#METABOLOMICS WORKBENCH ICOBicob337_20250920_034730 DATATRACK_ID:6462 STUDY_ID:ST004252 ANALYSIS_ID:AN007075 PROJECT_ID:PR002681 VERSION 1 CREATED_ON October 1, 2025, 10:43 pm #PROJECT PR:PROJECT_TITLE metabolomics in mcherryKD_vs_AccKD PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY PROJECT SUMMARY Reproduction requires the integration of nutrient availability PR:PROJECT_SUMMARY and lipid metabolism to sustain oogenesis and ensure fertility. Disruptions in PR:PROJECT_SUMMARY this metabolic balance are strongly associated with reproductive failure, yet PR:PROJECT_SUMMARY the mechanisms that coordinate these processes remain poorly defined. This PR:PROJECT_SUMMARY project aimed to dissect how lipid metabolic pathways intersect with PR:PROJECT_SUMMARY nutrient-sensing signals to govern germ cell differentiation. Specifically, we PR:PROJECT_SUMMARY focused on the role of Acetyl-CoA Carboxylase (ACC), the rate-limiting enzyme PR:PROJECT_SUMMARY for fatty acid synthesis, in maintaining oocyte development. We demonstrate that PR:PROJECT_SUMMARY ACC functions as a key metabolic regulator by sustaining endosomal trafficking PR:PROJECT_SUMMARY and modulating nutrient-responsive TOR signalling. Loss of ACC activity shifts PR:PROJECT_SUMMARY metabolism toward fatty acid oxidation (FAO), which increases flux through the PR:PROJECT_SUMMARY tricarboxylic acid (TCA) cycle and electron transport chain (ETC). This altered PR:PROJECT_SUMMARY metabolic state hyperactivates TOR signalling, leading to excessive protein PR:PROJECT_SUMMARY synthesis, disrupted endosomal trafficking, and impaired germ cell fate PR:PROJECT_SUMMARY determination. Importantly, we show that these defects are reversible. Targeted PR:PROJECT_SUMMARY interventions—including inhibition of FAO or TOR signalling, attenuation of PR:PROJECT_SUMMARY protein synthesis, or dietary protein adjustment—restore cellular homeostasis PR:PROJECT_SUMMARY and rescue oogenic defects. The findings from this study reveal a previously PR:PROJECT_SUMMARY unrecognised link between lipid metabolism, nutrient-sensing pathways, and PR:PROJECT_SUMMARY reproductive development. By defining ACC as a central coordinator of metabolic PR:PROJECT_SUMMARY and signalling networks in oogenesis, this work advances fundamental PR:PROJECT_SUMMARY understanding of how energy metabolism shapes reproductive outcomes. PR:PROJECT_SUMMARY Furthermore, the identification of corrective strategies highlights potential PR:PROJECT_SUMMARY therapeutic avenues for addressing infertility and reproductive disorders PR:PROJECT_SUMMARY associated with metabolic imbalance. PR:INSTITUTE Academia Sinica PR:DEPARTMENT Institute of Cellular and Organismic Biology PR:LABORATORY R337, Hsu Lab PR:LAST_NAME Amamrtuvshin PR:FIRST_NAME Oyundari PR:ADDRESS R337, ICOB, No. 128, Section 2, Academia Rd, Nangang District, Taipei City, PR:ADDRESS Taiwan 115 PR:EMAIL oyundariamar@gmail.com PR:PHONE 886-227871542 #STUDY ST:STUDY_TITLE Acetyl-CoA Carboxylase Maintains Energetic Balance for Functional Oogenesis ST:STUDY_SUMMARY Summary of the study Reproduction is closely tied to nutrient intake and lipid ST:STUDY_SUMMARY metabolism, with imbalances often leads to reproductive failure. We ST:STUDY_SUMMARY characterized the metabolic mechanisms mediated by Acetyl-CoA Carboxylase (ACC, ST:STUDY_SUMMARY a rate-limiting enzyme for fatty acid synthesis) that support oogenesis and ST:STUDY_SUMMARY discovered that ACC regulates nutrient-responsive TOR signaling to maintain ST:STUDY_SUMMARY endosomal trafficking, crucial for oocyte determination. ACC deficiency shifts ST:STUDY_SUMMARY metabolism toward fatty acid oxidation (FAO), fueling the TCA cycle and electron ST:STUDY_SUMMARY transport chain (ETC), which hyperactivates TOR signaling. This results in ST:STUDY_SUMMARY excessive protein synthesis, disrupting endosomal trafficking and impairing germ ST:STUDY_SUMMARY cell differentiation. Restoring balance through FAO or TOR inhibition, reducing ST:STUDY_SUMMARY protein synthesis, or adjusting dietary protein intake corrects these defects. ST:STUDY_SUMMARY Our findings reveal a critical link between lipid metabolism and ST:STUDY_SUMMARY nutrient-sensing pathways in oogenesis, offering potential therapeutic ST:STUDY_SUMMARY strategies for metabolic disorders affecting reproduction. ST:INSTITUTE Academia Sinica ST:DEPARTMENT ICOB ST:LABORATORY Hsu Lab ST:LAST_NAME Amartuvshin ST:FIRST_NAME Oyundari ST:ADDRESS No. 128, Section 2, Academia Rd, Nangang District, Taipei City, Taiwan 115 ST:EMAIL oyundariamar@gmail.com ST:PHONE 886-227871542 #SUBJECT SU:SUBJECT_TYPE Invertebrate SU:SUBJECT_SPECIES Drosophila melanogaster SU:TAXONOMY_ID 7227 SU:GENOTYPE_STRAIN nos>mCherryRNAi and nos>ACCRNAi #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 nos>mCherryRNAi mcherryKD1-1 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD1-1.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD1-2 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD1-2.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD2-1 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD2-1.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD2-2 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD2-2.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD3-1 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD3-1.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD3-2 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD3-2.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD4-1 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD4-1.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD4-2 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD4-2.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD5-1 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD5-1.mzML SUBJECT_SAMPLE_FACTORS nos>mCherryRNAi mcherryKD5-2 Sample source:Drosophila ovary | Treatment:control RAW_FILE_NAME(Raw File Name)=mcherryKD5-2.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD1-1 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD1-1.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD1-2 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD1-2.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD2-1 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD2-1.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD2-2 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD2-2.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD3-1 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD3-1.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD3-2 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD3-2.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD4-1 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD4-1.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD4-2 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD4-2.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD5-1 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD5-1.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD5-2 Sample source:Drosophila ovary | Treatment:Acc knockdown RAW_FILE_NAME(Raw File Name)=AccKD5-2.mzML #COLLECTION CO:COLLECTION_SUMMARY Collection summary Extraction solution (2:2:1 acetonitrile: methanol: ddH2O) was CO:COLLECTION_SUMMARY prepared and stored overnight at -20 degree before usage. Fifty pairs of ovaries CO:COLLECTION_SUMMARY from each genotype were dissected in cold 1xPBS. Since nos>ACC RNAi ovaries CO:COLLECTION_SUMMARY lacked vitellogenic egg chambers, the vitellogenic egg chambers from control CO:COLLECTION_SUMMARY ovaries were removed using forceps, leaving the transparent regions for CO:COLLECTION_SUMMARY metabolite extraction. Drosophila dissection was completed within 1 hour. Ovary CO:COLLECTION_SUMMARY samples for each genotype were collected in an Eppendorf and metabolites from CO:COLLECTION_SUMMARY each sample (containing about 12.5 µg DNA) were extracted using 200 µl CO:COLLECTION_SUMMARY metabolite extraction solvent. Samples were homogenized by vortexing for 5 sec CO:COLLECTION_SUMMARY followed by cold bath sonication for 5 min, repeated twice. Tissue debris was CO:COLLECTION_SUMMARY pelleted by centrifugation at 15871x g (rcf) for 10 min at 4°C. A total of 200 CO:COLLECTION_SUMMARY µl metabolite- containing supernatant was transferred into a new tube. The CO:COLLECTION_SUMMARY supernatant was freeze-dried for at least 3 h using a freeze drier (VirTis CO:COLLECTION_SUMMARY BenchTop K). Dried samples were kept at -80°C until analysed for amino acids CO:COLLECTION_SUMMARY and TCA cycle byproducts. CO:COLLECTION_PROTOCOL_FILENAME TCAcycle_AminoAcid_protocol.docx CO:SAMPLE_TYPE Ovaries CO:COLLECTION_METHOD Drosophila ovary dissection in 1xPBS CO:COLLECTION_LOCATION Taiwan CO:COLLECTION_DURATION within 1 hour CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS Eppendorf CO:STORAGE_VIALS Eppendorf #TREATMENT TR:TREATMENT_SUMMARY noGAL4 virgins were crossed with either mCherryRNAi or AccRNAi flies. Eggs were TR:TREATMENT_SUMMARY laid and allowed to develop into adults at 18 °C. Adult flies were then shifted TR:TREATMENT_SUMMARY to 29 °C to increase the expression of RNAi in the germline for 7 days. The TR:TREATMENT_SUMMARY food was changed daily. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fifty pairs of ovaries from each genotype were dissected in cold 1xPBS. Since SP:SAMPLEPREP_SUMMARY nos greater than ACC RNAi ovaries lacked vitellogenic egg chambers, the vitellogenic egg SP:SAMPLEPREP_SUMMARY chambers from control ovaries were removed using forceps, leaving the SP:SAMPLEPREP_SUMMARY transparent regions for metabolite extraction. Metabolites from each sample SP:SAMPLEPREP_SUMMARY (containing about 12.5 µg DNA) were extracted using 200 µl metabolite SP:SAMPLEPREP_SUMMARY extraction solvent (2:2:1 acetonitrile: methanol: ddH2O) and stored at -20°C SP:SAMPLEPREP_SUMMARY overnight. Samples were homogenized by vortexing for 5 sec followed by cold bath SP:SAMPLEPREP_SUMMARY sonication for 5 min, repeated twice. Tissue debris was pelleted by SP:SAMPLEPREP_SUMMARY centrifugation at 15871x g (rcf) for 10 min at 4°C. A total of 200 µl SP:SAMPLEPREP_SUMMARY metabolite- containing supernatant was transferred into a new tube. The SP:SAMPLEPREP_SUMMARY supernatant was freeze-dried for at least 3 h using a freeze drier (VirTis SP:SAMPLEPREP_SUMMARY BenchTop K). Dried samples were kept at -80°C until analyzed for amino acids SP:SAMPLEPREP_SUMMARY and TCA cycle byproducts. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The samples were derivatized by bis(trimethylsilyl)- trifluoroacetamide (BSTFA) CH:CHROMATOGRAPHY_SUMMARY containing 1% trimethylchlorosilane (TMCS) and analyzed using Agilent 7890B gas CH:CHROMATOGRAPHY_SUMMARY chromatography coupled with 7250 quadrupole time-of-flight mass spectrometer CH:CHROMATOGRAPHY_SUMMARY (GC-Q-TOF/MS) equipped with electron ionization (EI). The separation was CH:CHROMATOGRAPHY_SUMMARY performed on Zorbax DB5- MS+10 m Duragard Capillary Column (30 m x 0.25 mm x CH:CHROMATOGRAPHY_SUMMARY 0.25 mm, Agilent). The GC temperature profile was held at 60℃ for 1 minutes CH:CHROMATOGRAPHY_SUMMARY and then raised at 10℃/min to 325℃ and held at 325℃ for 10 minutes. The CH:CHROMATOGRAPHY_SUMMARY transfer line and the ion source temperature were set at 300 ºC and 280 ºC, CH:CHROMATOGRAPHY_SUMMARY respectively. The mass-range monitored was from 50 to 600 Daltons. The data CH:CHROMATOGRAPHY_SUMMARY acquisition and analysis were performed on MassHunter Workstation software. Mass CH:CHROMATOGRAPHY_SUMMARY spectra were compared against the NIST 2017, Fiehn and Wiley Registry 11th CH:CHROMATOGRAPHY_SUMMARY Edition mass spectral library. The Agilent MassHunter Unknows Analysis software CH:CHROMATOGRAPHY_SUMMARY was used for deconvolution of the signals. The results were imported into the CH:CHROMATOGRAPHY_SUMMARY Agilent Mass Profiler Professional software (Agilent Technologies,15.1, Santa CH:CHROMATOGRAPHY_SUMMARY Clara, CA, USA) for further peak alignment. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Zorbax DB5- MS+10 m Duragard Capillary Column (30 m x 0.25 mm x 0.25 mm) CH:SOLVENT_A Not Applicable CH:SOLVENT_B Not Applicable CH:FLOW_GRADIENT 60℃ for 1 minutes and then raised at 10℃/min to 325℃ and held at 325℃ CH:FLOW_GRADIENT for 10 minutes CH:FLOW_RATE 1ml/min CH:COLUMN_TEMPERATURE 300 ºC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7890B MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass-range monitored was from 50 to 600 Daltons. The data acquisition and MS:MS_COMMENTS analysis were performed on MassHunter Workstation software. Mass spectra were MS:MS_COMMENTS compared against the NIST 2017, Fiehn and Wiley Registry 11th Edition mass MS:MS_COMMENTS spectral library. The Agilent MassHunter Unknows Analysis software was used for MS:MS_COMMENTS deconvolution of the signals. The results were imported into the Agilent Mass MS:MS_COMMENTS Profiler Professional software (Agilent Technologies,15.1, Santa Clara, CA, USA) MS:MS_COMMENTS for further peak alignment. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples mcherryKD1-1 mcherryKD1-2 mcherryKD2-1 mcherryKD2-2 mcherryKD3-1 mcherryKD3-2 mcherryKD4-1 mcherryKD4-2 mcherryKD5-1 mcherryKD5-2 AccKD1-1 AccKD1-2 AccKD2-1 AccKD2-2 AccKD3-1 AccKD3-2 AccKD4-1 AccKD4-2 AccKD5-1 AccKD5-2 Factors Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:control Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Sample source:Drosophila ovary | Treatment:Acc knockdown Citrate 16.58 16.58 16.59 16.59 NA NA NA NA 16.646252 16.646252 16.58 16.58 16.59 16.59 NA NA NA NA 16.646252 16.646252 α-KG 13.84 13.84 NA NA 13.84 13.84 13.84 13.84 13.903 13.903 13.84 13.84 NA NA 13.84 13.84 13.84 13.84 13.903 13.903 Glutamate 14.38 14.38 13.35 13.35 NA NA NA NA 14.439 14.439 14.38 14.38 13.35 13.35 NA NA NA NA 14.439 14.439 Glutamine NA NA 13.38 13.38 13.38 13.38 13.38 13.38 NA NA NA NA 13.38 13.38 13.38 13.38 13.38 13.38 NA NA Fumarate 10.99 10.99 10.99 10.99 10.98 10.98 10.98 10.98 11.049 11.049 10.99 10.99 10.99 10.99 10.98 10.98 10.98 10.98 11.049 11.049 Malate 12.76 12.76 12.76 12.76 NA NA NA NA 12.827 12.827 12.76 12.76 12.76 12.76 NA NA NA NA 12.827 12.827 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID CAS number Citrate 31348 C00158 5949-29-1 α-KG 51 C00026 328-50-7 Glutamate 33032 C00217 56-86-0 Glutamine 5961 C00064 56-85-9 Fumarate 444972 C00122 17013-01-3 Malate 525 C00149 617-48-1 METABOLITES_END #END