Abstract Adipose tissue senescence is a precursor to organismal aging and understanding adipose remodelling contributes to discovering novel anti-aging targets. Glutathione peroxidase 3 (GPx3), a critical endogenous antioxidant enzyme, is diminished in the subcutaneous adipose tissue (sWAT) with white adipose expansion. Based on the active role of the antioxidant system in counteracting aging, we investigated the involvement of GPx3 in adipose senescence. We determined that knockdown of GPx3 in adipose tissue by adeno-associated viruses impaired mitochondrial function in mice, increased susceptibility to obesity, and exacerbated adipose tissue senescence. Impairment of GPx3 may cause mitochondrial dysfunction through inner mitochondrial membrane disruption. Adipose reshaping management (cold stimulation and intermittent diet) counteracted the aging of tissues, with an increase in GPx3 expression. Overall metabolic improvement induced by cold stimulation was partially attenuated when GPx3 was depleted. GPx3 may be involved in adipose browning by interacting with UCP1, and GPx3 may be a limiting factor for intracellular reactive oxygen species (ROS) accumulation during stem cell browning. Collectively, these findings emphasise the importance of restoring the imbalanced redox state in adipose tissue to counteract aging and that GPx3 may be a potential target for maintaining mitochondrial homeostasis and longevity. Keywords: Glutathione peroxidase 3, Adipose tissue, Mitochondrial, Anti-Senescence Highlights * • GPx3 deregulation by abnormal fat expansion may exacerbate adipose tissue senescence. * • Adipose-specific GPx3 deficiency causes metabolic abnormalities due to mitochondrial derangement. * • GPx3 deficiency partially restricts cold-induced adipose remodelling and anti-aging effect. * • GPx3 may affect adipocyte browning by interacting with UCP1. __________________________________________________________________ Abbreviations AUC Area under the curve CS Cold Stimulation DEG Differential Expressed Genes FBS Fetal Bovine Serum GO Gene Ontology GPx3 Glutathione Peroxidase 3 GSH Glutathione GSSG Oxidized Glutathione GTT Glucose tolerance test H[2]O[2] Hydrogen Peroxide HDL-C High-Density Lipoprotein Cholesterol H&E Hematoxylin-eosin HFD High-Fat Diet HOMA-IR Homeostatic Model Assessment for Insulin Resistance ITT Insulin tolerance test KEGG Kyoto Encyclopedia of The Genome LC-MS Liquid Chromatography-Mass Spectrometry LDL-C Low-Density Lipoprotein Cholesterol MDA Malondialdehyde NEFA Non-Esterified Fatty Acids rAAV Recombinant Adeno-Associated Virus S1P Sphingosine 1-Phosphate SA-β-gal β-galactosidase SOD Superoxide dismutase SVF Vascular Stromal Fractions sWAT Subcutaneous Adipose Tissue T-CHO Total Cholesterol TEM Transmission Electron Microscopy TG Triglycerides [39]Open in a new tab 1. Introduction Aging is an inevitable global upsurge, with an increasing population enjoying longer life expectancies. This has become a major demand and added to the economic burden of healthcare [[40]1,[41]2]. Over the past decade, aging has been intensively studied, and a multitude of aging hallmarks have been revealed, including metabolic decline [[42]3], mitochondrial disorders, and cellular senescence [[43]4]. Attention is drawn to the fact that adipose tissue is one of the most sensitive organs in the ageing process, with alterations in a wide range of biological and physiological processes that can affect the overall health of the organism [[44]5]. However, the mechanisms underlying adipose tissue senescence and the causes of accelerated systemic senescence remain poorly understood. Understanding the physiological changes that occur during adipose senescence may provide novel strategies to combat aging. Adipose senescence is characterised by fat redistribution and dysfunction [[45]6], including adipose hypertrophy, brown/beige fat loss, and reduced adipose progenitor/stem cell function, and is highly similar to obesity [[46]7]. The overexpansion of white adipose tissue may be an early risk factor for tissue aging. Metabolic and molecular reorganisation are activated in adipocytes, culminating in a hypertrophic and hypersecretory phenotype that accelerates ageing [[47]8]. Conversely, adipose remodelling strategies, such as cold stimulation and intermittent fasting [[48]9], favour mitochondrial function and mitochondrial reactive oxygen species ([mt]ROS)-mediated signalling with anti-aging potential [[49][10], [50][11], [51][12]], in which the activation of insulin and SIRT1 may be involved. A growing consensus exists that adipose browning, induced by different stresses such as a cold environment and intermittent calorie-restricted diet, represents an adaptive response to counteract exogenous or endogenous changes [[52]13]. In conjunction with the oxidative damage (free radical) theory of aging, which suggests the activation of endogenous antioxidant defences to prolong lifespan [[53]14], a possible hypothesis is that fat browning may ameliorate aging by orchestrating the antioxidant system. Evidence has shown that elevated antioxidant capacity protects against metabolic abnormalities in obesity and age-related diseases by activating SIRT1 and maintaining mitochondrial function [[54]15]. In addition, some of the browning-associated factors (such as UCP1 and PGC1α) are also correlated with antioxidant systems [[55]16]. In this study, we induced adipocyte browning and explored its anti-aging effects. Under normal conditions, excess superoxide during adipose expansion activates antioxidant systems such as superoxide dismutase (SOD), the glutathione reductase system, and mitophagy to maintain redox homeostasis [[56]17]. However, a decline in the antioxidant defense system leads to an imbalance in oxidative homeostasis, ultimately triggering the onset of aging. As the fat mass expands, white adipose tissue becomes hypoxic, exacerbating oxidative stress, which is thought to underlie the development of inflammation and cellular dysfunction. Under hypoxic conditions, glycolytic pathways are significantly upregulated in adipocytes, resulting in high levels of lactate production and overexpressing HIF-1α, which promotes insulin resistance. Alternatively, a decrease in substrate flux through lipid and oxidative metabolism, which is indicated by a decrease in PPARγ and PGC-1α, has been reported [[57]18]. However, randomised clinical trials on the antiaging effects of antioxidant supplements lack efficacy in numerous cases [[58]19]. This may be related to the lack of reliability and specificity of oxidative stress indicators; therefore, the identification of biomarkers in clinical samples for monitoring oxidative homeostasis is helpful for anti-aging studies. Glutathione peroxidase 3 (GPx3) is a vital antioxidant enzyme that is widely found in the plasma and other body fluids, especially in adipose tissue, and degrades various hydrogen peroxides through the catalysis of glutathione (GSH) conversion to oxidized glutathione (GSSG) [[59]20,[60]21]. One clinical study of adipose tissue pointed out that GPx3 expression in subcutaneous white adipose tissue (sWAT) was significantly lower in obese than in lean people and was even lower in insulin-resistant obese individuals [[61]22]. GPx3 is engaged in the regulation of insulin receptor expression in pre-adipocytes as a potential novel regulator of insulin sensitivity [[62]23]. Conversely, weight loss induced by bariatric surgery results in a significant reduction in serum GPx3 concentration [[63]22]. The dysregulation of GPx3 in obese individuals is typically accompanied by increased inflammatory signalling and oxidative stress [[64]24,[65]25], which are factors that exacerbate aging. Therefore, we investigated the role of GPx3 in adipose senescence to reveal the possible anti-aging mechanisms. Moreover, adipose browning was induced to further demonstrate the antiaging effects of GPx3. 2. Materials and methods 2.1. Mice Male C57/BL mice (20–22g) were purchased from Weitong Lihua Experimental Animal Technology Co., Ltd (Beijing, China). The experimental animals were housed in a specific pathogen-free environment at the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine (SHUTCM), providing 12h light/dark cycles and free diets. Mice were acclimatized for one week before the experiment. All animal experiments follow the requirements of the SHUTCM Laboratory Animal Ethics Committee. (Ethics No. PZSHUTCM2303030001). Adoption of an ad libitum high-fat diet (HFD) to induce obesity model in mice. 60 % fat supply HFD (D12492, Research diet) was changed at 3-day intervals during the modeling period, with regular recordings of mouse body weights. Cold stimulation (CS) and intermittent dietary induction were performed to observe the effects of the two weight loss measures on HFD-induced obese mice. The mice in the CS group were first subjected to one week of 18 °C cold acclimatization before being given 4 °C cold exposure [[66]26]. The mice in the intermittent diet group were then subjected to a cyclic cycle of 24 h ad libitum feeding and 24 h fasting, with free access to water [[67]27]. Recombinant adeno-associated virus (rAAV) was utilized to specifically knock down GPx3 expression in adipose tissue by subcutaneous site-specific injection. Briefly, in order to achieve the packageing of rAAV and replace its entire genomic genes, we produced AAV vectors based on the AAV 293T cell line in vitro and additionally supplied the required components for rAAV, which mainly included the packageing plasmid, assistant plasmid, and Helper plasmid. The plasmid backbone carries the AdipoQ promoter to act specifically on lipids, and GPx3 knockdown was achieved by inserting the mir30-shGPX3 sequence into the vector (AdipoQ-AAV8-shGPX3). After sufficient rAAV was produced from the cell supernatant, the cells were lysed and purified by ultracentrifugation before freezing at −80 °C. Adipose tissues were transfected with knockout GPx3 by subcutaneous targeted injection following the end of mouse acclimatization. Peer control groups were given injections of AdipoQ-AAV8-shVeh. 2.2. Serum biochemistry index assay Prior to the experimental endpoint, mice were fasted for 6h and subjected to tail-tip blood sampling for fasting blood glucose measurement using a glucometer. Measurement of blood glucose at different time points to reflect glucose tolerance and insulin tolerance in mice after gavage of insulin (0.75 U/kg) and glucose (1 g/kg) solutions, respectively [[68]28]. After anesthesia, mice were subjected to blood sampling and centrifuged to separate the serum following resting. Total Cholesterol (T-CHO), Triglycerides (TG), Non-esterified fatty acids (NEFA), High-density lipoprotein cholesterol (HDL-C), and Low-density lipoprotein cholesterol (LDL-C) were measured by using commercial kits (Nanjing Jiancheng, China) as described. Mouse serum insulin concentration was measured by ELISA, and HOMA-IR was calculated by comparing the measured values with the corresponding mouse blood glucose [[69]29]. 2.3. Malondialdehyde (MDA) and hydrogen peroxide (H[2]O[2]) assays After mice were sacrificed, the subcutaneous adipose tissue was rapidly isolated and then frozen. MDA and H[2]O[2] levels in tissues were measured by specific commercialized kits (Cat. S0131 M for MDA and Cat. S0038 for H[2]O[2]) from Beyotime. All tissues used in the assay were weighed and applied to the final concentration calibration. 2.4. Measurement of total GPx and SOD enzymes Antioxidant enzyme (GPX and SOD) activities were measured individually with specific commercial kits (Cat. S0101S for SOD and Cat. S0058 for GPX) from Beyotime. Briefly, the level of GPx enzyme activity was responded to by the reduction in NADPH during the GPx-catalysed production of GSSG from GSH. The disproportionation of superoxide anions was utilized for the detection of SOD by the WST-8 method of reaction. Furthermore, commercial ELISA kits were used to detect GPx3 concentration in serum and tissues. 3. Method detail 3.1. Gene Ontology (GO) and KEGG pathway enrichment analysis The mRNA profiles of adipose tissue from obese mice ([70]GSE24637) and the gene annotation platform of [71]GPL1261 were searched from the Gene Expression Omnibus (GEO) database ([72]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24637). Based on R Studio, differentially expressed genes (DEGs) were detected by applying the LIMMA algorithm according to exon-modeled fragments per kilobase per million mapped fragments (FPKM) data. In this case, genes with an adjusted P value of <0.05 and |log 2 fold change (FC)| >1.0 in obese mice were selected as obesity-related genes. Pathway enrichment analysis was performed for differential expressed genes (DEG), using the “clusterProfiler” packages to identify hub maps for selected Gene Ontology (GO) and Kyoto Encyclopedia of the Genome (KEGG) pathway enrichment analysis. 3.2. Real-time PCR analysis Total RNA was extracted from tissue or cells using TRIzol reagent (9108, Takara). Isolated RNA was measured for purity and concentration using a NanoDrop spectrophotometer, followed by cDNA synthesis using a reverse transcriptase kit (RR037A, Takara). The Real-Time PCR analysis was conducted using SYBR Green (QPK-201CH, TOYOB), of which were analyzed using the 2-^ΔΔCT method. The gene expressions were normalized to β-actin expression. Fold change values were calculated for genes expressed in experimental versus control conditions. Primer sequences used are listed in [73]Table S1. 3.3. Western blotting analysis Tissue or cell samples were lysed by RIPA buffer (Cat. WB0102 from Wellbi) for protein quantification and the protein structure was reverted with loading buffer. The adjusted concentration of the samples was added to SDS-PAGE gels for electrophoresis and followed by transferring the proteins to PVDF membranes. After sealing the membrane with 5 % BSA solution, incubation with the primary antibodies (overnight at 4 °C) and secondary antibodies (2h at room temperature) was performed sequentially. Finally, the ECL visualizer was added dropwise to the membrane and visualized by enhanced chemiluminescence. Protein bands were analyzed with Image J software and normalized to the expression of β-actin or β-tubulin. 3.4. Histological staining After anesthesia, the adipose and liver tissue were fixed in 4 % paraformaldehyde for 48 h. Tissues were paraffin-embedded and cut into 5 μm sections for hematoxylin-eosin staining (Cat. 0105 M from Beyotime). Whereas senescent β-galactosidase-stained tissues were not paraffin-embedded and were washed several times with PBS and stained as described in the commercial kit (Cat. C0602 from Beyotime). 3.5. Immunofluorescence staining Fat pads were removed and fixed in 4 % paraformaldehyde for 48 h. Tissues were paraffin-embedded and cut into 5 μm sections for immunofluorescence staining (IF). sWAT samples were immunofluorescence staining to detect GPx3 (primary antibody: Rabbit Anti GPx-3, abcam, CAT: ab256470, dilution 1:500; secondary antibody: Donkey anti-rabbit Alexa Fluor 488, Invitrogen, CAT: A21206, dilution 1:500), Ucp1 (primary antibody: Mouse anti-UCP1, R&D systems, CAT: MAB6158, dilution 1:100; secondary antibody: Donkey anti-mouse Alexa Fluor 555, Invitrogen, CAT: [74]A31570, dilution 1:500), Ucp2 (primary antibody: Mouse anti-UCP2, Santa cruz, CAT: sc-390189, dilution 1:100; secondary antibody: Donkey anti-mouse Alexa Fluor 555, Invitrogen, CAT: [75]A31570, dilution 1:500). For cell staining, 4 % paraformaldehyde was used to fix the cells for 20 min to cell slides. Pre-fixed cell slides were washed in 1 % FBS-PBS. Sections were incubated overnight at 4 °C with primary antibody, fully washed, and then incubated with secondary antibody for 1h at room temperature. All slides were covered with Antifade mounting medium containing DAPI. Stained sections were imaged using OLYMPUS IX83. The exposure time of each channel was kept consistent for different samples. The images were subsequently analyzed using Fiji image processing software. 3.6. Multiple immunohistochemistry Pre-fixed adipose tissue were paraffin-embedded and cut into 5 μm sections for multiple immunohistochemistry (mIHC). Briefly, the sections were incubated successively with primary and secondary antibodies after antigen repair. The staining is performed by TSA signal amplifying dye and heated for re-antigen repair to remove the existing antibody binding. The primary antibodies of GPx3 and Ucp1 are used in a manner consistent with that in the IF. Rabbit anti-Tom 20 (CST, CAT: 42406, dilution 1:200) were used to mark mitochondrial. Stained sections were imaged using nikon csu-w1 sora confocal microscopy, and the XY resolving power of nikon csu-w1 sora confocal microscopy is approx. 120 nm. The exposure time of each channel was kept consistent for different samples. The images were subsequently analyzed using Fiji image processing software. 3.7. Transmission electron microscopy (TEM) specimen preparation and analysis After mouse execution, adipose tissue was rapidly isolated and immediately cut into cubes (∼1 mm^3) on cold with a clean scalpel. Samples were transferred to a fixative (2.5 % glutaraldehyde and 0.1 M sodium phosphate, pH 7.4) and fixed at 4 °C for 24 h. Subsequent samples were dehydrated with graded alcohol and fixed in 1 % OsO[4] solution for 1 h before embedding in epoxy resin. Specimens were made into ultrathin sections (50 nm) using an ultramicrotome (Leica, 705902) stained with uranyl acetate and lead citrate, and then observed under TEM (FEI Tecnai G2 spirit). 3.8. Tandem mass tags (TMT)-labeled liquid chromatography-mass spectrometry (LC-MS) detection The sWAT in the shVeh-CS and shGpx3-CS groups were conducted TMT-based proteomics to elucidate the role of adipose GPx3 in browning. Briefly, tissue samples are lysed to extract proteins and then lyophilized. The lyophilized samples were re-suspended in 100 mM tetraethylammonium bromide and transferred to new tubes, acetonitrile was added and 10 μl of TMT reagent was added to each sample and incubated for 1 h for labeling. Hydroxylamine was added to each sample and incubated for 15 min to terminate the reaction and then lyophilized for subsequent LC-MS detection. 3.9. Off-target lipid metabolome Serum from HFD-fed shVeh and shGPx3 groups of mice was collected and detected by Q-TOF mass spectrometry after sample pre-treatment in order to analyze lipid metabolite changes. Mass spectrometry was performed using Agilent 6530B Q-TOF mass spectrometry in positive ion scan mode with the following parameters set for the electrospray ionization source: Mass Range (m/z): 50–1700; Acquisition Rate/Time: 3.00 spectra/sec; Gas Temp: 350 °C; Drying Gas: 11 L/min; Nebuilzer: 45 psi; Sheath Gas Temp: 350 °C; Sheath Gas Flow: 11 L/min; VCap: 4000 V; Nozzle Voltage: 500 V; Fragmentor: 140 V; Skimmer: 65 V, CE: 0–40 V. Data acquisition was performed using a Masshunter. Data acquisition was performed using Masshunter 8.0 software with a mass error of ≤20 ppm. To visualize the raw data, an analysis software (MS-Dial, version 4.24 [76]https://prime.psc.riken.jp/compms/msdial/main.html) was used. The calculated ions were constructed from the MS/MS spectra of the standard and detected internal compounds, where isomers were further distinguished by the abundance ratio of the MS/MS spectra. Using the MS-Finder, additional candidate ions were identified by searching Scifinder, HMDB, LipidMAPS and other databases. 3.10. Cell culture and treatment The multipotent stem cell C3H/10T1/2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 10 % fetal bovine serum (FBS) and 1 % antibiotic–antimycotic. This cell could be induced to differentiate to form beige adipocytes after stimulation by a mixed cocktail method. Briefly, after the cells had grown all over the culture disc, the induction medium containing indomethacin, dexamethasone, rosiglitazone, 3,3′,5-Triiodo-l-thyronine, 3-Isobutyl-1-methylxanthine, and insulin was replaced to induce cell differentiation for two days. Additional insulin, rosiglitazone, and 3,3′,5-Triiodo-l-thyronine were added to the medium to maintain cell differentiation status for six days [[77]30]. During induction, the state of cell differentiation was observed under the microscope. The siGPx3 was added to the cells into transfection overnight after incubation with Lipofectamine 3000 and Opti-MEM mix. Peer controls were given siNC. 3.11. Ribonucleic acid sequencing (RNA-seq) analysis RNA was extracted from cells using TRIzol reagent, and RNA samples from each set of triplicates were subjected to RNA sequencing and analysis. Purified RNA was quantified using the Qubit RNA wide range assay and RNA quality was assessed using an RNA nano-chip on an Agilent 2100 Bioanalyzer. The rRNA-removed RNA was converted to cDNA and a final sequence-ready library using the NEBNext Ultra II RNA Library Prep Kit. The final libraries were sequenced on a NextSeq 500 (Illumina) using paired 40 bp reads. 3.12. Flow cytometry analysis Cells were initially seeded in 12-well plates and after cell adhesion, siNC and siGPx3 were transfected separately. Cells were stained for 30 min at 4 °C, with antibodies including Mito-Tracker Red CMXRos (C1035, Beyotime), Bodipy 493/503 (C2053S, Beyotime), UCP1 Alexa Fluor® 647-conjugated antibody (IC6158R, R&D Systems), then washed and resuspended in FACS buffer. Finally, cells were analyzed immediately on a Beckman CytoFLEX LX flow cytometer for data acquisition. 3.13. Statistical analysis All data were expressed as the mean ± SEM, and statistical analysis was performed using GraphPad Prism software 10.0. Differences between the two groups were analyzed using an unpaired Student's t-test, and multiple comparisons were assessed using a one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. p < 0.05 was considered a significant difference and is labeled as ∗, while p < 0.01 is labeled as ∗∗ and p < 0.001 is labeled as ∗∗∗. In addition, no significant differences were labeled as “ns”. 4. Results 4.1. A weakened antioxidant system exacerbated adipose tissue senescence, especially GPx3 To elucidate the changes in the antioxidant system involved in accelerated tissue senescence caused by adipose expansion, we performed a combination of adipose Gene Expression Omnibus (GEO) database analyses and a variable-time high-hat diet (HFD)-fed mouse model. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that obesity affects “longevity regulatory pathways”, in addition to the insulin resistance and adipocytokine model pathways, which are closely associated with aging ([78]Fig. 1A). The aggravation of senescence by adipose expansion may occur through multiple pathways, such as upregulation of lipid synthesis, decline of the mitochondrial matrix, and attenuation of GTP binding ([79]Fig. 1B). We detected oxidative homeostasis in the adipose tissue of obese mice, in which the HFD group was fed a high-fat diet for 8 weeks and the prolonged HFD (P-HFD) group for 16 weeks ([80]Fig. 1C). Accompanying the HFD-induced fat mass surge ([81]Fig. 1D), oxidative contingency damage was significantly elevated in sWAT, which may be closely related to the downregulation of antioxidant enzymes (SOD and GPx) ([82]Fig. 1E). Moreover, as the HFD feeding cycle increased, fat mass increased, along with a further increase in MDA and a decrease in antioxidant enzyme profiles in sWAT. This suggests that adipose tissue expansion is typically coupled with an intra-tissue oxidative imbalance. Fig. 1. [83]Fig. 1 [84]Open in a new tab Bioinformatics analysis and experimental evaluation of GPx3 in obese mice; (A) KEGG pathway and (B) GO enrichment of DEG based on [85]GSE24637; (C) Body weight and (D) fat mass, where the HFD group was fed with high-fat diet for 8 weeks and the prolonged HFD (P-HFD) group for 16 weeks; (E) Malondialdehyde (MDA) concentration, total SOD and total GPx enzyme activity assays in sWAT; (F) Gene expression ([86]GSE24637) shown as a volcano plot, where down-regulated genes in the obese group are labeled in blue and up-regulated genes are labeled in red; (G) GPx3 concentration in sWAT and (H) serum; (I) Correlation analysis between body weight and serum GPx3 levels; (J) Gpx3 mRNA expression in Swat (fold change of Lean); (K) Senescence and antioxidant-related gene mRNA expressions in sWAT. Relative expression of target genes for β-actin (fold change of Lean); (L) β-galactosidase staining of sWAT to assess cellular senescence; (M) Ageing-related protein expressions in sWAT. (For interpretation of the references to