#METABOLOMICS WORKBENCH Kaori_20250611_192128 DATATRACK_ID:6029 STUDY_ID:ST003985 ANALYSIS_ID:AN006565 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; 300 mM diethylamine
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 6410 QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Agilent jet stream-ESI-MS/MS analysis was performed in negative ion mode using
MS:MS_COMMENTS                   	the following source parameters: dry gas temperature, 300°C; dry gas flow rate,
MS:MS_COMMENTS                   	10 L/min; nebulizer pressure, 10 psi. Data were acquired using dynamic multiple
MS:MS_COMMENTS                   	reaction monitoring (MRM) mode, and the MRM transition setting was based on the
MS:MS_COMMENTS                   	in-house MRM library. Peaks were extracted using automatic integration software
MS:MS_COMMENTS                   	MasterHands (Keio University, Tsuruoka, Japan)(Sugimoto et al., Metabolomics,
MS:MS_COMMENTS                   	2009).
#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
Glucose	0.67	0.14	0.16	0.15
Glucose (m+01)	0.020	N.D.	N.D.	N.D.
Glucose (m+02)	N.D.	N.D.	N.D.	N.D.
Glucose (m+04)	N.D.	N.D.	N.D.	N.D.
Glucose (m+05)	N.D.	0.031	0.089	0.043
Glucose (m+06)	0.005	1.1	1.3	0.81
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	PubChem
Glucose	C00031	5793
Glucose (m+01)	-	-
Glucose (m+02)	-	-
Glucose (m+04)	-	-
Glucose (m+05)	-	-
Glucose (m+06)	-	-
METABOLITES_END
#END