Clearance of p16Ink4a-positive cells in a mouse transgenic model does not change β-cell mass and has limited effects on their proliferative capacity

Removal of p16^Ink4a-expressing cells in non-metabolically challenged middle-aged mice had no effect on β-cell mass while proliferation inversely correlated with senescence load

INK ATTAC mice are a mouse transgenic model where p16^Ink4a cells can be specifically tracked through FLAG staining and removed upon B/B homodimerizer injection (Figure 1A) [21]. Validation of this deletion model in pancreatic islets has been previously published [19] and showed that senescent islet cells were deleted after administration of two 3-day courses of B/B homodimerizer, 14 days apart, in 8–9 months old mice. FLAG immunostaining was used as a senescence marker and treatment with B/B homodimerizer significantly increased the proportion of FLAG-negative (non-senescent) islets [19]. Herein, islets from B/B homodimerizer treated animals were isolated and compared to islets from non-treated animals. A significant decrease of transgene markers eGFP and Caspase8 were detected as well as a decrease in p16^Ink4a mRNA (Figure 1B). It is interesting to note that p16^Ink4a transcript levels decrease by 50% after B/B homodimerizer treatment whereas those of eGfp and Caspase 8 are almost undetectable. This can be due to lower expression of second and third cistrons secondary to internal in-frame AUGs. This phenomenon has been described both in Drosophila [23] and mammalian cells [24]. In this model, FLAG intensity is an indicator of cellular senescence because it is driven by the p16^Ink4a promoter. Therefore, confocal pictures of FLAG-stained islets were taken using the same settings such that differences in intensity reflected differences in protein quantity. Quantification of FLAG intensity in insulin positive islets, showed a significant decrease in pancreas from B/B homodimerizer treated animals when compared to those treated with vehicle (Figure 1C and 1D) consistent with decreased senescence. Additionally, increased apoptotic nuclei were counted after TUNEL staining of pancreatic sections from animals treated with B/B homodimerizer (Figure 1E and Supplementary Figure 1). Since, INK-ATTAC mice are whole body transgenics and to understand the expression of p16^Ink4a in other islet cell-types, analysis of scRNASeq data from mouse islets was used and showed that β-cells are the main islet-cell population expressing Cdkn2a, the gene encoding p16^Ink4a (Figure 1F) both in control and insulin resistance conditions (Supplementary Figure 2). However, other islet cell types can also express the transgene to a lower extent. These results confirm that INK-ATTAC animals are a valid model for studying the effect of removing p16^Ink4a expressing β-cells.

Figure 1. INK ATTAC mice as a model to remove p16Ink4a⁺ cells. (A) The INK ATTAC transgene is driven by the p16^Ink4a promoter and encodes a Caspase 8 moiety that upon B/B homodimerizer administration leads to specific apoptosis of this cell subpopulation and is compared to animals treated with vehicle (referred to as controls). FLAG tag and eGFP expression can be tracked to measure effectiveness of deletion and are a surrogate marker of senescence load. Diagram modified from [21]. (B) qPCR from islets of animals treated with B/B homodimerizer and compared to control, untreated animals. A significant decrease of the transgene transcripts Casp8 and eGFP and p16^Ink4a was observed after treatment with B/B. (C) Representative confocal picture of islets from B/B treated and untreated animals showing a significant decrease of FLAG staining. (D) Quantification of FLAG intensity using image analysis software (Image J) showed a significant decrease in senescence load in islets from animals treated with B/B homodimerizer; n_control = 5 animals, 290 islets analyzed; n_treated = 8 animals, 518 islets analyzed mean+/−SEM, ^*p = 0.0004 by unpaired t-test. (E) Increased number of apoptotic nuclei per field in pancreas from 8-month-old INK-ATTAC mice; n_control = 5 animals, 201 images analyzed; n_treated = 6 animals, 223 images analyzed; mean+/−SEM, ^*p = 0.02 by unpaired t-test. (F) Heatmap of islet scRNASeq expression of Cdkn2a (encodes p16^Ink4) in different islet cell types reveals enriched expression of Cdkn2a in β-cells. No significant changes of mean of (G) blood glucose levels (mg/dL), (H) body weight (g), (I) pancreatic weight (mg) of 8/9-month-old INK-ATTAC mice, n = 6 per group; and (J) Beta cell mass (mg) of 6-month-old INK-ATTAC mice, n_control = 7 and n_treated = 8.

We used male and female 6–9-month-old INK-ATTAC mice fed a chow diet to evaluate the effects of B/B homodimerizer in animals without a metabolic challenge. Analysis revealed no changes in circulating blood glucose (Figure 1G), body weight (Figure 1H), pancreatic weight (Figure 1I), or β-cell mass (Figure 1J). Transcriptional analysis of insulin-sensitive peripheral tissues (fat, liver, red and white muscle) showed no significant differences in transcription of senescence genes p16^Ink4a (encoded by Cdkn2a) (Supplementary Figure 3A) and p21^Cip1 (encoded by Cdkn1a) (Supplementary Figure 3B).

To quantify the proliferation in the same cohort of middle-aged mice, pancreatic sections were stained for BrdU and insulin. B/B homodimerizer did not have an effect on β-cell proliferation at this age (Figure 2A). Consistent with the literature [13], there was a proportion of mice with undetectable proliferation levels, 26% at 6 months and 33% at 8–9 months (Figure 2B). To further assess remnant proliferation and its relation to cellular senescence, the percentage of animals that maintained proliferative capacity and its correlation to FLAG staining were quantified. A significant direct correlation was found between the proportion of non-senescent FLAG negative islets (negative for FLAG staining) and the percentage of proliferation (positive nuclear staining of BrDU) in 6–9-month-old animals (Figure 2C) supporting the idea that higher loads of senescent cells decrease β-cell basal proliferation while maintaining glucose homeostasis.

Figure 2. Inverse correlation between cellular senescence load and proliferative capacity. (A) Overall proliferation quantification of 6–9-month-old INK-ATTAC animals, n_control = 14, and n_treated = 13; (B) Percentage of islets with proliferating cells in 6 and 8/9-month-old INK-ATTAC mice measured by the percentage of BrDU⁺ Ins⁺ cells, n_6month = 15, and n_8–9month = 12; (C) Correlation between percentage of non-senescent FLAG⁻ islets (negative for FLAG staining) and proliferation (positive nuclear staining of BrDU) in 6–9-month-old (n = 19) INK-ATTAC mice. Line of best fit is shown along with dotted lines indicating 95% confidence intervals. P-value was calculated using the null hypothesis that the slope of the best-fit line equals 0.

Improved β-cell function with removal of p16^Ink4a expressing cells in a HFD model in 1-year-old animals

In adult animals, β-cell proliferation is induced by increased metabolic demand. Therefore, the effects of removing p16^Ink4a cells in a high-fat diet (HFD) model were evaluated. One-year-old INK-ATTAC animals were maintained on a specific diet for 8 weeks in the following groups: control on a chow diet, HFD only, and HFD with four courses of B/B homodimerizer. Weekly body weight was monitored and showed a significant increase of 19% in both the HFD and HFD with B/B homodimerizer groups (Figure 3A). As previously reported [19], HFD accelerated β-cell senescence, which in this model was indicated as an increased Caspase8 (Figure 3B) and p16^Ink4a (Figure 3C) transcript in islets from animals fed a HFD. Representative confocal images of the pancreas from B/B treated animals showed a decrease in FLAG staining (Figure 3D) when compared with those treated with vehicle. HFD diet increased blood glucose by 27%, these levels were restored in the B/B treated group and comparable to chow animals (Figure 3E and 3F). Insulin tolerance test showed no significant differences among the groups (Supplementary Figure 4) even though results were different between males (Supplementary Figure 4A) and females (Supplementary Figure 4B). Fasting hyperinsulinemia was noted in the HFD group and restored after BB treatment (Figure 3G). Similarly, glucose stimulated insulin secretion (GSIS) in vivo during IPGTT, showed increased basal insulin in the HFD treated group which returned to normal after treatment with BB homodimerizer (Figure 3H). These results show that clearance of p16^Ink4a in a HFD model partially restored glucose homeostasis.

Figure 3. Specific deletion of p16^Ink4a-expressing cells improved metabolic profile. (A) Bodyweight changes compared to the beginning of the HFD. (B) HFD increased transcription of Caspase 8 by qPCR indicating acceleration of senescence in pancreatic islets. FC (fold change) to C (control). (C) HFD increased transcription of p16Ink4a by qPCR indicating acceleration of senescence in pancreatic islets. (D) Representative confocal images of HFD and HFD+ B/B homodimerizer treated islets. FC (fold change) to C (control). (E) Blood glucose percentage changes compared to the beginning of the treatment and (F) area under the curve for the three groups throughout the treatment period. (G) Fasting insulin (ng/mL) levels collected from tail blood during IPGTT (p = 0.08 by two-tailed unpaired t-test). (H) Glucose stimulated insulin secretion (GSIS) evaluated in vivo during IPGTT revealed restoration of basal insulin secretion after treatment with B/B homodimerizer. 1-year old INK-ATTAC mice; n = 8 control group, n = 8 HFD, n = 9 HFD = BB homodimerizer; males and females. ^*p < 0.05 after t-test.

Removal of p16^Ink4a expressing β-cells induced proliferation in a subset of HFD-treated adult mice

A significant correlation between the proliferation percentage and percentage of non-senescent β-cells was also seen at 12-months of age (Figure 4A) indicating that even at this age, removal of p16^Ink4a+ cells can have an impact on beta-cell proliferation.

Figure 4. Senolysis restored proliferative capacity in a subset of animals after a HFD metabolic challenge in 1-year old mice. (A) Correlation between percentage of non-senescent FLAG⁻ islets and beta-cell proliferation in 12-month-old animals. Line of best fit is shown along with dotted lines indicating 95% confidence intervals. P-value was calculated using the null hypothesis that the slope of the best-fit line equals 0. (B) Pancreatic weight of the groups; (C) beta-cell mass calculated in (mg). (D) Beta-cell proliferation calculated by %BrDU⁺ Ins⁺ cells. Mean+/− SEM; significance calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons. (E) Comparison between responders and non-responders to BB/homodimerizer treatment in the HFD group. AUC for IPGTT before treatment. (F) Blood glucose percentage changes compared to the beginning of the treatment and AUC (G) between responders and non-responders.

There were no significant differences in pancreatic weight in different treatment groups (Figure 4B). Systematic imaging of pancreatic sections revealed no significant changes in β-cell mass amongst groups (Figure 4C) and overall, clearance of p16^Ink4a expressing cells did not change the rate of β-cell proliferation. However, there was a subset of animals treated with B/B homodimerizer where BrdU incorporation was significantly increased and we termed this group “responders” which represented 44% (Figure 4D) of the total treated population. In the remainder 56% of the animals, proliferation did not increase in response to p16^Ink4a clearance (Figure 4D). Further analysis comparing the non-responders to the responders revealed that the latter had better glucose clearance in the initial IPGTT (Figure 4E) and maintained overall lower blood glucose levels than non-responders throughout the experiment (Figure 4F and 4G). However, no differences were found between the two groups in the following parameters: gender, initial ITT, weight gain, fed or fasting insulin and fasting blood glucose (Supplementary Figure 5). These results suggest that responders had better β-cell function at the beginning of the experiment but further experiments are required to fully understand these differences.

β-cell proliferation in a pharmacological model of insulin resistance

Insulin receptor antagonist S961 was used as an additional model to induce acute insulin resistance in 1.5-year-old male and female mice. S961 induced hyperglycemia (Figure 5A) and hyperinsulinemia (Figure 5B). S961 increased β-cell senescence as seen by an increase in Caspase8 transcript (Figure 5C). Proliferation was significantly induced by S961 even in older animals (Figure 5D, Supplementary Figure 6A) however this proliferative rate was not further increased by deletion if p16^Ink4a cells (Figure 5D, Supplementary Figure 6B) and no changes in beta cell mass were found after treatment (Figure 5E). Removal of p16^Ink4a expressing cells in this group worsened blood glucose levels (Figure 5A) indicating that the functional reserve was adversely affected by the removal of this cell population.

Figure 5. Senolysis in an acute insulin resistance model, S961, did not affect beta-cell proliferative capacity. (A) Area under the curve of blood glucose levels throughout the treatment period, n_control = 8, n_S961 = 3, n_S961+BB = 5. (B) Fed plasma insulin (ng/mL) levels in control, S961, and S961 + BB; significance calculated by repeated unpaired t-test. (C) qPCR of islets from S961 treated animals increased Caspase 8 transcription consistent with accelerated senescence. (D) Proliferation of beta cells (%) in 8–19-month-old mice. (E) Beta cell mass (mg) 18–19 month-old INK ATTAC mice, male and female. Mean +/− SEM, significance calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons.

The targeted p16^Ink4a subpopulation is non-proliferative and non-SASP producing

To further understand the lack of effects of the removal of p16^Ink4a cells in β-cell proliferation, scRNASeq analysis was performed in previously reported β-cells pooled from control and S961 treated animals [25]. t-SNE analysis revealed β-cell subpopulations that were divided into 6 clusters (Figure 6A). Analysis was concentrated in 4 of these subpopulations (Figure 6B). Two senescent subpopulations: p16Ink4a⁺, p21Cip1⁺, and two non-senescent subpopulations: p16Ink4a⁻/p21Cip1⁻ A and p16Ink4a⁻/p21Cip1⁻ B. In the INK-ATTAC model, only the p16Ink4a⁺ cells were removed (Figure 1B) while p21Cip1⁺ remained unchanged (Figure 6K). scRNASeq allowed further analysis of the proliferative capacity and SASP transcription of these specific cell populations. As previously described, p16Ink4a⁺ cells were non-proliferating (Figure 6C and 6D) whereas p21Cip1⁺ and p16Ink4a⁻/p21Cip1⁻ cells upregulated of proliferative genes (Figure 6C, 6D). We believe that the upregulation of proliferation genes in p21Cip1⁺ represents a population of growth arrest during early senescence [26, 27]. This is further supported by significant upregulation of Cdk inhibitors (Figure 6G and 6H) in this subpopulation. Other cyclins and Id were significantly upregulated in the p16Ink4a⁻/p21Cip1⁻ B subpopulation (Figure 6E and 6F). scRNASeq data was further inquired for the transcriptome of SASP factors known to be upregulated in β-cells (Figure 6I and 6J). These changes were confirmed by performing qPCR on islets from BB homodimerizer treated animals. The main SASP-producing β-cell subpopulation was the p21Cip1⁺ which persisted in the islets from mice treated with BB (Figure 6K).

Figure 6. p16^Ink4a-expressing cells do not proliferate or secrete SASP compared to other senescent cells. (A) tSNE of beta-cell clusters based on Ins2 expression and four subpopulations highlighted: p16^Ink4a+, p21^Cip1+ (senescent) and p16^Ink4a−/p21^Cip1− A and p16^Ink4a−/p21^Cip1− B (non-senescent). (B) Heat map showing beta cell senescence genes. (C, D) Heat map and violin plot showing beta-cell proliferation genes. (E, F). Heat map and violin plot showing beta-cell expression of cyclins and Id. (G, H) Heat map and violin plot of Cdk-inhibitors; (I, J) Heat map and violin plot of SASP genes. (K) qPCR of islets treated with B/B homodimerizer show no changes in the transcript of p21Cip1 and several transcription factors in the INK ATTAC model. Significance calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons.

These results highlight the existence of different senescence subpopulations of β-cells with varying proliferative and SASP producing characteristics which are differentially targeted in this transgenic model. In this particular case, the removal of p16^Ink4a+ cells did not result in the removal of SASP-producing subpopulations which might account for a lack of effects upon proliferative capacity.

In summary, a transgenic model that targets p16^Ink4+ senescent cells, did not alter β-cell mass and increased proliferation levels only in a subset of mice in response to HFD. This is potentially due to remaining senescent cells which are negative for p16^Ink4a and whose local SASP production continues to impair the proliferative capacity of the remaining cells. Additionally, a direct correlation between β-cell proliferative capacity and non-senescent islet cells was found.

Animals

All experiments were conducted at Joslin Diabetes Center with approval of its Animal Care and Use Committee. Mice were kept in a conventional facility in a 12-hour light/dark cycle with water and food ad libitum and a temperature between 22.2–22.7°C. When specified, a high-fat diet (HFD) 60kcal% fat (Research Diets; NJ, USA) was used for the specified amount of time. C57Bl6/J mice (Jackson Laboratory; ME, USA) were used for some experiments as specified. Breeding pairs of INK-ATTAC mice were a gift from Dr. Jan van Deursen [21] and all the animals used came from our colony. Middle-aged animals were ages 6–9 months with 15 animals 6-month-old (n_female = 7 and n_male = 8) and 12 animals 8–9-month-old (n_female = 6 and n_male = 6). 25 one-year-old animals were used for HFD (n_female = 13 and n_male = 12). 9 animals were used for S961 ages 18–19 months (n_female = 2 and n_male = 7). Animals of different ages were used because age is one of the main factors determining both beta-cell proliferation and senescence load. By analyzing animals of different ages, it is possible to evaluate whether the effects of p16^Ink4a removal are dependent on chronological age.

Assessment of glucose homeostasis

Bodyweight and morning fed glucose levels were monitored weekly. Blood glucose values were measured using a glucometer (Contour; NJ, USA) on blood from tail snip. For intraperitoneal glucose tolerance tests, blood samples for glucose levels from mice fasted for 6 hours were collected at 0, 15, 30, 60, 90, and 120 min after intraperitoneal injection of 10% glucose solution (Sigma Aldrich; MO, USA; 0.02 mL/g body weight). Insulin was measured from serum collected time of sacrifice using an insulin ELISA kit (Mercodia; NC, USA). For insulin tolerance tests, mice were fasted for 4 hours, insulin (Humulin R, Eli Lilly; IN, USA; 1 unit/g body weight) was injected intraperitoneally, and blood glucose was measured at 0, 15, 30, and 60 min.

Senolytic treatment

The deletion protocol of p16^Ink4a-expressing cells for INK-ATTAC mice consisted of the administration of 3-day courses of B/B homodimerizer (Takara Bio; CA, USA; 10 mg/kg) repeated every 14 days to activate the caspase-8 moiety. Mice were treated with an intraperitoneal injection of vehicle (Ethanol: polyethylene glycol 400: Tween 2% at 2:5:43) or B/B homodimerizer (in ethanol: polyethylene glycol 400: Tween 2% at 2:5:43).

S961 treatment

S961 was a generous gift from Dr. Lauge Schaffer (Novo Nordisk; Denmark) [39]. Vehicle (PBS) or 20 nmol S961 was loaded into an Alzet osmotic pump and surgically implanted subcutaneously in the back of the anesthetized mice [40] and changed weekly for a total of two weeks.

Pancreas isolation for quantification of mass and proliferation

Mice were injected with 10 mg/mL BrdU (Sigma Aldrich; MO, USA; 10 uL/g body weight) intraperitoneally, 6 hours before sacrifice. Under anesthesia, the pancreas was excised, weighed, and fixed in 4% (para)-formaldehyde (PFA) for 2 hours and embedded in paraffin for sectioning and immunostaining.

Immunostaining and morphometric evaluation

Paraffin sections were deparaffinized with xylene and ethanol gradients, washed with PBS, permeabilized with Triton-X 0.3%, antigen retrieval was completed with heated citric acid for all stains and blocked with normal donkey serum. After washing with PBS and 2% lamb serum, slides were incubated overnight at 4°C with the primary antibody (Table 1). This was followed by one wash with PBS + 2% lamb serum and incubations for 1hr with secondary antibodies (Table 1). The slides were mounted with Fluoroshield + DAPI for nuclear localization (Sigma Aldrich; MO, USA).

For quantification, islet images were captured systematically covering the whole section in confocal mode on a Zeiss LSM 710 microscope. For mass quantification, the entire section was pictured using the tile-scan system and images were quantified using ImageJ (https://imagej.net/ij/index.html) and Adobe Photoshop (https://www.adobe.com/products/photoshop.html). Beta-cell mass was calculated by multiplying the relative area of beta cells by the pancreatic weight. For all other stains, islets were pictured, coded, and read blindly. For BrdU, 589–3295 cells were counted from at least 10 islets per animal. Quantification of FLAG staining was done using ImageJ and selecting the islet area through insulin staining.

TUNEL stain and quantification

TUNEL staining was done using TUNEL Assay Kit-HRP-DAB (ab206386) from Abcam following their protocols. Positive staining, indicating apoptotic nuclei, was determined by a dark brown color (Supplementary Figure 1). Quantification was done using a brightfield microscope and Ocular^® Scientific Image Acquisition Software. For quantification, systematic alternating pictures were taken at 20× magnification of the whole tissue and positive nuclei were manually counted.

Quantitative real-time PCR

RNA was extracted from cells using the RNEasy Plus Mini Kit (QIAGEN; Germany); SuperScript reverse transcriptase (Invitrogen; MA, USA) was used to reverse transcribe RNA and generate cDNA for quantitative PCRs (Table 2). To measure gene expression levels, we used Fast SYBR green (ThermoFisher; MA, USA) and ΔCT values to βActin were calculated.

Single-cell RNA-seq

Single-cell RNA-seq data presented in this article is an analysis of pooled beta-cells from islets from C57BL6/J mice treated with PBS and S961. Beta cells were identified as having high Ins2 expression and raw data was previously published [25] and deposited under accession number GSE149984. Briefly, islets from animals with S961 or PBS pumps (described above) were isolated from mice for scRNA-seq, cultured overnight, and dispersed. Transcriptomic analysis was performed using the 10× Genomics Chromium Single Cell Gene Expression Assay core at Brigham and Women's Hospital. The Illumina NextSeq500 was used for sequencing, and the two libraries were pooled evenly on one lane. Data analysis was performed by the Bioinformatics and Biostatistics Core at Joslin Diabetes Center. Raw sequencing data were demultiplexed, aligned to the mouse genome and UMIcollapsed using CellRanger [41]. The inclusion criteria were: UMI >500, detected genes >1000, and mitochondrial genes < 20%. Data were analyzed using R. Deconvolution of size factors from cell pools, estimation of technical noise, and denoised Principal Component Analysis (PCA) were done using scran [42, 43]. t-Distributed Stochastic Neighbor Embedding (t-SNE) plots were made using scatter [44]. Cells were clustered into putative subpopulations using a shared-nearest-neighbor graph constructed from the PCA coordinates, and the clusters were found using a spin-glass algorithm [45, 46]. High Ins2 expression was used to identify beta cell clusters. Differential gene expression was assessed using linear modeling with limma [47].

Quantification and statistical analysis

Data are shown as mean ± SEM. For statistical analysis, unpaired Student’s t-tests were used to compare two groups and one-way ANOVA followed by post hoc test for more than two groups. A normality test was performed and when not passed non-parametric statistics were run. A p-value ≤ 0.05 was considered significant. Prism 9.0 software by GraphPad (https://www.graphpad.com/scientific-software/prism/) was used for graphs and statistical analysis (significance and distribution). Animals were assigned to either control, intervention, or treatment groups to have equal age and gender distribution among all groups. The B/B homodimerizer treatment was completely composed of animals positive for the INK ATTAC transgene while the intervention and control groups had a mixture of positive and negative animals. No differences have been shown between animals with and without the transgene. Animals were excluded from the analysis if they became sick or developed physical anomalies.

Data availability

scRNASeq data reported in this article can be obtained with the accession number GSE149984.