Senescence markers in subcutaneous preadipocytes differ in childhood- versus adult-onset obesity before and after weight loss.

OBJECTIVE: The aim of this study was to determine the effect of age of obesity onset on senescence-related markers in abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) before and after moderate (~10%) weight loss. METHODS: AB and FEM SAT were collected from human females with childhood-onset obesity (CO) or adult-onset obesity (AO) before and after diet- and exercise-induced weight loss. Immunofluorescence analysis of γH2AX/RAD51 (DNA damage/repair markers) and p53/p21 (senescence markers) was conducted in cultured preadipocytes, and senescence-associated ß-galactosidase (SA-ß-gal) activity was measured in SAT. RESULTS: CO had proportionately more AB and FEM preadipocytes with DNA damage (γH2AX+ ) and senescence markers (p53+ and/or p21+ ) than AO at baseline. The proportion of γH2AX+ FEM preadipocytes declined with weight loss in CO and was similar between groups after weight loss. The number of γH2AX foci in γH2AX+ preadipocytes decreased similarly between groups and regions with weight loss in parallel with an increase in RAD51. The proportion of p53+ and p21+ preadipocytes and SA-ß-gal+ cells in SAT did not change with weight loss, but the total p21 intensity in p53+ /p21+ FEM preadipocytes declined in AO. CONCLUSIONS: These results provide preliminary evidence that females with CO have an accelerated preadipocyte aging state that improves with weight loss in terms of DNA damage but not senescence.

SARS-CoV-2 causes senescence in human cells and exacerbates the senescence-associated secretory phenotype through TLR-3.

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse ß-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.

Whole-body senescent cell clearance alleviates age-related brain inflammation and cognitive impairment in mice.

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro-inflammatory senescence-associated secretory phenotype (SASP), which is a major contributor to aging and age-related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single-nuclei and single-cell RNA-seq in the hippocampus from young and aged mice. We observed an age-dependent increase in p16Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK-ATTAC mice, in which p16Ink4a -positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof-of-concept for senolytic interventions' being a potential therapeutic avenue for alleviating age-associated cognitive impairment.

Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease.

BACKGROUND: Senescent cells, which can release factors that cause inflammation and dysfunction, the senescence-associated secretory phenotype (SASP), accumulate with ageing and at etiological sites in multiple chronic diseases. Senolytics, including the combination of Dasatinib and Quercetin (D + Q), selectively eliminate senescent cells by transiently disabling pro-survival networks that defend them against their own apoptotic environment. In the first clinical trial of senolytics, D + Q improved physical function in patients with idiopathic pulmonary fibrosis (IPF), a fatal senescence-associated disease, but to date, no peer-reviewed study has directly demonstrated that senolytics decrease senescent cells in humans. METHODS: In an open label Phase 1 pilot study, we administered 3 days of oral D 100 mg and Q 1000 mg to subjects with diabetic kidney disease (N = 9; 68·7 ±â€¯3·1 years old; 2 female; BMI:33·9 ±â€¯2·3 kg/m2; eGFR:27·0 ±â€¯2·1 mL/min/1·73m2). Adipose tissue, skin biopsies, and blood were collected before and 11 days after completing senolytic treatment. Senescent cell and macrophage/Langerhans cell markers and circulating SASP factors were assayed. FINDINGS: D + Q reduced adipose tissue senescent cell burden within 11 days, with decreases in p16INK4A-and p21CIP1-expressing cells, cells with senescence-associated ß-galactosidase activity, and adipocyte progenitors with limited replicative potential. Adipose tissue macrophages, which are attracted, anchored, and activated by senescent cells, and crown-like structures were decreased. Skin epidermal p16INK4A+ and p21CIP1+ cells were reduced, as were circulating SASP factors, including IL-1α, IL-6, and MMPs-9 and -12. INTERPRETATION: "Hit-and-run" treatment with senolytics, which in the case of D + Q have elimination half-lives <11 h, significantly decreases senescent cell burden in humans. FUND: NIH and Foundations. ClinicalTrials.gov Identifier: NCT02848131. Senescence, Frailty, and Mesenchymal Stem Cell Functionality in Chronic Kidney Disease: Effect of Senolytic Agents.

Targeting senescent cells alleviates obesity-induced metabolic dysfunction.

Adipose tissue inflammation and dysfunction are associated with obesity-related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug-inducible "suicide" genes driven by the p16Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra-abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity-related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity-related metabolic dysfunction and its complications.

Obesity-Induced Cellular Senescence Drives Anxiety and Impairs Neurogenesis.

Cellular senescence entails a stable cell-cycle arrest and a pro-inflammatory secretory phenotype, which contributes to aging and age-related diseases. Obesity is associated with increased senescent cell burden and neuropsychiatric disorders, including anxiety and depression. To investigate the role of senescence in obesity-related neuropsychiatric dysfunction, we used the INK-ATTAC mouse model, from which p16Ink4a-expressing senescent cells can be eliminated, and senolytic drugs dasatinib and quercetin. We found that obesity results in the accumulation of senescent glial cells in proximity to the lateral ventricle, a region in which adult neurogenesis occurs. Furthermore, senescent glial cells exhibit excessive fat deposits, a phenotype we termed "accumulation of lipids in senescence." Clearing senescent cells from high fat-fed or leptin receptor-deficient obese mice restored neurogenesis and alleviated anxiety-related behavior. Our study provides proof-of-concept evidence that senescent cells are major contributors to obesity-induced anxiety and that senolytics are a potential new therapeutic avenue for treating neuropsychiatric disorders.

Fisetin is a senotherapeutic that extends health and lifespan.

BACKGROUND: Senescence is a tumor suppressor mechanism activated in stressed cells to prevent replication of damaged DNA. Senescent cells have been demonstrated to play a causal role in driving aging and age-related diseases using genetic and pharmacologic approaches. We previously demonstrated that the combination of dasatinib and the flavonoid quercetin is a potent senolytic improving numerous age-related conditions including frailty, osteoporosis and cardiovascular disease. The goal of this study was to identify flavonoids with more potent senolytic activity. METHODS: A panel of flavonoid polyphenols was screened for senolytic activity using senescent murine and human fibroblasts, driven by oxidative and genotoxic stress, respectively. The top senotherapeutic flavonoid was tested in mice modeling a progeroid syndrome carrying a p16INK4a-luciferase reporter and aged wild-type mice to determine the effects of fisetin on senescence markers, age-related histopathology, disease markers, health span and lifespan. Human adipose tissue explants were used to determine if results translated. FINDINGS: Of the 10 flavonoids tested, fisetin was the most potent senolytic. Acute or intermittent treatment of progeroid and old mice with fisetin reduced senescence markers in multiple tissues, consistent with a hit-and-run senolytic mechanism. Fisetin reduced senescence in a subset of cells in murine and human adipose tissue, demonstrating cell-type specificity. Administration of fisetin to wild-type mice late in life restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan. INTERPRETATION: The natural product fisetin has senotherapeutic activity in mice and in human tissues. Late life intervention was sufficient to yield a potent health benefit. These characteristics suggest the feasibility to translation to human clinical studies. FUND: NIH grants P01 AG043376 (PDR, LJN), U19 AG056278 (PDR, LJN, WLL), R24 AG047115 (WLL), R37 AG013925 (JLK), R21 AG047984 (JLK), P30 DK050456 (Adipocyte Subcore, JLK), a Glenn Foundation/American Federation for Aging Research (AFAR) BIG Award (JLK), Glenn/AFAR (LJN, CEB), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK), Robert J. and Theresa W. Ryan (JLK), and a Minnesota Partnership Grant (AMAY-UMN#99)-P004610401-1 (JLK, EAA).

New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463.

Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine. Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-XL inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes. A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.

Cellular senescence mediates fibrotic pulmonary disease.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by interstitial remodelling, leading to compromised lung function. Cellular senescence markers are detectable within IPF lung tissue and senescent cell deletion rejuvenates pulmonary health in aged mice. Whether and how senescent cells regulate IPF or if their removal may be an efficacious intervention strategy is unknown. Here we demonstrate elevated abundance of senescence biomarkers in IPF lung, with p16 expression increasing with disease severity. We show that the secretome of senescent fibroblasts, which are selectively killed by a senolytic cocktail, dasatinib plus quercetin (DQ), is fibrogenic. Leveraging the bleomycin-injury IPF model, we demonstrate that early-intervention suicide-gene-mediated senescent cell ablation improves pulmonary function and physical health, although lung fibrosis is visibly unaltered. DQ treatment replicates benefits of transgenic clearance. Thus, our findings establish that fibrotic lung disease is mediated, in part, by senescent cells, which can be targeted to improve health and function.

17α-Estradiol Alleviates Age-related Metabolic and Inflammatory Dysfunction in Male Mice Without Inducing Feminization.

Aging is associated with visceral adiposity, metabolic disorders, and chronic low-grade inflammation. 17α-estradiol (17α-E2), a naturally occurring enantiomer of 17ß-estradiol (17ß-E2), extends life span in male mice through unresolved mechanisms. We tested whether 17α-E2 could alleviate age-related metabolic dysfunction and inflammation. 17α-E2 reduced body mass, visceral adiposity, and ectopic lipid deposition without decreasing lean mass. These declines were associated with reductions in energy intake due to the activation of hypothalamic anorexigenic pathways and direct effects of 17α-E2 on nutrient-sensing pathways in visceral adipose tissue. 17α-E2 did not alter energy expenditure or excretion. Fasting glucose, insulin, and glycosylated hemoglobin were also reduced by 17α-E2, and hyperinsulinemic-euglycemic clamps revealed improvements in peripheral glucose disposal and hepatic glucose production. Inflammatory mediators in visceral adipose tissue and the circulation were reduced by 17α-E2. 17α-E2 increased AMPKα and reduced mTOR complex 1 activity in visceral adipose tissue but not in liver or quadriceps muscle, which is in contrast to the generalized systemic effects of caloric restriction. These beneficial phenotypic changes occurred in the absence of feminization or cardiac dysfunction, two commonly observed deleterious effects of exogenous estrogen administration. Thus, 17α-E2 holds potential as a novel therapeutic for alleviating age-related metabolic dysfunction through tissue-specific effects.

Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors.

Clearing senescent cells extends healthspan in mice. Using a hypothesis-driven bioinformatics-based approach, we recently identified pro-survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro-survival regulators identified was Bcl-xl. Here, we tested whether the Bcl-2 family inhibitors, navitoclax (N) and TW-37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl-xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl-2, Bcl-xl, and Bcl-w, while T targets Bcl-2, Bcl-xl, and Mcl-1. The combination of Bcl-2, Bcl-xl, and Bcl-w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl-2, Bcl-xl, and Mcl-1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl-2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type-restricted manner. The hypothesis-driven, bioinformatics-based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type-specific senolytic agents.

JAK inhibition alleviates the cellular senescence-associated secretory phenotype and frailty in old age.

Chronic, low grade, sterile inflammation frequently accompanies aging and age-related diseases. Cellular senescence is associated with the production of proinflammatory chemokines, cytokines, and extracellular matrix (ECM) remodeling proteases, which comprise the senescence-associated secretory phenotype (SASP). We found a higher burden of senescent cells in adipose tissue with aging. Senescent human primary preadipocytes as well as human umbilical vein endothelial cells (HUVECs) developed a SASP that could be suppressed by targeting the JAK pathway using RNAi or JAK inhibitors. Conditioned medium (CM) from senescent human preadipocytes induced macrophage migration in vitro and inflammation in healthy adipose tissue and preadipocytes. When the senescent cells from which CM was derived had been treated with JAK inhibitors, the resulting CM was much less proinflammatory. The administration of JAK inhibitor to aged mice for 10 wk alleviated both adipose tissue and systemic inflammation and enhanced physical function. Our findings are consistent with a possible contribution of senescent cells and the SASP to age-related inflammation and frailty. We speculate that SASP inhibition by JAK inhibitors may contribute to alleviating frailty. Targeting the JAK pathway holds promise for treating age-related dysfunction.

The Achilles' heel of senescent cells: from transcriptome to senolytic drugs.

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

Deleted in breast cancer 1 limits adipose tissue fat accumulation and plays a key role in the development of metabolic syndrome phenotype.

Obesity is often regarded as the primary cause of metabolic syndrome. However, many lines of evidence suggest that obesity may develop as a protective mechanism against tissue damage during caloric surplus and that it is only when the maximum fat accumulation capacity is reached and fatty acid spillover occurs into to peripheral tissues that metabolic diseases develop. In this regard, identifying the molecular mechanisms that modulate adipocyte fat accumulation and fatty acid spillover is imperative. Here we identify the deleted in breast cancer 1 (DBC1) protein as a key regulator of fat storage capacity of adipocytes. We found that knockout (KO) of DBC1 facilitated fat cell differentiation and lipid accumulation and increased fat storage capacity of adipocytes in vitro and in vivo. This effect resulted in a "healthy obesity" phenotype. DBC1 KO mice fed a high-fat diet, although obese, remained insulin sensitive, had lower free fatty acid in plasma, were protected against atherosclerosis and liver steatosis, and lived longer. We propose that DBC1 is part of the molecular machinery that regulates fat storage capacity in adipocytes and participates in the "turn-off" switch that limits adipocyte fat accumulation and leads to fat spillover into peripheral tissues, leading to the deleterious effects of caloric surplus.

TRAIL receptor deletion in mice suppresses the inflammation of nutrient excess.

BACKGROUND & AIMS: Low-grade chronic inflammation is a cardinal feature of the metabolic syndrome, yet its pathogenesis is not well defined. The purpose of this study was to examine the role of TRAIL receptor (TR) signaling in the pathogenesis of obesity-associated inflammation using mice with the genetic deletion of TR. METHODS: TR knockout (TR(-/-)) mice and their littermate wild-type (WT) mice were fed a diet high in saturated fat, cholesterol and fructose (FFC) or chow. Metabolic phenotyping, liver injury, and liver and adipose tissue inflammation were assessed. Chemotaxis and activation of mouse bone marrow-derived macrophages (BMDMϕ) was measured. RESULTS: Genetic deletion of TR completely repressed weight gain, adiposity and insulin resistance in FFC-fed mice. Moreover, TR(-/-) mice suppressed steatohepatitis, with essentially normal serum ALT, hepatocyte apoptosis and liver triglyceride accumulation. Gene array data implicated inhibition of macrophage-associated hepatic inflammation in the absence of the TR. In keeping with this, there was diminished accumulation and activation of inflammatory macrophages in liver and adipose tissue. TR(-/-) BMDMϕ manifest reduced chemotaxis and diminished activation of nuclear factor-κ B signaling upon activation by palmitate and lipopolysaccharide. CONCLUSIONS: These data advance the concept that macrophage-associated hepatic and adipose tissue inflammation of nutrient excess requires TR signaling.

Deleted in Breast Cancer 1 regulates cellular senescence during obesity.

Chronic obesity leads to inflammation, tissue dysfunction, and cellular senescence. It was proposed that cellular senescence during obesity and aging drives inflammation and dysfunction. Consistent with this, clearance of senescent cells increases healthspan in progeroid mice. Here, we show that the protein Deleted in Breast Cancer-1 (DBC1) regulates cellular senescence during obesity. Deletion of DBC1 protects preadipocytes against cellular senescence and senescence-driven inflammation. Furthermore, we show protection against cellular senescence in DBC1 KO mice during obesity. Finally, we found that DBC1 participates in the onset of cellular senescence in response to cell damage by mechanism that involves binding and inhibition of HDAC3. We propose that by regulating HDAC3 activity during cellular damage, DBC1 participates in the fate decision that leads to the establishment of cellular senescence and consequently to inflammation and tissue dysfunction during obesity.

Increased TNFalpha and CCAAT/enhancer-binding protein homologous protein with aging predispose preadipocytes to resist adipogenesis.

Fat depot sizes peak in middle age but decrease by advanced old age. This phenomenon is associated with ectopic fat deposition, decreased adipocyte size, impaired differentiation of preadipocytes into fat cells, decreased adipogenic transcription factor expression, and increased fat tissue inflammatory cytokine generation. To define the mechanisms contributing to impaired adipogenesis with aging, we examined the release of TNFalpha, which inhibits adipogenesis, and the expression of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), which blocks activity of adipogenic C/EBP family members, in preadipocytes cultured from young, middle-aged, and old rats. Medium conditioned by fat tissue, as well as preadipocytes, from old rats impeded lipid accumulation by preadipocytes from young animals. More TNFalpha was released by preadipocytes from old than young rats. Differences in TNFalpha-converting enzyme, TNFalpha degradation, or the presence of macrophages in cultures were not responsible. TNFalpha induced rat preadipocyte CHOP expression. CHOP was higher in undifferentiated preadipocytes from old than younger animals. Overexpression of CHOP in young rat preadipocytes inhibited lipid accumulation. TNFalpha short interference RNA reduced CHOP and partially restored lipid accumulation in old rat preadipocytes. CHOP normally increases during late differentiation, potentially modulating the process. This late increase in CHOP was not affected substantially by aging: CHOP was similar in differentiating preadipocytes and fat tissue from old and young animals. Hypoglycemia, which normally causes an adaptive increase in CHOP, was less effective in inducing CHOP in preadipocytes from old than younger animals. Thus increased TNFalpha release by undifferentiated preadipocytes with elevated basal CHOP contributes to impaired adipogenesis with aging.

Fat depot-specific characteristics are retained in strains derived from single human preadipocytes.

Fat depots vary in size, function, and potential contribution to disease. Since fat tissue turns over throughout life, preadipocyte characteristics could contribute to this regional variation. To address whether preadipocytes from different depots are distinct, we produced preadipocyte strains from single abdominal subcutaneous, mesenteric, and omental human preadipocytes by stably expressing human telomere reverse transcriptase (hTERT). These strains could be subcultured repeatedly and retained capacity for differentiation, while primary preadipocyte adipogenesis and replication declined with subculturing. Primary omental preadipocytes, in which telomeres were longest, replicated more slowly than mesenteric or abdominal subcutaneous preadipocytes. Even after 40 population doublings, replication, abundance of the rapidly replicating preadipocyte subtype, and resistance to tumor necrosis factor alpha-induced apoptosis were highest in subcutaneous, intermediate in mesenteric, and lowest in omental hTERT-expressing strains, as in primary preadipocytes. Subcutaneous hTERT-expressing strains accumulated more lipid and expressed more adipocyte fatty acid-binding protein (aP2), peroxisome proliferator-activated receptor gamma2, and CCAAT/enhancer-binding protein alpha than omental cells, as in primary preadipocytes, while hTERT abundance was similar. Thus, despite dividing 40 population doublings, hTERT strains derived from single preadipocytes retained fat depot-specific cell dynamic characteristics, consistent with heritable processes contributing to regional variation in fat tissue function.

Increased CUG triplet repeat-binding protein-1 predisposes to impaired adipogenesis with aging.

Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor gamma (PPARgamma), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBPbeta mRNA, which is upstream of C/EBPalpha and PPARgamma. C/EBPbeta-liver-enriched inhibitory protein (C/EBPbeta-LIP), a truncated C/EBPbeta isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBPbeta mRNA, increasing C/EBPbeta-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBPbeta-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBPbeta-LIP abundance and promoted adipogenesis. Tumor necrosis factor-alpha, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBPbeta-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-alpha. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBPbeta-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.

Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots.

Fat depots vary in function and size. The preadipocytes that fat cells develop from exhibit distinct regional characteristics that persist in culture. Human abdominal subcutaneous cultured preadipocytes undergo more extensive lipid accumulation, higher adipogenic transcription factor expression, and less TNF-alpha-induced apoptosis than omental preadipocytes. We found higher replicative potential in subcutaneous and mesenteric than in omental preadipocytes. In studies of colonies arising from single preadipocytes, two preadipocyte subtypes were found, one capable of more extensive replication, differentiation, and adipogenic transcription factor expression and less apoptosis in response to TNF-alpha than the other. The former was more abundant in subcutaneous and mesenteric than in omental preadipocyte populations, potentially contributing to regional variation in replication, differentiation, and apoptosis. Both subtypes were found in strains derived from single human preadipocytes stably expressing telomerase, confirming that both subtypes are of preadipocyte lineage. After subcloning of cells of either subtype, both subtypes were found, indicating that switching can occur between subtypes. Thus proportions of preadipocyte subtypes with distinct cell-dynamic properties vary among depots, potentially permitting tissue plasticity through subtype selection during development. Furthermore, mesenteric preadipocyte cell-dynamic characteristics are distinct from omental cells, indicating that visceral fat depots are not functionally uniform.