Animal experiments

All animal experiment conducted in this study were complied with the guidelines of the Committee on the Use and Care of Animal of the Nanjing Drum Tower Hospital, Nanjing University Medical School (Nanjing, China). Five-week-old mice were purchased from GemPharmatech Co., Ltd and housed in individually ventilated cages under specific pathogen-free conditions. The mice were provided sterile feed and maintained under a 12 h light/dark cycle, with the room temperature controlled at 24 ± 2 °C and relative humidity regulated within the range of (55 ± 5)%.

To establish a mouse model for human T2DM, 6-week-old C57BL/6 J male mice were randomly divided into two groups after a one-week acclimatization period: a CTRL group (CTRL, n = 6) and a T2DM group (HFD&STZ, n = 6). To assess the potential therapeutic effect of Sirt3-overexpression on osteoporosis in diabetic mice, the animals were further divided into four groups: Ctrl group (n = 6), Ctrl+AAV-Sirt3 group (n = 6), HFD&STZ group (n = 6), HFD&STZ + AAV-Sirt3 group (n = 6). Mice in the Ctrl and Ctrl+ AAV-Sirt3 groups were given standard feed (D12450B, Research Diet) and received a citrate buffer (HY-B1610N, MedChemExpress, Monmouth Junction, NJ, USA) injection, while those in the HFD&STZ and HFD&STZ + AAV-Sirt3 groups were fed a high-fat diet (HFD, D12492, Research Diet) for 6 weeks followed by intraperitoneal injections of 35 mg/kg STZ (Cat No.60256ES80; Yeasen, Shanghai, China) diluted using citrate buffer once daily for 3 consecutive days. Subsequently, mice in the HFD&STZ + AAV-Sirt3 and Ctrl+AAV-Sirt3 groups were administered with AAV9-delivered SIRT3 (Life-iLab, Shanghai, China), while the remaining two groups of mice received injections of a control virus (AAV-ctrl). Body weight and fasting blood glucose levels were measured weekly or biweekly. Only mice with fasting blood glucose levels exceeding 11.1 mmol/L, as measured from a tail blood sample, were verified as diabetic for further investigation. All mice were euthanized 10 weeks post-STZ injection for subsequent histological and biochemical analyses.

Cell culture

The osteoblastic cell line MC3T3-E1 (IM-M080) was purchased from IMMOCELL (Xiamen, Fujian, China) and cultured in α-MEM (A19511, HAKATA) supplemented with 10% FBS (BC-SE-FBS06C, BioChannel Biological Technology Co., Ltd) and 1% Penicillin-Streptomycin Solution (PB180120, Pricella Life Science&Technology Co., Ltd). Cell cultures were either passaged using trypsin-EDTA solution (H0516, HAKATA) or cryopreserved with a serum-free cell freezing medium (CSP042, ZQXZ-bio) with cryovial (606802, NEST Biotechnology). To mimic an in vivo hyperglycemic and hyperlipidemic condition, 200 μmol/L palmitic acid (PA, Kunchuang Technology, Xi’an, Shaanxi, China) and 25.5 mmol/L glucose (G7021, Merck) were supplemented. Upon reaching around (70–80)% confluence in culture medium, cells were replaced induction medium to stimulate osteoblast differentiation and added with or without HGPA. The induction medium was replaced every two days along with the addition of HGPA. Honokiol (10 μmol/L, HKL, T3001) was purchased from TargetMol (USA) to serve as the agonist of SIRT3. Additionally, mdivi-1 (10 μmol/L, HY-15886), Bafilomycin A1 (10 nm, Baf-A1), CCCP (10 μmol/L, HY-100941), dexamethasone (HY-14648), β-Glycerophosphate disodium salt pentahydrate (HY-D0886), and vitamin C (HY-B0166) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Isolation of primary osteoblasts

For mice receiving various interventions, the bilateral hindlimbs were collected and meticulously cleaned, removing the adjacent muscle and adipose tissue. Subsequently, the limbs were finely chopped using scissors and washed thrice with phosphate-buffered saline (PBS, SP02020500, Sperikon Life Science & Biotechnology co.,ltd). Following this, the tissue was immersed in a shaking incubator at 37 °C for 2 h in diluted type I collagenase (C917425, Macklin). The digested tissue was then suspended in complete culture medium (α-MEM) supplemented with 10% FBS. Upon reaching approximately (70–80)% confluence, cells at passage 2 were employed for subsequent experiments. The osteogenic induction process is the same as that for MC3T3-E1 cells.

Stable cell line generation

Sirt3 lentiviral vectors were purchased from PackGene Biotech. A preliminary experiment was conducted in accordance with the manufacturer’s instructions to determine the optimal multiplicity of infection (MOI) for Lv-Sirt3 transduction. Subsequently, 8 × 104 cells per well were seeded in a 12-well plate. On the following day, the appropriate volume of virus at the optimized MOI was added to the medium containing 5 μg/mL polybrene (HY-112735, MedChemExpress, Monmouth Junction, NJ, USA). The medium was replaced after 24 h and incubated for an additional 48 hours. Puromycin (5 μg/mL, HY-B1743A, MedChemExpress) was utilized to screen and establish stable transformants.

Osteogenic differentiation and mineralization

Cells were seeded into 12-well plates (Jet Biofil) at a density of 2×105 cells per well (counted with Countstar, IE1000, China) and cultured in osteogenic medium containing 10% FBS, 10 nmol/L dexamethasone, 10 mmol/L β-Glycerophosphate disodium salt pentahydrate and 50 μg/mL vitamin C. Osteogenic differentiation and mineralization were evaluated on day 14 and 21 using the BCIP/NBT Alkaline Phosphatase Color Development Kit (C3206, Beyotime) and Alizarin Red S Solution (ALIR-10001, cyagen). HGPA is continuously maintained in the induction medium to mimic the chronic progression of diabetes.

Osteoclast differentiation

The primary osteoclast was induced from bone marrow-derived monocytes with the presence of Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL, 50343-M07H, Sino Biological Inc). Bone marrow was flushed out after removing the muscles surrounding femurs in CTRL and HFD&STZ mice. Subsequently, the bone marrow was cultured in medium (L1051, BDBIO HangZhou China) containing 25 ng/mL M-CSF (CK02, Novoprotein, Shanghai, China) for 3 days. The cells were resuspneded using a cell scraper (CSC011025, Jet Biofil) and then plated. Afterward, 50 ng/mL RANKL was introduced to induce osteoclast differentiation. The culture medium was changed every two days until mature multinucleated osteoclasts formation.

Micro-CT analysis

Mouse femurs were collected and fixed in 4% paraformaldehyde (PFA, HY-Y0333, MedChemExpress) for scanning as previously described in ref. 56. Microscopic computed tomography (micro-CT) was used to evaluate bone mass using a micro-CT scanner (VivaCT80; Scanco Medical AG, Swizerland) at a voxel resolution of 15.6 μm. The ratio of bone volume to total volume (BV/TV), the number of trabecular bones (Tb.N), bone surface per tissue volume (BS/TV), and trabecular spacing (Tb.Sp) were performed to analysis bone morphology.

Hematoxylin and Eosin (H&E) and Immunohistochemical (IHC) staining

Femur samples were fixed with 4% PFA, decalcified for 28 days using EDTA (HY-Y0682, MedChemExpress), followed by dehydration and embedded in paraffin. Then embedded specimens were sectioned at a thickness of 5 μm and stained with hematoxylin and eosin (H&E, G4520, Beijing Solarbio Science & Technology Co., Ltd.) after deparaffinized and rehydrated. For IHC staining, deparaffinized and rehydrated sections were antigen-retrieved and incubated with 3% hydrogen peroxide. After primary antibody incubation, the slides were exposed to an HRP-conjugated secondary antibody (abs957, absin) and stained with 3,3’-diaminobenzidine at room temperature. The primary antibodies used were against OCN (bs0470R, Bioss USA), RUNX2 (R25634, Zen-bio), PINK1 (23274-1-AP, proteintech), PRKN (14060-1-AP, proteintech), and SIRT3 (HA722251, HUABIO).

In vivo bone formation analysis

For in vivo bone formation analysis, xylenol orange (30 mg/kg, HY-W110883, MedChemExpress, Monmouth Junction, NJ, USA) was injected on the 8th day and 2th day prior to sacrifice. Following fixation and dehydration, the femurs were embedded in polymethyl methacrylate resin. The sections at a thickness of 5 μm were generated with a microtome for hard tissue and the images were captured with Olympus FV3000 laser confocal microscope. Bone dynamic histomorphometric assessment was performed utilizing Image J software.

Immunofluorescence (IF) staining

MC3T3-E1 cells were cultured on glass bottom culture dishes (801002, NEST Biotechnology) and subjected to corresponding treatments. The cells were fixed by 4% PFA for 10 minutes and incubated with 0.1% Triton X-100 (E-IR-R122, Elabscience Biotechnology Co., Ltd). Subsequently, cells were blocked with goat serum (E-IR-R110, Elabscience Biotechnology Co., Ltd) at room temperature for 60 min, followed by overnight incubation with primary antibodies at 4 °C. The next day, secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (A-11094 and 331594, Invitrogen) were utilized, and the cell nuclei were stained with DAPI solution (HY-K1048, MedChemExpress).

For femur sections, the same procedure was applied as described for IHC staining. Finally, images were acquired using Olympus FV3000 laser confocal microscope or Leica DMi8 THUNDER Imaging Systems. The primary antibodies used for IF staining were as follows: OCN (bs0470R, Bioss USA), RUNX2 (R25634, Zen-bio), TOM20 (A19403, ABclonal, China), PRKN (66674-1, Proteintech), PINK1 (A7131, ABclonal, China), SIRT3 (HA722251, HUABIO), CTSK (bs-1611R, Bioss), FOXO3 (66428-1, Proteintech).

Western blot analysis

Following specific treatment, cell samples were lysed in RIPA lysis buffer (PL102, Beijing Genesand Biotech Co., Ltd) supplemented with phosphatase inhibitor cocktail (C0104, Beijing LABLEAD Inc), protease inhibitor cocktail (DI111; TransGen Biotech, China), and PMSF (HY-B0496, MedChemExpress) for 30 min at 4 °C. Bone samples were homogenated in liquid nitrogen post muscle removal, and then mixed with lysis buffer. Subsequently, the lysates underwent centrifugation at 12 000 g for 10 min using a high-speed microcentrifuge (D3024, DLAB Scientific Co. Ltd) to obtain protein. Mitochondrial protein from MC3T3-E1 cells was isolated with a Mitochondrial Isolation Kit (K1138, APExBIO, Houston, USA). Nuclear and cytoplasmic proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime). Protein concentrations were quantitatively determined according to the instructions provided with the BCA Protein Assay kit (PA002, Novoprotein, Shanghai, China). Then SDS-PAGE Sample Loading Buffer (452343, Sperikon Life Science & Biotechnology Co., ltd) were added and heated at 100 °C for denaturation.

The protein samples were separated by SDS-PAGE (M00666, GenScript Corporation), and transferred to PVDF membranes (Cobetter). These membranes were blocked using quick blocking buffer (P30500, New Cell & Molecular Biotech, China) for 15 min and incubated with primary antibodies overnight, followed by the secondary antibody conjugated with horseradish peroxidase. Western blotting bands were detected with enhanced chemiluminescence kit (A508, BDBIO HangZhou China). The primary antibodies used were as follows: ALP (A0514, ABclonal, China), RUNX2 (R25634, Zen-bio), COL1A1 (A1352, ABclonal, China), OPN (FNab06033, FineTest), LC3 (ET1701-65, HUABIO), PRKN (66674-1, Proteintech), PINK1 (A7131, ABclonal, China), SQSTM1 (HA721171, HUABIO), SIRT3 (HA722251, HUABIO), Anti-Acetyllysine Rabbit mAb (PTM-105RM, PTM BIO), FOXO3 (66428-1, Proteintech), ACTB (AC026, ABclonal, China), Histone H3 (68345-1, Proteintech) and VDAC1 (41776-1, Signalway Antibody).

RNA extraction and qPCR analysis

RNA samples were extracted utilizing a cell total RNA isolation kit (AN51L518, Life-iLab, China). For bone samples, RNA extraction was carried out using the standard Trizol reagent (R1000, LABLEAD Inc.). Briefly, surrounding muscles were removed from fresh bones and homogenized using liquid nitrogen. Then the samples resuspended in Trizol reagent and lysed for 30 min. Following this, chloroform was added to facilitate phase separation and the mixture was subjected to high-speed centrifugation at 12 000 g. After precipitation with isopropanol, the RNA pellet was washed with 75% ethanol and dissolved in DEPC (JDW0101, BiOligo Biotechnology Shanghai). Subsequently, cDNA synthesis was performed using the Evo M-MLV RT kit for qPCR (AG11707, ACCURATE BIOTECHNOLOGY(HUNAN) CO.,LTD, ChangSha, China). Gene expression level relative to Actb was determined using gene-specific primers (Generay Biotechnology, Shanghai, China) and SYBR Green (B110032, Sangon Biotech). The primer sequences used were detailed in Table S1.

RNA sequencing

Transcriptome sequencing was conducted for bone RNA samples obtained from the CTRL and HFD&STZ mice. Each group consisted of three independent biological replicates. The total RNA was extracted and sequenced utilizing an Illumina Novaseq platform by Seqhealth Technology Co., LTD (Wuhan, China). Subsequently, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed online in BioLadder (BioLadder.cn), with a significance level of P < 0.05 indicating a statistically significant difference.

Transmission electron microscopy

Fresh bone or cell samples were fixed in 2.5% glutaraldehyde (sigma-Aldrich, G5882) at 4 °C for 24 h, followed by three washes with PBS. The specimens were then post-fixed with 1% osmium tetroxide, dehydrated, and embedded. Subsequently, the samples were sectioned into 70–90 nm utilizing a LEICA EM UC7 ultramicrotome. These sections were stained with a combination of lead citrate and uranyl acetate. Finally, the mitochondrial ultrastructural changes were visualized using a HITACHI HT7700 transmission electron microscope.

ROS measurements

Intracellular and mitochondrial superoxide (MitoSox) were measured with CheKintonge™ reactive oxygen species (ROS) detection fluorometric assay Kit (KTB1910, Abbkine) and MitoSOX Red Mitochondrial Superoxide Indicator dye (M36009, Invitrogen™). Briefly, MC3T3-E1 cells were cultured in chamber slides (1092000, SAINING Biotechnology) and treated with or without HGPA. Then, cells were incubated with 10 μmol/L fluorescent probes for 30 minutes at 37 °C. Then the nuclei of cells were stained with DAPI. The fluorescence of the cells was visualized using Leica DMi8 THUNDER Imaging Systems.

Enzyme-Linked Immunosorbent Assay (ELISA)

Blood samples were obtained and subsequently centrifuged at 3 000 rpm for 15 min to isolate serum. The N-Propeptide of Type I Procollagen (PINP) concentration in the serum was quantified using the ELISA kit (JL20174) obtained from Jianglai biology (Shanghai, China) according to the manufacturer’s instructions. Absorbance was measured using the Varioskan LUX multifunctional microplate reader (Thermo Fisher ScientificTM).

MitoTracker staining assay and Mitochondrial membrane potential detection

Cells were seeded onto chamber slides (1092000, SAINING Biotechnology) and treated with HGPA for 48 hours. Mitochondrial morphology and mitochondrial membrane potential were assessed using Mito-Tracker Green (KGE2704, Keygen BioTECH) and Mitochondrial Membrane Potential Assay Kit (AKOP013-1, Beijing Boxbio Science & Technology Co., Ltd). According to the manufacturer’s instructions, cells were incubated with 100 nmol/L probes for 30 min at 37 °C in the dark. Subsequently, the cells were rinsed with balanced salt solution and stained with DAPI. The images were captured using an Olympus FV3000 laser confocal microscope and analyzed by Image J.

Co-immunoprecipitation and molecular docking

After cell lysis using immunoprecipitation (IP) lysis buffer (W6001, US EVERBRIGHT, Suzhou, China), the cell lysates were incubated at 4 °C overnight with an FOXO3 antibody (66428-1, Proteintech) or SIRT3 antibody (HA722251, HUABIO) for antigen-antibody interactions. Subsequently, Protein A/G magnetic beads (HY-K0202, MedChemExpress, Monmouth Junction, NJ, USA) were introduced into the antigen-antibody mixture and incubated at room temperature for 30 min to facilitate the formation of antigen-antibody-bead complexes. Following thorough washing to eliminate non-specifically bound proteins, the beads were separated, and the supernatant containing the immunoprecipitated complexes was carefully collected for analysis via SDS-PAGE, allowing for the evaluation of protein acetylation status using pan-acetyl lysine antibody (PTM-105RM, PTM BIO), FOXO3 antibody (66428-1, Proteintech), and SIRT3 antibody (HA722251, HUABIO). To validate the regulatory relationship between SIRT3 and FOXO3, we acquired the protein structural data from AlphaFold DB (created with the AlphaFold Monomer v2.0 pipeline) and performed molecular docking using the GRAMM Web Server platform. The results were then visualized using PyMOL.

Cleavage Under Targets and Tagmentation (CUT&Tag) assay

The CUT&Tag assay was conducted utilizing Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (TD904, Vazyme Biotech Co., Ltd). Briefly, cells were harvested and incubated with ConA Beads Pro at room temperature for 10 minutes. Afterwards, primary antibody against FOXO3 were added and allowed to incubate overnight at 4°C. The next day, pA/G-Tnp Pro reagent was employed to facilitate the transposon integration with the cell-beads complexes, thereby enhancing capture efficiency. The quantitative analysis of the captured DNA fragments was carried out using real-time quantitative polymerase chain reaction (qPCR). The primer sequences used were detailed in Table S2.

Statistical analysis

All quantitative data are presented as the mean ± SD values of at least three separate experiments. Graphpad Prism 9 software was used for Student’s test to compare two groups or one-way ANOVA with Tukey’s post hoc test to compare among more than two groups. Significance levels P-value < 0.05 was considered as relatively significant levels.