#METABOLOMICS WORKBENCH ICOBicob337_20250922_012905 DATATRACK_ID:6463 STUDY_ID:ST004284 ANALYSIS_ID:AN007124 PROJECT_ID:PR002681 VERSION 1 CREATED_ON October 4, 2025, 3:35 am #PROJECT PR:PROJECT_TITLE Acetyl-CoA Carboxylase Maintains Energetic Balance for Functional Oogenesis 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 unrecognized 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 Institute of Cellular and Organismic Biology, Academia Sinica PR:DEPARTMENT ICOB PR:LABORATORY R337, Hsu Lab PR:LAST_NAME Hsu PR:FIRST_NAME Hweijan PR:ADDRESS no 128, sec 2, academia rd, Academia Sinica, ICOB 337, Taipei, Taipei, 11529, PR:ADDRESS Taiwan PR:EMAIL cohsu@gate.sinica.edu.tw PR:PHONE 886-227871542 #STUDY ST:STUDY_TITLE Acetyl-CoA Carboxylase depletion in germline results in increased Acetyl-CoA ST:STUDY_TITLE level ST:STUDY_SUMMARY Summary of the study: Reproduction is tightly linked to nutrient availability ST:STUDY_SUMMARY and lipid metabolism, and disturbances in these processes often lead to ST:STUDY_SUMMARY reproductive failure. In our study, we investigated the metabolic mechanisms ST:STUDY_SUMMARY governed by Acetyl-CoA Carboxylase (ACC), a rate-limiting enzyme in fatty acid ST:STUDY_SUMMARY synthesis, that support oogenesis. We found that ACC modulates ST:STUDY_SUMMARY nutrient-responsive TOR signaling to sustain endosomal trafficking, a process ST:STUDY_SUMMARY essential for oocyte specification. Loss of ACC shifts cellular metabolism ST:STUDY_SUMMARY toward fatty acid oxidation (FAO), elevating Acetyl-CoA levels and enhancing ST:STUDY_SUMMARY flux through the TCA cycle and electron transport chain (ETC). This metabolic ST:STUDY_SUMMARY reprogramming results in hyperactivation of TOR signaling, leading to excessive ST:STUDY_SUMMARY protein synthesis, disrupted endosomal trafficking, and defective germ cell ST:STUDY_SUMMARY differentiation. ST:INSTITUTE Institute of Cellular and Organismic Biology, Academia Sinica ST:DEPARTMENT ICOB ST:LABORATORY R337, Hsu Lab ST:LAST_NAME Hsu ST:FIRST_NAME Hweijan ST:ADDRESS no 128, sec 2, academia rd, Academia Sinica, ICOB 337, Taipei, Taipei, 11529, ST:ADDRESS Taiwan ST:EMAIL cohsu@gate.sinica.edu.tw 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 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>AccRNAi AccKD1-1 Sample source:Drosophila Ovary | Treatment:Acc-depleted RAW_FILE_NAME(Raw_file name)=AccKD1-1.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD2-1 Sample source:Drosophila Ovary | Treatment:Acc-depleted RAW_FILE_NAME(Raw_file name)=AccKD2-1.mzML SUBJECT_SAMPLE_FACTORS nos>AccRNAi AccKD2-2 Sample source:Drosophila Ovary | Treatment:Acc-depleted RAW_FILE_NAME(Raw_file name)=AccKD2-2.mzML #COLLECTION CO:COLLECTION_SUMMARY Collection summary An extraction solution (2:2:1 acetonitrile: methanol: ddH2O) CO:COLLECTION_SUMMARY was prepared and stored overnight at -20 °C before use. Hundred pairs of CO:COLLECTION_SUMMARY ovaries from each genotype were dissected in cold 1xPBS. Since nos>ACC RNAi CO:COLLECTION_SUMMARY ovaries lacked vitellogenic egg chambers, the vitellogenic egg chambers from CO:COLLECTION_SUMMARY control 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 tube, and metabolites CO:COLLECTION_SUMMARY from 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:SAMPLE_TYPE Ovaries #TREATMENT TR:TREATMENT_SUMMARY nosGAL4 virgins were crossed with either mCherryRNAi or AccRNAi flies. Eggs were TR:TREATMENT_SUMMARY laid and allowed to develop into adults at 18 degrees. Adult flies were then TR:TREATMENT_SUMMARY shifted to 29 degrees to increase the expression of RNAi in the germline for 7 TR:TREATMENT_SUMMARY days. The food was changed daily #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fifty pairs of ovaries from each genotype were dissected in cold 1xPBS. Since SP:SAMPLEPREP_SUMMARY nosGAL4>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 sample was separated with ACQUITY BEH Amide column (1.7μm particle size, CH:CHROMATOGRAPHY_SUMMARY 2.1 × 100 mm, Waters). The UPLC was operated at a flow rate of 0.3 mL/min and CH:CHROMATOGRAPHY_SUMMARY column temperature of 40℃. The composition of mobile phase A was water CH:CHROMATOGRAPHY_SUMMARY containing 20mM ammonium acetate and 0.3% ammonium hydroxide, mobile phase B was CH:CHROMATOGRAPHY_SUMMARY 90% acetonitrile containing containing 20mM ammonium acetate and 0.3% ammonium CH:CHROMATOGRAPHY_SUMMARY hydroxide. Characteristic MS transitions were monitored using positive multiple CH:CHROMATOGRAPHY_SUMMARY reaction monitoring (MRM) mode for acetyl-CoA (m/z, 810>303). Data acquisition CH:CHROMATOGRAPHY_SUMMARY and processing were performed using MassLynx version 4.1 and TargetLynx software CH:CHROMATOGRAPHY_SUMMARY (Waters Corp.). CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:SOLVENT_A Not Applicable CH:SOLVENT_B Not Applicable CH:FLOW_GRADIENT NA CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 40℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters AXQUITY UPLC MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The composition of mobile phase A was water containing 20mM ammonium acetate and MS:MS_COMMENTS 0.3% ammonium hydroxide, and mobile phase B was 90% acetonitrile containing 20mM MS:MS_COMMENTS ammonium acetate and 0.3% ammonium hydroxide. Characteristic MS transitions were MS:MS_COMMENTS monitored using positive multiple reaction monitoring (MRM) mode for acetyl-CoA MS:MS_COMMENTS (m/z, 810>303). Data acquisition and processing were performed using MassLynx MS:MS_COMMENTS version 4.1 and TargetLynx software (Waters Corp.). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples mcherryKD1-1 mcherryKD2-1 mcherryKD2-2 AccKD1-1 AccKD2-1 AccKD2-2 Factors Sample source:Drosophila Ovary | Treatment:control Sample source:Drosophila Ovary | Treatment:control Sample source:Drosophila Ovary | Treatment:control Sample source:Drosophila Ovary | Treatment:Acc-depleted Sample source:Drosophila Ovary | Treatment:Acc-depleted Sample source:Drosophila Ovary | Treatment:Acc-depleted Acetyl-CoA 4.6 4.6 4.6 4.6 4.6 4.6 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID CAS ID Acetyl-CoA 444493 C0024 72-89-9 METABOLITES_END #END