#METABOLOMICS WORKBENCH kliu2017_20250825_012101 DATATRACK_ID:6329 STUDY_ID:ST004149 ANALYSIS_ID:AN006879 PROJECT_ID:PR002611 VERSION 1 CREATED_ON August 26, 2025, 8:11 am #PROJECT PR:PROJECT_TITLE Nicotinamide mononucleotide treatment improves spermatogenesis in obese mice by PR:PROJECT_TITLE reducing lysine acetylation of lactate dehydrogenase C PR:PROJECT_SUMMARY Obesity is associated with impaired spermatogenesis and decreased sperm quality, PR:PROJECT_SUMMARY in part through reducing Sertoli cells (SCs) lactate and nicotinamide adenine PR:PROJECT_SUMMARY dinucleotide (NAD+) production. It is not known whether nicotinamide PR:PROJECT_SUMMARY mononucleotide (NMN) treatment improves spermatogenesis. In the present study, PR:PROJECT_SUMMARY NMN improved lipid metabolism and enhanced spermatogenesis in obese mice, PR:PROJECT_SUMMARY alleviated SCs dysfunction in vivo and in vitro. Label-free quantitative PR:PROJECT_SUMMARY acetylomics analysis of mouse testes suggested that protein acetylation PR:PROJECT_SUMMARY influenced both the structural and functional properties of metabolic proteins. PR:PROJECT_SUMMARY The beneficial effects of NMN were due in part to changes in the acetylation of PR:PROJECT_SUMMARY glycolysis-related proteins. Furthermore, multi-omics and correlation analyses PR:PROJECT_SUMMARY demonstrated that interactions among the gut microbiota, metabolites, PR:PROJECT_SUMMARY spermatogenesis, and LDHC acetylation mediated the beneficial effects of NMN. PR:PROJECT_SUMMARY Importantly, we found that NMN treatment reduced acetylation of the lysine PR:PROJECT_SUMMARY residues 5, 17, and 90 of LDHC, which plays a critical role in SC lactate PR:PROJECT_SUMMARY production in obesity. Collectively, our findings show that NMN supplementation PR:PROJECT_SUMMARY improves sperm quality in obese mice by decreasing LDHC acetylation, thereby PR:PROJECT_SUMMARY increasing Sertoli cell lactate and NAD+ production. PR:INSTITUTE University of South China PR:LAST_NAME Liu PR:FIRST_NAME Ke PR:ADDRESS Hongxiang street, Hunan, Hengyang, 421001, China PR:EMAIL kliu2017@163.com PR:PHONE 15358930855 #STUDY ST:STUDY_TITLE Nicotinamide mononucleotide treatment improves spermatogenesis in obese mice by ST:STUDY_TITLE reducing lysine acetylation of lactate dehydrogenase C ST:STUDY_SUMMARY Objective: Does increasing nicotinamide adenine dinucleotide (NAD+) levels via ST:STUDY_SUMMARY nicotinamide mononucleotide (NMN) supplementation improve spermatogenic function ST:STUDY_SUMMARY in a high fat diet (HFD)–induced mouse model of obesity? Design: ST:STUDY_SUMMARY Three-week-old male C57BL/6 mice (n = 48) were fed a normal diet or HFD for 12 ST:STUDY_SUMMARY weeks to serve as a control group or induce obesity, respectively. Beginning on ST:STUDY_SUMMARY week 13, half of the mice in the HFD group were subjected to oral gavage daily ST:STUDY_SUMMARY with NMN for 7 weeks, while the other half received saline gavage. At the end of ST:STUDY_SUMMARY the 19-week experimental period, the animals were sacrificed, and samples were ST:STUDY_SUMMARY collected to analyze tissue morphology, serum lipid levels, sperm quality, lipid ST:STUDY_SUMMARY deposition, the gut microbiota, serum metabolites, protein acetylation, NAD+ ST:STUDY_SUMMARY levels, and testicular mRNA and protein expression profiles. Results: NMN ST:STUDY_SUMMARY improved lipid metabolism and enhanced spermatogenesis in obese mice, alleviated ST:STUDY_SUMMARY Sertoli cell (SC) dysfunction in vivo and vitro, and up-regulated the expression ST:STUDY_SUMMARY of glycolysis rate-limiting enzymes. Label-free quantitative acetylomics ST:STUDY_SUMMARY analysis of mouse testes suggested that protein acetylation influenced both the ST:STUDY_SUMMARY structural and functional properties of metabolic proteins. The beneficial ST:STUDY_SUMMARY effects of NMN were due in part to changes in the acetylation of ST:STUDY_SUMMARY glycolysis-related proteins. Furthermore, treatment with NMN increased the ST:STUDY_SUMMARY levels of lactate and NAD+ produced by SCs stimulated with palmitic acid in ST:STUDY_SUMMARY vitro. Multi-omics and correlation analyses demonstrated that interactions among ST:STUDY_SUMMARY the gut microbiota, metabolites, spermatogenesis, and LDHC acetylation mediated ST:STUDY_SUMMARY the beneficial effects of NMN. Importantly, we found that NMN treatment reduced ST:STUDY_SUMMARY acetylation of the lysine (K) residues 5, 17, and 90 of LDHC, which plays a ST:STUDY_SUMMARY critical role in SC lactate production in obesity. Conclusion: NMN ST:STUDY_SUMMARY supplementation improves sperm quality in obese mice by decreasing lactate ST:STUDY_SUMMARY dehydrogenase C (LDHC) acetylation, thereby increasing Sertoli cell lactate and ST:STUDY_SUMMARY NAD+ production. ST:INSTITUTE University of South China ST:LAST_NAME Liu ST:FIRST_NAME Ke ST:ADDRESS Hongxiang street, Hunan, Hengyang, 421001, China ST:EMAIL kliu2017@163.com ST:PHONE 15358930855 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE Three-week-old SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Laboratory Animal Center of the University of South China #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 C1 C1-1 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023883-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023883-1A.raw SUBJECT_SAMPLE_FACTORS C2 C1-2 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023884-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023884-1A.raw SUBJECT_SAMPLE_FACTORS C3 C1-3 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023885-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023885-1A.raw SUBJECT_SAMPLE_FACTORS C4 C1-4 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023886-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023886-1A.raw SUBJECT_SAMPLE_FACTORS C5 C1-5 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023887-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023887-1A.raw SUBJECT_SAMPLE_FACTORS C6 C1-6 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023888-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023888-1A.raw SUBJECT_SAMPLE_FACTORS C7 C1-7 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023889-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023889-1A.raw SUBJECT_SAMPLE_FACTORS C8 C1-8 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023890-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023890-1A.raw SUBJECT_SAMPLE_FACTORS C9 C1-9 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023891-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023891-1A.raw SUBJECT_SAMPLE_FACTORS C10 C1-10 Sample source:blood | treatment:normal saline RAW_FILE_NAME(Raw file name(POS))=HFX5_CP1_FZTM240023892-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN1_FZTM240023892-1A.raw SUBJECT_SAMPLE_FACTORS M1 M1-1 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023893-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023893-1A.raw SUBJECT_SAMPLE_FACTORS M2 M1-2 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023894-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023894-1A.raw SUBJECT_SAMPLE_FACTORS M3 M1-3 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023895-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023895-1A.raw SUBJECT_SAMPLE_FACTORS M4 M1-4 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023896-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023896-1A.raw SUBJECT_SAMPLE_FACTORS M5 M1-5 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023897-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023897-1A.raw SUBJECT_SAMPLE_FACTORS M6 M1-6 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023898-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023898-1A.raw SUBJECT_SAMPLE_FACTORS M7 M1-7 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023899-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023899-1A.raw SUBJECT_SAMPLE_FACTORS M8 M1-8 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023900-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023900-1A.raw SUBJECT_SAMPLE_FACTORS M9 M1-9 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023901-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023901-1A.raw SUBJECT_SAMPLE_FACTORS M10 M1-10 Sample source:blood | treatment:HFD RAW_FILE_NAME(Raw file name(POS))=HFX5_CP2_FZTM240023902-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN2_FZTM240023902-1A.raw SUBJECT_SAMPLE_FACTORS N1 N1-1 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023913-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023913-1A.raw SUBJECT_SAMPLE_FACTORS N2 N1-2 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023914-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023914-1A.raw SUBJECT_SAMPLE_FACTORS N3 N1-3 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023915-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023915-1A.raw SUBJECT_SAMPLE_FACTORS N4 N1-4 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023916-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023916-1A.raw SUBJECT_SAMPLE_FACTORS N5 N1-5 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023917-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023917-1A.raw SUBJECT_SAMPLE_FACTORS N6 N1-6 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023918-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023918-1A.raw SUBJECT_SAMPLE_FACTORS N7 N1-7 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023919-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023919-1A.raw SUBJECT_SAMPLE_FACTORS N8 N1-8 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023920-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023920-1A.raw SUBJECT_SAMPLE_FACTORS N9 N1-9 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023921-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023921-1A.raw SUBJECT_SAMPLE_FACTORS N10 N1-10 Sample source:blood | treatment:NMN RAW_FILE_NAME(Raw file name(POS))=HFX5_CP4_FZTM240023922-1A.raw; RAW_FILE_NAME(Raw file name(NEG))=HFX5_CN4_FZTM240023922-1A.raw #COLLECTION CO:COLLECTION_SUMMARY At the end of the 19-week experimental period, the animals were anesthetized by CO:COLLECTION_SUMMARY intraperitoneal administration of 0.6 mg/kg urethane, weighed, and CO:COLLECTION_SUMMARY sacrificed.After anesthesia, blood was collected from the mice via the abdominal CO:COLLECTION_SUMMARY aorta and allowed to rest for 30 min at room temperature. Then, the samples were CO:COLLECTION_SUMMARY centrifuged at 1,200 × g for 15 min at 4°C, and the supernatants containing CO:COLLECTION_SUMMARY the serum were frozen at −20°C. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY Three-week-old male C57BL/6 mice (n = 48) were purchased from the Laboratory TR:TREATMENT_SUMMARY Animal Center of the University of South China (Hengyang, China; permit number: TR:TREATMENT_SUMMARY SYXK (Xiang) 2020-0002). The animals were maintained in a facility with a room TR:TREATMENT_SUMMARY temperature of 23 ± 2°C, a 12/12-h light/dark cycle (7 a.m./7 p.m.), and 50% TR:TREATMENT_SUMMARY to 60% relative humidity. After 1 week of adaption, the mice were randomly TR:TREATMENT_SUMMARY divided into two groups. One group (normal control, NC, n = 18) received a TR:TREATMENT_SUMMARY normal diet, while the other group (obese group, HFD, n = 30) were fed an HFD. TR:TREATMENT_SUMMARY After 12 weeks, the mice in the HFD group exhibited body weights 20% higher than TR:TREATMENT_SUMMARY those seen in the control group. The obese mice were further divided into two TR:TREATMENT_SUMMARY subgroups: the obesity model group (Obe, n = 18), which was fed the HFD and TR:TREATMENT_SUMMARY subjected to oral gavage once a day with normal saline, and the NMN intervention TR:TREATMENT_SUMMARY group (Obe+NMN, n = 12), which was fed the HFD and subjected to oral gavage once TR:TREATMENT_SUMMARY a day with 500 mg/kg/d NMN. The mice were sacrificed after 19 weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY UPLC-MS/MS serum analysis was carried out by Novogene (Beijing, China). Briefly, SP:SAMPLEPREP_SUMMARY the serum samples (100 μL) were mixed with prechilled 80% methanol by SP:SAMPLEPREP_SUMMARY vortexing, incubated on ice for 5 min, and centrifuged at 15,000 × g at 4°C SP:SAMPLEPREP_SUMMARY for 20 min. The supernatant was diluted with LC-MS–grade water to final SP:SAMPLEPREP_SUMMARY concentration of 53% methanol. The diluted samples were subsequently transferred SP:SAMPLEPREP_SUMMARY to a fresh Eppendorf tube and centrifuged at 15,000 × g at 4°C for 20 min. SP:SAMPLEPREP_SUMMARY Finally, the supernatant was injected into the LC-MS/MS system for analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Hypersil GOLD aQ (100 x 2.1 mm,1.9 μm) CH:SOLVENT_A 100% Water; 0.1% Formic acid CH:SOLVENT_B 100% Methyl alcohol CH:FLOW_GRADIENT 0 98%A-5%B; 1.5 98%A-5%B;3 15%A-85%B;10 0%A-100%B; 10.1 98%A-2%B; 11 CH:FLOW_GRADIENT 98%A-2%B;1298%A-2%B CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 40℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system (ThermoFisher, MS:MS_COMMENTS Germany) coupled with an Orbitrap Q ExactiveTM HF mass spectrometer or Orbitrap MS:MS_COMMENTS Q ExactiveTMHF-X mass spectrometer (Thermo Fisher, Germany) in Novogene Co., MS:MS_COMMENTS Ltd. (Beijing, China). Samples were injected onto a Hypersil Goldcolumn MS:MS_COMMENTS (100×2.1 mm, 1.9μm) using a 12-min linear gradient at a flow rate of 0.2 MS:MS_COMMENTS mL/min. The eluents for the positive and negative polarity modes were eluent A MS:MS_COMMENTS (0.1% FA in Water) and eluent B (Methanol). The solvent gradient was set as MS:MS_COMMENTS follows: 2% B, 1.5 min; 2-85% B, 3 min; 85-100% B, 10 min；100-2% B, 10.1 MS:MS_COMMENTS min； 2% B, 12 min. Q ExactiveTM HF mass spectrometer was operated in MS:MS_COMMENTS positive/negative polarity mode with spray voltage of 3.5 kV, capillary MS:MS_COMMENTS temperature of 320°C, sheath gas flow rate of 35 psi and aux gas flow rate of MS:MS_COMMENTS 10 L/min, S-lens RF level of 60, Aux gas heater temperature of 350°C. MS:MS_RESULTS_FILE ST004149_AN006879_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END