RESUMO
OBJECTIVE: Arterial thrombosis may be initiated by endothelial inflammation or denudation, activation of blood-borne elements or the coagulation system. Tissue factor (TF), a central trigger of the coagulation cascade, is regulated by the pro-inflammatory NF-κB-dependent pathways. Sirtuin 6 (SIRT6) is a nuclear member of the sirtuin family of NAD+-dependent deacetylases and is known to inhibit NF-κB signaling. Its constitutive deletion in mice shows early lethality with hypoglycemia and accelerated aging. Of note, the role of SIRT6 in arterial thrombosis remains unknown. Thus, we hypothesized that endothelial SIRT6 protects from arterial thrombosis by modulating inhibition of NF-κB-associated pathways. APPROACH AND RESULTS: Using a laser-induced carotid thrombosis model, in vivo arterial occlusion occurred 45% faster in 12-week-old male endothelial-specific Sirt6-/- mice as compared to Sirt6fl/fl controls (n ≥ 9 per group; p = 0.0012). Levels of procoagulant TF were increased in animals lacking endothelial SIRT6 as compared to control littermates. Similarly, in cultured human aortic endothelial cells, SIRT6 knockdown increased TF mRNA, protein and activity. Moreover, SIRT6 knockdown increased mRNA levels of NF-κB-associated genes tumor necrosis factor alpha (TNF-α), poly [ADP-ribose] polymerase 1 (PARP-1), vascular cell adhesion molecule 1 (VCAM-1), and cyclooxygenase-2 (COX-2); at the protein level, COX-2, VCAM-1, TNF-α, and cleaved PARP-1 remained increased after Sirt6 knockdown. CONCLUSIONS: Endothelium-specific Sirt6 deletion promotes arterial thrombosis in mice. In cultured human aortic endothelial cells, SIRT6 silencing enhances TF expression and activates pro-inflammatory pathways including TNF-α, cleaved PARP-1, VCAM-1 and COX-2. Hence, endogenous endothelial SIRT6 exerts a protective role in experimental arterial thrombosis.
Assuntos
Sirtuínas , Trombose , Animais , Humanos , Masculino , Camundongos , Células Cultivadas , Ciclo-Oxigenase 2 , Células Endoteliais , NF-kappa B , Inibidores de Poli(ADP-Ribose) Polimerases , Sirtuínas/genética , Trombose/genética , Fator de Necrose Tumoral alfa , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
AIMS: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. METHODS AND RESULTS: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe-/- mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. CONCLUSIONS: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Medula Óssea/patologia , Deleção de Genes , Receptores Depuradores Classe A/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Transplante de Medula Óssea , Regulação para Baixo , Técnicas de Silenciamento de Genes , Hematopoese , Homozigoto , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Modelos Biológicos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células RAW 264.7RESUMO
Aims: Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI). Methods and results: Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3-/- mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3-/- compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3-/- mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3-/- bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3-/- mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3-/- neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01). Conclusions: Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.
Assuntos
Coagulação Sanguínea , Doenças das Artérias Carótidas/enzimologia , Armadilhas Extracelulares/enzimologia , Neutrófilos/enzimologia , Sirtuína 3/deficiência , Tromboplastina/metabolismo , Trombose/enzimologia , Animais , Coagulação Sanguínea/genética , Doenças das Artérias Carótidas/sangue , Doenças das Artérias Carótidas/genética , Estudos de Casos e Controles , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Estudos Prospectivos , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Infarto do Miocárdio com Supradesnível do Segmento ST/enzimologia , Sirtuína 3/sangue , Sirtuína 3/genética , Superóxido Dismutase/metabolismo , Trombose/sangue , Trombose/genética , Fatores de TempoRESUMO
Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress. We have reported accelerated weight gain and impaired metabolic flexibility in atherosclerotic Sirt3 (-/-) mice. Oxidative stress is a hallmark of endothelial dysfunction. Yet, the role of Sirt3 in this context remains unknown. Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress. Knockdown of Sirt3 in human aortic endothelial cells (HAEC) increased intracellular mitochondrial superoxide accumulation, as assessed by electron spin resonance spectroscopy and fluorescence imaging. Endothelium-dependent relaxation of aortic rings from Sirt3 (-/-) mice exposed to a normal diet did not differ from wild-type controls. However, following 12 weeks of high-cholesterol diet and increasing oxidative stress, endothelial function of Sirt3 (-/-) mice was mildly impaired compared with wild-type controls. Relaxation was restored upon enhanced superoxide scavenging using pegylated superoxide dismutase. Knockdown of Sirt3 in cultured HAEC diminished SOD2 specific activity, which was compensated for by a CCAAT/enhancer binding protein beta (C/EBP-ß)-dependent transcriptional induction of SOD2. Abrogation of this feedback regulation by simultaneous knockdown of C/EBP-ß and Sirt3 exacerbated mitochondrial superoxide accumulation and culminated into endothelial cell death upon prolonged culture. Taken together, Sirt3 deficiency induces a mild, superoxide-dependent endothelial dysfunction in mice fed a high-cholesterol diet. In cultured endothelial cells, a novel C/EBP-ß-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Dieta Hiperlipídica , Células Endoteliais/metabolismo , Estresse Oxidativo/fisiologia , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Espectroscopia de Ressonância de Spin Eletrônica , Retroalimentação Fisiológica/fisiologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Sirtuína 3/deficiência , TransfecçãoRESUMO
OBJECTIVES: Levels of inflammatory biomarkers associate with changes of coronary atheroma burden in statin-treated patients with stable coronary artery disease. This study sought to determine changes of plaque composition in vivo in relation to high-sensitivity C-reactive protein (hs-CRP) levels in patients with ST-elevation myocardial infarction (STEMI) receiving high-intensity statin therapy. METHODS: The IBIS-4 study performed serial (baseline and 13-month), 2-vessel intravascular ultrasound (IVUS) and radiofrequency-IVUS of the non-infarct-related arteries in patients with STEMI treated with high-intensity statin therapy. The present analysis included 44 patients (80 arteries) with serial measurements of hs-CRP. RESULTS: At follow-up, median low-density lipoprotein cholesterol (LDL-C) levels decreased from 126 to 77 mg/dl, HDL-C increased from 44 to 47 mg/dl, and hs-CRP decreased from 1.6 to 0.7 mg/L. Regression of percent atheroma volume (-0.99%, 95% CI -1.84 to -0.14, p = 0.024) was accompanied by reduction of percent fibro-fatty (p = 0.04) and fibrous tissue (p < 0.001), and increase in percent necrotic core (p = 0.006) and dense calcium (p < 0.001). Follow-up levels of hs-CRP, but not LDL-C, correlated with changes in percent necrotic core (p = 0.001) and inversely with percent fibrous tissue volume (p = 0.008). Similarly, baseline-to-follow-up change of hs-CRP correlated with the change in percent necrotic core volume (p = 0.02). CONCLUSIONS: In STEMI patients receiving high-intensity statin therapy, stabilization of VH-IVUS-defined necrotic core was confined to patients with lowest on-treatment levels and greatest reduction of hs-CRP. Elevated CRP levels at follow-up may identify progression of high-risk coronary plaque composition despite intensive statin therapy and overall regression of atheroma volume.
Assuntos
Anti-Inflamatórios/uso terapêutico , Proteína C-Reativa/metabolismo , Doença da Artéria Coronariana/tratamento farmacológico , Vasos Coronários/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Mediadores da Inflamação/sangue , Infarto do Miocárdio/tratamento farmacológico , Placa Aterosclerótica , Idoso , Biomarcadores/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Feminino , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico por imagem , Necrose , Estudos Prospectivos , Fatores de Tempo , Resultado do Tratamento , Ultrassonografia de IntervençãoRESUMO
AIMS: The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture. METHODS AND RESULTS: Apolipoprotein E-deficient (Apoe(-/-)) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg(-1) diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr(-/-) mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis. CONCLUSION: We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis.
Assuntos
Arteriosclerose/prevenção & controle , Hepatócitos/metabolismo , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Sirtuína 1/metabolismo , Anilidas/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Apolipoproteínas E/deficiência , Células Cultivadas , LDL-Colesterol/metabolismo , Inibidores Enzimáticos/farmacologia , Hepatócitos/efeitos dos fármacos , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , RNA Mensageiro/metabolismo , Receptores de LDL/efeitos dos fármacos , Tiazóis/farmacologiaRESUMO
Sirt3 is a mitochondrial NAD(+)-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR(-/-)) and LDLR/Sirt3 double-knockout (Sirt3(-/-)LDLR(-/-)) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR(-/-) mice. Sirt3 deficiency does not affect atherosclerosis in LDLR(-/-) mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.
Assuntos
Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/fisiopatologia , Sirtuína 3/deficiência , Animais , Western Blotting , Modelos Animais de Doenças , Imunofluorescência , Homeostase , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Receptores de LDL/deficiência , Receptores de LDL/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de RiscoRESUMO
AIMS: Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. METHODS AND RESULTS: C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. CONCLUSION: Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Endotélio Vascular/efeitos dos fármacos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Obesidade/etiologia , Receptores de Mineralocorticoides/fisiologia , Espironolactona/análogos & derivados , Tecido Adiposo/efeitos dos fármacos , Aldosterona/metabolismo , Animais , Antioxidantes/metabolismo , Aorta/efeitos dos fármacos , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Eplerenona , Glutationa Peroxidase/metabolismo , Hiperglicemia/tratamento farmacológico , Inflamação/tratamento farmacológico , Oxirredutases Intramoleculares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Espironolactona/farmacologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Regulação para Cima , Glutationa Peroxidase GPX1RESUMO
The immune competent abdominal adipose tissue, either stored viscerally [visceral adipose tissue (VAT)] or sc [sc adipose tissue (SAT)], has been identified as a source of IL-1ß and IL-18. To become active, the proforms of these cytokines require processing by caspase-1, which itself is mediated by the inflammasome. In this descriptive study, we investigate the expression of inflammasome components and caspase-1 in human fat and determine whether caspase-1 activity contributes to the enhanced inflammatory status of VAT. Paired SAT and VAT biopsies from 10 overweight subjects (body mass index, 25-28 kg/m(2)) were used to study the cellular composition and the intrinsic inflammatory capacity of both adipose tissue depots. The percentage of CD8(+) T cells within the lymphocyte fraction was significantly higher in VAT compared with SAT (41.6 vs. 30.4%; P < 0.05). Adipose tissue cultures showed a higher release of IL-1ß (10-fold; P < 0.05), IL-18 (3-fold; P < 0.05), and IL-6 and IL-8 (3-fold, P < 0.05; and 4-fold, P < 0.05, respectively) from VAT compared with SAT that was significantly reduced by inhibiting caspase-1 activity. In addition, caspase-1 activity was 3-fold (P < 0.05) higher in VAT compared with SAT, together with an increase in the protein levels of the inflammasome members apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain (2-fold; P < 0.05) and nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (2-fold; nonsignificant). Finally, caspase-1 activity levels were positively correlated with the percentage of CD8(+) T cells present in adipose tissue. Our results show that caspase-1 and nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 inflammasome members are abundantly present in human VAT. The increased intrinsic caspase-1 activity in VAT represents a novel and specific inflammatory pathway that may determine the proinflammatory character of this specific depot.
Assuntos
Caspase 1/fisiologia , Inflamassomos/fisiologia , Inflamação/etiologia , Gordura Intra-Abdominal/imunologia , Adulto , Linfócitos T CD8-Positivos/imunologia , Ativação Enzimática , Feminino , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Gordura Intra-Abdominal/enzimologia , Masculino , Pessoa de Meia-Idade , Gordura Subcutânea/imunologiaRESUMO
OBJECTIVE: Obesity is characterized by elevated levels of proinflammatory cytokines, including interleukin (IL)-1ß, that contribute to the development of insulin resistance. In this study, we set out to investigate whether hyperglycemia drives IL-1ß production and caspase-1 activation in murine and human adipose tissue, thus inducing insulin resistance. RESEARCH DESIGN AND METHODS: ob/ob animals were used as a model to study obesity and hyperglycemia. Human adipose tissue fragments or adipocytes were cultured in medium containing normal or high glucose levels. Additionally, the role of thioredoxin interacting protein (TXNIP) in glucose-induced IL-1ß production was assessed. RESULTS: TXNIP and caspase-1 protein levels were more abundantly expressed in adipose tissue of hyperglycemic ob/ob animals as compared with wild-type mice. In human adipose tissue, high glucose resulted in a 10-fold upregulation of TXNIP gene expression levels (P < 0.01) and a 10% elevation of caspase-1 activity (P < 0.05), together with induction of IL-1ß transcription (twofold, P < 0.01) and a significant increase in IL-1ß secretion. TXNIP suppression in human adipocytes, either by a small interfering RNA approach or a peroxisome proliferator-activated receptor-γ agonist, counteracted the effects of high glucose on bioactive IL-1 production (P < 0.01) mainly through a decrease in transcription levels paralleled by reduced intracellular pro-IL-1ß levels. CONCLUSIONS: High glucose activates caspase-1 in human and murine adipose tissue. Glucose-induced activation of TXNIP mediates IL-1ß mRNA expression levels and intracellular pro-IL-1ß accumulation in adipose tissue. The concerted actions lead to enhanced secretion of IL-1ß in adipose tissue that may contribute to the development of insulin resistance.
Assuntos
Tecido Adiposo/metabolismo , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Hiperglicemia/genética , Interleucina-1beta/genética , Obesidade/metabolismo , Análise de Variância , Animais , Western Blotting , Glucose/metabolismo , Glucose/farmacologia , Humanos , Hiperglicemia/metabolismo , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Obesos , Obesidade/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Obesity is characterized by chronic low-grade inflammation originating from expanding adipose tissue. In the present study, we examined the adipogenic expression levels of IL-1F6 and IL-1F8, both members of the IL-1 family of cytokines, and their effects on adipose tissue gene expression. Although IL-1F6 is primarily present in adipose tissue resident macrophages and induced by inflammation, IL-1F8 is absent. IL-1F6, but not IL-1F8, reduces adipocyte differentiation, as shown by a significant decrease in PPARγ gene expression. Finally, both IL-1F6 and IL-1F8 are able to induce inflammatory gene expression in mature adipocytes. In conclusion, we demonstrate for the first time that IL-1F6 is present in adipose tissue and that IL-1F6 and IL-1F8 are involved in the regulation of adipose tissue gene expression. Importantly, IL-1F6 inhibits PPARγ expression which may lead to reduced adipocyte differentiation suggesting metabolic effects of this cytokine.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Regulação da Expressão Gênica , Inflamação/metabolismo , Interleucinas/metabolismo , Obesidade/metabolismo , PPAR gama/metabolismo , Adipócitos/citologia , Animais , Diferenciação Celular , Linhagem Celular , Humanos , Inflamação/etiologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/complicações , Obesidade/genética , PPAR gama/genéticaRESUMO
OBJECTIVE: Familial combined hyperlipidemia (FCH) is a common familial lipid disorder characterized by increases in plasma total cholesterol, triglyceride, and apolipoprotein B-100 levels. In light of prior metabolic and genetic research, our purpose was to ascertain whether FCH cases had significant abnormalities of plasma markers of cholesterol synthesis and absorption as compared to unaffected kindred members. METHODS AND RESULTS: Plasma levels of squalene, desmosterol, and lathosterol (cholesterol synthesis markers) and campesterol, sitosterol, and cholestanol (cholesterol absorption markers) were measured by gas-liquid chromatography in 103 FCH patients and 240 normolipidemic relatives (NLR). Squalene, desmosterol, and lathosterol levels were 6% (0.078), 31%, (P<0.001) and 51% (P<0.001) higher in FCH as compared to NLR, and these differences were especially pronounced in women. An interaction with obesity was also noted for a subset of these markers. We did not observe any apparent differences for the cholesterol absorption markers among FCH patients and NLR. CONCLUSIONS: Our data indicate that both men and women with FCH have alterations in the cholesterol synthesis pathway, resulting in 51% higher levels of lathosterol (and additionally desmosterol in women). Plasma levels of the cholesterol precursor sterol squalene were only slightly increased (6%), suggesting enhanced conversion of squalene to lathosterol in this disorder.
Assuntos
Biomarcadores/sangue , Colesterol/biossíntese , Colesterol/sangue , Hiperlipidemia Familiar Combinada/metabolismo , Absorção Intestinal/fisiologia , Adulto , Idoso , Colestanol/sangue , Colesterol/análogos & derivados , Desmosterol/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fitosteróis/sangue , Caracteres Sexuais , Sitosteroides/sangue , Esqualeno/sangueRESUMO
CONTEXT: Obesity-related insulin resistance is associated with an increase in adipocyte size. In rodent models, treatment with the insulin-sensitizers thiazolidinediones (TZDs) leads to the appearance of small, insulin-sensitive adipocytes. Whether such TZD-dependent morphological changes occur in adipose tissue of insulin-resistant patients is unclear. OBJECTIVE: The objective of the study was to study the effects of treatment with the TZD pioglitazone on sc adipose tissue morphology and function in insulin-resistant subjects. DESIGN: This was a placebo-controlled, randomized crossover study. SETTING: The study was conducted at a university medical center. PATIENTS: Twelve adult patients with congenital adrenal hyperplasia (CAH) characterized by insulin resistance were included in this study. INTERVENTION: After a 4-wk run-in phase, patients were treated with pioglitazone (45 mg/d) followed by placebo, each for 16 wk or vice versa. MAIN OUTCOME MEASURES: After both placebo and pioglitazone treatment, insulin sensitivity was determined by hyperinsulinemic euglycemic clamp and abdominal sc adipose tissue was obtained to measure adipocyte cell surface and expression of genes involved in glucose uptake and inflammation. RESULTS: Pioglitazone treatment significantly improved the insulin sensitivity index (placebo: 0.35 +/- 0.16 micromol/kg . min per milliunit per liter; pioglitazone 0.53 +/- 0.16 micromol/kg . min per milliunit per liter, P < 0.001) and increased mRNA expression levels of adiponectin and glucose transporter-4 in adipose tissue. The increase in insulin sensitivity was accompanied by a significant enlargement of the sc adipocyte cell surface (placebo: 2323 +/- 725 microm(2); pioglitazone 2821 +/- 885 microm(2), P = 0.03). CONCLUSIONS: In the human situation, treatment of insulin-resistant subjects with pioglitazone improves insulin sensitivity, whereas at the same time, sc adipocyte cell surface increases.
Assuntos
Adipócitos/patologia , Hiperplasia Suprarrenal Congênita/tratamento farmacológico , Hipoglicemiantes/farmacologia , Resistência à Insulina/fisiologia , Tiazolidinedionas/uso terapêutico , Adipócitos/efeitos dos fármacos , Hiperplasia Suprarrenal Congênita/patologia , Adulto , Estudos Cross-Over , Técnica Clamp de Glucose/métodos , Humanos , Obesidade/patologia , Pioglitazona , PlacebosRESUMO
Adiponectin is secreted from adipocytes in different multimers, of which the high molecular weight (HMW) form is supposed to mediate favorable metabolic and anti-atherogenic effects. We determined adiponectin multimers in 29 female and 22 male patients with familial combined hyperlipidemia (FCH) and 51 age-, gender-, and BMI-matched controls in relation to cardiovascular disease (CVD). We observed a clear sexual dimorphism of total adiponectin and its multimers. Female, but not male, FCH patients had significant lower total adiponectin and both HMW and low molecular weight (LMW) adiponectin than controls. The adiponectin sensitivity index (ASI), reflected by HMW/total adiponectin, and the LMW/HMW adiponectin ratio did not differ significantly between FCH females and control females. However, FCH females with CVD exhibited significantly lower ASI (34.2+/-10.1% vs 46.0+/-7.1%) and higher LMW/HMW ratio (1.5+/-0.8 vs 0.7+/-0.3) compared to FCH females without CVD, reflecting a more atherogenic adiponectin multimer distribution.
Assuntos
Adipócitos/metabolismo , Adiponectina/metabolismo , Hiperlipidemia Familiar Combinada/metabolismo , Caracteres Sexuais , Adiponectina/sangue , Idoso , Doenças Cardiovasculares/metabolismo , Feminino , Humanos , Hiperlipidemia Familiar Combinada/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
HFE C282Y-homozygosity has been associated with low hepcidin expression, leading to increased ferritin levels. However, serum hepcidin protein levels have not been documented in humans. In the current study, we compared serum hepcidin levels of newly diagnosed HFE C282Y-homozygotes with (N = 15) and without (N = 7) elevated serum ferritin levels to levels of 40 controls (20 heterozygotes and 20 wild types). In addition, hepcidin levels of four C282Y homozygotes were investigated during the course of all phlebotomy treatment phases. Serum hepcidin levels were lower in HFE C282Y-homozygotes (median; 25th-75th percentile: 1.88; 0.78-2.77 nmol/l) compared to controls (2.74; 1.45-5.39). Hepcidin/ferritin ratios were also lower in homozygotes. Homozygotes with an elevated serum ferritin had a higher serum hepcidin but a lower hepcidin/ferritin ratio than those with normal ferritin (2.28; 1.62-3.23 nmol/l hepcidin vs. 0.80; 0.60-1.29 and 3.63; 2.72-7.59 pmol hepcidin/microg ferritin vs. 13.2; 5.15-14.2). Serum hepcidin decreased during the depletion phase of phlebotomy and remained low during maintenance. This study showed that serum hepcidin is innately low in HFE-related haemochromatosis. Elevated ferritin levels were associated with increased hepcidin levels while erythropoiesis lead to lower hepcidin levels. During depletion, therefore, hepcidin levels are decreased, which may exacerbate intestinal iron absorption.
Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Hemocromatose/sangue , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Adulto , Idoso , Eritropoese , Feminino , Ferritinas/sangue , Hemocromatose/genética , Hemocromatose/cirurgia , Proteína da Hemocromatose , Hepcidinas , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , FlebotomiaRESUMO
There is strong evidence from both animal- and in vitro-models that paraoxonase (PON1) is involved in the onset of cardiovascular disease. In humans there is no consensus on this issue and therefore we investigated the effect of PON1 genotype and activity on the incidence of coronary heart disease (CHD) and acute myocardial infarction (AMI) in a large prospective cohort of 17,357 middle-aged women. We applied a case-cohort design using the CHD (n=211) and AMI cases (n=71) and a random sample from the baseline cohort (n=1527). A weighted Cox proportional hazards model was used to estimate age- and multivariate-adjusted hazard ratios (HR) for the PON1 genetic variants (192Q > R and -107C > T) and tertiles of the PON1 arylesterase- and paraoxonase activities. Neither the PON1 genetic variants, nor the PON1 activities affected the incidence of CHD in general, but, an increased paraoxonase activity was associated with a higher risk of AMI: the second and third tertile HR were 1.31 and 2.07, respectively (P-trend=0.029, multivariate model). In the subgroup of never-smokers, paraoxonase activity was associated with an increased risk for AMI: the second and third tertile HR were 4.1 and 4.7, respectively (P-trend=0.009, multivariate model). Additionally, when compared to the lowest paraoxonase tertile in never-smokers, the highest paraoxonase tertile in current-smokers showed a 19.2-fold higher risk for AMI (95%CI: 5.3-69.5, P < 0.0001, multivariate model). In conclusion, this study shows that in middle-aged women paraoxonase activity was associated with an increased risk for AMI and that the risk was modified by the effects of smoking.
Assuntos
Arildialquilfosfatase/genética , Doença das Coronárias/genética , Infarto do Miocárdio/genética , Idoso , Arildialquilfosfatase/fisiologia , Estudos de Coortes , Doença das Coronárias/epidemiologia , Doença das Coronárias/metabolismo , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/metabolismo , Países Baixos , Modelos de Riscos Proporcionais , Estudos Prospectivos , Risco , FumarRESUMO
Knowledge of hepcidin regulation is foremost gained by in vitro studies. We aimed to translate this knowledge into the human in vivo situation. Therefore, we measured serum markers as transferrin saturation (TS), soluble transferrin receptor (sTfR), and C-reactive protein (CRP) in parallel with hepcidin and prohepcidin in patients with iron metabolism disorders and controls. To assess sTfR as erythropoietic activity-associated factor in hepcidin regulation, we studied its influence on hepcidin expression in HepG2 cells. Results showed that sTfR highly associates with erythropoietic activity that strongly interfered with the iron store regulation of hepcidin. HepG2 expression results display an inverse association between hepcidin and sTfR. Inflammation was strongly related to increased hepcidin levels regardless of the iron store and erythropoietic activity status. In contrast, prohepcidin failed to correlate to any other parameter. In conclusion, these studies verify that previous conclusions based on in vitro studies on hepcidin regulation are also likely to apply to human patients. This is underscored by a simple algorithm, based on parameters reflecting the main regulating pathways, that accurately predict the actual measured hepcidin levels. Future studies are needed to validate the combined utility of this predictive algorithm together with actual measured hepcidin levels in clinical diagnosis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Proteína C-Reativa/análise , Distúrbios do Metabolismo do Ferro/metabolismo , Precursores de Proteínas/sangue , Receptores da Transferrina/sangue , Transferrina/análise , Algoritmos , Anemia Ferropriva/sangue , Anemia Ferropriva/metabolismo , Linhagem Celular , Eritropoese , Feminino , Ferritinas/sangue , Hepcidinas , Humanos , Inflamação/sangue , Inflamação/metabolismo , Ferro/sangue , Distúrbios do Metabolismo do Ferro/sangue , Masculino , Redes e Vias Metabólicas , Talassemia beta/sangue , Talassemia beta/metabolismoRESUMO
Familial combined hyperlipidemia (FCH) is a common genetic lipid disorder of which the molecular basis still remains to be elucidated. Since the HDL-associated enzyme serum paraoxonase (PON1) is associated with variation in serum lipids and lipoproteins, we determined whether variation in PON1 also contributes to the FCH phenotype. The study population consisted of 32 well-defined families with FCH, including 103 FCH patients and 240 normolipidemic relatives (NLR). In addition to plasma lipids and lipoproteins we determined PON1 activity (arylesterase- and paraoxonase activity) as well as the common genetic variants -107C>T, 55L>M and 192Q>R in the PON1 gene. The arylesterase activity was significantly higher in FCH patients when compared to NLR (P<0.001). In the total population, the PON1 genetic variants associated with the highest arylesterase activity (-107CC and 55LL) also associated with higher levels of total cholesterol, apolipoprotein B, triglycerides and VLDL-cholesterol and decreased levels of HDL-cholesterol. In support, the combination of the -107CC with the 55LL genotype associated with a significant increased risk for FCH when compared to the -107TT/55MM genotype (odds ratio 5.0 (95% CI, 1.3-19.1, P=0.02)). In conclusion, in this population of subjects from well-defined families with FCH, PON1 is biochemically and genetically associated with FCH.
Assuntos
Arildialquilfosfatase/sangue , Arildialquilfosfatase/genética , Hiperlipidemia Familiar Combinada/genética , Hiperlipidemia Familiar Combinada/metabolismo , Adulto , Idoso , Colesterol/sangue , Ativação Enzimática , Feminino , Predisposição Genética para Doença/epidemiologia , Variação Genética , Genótipo , Humanos , Hiperlipidemia Familiar Combinada/epidemiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Triglicerídeos/sangueRESUMO
Exogenous radiolabeled annexin A5 is taken up by atherosclerotic tissue. We measured endogenous plasma annexin A5 and circulating oxidized low-density lipoprotein (oxLDL), a biochemical marker of atherosclerosis, in men with either severe angiographically determined coronary stenosis (n=90) or no or only minor stenosis (n=96). Men without history of cardiac disease or treatment and free of plaques in the carotid artery (by ultrasonography) were taken as controls (n=87). Opposite to oxLDL, annexin A5 decreased at increasing severity of stenosis. OxLDL was lowest and annexin A5 was highest in controls. Percentage differences between groups were higher for annexin A5 than for oxLDL, and highest for oxLDL/annexin A5 ratio. The oxLDL/annexin A5 ratio is a better marker of the severity of coronary stenosis than oxLDL alone, may reflect the presence and extent of the atherosclerotic cardiovascular disease, and might prove useful for preclinical screening purposes.