SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

PAHSAs reduce cellular senescence and protect pancreatic beta cells from metabolic stress through regulation of Mdm2/p53.

Senescence in pancreatic beta cells plays a major role in beta cell dysfunction, which leads to impaired glucose homeostasis and diabetes. Therefore, prevention of beta cell senescence could reduce the risk of diabetes. Treatment of nonobese diabetic (NOD) mice, a model of type 1 autoimmune diabetes (T1D), with palmitic acid hydroxy stearic acids (PAHSAs), a novel class of endogenous lipids with antidiabetic and antiinflammatory effects, delays the onset and reduces the incidence of T1D from 82% with vehicle treatment to 35% with PAHSAs. Here, we show that a major mechanism by which PAHSAs protect islets of the NOD mice is by directly preventing and reversing the initial steps of metabolic stress-induced senescence. In vitro PAHSAs increased Mdm2 expression, which decreases the stability of p53, a key inducer of senescence-related genes. In addition, PAHSAs enhanced expression of protective genes, such as those regulating DNA repair and glutathione metabolism and promoting autophagy. We demonstrate the translational relevance by showing that PAHSAs prevent and reverse early stages of senescence in metabolically stressed human islets by the same Mdm2 mechanism. Thus, a major mechanism for the dramatic effect of PAHSAs in reducing the incidence of type 1 diabetes in NOD mice is decreasing cellular senescence; PAHSAs may have a similar benefit in humans.

Functional changes in beta cells during ageing and senescence.

Insulin secretion from beta cells is crucial for maintaining euglycaemia and preventing type 2 diabetes, a disease correlated with ageing. Therefore, understanding the functional changes that beta cell function undergoes with age can reveal new therapeutic targets and strategies to delay or revert the disease. Herein, a systematic review of the literature agrees that, as humans age, their beta cell function declines, independently of peripheral insulin resistance, BMI and waist circumference. Rodent studies reveal that, with age, basal insulin secretion increases with either no change or an increase in stimulated insulin secretion, but the biological significance of this is unclear. The accumulation of senescent beta cells could explain some of these functional changes: transcriptional analysis of senescent and aged beta cells revealed parallel downregulation of several steps along the pathway linking glucose stimulation and insulin secretion. Moreover, specific deletion of senescent cells (senolysis) improved residual beta cell function, gene expression profile and blood glucose levels. In conclusion, cellular senescence could underlie the functional decline of beta cells during ageing and could represent a novel and promising approach for recovering insulin secretion. Graphical abstract.

Understanding the growing epidemic of type 2 diabetes in the Hispanic population living in the United States.

The prevalence and incidence of type 2 diabetes (T2D) among the Hispanic population in the United States are higher than the national average. This is partly due to sociocultural factors, such as lower income and decreased access to education and health care, as well as a genetic susceptibility to obesity and higher insulin resistance. This review focuses on understanding the Hispanic population living in the United States from a multidisciplinary approach and underlines the importance of cultural, social, and biological factors in determining the increased risk of T2D in this population. An overview of the acute and chronic complications of T2D upon this population is included, which is of paramount importance to understand the toll that diabetes has upon this population, the health system, and society as a whole. Specific interventions directed to the Hispanic populations are needed to prevent and alleviate some of the burdens of T2D. Different prevention strategies based on medications, lifestyle modifications, and educational programmes are discussed herein. Diabetes self-management education (DSME) is a critical element of care of all people with diabetes and is considered necessary to improve patient outcomes. To be more effective, programmes should take into consideration cultural factors that influence the development and progression of diabetes. These interventions aim to enhance long-term effects by reducing the incidence, morbidity, and mortality of T2D in the Hispanic population of the United States.

Tissue-specific disallowance of housekeeping genes: the other face of cell differentiation.

We report on a hitherto poorly characterized class of genes that are expressed in all tissues, except in one. Often, these genes have been classified as housekeeping genes, based on their nearly ubiquitous expression. However, the specific repression in one tissue defines a special class of "disallowed genes." In this paper, we used the intersection-union test to screen for such genes in a multi-tissue panel of genome-wide mRNA expression data. We propose that disallowed genes need to be repressed in the specific target tissue to ensure correct tissue function. We provide mechanistic data of repression with two metabolic examples, exercise-induced inappropriate insulin release and interference with ketogenesis in liver. Developmentally, this repression is established during tissue maturation in the early postnatal period involving epigenetic changes in histone methylation. In addition, tissue-specific expression of microRNAs can further diminish these repressed mRNAs. Together, we provide a systematic analysis of tissue-specific repression of housekeeping genes, a phenomenon that has not been studied so far on a genome-wide basis and, when perturbed, can lead to human disease.

Heterogeneity of increased biological age in type 2 diabetes correlates with differential tissue DNA methylation, biological variables, and pharmacological treatments.

Biological age (BA) closely depicts age-related changes at a cellular level. Type 2 diabetes mellitus (T2D) accelerates BA when calculated using clinical biomarkers, but there is a large spread in the magnitude of individuals' age acceleration in T2D suggesting additional factors contributing to BA. Additionally, it is unknown whether BA can be changed with treatment. We hypothesized that potential determinants of the heterogeneous BA distribution in T2D could be due to differential tissue aging as reflected at the DNA methylation (DNAm) level, or biological variables and their respective therapeutic treatments. Publicly available DNAm samples were obtained to calculate BA using the DNAm phenotypic age (DNAmPhenoAge) algorithm. DNAmPhenoAge showed age acceleration in T2D samples of whole blood, pancreatic islets, and liver, but not in adipose tissue or skeletal muscle. Analysis of genes associated with differentially methylated CpG sites found a significant correlation between eight individual CpG methylation sites and gene expression. Clinical biomarkers from participants in the NHANES 2017-2018 and ACCORD cohorts were used to calculate BA using the Klemera and Doubal (KDM) method. Cardiovascular and glycemic biomarkers associated with increased BA while intensive blood pressure and glycemic management reduced BA to CA levels, demonstrating that accelerated BA can be restored in the setting of T2D.

Exercise activates AMPK in mouse and human pancreatic islets to decrease senescence.

Beta (ß)-cell senescence contributes to type 2 diabetes mellitus (T2DM). While exercise is vital for T2DM management and significantly affects cellular ageing markers, its effect on ß-cell senescence remains unexplored. Here, we show that short-term endurance exercise training (treadmill running, 1 h per day for 10 days) in two male and female mouse models of insulin resistance decreases ß-cell senescence. In vivo and in vitro experiments revealed that this effect is mediated, at least in part, by training-induced increases in serum glucagon, leading to activation of 5'-AMP-activated protein kinase (AMPK) signalling in ß-cells. AMPK activation resulted in the nuclear translocation of NRF2 and decreased expression of senescence markers and effectors. Remarkably, human islets from male and female donors with T2DM treated with serum collected after a 10-week endurance exercise training programme showed a significant decrease in the levels of senescence markers. These findings indicate that exercise training decreases senescence in pancreatic islets, offering promising therapeutic implications for T2DM.

Interaction between Autophagy and Senescence in Pancreatic Beta Cells.

Aging leads to an increase in cellular stress due to the fragility of the organism and the inability to cope with it. In this setting, there is a higher chance of developing different cardiometabolic diseases like diabetes. Cellular senescence and autophagy, both hallmarks of aging and stress-coping mechanisms, have gained increased attention for their role in the pathophysiology of diabetes. Studies show that impairing senescence dampens and even prevents diabetes while the role of autophagy is more contradictory, implying a context- and disease-stage-dependent effect. Reports show conflicting data about the effect of autophagy on senescence while the knowledge about this interaction in beta cells remains scarce. Elucidating this interaction between autophagy and senescence in pancreatic beta cells will lead to an identification of their respective roles and the extent of the effect each mechanism has on beta cells and open new horizons for developing novel therapeutic agents. To help illuminate this relationship we will review the latest findings of cellular senescence and autophagy with a special emphasis on pancreatic beta cells and diabetes.

Regulation of Cellular Senescence in Type 2 Diabetes Mellitus: From Mechanisms to Clinical Applications.

Cellular senescence is accelerated by hyperglycemia through multiple pathways. Therefore, senescence is an important cellular mechanism to consider in the pathophysiology of type 2 diabetes mellitus (T2DM) and an additional therapeutic target. The use of drugs that remove senescent cells has led to improvements in blood glucose levels and diabetic complications in animal studies. Although the removal of senescent cells is a promising approach for the treatment of T2DM, two main challenges limit its clinical application: the molecular basis of cellular senescence in each organ is yet to be understood, and the specific effect of removing senescent cells in each organ has to be determined. This review aims to discuss future applications of targeting senescence as a therapeutic option in T2DM and elucidate the characteristics of cellular senescence and senescence-associated secretory phenotype in the tissues important for regulating glucose levels: pancreas, liver, adipocytes, and skeletal muscle.

Pancreatic ß-cell senescence in diabetes: mechanisms, markers and therapies.

Cellular senescence is a response to a wide variety of stressors, including DNA damage, oncogene activation and physiologic aging, and pathologically accelerated senescence contributes to human disease, including diabetes mellitus. Indeed, recent work in this field has demonstrated a role for pancreatic ß-cell senescence in the pathogenesis of Type 1 Diabetes, Type 2 Diabetes and monogenic diabetes. Small molecule or genetic targeting of senescent ß-cells has shown promise as a novel therapeutic approach for preventing and treating diabetes. Despite these advances, major questions remain around the molecular mechanisms driving senescence in the ß-cell, identification of molecular markers that distinguish senescent from non-senescent ß-cell subpopulations, and translation of proof-of-concept therapies into novel treatments for diabetes in humans. Here, we summarize the current state of the field of ß-cell senescence, highlighting insights from mouse models as well as studies on human islets and ß-cells. We identify markers that have been used to detect ß-cell senescence to unify future research efforts in this field. We discuss emerging concepts of the natural history of senescence in ß-cells, heterogeneity of senescent ß-cells subpopulations, role of sex differences in senescent responses, and the consequences of senescence on integrated islet function and microenvironment. As a young and developing field, there remain many open research questions which need to be addressed to move senescence-targeted approaches towards clinical investigation.

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

Type 2 diabetes is partly characterized by decreased ß-cell mass and function which have been linked to cellular senescence. Despite a low basal proliferative rate of adult ß-cells, they can respond to growth stimuli, but this proliferative capacity decreases with age and correlates with increased expression of senescence effector, p16Ink4a. We hypothesized that selective deletion of p16Ink4a-positive cells would enhance the proliferative capacity of the remaining ß-cells due to the elimination of the local senescence-associated secretory phenotype (SASP). We aimed to investigate the effects of p16Ink4a-positive cell removal on the mass and proliferative capacity of remaining ß-cells using INK-ATTAC mice as a transgenic model of senolysis. Clearance of p16Ink4a positive subpopulation was tested in mice of different ages, males and females, and with two different insulin resistance models: high-fat diet (HFD) and insulin receptor antagonist (S961). Clearance of p16Ink4a-positive cells did not affect the overall ß-cell mass. ß-cell proliferative capacity negatively correlated with cellular senescence load and clearance of p16Ink4a positive cells in 1-year-old HFD mice improved ß-cell function and increased proliferative capacity in a subset of animals. Single-cell sequencing revealed that the targeted p16Ink4a subpopulation of ß-cells is non-proliferative and non-SASP producing whereas additional senescent subpopulations remained contributing to continued local SASP secretion. In conclusion, deletion of p16Ink4a cells did not negatively impact beta-cell mass and blood glucose under basal and HFD conditions and proliferation was restored in a subset of HFD mice opening further therapeutic targets in the treatment of diabetes.

Decreased IGF1R attenuates senescence and improves function in pancreatic ß-cells.

Introduction: The enhanced ß-cell senescence that accompanies insulin resistance and aging contributes to cellular dysfunction and loss of transcriptional identity leading to type 2 diabetes (T2D). While senescence is among the 12 recognized hallmarks of aging, its relation to other hallmarks including altered nutrient sensing (insulin/IGF1 pathway) in ß-cells is not fully understood. We previously reported that an increased expression of IGF1R in mouse and human ß-cells is a marker of older ß-cells; however, its contribution to age-related dysfunction and cellular senescence remains to be determined. Methods: In this study, we explored the direct role of IGF1R in ß-cell function and senescence using two independent mouse models with decreased IGF1/IGF1R signaling: a) Ames Dwarf mice (Dwarf +/+), which lack growth hormone and therefore have reduced circulating levels of IGF1, and b) inducible ß-cell-specific IGF1R knockdown (ßIgf1rKD) mice. Results: Compared to Dwarf+/- mice, Dwarf+/+ mice had lower body and pancreas weight, lower circulating IGF1 and insulin levels, and lower IGF1R and p21Cip1 protein expression in ß-cells, suggesting the suppression of senescence. Adult ßIgf1rKD mice showed improved glucose clearance and glucose-induced insulin secretion, accompanied by decreased p21Cip1 protein expression in ß-cells. RNA-Seq of islets isolated from these ßIgf1rKD mice revealed the restoration of three signaling pathways known to be downregulated by aging: sulfide oxidation, autophagy, and mTOR signaling. Additionally, deletion of IGF1R in mouse ß-cells increased transcription of genes important for maintaining ß-cell identity and function, such as Mafa, Nkx6.1, and Kcnj11, while decreasing senescence-related genes, such as Cdkn2a, Il1b, and Serpine 1. Decreased senescence and improved insulin-secretory function of ß-cells were also evident when the ßIgf1rKD mice were fed a high-fat diet (HFD; 60% kcal from fat, for 5 weeks). Discussion: These results suggest that IGF1R signaling plays a causal role in aging-induced ß-cell dysfunction. Our data also demonstrate a relationship between decreased IGF1R signaling and suppressed cellular senescence in pancreatic ß-cells. Future studies can further our understanding of the interaction between senescence and aging, developing interventions that restore ß-cell function and identity, therefore preventing the progression to T2D.

Diabetes mellitus correlates with increased biological age as indicated by clinical biomarkers.

Chronological age (CA) is determined by time of birth, whereas biological age (BA) is based on changes on a cellular level and strongly correlates with morbidity, mortality, and longevity. Type 2 diabetes (T2D) associates with increased morbidity and mortality; thus, we hypothesized that BA would be increased and calculated it from biomarkers collected at routine clinical visits. Deidentified data was obtained from three cohorts of patients (20-80 years old)-T2D, type 1 diabetes (T1D), and prediabetes-and compared to gender- and age-matched non-diabetics. Eight clinical biomarkers that correlated with CA in people without diabetes were used to calculate BA using the Klemera and Doubal method 1 (KDM1) and multiple linear regression (MLR). The phenotypic age (PhAge) formula was used with its predetermined biomarkers. BA of people with T2D was, on average, 12.02 years higher than people without diabetes (p < 0.0001), while BA in T1D was 16.32 years higher (p < 0.0001). Results were corroborated using MLR and PhAge. The biomarkers with the strongest correlation to increased BA in T2D using KDM were A1c (R2 = 0.23, p < 0.0001) and systolic blood pressure (R2 = 0.21, p < 0.0001). BMI had a positive correlation to BA in non-diabetes subjects but disappeared in those with diabetes. Mortality data using the ACCORD trial was used to validate our results and showed a significant correlation between higher BA and decreased survival. In conclusion, BA is increased in people with diabetes, irrespective of pathophysiology, and to a lesser extent in prediabetes.

Extracellular Nicotinamide Phosphoribosyltransferase Is a Component of the Senescence-Associated Secretory Phenotype.

Cellular senescence is a stress or damage response by which a cell adopts of state of essentially permanent proliferative arrest, coupled to the secretion of a number of biologically active molecules. This senescence-associated secretory phenotype (SASP) underlies many of the degenerative and regenerative aspects of cellular senescence - including promoting wound healing and development, but also driving diabetes and multiple age-associated diseases. We find that nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in nicotinamide adenine dinucleotide (NAD) biosynthesis, is elevated in senescent cells without a commensurate increase in NAD levels. This elevation is distinct from the acute DNA damage response, in which NAD is depleted, and recovery of NAD by NAMPT elevation is AMPK-activated protein kinase (AMPK)-dependent. Instead, we find that senescent cells release extracellular NAMPT (eNAMPT) as part of the SASP. eNAMPT has been reported to be released as a catalytically active extracellular vesicle-contained dimer that promotes NAD increases in other cells and extends lifespan, and also as free monomer that acts as a damage-associated molecular pattern and promotes conditions such as diabetes and fibrosis. Senescent cells released eNAMPT as dimer, but surprisingly eNAMPT appeared in the soluble secretome while being depleted from exosomes. Finally, diabetic mice showed elevated levels of eNAMPT, and this was lowered by treatment with the senolytic drug, ABT-263. Together, these data reveal a new SASP factor with implications for NAD metabolism.

Bromodomain Inhibition Reveals FGF15/19 As a Target of Epigenetic Regulation and Metabolic Control.

Epigenetic regulation is an important factor in glucose metabolism, but underlying mechanisms remain largely unknown. Here we investigated epigenetic control of systemic metabolism by bromodomain-containing proteins (Brds), which are transcriptional regulators binding to acetylated histone, in both intestinal cells and mice treated with the bromodomain inhibitor JQ-1. In vivo treatment with JQ-1 resulted in hyperglycemia and severe glucose intolerance. Whole-body or tissue-specific insulin sensitivity was not altered by JQ-1; however, JQ-1 treatment reduced insulin secretion during both in vivo glucose tolerance testing and ex vivo incubation of isolated islets. JQ-1 also inhibited expression of fibroblast growth factor (FGF) 15 in the ileum and decreased FGF receptor 4-related signaling in the liver. These adverse metabolic effects of Brd4 inhibition were fully reversed by in vivo overexpression of FGF19, with normalization of hyperglycemia. At a cellular level, we demonstrate Brd4 binds to the promoter region of FGF19 in human intestinal cells; Brd inhibition by JQ-1 reduces FGF19 promoter binding and downregulates FGF19 expression. Thus, we identify Brd4 as a novel transcriptional regulator of intestinal FGF15/19 in ileum and FGF signaling in the liver and a contributor to the gut-liver axis and systemic glucose metabolism.

Effects of exercise on cellular and tissue aging.

The natural aging process is carried out by a progressive loss of homeostasis leading to a functional decline in cells and tissues. The accumulation of these changes stem from a multifactorial process on which both external (environmental and social) and internal (genetic and biological) risk factors contribute to the development of adult chronic diseases, including type 2 diabetes mellitus (T2D). Strategies that can slow cellular aging include changes in diet, lifestyle and drugs that modulate intracellular signaling. Exercise is a promising lifestyle intervention that has shown antiaging effects by extending lifespan and healthspan through decreasing the nine hallmarks of aging and age-associated inflammation. Herein, we review the effects of exercise to attenuate aging from a clinical to a cellular level, listing its effects upon various tissues and systems as well as its capacity to reverse many of the hallmarks of aging. Additionally, we suggest AMPK as a central regulator of the cellular effects of exercise due to its integrative effects in different tissues. These concepts are especially relevant in the setting of T2D, where cellular aging is accelerated and exercise can counteract these effects through the reviewed antiaging mechanisms.

Unique Human and Mouse ß-Cell Senescence-Associated Secretory Phenotype (SASP) Reveal Conserved Signaling Pathways and Heterogeneous Factors.

The aging of pancreatic ß-cells may undermine their ability to compensate for insulin resistance, leading to the development of type 2 diabetes (T2D). Aging ß-cells acquire markers of cellular senescence and develop a senescence-associated secretory phenotype (SASP) that can lead to senescence and dysfunction of neighboring cells through paracrine actions, contributing to ß-cell failure. In this study, we defined the ß-cell SASP signature based on unbiased proteomic analysis of conditioned media of cells obtained from mouse and human senescent ß-cells and a chemically induced mouse model of DNA damage capable of inducing SASP. These experiments revealed that the ß-cell SASP is enriched for factors associated with inflammation, cellular stress response, and extracellular matrix remodeling across species. Multiple SASP factors were transcriptionally upregulated in models of ß-cell senescence, aging, insulin resistance, and T2D. Single-cell transcriptomic analysis of islets from an in vivo mouse model of reversible insulin resistance indicated unique and partly reversible changes in ß-cell subpopulations associated with senescence. Collectively, these results demonstrate the unique secretory profile of senescent ß-cells and its potential implication in health and disease.

Acceleration of ß Cell Aging Determines Diabetes and Senolysis Improves Disease Outcomes.

Type 2 diabetes (T2D) is an age-related disease. Although changes in function and proliferation of aged ß cells resemble those preceding the development of diabetes, the contribution of ß cell aging and senescence remains unclear. We generated a ß cell senescence signature and found that insulin resistance accelerates ß 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 ß 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 ß cells also responded to senolysis, establishing the foundation for translation. These novel findings lay the framework to pursue senolysis of ß cells as a preventive and alleviating strategy for T2D.

Pancreatic ß Cell Regeneration as a Possible Therapy for Diabetes.

Diabetes is the result of having inadequate supply of functional insulin-producing ß cells. Two possible approaches for replenishing the ß cells are: (1) replacement by transplanting cadaveric islets or ß cells derived from human embryonic stem cells/induced pluripotent stem cells and (2) induction of endogenous regeneration. This review focuses on endogenous regeneration, which can follow two pathways: enhanced replication of existing ß cells and formation of new ß cells from cells not expressing insulin, either by conversion from a differentiated cell type (transdifferentiation) or differentiation from progenitors (neogenesis). Exciting progress on both pathways suggest that regeneration may have therapeutic promise.