RESUMO
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.
Assuntos
Senescência Celular , Proteína Supressora de Tumor p53 , Feminino , Humanos , Adulto , Proteína Supressora de Tumor p53/farmacologia , Envelhecimento , Obesidade , Gordura SubcutâneaRESUMO
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.
Assuntos
COVID-19/virologia , Senescência Celular , SARS-CoV-2/patogenicidade , Receptor 3 Toll-Like/metabolismo , Envelhecimento , Animais , Apoptose , COVID-19/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Inflamação , Pulmão/metabolismo , Camundongos , Fenótipo , Proteínas Virais , Tratamento Farmacológico da COVID-19RESUMO
Preadipocytes dynamically produce sensory cilia. However, the role of primary cilia in preadipocyte differentiation and adipose homeostasis remains poorly understood. We previously identified transition fiber component FBF1 as an essential player in controlling selective cilia import. Here, we establish Fbf1tm1a/tm1a mice and discover that Fbf1tm1a/tm1a mice develop severe obesity, but surprisingly, are not predisposed to adverse metabolic complications. Obese Fbf1tm1a/tm1a mice possess unexpectedly healthy white fat tissue characterized by spontaneous upregulated beiging, hyperplasia but not hypertrophy, and low inflammation along the lifetime. Mechanistically, FBF1 governs preadipocyte differentiation by constraining the beiging program through an AKAP9-dependent, cilia-regulated PKA signaling, while recruiting the BBS chaperonin to transition fibers to suppress the hedgehog signaling-dependent adipogenic program. Remarkably, obese Fbf1tm1a/tm1a mice further fed a high-fat diet are protected from diabetes and premature death. We reveal a central role for primary cilia in the fate determination of preadipocytes and the generation of metabolically healthy fat tissue.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Tecido Adiposo Bege/metabolismo , Tecido Adiposo Branco/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/metabolismo , Adipogenia , Animais , Respiração Celular , Cílios/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Homozigoto , Humanos , Hiperfagia/complicações , Hiperfagia/patologia , Hiperplasia , Inflamação/patologia , Masculino , Síndrome Metabólica/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Obesidade/complicações , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
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 (Dâ¯+â¯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, Dâ¯+â¯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 3â¯days of oral D 100â¯mg and Q 1000â¯mg to subjects with diabetic kidney disease (Nâ¯=â¯9; 68·7⯱â¯3·1â¯years old; 2 female; BMI:33·9⯱â¯2·3â¯kg/m2; eGFR:27·0⯱â¯2·1â¯mL/min/1·73m2). Adipose tissue, skin biopsies, and blood were collected before and 11â¯days after completing senolytic treatment. Senescent cell and macrophage/Langerhans cell markers and circulating SASP factors were assayed. FINDINGS: Dâ¯+â¯Q reduced adipose tissue senescent cell burden within 11â¯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 Dâ¯+â¯Q have elimination half-lives <11â¯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.
Assuntos
Senescência Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Nefropatias Diabéticas/metabolismo , Quercetina/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Idoso , Biomarcadores , Biópsia , Ensaios Clínicos Fase I como Assunto , Dasatinibe/uso terapêutico , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/tratamento farmacológico , Quimioterapia Combinada , Feminino , Humanos , Imuno-Histoquímica , Testes de Função Renal , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Quercetina/uso terapêuticoRESUMO
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.
Assuntos
Ansiedade/etiologia , Senescência Celular/efeitos dos fármacos , Neurogênese , Obesidade/complicações , Animais , Ansiedade/tratamento farmacológico , Astrócitos/metabolismo , Comportamento Animal/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/embriologia , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dasatinibe/farmacologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Gotículas Lipídicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/etiologia , Quercetina/farmacologia , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Tacrolimo/uso terapêuticoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Flavonoides/farmacologia , Proteína bcl-X/antagonistas & inibidores , Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Flavonóis , Humanos , Sulfonamidas/farmacologiaRESUMO
BACKGROUND AND OBJECTIVES: New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. DESIGN: Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. RESULTS: In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. CONCLUSIONS: PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.
Assuntos
Adipócitos/metabolismo , Adrenomedulina/metabolismo , Exossomos/metabolismo , Lipólise , Sistema de Sinalização das MAP Quinases , Neoplasias Pancreáticas/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Endocitose/fisiologia , Glicerol/metabolismo , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Adrenomedulina/antagonistas & inibidores , Receptores de Adrenomedulina/metabolismo , Gordura Subcutânea/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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.
Assuntos
Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Dasatinibe/farmacologia , Embrião de Mamíferos/citologia , Endonucleases/deficiência , Endonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Modelos Biológicos , Quercetina/farmacologia , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Adipócitos/enzimologia , Senescência Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Janus Quinases/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adipócitos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/enzimologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Senescência Celular/genética , Matriz Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Macrófagos/citologia , Macrófagos/enzimologia , Camundongos , RNA Interferente Pequeno/genética , Transdução de Sinais/genéticaRESUMO
OBJECTIVE: Visceral white adipose tissue (WAT) expansion and macrophage accumulation are associated with metabolic dysfunction. Visceral WAT typically shows greater macrophage infiltration. Preadipocytes show varying proinflammatory expression profiles among WAT depots. The objective was to examine the secretomes and chemoattractive properties of preadipocytes from visceral and subcutaneous WAT. METHODS: A label-free quantitative proteomics approach was applied to study secretomes of subcutaneous and omental preadipocytes from obese subjects. Enzyme-linked immunosorbent assays and chemotaxis assays were used to confirm proinflammatory chemokine secretion between depots. RESULTS: Preadipocyte secretomes showed greater variation between depots than did intracellular protein expression. Chemokines were the most differentially secreted proteins. Omental preadipocytes induced chemoattraction of macrophages and monocytes. Neutralizing antibodies to the identified chemokines reduced macrophage/monocyte chemoattraction. Subcutaneous preadipocytes treated with interleukin-6 (IL-6) resembled omental preadipocytes in terms of chemokine secretion and macrophage/monocyte chemoattraction. Janus-activated kinase (JAK1/2) protein expression, which transduces IL-6 signaling, was higher in omental than subcutaneous preadipocytes and WAT. Inhibiting JAK in omental preadipocytes decreased chemokine secretion and macrophage/monocyte chemoattraction to levels closer to that observed in subcutaneous preadipocytes. CONCLUSIONS: Secretomes of omental and subcutaneous preadipocytes are distinct, with the former inducing more macrophage/monocyte chemoattraction, in part through IL-6/JAK-mediated signaling.
Assuntos
Adipócitos Brancos/metabolismo , Inflamação/metabolismo , Obesidade/metabolismo , Omento/metabolismo , Gordura Subcutânea/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-6/metabolismo , Gordura Intra-Abdominal/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Proteínas/metabolismoRESUMO
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.
Assuntos
Envelhecimento/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Osteoporose/prevenção & controle , Quercetina/farmacologia , Transcriptoma , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Senescência Celular/genética , Classe I de Fosfatidilinositol 3-Quinases , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Combinação de Medicamentos , Endonucleases/genética , Endonucleases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Efrinas/genética , Efrinas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Coração/efeitos dos fármacos , Coração/fisiopatologia , Disco Intervertebral/química , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Knockout , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Body fat distribution is an important predictor of the metabolic consequences of obesity, but the cellular mechanisms regulating regional fat accumulation are unknown. We assessed the changes in adipocyte size (photomicrographs) and number in response to overfeeding in upper- and lower-body s.c. fat depots of 28 healthy, normal weight adults (15 men) age 29 ± 2 y. We analyzed how these changes relate to regional fat gain (dual energy X-ray absorptiometry and computed tomography) and baseline preadipocyte proliferation, differentiation [peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α (C/EBPα) mRNA]), and apoptotic response to TNF-α. Fat mass increased by 1.9 ± 0.2 kg in the upper body and 1.6 ± 0.1 kg in the lower body. Average abdominal s.c. adipocyte size increased by 0.16 ± 0.06 µg lipid per cell and correlated with relative upper-body fat gain (r = 0.74, P < 0.0001). However, lower-body fat responded to overfeeding by fat-cell hyperplasia, with adipocyte number increasing by 2.6 ± 0.9 × 10(9) cells (P < 0.01). We found no depot-differences in preadipocyte replication or apoptosis that would explain lower-body adipocyte hyperplasia and abdominal s.c. adipocyte hypertrophy. However, baseline PPARγ2 and C/EBPα mRNA were higher in abdominal than femoral s.c. preadipocytes (P < 0.005 and P < 0.03, respectively), consistent with the ability of abdominal s.c. adipocytes to achieve a larger size. Inherent differences in preadipocyte cell dynamics may contribute to the distinct responses of different fat depots to overfeeding, and fat-cell number increases in certain depots in adults after only 8 wk of increased food intake.
Assuntos
Tecido Adiposo/metabolismo , Ingestão de Energia , Absorciometria de Fóton , Adipócitos/metabolismo , Tecido Adiposo/citologia , Adulto , Composição Corporal , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Feminino , Humanos , Masculino , PPAR gama/genética , PPAR gama/metabolismo , Tomografia Computadorizada por Raios X , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To elucidate cellular mechanisms of sex-related differences in fat distribution, we determined body fat distribution (dual-energy X-ray absorptiometry and single-slice abdominal computed tomography (CT)), adipocyte size, adipocyte number, and proportion of early-differentiated adipocytes (aP2(+)CD68(-)) in the stromovascular fraction (SVF) in the upper and lower body of normal-weight healthy men (n = 12) and premenopausal women (n = 20) (age: 18-49 years, BMI: 18-26 kg/m(2)). Women had more subcutaneous and less visceral fat than men. The proportion of early differentiated adipocytes in the subcutaneous adipose tissue SVF of women was greater than in men (P = 0.01), especially in the femoral depot, although in vitro adipogenesis, as assessed by peroxisome proliferator activated receptor-γ (PPARγ) expression, was not increased in femoral preadipocytes cultured from women compared with men. In women, differentiation of femoral preadipocytes was less than that of abdominal subcutaneous preadipocytes (P = 0.04), and femoral subcutaneous preadipocytes tended to be more resistant to tumor necrosis factor-α (TNFα)-induced apoptosis (P = 0.06). Thus, turnover and utilization of the preadipocyte pool may be reduced in lower vs. the upper-body fat in women. Collectively, these data indicate that the microenvironment, rather than differences in inherent properties of preadipocytes between genders, may explain the gynoid obesity phenotype and higher percent body fat in women compared to men.
Assuntos
Adipócitos , Adipogenia , Distribuição da Gordura Corporal , Gordura Intra-Abdominal , Obesidade , Caracteres Sexuais , Gordura Subcutânea , Adipócitos/citologia , Adipócitos/metabolismo , Adiposidade , Adolescente , Adulto , Apoptose , Feminino , Fêmur , Humanos , Gordura Intra-Abdominal/citologia , Gordura Intra-Abdominal/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/patologia , PPAR gama/metabolismo , Valores de Referência , Gordura Subcutânea/citologia , Gordura Subcutânea/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
White adipose tissue is intimately involved in the regulation of immunity and inflammation. We reported that human mesenteric preadipocytes express the substance P (SP)-mediated neurokinin-1 receptor (NK-1R), which signals proinflammatory responses. Here we tested the hypothesis that SP promotes proliferation and survival of human mesenteric preadipocytes and investigated responsible mechanism(s). Preadipocytes were isolated from mesenteric fat biopsies during gastric bypass surgery. Proliferative and antiapoptotic responses were delineated in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), bromodeoxyuridine (BrdU), caspase-3, and TUNEL assays, as well as Western immunoanalysis. SP (10(-7) M) increased MTS and proliferation (BrdU) and time dependently (15-30 min) induced Akt, EGF receptor, IGF receptor, integrin alphaVbeta3, phosphatidylinositol 3-kinase, and PKC-theta phosphorylation. Furthermore, pharmacological antagonism of Akt and PKC-theta activation significantly attenuated SP-induced preadipocyte proliferation. Exposure of preadipocytes to the proapoptotic Fas ligand (FasL, 100 microM) resulted in nuclear DNA fragmentation (TUNEL assay), as well as increased cleaved poly (ADP-ribose) polymerase, cleaved caspase-7, and caspase-3 expression. Cotreatment with SP almost completely abolished these responses in a NK-1R-dependent fashion. SP (10(-7) M) also time dependently stimulated expression 4E binding protein 1 and phosphorylation of p70 S6 kinase, which increased protein translation efficiency. SP increases preadipocyte viability, reduces apoptosis, and stimulates proliferation, possibly via cell cycle upregulation and increased protein translation efficiency. SP-induced proliferative and antiapoptotic pathways in fat depots may contribute to development of the creeping fat and inflammation characteristic of Crohn's disease.
Assuntos
Adipócitos/metabolismo , Apoptose , Proliferação de Células , Gordura Intra-Abdominal/metabolismo , Transdução de Sinais , Substância P/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Adipócitos/patologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Receptores ErbB/metabolismo , Proteína Ligante Fas/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/enzimologia , Gordura Intra-Abdominal/patologia , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores da Neurocinina-1/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Adipócitos/citologia , Gordura Abdominal/citologia , Tecido Adiposo/citologia , Adolescente , Adulto , Idoso , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Mesentério/citologia , Pessoa de Meia-Idade , Omento/citologia , Células-Tronco/citologia , Gordura Subcutânea/citologia , Telomerase/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
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.
Assuntos
Adipócitos/citologia , Tecido Adiposo/citologia , Células-Tronco/citologia , Adulto , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Comunicação Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco/classificação , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Fat distribution varies among individuals with similar body fat content. Innate differences in adipose cell characteristics may contribute because lipid accumulation and lipogenic enzyme activities vary among preadipocytes cultured from different fat depots. We determined expression of the adipogenic transcription factors peroxisome proliferator activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer binding protein-alpha (C/EBP-alpha) and their targets in abdominal subcutaneous, mesenteric, and omental preadipocytes cultured in parallel from obese subjects. Subcutaneous preadipocytes, which had the highest lipid accumulation, glycerol-3-phosphate dehydrogenase (G3PD) activity, and adipocyte fatty acid binding protein (aP2) abundance, had highest PPAR-gamma and C/EBP-alpha expression. Levels were intermediate in mesenteric and lowest in omental preadipocytes. Overexpression of C/EBP-alpha in transfected omental preadipocytes enhanced differentiation. The proportion of differentiated cells in colonies derived from single subcutaneous preadipocytes was higher than in mesenteric or omental clones. Only cells that acquired lipid inclusions exhibited C/EBP-alpha upregulation, irrespective of depot origin. Thus regional variation in adipogenesis depends on differences at the level of transcription factor expression and is a trait conferred on daughter cells.