Heterogeneity and Asynchrony of p16- and p21-related Aging in Type 2 Diabetic Mouse Tissues

Heterogeneity and Asynchrony of p16- and p21-related Aging in Type 2 Diabetic Mouse Tissues | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 19 March 2025 V1 Latest version Share on Heterogeneity and Asynchrony of p16- and p21-related Aging in Type 2 Diabetic Mouse Tissues Authors : Miss Jiayu Yan , Zimei Yi , Siyi Chen , Miss Ruowen Zhao , Jiaying Shi , Shuwen Ding , Jiayu Zhu , and Junhua Wu 0000-0001-8442-2339 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174236407.79869079/v1 297 views 160 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract This study investigates aging heterogeneity in diabetes-related tissues using the p21-3MR mouse model, focusing on p16 and p21 as markers of senescence. Type 2 diabetes, a common age-related disease, impacts multiple organs, and the study examines aging patterns in tissues such as the pancreas, kidney, heart, adipose tissue, femur, spleen, thymus, liver, and lungs. The results reveal significant aging heterogeneity and asynchrony across different organs during diabetes progression, with varying responses to anti-aging treatments. Specifically, the combination of dasatinib and quercetin demonstrated superior anti-aging effects in several tissues compared to p21 intervention alone, while p21 intervention showed distinct responses in adipose tissue and bone marrow. Immune organs displayed immunosenescence heterogeneity, and the liver and lungs showed greater sensitivity to vascular aging. This is the first study to use the p21-3MR model to explore aging heterogeneity and the differential effects of anti-aging treatments in diabetic tissues. The findings highlight the need for personalized anti-aging strategies, particularly in adipocytes and metabolic disorders. Future research should focus on understanding the mechanisms of p21 high cells in aging-related diseases, offering insights for more effective treatments. Heterogeneity and Asynchrony of p16- and p21-related Aging in Type 2 Diabetic Mouse Tissues Jiayu Yan 1 ,Zimei Yi 1 ,Siyi Chen 1 ,Ruowen Zhao 1 ,Jiaying Shi 1 ,Shuwen Ding 1 ,Jiayu Zhu 1 ,Junhua Wu 1* 1 Shanghai Engineering Research Center of Tooth Restoration and Regeneration & Tongji Research Institute of Stomatology & Department of Prosthodontics, Shanghai Tongji Stomatological Hospital and Dental School, Tongji University, Shanghai, 200072, China. * Correspondence: [email protected] ; Tel: +86-135-8587-3929； ORCID：0000-0001-8442-2339 Jiayu Yan: [email protected] Zimei Yi: [email protected] Siyi Chen: [email protected] Ruowen Zhao: [email protected] Jiaying Shi: [email protected] Shuwen Ding: [email protected] Jiayu Zhu: [email protected] Junhua Wu: [email protected] Funding information： Science and Technology Committee Foundation of Shanghai, Grant/Award Number: 23141902500; Natural Science Foundation of Shanghai, Grant/Award Number: 23ZR1469100. Abstract： This study investigates aging heterogeneity in diabetes-related tissues using the p21-3MR mouse model, focusing on p16 and p21 as markers of senescence. Type 2 diabetes, a common age-related disease, impacts multiple organs, and the study examines aging patterns in tissues such as the pancreas, kidney, heart, adipose tissue, femur, spleen, thymus, liver, and lungs. The results reveal significant aging heterogeneity and asynchrony across different organs during diabetes progression, with varying responses to anti-aging treatments. Specifically, the combination of dasatinib and quercetin demonstrated superior anti-aging effects in several tissues compared to p21 intervention alone, while p21 intervention showed distinct responses in adipose tissue and bone marrow. Immune organs displayed immunosenescence heterogeneity, and the liver and lungs showed greater sensitivity to vascular aging. This is the first study to use the p21-3MR model to explore aging heterogeneity and the differential effects of anti-aging treatments in diabetic tissues. The findings highlight the need for personalized anti-aging strategies, particularly in adipocytes and metabolic disorders. Future research should focus on understanding the mechanisms of p21 high cells in aging-related diseases, offering insights for more effective treatments. Keywords : type 2 diabetes;cellular senescence;heterogeneous aging;asynchronous aging;p21 Heterogeneity and Asynchrony of p16- and p21-related Aging in Type 2 Diabetic Mouse Tissues Abstract： This study investigates aging heterogeneity in diabetes-related tissues using the p21-3MR mouse model, focusing on p16 and p21 as markers of senescence. Type 2 diabetes, a common age-related disease, impacts multiple organs, and the study examines aging patterns in tissues such as the pancreas, kidney, heart, adipose tissue, femur, spleen, thymus, liver, and lungs. The results reveal significant aging heterogeneity and asynchrony across different organs during diabetes progression, with varying responses to anti-aging treatments. Specifically, the combination of dasatinib and quercetin demonstrated superior anti-aging effects in several tissues compared to p21 intervention alone, while p21 intervention showed distinct responses in adipose tissue and bone marrow. Immune organs displayed immunosenescence heterogeneity, and the liver and lungs showed greater sensitivity to vascular aging. This is the first study to use the p21-3MR model to explore aging heterogeneity and the differential effects of anti-aging treatments in diabetic tissues. The findings highlight the need for personalized anti-aging strategies, particularly in adipocytes and metabolic disorders. Future research should focus on understanding the mechanisms of p21 high cells in aging-related diseases, offering insights for more effective treatments. Keywords : type 2 diabetes;cellular senescence;aging heterogeneity;asynchronous aging;p21 Significance Statement: This study employs the p21-3MR mouse model to map spatiotemporal heterogeneity in cellular senescence across diabetic tissues. Analysis of 11 organs reveals distinct tissue-specific aging patterns mediated by differential p16/p21 activities. While Dasatinib and Quercetin therapy alleviates multi-organ senescence, it paradoxically aggravates beige adipose tissue aging, underscoring the need for precision interventions. Crucially, p21 high adipocytes are identified as drivers of diabetic osteoporosis via paracrine mechanisms, linking metabolic dysfunction to skeletal aging. These findings establish a framework for organ-selective senotherapies and highlight the necessity of spatial resolution in aging regulation. The work bridges systemic aging modulation with personalized therapeutic strategies, emphasizing context-dependent senescence dynamics in diabetic complications. 1.Introduction Cellular senescence is a state of stable cell cycle arrest induced by acute or chronic injury and is often closely associated with aging and inflammation-related diseases[1].The mechanisms underlying cellular senescence are complex and involve multiple biological and signaling pathways, with p16 and p21 serving as significant cell cycle inhibitory factors. At the transcriptional level, p16 and p21 act as common markers for senescent cells, playing a crucial role in the initiation and maintenance of cellular senescence by regulating cell cycle arrest. Although these markers have been extensively utilized to identify senescent cells across various tissues, their expression is not universally conserved among all senescent cells, and our current understanding of the heterogeneity of aging remains incomplete. The current understanding of cellular senescence remains somewhat incomplete, with one of the primary challenges being the absence of specific aging markers. While p16 has historically served as a significant marker in aging research, recent studies suggest that p16 may lack both sensitivity and specificity as a reliable indicator of aging[2,3]. Increasingly, research has highlighted the significant heterogeneity of cellular senescence across various tissues, underscoring the necessity for additional aging markers beyond p16, particularly p21. Our comparison of p16 and p21 reveals that their distribution within cells and tissues is heterogeneous, their regulatory mechanisms differ, and their secretion phenotypes, along with their effects on aging and lifespan, are distinct[4]. Furthermore, an increasing number of studies indicate that p16 high and p21 high represent two different cell populations[5]. However, the specific functional differences of these populations in various tissues and cell types remain incompletely understood. In recent years,increasing studies have demonstrated that cellular senescence plays a crucial role in the pathogenesis of diabetes, particularly type 2 diabetes(T2DM)[6].The aging-related protein p16 is closely linked to the enhancement of β-cell insulin secretion and the regulation of blood glucose levels 〔7,8〕 . Furthermore, the accumulation of p21 high senescent cells in adipose tissue associated with obesity is considered a significant mechanism underlying insulin resistance[5]. Research indicates that senescent islet β-cells exhibit elevated p16 expression[9], while aging adipocytes show increased levels of p21[10]. The heterogeneity of cellular senescence is a notable characteristic of the aging process in T2DM. However, there is currently a lack of simultaneous observations of critical tissue aging phenotypes in mice within the context of T2DM. To address this gap, we employed the p21-3MR mouse model to investigate the spatiotemporal distribution of p16 high and p21 high cells across multiple vital organs, with a particular focus on the expression of p16 and p21 senescence markers and their variations in different organs throughout the progression of T2DM. Our findings reveal that multiple organs exhibit significant aging heterogeneity and asynchrony during the course of T2DM, offering a novel perspective for further exploration of the pathological processes associated with aging in diabetes. Currently, all anti-aging drugs exhibit varying effects on different populations of senescent cells, and the sensitivity of these cells to anti-aging treatments, such as senolytics, also demonstrates significant variability. This observation has led to the proposal of the concept of heterogeneity of cellular senescence[11]. However, the markers of senescent cells have not yet been completely and precisely defined. In most relevant studies, senescent cells are distributed across various cell populations rather than being concentrated in specific clusters, further underscoring the heterogeneity of cellular senescence. For instance, dasatinib is particularly effective at eliminating senescent preadipocytes, while quercetin demonstrates greater efficacy in targeting endothelial cells[12].Asynchronous aging refers to the variations and imbalances in the aging process among different cell types, tissues, or individuals[13]. Studies have shown that, following glucocorticoid treatment in adult mice, adipocytes in the bone marrow initially undergo primary aging. Subsequently, these adipocytes propagate the aging process to endothelial cells and osteoblasts through the secretion of senescence-associated secretory phenotype (SASP) factors, leading to secondary aging[14].The removal of p16 high macrophages has been shown to significantly reduce liver cell damage, whereas the removal of p16 high endothelial cells may exacerbate liver damage[15]. Furthermore, regular removal of a small number of p21 high cells enhances cardiac and metabolic functions, significantly extending both the average and maximum lifespan of mice[16]. Evaluating the efficacy of various anti-aging treatments across different tissues is essential for optimizing treatment strategies. In this study, we implemented several interventions in diabetic mice and assessed the effects of two anti-aging treatments: a combination of dasatinib quercetin(DQ) and p21 administered individually. The results indicate significant differences in the effects of these two treatments across various tissues. Notably, DQ treatment demonstrates superior anti-aging effects compared to p21 alone in most tissues; however, p21 alone exhibits a more pronounced response in adipose tissue and bone marrow. Therefore, developing personalized treatment strategies tailored to the aging characteristics of different tissues may provide novel approaches to addressing aging-related diseases, such as diabetes. A deeper understanding of the heterogeneity and asynchrony of cellular senescence will be crucial for the development of effective treatments. 2.Materials and Methods 2.1.Animals Eight-week-old SPF-grade male C57BL/6 mice, weighing between 23 and 25 grams, were acquired from Hangzhou Ziyuan Experimental Technology Co., Ltd. The p21-3MR transgenic mice were developed by Shanghai Nanmo Biology Co., Ltd. This research group has been consistently breeding and maintaining these mice at the SPF Animal Experiment Center of Tongji University. In accordance with the ”Care and Use of Laboratory Animals” guidelines established by the National Institutes of Health (NIH), humane care was ensured for each animal throughout the experiments, in compliance with the specified standards. The identification results of the partially bred p21-3MR homozygous mice are illustrated in Supplementary Figure 1b.The corresponding ethical approval code is [2023]-DW-85. 2.2 Animal modeling and grouping of diabetic mice. Ninety-six WT C57BL/6 mice were randomly assigned to either a control group or a diabetes group. After a 4-week period during which the diabetic group was fed a high-fat and high-sugar diet, the mice underwent a fasting period of 6-8 hours for 5 consecutive days. Following this, they were intraperitoneally injected with 1% STZ at a mass concentration of 40 mg/kg in sodium citrate solution. Post-injection, random blood glucose levels were monitored regularly. A diabetes model was considered successfully established if the random blood glucose concentration in the tail vein was ≥16.7 mmol/L for three consecutive measurements. The control group mice were fed a standard diet and received an equivalent volume of sodium citrate buffer as the diabetic group. After successful modeling, the progression of the disease was documented, with the weight and blood glucose levels of the mice recorded at 1 week, 2 weeks, 4 weeks, and 6 weeks following the onset of diabetes. Subsequently, tissue samples, including those from the heart, liver, spleen, lung, kidney, epididymal fat, inguinal fat, femoral bone marrow, femoral bone cortex, pancreas, and thymus, were collected for histological and molecular biology analyses. 2.3 RNA extraction and qPCR detection. Total RNA was extracted from mouse tissue using the RNAiso Plus reagent (Takara Company, Japan). Peel the muscles and fascia from the surface of the mouse femur, ensuring the bone remains intact. Cut both ends of the femur, and repeatedly rinse the bone marrow into a centrifuge tube using 1 mL of RNAiso Plus reagent, thoroughly washing until the bone marrow cavity is clear and the bone appears white, ensuring complete separation of the bone marrow from the cortical bone. Subsequently, chop the cortical bone into pieces in the RNAiso Plus solution. Add small steel beads and vortex the tissues thoroughly on a tissue vortex to extract total RNA from each tissue. Reverse transcription was performed with the PrimeScript™ RT kit (Takara Company, Japan), followed by the configuration of the reaction system using SYBR Green fluorescent dye reagent. Amplification reactions and analyses were conducted with a LightCycler qPCR instrument (Roche Company, Switzerland). β-actin served as the internal reference, and the fold change of mRNA was calculated using the 2 -△△Ct method. The primer sequences for all genes utilized in this study were verified through the literature, with specific sequences provided in Additional Table 2. 2.4 Monomeric red fluorescent protein tracks senescent cells. The hard tissues of p21-3MR mice were decalcified and subsequently immersed in a 15% sucrose solution for 24 hours, followed by a 30% sucrose solution for an additional 24 hours. The soft tissues did not require decalcification and were directly placed into the sucrose solution to initiate dehydration. The embedding mold was prepared by allowing the OCT embedding agent to reach room temperature for later use. The sample was then positioned in the mold, adjusted to the appropriate angle, and placed in an ice cutter to solidify and fix the sample. The temperature in the ice cutting machine was set to -30°C, and sections were cut to a thickness of 10 microns, followed by a 5-minute stabilization period at room temperature. Prior to staining, the frozen sections were placed on a baking machine and incubated at 37°C for 5 minutes to stabilize the tissue. The target tissue was outlined with a specialized pen for immunohistochemistry and washed three times with PBS. Finally, DAPI was diluted to a ratio of 1:2000 and incubated at room temperature for 5 minutes in the dark. The slide was then sealed with an anti-fade mounting agent, and images were captured under a fluorescence microscope to observe the distribution of p21 high cells. 2.5 Immunofluorescence Staining Frozen tissue sections were baked in a baking machine for 5 minutes, fixed in 4% paraformaldehyde for 15 minutes, and infiltrated with a 0.2% Triton X-100 (Sigma, USA) solution for 20 minutes. Subsequently, the sections were treated with a 5% bovine serum albumin solution in PBS to block non-specific staining for 60 minutes. Following this, the sections were incubated overnight at 4°C with primary antibodies against p16 (1:100, affinity) and p21 (1:100, Sevier). Afterward, they were incubated with fluorescent secondary antibodies, followed by a 5-minute incubation with DAPI. Finally, the sections were examined under a fluorescence microscope, and ImageJ was used to analyze the distribution of fluorescence signals.Using ImageJ to remove background signals and select regions of interest, the pixel fluorescence intensity profiles of the red and green channels are plotted to visually demonstrate the colocalization of the two signals. 2.6 Senolytic and senostatic treatments The mice in the DQ group were orally administered 5 mg/kg of dasatinib and 50 mg/kg of quercetin, both dissolved in 10% polyethylene glycol (PEG400), for five consecutive days over a two-week period, repeated for two months. The diabetic group received only the 10% PEG400 solvent orally. Dasatinib (CDS23389), quercetin (1592409), and PEG400 (8074851000) were procured from Sigma-Aldrich. 2.7 P21-3MR mouse model combined with intraperitoneal injection of GCV. The team employed CRISPR-Cas9 technology in the initial stages to develop p21-3MR mice, which can be targeted by GCV to eliminate p21 high cells. These mice serve as a model for investigating the role of p21. A total of forty-eight 8-week-old p21-3MR mice were randomly assigned to four groups: the control group, which received a normal diet; the diabetes group, which established a diabetes model as previously described and was administered a 10% PEG solution two weeks post-successful modeling; the DQ group, which received 0.5% quercetin and 0.05% Dasatinib in a 10% PEG solution via intragastric administration two weeks after the establishment of the diabetes model, with dosages calculated based on body weight (100 µL/10 g) over a duration of 5 days within two weeks, continuing for 2 months; and the GCV group, which began intraperitoneal injections two weeks after successful diabetes modeling, with a dosage of 100 µL/10 g. Injections were administered continuously for 5 days within a week, with each injection delivering 15 mg/kg, lasting for 2 months. Changes in body weight and blood glucose levels were recorded, and samples from the heart, liver, spleen, lungs, kidneys, epididymal fat, inguinal fat, femoral bone cortex, femoral bone marrow, pancreas, and thymus were collected for histological and molecular biology analyses. 2.8 Statistical analysis. Statistical analysis of experimental data was conducted using SPSS 20.0. For continuous data that followed a normal distribution, the mean ± standard deviation (mean ± SD) was used to represent the data, and group mean differences were assessed using independent two-sample t-tests and one-way ANOVA, with a significance level set at 5% (P < 0.05). For data that did not follow a normal distribution, non-parametric analyses such as Wilcoxon rank-sum test or Kruskal-Wallis H test were employed. In multiple group comparisons, if the assumption of homogeneity of variance was met, a one-way ANOVA with a completely randomized design was used; if the assumption of homogeneity of variance was not met, the Kruskal-Wallis H test was used, followed by Bonferroni correction for post-hoc tests. All statistical results were considered statistically significant at P < 0.05. 3.Results 3.1 Aging heterogeneity and aging asynchrony in different tissues during the progression of mouse diabetes. We selected 96 wild-type (WT) mice, which were then randomly divided into control and diabetic groups. The diabetic group established a T2DM model through a high-fat diet combined with streptozotocin(STZ) injection. Prior to modeling, we ensured that the fasting blood glucose levels of all mice were approximately 8 mmol/L. Mice in the diabetic group were monitored for 2 weeks following the STZ injection. If random blood sugar levels exceeded 16.7 mmol/L for three consecutive measurements, the diabetes model was considered successfully established, and the duration of diabetes was calculated from that point. Subsequently, the diabetic group exhibited pronounced symptoms of diabetes, including polydipsia, polyphagia, and polyuria, while the control group showed no significant fluctuations in fasting blood sugar. In addition to recording body weight, blood glucose levels, and results from the oral glucose tolerance test(OGTT) (Supplement Figure 1D), we also collected tissue samples from the heart, liver, spleen, lung, kidney, epididymal fat, inguinal fat, femoral bone cortex, femoral bone marrow, pancreas, and thymus for analysis through histology and molecular biology. (1) Aging heterogeneity and asynchrony in organs with major pathological changes in diabetes. Diabetes induces pathological changes associated with aging by affecting various organs, particularly the pancreas, kidneys, and heart. These alterations typically manifest as a decline in cellular function, tissue fibrosis, structural remodeling of organs, and an upregulation of aging markers[17]. As diabetes progresses, aging-related changes accelerate the loss of organ function and exacerbate the occurrence of diabetic complications. We collected relevant tissues from diabetic mice at different stages of the disease and assessed the transcriptional expression levels of aging-related genes p16, p21, and p53. The results indicate that in tissues exhibiting severe diabetic damage, p16 is strongly correlated with aging (Figure 1-3), suggesting that glucose metabolism may be more closely related to the expression of p16. However, the spatiotemporal expression of aging markers across the three tissues is not entirely consistent. Co-staining immunofluorescence analysis of p16 and p21 revealed that p16 expression predominates in the pancreas and heart during the progression of diabetes. In the pancreas, p16 is primarily concentrated around the gland (Figure 1b-c), while in the heart, p16 expression is prominent in the muscle area, with p21 not showing significant peaks in the early and late stages (Figure 3b-c). In the kidney, high expression of p16 is observed predominantly in the early stage of prediabetes. However, as the disease progresses to six weeks, the expression of p16 decreases significantly while p21 increases markedly. Notably, p21 appears to have a strong association with the glomerular unit (Figure 2b–c). By assessing aging progression at the transcriptome level through the senescent secretory phenotype (SASP), we determined that the aging process begins earliest in the pancreas. At week 2 of diabetes, levels of IL-6 and TNF-α in the pancreas were significantly elevated compared to the control group (Figure 1a). The aging process in the kidney occurs relatively later, with marked increases in TNF-α and TGF-β levels observed during the 4th week of diabetes (Figure 2a). In contrast, the aging state of the heart lags behind, with significant increases in IL-6, TNF-α, and TGF-β typically commencing after the 4th week of diabetes (Figure 3a). A comparison of aging progression across various tissues during diabetes revealed that the aging process aligns closely with the pathological changes associated with the disease. This suggests that aging may represent a sub-health state in the progression of diabetes and could serve as a critical stage for effective intervention or even reversal of diabetes progression. (2) Aging heterogeneity and asynchrony of adipose tissue. As our understanding of adipose tissue continues to evolve, research has revealed significant heterogeneity in its cell composition and development[18]. In this study, we collected bone marrow and bone cortex samples from the femur, in addition to three distinct types of adipose tissue: epididymal fat, inguinal fat, and bone marrow tissue. We subsequently assessed the transcriptional expression levels of aging-related genes, specifically p16, p21, and p53. Our results indicated that white fat, particularly epididymal fat, exhibited a strong correlation with p21, with expression levels significantly elevated compared to the control group by the fourth week of diabetes (Figure 4a). Furthermore, bone marrow tissue, which remains incompletely characterized, also demonstrated a strong correlation with p21, while the expression pattern of p53 closely mirrored that of p21 (Figure 6a). In contrast, beige fat, represented by inguinal fat, revealed a more pronounced correlation with p16, with expression levels significantly higher than those in the control group by the second week of diabetes (Figure 5a). Combined with co-staining immunofluorescence and distribution analysis of p16 and p21, the results indicated that the number of p21 high cells was greater in epididymal fat, and the expression distribution of p21 was broader compared to that of p16 (Figure 4b-c). In contrast, p16 exhibited a wider distribution in inguinal fat; however, p21 and p16 predominantly overlapped, resulting in the presence of double-positive (p16 high p21 high ) cells (Figure 5b-c). At the epiphysis of the femur, p21 high cells appeared earlier near the growth plate, with the distribution range of p21 being greater than that of p16. Nevertheless, as diabetes progressed, the obesity phenotype in the bone marrow deteriorated, leading to a gradual accumulation of lipid droplets in the femoral epiphysis. A notable correlation was observed between p16 high p21 high cells and lipid droplets (Figure 6c-d). These findings suggest that senescent adipocytes associated with the obese phenotype in femoral bone marrow may play a crucial role in the pathogenesis of the disease during the progression of diabetes. (3) Aging heterogeneity and asynchrony of bone and bone marrow tissues. In recent years, the differences between bone and fat in the aging process have been studied in greater depth. Building on this background, we collected bone marrow and bone cortex from the femur to analyze the aging changes in these two tissues. The results indicated that bone marrow exhibited a stronger correlation with p21 and p53, showing significant differences from the control group during the first week of diabetes (Figure 6a). In contrast, the bone cortex demonstrated a more pronounced correlation with p16. By the second week of diabetes, the expression of p16 was significantly elevated compared to the control group, with an expression fold reaching as high as 70 times (Figure 6b). These findings suggest that p16 and p21 may play distinct roles in the aging processes of fat and bone, further validating the heterogeneity of aging in psoriasis. By comparing SASP transcript levels, we observed that all three adipose tissues exhibited significantly elevated expression after two weeks of diabetes progression, which was markedly higher than that observed in the control group (Figure 4a, Figure 5a, Figure 6a). In contrast, the SASP expression level in the bone cortex remained low, showing minimal differences from the control group. The highest expression fold was observed at 30 times, with most tissues demonstrating increased expression levels only after six weeks of diabetes progression (Figure 6b). These results indicate that the aging process in adipose tissue occurs significantly earlier than in the bone cortex. (4) Aging heterogeneity and asynchrony of immune-related tissues. Research on the phenomenon of immunosenescence in metabolic diseases has increasingly garnered attention[19]. To investigate aging-related changes in immune organs during the progression of diabetes, we collected thymus and spleen tissues. Transcript level analysis of aging-related genes revealed a significant increase in the expression of p53 in the thymus at 6 weeks post-diabetes onset, which exhibited a lack of synchronization with the expression of p21 (Figure 7a). In the spleen, one week after the onset of diabetes, the expression level of p16 was significantly elevated compared to the control group, showing a weak correlation with p53 (Figure 8a). Consistent with the immunofluorescence results, the senescence markers in the thymus displayed a strong correlation with p53, whereas the spleen predominantly showed a strong correlation with p16 (Figures 7b-c, 8b-c). A comparison of SASP transcript levels revealed that the expression of TNFα and TGFβ in the spleen significantly increased after one week of diabetes progression, while IL-6 exhibited significant differences at four weeks of diabetes progression (Figure 8a). In contrast, IL-6, TNFα, and TGFβ showed significant increases in the thymus at four weeks of diabetes progression (Figure 7a). These results suggest that immunosenescence displays heterogeneity and asynchrony across various immune organs. Specifically, the spleen is associated with p16, whereas the thymus is linked to p53, indicating that the spleen ages more rapidly than the thymus. (5) Aging heterogeneity and asynchrony in other major organs. Diabetes is a prevalent metabolic disorder characterized by disturbances in glucose metabolism, which are closely linked to lipid metabolism in the liver[20]. This study focused on the aging changes occurring in liver tissue. The qPCR results indicated a significant increase in the expression levels of p21 and p53 in the liver, with both proteins being upregulated synchronously (Figure 9a). Furthermore, immunofluorescence analysis revealed that p21 exhibited a broader distribution compared to p16 (Figures 9b–c). These findings, in conjunction with the previously discussed results concerning adipose tissue, suggest that p21 may play a crucial role in the aging process associated with lipid metabolism. Additionally, the analysis of SASP transcript levels demonstrated that the liver entered a state of elevated SASP expression one week after the onset of diabetes, indicating that the aging process may have commenced earlier (Figure 9a). In aging-related diseases such as chronic obstructive pulmonary disease, lung tissue exhibits a strong correlation with p21[21]. Therefore, we examined lung aging in the context of metabolic diseases. Notably, diabetes displays signs of lung aging later in the disease progression. By the sixth week of diabetes, the expression of p16 in lung tissue was significantly elevated compared to the control group (Figure 10a). Immunofluorescence results further indicated an accumulation of p16 high p21 high cells in the ciliated area surrounding the bronchus. Additionally, mRNA and fluorescence distribution analyses revealed a stronger association of p16 with lung progression in diabetes (Figure 10b-c). We hypothesize that this association may be linked to microcirculation disorders and elevated blood sugar levels induced by diabetes. 3.2 Compare the efficacy of different anti-aging treatments on diabetic mice. We utilized CRISPR-Cas9 technology to insert the exogenous gene 3M into the p21 promoter located on chromosome 6 (Supplement Figure 1a). This module is capable of targeting, detecting, imaging, and eliminating p21 high cells in vivo[22]. To successfully screen for knock-in homozygous mice, WT gene sequences (268 bp) and 3MR gene sequences (611 bp) were designed. qPCR amplification followed by gel electrophoresis analysis revealed that the PCR product from p21-3MR homozygous mouse DNA displayed a band exclusively at 611 bp. A total of 48 p21-3MR homozygous mice were identified (Supplement Figure 1b) and were randomly divided into four groups: control, diabetes, DQ, and GCV (Supplement Figure 1c). The diabetes, DQ, and GCV groups underwent diabetes modeling via a high-fat diet combined with STZ injection, with the progression of diabetes monitored following successful modeling. The diabetic group received oral administration of a 10% PEG solution, while the DQ group was administered a 10% PEG solution containing quercetin and dasatinib, and the GCV group received intraperitoneal injections of ganciclovir(GCV) to selectively eliminate p21 high cells. All treatments were conducted over a two-week period, five days a week, for a total duration of two months. This grouping scheme will facilitate a comparative analysis of the efficacy of various anti-aging treatments on diabetes-related pathologies. (1) Comparison of anti-aging effects on organs that undergo major pathological changes in diabetes. In our preliminary results, we observed that tissues such as the pancreas and kidney, which are involved in the progression of diabetes, exhibit age-related changes associated with p16. Following treatment with DQ and GCV, these tissues displayed varying responses to the interventions. Notably, both DQ and GCV treatments significantly reduced the transcription levels of p16, p21, and p53 in the pancreas. Furthermore, different disease backgrounds and senescence inducers led to alterations in the components of the SASP. Among these components, the bioactive secretions linked to p21 are referred to as p21-activated secretion phenotypes(PASP)[23,24].To investigate the effect of selectively clearing p21, we assessed the levels of the PASP factor. Transcript levels of SASP and PASP indicated that both treatments effectively inhibited the expression of the majority of SASP and PASP factors (Figures 11a and 11d). Additionally, immunofluorescence analysis revealed that while a small number of p21 high cells were present in the DQ treatment group, p16 high cells persisted in the GCV group (Figures 11b-c). Given that the pancreas is the primary organ associated with p16-related aging, we propose that DQ treatment has a more favorable effect in this organ. GCV treatment appears to selectively eliminate p21 high cells from the kidney without significantly affecting p16 levels. Transcriptional analysis revealed that, following DQ treatment, the transcription levels of p16, p21, and p53 in the kidney were significantly reduced. In contrast, GCV treatment resulted in decreased expression of p21 and p53, with no significant difference in p16 levels observed between the diabetes group and the GCV treatment group (Figure 12a). Immunofluorescence results further demonstrated that the fluorescence intensity of both p16 and p21 was significantly diminished in the DQ-treated group, whereas a significant decrease in p21 fluorescence was observed in the GCV-treated group alone (Figures 12b-c). Analysis of SASP and PASP indicated that, with the exception of Cxcl10 and IL-1β, most SASP were significantly reduced following DQ treatment, while IL-6, TGFβ, and Cxcl10 levels were decreased after GCV treatment (Figure 12d). These findings suggest that DQ treatment effectively decreases the expression of both p16 and p21, indicating a more favorable therapeutic effect in the kidney. In cardiac tissue, both DQ and GCV treatments resulted in the simultaneous downregulation of the transcription levels of p16, p21, and p53. Detection results of SASP indicated that multiple inflammation-related factors, including IL-1β, IL-8, Cxcl10, and others, were also significantly downregulated (Figure 13a). Immunofluorescence analysis further demonstrated that p16 expression was elevated in the diabetic group, with a significant decrease in p16 expression observed following both treatments (Figure 13b-c). The transcription results of PASP factors revealed a decrease in p21 expression post-treatment, and subsequently, the PASP exhibited a significant decline (Figure 13d). These findings suggest that the deletion of p21 by DQ or GCV can effectively mitigate the aging phenomenon; however, in the context of p16-related aging in the heart, the effects of DQ treatment appear to be more comprehensive. (2) Comparison of anti-aging effects of adipose tissue. We observed significant differences in three distinct types of adipose tissue following various anti-aging treatments. Notably, epididymal fat exhibited a strong association with p21-related aging. After GCV treatment, the expression levels of p21 and p53 were markedly reduced. In contrast, DQ treatment effectively decreased the transcription levels of p16, p21, and p53; however, the levels of p21 and p53 in the GCV group were significantly lower than those in the DQ group (Figure 14a). This finding indicates that the regulatory effect of DQ treatment on p21 in epididymal fat is limited. Additionally, immunofluorescence results revealed the presence of p21 high cells in the DQ group, while only a small number of p16 high cells were observed in the GCV group (Figures 14b-c). Analysis of the transcription levels of SASP and PASP factors demonstrated that both treatments could reduce the expression of most aging-related factors (Figure 14d). These results suggest that, although both treatments are effective in alleviating aging, GCV is more precise in targeting p21 in epididymal fat associated with p21-related aging. The results indicate that inguinal fat, a tissue characterized by pronounced p16-related aging, exhibits a significantly different response compared to epididymal fat. In the DQ treatment group, there were increases in the expressions of p16 and p21, along with elevated levels of SASP and PASP factors. Conversely, the GCV treatment group demonstrated a significant reduction in the expression of p21 and PASP factors, with TGFβ levels notably lower than those observed in the diabetes group (Figures 15a and 15d). Additionally, immunofluorescence results revealed an increase in the number of p16 high and p21 high cells in the DQ treatment group, while the GCV group primarily exhibited an increase in p16 high cells (Figures 15b-c). These findings suggest that DQ treatment may not be an optimal approach for mitigating the aging of inguinal fat and could potentially exacerbate aging levels. Consequently, the application of DQ may prove ineffective in addressing the aging of inguinal fat. (3) Comparison of anti-aging effects of bone and bone marrow tissues. In bone marrow tissue exhibiting pronounced p21-related aging, both the DQ and GCV groups effectively reduced the expression levels of p16, p21, and p53. However, GCV treatment demonstrated superior control over SASP compared to the DQ group, particularly regarding TGF-β, IL-1β, and Cxcl10, which were elevated in the DQ group (Figure 16a). In cortical bone associated with significant p16-related aging, both treatments successfully down-regulated p16 expression; however, p53 expression increased in the DQ group, while the GCV group exhibited a down-regulation of p53 (Figure 16b). Additionally, both methods were effective in reducing the levels of IL-6, TNF-α, and IL-1β. Nonetheless, TGF-β levels in the DQ group remained significantly different from those in the control group, and the GCV group was less effective in controlling IL-8 compared to the DQ group (Figure 16b). In the DQ group, the expression levels of IL-7 and Cxcl14 were elevated compared to those in the diabetes group, whereas the levels of the PASP factor in the GCV group were more effectively regulated (Supplement Figure 1e-f). These results suggest that DQ and GCV have distinct roles in different tissues for various types of aging, each exerting unique regulatory effects on specific factors. Immunofluorescence results indicated that the numbers of p21 high and p16 high cells were significantly elevated in the DQ group compared to the GCV group (Figure 16c-d). This finding suggests that GCV treatment is more effective in mitigating bone marrow obesity. In addition, we conducted MicroCT scans of the femurs from the four groups of mice, reconstructed the images (Figure 16e), and analyzed the bone parameters for each group. The results indicated that both the DQ and GCV groups significantly increased the bone volume fraction (BV/TV, %) (Additional Figure 1f), the number of bone trabeculae (Tb.N, 1mm) (Supplement Figure 1f), and reduced the bone size, as measured by beam spacing (Tb.Sp, mm) (Supplement Figure 1f). These findings suggest that both treatments are effective in alleviating osteoporosis and trabecular bone loss in diabetic mice. However, statistical analysis revealed no significant difference in the therapeutic effects of DQ and GCV on these bone parameters, indicating that the two treatments exhibit similar efficacy. (4) Comparison of anti-aging efficacy of immune-related tissues. In the thymus displaying pronounced p53-related senescence, both DQ and GCV treatments significantly reduce the transcription levels of p16, p21, and p53, along with SASP and PASP factors (Figures 17a and 17d). Immunofluorescence results indicate that both treatments markedly downregulate the distribution of p16 high cells (Figures 17b-c). These findings suggest that both treatment modalities exert comparable effects in ameliorating the aging state of the thymus, leading to favorable outcomes. In the spleen, there is a pronounced expression of p16-related senescence during the early stages, alongside a marked expression of p21-related senescence in the later stages. The DQ group significantly reduced the expression level of p21, while GCV simultaneously decreased the expression levels of p16, p21, and p53 (Figure 18a). Furthermore, the Cxcl10 level in the DQ group increased, whereas the levels of most SASP and PASP factors in the GCV group significantly decreased compared to the diabetes group (Figure 18d). Immunofluorescence results indicated that the DQ group retained a distribution of p16 high and p21 high cells, while the GCV group exhibited a significant reduction in the distribution of these cells (Figure 18b-c). These findings suggest that, although DQ can slow the aging process of the spleen, GCV is more effective in alleviating spleen aging by directly clearing p21 high cells. (5) Comparison of anti-aging effects on other major organs. In the liver, pronounced p21-related senescence was observed during the early stage. Both the DQ group and the GCV group significantly down-regulated the expression levels of p16, p21, p53, as well as SASP and PASP factors (Figures 19a and 19d). Additionally, immunofluorescence results indicated that both the DQ and GCV groups effectively reduced the number of p16 high and p21 high cells (Figures 19b-c). These findings suggest that both anti-aging treatment options demonstrate considerable efficacy in mitigating diabetes-induced liver metabolic aging. In lung tissue exhibiting pronounced p16-related aging, both the DQ group and the GCV group demonstrated significant reductions in the expression levels of p16, p21, and p53. With the exception of IL-6, the DQ group significantly decreased the expression of most SASP and PASP factors, while the GCV group also exhibited significant decreases in most SASP and PASP factors, with the exception of MMP9 (Fig. 20a, 20d). Immunofluorescence results indicated that both the DQ and GCV groups significantly diminished the distribution of p16 high and p21 high cells; however, a small number of p21 high cells were still observed in the DQ group (Figures 20b-c). These findings suggest that both DQ and GCV treatments can effectively mitigate the aging of lung tissue, although the DQ group may be slightly less effective in alleviating p21-related aging. 4.Discussion Our study utilized the p21-3MR mouse model to investigate the heterogeneity and asynchrony of p16 and p21 expression across various tissues during the progression of diabetes, as well as to evaluate the efficacy of different anti-aging treatments. We observed that throughout the development of diabetes, most organs exhibit signs of aging, with notable variations in the expression patterns of p16 and p21 among different tissues. Specifically, in organs primarily affected by the pathological changes associated with diabetes, such as the pancreas, kidney, and heart, p16-related aging is more prominent. The sequence of aging generally follows the pattern of organ damage, although some discrepancies were noted. For example, the kidney predominantly exhibits p16-related senescence in the early stages of diabetes, but transitions to p21-related senescence in the later stages of the disease. Upon administering DQ or GCV anti-aging treatments, we found that DQ treatment was more effective than targeting p21 alone in alleviating the aging effects observed in these tissues. Furthermore, we found that adipose tissue exhibits significant heterogeneity during aging. Specifically, white fat and bone marrow showed a strong correlation with p21, while beige fat exhibited a more prominent correlation with p16. Moreover, DQ anti-aging treatment produced markedly different effects across various types of adipose tissue. In white fat and bone marrow, DQ treatment demonstrated a relatively favorable anti-aging effect; however, in inguinal fat, DQ treatment exacerbated the aging condition. This finding highlights the need for future anti-aging treatments to be more targeted and precise, emphasizing personalized interventions based on the distinct aging characteristics of different tissues. Additionally, p16 high and p21 high cells within adipose tissue appear to represent two distinct cell populations in the context of diabetes, potentially playing different roles in the aging process. During the progression of diabetes, bone marrow obesity and osteoporosis were observed in the femur, with the emergence of p21 high adipocytes occurring at an earlier stage. We hypothesize that p21 high adipocytes may play a critical role in promoting the development of diabetes-related osteoporosis; however, this hypothesis requires further validation through additional studies. Furthermore, we noted aging heterogeneity in both cortical bone and bone marrow: cortical bone exhibited a strong association with p16, whereas bone marrow was more closely linked to p21. Although the two treatments, DQ and GCV, did not show significant differences in improving femoral bone parameters, we found that the efficacy of DQ in ameliorating bone marrow aging was notably inferior to that of GCV, which effectively cleared p21 high cells. Therefore, the role of p21 in driving bone marrow aging warrants further investigation to develop more effective anti-aging treatments. During the course of diabetes, we observed that immune organs exhibit heterogeneity in immunosenescence. The spleen is primarily associated with p16 in the early stage and p21 in the later stage, while the thymus is strongly related to p53. Notably, in the spleen, the anti-aging effect of GCV, achieved by clearing p21 high cells, was significantly superior to that of DQ. In contrast, both DQ and GCV treatments demonstrated relatively favorable effects on thymus aging. These findings suggest that more precise, targeted treatment strategies may be necessary to address the distinct aging characteristics of different immune organs. Our findings indicate that the liver is an organ closely associated with p21, likely due to its pivotal role in fat metabolism. This reinforces the idea that p21 may play a significant role in regulating lipid metabolism throughout the aging process. Additionally, both DQ and GCV have been identified as effective anti-aging treatments that can improve liver aging. Furthermore, our observations regarding the lungs were somewhat unexpected. Despite limited research on lung involvement in diabetic complications, our results demonstrate that the lungs also exhibit signs of aging during diabetes, with a strong correlation to p16. Both DQ and GCV treatments were relatively effective in ameliorating lung aging as well.Immunofluorescence analysis of the liver and lungs revealed that p16 high and p21 high cells first appeared near blood vessels. This suggests that, in the pathological state of diabetes, vascular aging may be particularly sensitive. Consequently, future interventions targeting vascular aging could represent a promising strategy for mitigating aging in diabetes. 5.Conclusion In conclusion, our study investigates the heterogeneity of diabetes-related tissue aging and the varying effects of anti-aging treatments, focusing on the aging characteristics of multiple vital organs and the roles of the p16 and p21 aging markers. Overall, different tissues exhibit distinct aging patterns during the progression of diabetes, leading to significantly different outcomes from anti-aging interventions. Notably, DQ treatment proves to be more effective than the simple intervention of p21 in most tissues, particularly in the anti-aging of adipose tissue and bone marrow, where responses differ among various fat types. In our exploration of bone marrow, we identified that p21 high adipocytes may play a key role in the development of diabetic osteoporosis, which warrants further investigation. Additionally, we observed the heterogeneity of immunosenescence within immune organs, with varying associations between the spleen and thymus and different aging markers at different stages. The effectiveness of DQ and GCV in addressing immunosenescence also varied across these organs. Moreover, the aging patterns in the liver and lungs, along with the role of vascular aging in diabetes, provide new insights into future anti-aging strategies. Specifically, targeted therapies focused on the vasculature may offer an effective approach to mitigating diabetic aging. This study represents our first use of the p21-3MR mouse model to explore diabetic aging and its associated heterogeneity. Future research should further investigate the p21-3MR model, deepening our understanding of aging heterogeneity, the role of p21 high cells, and the impact of anti-aging treatments, with an emphasis on personalized interventions, especially in adipocytes, and their mechanisms in metabolic disorders. Author Contributions: Conceptualization, J.Y. and J.W.;Methodology, S.C, R.Z., J.S. and S.D.;Validation, J.Y. and J.S.; Formal analysis, J.Y., Z.Y,. S.C. and R.Z.; Investigation, J.Y. and J.W.; Resources, J.Z.; Writing—original draft, J.Y. and Z.Y.; Writing—review & editing, J.Y,.Y.S.,R.Z. and J.Z.; Supervision, S.C. and J.W.; Project administration,J.Y.; Funding acquisition, J.W. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Science and Technology Committee Foundation of Shanghai (grant no. 23141902500) and the Natural Science Foundation of Shanghai (grant no. 23ZR1469100). Institutional Review Board Statement : The procedures performed on the mice were approved by the Institute of Animal Care and Use Committee of Tongji University (no. TJLAC-018-029). Informed Consent Statement: Not applicable. Data Availability Statement: All data included in this study are available upon request by contact with the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References 1.Wang B, Han J, Elisseeff JH, Demaria M. The senescence-associated secretory phenotype and its physiological and pathological implications. 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Keywords asynchronous aging； cellular senescence heterogeneous aging p21 type 2 diabetes Authors Affiliations Miss Jiayu Yan Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Zimei Yi Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Siyi Chen Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Miss Ruowen Zhao Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Jiaying Shi Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Shuwen Ding Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Jiayu Zhu Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Junhua Wu 0000-0001-8442-2339 [email protected] Tongji University Shanghai Engineering Research Center of Tooth Restoration and Regeneration View all articles by this author Metrics & Citations Metrics Article Usage 297 views 160 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Miss Jiayu Yan, Zimei Yi, Siyi Chen, et al. Heterogeneity and Asynchrony of p16- and p21-related Aging in Type 2 Diabetic Mouse Tissues. Authorea . 19 March 2025. DOI: https://doi.org/10.22541/au.174236407.79869079/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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