylhistidine	C01152	64969
Arg	C00062	28782
Citrulline	C00327	6992098
Tyr	C00082	6057
Phosphorylcholine	C00588	1014
N1-Acetylspermidine	C00612	496
N6,N6,N6-Trimethyllysine	C03793	440120
SAMm+	C00019	24762165
o-Acetylcarnitine	C02571	439756
Trp	C00078	6305
Cysteine-glutathione disulphide	-	10455148
Thymidine	C00214	5789
Pyridoxamine 5'-phosphate	C00647	1053
Glycerophosphorylcholine	C00670	657272
Thiamine	C00378	1130
1-Methyladenosine	C02494	27476
Guanosine	C00387	6802
Ophthalmate	C21016	7018721
Argininosuccinate	C03406	16950
5-Methylthioadenosine	C00170	439176
Glutathione(ox)	C00127	11215652
Glutathione(red)	C00051	124886
S-Lactoylglutathione	C03451	440018
METABOLITES_END
#END