#METABOLOMICS WORKBENCH metabolic_charlie_20220513_001750 DATATRACK_ID:3247 STUDY_ID:ST002167 ANALYSIS_ID:AN003551 PROJECT_ID:PR001376
VERSION             	1
CREATED_ON             	May 16, 2022, 7:14 am
#PROJECT
PR:PROJECT_TITLE                 	Remote solid cancers rewire hepatic nitrogen metabolism via host
PR:PROJECT_TITLE                 	nicotinamide-N-methyltransferase
PR:PROJECT_SUMMARY               	Cancers disrupt host homeostasis in various manners but the identity of host
PR:PROJECT_SUMMARY               	factors underlying such disruption remains largely unknown. Here we show that
PR:PROJECT_SUMMARY               	nicotinamide-N-methyltransferase (NNMT) is a novel host factor that mediates
PR:PROJECT_SUMMARY               	metabolic dysfunction in the livers of cancer-bearing mice. Multiple solid
PR:PROJECT_SUMMARY               	cancers distantly increase expression of Nnmt and its product
PR:PROJECT_SUMMARY               	1-methylnicotinamide (MNAM) in the liver. Multi-omics analyses reveal
PR:PROJECT_SUMMARY               	suppression of the urea cycle accompanied by accumulation of amino acids, and
PR:PROJECT_SUMMARY               	enhancement of uracil biogenesis in the livers of cancer-bearing mice.
PR:PROJECT_SUMMARY               	Importantly, genetic deletion of Nnmt leads to alleviation of these metabolic
PR:PROJECT_SUMMARY               	abnormalities, and buffers cancer-dependent weight loss and reduction of the
PR:PROJECT_SUMMARY               	voluntary wheel-running activity. Our data also demonstrate that MNAM is capable
PR:PROJECT_SUMMARY               	of affecting urea cycle metabolites in the liver. These results suggest that
PR:PROJECT_SUMMARY               	cancers up-regulate the hepatic NNMT pathway to rewire liver metabolism towards
PR:PROJECT_SUMMARY               	uracil biogenesis rather than nitrogen disposal via the urea cycle, thereby
PR:PROJECT_SUMMARY               	disrupting host homeostasis.
PR:INSTITUTE                     	Tohoku University
PR:LAST_NAME                     	Kawaoka
PR:FIRST_NAME                    	Shinpei
PR:ADDRESS                       	4-1 Seiryo-cho, Sendai, Miyagi, 9808575, Japan
PR:EMAIL                         	kawaokashinpei@gmail.com
PR:PHONE                         	0227178568
#STUDY
ST:STUDY_TITLE                   	Remote solid cancers rewire hepatic nitrogen metabolism via host
ST:STUDY_TITLE                   	nicotinamide-N-methyltransferase (AML cells)
ST:STUDY_SUMMARY                 	Cancers disrupt host homeostasis in various manners but the identity of host
ST:STUDY_SUMMARY                 	factors underlying such disruption remains largely unknown. Here we show that
ST:STUDY_SUMMARY                 	nicotinamide-N-methyltransferase (NNMT) is a novel host factor that mediates
ST:STUDY_SUMMARY                 	metabolic dysfunction in the livers of cancer-bearing mice. Multiple solid
ST:STUDY_SUMMARY                 	cancers distantly increase expression of Nnmt and its product
ST:STUDY_SUMMARY                 	1-methylnicotinamide (MNAM) in the liver. Multi-omics analyses reveal
ST:STUDY_SUMMARY                 	suppression of the urea cycle accompanied by accumulation of amino acids, and
ST:STUDY_SUMMARY                 	enhancement of uracil biogenesis in the livers of cancer-bearing mice.
ST:STUDY_SUMMARY                 	Importantly, genetic deletion of Nnmt leads to alleviation of these metabolic
ST:STUDY_SUMMARY                 	abnormalities, and buffers cancer-dependent weight loss and reduction of the
ST:STUDY_SUMMARY                 	voluntary wheel-running activity. Our data also demonstrate that MNAM is capable
ST:STUDY_SUMMARY                 	of affecting urea cycle metabolites in the liver. These results suggest that
ST:STUDY_SUMMARY                 	cancers up-regulate the hepatic NNMT pathway to rewire liver metabolism towards
ST:STUDY_SUMMARY                 	uracil biogenesis rather than nitrogen disposal via the urea cycle, thereby
ST:STUDY_SUMMARY                 	disrupting host homeostasis. Anionic polar metabolites (i.e., organic acids,
ST:STUDY_SUMMARY                 	sugar phosphates, nucleotides, etc.) were analyzed via IC/HR/MS/MS. Cationic
ST:STUDY_SUMMARY                 	polar metabolites (i.e., amino acids, bases, nucleosides, NAM, SAM, MNAM, SAH,
ST:STUDY_SUMMARY                 	me2PY, me4PY, etc) were analyzed via PFPP-LC/HR/MS/MS.
ST:INSTITUTE                     	Tohoku University
ST:LAST_NAME                     	Kawaoka
ST:FIRST_NAME                    	Shinpei
ST:ADDRESS                       	4-1 Seiryo-cho, Sendai, Miyagi, 9808575, Japan
ST:EMAIL                         	kawaokashinpei@gmail.com
ST:PHONE                         	0227178568
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
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           	191015	control_1	Culture conditions:Control | Treatment:No treatment	RAW_FILE_NAME=1
SUBJECT_SAMPLE_FACTORS           	191015	control_2	Culture conditions:Control | Treatment:No treatment	RAW_FILE_NAME=3
SUBJECT_SAMPLE_FACTORS           	191015	control_3	Culture conditions:Control | Treatment:No treatment	RAW_FILE_NAME=5
SUBJECT_SAMPLE_FACTORS           	191015	control_4	Culture conditions:Control | Treatment:No treatment	RAW_FILE_NAME=7
SUBJECT_SAMPLE_FACTORS           	191015	4T1_1	Culture conditions:4T1-conditioned media | Treatment:No treatment	RAW_FILE_NAME=2
SUBJECT_SAMPLE_FACTORS           	191015	4T1_2	Culture conditions:4T1-conditioned media | Treatment:No treatment	RAW_FILE_NAME=4
SUBJECT_SAMPLE_FACTORS           	191015	4T1_3	Culture conditions:4T1-conditioned media | Treatment:No treatment	RAW_FILE_NAME=6
SUBJECT_SAMPLE_FACTORS           	191015	4T1_4	Culture conditions:4T1-conditioned media | Treatment:No treatment	RAW_FILE_NAME=8
SUBJECT_SAMPLE_FACTORS           	200401	cont_1	Culture conditions:Control | Treatment:No treatment	RAW_FILE_NAME=cell1
SUBJECT_SAMPLE_FACTORS           	200401	cont_2	Culture conditions:Control | Treatment:No treatment	RAW_FILE_NAME=cell4
SUBJECT_SAMPLE_FACTORS           	200401	cont_3	Culture conditions:Control | Treatment:No treatment	RAW_FILE_NAME=cell7
SUBJECT_SAMPLE_FACTORS           	200401	TNF20_1	Culture conditions:Control | Treatment:TNFalpha (20 ng/mL) treatment	RAW_FILE_NAME=cell3
SUBJECT_SAMPLE_FACTORS           	200401	TNF20_2	Culture conditions:Control | Treatment:TNFalpha (20 ng/mL) treatment	RAW_FILE_NAME=cell6
SUBJECT_SAMPLE_FACTORS           	200401	TNF20_3	Culture conditions:Control | Treatment:TNFalpha (20 ng/mL) treatment	RAW_FILE_NAME=cell9
SUBJECT_SAMPLE_FACTORS           	200401	TNF200_1	Culture conditions:Control | Treatment:TNFalpha (200 ng/mL) treatment	RAW_FILE_NAME=cell2
SUBJECT_SAMPLE_FACTORS           	200401	TNF200_2	Culture conditions:Control | Treatment:TNFalpha (200 ng/mL) treatment	RAW_FILE_NAME=cell5
SUBJECT_SAMPLE_FACTORS           	200401	TNF200_3	Culture conditions:Control | Treatment:TNFalpha (200 ng/mL) treatment	RAW_FILE_NAME=cell8
#COLLECTION
CO:COLLECTION_SUMMARY            	4T1 cells were cultured in 10 cm dishes for 48 hours and the culture supernatant
CO:COLLECTION_SUMMARY            	was collected. The supernatant was stored as the 4T1-conditioned media at 4°C
CO:COLLECTION_SUMMARY            	until use. AML cells per a well were cultured in a 6 well plate for 24 hours,
CO:COLLECTION_SUMMARY            	and then the media was switched to the 4T1-conditioned media. After 24 hours,
CO:COLLECTION_SUMMARY            	the treated AML12 cells were collected.
CO:SAMPLE_TYPE                   	AML cells
#TREATMENT
TR:TREATMENT_SUMMARY             	AML cells per well were cultured in a 24 well plate for 24 hours. The media was
TR:TREATMENT_SUMMARY             	then switched to the bovine-serum free media, and TNF alpha was added at the
TR:TREATMENT_SUMMARY             	concentration of 20 ng/ml or 200 ng/ml (Roche). After 24 hours, the treated
TR:TREATMENT_SUMMARY             	AML12 cells were collected.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites were extracted from AML12 cells (less than 6 × 10E5 cells/well (6
SP:SAMPLEPREP_SUMMARY            	well plate)) using the Bligh and Dyer’s method with some modifications.
SP:SAMPLEPREP_SUMMARY            	Briefly, each sample was mixed with 1 mL of cold methanol containing
SP:SAMPLEPREP_SUMMARY            	10-camphorsulfonic acid (1.5 nmol) and piperazine-1,4-bis (2-ethanesulfonic
SP:SAMPLEPREP_SUMMARY            	acid) (PIPES, 1.5 nmol) as internal standards for mass spectrometry-based
SP:SAMPLEPREP_SUMMARY            	metabolomic analysis. The samples were vigorously mixed by vortexing for 1 min
SP:SAMPLEPREP_SUMMARY            	followed by 5 min of sonication. The extracts were then centrifuged at 16,000 ×
SP:SAMPLEPREP_SUMMARY            	g for 5 min at 4 °C, and the resultant supernatant (400 uL) was collected.
SP:SAMPLEPREP_SUMMARY            	After mixing 400 uL of supernatant with 400 uL of chloroform and 320 uL of
SP:SAMPLEPREP_SUMMARY            	water, the aqueous and organic layers were separated by vortexing and subsequent
SP:SAMPLEPREP_SUMMARY            	centrifugation at 16,000 × g and 4 °C for 5 min. The aqueous (upper) layer
SP:SAMPLEPREP_SUMMARY            	(500 uL) was transferred into a clean tube. After the aqueous layer extracts
SP:SAMPLEPREP_SUMMARY            	were evaporated under vacuum, the dried extracts were stored at −80 °C until
SP:SAMPLEPREP_SUMMARY            	the analysis of hydrophilic metabolites. Prior to analysis, the dried aqueous
SP:SAMPLEPREP_SUMMARY            	layer was reconstituted in 50 uL of water.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Cationic polar metabolites (i.e., amino acids, bases, nucleosides, NAM, SAM,
CH:CHROMATOGRAPHY_SUMMARY        	MNAM, SAH, me2PY, me4PY, etc) were analyzed via PFPP-LC/HRMS/MS.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Shimadzu Nexera X2
CH:COLUMN_NAME                   	Discovery HS (2.1 mm i.d. × 150 mm, 3 um particle size, Merck)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	-
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	cont_1	cont_2	cont_3	TNF20_1	TNF20_2	TNF20_3	TNF200_1	TNF200_2	TNF200_3	control_1	control_2	control_3	control_4	4T1_1	4T1_2	4T1_3	4T1_4
Factors	Culture conditions:Control | Treatment:No treatment	Culture conditions:Control | Treatment:No treatment	Culture conditions:Control | Treatment:No treatment	Culture conditions:Control | Treatment:TNFalpha (20 ng/mL) treatment	Culture conditions:Control | Treatment:TNFalpha (20 ng/mL) treatment	Culture conditions:Control | Treatment:TNFalpha (20 ng/mL) treatment	Culture conditions:Control | Treatment:TNFalpha (200 ng/mL) treatment	Culture conditions:Control | Treatment:TNFalpha (200 ng/mL) treatment	Culture conditions:Control | Treatment:TNFalpha (200 ng/mL) treatment	Culture conditions:Control | Treatment:No treatment	Culture conditions:Control | Treatment:No treatment	Culture conditions:Control | Treatment:No treatment	Culture conditions:Control | Treatment:No treatment	Culture conditions:4T1-conditioned media | Treatment:No treatment	Culture conditions:4T1-conditioned media | Treatment:No treatment	Culture conditions:4T1-conditioned media | Treatment:No treatment	Culture conditions:4T1-conditioned media | Treatment:No treatment
10-Camphorsulfonic acid (IS)	2.E+07	2.E+07	1.E+07	2.E+07	2.E+07	7.E+06	2.E+07	1.E+07	1.E+07	3.E+07	2.E+07	2.E+07	2.E+07	2.E+07	3.E+07	2.E+07	2.E+07
PIPES (IS)	6.E+06	6.E+06	5.E+06	6.E+06	5.E+06	5.E+06	6.E+06	6.E+06	5.E+06	4.E+06	3.E+06	2.E+06	3.E+06	4.E+06	4.E+06	3.E+06	2.E+06
SAM	2.E+06	2.E+06	2.E+06	2.E+06	1.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	1.E+06	2.E+06	1.E+06	1.E+06
NAM	3.E+07	8.E+06	7.E+06	8.E+06	9.E+06	5.E+06	8.E+06	1.E+07	8.E+06	3.E+07	2.E+07	2.E+07	2.E+07	2.E+07	4.E+07	2.E+07	3.E+07
MNAM	2.E+07	2.E+07	1.E+07	2.E+07	2.E+07	2.E+07	2.E+07	2.E+07	2.E+07	4.E+07	2.E+07	2.E+07	3.E+07	5.E+07	5.E+07	5.E+07	4.E+07
SAH	1.E+05	0.E+00	3.E+05	2.E+05	9.E+04	0.E+00	1.E+05	0.E+00	1.E+05	1.E+05	2.E+05	1.E+05	4.E+04	4.E+04	1.E+05	3.E+04	0.E+00
Citrulline	1.E+06	7.E+05	1.E+06	5.E+05	1.E+06	1.E+06	8.E+05	3.E+05	6.E+05	2.E+05	5.E+04	2.E+04	2.E+05	9.E+04	3.E+05	1.E+05	5.E+04
Asp	4.E+06	5.E+06	1.E+06	3.E+06	1.E+06	2.E+06	4.E+06	2.E+06	2.E+06	7.E+05	1.E+06	5.E+05	1.E+06	4.E+05	6.E+05	4.E+05	4.E+05
Argininosuccinate	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00
Arg	1.E+09	1.E+08	1.E+08	2.E+08	2.E+08	7.E+07	1.E+08	2.E+08	1.E+08	3.E+08	2.E+08	2.E+08	4.E+08	1.E+08	5.E+08	3.E+08	2.E+08
Orn	3.E+06	4.E+05	1.E+06	3.E+05	6.E+05	3.E+05	5.E+05	2.E+05	4.E+05	6.E+05	7.E+04	5.E+04	2.E+05	4.E+05	4.E+05	1.E+05	2.E+05
Cytidine	2.E+06	3.E+05	6.E+05	7.E+05	5.E+05	3.E+05	4.E+05	2.E+05	5.E+05	1.E+06	5.E+05	5.E+05	6.E+05	6.E+05	7.E+05	1.E+05	1.E+05
Uridine	8.E+05	5.E+04	2.E+05	0.E+00	1.E+05	0.E+00	1.E+05	6.E+04	1.E+05	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00
Beta-Ala	2.E+07	2.E+07	2.E+07	2.E+07	1.E+07	1.E+07	2.E+07	1.E+07	2.E+07	7.E+07	7.E+07	4.E+07	6.E+07	3.E+07	3.E+07	3.E+07	3.E+07
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	cont_1	cont_2	cont_3	TNF20_1	TNF20_2	TNF20_3	TNF200_1	TNF200_2	TNF200_3	control_1	control_2	control_3	control_4	4T1_1	4T1_2	4T1_3	4T1_4
10-Camphorsulfonic acid (IS)	2.E+07	2.E+07	1.E+07	2.E+07	2.E+07	7.E+06	2.E+07	1.E+07	1.E+07	3.E+07	2.E+07	2.E+07	2.E+07	2.E+07	3.E+07	2.E+07	2.E+07
PIPES (IS)	6.E+06	6.E+06	5.E+06	6.E+06	5.E+06	5.E+06	6.E+06	6.E+06	5.E+06	4.E+06	3.E+06	2.E+06	3.E+06	4.E+06	4.E+06	3.E+06	2.E+06
SAM	2.E+06	2.E+06	2.E+06	2.E+06	1.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	2.E+06	1.E+06	2.E+06	1.E+06	1.E+06
NAM	3.E+07	8.E+06	7.E+06	8.E+06	9.E+06	5.E+06	8.E+06	1.E+07	8.E+06	3.E+07	2.E+07	2.E+07	2.E+07	2.E+07	4.E+07	2.E+07	3.E+07
MNAM	2.E+07	2.E+07	1.E+07	2.E+07	2.E+07	2.E+07	2.E+07	2.E+07	2.E+07	4.E+07	2.E+07	2.E+07	3.E+07	5.E+07	5.E+07	5.E+07	4.E+07
SAH	1.E+05	0.E+00	3.E+05	2.E+05	9.E+04	0.E+00	1.E+05	0.E+00	1.E+05	1.E+05	2.E+05	1.E+05	4.E+04	4.E+04	1.E+05	3.E+04	0.E+00
Citrulline	1.E+06	7.E+05	1.E+06	5.E+05	1.E+06	1.E+06	8.E+05	3.E+05	6.E+05	2.E+05	5.E+04	2.E+04	2.E+05	9.E+04	3.E+05	1.E+05	5.E+04
Asp	4.E+06	5.E+06	1.E+06	3.E+06	1.E+06	2.E+06	4.E+06	2.E+06	2.E+06	7.E+05	1.E+06	5.E+05	1.E+06	4.E+05	6.E+05	4.E+05	4.E+05
Argininosuccinate	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00
Arg	1.E+09	1.E+08	1.E+08	2.E+08	2.E+08	7.E+07	1.E+08	2.E+08	1.E+08	3.E+08	2.E+08	2.E+08	4.E+08	1.E+08	5.E+08	3.E+08	2.E+08
Orn	3.E+06	4.E+05	1.E+06	3.E+05	6.E+05	3.E+05	5.E+05	2.E+05	4.E+05	6.E+05	7.E+04	5.E+04	2.E+05	4.E+05	4.E+05	1.E+05	2.E+05
Cytidine	2.E+06	3.E+05	6.E+05	7.E+05	5.E+05	3.E+05	4.E+05	2.E+05	5.E+05	1.E+06	5.E+05	5.E+05	6.E+05	6.E+05	7.E+05	1.E+05	1.E+05
Uridine	8.E+05	5.E+04	2.E+05	0.E+00	1.E+05	0.E+00	1.E+05	6.E+04	1.E+05	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00	0.E+00
Beta-Ala	2.E+07	2.E+07	2.E+07	2.E+07	1.E+07	1.E+07	2.E+07	1.E+07	2.E+07	7.E+07	7.E+07	4.E+07	6.E+07	3.E+07	3.E+07	3.E+07	3.E+07
METABOLITES_END
#END