Type 2 diabetes (T2D) is an age-related disease. Although changes in function and proliferation of aged β cells resemble those preceding the development of diabetes, the contribution of β cell aging and senescence remains unclear. We generated a β cell senescence signature and found that insulin resistance accelerates β cell senescence leading to loss of function and cellular identity and worsening metabolic profile. Senolysis (removal of senescent cells), using either a transgenic INK-ATTAC model or oral ABT263, improved glucose metabolism and β cell function while decreasing expression of markers of aging, senescence, and senescence-associated secretory profile (SASP). Beneficial effects of senolysis were observed in an aging model as well as with insulin resistance induced both pharmacologically (S961) and physiologically (high-fat diet). Human senescent β cells also responded to senolysis, establishing the foundation for translation. These novel findings lay the framework to pursue senolysis of β cells as a preventive and alleviating strategy for T2D.

Type 2 diabetes is a disease that increases with age, and defective function of the insulin-producing pancreatic β cells has a decisive role in its development. However, treatments that target the aging component are currently lacking. This work sheds light on the role of β cell aging, or senescence, in the development of diabetes by identifying gene expression changes associated with β cell aging and demonstrating that insulin resistance directly increases the proportion of senescent β cells. Decreasing the number of aged β cells is an effective strategy to restore β cell function and identity and improve glucose metabolism. These results open novel therapeutic approaches against type 2 diabetes.

To this end, we developed a β cell senescence signature, which characterizes senescent β cells that actively secrete SASP factors. As hypothesized, metabolic stressors, such as S961-induced insulin resistance and high-fat diet (HFD), accelerated the appearance of aging and senescence markers in β cells and led to their loss of function and impaired glucose tolerance. Clearance of p16 Ink4a + cells, using the INK-ATTAC mouse, ameliorated glucose metabolism, improved insulin secretion, and decreased expression of aging, senescence, and SASP genes in islets from models of aging and insulin resistance. Additionally, an oral senolytic compound, ABT263, ameliorated hyperglycemia and improved the β cell gene expression profile in animals challenged with insulin receptor antagonist S961. Human β cells share the same biology: the load of senescent cells increased with age and diabetes, and they overexpress p16 Ink4a . Our work provides the biological and cellular framework to pursue senolysis of β cells as a potential therapy for inhibiting the progression of T2D.

We have previously found () that even in young (3–4-month-old) mice, a population of β cells express known aging markers (senescence-associated acidic β-galactosidase activity [β-Gal], p16, and p53BP1) and that this population increased with age. Aged β cells had impaired function, characterized by higher basal insulin secretion and a lower recruitment to glucose challenges. Moreover, acute insulin resistance, induced by the insulin receptor antagonist S961, resulted in expression of aging markers p16and Bambi, suggesting that insulin resistance was a driver of accelerated β cell aging. In the present study, we address the relationship between β cell aging, the development of diabetes, and whether strategies aimed at decreasing the load of aged β cells can improve cellular identity, function, and overall metabolic parameters.

With age, accumulation of dysfunctional senescent β cells likely contributes to impaired glucose tolerance and diabetes. Yet, the specific contribution of β cell aging and senescence to diabetes has received little attention, and the specific SASP profile of β cells remains to be determined.

Cellular senescence is a state in which cells cease to divide but remain metabolically active with an altered phenotype (). There are no universal markers of senescence, and the markers that exist are not consistent in every senescent tissue (). p16, a cyclin-dependent kinase inhibitor encoded by the Cdkn2a locus, has been identified as both marker and effector of β cell senescence (). An increase in p21, another effector of cellular senescence, is thought to mark the entry into early senescence leading to increased p16expression, which then maintains senescence, resulting in the expression of the senescence-associated secretory profile (SASP) (). SASP profiles differ with tissue type and can include soluble and insoluble factors (chemokines, cytokines, and ECM) that affect surrounding cells and contribute to multiple pathologies ().

Type 2 diabetes (T2D) is an age-related disease characterized by a decrease of β cell mass and function, representing a failure to compensate for the high insulin demand of insulin-resistant states (). Yet, the role of aging as it pertains to pancreatic β cells is poorly understood, and therapies that target the aging aspect of the disease are virtually non-existent. For many years, β cells can compensate for increased metabolic demands with increased insulin secretion, keeping hyperglycemia at bay. This compensation may be limited by the age-related decline in β cell proliferation seen in rodents () and humans (). This deficiency in proliferative response to increased demand may arise partly from the accumulation of senescent β cells.

Clinical importance of insulin secretion and its interaction with insulin resistance in the treatment of type 2 diabetes mellitus and its complications.

To further our understanding about how these findings translate into human β cell biology, aging, and type 2 diabetes (T2D), we analyzed islets isolated from donors of different ages with and without T2D. As with rodents, the percentage of β-Galislet cells increased in islets isolated from older donors compared to younger ones ( Figure 7 A), and this proportion seems to be increased further in islets from T2D donors, suggesting that there is a component of β cell senescence in this disease. Furthermore, we confirmed increased expression of P16 Figure 7 B) and SASP factors CCL4 and IL6 mRNA ( Figure 7 C) in the β-Galhuman subpopulation. When human islets were sorted into β-Galand β-Galcells and treated with ABT263, the β-Galsubpopulation had a significantly higher cell mortality, as reflected by propidium iodide incorporation ( Figure 7 D). To further characterize the correlation between β cell aging, senescence, and diabetes, pancreatic sections from different aged donors with and without diabetes were stained for β cell aging marker IGF1R and for DNA damage and senescence marker nuclear P53BP1. In donors younger than 40 years of age, the presence of T2D was associated with a higher intensity of IGF1R in β cells ( Figures 7 E and 7F), suggesting early aging phenomena associated with diabetes. For P53BP1 ( Figure 7 G), in non-diabetic donors, there was a direct correlation between nuclear P53BP1 and BMI ( Figure 7 H), suggesting that states of higher insulin resistance, such as those associated with obesity, correlated with higher expressions of P53BP1. Finally, when donors with high BMIs (>33) were excluded, an increase in P653BP1 intensity was observed in islets within pancreas from patients with T2D compared with those from non-diabetics ( Figure 7 I). These results are consistent with our animal models and suggest that human β cells are a potential target for the senolytic drug therapies in the context of insulin resistance and early diagnosed diabetes.

(E) Representative pictures of IGF1R-stained islets in sections of a human pancreas. Sections were stained in parallel and pictures taken in the confocal microscope under the same setting such that differences in intensity reflect differences in protein concentration.

(A) The percentage of β-Gal + cells in human islets increased as the age of the donor increased. Samples from T2D were enriched for senescent cells compared to age-matched non-diabetic donors. Linear regression analysis.

Senescent cells have upregulated anti-apoptotic pathways that conserve their presence in otherwise healthy tissues (reviewed in). Their deleterious functional effects are then amplified by their secreted SASP that can lead to impairment of neighboring cells. At least 5 senescent cell anti-apoptotic pathways have been identified in different tissues. Senolytic therapies that specifically target these pathways are a promising approach to alleviate some of the conditions associated with an increased load of senescent cells. Based on pathway analysis of our RNASeq data, β-Galβ cells had upregulation of two of these pathways: the HIF1α pathway (FDR = < 0.0001) that can be targeted with quercetin and dasatinib, and anti-apoptotic members of the BCL2 pathway, such as A1/Bfl1 (FDR < 0.0001) or Mcl1 (FDR < 0.001), that can be targeted with ABT263 (navitoclax). We tested the in vitro effects of ABT 263 on sorted β-Galand β-Galβ cells and found that ABT263 killed a significant portion of β-Galsubpopulation at a dose of 5 μM after 4 days of treatment ( Figure 6 A). Based on these results, we selected ABT263 for in vivo treatment of S961-treated (6–9-month-old) INK-ATTAC male mice. S961-induced insulin-resistant mice that were simultaneously treated with ABT263 had only a 3-fold increase in blood glucose levels, as compared to a 5-fold increase of S961-only-treated mice ( Figures 6 B and 6C). The load of senescent β cells decreased by 25%, as revealed by β-GalFACS sorted population analysis ( Figure 6 D). When analyzing gene expression, the aging index of islets from ABT263-treated mice did not change compared to the untreated insulin resistant mice, although their p16levels decreased ( Figures S6 B and S6E). ABT263 treatment decreased the SASP index of islets but had little effect on aging or β cell indices ( Figures 6 E–6G, S6 A, and S6B). The main caveat of senolytic therapies is that they can act broadly across all cells and tissues. To evaluate which of the main tissues that contributes to glucose metabolism were targeted by the ABT263 treatment, we measured p16and p21transcripts in islets, liver, white fat, and red and white muscle of treated versus untreated animals. p16expression decreased only in islets and liver ( Figure S6 E), while p21was unchanged in all tissues ( Figure S6 F). ABT263 administration was also tested in an HFD model. 5–6-month-old INK-ATTAC female mice were placed on an HFD for 12 weeks and oral ABT administered during 5-day courses every 3 weeks. Although there were no effects on fed blood glucose ( Figure 6 H), the percentage of β-Galcells, aging, and SASP indices decreased with respect to the HFD group, with no change in the β cell index ( Figures 6 I–6K, S6 C, and S6D). Their peripheral tissues had significantly decreased p16 Figure S6 G), with only red muscle having significantly decreased p21 Figure S6 H). We also tested in vitro the effects of quercetin and quercetin + dasatinib on sorted β-Galand β-Galβ cells. While quercetin alone did not have effects on senescent β cell mortality ( Figure S7 A), the combination of quercetin and dasatinib decreased the senescent cell number by 40% ( Figure S7 B). In vivo, acute insulin resistance was induced in mice using S961, and combined quercetin (50 mg/kg) and dasatinib (5 mg/kg) was given orally by gavage once per week. By the end of the two weeks, blood glucose levels decreased in the treated group ( Figures S7 C and S7D), underlying the physiological value of senolytic therapies in the treatment of diabetes. There were few if any CD45immune cells in the islets; this percentage did not change from the 0.5% control values with S961 or senolytic treatment ( Figure S7 E). These results show that oral administration of a senolytic agent during either acute or chronic insulin resistance was able to partially reverse the adverse metabolic effects as well as provide some restoration of β cell identity.

(H–L) Administration of ABT263 during 12 weeks of HFD had no effects on fed glucose levels (H) but decreased the percentage of β-Galcells (I) as well as the aging and SASP indices (J and K) and unchanged β cell index (L) compared to those animals receiving HFD without ABT263 treatment (see Figure S6 for individual values). INK-ATTAC 6–9-month-old female mice; n = 3–4 animals/group.

(B-G) When ABT263 was administered in vivo by daily gavage to INK-ATTAC mice treated with S961, circulating blood glucose levels significantly improved (B and C), the percentage of β-Galcells decreased (D) (31,684–64,940 events per data point) with no change in aging index, SASP, and β cell indices (E, F, and G) (see Figure S6 for individual values). INK-ATTAC male mice were 6–9 months old. n = 3–5 animals/group.

INK-ATTAC animals are whole-body transgenics, and B/B treatment will have effects in all tissues in which p16is expressed, raising the possibility that the improvements in glucose metabolism were due to improved insulin action in fat, liver, and/or muscle. However, the ITTs (which evaluate peripheral insulin resistance) of the treated and untreated groups did not differ but tended to normalization ( Figure S5 C). Additionally, in peripheral tissues important for glucose homeostasis (fat, liver, and white and red muscle), B/B treatment resulted in no significant changes in p16and p21mRNA ( Figures S5 D and S5E). These results underline the importance of β cell senescence in glucose homeostasis and suggest that targeting this cell population is a strategy to consider in diabetes.

Finally, HFD administered to 9-month-old INK-ATTAC mice induced significantly increased body weight and fed glucose levels ( Figures 5 A and 5B ) after 8 weeks, which were blunted by B/B homodimerizer administration in courses of 3 days followed by 14 days in between. After 12 weeks HFD, B/B-treated female INK-ATTAC mice had improved glucose tolerance ( Figures 5 C and 5D), improved β cell function as reflected by in vivo GSIS ( Figure 5 E), and improved aging and β cell indices, yet an increase in the SASP index ( Figures 5 F–5H, S5 A, and S5B), most likely due to the length of the metabolic stress and induction of irreversible late senescence. The improved genetic changes in β cells induced by B/B senolysis likely explain their improved function.

(E–H) In vivo insulin secretion parameters improved after B/B treatment (E) and although no beneficial changes were seen in aging (F) and SASP (G) indices, islets had an enhanced β cell index (H). (See Figure S5 for individual values.) INK-ATTAC female mice were 9 months old; n = 6–7 animals/group at start of the study; at isolation, 4–7 animals/group remained.p < 0.05 respect to control; +p < 0.05 with respect to HFD.

(A–D) Changes induced with HFD, such as increased body weight (A), fed hyperglycemia (B), and glucose intolerance (C and D), were blunted after B/B homodimerizer.

With the second model, acute insulin resistance was induced by S961 over two weeks in 9–14-month-old INK-ATTAC male mice. Two 3-day courses of B/B homodimerizer treatment, separated by 7 days, significantly decreased fed blood glucose levels ( Figures 4 H and 4I), islet indices for aging, and SASP expression ( Figures 4 S4 B, and S4C).

To test whether the deletion of senescent cells had beneficial effects, we tested the 3 models (aging, S961 treatment, and HFD) in INK-ATTAC mice, a whole-body FLAG-tagged transgenic that allows deletion of cells expressing p16upon administration of B/B homodimerizer (). First, we verified that senescent islet cells were deleted after administration of two 3-day courses of B/B homodimerizer (10 mg/kg) with 14 days between courses () in 1–1.9-year-old animals by measuring eGFP and Caspase 8 levels by qPCR ( Figure 4 A). At the protein level, this deletion in pancreatic islets was confirmed by staining the pancreas for the transgenic FLAG and insulin ( Figures 4 B and 4C). Quantification of FLAG staining ( Figure 4 B) showed a significantly increased proportion of FLAG-negative islets and decreased proportion of those with medium/high FLAG staining after B/B treatment. Then, in aged (1.3–1.6-year-old female) INK-ATTAC mice, treatment with B/B homodimerizer improved β cell aging and SASP indices ( Figures 4 E, 4F, and S4 A) without significant changes in glucose tolerance ( Figure 4 D). However, β cell function was improved, as seen by in vivo GSIS ( Figure 4 G), which showed decreased basal insulin levels after an overnight fast followed by a significant increase of insulin levels 15 min after a glucose load.

(J and K) Improvement of aging (J) and SASP (K) indices was also observed. 9–14-month-old INK-ATTAC male S961 (20 nM/week) treatment for 2 weeks. n = 3–4 per group; S961p < 0.05. (See Figure S4 for individual values.).

(H and I) 1.3–1.6-year-old INK-ATTAC female mice. n = 7–8 per group; ∗ p < 0.05. INK-ATTAC mice treated with insulin receptor antagonist S961 and B/B homodimerizer had an improved metabolic profile (H), as shown by the AUC of their fed glucose levels (I).

(D–G) Old INK-ATTAC mice partially improved glucose metabolism (D) and recovered β cell function (G) after treatment with B/B homodimerizer. Isolated islets had decreased expression of genes of the aging (E) and SASP (F) indices.

(B and C) Effects of B/B homodimerizer treatment on deletion of p16 Ink4a β cells were evaluated by quantification of FLAG and insulin co-staining. Pancreases from 5-month-old animals (n = 6 per group) were stained in parallel, and confocal pictures were taken under the same settings, such that differences in intensity reflect differences in protein concentration. The magnification bar represents 50 μm; ∗ p < 0.05.

(A) Evaluation of deletion protocol with B/B Homodimerizer (10 mg/kg) revealed decreased eGFP and Casp8 mRNA in INK-ATTAC islets. Treatment was two courses of 3 days with 14 days between courses to activate the transgene caspase-8 moiety and lead to cell deletion of p16 Ink4a -expressing cells in the INK-ATTAC mice. Each dot represents the islets from an individual animal.

The second model of insulin resistance used HFD. After 8-week HFD started at 8 weeks of age, body weight and fasting blood glucose levels increased, glucose tolerance deteriorated, as judged by IPGTT ( Figures 3 M–3O), and peripheral insulin resistance was modestly increased, as determined by an insulin tolerance test (ITT) ( Figure S3 E), all consistent with a higher demand on the mass and function of pancreatic β cells. Under these conditions, the percentage of β-Galcells in dispersed islets increased from 2% to 8% ( Figure 3 P); additionally, there were significant increases in the aging and SASP indices ( Figures 3 Q, 3R, and S3 B), indicating increased β cell senescence.

To study the effects of insulin resistance, we used both the insulin receptor antagonist S961 and high-fat diet (HFD). Both approaches increased the proportion of β-Galcells as well as increased the aging and SASP indices of gene expression ( Figures 3 and S3 A–S3C). First, an acute and severe model of insulin resistance was induced in mice using the insulin receptor antagonist S961 (osmotic minipump administration) that induced marked hyperglycemia and hyperinsulinemia ( Figures 3 A, 3B, 3H, and 3I). By qPCR and immunostaining, p21was significantly increased at both the mRNA and protein level ( Figures 3 F, 3G, and S3 ). Blood glucose and insulin levels were completely reversed two weeks after removal of the minipumps ( Figures 3 H and 3I). Interestingly, normalization of hyperglycemia also reversed the aging and SASP indices ( Figures 3 J, 3K, and S3 D), suggesting that cellular senescence is at least partially reversible. After the recovery, β cell-specific genes (comprising the β cell index) were also upregulated, suggesting improved cellular health of β cells.

(H–O) Two weeks after minipump excision and normalization of blood glucose (H) and insulin (I) levels, some changes induced by S961 were reversed: both the aging (J) and SASP (K) indices decreased with respect to S961 islets, with no change on β cell-related genes (L) (see Figure S3 for individual values). 7-month-old C57Bl6/J male; n = 5 per group. Eight weeks of high-fat diet (HFD) starting at 8 weeks increased body weight (M), fasting glucose (N), and induced glucose intolerance (O).

(G) Quantification of cellular P21 CIS1 staining intensity presented as mean per β cells of each islet, at least 30 islets counted per pancreas, n = 3 control, 6 S961 animals. Box and violin plot, line at median, ∗p = 0.02 by Mann-Whitney.

It is worth noting that the SASP profile differed between primary β cells ( Figure 2 B) and MIN6 cells ( Figures 2 E and 2G). This suggests that β cell senescence is a complex stepwise process (as recently suggested by) and that more research regarding the timeline and molecular mechanisms behind β cell SASP is needed. To evaluate the correlation between senescence and SASP in a pure β cell model, we knocked down p16Ink4a in MIN6 cells using siRNA. A 50% decreased p16Ink4a expression resulted in significantly decreased Il6, Il1a, Igfbp5, Lamb1, and Lamc1 mRNA and increased Cxcl2, Cxcr4, and Ccl2 mRNA ( Figure 2 H). These data suggest that these factors are downstream of p16Ink4a, although the mechanism of action behind this relationship remains to be determined.

Analysis revealed an upregulation of SASP genes in the β-Galsubpopulation ( Figure 2 A), and, as part of their SASP profile, primary β cells secreted more IL6, TNF, and CXCL1 than did non-senescent cells ( Figure 2 B). Conditioned media (CM) generated by collecting media from cultured sorted β-Galand β-Galβ cell populations was used to culture dispersed isolated islets. Cells exposed to CM from β-Galcells increased expression of p16Ink4a with no change in p21 Figure 2 C) compared with those cultured with CM from β-Galcells, suggesting that SASP from senescent primary β cells was functional. Given that β cells are not an inflammatory cell type, and there was potential participation of resident macrophages (<1%), we tested the effect of CM from senescence-induced MIN6 cells, a mouse β cell-immortalized cell line. Senescence was induced by treating these cells with 200 nM doxorubicin or 450 μM Hfor 24 h and then collecting conditioned media (CM) after 24–48 h to measure SASP protein secretion by a β cell-derived line without contamination by other cell types. Induction of senescence was confirmed by increased expression of p16and p21mRNA ( Figures 2 D and 2F); SASP factor proteins were measured in CM showing greater secretion from senescent cells ( Figures 2 E and 2G). Viability of MIN6 cells was not affected by doxorubicin but was greatly diminished by H Figure S2 ).

(H) MIN6 cells were treated with p16 Ink4a siRNA, decreasing expression by 50%. Several SASP factor mRNAs were significantly changed (red bars) compared to cells treated with Scr siRNA, suggesting their regulation in β cells is downstream of p16 Ink4a . n = 4 experiments in triplicate. Means are plotted with each point representing a single sample. p < 0.05.

(F) MIN6 cell senescence was induced by exposing them to 450 μM H 2 O 2 for 24 h; senescence was confirmed by upregulation of p16 Ink4a and p21 Cis1 mRNA.

(C) Isolated mouse islets that were cultured for 4 days in the presence of CM from β-Gal + β cells had regulation of p16 Ink4a compared to those in CM form exposed to CM from β-Gal − β cells, indicating a functional β cell SASP. CM from 5 FACS sorts were used on 2–3 separate islet isolations.

(A and B) Senescent β cells were characterized by an upregulation of SASP factors (A), some of which are significantly increased in conditioned media from β-Gal + cells (B). Each point represents a FACS sorting experiment. Mean concentrations of proteins were IL1a, 6pg/mL; TNFa, 23 pg/mL; IL6, 83 pg/mL; CCL5, 5 pg/mL; CSCL1, 22 pg/mL; CCL3, 6 pg/mL; CCL4, 4 pg/mL; and CXCL10, 2 pg/mL.

Pancreatic islets isolated from C57Bl6/J male retired breeders (7–8 months old) were dispersed into single cells and FACS sorted ( Figure S1 ) based on β-Galactosidase (β-Gal) activity as previously described () ( Figure 1 A and Figure S1 D) and gated for an enriched β cell subpopulation ( Figure 1 B). β cell enrichment was confirmed using zinc-selective indicator FluoZin-3AM that specifically labels β cells ( Figure S1 E). To evaluate the potential presence of immune cells in our FACS sorted populations, a sort for CD45revealed that about 0.5% of our population were immune cells mainly represented by resident macrophages F4/80CD11b Figures S1 F and S1G) and were distributed within β-Galand β-Galfractions ( Figures S1 H and S1I). When quantified, our FACS-sorted population was composed of 90% pure β cells and 0.5% resident macrophages ( Figure 1 C). As is characteristic for senescent cells, β-Galcells were significantly larger than β-Galcells ( Figure 1 D), and their diameter did not overlap with that of resident macrophages (>20 μm). RNA-seq analysis showed that 3,732 genes out of 15,500 were differentially regulated between β-Galand β-Galβ cell samples from the same animals. Importantly, we observed a downregulation of key hallmark β cell identity genes () ( Figure 1 E), including Insulin 1, Mafa, Nkx6.1, and Pdx1. Simultaneously, there was an upregulation of genes whose expression are usually repressed in β cell “disallowed genes” (), such as Ldha and catalase ( Figure 1 F). Specifically curated molecular signature databases () for aging ( Figure 1 G), and senescence ( Figure 1 H) genes showed an upregulation of these genes in β-Galas compared to β-Galcells. Using these data, we generated indices for assessment of β cell identity, aging, and senescence (see STAR Methods for specific genes).

(D) As described for senescent cells, β-Gal + β cells were significantly larger than β-Gal − cells. At least 100 cells were counted from 3 different fields. For RNA-seq data, there were 7 sets of paired samples, each set from islets pooled of 30 mice. Mean ± SEM; p = 4 × 10 −12

(A and B) Islets isolated from 7–8-month-old C57BL/6J male retired breeders were FACS sorted into non-senescent (β-Gal negative ) and senescent (β-Gal positive ) subpopulations (A) for RNASeq after gating for enrichment of β cells (B).

Pathway analysis of our previously published microarray data () comparing β cells from old and young mice revealed that of the 9 described pathways of aging (), the old β cells were enriched for genes related to cellular senescence (positive regulation of cell cycle, negative regulation of cell cycle, cell cycle arrest, regulation of mitotic cell cycle) and altered intercellular communication or SASP (cytokine- and chemokine-mediated signaling pathway) ( Table S1 ). Therefore, our focus to understand β cell aging was mostly centered on senescence and its SASP aspect.

Discussion

It is widely accepted that T2D has an important aging component; however, no therapies to date have been directed at this aspect. Our work demonstrates that markers of β cell aging and senescence are increased by metabolic stressors of insulin resistance, suggesting that targeting the senescent β cell population may have a therapeutic benefit.

Cis1 and Igf1r. This loss of identity in senescent β cells resembles the changes found with glucose toxicity ( Jonas et al., 1999 Jonas J.C.

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Weir G.C. Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes. By generating a novel β cell senescence signature, we showed that as they senesce, β cells downregulate expression of key genes vital to their function and identity, such as insulin, Mafa, Pdx1, and Neurod1. At the same time, the “disallowed,” or usually suppressed genes, such as Ldha, become expressed, as are genes directly related to both aging and senescence, like p21and Igf1r. This loss of identity in senescent β cells resembles the changes found with glucose toxicity ().

Cis1 followed by that of p16Ink4a ( De Cecco et al., 2019 De Cecco M.

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et al. L1 drives IFN in senescent cells and promotes age-associated inflammation. Cis1 than of p16Ink4a, suggesting that we might still be within a critical window within which senescence could be reversed if the metabolic stressor were removed. Yet, many cases of insulin resistance and type 2 diabetes are long standing; therefore, interventions aimed at decreasing the load of senescent β cells could be beneficial at various time points, as shown by our aged INK-ATTAC model. When these mice were treated with B/B homodimerizer, their glucose metabolism improved, as did their β cell function and gene profile. Understanding from an aging point of view the changes in gene expression of β cells with insulin resistance and detecting a window of reversibility open up an exciting new therapeutic opportunity to inhibit progression of diabetes. We show how β cell senescence is a dynamic process that can be accelerated by insulin resistance and be partially reversed. As shown by our S961 experiment, once the induction of insulin resistance is removed and islets allowed to recover for 2 weeks, many of the gene expression changes associated with early senescence reverted toward normal. Given that senescence is a multistep process characterized by the initial upregulation of p21followed by that of p16) and then SASP factors, our results suggest that some of these initial steps (e.g., p21increase) might be reversible. However, if the stressor is continued, the senescence program might become irreversible and late senescence SASP factors turned on, as suggested by (). Interestingly, some of our models (mainly the acute insulin resistance with S961) showed greater upregulation of p21than of p16, suggesting that we might still be within a critical window within which senescence could be reversed if the metabolic stressor were removed. Yet, many cases of insulin resistance and type 2 diabetes are long standing; therefore, interventions aimed at decreasing the load of senescent β cells could be beneficial at various time points, as shown by our aged INK-ATTAC model. When these mice were treated with B/B homodimerizer, their glucose metabolism improved, as did their β cell function and gene profile. Understanding from an aging point of view the changes in gene expression of β cells with insulin resistance and detecting a window of reversibility open up an exciting new therapeutic opportunity to inhibit progression of diabetes.

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+) was only 8%–10% in our models, SASP secretion, part of the senescent phenotype, can contribute to multiple pathologies associated with diabetes ( Coppé et al., 2010 Coppé J.P.

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Van Gool F.

Atkinson M.

Bhushan A. Targeted elimination of senescent beta cells prevents type 1 diabetes. + cells than from β-Gal− ones. We also show that these secreted factors were able to upregulate p16Ink4a in islet cells, meaning that the SASP from β cells is functional. Importantly, we saw different SASP profiles between primary rodent and human β cells and with β cell-derived MIN6 cells. This should not be surprising, since, as recently shown by ( De Cecco et al., 2019 De Cecco M.

Ito T.

Petrashen A.P.

Elias A.E.

Skvir N.J.

Criscione S.W.

Caligiana A.

Brocculi G.

Adney E.M.

Boeke J.D.

et al. L1 drives IFN in senescent cells and promotes age-associated inflammation. Although the population of senescent β cells (β-Gal) was only 8%–10% in our models, SASP secretion, part of the senescent phenotype, can contribute to multiple pathologies associated with diabetes (). Here, we have characterized a specific SASP profile of β cells and show that some of these factors are detectable in higher concentrations in the conditioned media (CM) obtained from cultured β-Galcells than from β-Galones. We also show that these secreted factors were able to upregulate p16in islet cells, meaning that the SASP from β cells is functional. Importantly, we saw different SASP profiles between primary rodent and human β cells and with β cell-derived MIN6 cells. This should not be surprising, since, as recently shown by (), senescence has a progressive development, and our experiments document cross sectionally the stage of senescence at a given point. In addition, differences may also be due to distinct experimental approaches in our two in vitro models. In primary β cells, we observed a correlation between β-Gal activity and SASP expression without any experimental intervention. In MIN6 cells, we sought to identify a causal relationship between senescence-inducing chemicals (H2O2 and doxorubicin) and SASP expression. These chemicals may have also induced other cellular changes that modified SASP expression. Further experiments should be performed to understand the whole senescence process in different models of β cells, and only then will comparisons between models and species be valid.

Chang et al., 2016 Chang J.

Wang Y.

Shao L.

Laberge R.M.

Demaria M.

Campisi J.

Janakiraman K.

Sharpless N.E.

Ding S.

Feng W.

et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Xu et al., 2018 Xu M.

Pirtskhalava T.

Farr J.N.

Weigand B.M.

Palmer A.K.

Weivoda M.M.

Inman C.L.

Ogrodnik M.B.

Hachfeld C.M.

Fraser D.G.

et al. Senolytics improve physical function and increase lifespan in old age. Ink4a+ cells in models of aging and insulin resistance (S961 and HFD) through the administration of B/B homodimerizer. In all three models, this treatment improved glucose homeostasis, β cell function, and β cell gene expression profile. Senolytic therapies, which specifically target senescent cells, have recently been shown to be beneficial to an array of age-related conditions, such as hepatic steatosis, stem cell biology, and longevity (). One of the hallmarks of senescent cells is their resistance to apoptosis, and, coupled with their secretion of SASP, they represent a cell population that can induce dysfunction and senescence in their neighboring cells. Using the INK-ATTAC transgenic mouse, we were able to specifically delete p16cells in models of aging and insulin resistance (S961 and HFD) through the administration of B/B homodimerizer. In all three models, this treatment improved glucose homeostasis, β cell function, and β cell gene expression profile.

Kirkland et al., 2017 Kirkland J.L.

Tchkonia T.

Zhu Y.

Niedernhofer L.J.

Robbins P.D. The clinical potential of senolytic drugs. Ogrodnik et al., 2017 Ogrodnik M.

Miwa S.

Tchkonia T.

Tiniakos D.

Wilson C.L.

Lahat A.

Day C.P.

Burt A.

Palmer A.

Anstee Q.M.

et al. Cellular senescence drives age-dependent hepatic steatosis. Xu et al., 2018 Xu M.

Pirtskhalava T.

Farr J.N.

Weigand B.M.

Palmer A.K.

Weivoda M.M.

Inman C.L.

Ogrodnik M.B.

Hachfeld C.M.

Fraser D.G.

et al. Senolytics improve physical function and increase lifespan in old age. + β cells and improved blood glucose levels in animals treated with S961 without affecting the proportion of immune cells. ABT 263, which has been shown to rejuvenate aged hematopoietic stem cells in mice ( Chang et al., 2016 Chang J.

Wang Y.

Shao L.

Laberge R.M.

Demaria M.

Campisi J.

Janakiraman K.

Sharpless N.E.

Ding S.

Feng W.

et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. + β cells. When administered in vivo in mice with S961 and HFD, ABT263 improved their glucose metabolic profile and β cell genetic identity and specifically reduced p16Ink4a in islets of treated animals. Even though parallel effects were shown by clearing cells with high p16Ink4a expression from INK-ATTAC mice and using senolytics to target an antiapoptotic pathway upregulated in senescence cells, it should be kept in mind that the mechanisms behind these approaches are not the same. One way to target senescent cells is through drugs that focus on upregulated antiapoptotic pathways. At least 5 anti-apoptotic pathways have been described in senescent cells (reviewed in). Our RNA-seq data indicated that at least two of these pathways HIF1α and Bcl2 upregulated. We tested drugs specific for each of these two pathways in vitro: quercetin and quercetin + dasatinib for HIF1α and ABT263 for Bcl2 pathway. In line with previously published work regarding beneficial effects of quercetin + dasatinib in hepatic steatosis (), physical function, and lifespan (), quercetin + dasatinib selectively cleared β-Galβ cells and improved blood glucose levels in animals treated with S961 without affecting the proportion of immune cells. ABT 263, which has been shown to rejuvenate aged hematopoietic stem cells in mice (), was effective in selectively reducing the percentage of β-Galβ cells. When administered in vivo in mice with S961 and HFD, ABT263 improved their glucose metabolic profile and β cell genetic identity and specifically reduced p16in islets of treated animals. Even though parallel effects were shown by clearing cells with high p16expression from INK-ATTAC mice and using senolytics to target an antiapoptotic pathway upregulated in senescence cells, it should be kept in mind that the mechanisms behind these approaches are not the same.

To further the translation potential of our studies into humans, we obtained human islets from donors of different ages and found that β cell senescence load is age dependent, and T2D seems to accelerate this process. Moreover, β-Gal+ human β cells express higher levels of P16INK4A than do the β-Gal−-sorted cells. In mice, ABT263 specifically reduced the load of p16Ink4a transcripts in islets of treated animals, which opens up the possibility that β-Gal+ human cells may respond to senolytic therapies.

Ink4a mRNA only in β cells and liver, while p21Cis1 was unchanged in all tested tissues. This should not be surprising since, as previously mentioned, several different antiapoptotic pathways can be upregulated in senescent cells, and which ones are upregulated vary from one tissue to another. Moreover, it has been shown that ABT263 causes apoptosis of senescent endothelial cells but has little effect on senescent fat cell precursors ( Zhu et al., 2015 Zhu Y.

Tchkonia T.

Pirtskhalava T.

Gower A.C.

Ding H.

Giorgadze N.

Palmer A.K.

Ikeno Y.

Hubbard G.B.

Lenburg M.

et al. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Ink4a in most peripheral tissues, suggesting that tissue specificity of senolytics might also be achieved by the duration of treatment, with some tissues being more sensitive than others. Identifying cell-specific pathways may render some tissue specificity to senolytic therapies. Moreover, the deletion of senescent cells has been shown to be beneficial for different pathologies, including hepatic steatosis, autoimmune diabetes, hematopoietic stem cells, and longevity; therefore, even if not specific, off-target effects may not represent a clinical problem. The main caveat of senolytic therapies is that they target cells and tissues indiscriminately and broadly. Our model shows that a short-term treatment with ABT263 decreased p16mRNA only in β cells and liver, while p21was unchanged in all tested tissues. This should not be surprising since, as previously mentioned, several different antiapoptotic pathways can be upregulated in senescent cells, and which ones are upregulated vary from one tissue to another. Moreover, it has been shown that ABT263 causes apoptosis of senescent endothelial cells but has little effect on senescent fat cell precursors (); therefore, it might not be expected to have as big an effect on fat tissue as other senolytics. However, in our long-term HFD model, ABT263, decreased levels of p16in most peripheral tissues, suggesting that tissue specificity of senolytics might also be achieved by the duration of treatment, with some tissues being more sensitive than others. Identifying cell-specific pathways may render some tissue specificity to senolytic therapies. Moreover, the deletion of senescent cells has been shown to be beneficial for different pathologies, including hepatic steatosis, autoimmune diabetes, hematopoietic stem cells, and longevity; therefore, even if not specific, off-target effects may not represent a clinical problem.

Gandhi et al., 2011 Gandhi L.

Camidge D.R.

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Gandara D.

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Hemken P.M.

et al. Phase I study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in patients with small-cell lung cancer and other solid tumors. Wilson et al., 2010 Wilson W.H.

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et al. Navitoclax, a targeted high-affinity inhibitor of BCL-2, in lymphoid malignancies: a phase 1 dose-escalation study of safety, pharmacokinetics, pharmacodynamics, and antitumour activity. Eisenbarth, 1986 Eisenbarth G.S. Type I diabetes mellitus. A chronic autoimmune disease. Thompson et al., 2019 Thompson P.J.

Shah A.

Ntranos V.

Van Gool F.

Atkinson M.

Bhushan A. Targeted elimination of senescent beta cells prevents type 1 diabetes. ABT263 was originally tested as a chemotherapeutic agent for its anti-neoplastic effects; however, its oncologic uses are limited by thrombocytopenia (). While its use as a senolytic agent would necessitate lower and less frequent doses to limit some of its side effects, evaluation of other senolytic compounds with greater potency, specificity, and fewer side effects is necessary. Even so, as a proof of concept, this approach is a potential new therapeutic avenue that should be further explored in diabetes. We believe that β cell senolysis might also be applicable for new onset of autoimmune type 1 diabetes (T1D) where rising blood glucose levels are likely to induce glucotoxicity and lead to loss of β cells (), a concept supported by (). That paper, based on a very aggressive model of autoimmune Type 1 diabetes, the NOD mouse, reveals the presence of a senescent β cell population. Although the pathophysiology of type 1 and type 2 diabetes is very different, both studies support the presence of a β cell senescent subpopulation capable of secreting SASP.

An important consideration in assessing β cell senescence is the potential role of immune cells infiltrating the islets and contributing to the changes in SASP factors and β cell dysfunction and even whether a reduction of these immune cells could be responsible for the beneficial effects of ABT263, which is known to cause neutropenia. To evaluate the potential participation of the immune system, we characterized the presence of immune cells in the islets from the mice we were working with: they represent only 0.5% of the total cell population, while 90% are β cells. Also, the diameters of the β-Gal+ (14 μm) and β-Gal− (12 μm) cells did not overlap with those of resident macrophages (20–80 μm). Therefore, we believe that the effects we saw were not due to immune cells but were specific to changes in the number and phenotype of the β cells.

In summary, we have established a β cell senescence signature and shown that β cell senescence plays a role in the loss of function and identity, and this process is accelerated by insulin resistance. Using both transgenic and pharmacological senolytic models, we showed that these changes can be delayed and even reversed, leading to a recovery of β cell function and identity. These pathways are preserved in human β cells, opening up a new and exciting approach to address the decline of β cell compensation in T2D.