Senolytics: Potential for Alleviating Diabetes and Its Complications.

Therapeutics that target cellular senescence, including novel "senolytic" compounds, hold significant promise for treating or preventing obesity-induced metabolic dysfunction, type 2 diabetes, and the multiple complications of diabetes and obesity. Senolytics selectively clear senescent cells, which accumulate with aging and obesity and represent a fundamental mechanism of aging that contributes to metabolic dysfunction and diabetes pathogenesis. In addition to improving metabolic function, targeting senescent cells holds promise as a preventive strategy to reduce the incidence and severity of diabetes complications. The intermittent administration schedule used for senolytic therapy may confer benefits in terms of improving adherence and limiting adverse effects. It is necessary to design effective clinical trials that will safely translate discoveries from preclinical models into human studies that may pave the way for a novel therapeutic class for treating obesity, diabetes, and their complications. In this review, we outline what is known regarding the role of cellular senescence in the pathogenesis of type 2 diabetes and its complications, present evidence from preclinical models that targeting cellular senescence is beneficial, review senolytic drugs, and outline the features of clinical trials investigating the role of targeting senescent cells for diabetes.

Targeting fundamental aging mechanisms such as cellular senescence has broad potential to prevent, treat, or alleviate multiple age-related conditions simultaneously rather than individually. This concept is termed the geoscience hypothesis. Diabetes itself is a major risk factor for premature development of age-related conditions, including cognitive impairment, cardiovascular disease, renal dysfunction, and others. Current therapeutic options focus mainly on glycemic control as a preventive measure and do not target end-organ damage once present. Therefore, novel therapeutics are needed to target diabetes and its complications. Senescent cells accumulate with obesity, atherosclerosis, and aging. Senescent cells, which appear to play a role in development of diabetes and its associated conditions, are a promising therapeutic target based on mounting preclinical data. Senolytic, or senescence targeting, drugs are under development and are being investigated in early clinical trials across multiple diseases. Senolytics hold particular promise for obesity-related metabolic dysfunction and diabetes. Well-designed trials are needed to investigate senolytic agents in diabetes and its complications.

Cellular Senescence in Obesity, Aging, and Diabetes: Features and Consequences

Cellular senescence is a cell fate that entails cell cycle arrest and occurs in response to a variety of stressors including metabolic signals (including hyperglycemia, saturated lipids, and reactive oxygen species), DNA damage, oncogene activation, and telomere shortening. Some senescent cells become enlarged, in part due to increased protein synthesis, and undergo changes in gene expression to produce the senescence-associated secretory phenotype, or SASP, which includes inflammatory factors, matrix remodeling enzymes, chemokines, cytokines, bioactive lipids (prostenoids, bradykines, ceramides), and nucleotides (including microRNAs and immune-modulatory mitochondrial DNA) (1-3). Aging and obesity, the major risk factors for type 2 diabetes, are both associated with increased burden of senescent cells, which accumulate in many tissues of the body (4-6). Despite representing a small percentage of cells in any particular tissue, senescent cells have been implicated in a wide variety of diseases and organ dysfunctions, as well as reduced physical activity and early mortality (7-9).

Cellular senescence is thought to play a role in development of insulin resistance and diabetic complications; however, the diabetic state can also cause formation of additional senescent cells, for example, through exposure to high glucose or lipid levels, which can themselves cause cellular senescence (10, 11). Once senescent cells form, the SASP can spread senescence in a paracrine or endocrine manner to previously normal local or distant cells (2, 9, 12-14). Senescent cells possess the ability to evade apoptosis through upregulation of antiapoptotic pathways, including the PI3K/AKT, p53/p21CIP1/serpine, HIF-1α, and BCL-2/BCL-XL pathways (15). Reliance on these pathways, termed senescent cell antiapoptotic pathways, or SCAPs, provides senescent cells with the ability to resist the proapoptotic and tissue-damaging factors that are components of their own SASP (15, 16). Through these mechanisms, a pathogenic loop is formed that begets more cellular senescence. According to our “Threshold Theory of Cellular Senescence,” when the senescent cell population surpasses the immune system’s ability to clear them, senescent cells accumulate more rapidly and cause increased pathology (17). Consistent with this theory, while transplantation of relatively few senescent cells into healthy younger animals induces early onset of age-related disease and frailty, a smaller number of cells is needed to exert this effect in aged animals, on top of their higher preexisting burden of senescent cells (9, 18). Obesity, like aging, is associated with senescent cell accumulation and therefore may lower the threshold of additional senescent cells needed to cause systemic dysfunction.

Adipose tissue can constitute a significant reservoir of senescent cells, particularly in obesity and diabetes (4, 5, 18). Visceral adipose tissue contains the highest burden of senescent cells in diet-induced obese mice, and tends to be the location to which senescent cells migrate when transplanted intraperitoneally into experimental animals (9). Senescent cell burden correlates with adipose cell size, even in nondiabetic individuals, and may be increased even before the development of type 2 diabetes in humans with a genetic predisposition to diabetes (4, 18, 19). Polymorphisms in markers of cellular senescence, for example CDKN2A (which encodes p16INK2A), confer increased risk of developing type 2 diabetes and cardiovascular disease (20). Similarly, individuals with some progeroid syndromes such as those associated with lamin-A (LMNA) mutations, which involve increased senescent cell burden, are also associated with lipodystrophy, metabolic syndrome, and diabetes (21, 22). The tumor suppressor p53 is associated with cellular senescence and is upregulated in adipose tissue in obesity, type 2 diabetes, and aging (5, 23, 24). p53 plays an important role in suppressing normal adipogenesis in obesity and blunts insulin-dependent glucose transport and induction of lipolysis (25). These actions contribute to inflammation and insulin resistance. Cellular senescence also limits adipogenesis in a cell-autonomous manner, as well as through paracrine and endocrine effects of SASP factors such as activin A, tumor necrosis factor-α, and interleukin 6 (26-28). Reduced adipogenesis limits adipose tissue capacity to store excess lipids and likely contributes to increased ectopic lipid deposition in other tissues, which can promote insulin resistance (29). Additionally, senescent cells appear to play a role in the chemoattraction of macrophages into visceral adipose tissue, which is associated with insulin resistance (18).

Senescent cell accumulation in other metabolic organs, such as muscle and liver, likely also plays a role in the development of insulin resistance (18, 30). In the pancreas, genes related to cellular senescence, such as p16Ink4a, are increased with aging, and this is associated with reduced β-cell proliferative capacity. However, rather than having reduced insulin secretion, p16Ink4a-positive β cells actually were found to secrete more insulin, consistent with the observation that aged animals have increased basal insulin secretion (31). Other studies have shown that clearance of senescent β cells in a mouse model of type 1 diabetes actually improves insulin secretion, an effect that may be more relevant to the severe insulin deficiency seen in type 1 diabetes (32). More work is needed to further elucidate the relationship between cellular senescence and insulin secretion by the pancreas.

Senolytics: Drugs That Target Senescent Cells

In 2004, caloric restriction, an intervention that extends life and health span and delays age-related pathologies in mice, was found to be linked to delayed age-related senescent cell accumulation, indicating that senescent cell burden and health span are associated (33). This prompted efforts to identify pharmacologic agents that target senescent cells to reduce the burden of age-related disorders and diseases (17, 34). Successful identification of senolytics began with the discovery of senescent cell antiapoptotic pathways, or SCAPs. Senescent cells were previously known to be resistant to apoptosis (35). The necessity of each SCAP for senescent cell survival was tested using RNA interference, comparing the survival of senescent vs nonsenescent cells after knockdown of key nodes on different SCAPs. Drugs were identified by bioinformatics approaches that target these key SCAP nodes identified by RNA interference (15). Using this mechanism-based approach, the first senolytics discovered were dasatinib (D) and quercetin (Q), first published in early 2015 (15). These drugs were found to preferentially cause apoptosis in senescent cells without significant effects on proliferating or quiescent cells. D is a tyrosine kinase inhibitor used for treating cancers, but unlike most other tyrosine kinase inhibitors, it inhibits Src kinases (36). Q is a natural flavonoid and an inhibitor of PI3K, other kinases, and serpines (37, 38). Subsequently, additional senolytic drugs including navitoclax (ABT263) (16, 39), and fisetin (40) were discovered. Senolytic drugs have differential effects on various senescent cell types. For example, D is relatively selective for senescent human adipose progenitor cells, whereas Q causes apoptosis of senescent human endothelial cells. Therefore, combinations of senolytic drugs (such as D + Q) target a broader range of senescent cell types than single agents, especially agents that have a single molecular target, which tend to target only a restricted range of senescent cells and may have more side effects than agents or combinations that act across SCAP networks (17, 34, 41, 42).

Senolytics do not prevent formation of senescent cells. This is important because generation of senescent cells is in large part an anticancer mechanism, limiting the ability of precancerous or cancerous cells from dividing further. In addition, senescent cells have some beneficial effects, including in early wound healing (43). Therefore, it would not be prudent to develop therapies that inhibit the ability of cells to become senescent. Therapies to target cellular senescence must act once senescent cells are already formed, particularly on the 30% to 70% of senescent cells that have a tissue-destructive SASP. This can be achieved by causing their removal with senolytics or limiting their effects on other cells and tissues, such as by inhibiting their SASP (2, 7).

Since senescent cells can take weeks to develop and acquire an SASP, and because only a brief exposure of senescent cells to senolytics is sufficient to cause them to be removed by apoptosis, administration of senolytic drugs can be in a “hit and run” fashion, much like antibiotics or chemotherapeutics. In effect, senescent cells can be cleared in waves during each round of senolytic therapy. This may be superior to approaches that only mitigate the effects of senescent cells, for example, SASP inhibitors, as SASP inhibitors and related agents need to be administered continuously. The effect of senolytics is durable because senescent cells undergo apoptosis in response to senolytic therapy and are therefore removed from tissue. As an example of the durability of response to senolytic therapy, positive effects of one round of senolytic treatment on the mobility of mice that had undergone radiation of one hind leg lasted for at least 7 months, despite the elimination half-life of each drug being only a few hours (15, 44-46). The rate of accumulation of senescent cells will likely determine the optimal administration schedule of senolytics for any particular indication. For example, one course of senolytic treatment may be sufficient for dysfunction caused by senescent cells induced by therapeutic radiation or chemotherapy; however, more frequent courses of treatment may be needed in diabetes because of ongoing metabolic insults causing ongoing formation of new senescent cells.

Like all drugs, senolytics are expected to have side effects. These are well characterized for some senolytic drugs, such as D and Q, which both are approved for use in humans. D is used as chemotherapy continuously, in some cases for years, with a tolerable side-effect profile. Pleural effusions and pulmonary hypertension can be seen with D therapy and are typically reversible on discontinuation of the drug or preventable with dose reduction (47). Q has been used as a dietary supplement with rare side effects; however, it has ability to cause nephrotoxicity at higher doses or in combination with other nephrotoxic agents (48). Navitoclax, another senolytic drug, can cause transient thrombocytopenia and direct cytotoxicity to osteoblasts (49). Fisetin has not been associated with any adverse effects (50). As senolytics, these drugs would be administered in an intermittent fashion, for example, once every few weeks. This intermittent dosing strategy, when combined with the short elimination half-lives of senolytic drugs, may limit drug toxicity and those off-target effects that arise through continuous drug occupancy of a receptor or inhibition of an enzyme (45, 46). In fact, an intermittent dosing schedule was shown to improve tolerability of D in patients with chronic myeloid leukemia (51). Intermittent dosing may also be beneficial for medication adherence, given that senolytics can be taken for only a fraction of days out of a week or month. Theoretical adverse effects due to the clearance of senescent cells, rather than due to off-targets of the drugs themselves, are at this time theoretical. However, based on preclinical data these might include negative effects on wound healing or fibrosis during liver regeneration (43, 52).

Initial clinical trials of senolytics in humans are under way, and the results of several small studies have already been published (17, 53, 54). In an open-label pilot study of patients with idiopathic pulmonary fibrosis, senolytic (D + Q) therapy improved physical function, including 6-minute walk distance, gait speed, chair stand time, and the Short Physical Performance Battery (54). In another open-label, phase 2 study of patients with diabetic kidney disease, senolytic therapy reduced senescent cell burden, macrophages, and crown-like structures in adipose tissue as well as blood SASP factors (53). Additional studies are beginning or are ongoing for osteoarthritis, osteoporosis, Alzheimer disease, coronavirus disease 2019 in hospitalized and nursing home patients, frailty in elderly women, and childhood cancer survivors, among others (ClinicalTrials.gov identifiers: NCT 04210986, 0431363, 04685590, 04476953, 04476953, 03430037, and 04733534, respectively).

Targeting Senescent Cells Improves Diabetic Phenotypes and Complications

In diet-induced obese mice, senescent cell clearance has many beneficial effects on adipose tissue function and systemic metabolism. After senescent cell clearance, mean adipocyte size is smaller, and adipose tissue distribution favors subcutaneous depots rather than visceral ones, without reduction of body weight (18). Ectopic lipid deposition in muscle and liver is also decreased (18, 30). These features are correlated with insulin sensitivity, which is improved after senescent cell clearance (18). Senescent cell clearance in aged mice mitigates age-related fat tissue loss due to improved adipogenesis (27). In addition to these metabolic benefits, some complications of diabetes were also improved or prevented in obese mice, including microalbuminuria, diastolic cardiac dysfunction, hepatic steatosis, and obesity-induced anxiety (18, 27, 55, 56). Osteoarthritis, atherosclerosis, and vascular reactivity have also been shown to improve after senolytic treatment in experimental animals (57, 58). As previously mentioned, reduction of senescent cells in adipose tissue has been seen in a small human study of senolytics, providing an indication that senolytic treatment may alleviate diabetic phenotypes in humans (53).

Little is known about the effect of current diabetes therapies on senescent cell quantity or function in humans. Certain existing therapies, including metformin and acarbose, have been shown to extend life span in nondiabetic mice, to a greater extent in males than females, although senescent cell burden was not measured in these studies (59). In cell culture experiments, metformin can alleviate the SASP via activation of Nrf2-Gpx7 and nuclear factor κB inhibition (60, 61). Metformin has also been shown to improve proliferative capacity of cells in culture, including mesenchymal stem cells isolated from mice with chronic kidney disease (62). In addition, metformin is known to alleviate diabetic complications and metabolic syndrome, and may confer lower cancer risk in diabetic patients (63). However, more studies are needed to explore whether these effects are mediated by prevention of senescence, inhibition of the SASP, or some combination of these mechanisms. Additionally, more investigation is needed to determine the impact that other diabetes therapies (ie, sodium-glucose cotransporter-2 inhibitors, sulfonylureas, glucagon-like peptide 1 agonists, dipeptidyl peptidase-4 inhibitors) may have on cellular senescence and its downstream effects.

Senolytic Clinical Trial Design

Senolytic drugs show a great deal of promise in preclinical studies for obesity-induced metabolic dysfunction and diabetes. Careful clinical trial design is needed to test whether these benefits also occur in humans and if senolytics are safe for use in humans. Studies to determine whether senolytic drugs can prevent the onset of diabetes are not feasible as a first step given the length of time needed to test that hypothesis. Optimally, initial trials should focus on conditions for which current therapies are ineffective, given that the risk-benefit profile of senolytic drugs in humans is not yet known. In the setting of diabetes, indications that could make sense to target in early trials are diabetic complications such as retinopathy, diabetic ulcers, and diabetic kidney disease, because there are limited or no current therapies to specifically treat several such complications, and these are difficult to manage once present. Investigations of senolytic effects on diabetic complications could be completed within a reasonable timeline. Because senolytic drugs remove senescent, dysfunctional cells that may exacerbate diabetic complications, they may have significant therapeutic potential for these indications.

If clinical trials for serious diabetic complications, such as nonhealing diabetic skin wounds, heart failure with preserved ejection fraction, nephropathy, or retinopathy, show efficacy and senolytic drug risk profiles are favorable, then additional studies could be pursued. For example, the ability of senolytic therapy to prevent, rather than treat, diabetic complications could be explored. Direct measures of insulin sensitivity, such as glucose or insulin tolerance testing or hyperglycemic clamping, or clinical measures, such as changes in required insulin dose following senolytic treatment, could be of interest. If successful, then trials focusing on the prevention of senescence-associated conditions, such as prevention of insulin resistance in obesity or prevention of diabetic chronic kidney disease, could be pursued. Of course, judicious safety monitoring is necessary, as the long-term effects of clearing senescent cells in humans are not known. The results of randomized, controlled trials must be available before senolytics are prescribed to patients outside clinical trials.

As previously mentioned, significant investigation is needed to determine the optimal administration schedule of senolytics (ie, weekly, monthly, bimonthly) for each individual disease state according to the rate of reaccumulation of senescent cells in that disease. Different senolytics and combinations of senolytics may need to be investigated to achieve optimal senescent cell clearance in particular tissues or diseases. There may also be patient factors, such as pharmacokinetic differences, that affect the efficacy of different senolytics for a particular subset of individuals. Targeted delivery of senolytic drugs to specific tissues could be explored in the future as a strategy. This may be especially useful in the case of localized pathologies, such as diabetic retinopathy.

Conclusions

Senescent cell burden is increased in obesity and diabetes and likely plays a role in the development of diabetes and its complications. Novel senolytic drugs have been identified that can remove senescent cells by disarming their prosurvival pathways called SCAPs. Senolytics have been shown to alleviate multiple diseases in preclinical studies, including insulin resistance and diabetic complications in animal models, and can reduce senescent cell abundance in human adipose tissue. Further preclinical studies are needed to define the advantages or noninferiority of senolytics when compared with current diabetes therapies, as well as to demonstrate their safety. Eventually, randomized, controlled clinical trials are needed to further investigate the utility of senolytics in human obesity and diabetes. Because there are currently no specific therapies to alleviate diabetic complications, these might be appropriate targets for early trials of senolytic drugs in diabetic patients. Data from well-designed clinical trials are needed prior to patients receiving senolytic drugs outside clinical trials. Senolytics represent a potential new therapeutic class for the prevention and treatment of diabetes and its complications.