RESUMO
Protein S-nitrosylation is a reversible protein modification implicated in both physiological and pathophysiological regulation of protein function. In obesity, skeletal muscle insulin resistance is associated with increased S-nitrosylation of insulin-signaling proteins. However, whether adipose tissue is similarly affected in obesity and, if so, what are the causes and functional consequences of increased S-nitrosylation in this tissue are unknown. Total protein S-nitrosylation was increased in intra-abdominal adipose tissue of obese humans and in high fat-fed or leptin-deficient ob/ob mice. Both the insulin receptor ß-subunit and Akt were S-nitrosylated, correlating with body weight. Elevated protein and mRNA expression of inducible NO synthase and decreased protein levels of thioredoxin reductase were associated with increased adipose tissue S-nitrosylation. Cultured differentiated pre-adipocyte cell lines exposed to the NO donors S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine exhibited diminished insulin-stimulated phosphorylation of Akt but not of GSK3 nor of insulin-stimulated glucose uptake. Yet the anti-lipolytic action of insulin was markedly impaired in both cultured adipocytes and in mice injected with GSNO prior to administration of insulin. In cells, impaired ability of insulin to diminish phosphorylated PKA substrates in response to isoproterenol suggested impaired insulin-induced activation of PDE3B. Consistently, increased S-nitrosylation of PDE3B was detected in adipose tissue of high fat-fed obese mice. Site-directed mutagenesis revealed that Cys-768 and Cys-1040, two putative sites for S-nitrosylation adjacent to the substrate-binding site of PDE3B, accounted for â¼50% of its GSNO-induced S-nitrosylation. Collectively, PDE3B and the anti-lipolytic action of insulin may constitute novel targets for increased S-nitrosylation of adipose tissue in obesity.
Assuntos
Adipócitos/citologia , Tecido Adiposo/metabolismo , Insulina/metabolismo , Nitrogênio/química , Obesidade/metabolismo , Animais , Cisteína/química , Feminino , Humanos , Resistência à Insulina/fisiologia , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Estresse Oxidativo , FosforilaçãoRESUMO
Obesity is a heterogenous condition that affects the life and health of patients to different degrees and in different ways. Yet, most approaches to treat obesity are not currently prescribed, at least in a systematic manner, based on individual obesity sub-phenotypes or specifically-predicted health risks. Adipose tissue is one of the most evidently affected tissues in obesity. The degree of adipose tissue changes - "adiposopathy", or as we propose to relate to herein as Obesity-related Adipose tissue Disease (OrAD), correspond, at least cross-sectionally, to the extent of obesity-related complications inflicted on an individual patient. This potentially provides an opportunity to better personalize anti-obesity management by utilizing the information that can be retrieved by assessing OrAD. This review article will summarize current knowledge on histopathological OrAD features which, beyond cross-sectional analyses, had been shown to predict future obesity-related endpoints and/or the response to specific anti-obesity interventions. In particular, the review explores adipocyte cell size, adipose tissue inflammation, and fibrosis. Rather than highly-specialized methods, we emphasize standard pathology laboratory approaches to assess OrAD, which are readily-available in most clinical settings. We then discuss how OrAD assessment can be streamlined in the obesity/weight-management clinic. We propose that current studies provide sufficient evidence to inspire concerted efforts to better explore the possibility of predicting obesity related clinical endpoints and response to interventions by histological OrAD assessment, in the quest to improve precision medicine in obesity.
Assuntos
Deficiência de Vitaminas , Medicina de Precisão , Adipócitos/patologia , Tecido Adiposo/patologia , Estudos Transversais , Humanos , Obesidade/complicações , Obesidade/patologia , Obesidade/terapiaRESUMO
Objective: Up-regulated expression of transcription-factor E2F1 in human visceral adipose tissue (VAT) characterizes a dysmetabolic obesity sub-phenotype. An E2F1-miRNA network has been described in multiple cancers. Here we investigated whether elevated VAT-E2F1 in obesity is associated with VAT-miRNA alterations similar to, or distinct from, those described in cancer. Furthermore, we assessed if E2F1-associated miRNA changes may contribute to the link between high- VAT-E2F1 and a dysmetabolic obesity phenotype. Methods: We assembled a cohort of patients with obesity and high-VAT-E2F1, matched by age, sex, ±BMI to patients with low-VAT-E2F1, with and without obesity (8 patients/groupX3 groups). We performed Nanostring©-based miRNA profiling of VAT samples from all 24 patients. Candidate E2F1-related miRNAs were validated by qPCR in an independent cohort of patients with extreme obesity, with or without type-2-diabetes (T2DM) (n = 20). Bioinformatic tools and manipulation of E2F1 expression in cells were used to establish the plausibility of the functional VAT-E2F1-miRNA network in obesity. Results: Among n = 798 identified miRNAs, 17 were differentially expressed in relation to E2F1 and not to obesity itself. No evidence for the cancer-related E2F1-miRNA network was identified in human VAT in obesity. In HEK293-cells, overexpression/downregulation of E2F1 correspondingly altered the expression of miRNA-206 and miRNA-210-5p, two miRNAs with reported metabolic functions consistent with those of E2F1. In VAT from both cohorts, the expression of both miRNA-206 and 210-5p intercorrelated, and correlated with the expression of E2F1. In cohort 1 we did not detect significant associations with biochemical parameters. In cohort 2 of patients with extreme obesity, all those with high VAT-E2F1 showed a diabetes-complicated obesity phenotype and higher expression of miRNA-206 and miRNA-210-5p, which also correlated with fasting glucose levels (both miRNAs) and fasting insulin (miRNA-210-5p). Conclusions: Whilst the previously described cancer-related E2F1-miRNA network does not appear to operate in VAT in obesity, miRNAs-206 and 210-5p may link high-E2F1 expression in VAT with diabetes-complicated extreme obesity phenotype.
Assuntos
Diabetes Mellitus Tipo 2 , MicroRNAs , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Glucose/metabolismo , Células HEK293 , Humanos , Insulina/metabolismo , Gordura Intra-Abdominal/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/genética , Obesidade/metabolismoRESUMO
Autophagy is a major degradative pathway(s) by which intracellular components are delivered into the lysosomes. It is largely implicated in determining cell death and survival because it eliminates unnecessary, damaged, and/or potentially harmful cellular products and organelles and is an important source for nutrients and energy production under conditions of external nutrient deficiency. As such, autophagy has been suggested to contribute to the regulation of carbohydrate and protein metabolism during fasting. Recently, three papers implicated a role for autophagy in cellular lipid metabolism as well. This Perspectives article presents these novel findings in the context of prior studies on the role of autophagy and lysosomes in metabolic and energy regulation, discusses their points of agreement and opposing propositions, and outlines key outstanding questions.
Assuntos
Autofagia/fisiologia , Metabolismo Energético/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lisossomos/metabolismo , Animais , Metabolismo dos Carboidratos/fisiologia , Humanos , Proteínas/metabolismoRESUMO
Elevated expression of E2F1 in adipocyte fraction of human visceral adipose tissue (hVAT) associates with a poor cardiometabolic profile. We hypothesized that beyond directly activating autophagy and MAP3K5 (ASK)-MAP kinase signaling, E2F1 governs a distinct transcriptome that contributes to adipose tissue and metabolic dysfunction in obesity. We performed RNA sequencing of hVAT samples from age-, sex-, and BMI-matched patients, all obese, whose visceral E2F1 protein expression was either high (E2F1high) or low (E2F1low). Tumor necrosis factor superfamily (TNFSF) members, including TRAIL (TNFSF10), TL1A (TNFSF15), and their receptors, were enriched in E2F1high While TRAIL was equally expressed in adipocytes and stromal vascular fraction (SVF), TL1A was mainly expressed in SVF, and TRAIL-induced TL1A was attributed to CD4+ and CD8+ subclasses of hVAT T cells. In human adipocytes, TL1A enhanced basal and impaired insulin-inhibitable lipolysis and altered adipokine secretion, and in human macrophages it induced foam cell biogenesis and M1 polarization. Two independent human cohorts confirmed associations between TL1A and TRAIL expression in hVAT and higher leptin and IL6 serum concentrations, diabetes status, and hVAT-macrophage lipid content. Jointly, we propose an intra-adipose tissue E2F1-associated TNFSF paracrine loop engaging lymphocytes, macrophages, and adipocytes, ultimately contributing to adipose tissue dysfunction in obesity.
Assuntos
Adipócitos/fisiologia , Fator de Transcrição E2F1/metabolismo , Linfócitos/fisiologia , Macrófagos/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Tecido Adiposo/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Técnicas de Cocultura , Fator de Transcrição E2F1/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Comunicação Parácrina , Ligante Indutor de Apoptose Relacionado a TNF/genética , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Adulto JovemRESUMO
Obesity has been proposed to inflict a variety of stresses on adipose tissue, including inflammatory, metabolic, oxidative and endoplasmic reticulum stress. Through the activation of 'stress-sensing pathways', metabolic and endocrine alterations are produced, which probably contribute to the co-morbidities associated with obesity. Here, we review the evidence supporting the development of various obesity-related stresses and the activation of several stress-sensing pathways, specifically in adipocytes and/or adipose tissue, which manifest metabolic and endocrine dysfunction frequently in obesity. As the central role of adipose tissue in regulating whole-body metabolism is elucidated, understanding adipose tissue stress-sensing pathways might provide potential new therapeutic targets to attenuate obesity-related morbidity.
Assuntos
Tecido Adiposo/enzimologia , Obesidade/etiologia , Fosfotransferases/fisiologia , Estresse Fisiológico/enzimologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Humanos , Hipertrofia , Inflamação/enzimologia , Inflamação/etiologia , Modelos Biológicos , Obesidade/complicações , Obesidade/enzimologia , Obesidade/metabolismo , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologiaRESUMO
MAPKs and inhibitory-kappaB kinase (IKK) were suggested to link various conditions thought to develop in adipose tissue in obesity (oxidative, endoplasmic reticulum stress, inflammation) with insulin resistance. Yet whether in obesity these kinases are affected in a fat-depot-differential manner is unknown. We assessed the expression and phosphorylation of these kinases in paired omental and abdominal-sc fat biopsies from 48 severely obese women (body mass index > 32 kg/m(2)). Protein and mRNAs of p38MAPK, ERK, c-Jun kinase-1, and IKKbeta were increased 1.5-2.5-fold in omental vs. sc fat. The phosphorylated (activated) forms of these kinases were also increased to similar magnitudes as the total expression. However, phosphorylation of insulin receptor substrate-1 on Ser312 (equivalent of murine Ser307) was not increased in omental, compared with sc, fat. Consistently, fat tissue fragments stimulated with insulin demonstrated that tyrosine phosphorylation and signal transduction to Akt/protein kinase B in omental fat was not inferior to that observable in sc fat. Comparison with lean women (body mass index 23.2 +/- 2.9 kg/m(2)) revealed similar ERK2 and IKKbeta expression and phosphorylation in both fat depots. However, as compared with lean controls, obese women exhibited 480 and 270% higher amount of the phosphorylated forms of p38MAPK and c-Jun kinase, respectively, in omental, but not sc, fat, and this expression level correlated with clinical parameters of glycemia and insulin sensitivity. Increased expression of stress-activated kinases and IKK and their phosphorylated forms in omental fat occurs in obesity, potentially contributing to differential roles of omental and sc fat in the pathophysiology of obesity.
Assuntos
Quinase I-kappa B/metabolismo , Insulina/farmacologia , Gordura Intra-Abdominal/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Obesidade/metabolismo , Omento/metabolismo , Gordura Subcutânea/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Técnicas In Vitro , Proteínas Substratos do Receptor de Insulina , Gordura Intra-Abdominal/enzimologia , Gordura Intra-Abdominal/patologia , Pessoa de Meia-Idade , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Obesidade/enzimologia , Obesidade/patologia , Omento/enzimologia , Omento/patologia , Proteína Oncogênica v-akt/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Gordura Subcutânea/enzimologia , Gordura Subcutânea/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
CONTEXT: Macrophage infiltration into adipose tissue has been demonstrated to accompany obesity, with a potential preferential infiltration into intraabdominal vs. sc fat. OBJECTIVE: Our objective was to determine whether this occurs across different populations with a range of body mass indexes and to assess the relationship with regional adiposity and comorbidity of obesity. SETTING AND PATIENTS: In two independent cohorts, we used paired omental (OM) and sc fat biopsies from lean controls or predominantly sc or intraabdominally obese persons with minimal comorbidity (n = 60, cohort 1), or from severely obese women with a significant rate of comorbidity (n = 29, cohort 2). RESULTS: Elevated macrophage infiltration into OM vs. sc fat was observable in lean subjects and exaggerated by obesity, particularly if predominantly intraabdominal. This was paralleled by increased monocyte chemoattractant protein-1 (MCP1) and colony-stimulating factor-1 (CSF1) mRNA levels. Level of CSF1 and MCP1 mRNA correlated with the number of OM macrophages (r = 0.521, P < 0.0001 and r = 0.258, P < 0.051, respectively). In severely obese women (mean body mass index = 43.0 +/- 1.1 kg/m(2)), higher protein expression of both MCP1 and CSF1 was detected in OM vs. sc fat. Number of OM macrophages, but not of sc macrophages, correlated with waist circumference (r = 0.636, P = 0.001 vs. r = 0.170, P = 0.427) and with the number of metabolic syndrome parameters (r = 0.385, P = 0.065 vs. r = -0.158, P = 0.472, respectively). Preferential macrophage infiltration into OM fat was mainly observed in a subgroup in whom obesity was associated with impaired glucose homeostasis. CONCLUSIONS: Preferential macrophage infiltration into OM fat is a general phenomenon exaggerated by central obesity, potentially linking central adiposity with increased risk of diabetes and coronary heart disease.
Assuntos
Macrófagos/citologia , Obesidade/imunologia , Obesidade/mortalidade , Omento/citologia , Gordura Subcutânea Abdominal/citologia , Adulto , Idoso , Biópsia , Índice de Massa Corporal , Movimento Celular/imunologia , Quimiocina CCL2/genética , Estudos de Coortes , Comorbidade , Feminino , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Obesidade/patologia , Omento/imunologia , RNA Mensageiro/metabolismo , Gordura Subcutânea Abdominal/imunologiaRESUMO
Adipose tissue inflammation and dysfunction are considered central in the pathogenesis of obesity-related dysmetabolism, but their role in the rapid metabolic recovery upon obesity reversal is less well defined. We hypothesized that changes in adipose tissue endocrine and paracrine mechanisms may support the rapid improvement of obesity-induced impairment in cellular lipid handling. C57Bl-6J mice were fed ad libitum either normal chow (NC) or high-fat diet (HFF) for 10 weeks. A dietary obesity reversal group was fed HFF for 8 weeks and then switched to NC for 2 weeks (HFFâNC). Whole-body glucose homeostasis rapidly nearly normalized in the HFFâNC mice (fasting glucose and insulin fully normalized, glucose and insulin tolerance tests reversed 82% to the NC group levels). During 2 weeks of the dietary reversal, the liver was significantly cleared from ectopic fat, and functionally, glucose production from pyruvate, alanine or fructose was normalized. In contrast, adipose tissue inflammation (macrophage infiltration and polarization) largely remained as in HFF, though obesity-induced adipose tissue macrophage lipid accumulation decreased by ~50%, and adipose tissue MAP kinase hyperactivation was reversed. Ex vivo, mild changes in adipose tissue adipocytokine secretion profile were noted. These corresponded to partial or full reversal of the excess cellular lipid droplet accumulation induced by HFF adipose tissue conditioned media in hepatoma or macrophage cells, respectively. We propose that early after initiating reversal of nutritional obesity, rapid metabolic normalization largely precedes resolution of adipose tissue inflammation. Nevertheless, we demonstrate a hitherto unrecognized contribution of adipose tissue to the rapid improvement in lipid handling by the liver and by macrophages.
Assuntos
Tecido Adiposo/fisiologia , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Macrófagos/fisiologia , Obesidade/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Redução de PesoRESUMO
BACKGROUND: Antiretroviral therapy is frequently associated with adverse metabolic effects and lipodystrophy, but the role of HIV protease inhibitors and the mechanisms involved are poorly understood. The HIV protease inhibitor nelfinavir (NFV) impairs insulin signal propagation by inducing similar signalling defects to those induced by exposure to oxidative stress. AIM: We set out to determine if oxidative stress is involved in NFV-induced insulin resistance in 3T3-L1 adipocytes, and whether antioxidant agents with unique modes of action can prevent this effect. RESULTS: Cells exposed to NFV exhibited the following markers of increased oxidative stress: a decrease in both total and low molecular weight reduced thiols, a 20-fold increase in haem oxygenase 1 (HO-1) mRNA, an increase in intracellular reactive oxygen species production (determined by 2',7'-dichlorofluorescein fluorescence), and increased markers of apoptosis. Enhancing cellular thiols with N-acetylcystein prevented the NFV-induced drop in reduced thiols and partially protected against the induction in HO-1, but failed to prevent insulin resistance or cleavage of poly ADP ribose polymerase (PARP), a process indicative of activation of pro-apoptotic caspases. Conversely, the superoxide dismutase-mimetic antioxidant MnTBAP had no effect on cellular thiols in response to NFV, but protected against HO-1 induction and against the impairment in insulin-stimulated Akt/protein kinase B activation and PARP cleavage. CONCLUSIONS: Induction of oxidative stress plays a role in adipocyte insulin resistance and apoptosis induced by NFV through a radical-dependent but thiol-independent mechanism(s). The results may suggest a new mechanism for the adverse effects of NFV on fat cells, and offer potential new intervention approaches.
Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Antioxidantes/farmacologia , Resistência à Insulina/fisiologia , Nelfinavir/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células 3T3-L1 , Acetilcisteína/farmacologia , Adipócitos/metabolismo , Animais , Insulina/farmacologia , Metaloporfirinas/farmacologia , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Transdução de SinaisRESUMO
Insulin responsiveness of adipocytes is acquired during normal adipogenesis, and is essential for maintaining whole-body insulin sensitivity. Differentiated adipocytes exposed to oxidative stress become insulin resistant, exhibiting decreased expression of genes like the insulin-responsive glucose transporter GLUT4. Here we assessed the effect of oxidative stress on DNA binding capacity of C/EBP isoforms known to participate in adipocyte differentiation, and determine the relevance for GLUT4 gene regulation. By electrophoretic mobility shift assay, nuclear proteins from oxidized adipocytes exhibited decreased binding of C/EBPalpha-containing dimers to a DNA oligonucleotide harboring the C/EBP binding sequence from the murine GLUT4 promoter. C/EBPdelta-containing dimers were increased, while C/EBPbeta-dimers were unchanged. These alterations were mirrored by a 50% decrease and a 2-fold increase in the protein content of C/EBPalpha and C/EBPdelta, respectively. In oxidized cells, GLUT4 protein and mRNA levels were decreased, and a GLUT4 promoter segment containing the C/EBP binding site partially mediated oxidative stress-induced repression of a reported gene. The antioxidant lipoic acid protected against oxidation-induced decrease in GLUT4 and C/EBPalpha mRNA, but did not prevent the increase in C/EBPdelta mRNA. We propose that oxidative stress induces adipocyte insulin resistance partially by affecting the expression of C/EBPalpha and delta, resulting in altered C/EBP-dimer composition potentially occupying the GLUT4 promoter.
Assuntos
Adipócitos/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Estresse Oxidativo/fisiologia , Fatores de Transcrição/metabolismo , Células 3T3-L1/efeitos dos fármacos , Células 3T3-L1/metabolismo , Adipócitos/efeitos dos fármacos , Adipogenia/fisiologia , Animais , Células CHO , Cricetinae , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Peróxido de Hidrogênio/farmacologia , Resistência à Insulina , Camundongos , Estresse Oxidativo/efeitos dos fármacosRESUMO
BACKGROUND: Visceral adipose tissue foam cells are increased in human obesity, and were implicated in adipose dysfunction and increased cardio-metabolic risk. In the circulation, non-classical monocytes (NCM) are elevated in obesity and associate with atherosclerosis and type 2 diabetes. We hypothesized that circulating NCM correlate and/or are functionally linked to visceral adipose tissue foam cells in obesity, potentially providing an approach to estimate visceral adipose tissue status in the non-surgical obese patient. METHODS: We preformed ex-vivo functional studies utilizing sorted monocyte subclasses from healthy donors. Moreover, we assessed circulating blood monocyte subclasses and visceral fat adipose tissue macrophage (ATM) lipid content by flow-cytometry in paired blood and omental-fat samples collected from patients (n = 65) undergoing elective abdominal surgery. RESULTS: Ex-vivo, NCM and NCM-derived macrophages exhibited lower lipid accumulation capacity compared to classical or intermediate monocytes/-derived macrophages. Moreover, of the three subclasses, NCM exhibited the lowest migration towards adipose tissue conditioned-media. In a cohort of n = 65, increased %NCM associated with higher BMI (r = 0.250,p<0.05) and ATM lipid content (r = 0.303,p<0.05). Among patients with BMI≥25Kg/m2, linear regression models adjusted for age, sex or BMI revealed that NCM independently associate with ATM lipid content, particularly in men. CONCLUSIONS: Collectively, although circulating blood NCM are unlikely direct functional precursor cells for adipose tissue foam cells, their increased percentage in the circulation may clinically reflect higher lipid content in visceral ATMs.
Assuntos
Tecido Adiposo/patologia , Lipídeos/química , Macrófagos/metabolismo , Monócitos/metabolismo , Obesidade/sangue , Obesidade/patologia , Adulto , Movimento Celular/efeitos dos fármacos , Estudos de Coortes , Meios de Cultivo Condicionados/farmacologia , Citometria de Fluxo , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Monócitos/citologia , Monócitos/efeitos dos fármacos , Omento/efeitos dos fármacos , Omento/metabolismoRESUMO
In diabetes (type 1 and type 2), increased flux of free fatty acids and glucose is associated with increased mitochondrial reactive oxygen species (ROS) production and, as a consequence, increased oxidative stress. ROS have been shown to activate various cellular stress-sensitive pathways, which can interfere with cellular signaling pathways. Exposure of different cell lines to micromolar concentrations of hydrogen peroxide leads to the activation of stress kinases such as c-Jun N-terminal kinase, p38, I kappaB kinase, and extracellular receptor kinase 1/2. This activation is accompanied by a down-regulation of the cellular response to insulin, leading to a reduced ability of insulin to promote glucose uptake, and glycogen and protein synthesis. The mechanisms leading to this down-regulation in oxidized cells are complicated, involving increased serine/threonine phosphorylation of insulin receptor substrate-1 (IRS1), impaired insulin-stimulated redistribution of IRS1 and phosphatidylinositol-kinase between cytosol and low-density microsomal fraction, followed by a reduced protein kinase-B phosphorylation and GLUT4 translocation to the plasma membrane. In addition, prolonged exposure to ROS affects transcription of glucose transporters: whereas the level of GLUT1 is increased, GLUT4 level is reduced. As can be expected, administration of antioxidants such as lipoic acid in oxidized cells, in animal models of diabetes, and in type 2 diabetes shows improved insulin sensitivity. Thus, oxidative stress is presently accepted as a likely causative factor in the development of insulin resistance.
Assuntos
Resistência à Insulina/fisiologia , Estresse Oxidativo , Animais , Diabetes Mellitus/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Insulina/metabolismo , Fosfotransferases/metabolismoRESUMO
BACKGROUND: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is a major lipid second messenger in insulin-mediated signalling towards the metabolic actions of this hormone in muscle and fat. PURPOSE: Assessing the intracellular transport of exogenous PIP3 attached to a polymeric carrier in an attempt to overcome cellular insulin resistance. METHODS: Artificial chromatic bio-mimetic membrane vesicles composed of dimyristoylphosphatidylcholine and polydiacetylene were applied to screen the polymeric carriers. PIP3 cellular localization and bio-activity was assessed by fluorescent and live-cell microscopy in L6 muscle cells and in 3T3-L1 adipocytes. RESULTS AND DISCUSSION: We demonstrate that a specific-branched polyethylenimine (PEI-25, 25 kDa) carrier forms complexes with PIP3 that interact with the bio-mimetic membrane vesicles in a manner predictive of their interaction with cells: In L6 muscle cells, PEI-25/fluorescent-PIP3 complexes are retarded at the cell perimeter. PEI-25/PIP3 complexes retain their bio-activity, engaging signalling steps downstream of PIP3, even in muscle cells rendered insulin resistant by exposure to high glucose/high insulin. CONCLUSIONS: Inducing insulin actions by intracellular PIP3 delivery (PEI-25/PIP3 complexes) in some forms of insulin-resistant cells provides the first proof-of-principle for the potential therapeutic use of PIP3 in a "second-messenger agonist" approach. In addition, utilization of an artificial bio-mimetic membrane platform to screen for highly efficient PIP3 delivery predicts biological function in cells.
Assuntos
Sistemas de Liberação de Medicamentos , Resistência à Insulina , Insulina/metabolismo , Fosfatos de Fosfatidilinositol/administração & dosagem , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Células Cultivadas , Portadores de Fármacos/química , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fosfatos de Fosfatidilinositol/farmacologia , Polietilenoimina/química , Polímeros/química , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Autophagy genes' expression is upregulated in visceral fat in human obesity, associating with obesity-related cardio-metabolic risk. E2F1 (E2F transcription factor 1) was shown in cancer cells to transcriptionally regulate autophagy. We hypothesize that E2F1 regulates adipocyte autophagy in obesity, associating with endocrine/metabolic dysfunction, thereby, representing non-cell-cycle function of this transcription factor. E2F1 protein (N=69) and mRNA (N=437) were elevated in visceral fat of obese humans, correlating with increased expression of ATG5 (autophagy-related 5), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 ß), but not with proliferation/cell-cycle markers. Elevated E2F1 mainly characterized the adipocyte fraction, whereas MKI67 (marker of proliferation Ki-67) was elevated in the stromal-vascular fraction of adipose tissue. In human visceral fat explants, chromatin-immunoprecipitation revealed body mass index (BMI)-correlated increase in E2F1 binding to the promoter of MAP1LC3B, but not to the classical cell cycle E2F1 target, CCND1 (cyclin D1). Clinically, omental fat E2F1 expression correlated with insulin resistance, circulating free-fatty-acids (FFA), and with decreased circulating ADIPOQ/adiponectin, associations attenuated by adjustment for autophagy genes. Overexpression of E2F1 in HEK293 cells enhanced promoter activity of several autophagy genes and autophagic flux, and sensitized to further activation of autophagy by TNF. Conversely, mouse embryonic fibroblast (MEF)-derived adipocytes from e2f1 knockout mice (e2f1-/-) exhibited lower autophagy gene expression and flux, were more insulin sensitive, and secreted more ADIPOQ. Furthermore, e2f1-/- MEF-derived adipocytes, and autophagy-deficient (by Atg7 siRNA) adipocytes were resistant to cytokines-induced decrease in ADIPOQ secretion. Jointly, upregulated E2F1 sensitizes adipose tissue autophagy to inflammatory stimuli, linking visceral obesity to adipose and systemic metabolic-endocrine dysfunction.
RESUMO
OBJECTIVE: To test agent and cell-type specificity in insulin resistance induced by prolonged exposure to HIV protease inhibitors (HPI), and to assess its relation to the direct, short-term inhibition of insulin-stimulated glucose uptake. METHODS: Following prolonged (18 h) and short (5-10 min) exposure to HPI, insulin-stimulated glucose transport, protein kinase B (PKB) phosphorylation, and GLUT4 translocation were evaluated in 3T3-L1 adipocytes, fibroblasts, L6 myotubes, and L6 cells overexpressing a myc tag on the first exofacial loop of GLUT4 or GLUT1. RESULTS: Prolonged exposure of 3T3-L1 adipocytes to nelfinavir, but not to indinavir or saquinavir, resulted in increased basal lipolysis but decreased insulin-stimulated glucose transport and PKB phosphorylation. In addition, impaired insulin-stimulated glucose uptake and PKB phosphorylation were also observed in the skeletal muscle cell line L6, and in 3T3-L1 fibroblasts. Interestingly, this coincided with increased basal glucose uptake as well as with elevated total-membrane glucose transporter GLUT1 protein content. In contrast to these unique effects of nelfinavir, the mere presence of any of the agents in the 5 min transport assay inhibited insulin-stimulated glucose-uptake activity. This appeared to be caused by direct and specific interaction of the drugs with GLUT4 fully assembled at the plasma membrane, since insulin-stimulated cell-surface exposure of an exofacial myc epitope on GLUT4 was normal. CONCLUSIONS: Independent mechanisms for HPI-induced insulin resistance exist: prolonged exposure to nelfinavir interferes with insulin signaling and alters cellular metabolism of adipocytes and muscle cells, whereas a direct inhibitory effect on insulin-stimulated glucose uptake may occurs through specific interaction of HPI with GLUT4.
Assuntos
Inibidores da Protease de HIV/farmacologia , Resistência à Insulina , Proteínas Serina-Treonina Quinases , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Linhagem Celular , Desoxiglucose/metabolismo , Esquema de Medicação , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Transportador de Glucose Tipo 1 , Humanos , Indinavir/farmacologia , Lipólise/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Nelfinavir/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Saquinavir/farmacologiaRESUMO
OBJECTIVE: To examine if, as in obesity, pregnancies complicated by gestational diabetes mellitus (GDM) exhibit increased macrophage infiltration and activated MAP-kinases in omental adipose tissue. METHODS: Paired omental (OM) and abdominal subcutaneous (SC) fat samples were collected from 11 GDM and 20 normal pregnancies during cesarean delivery. Tissues were stained to detect macrophages, and analyzed to assess MAP-kinases. RESULTS: OM had higher macrophage counts than SC in GDM (6.10 ± 2.20 versus 2.53 ± 1.45, p = 0.04), but not in normal pregnancies (p = 0.346). GDM pregnancies had more macrophages than normal pregnancies in OM (6.10 ± 2.20 versus 1.29 ± 0.55, p = 0.01), while only a trend was observed in SC fat (p = 0.08). Significant correlation (R = 0.619, p = 0.005) was observed between OM-macrophage infiltration and insulin resistance. Using multivariate analysis, only obesity independently associated with GDM. Expression of total p38MAP-kinase was higher in OM versus SC in both normal and GDM pregnancies, without significant differences between these groups. However, expression of activated p-p38MAP-kinase, and its upstream kinase MKK4, was comparable between fat depots. CONCLUSION: GDM pregnancies demonstrate increased macrophage infiltration to OM fat, correlating with higher insulin resistance. As in non-pregnant-patients obesity and OM macrophage infiltration may be on the same causal pathway, leading to GDM. Yet, this occurs without activation of p38MAP-kinase signaling.
Assuntos
Gordura Abdominal , Movimento Celular , Diabetes Gestacional , Macrófagos/fisiologia , Estresse Fisiológico , Gordura Subcutânea , Gordura Abdominal/imunologia , Gordura Abdominal/metabolismo , Adulto , Estudos de Casos e Controles , Movimento Celular/imunologia , Diabetes Gestacional/imunologia , Diabetes Gestacional/metabolismo , Feminino , Humanos , MAP Quinase Quinase 4/metabolismo , Omento , Gravidez , Transdução de Sinais , Gordura Subcutânea/imunologia , Gordura Subcutânea/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1ß was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1ß, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1ß in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1ßKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1ß in regulating hepatic gluconeogenic flux. IL-1ßKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1ß within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1ßKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1ß on hepatocyte insulin resistance. Importantly, although IL-1ßKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1ßKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1ß supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.
Assuntos
Tecido Adiposo/patologia , Comunicação Autócrina , Fígado Gorduroso/patologia , Inflamação/patologia , Interleucina-1beta/metabolismo , Obesidade/complicações , Obesidade/patologia , Tecido Adiposo/transplante , Animais , Técnicas de Cocultura , Dieta Hiperlipídica , Fígado Gorduroso/complicações , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Resistência à Insulina , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismoRESUMO
CONTEXT: Autophagy is a housekeeping mechanism, involved in metabolic regulation and stress response, shown recently to regulate lipid droplets biogenesis/breakdown and adipose tissue phenotype. OBJECTIVE: We hypothesized that in human obesity autophagy may be altered in adipose tissue in a fat depot and distribution-dependent manner. SETTING AND PATIENTS: Paired omental (Om) and subcutaneous (Sc) adipose tissue samples were used from obese and nonobese (n = 65, cohort 1); lean, Sc-obese and intraabdominally obese (n = 196, cohort 2); severely obese persons without diabetes or obesity-associated morbidity, matched for being insulin sensitive or resistant (n = 60, cohort 3). RESULTS: Protein and mRNA levels of the autophagy genes Atg5, LC3A, and LC3B were increased in Om compared with Sc, more pronounced among obese persons, particularly with intraabdominal fat accumulation. Both adipocytes and stromal-vascular cells contribute to the expression of autophagy genes. An increased number of autophagosomes and elevated autophagic flux assessed in fat explants incubated with lysosomal inhibitors were observed in obesity, particularly in Om. The degree of visceral adiposity and adipocyte hypertrophy accounted for approximately 50% of the variance in omental Atg5 mRNA levels by multivariate regression analysis, whereas age, sex, measures of insulin sensitivity, inflammation, and adipose tissue stress were excluded from the model. Moreover, in cohort 3, the autophagy marker genes were increased in those who were insulin resistant compared with insulin sensitive, particularly in Om. CONCLUSIONS: Autophagy is up-regulated in adipose tissue of obese persons, especially in Om, correlating with the degree of obesity, visceral fat distribution, and adipocyte hypertrophy. This may co-occur with insulin resistance but precede the occurrence of obesity-associated morbidity.
Assuntos
Tecido Adiposo/patologia , Autofagia/fisiologia , Obesidade/patologia , Adulto , Biomarcadores , Biópsia , Western Blotting , Índice de Massa Corporal , Estudos de Coortes , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Omento/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Gordura Subcutânea/patologia , Bancos de TecidosRESUMO
Central obesity is frequently associated with adipose tissue inflammation and hepatic insulin resistance. To identify potential individual mediators in this process, we used in vitro systems and assessed if insulin resistance in liver cells could be induced by secreted products from adipocytes preexposed to an inflammatory stimulus. Conditioned medium from 3T3-L1 adipocytes pretreated without (CM) or with TNFalpha (CM-TNFalpha) was used to treat Fao hepatoma cells. ELISAs were used to assess the concentration of several inflammatory mediators in CM-TNFalpha. CM-TNFalpha-treated Fao cells exhibited about 45% diminution in insulin-stimulated phosphorylation of insulin receptor, insulin receptor substrate proteins, protein kinase B, and glycogen synthase kinase-3 as compared with CM-treated cells, without changes in the total abundance of these protein. Insulin increased glycogenesis by 2-fold in CM-treated Fao cells but not in cells exposed to CM-TNFalpha. Expression of IL-1beta mRNA was elevated 3-fold in TNFalpha-treated adipocytes, and CM-TNFalpha had 10-fold higher concentrations of IL-1beta but not TNFalpha or IL-1alpha. IL-1beta directly induced insulin resistance in Fao, HepG2, and in primary rat hepatocytes. Moreover, when TNFalpha-induced secretion/production of IL-1beta from adipocytes was inhibited by the IL-1 converting enzyme (ICE-1) inhibitor II (Ac-YVAD-CMK), insulin resistance was prevented. Furthermore, liver-derived cells treated with IL-1 receptor antagonist were protected against insulin resistance induced by CM-TNFalpha. Finally, IL-1beta secretion from human omental fat explants correlated with body mass index (R(2) = 0.639, P < 0.01), and the resulting CM induced insulin resistance in HepG2 cells, inhibitable by IL-1 receptor antagonist. Our results suggest that adipocyte-derived IL-1beta may constitute a mediator in the perturbed cross talk between adipocytes and liver cells in response to adipose tissue inflammation.