RESUMO
The secretin-like, class B1 sub-family of seven transmembrane-spanning G protein coupled receptors (GPCRs) consists of 15 members that coordinate important physiological processes. These receptors bind peptide ligands and utilize a distinct mechanism of activation that is driven by evolutionarily conserved structural features. For the class B1 receptors, the C-terminus of the cognate ligand is initially recognized by the receptor via a large N-terminal extracellular domain that forms a hydrophobic ligand binding groove. This binding enables the N-terminus of the ligand to engage deep into a large volume, open transmembrane pocket of the receptor. Importantly, the phylogenetic basis of this ligand-receptor activation mechanism has provided opportunities to engineer analogues of several class B1 ligands for therapeutic use. Among the most successful of these are drugs targeting the glucagon-like peptide-1 (GLP-1) receptor for the treatment of type 2 diabetes and obesity. Recently, multi-functional agonists possessing activity at the GLP-1 receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor, such as tirzepatide, and others that also contain glucagon receptor activity, have been developed. In this article, we review members of the class B1 GPCR family with focus on receptors for GLP-1, GIP, and glucagon, including their signal transduction and receptor trafficking characteristics. The metabolic importance of these receptors is also highlighted, along with the benefit of poly-pharmacologic ligands. Further, key structural features and comparative analyses of high-resolution cryogenic electron microscopy structures for these receptors in active-state complex with either native ligands or multi-functional agonists are provided, supporting the pharmacological basis of such therapeutic agents.
RESUMO
BACKGROUND & AIMS: Liver sinusoidal endothelial cell (LSEC) dysfunction has been reported in alcohol-related liver disease, yet it is not known whether LSECs metabolize alcohol. Thus, we investigated this, as well as the mechanisms of alcohol-induced LSEC dysfunction and a potential therapeutic approach for alcohol-induced liver injury. METHODS: Primary human, rat and mouse LSECs were used. Histone deacetylase 6 (HDAC6) was overexpressed specifically in liver ECs via adeno-associated virus (AAV)-mediated gene delivery to decrease heat shock protein 90 (Hsp90) acetylation in ethanol-fed mice. RESULTS: LSECs expressed CYP2E1 and alcohol dehydrogenase 1 (ADH1) and metabolized alcohol. Ethanol induced CYP2E1 in LSECs, but not ADH1. Alcohol metabolism by CYP2E1 increased Hsp90 acetylation and decreased its interaction with endothelial nitric oxide synthase (eNOS) leading to a decrease in nitric oxide (NO) production. A non-acetylation mutant of Hsp90 increased its interaction with eNOS and NO production, whereas a hyperacetylation mutant decreased NO production. These results indicate that Hsp90 acetylation is responsible for decreases in its interaction with eNOS and eNOS-derived NO production. AAV8-driven HDAC6 overexpression specifically in liver ECs deacetylated Hsp90, restored Hsp90's interaction with eNOS and ameliorated alcohol-induced liver injury in mice. CONCLUSION: Restoring LSEC function is important for ameliorating alcohol-induced liver injury. To this end, blocking acetylation of Hsp90 specifically in LSECs via AAV-mediated gene delivery has the potential to be a new therapeutic strategy. LAY SUMMARY: Alcohol metabolism in liver sinusoidal endothelial cells (LSECs) and the mechanism of alcohol-induced LSEC dysfunction are largely unknown. Herein, we demonstrate that LSECs can metabolize alcohol. We also uncover a mechanism by which alcohol induces LSEC dysfunction and liver injury, and we identify a potential therapeutic strategy to prevent this.
Assuntos
Acetilação/efeitos dos fármacos , Hepatopatias Alcoólicas/genética , Adulto , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/fisiopatologia , Análise de Variância , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Proteínas de Choque Térmico HSP90 , Humanos , Hepatopatias Alcoólicas/etiologia , Camundongos , RatosRESUMO
RATIONALE: Endothelial-mesenchymal transition (EndoMT) is implicated in myofibroblast-like cell-mediated damage to the coronary arterial wall in acute Kawasaki disease (KD) patients, as evidenced by positive staining for connective tissue growth factor (CTGF) and EndoMT markers in KD autopsy tissues. However, little is known about the molecular basis of EndoMT involved in KD. OBJECTIVE: We investigated the microRNA (miRNA) regulation of CTGF and the consequent EndoMT in KD pathogenesis. As well, the modulation of this process by statin therapy was studied. METHODS AND RESULTS: Sera from healthy children and KD subjects were incubated with human umbilical vein endothelial cells. Cardiovascular disease-related miRNAs, CTGF, and EndoMT markers were quantified using reverse transcriptase quantitative polymerase chain reaction, ELISA, and Western blotting. Compared with healthy controls, human umbilical vein endothelial cell incubated with sera from acute KD patients had decreased miR-483, increased CTGF, and increased EndoMT markers. Bioinformatics analysis followed by functional validation demonstrated that Krüppel-like factor 4 (KLF4) transactivates miR-483, which in turn targets the 3' untranslated region of CTGF mRNA. Overexpression of KLF4 or pre-miR-483 suppressed, whereas knockdown of KLF4 or anti-miR-483 enhanced, CTGF expression in endothelial cells in vitro and in vivo. Furthermore, atorvastatin, currently being tested in a phase I/IIa clinical trial in KD children, induced KLF4-miR-483, which suppressed CTGF and EndoMT in endothelial cells. CONCLUSIONS: KD sera suppress the KLF4-miR-483 axis in endothelial cells, leading to increased expression of CTGF and induction of EndoMT. This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. Statin therapy may benefit acute KD patients, in part, through the restoration of KLF4-miR-483 expression. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01431105.
Assuntos
Atorvastatina/administração & dosagem , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Transição Epitelial-Mesenquimal/fisiologia , Marcação de Genes/métodos , MicroRNAs/biossíntese , Síndrome de Linfonodos Mucocutâneos/sangue , Síndrome de Linfonodos Mucocutâneos/terapia , Animais , Bovinos , Pré-Escolar , Fator de Crescimento do Tecido Conjuntivo/genética , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Lactente , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/administração & dosagem , MicroRNAs/genética , Síndrome de Linfonodos Mucocutâneos/genéticaRESUMO
BACKGROUND: Atherosclerosis is a multifaceted inflammatory disease involving cells in the vascular wall (eg, endothelial cells [ECs]), as well as circulating and resident immunogenic cells (eg, monocytes/macrophages). Acting as a ligand for liver X receptor (LXR), but an inhibitor of SREBP2 (sterol regulatory element-binding protein 2), 25-hydroxycholesterol, and its catalyzing enzyme cholesterol-25-hydroxylase (Ch25h) are important in regulating cellular inflammatory status and cholesterol biosynthesis in both ECs and monocytes/macrophages. METHODS: Bioinformatic analyses were used to investigate RNA-sequencing data to identify cholesterol oxidation and efflux genes regulated by Krüppel-like factor 4 (KLF4). In vitro experiments involving cultured ECs and macrophages and in vivo methods involving mice with Ch25h ablation were then used to explore the atheroprotective role of KLF4-Ch25h/LXR. RESULTS: Vasoprotective stimuli increased the expression of Ch25h and LXR via KLF4. The KLF4-Ch25h/LXR homeostatic axis functions through suppressing inflammation, evidenced by the reduction of inflammasome activity in ECs and the promotion of M1 to M2 phenotypic transition in macrophages. The increased atherosclerosis in apolipoprotein E-/-/Ch25h-/- mice further demonstrates the beneficial role of the KLF4-Ch25h/LXR axis in vascular function and disease. CONCLUSIONS: KLF4 transactivates Ch25h and LXR, thereby promoting the synergistic effects between ECs and macrophages to protect against atherosclerosis susceptibility.
Assuntos
Aterosclerose/etiologia , Expressão Gênica/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Receptores X do Fígado/metabolismo , Animais , Humanos , Hidroxicolesteróis , Fator 4 Semelhante a Kruppel , Receptores X do Fígado/análise , Masculino , CamundongosRESUMO
Heart failure is associated with mitochondrial dysfunction so that restoring or improving mitochondrial health is of therapeutic importance. Recently, reduction in NAD+ levels and NAD+-mediated deacetylase activity has been recognized as negative regulators of mitochondrial function. Using a cardiac specific KLF4 deficient mouse line that is sensitive to stress, we found mitochondrial protein hyperacetylation coupled with reduced Sirt3 and NAD+ levels in the heart before stress, suggesting that the KLF4-deficient heart is predisposed to NAD+-associated defects. Further, we demonstrated that short-term administration of Nicotinamide Mononucleotide (NMN) successfully protected the mutant mice from pressure overload-induced heart failure. Mechanically, we showed that NMN preserved mitochondrial ultrastructure, reduced ROS and prevented cell death in the heart. In cultured cardiomyocytes, NMN treatment significantly increased long-chain fatty acid oxidation despite no direct effect on pyruvate oxidation. Collectively, these results provide cogent evidence that hyperacetylation of mitochondrial proteins is critical in the pathogenesis of cardiac disease and that administration of NMN may serve as a promising therapy.
Assuntos
Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/prevenção & controle , Homeostase , Mononucleotídeo de Nicotinamida/administração & dosagem , Mononucleotídeo de Nicotinamida/uso terapêutico , Acetilação , Animais , Morte Celular , Ácidos Graxos/metabolismo , Insuficiência Cardíaca/patologia , Homeostase/efeitos dos fármacos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , NAD/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Nicotinamida Fosforribosiltransferase/metabolismo , Oxirredução , Pressão , Ratos , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/metabolismoRESUMO
Hemoglobin subunit alpha (HBA) expression in endothelial cells (ECs) has recently been shown to control vascular tone and function. We sought to elucidate the transcriptional regulation of HBA expression in the EC. Gain of KLF2 or KLF4 function studies led to significant induction of HBA in ECs. An opposite effect was observed in ECs isolated from animals with endothelial-specific ablation of Klf2, Klf4 or both. Promoter reporter assays demonstrated that KLF2/KLF4 transactivated the hemoglobin alpha promoter, an effect that was abrogated following mutation of all four putative KLF-binding sites. Fine promoter mutational studies localized three out of four KLF-binding sites (sites 2, 3, and 4) as critical for the transactivation of the HBA promoter by KLF2/KLF4. Chromatin immunoprecipitation studies showed that KLF4 bound to the HBA promoter in ECs. Thus, KLF2 and KLF4 serve as important regulators that promote HBA expression in the endothelium.
Assuntos
Células Endoteliais/metabolismo , Hemoglobinas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Sítios de Ligação , Bovinos , Células Cultivadas , Genótipo , Hemoglobinas/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fragmentos de Peptídeos/genética , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição Gênica , Ativação Transcricional , Transfecção , Regulação para CimaRESUMO
This invited review summarizes work presented in the Russell Ross lecture delivered at the 2012 proceedings of the American Heart Association. We begin with a brief overview of the structural, cellular, and molecular biology of Krüppel-like factors. We then focus on discoveries during the past decade, implicating Krüppel-like factors as key determinants of vascular cell function in atherosclerotic vascular disease.
Assuntos
Aterosclerose/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , DNA/metabolismo , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Células Espumosas/metabolismo , Regulação da Expressão Gênica , Hemorreologia , Humanos , Inflamação , Fatores de Transcrição Kruppel-Like/química , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Modelos Cardiovasculares , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. OBJECTIVE: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. METHODS: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. RESULTS: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8(+) suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH(-/-) or miRNA-150(-/-) mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. CONCLUSIONS: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.
Assuntos
Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dermatite de Contato/prevenção & controle , Epitopos , Exossomos/fisiologia , Tolerância Imunológica , MicroRNAs/fisiologia , Animais , Humanos , Camundongos , Biossíntese de Proteínas , Linfócitos T Reguladores/imunologiaRESUMO
UNLABELLED: Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo-B in liver fibrosis and cirrhosis. Nogo-B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild-type (WT) and Nogo-A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo-B's involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo-B gene deletion could ameliorate portal hypertension. In normal livers, Nogo-B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo-B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo-B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor ß (TGF-ß) stimulation. Reconstitution of the Nogo-B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham-operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS). CONCLUSION: Nogo-B regulates liver fibrosis, at least in part, by facilitating the TGFß/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo-B ameliorates liver fibrosis and portal hypertension, Nogo-B blockade may be a potential therapeutic target in fibrosis/cirrhosis.
Assuntos
Cirrose Hepática/etiologia , Proteínas da Mielina/fisiologia , Animais , Ductos Biliares/cirurgia , Células Estreladas do Fígado/metabolismo , Humanos , Hipertensão Portal/prevenção & controle , Fígado/química , Cirrose Hepática/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Proteínas da Mielina/genética , Proteínas Nogo , Ratos , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Human genetics has been instrumental in identification of genetic variants linked to type 2 diabetes. Recently a rare, putative loss-of-function mutation in the orphan G-protein coupled receptor 151 (GPR151) was found to be associated with lower odds ratio for type 2 diabetes, but the mechanism behind this association has remained elusive. Here we show that Gpr151 is a fasting- and glucagon-responsive hepatic gene which regulates hepatic gluconeogenesis. Gpr151 ablation in mice leads to suppression of hepatic gluconeogenesis genes and reduced hepatic glucose production in response to pyruvate. Importantly, the restoration of hepatic Gpr151 levels in the Gpr151 knockout mice reverses the reduced hepatic glucose production. In this work, we establish a previously unknown role of Gpr151 in the liver that provides an explanation to the lowered type 2 diabetes risk in individuals with nonsynonymous mutations in GPR151.
Assuntos
Diabetes Mellitus Tipo 2 , Gluconeogênese , Humanos , Camundongos , Animais , Gluconeogênese/genética , Diabetes Mellitus Tipo 2/genética , Fígado , Ácido Pirúvico , Camundongos Knockout , GlucoseRESUMO
Proteostasis deficiency in mutated ion channels leads to a variety of ion channel diseases that are caused by excessive endoplasmic reticulum-associated degradation (ERAD) and inefficient membrane trafficking. We investigated proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors, the primary mediators of neuronal inhibition in the mammalian central nervous system. We screened a structurally diverse, Food and Drug Administration-approved drug library and identified dinoprost (DNP) and dihydroergocristine (DHEC) as highly efficacious enhancers of surface expression of four epilepsy-causing trafficking-deficient mutant receptors. Furthermore, DNP and DHEC restore whole-cell and synaptic currents by incorporating mutated subunits into functional receptors. Mechanistic studies revealed that both drugs reduce subunit degradation by attenuating the Grp94/Hrd1/Sel1L/VCP-mediated ERAD pathway and enhance the subunit folding by promoting subunit interactions with major GABAA receptors-interacting chaperones, BiP and calnexin. In summary, we report that DNP and DHEC remodel the endoplasmic reticulum proteostasis network to restore the functional surface expression of mutant GABAA receptors.
Assuntos
Di-Hidroergocristina/farmacologia , Dinoprosta/farmacologia , Epilepsia/tratamento farmacológico , Proteostase/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Linhagem Celular , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Epilepsia/metabolismo , Feminino , Humanos , Masculino , Receptores de GABA-A/genéticaRESUMO
Insulin resistance (IR) is fundamental to the development of type 2 diabetes (T2D) and is present in most prediabetic (preDM) individuals. Insulin resistance has both heritable and environmental determinants centered on energy storage and metabolism. Recent insights from human genetic studies, coupled with comprehensive in vivo and ex vivo metabolic studies in humans and rodents, have highlighted the critical role of reduced mitochondrial function as a predisposing condition for ectopic lipid deposition and IR. These studies support the hypothesis that reduced mitochondrial function, particularly in insulin-responsive tissues such as skeletal muscle, white adipose tissue, and the liver, is inextricably linked to tissue and whole body IR through the effects on cellular energy balance. Here we discuss these findings as well as address potential mechanisms that serve as the nexus between mitochondrial malfunction and IR.
Assuntos
Tecido Adiposo Branco/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina/fisiologia , Fígado/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Estado Pré-Diabético/metabolismo , Diabetes Mellitus Tipo 2/genética , Humanos , Metabolismo dos Lipídeos/fisiologia , Mitocôndrias/genética , Estado Pré-Diabético/genéticaRESUMO
Substantial evidence implicates crosstalk between metabolic tissues and the immune system in the inception and progression of obesity. However, molecular regulators that orchestrate metaflammation both centrally and peripherally remains incompletely understood. Here, we identify myeloid Krüppel-like factor 2 (KLF2) as an essential regulator of obesity and its sequelae. In mice and humans, consumption of a fatty diet downregulates myeloid KLF2 levels. Under basal conditions, myeloid-specific KLF2 knockout mice (K2KO) exhibit increased feeding and weight gain. High-fat diet (HFD) feeding further exacerbates the K2KO metabolic disease phenotype. Mechanistically, loss of myeloid KLF2 increases metaflammation in peripheral and central tissues. A combination of pair-feeding, bone marrow-transplant, and microglial ablation implicate central and peripheral contributions to K2KO-induced metabolic dysfunction observed. Finally, overexpression of myeloid KLF2 protects mice from HFD-induced obesity and insulin resistance. Together, these data establish myeloid KLF2 as a nodal regulator of central and peripheral metabolic inflammation in homeostasis and disease.
Assuntos
Fatores de Transcrição Kruppel-Like/imunologia , Doenças Metabólicas/imunologia , Células Mieloides/imunologia , Obesidade/imunologia , Animais , Sistema Nervoso Central/imunologia , Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos , Humanos , Inflamação , Resistência à Insulina , Fatores de Transcrição Kruppel-Like/genética , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Doenças Metabólicas/fisiopatologia , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/genética , Obesidade/fisiopatologia , Sistema Nervoso Periférico/imunologiaRESUMO
Genetic variation in the FAM13A (Family with Sequence Similarity 13 Member A) locus has been associated with several glycemic and metabolic traits in genome-wide association studies (GWAS). Here, we demonstrate that in humans, FAM13A alleles are associated with increased FAM13A expression in subcutaneous adipose tissue (SAT) and an insulin resistance-related phenotype (e.g. higher waist-to-hip ratio and fasting insulin levels, but lower body fat). In human adipocyte models, knockdown of FAM13A in preadipocytes accelerates adipocyte differentiation. In mice, Fam13a knockout (KO) have a lower visceral to subcutaneous fat (VAT/SAT) ratio after high-fat diet challenge, in comparison to their wild-type counterparts. Subcutaneous adipocytes in KO mice show a size distribution shift toward an increased number of smaller adipocytes, along with an improved adipogenic potential. Our results indicate that GWAS-associated variants within the FAM13A locus alter adipose FAM13A expression, which in turn, regulates adipocyte differentiation and contribute to changes in body fat distribution.
Assuntos
Adipócitos/metabolismo , Distribuição da Gordura Corporal , Proteínas Ativadoras de GTPase/genética , Adipogenia/genética , Animais , Diferenciação Celular/genética , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Silenciamento de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Resistência à Insulina/genética , Gordura Intra-Abdominal/metabolismo , Masculino , Metabolômica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Gordura Subcutânea/metabolismoRESUMO
BACKGROUND: Cardiovascular disease is the leading cause of morbidity and mortality in patients with end-stage renal disease. The accumulation of uremic solutes in this patient population is associated with endothelial dysfunction and accelerated cardiovascular disease. In this study, we examined the impact of the uremic milieu on the endothelial transcription factor, Krüppel-like factor 2 (KLF2), a key regulator of endothelial function and activation. METHODS AND RESULTS: Using serum from uremic pigs with chronic renal insufficiency, our results show that KLF2 expression is suppressed by the uremic milieu and individual uremic solutes in vitro. Specifically, KLF2 expression is significantly decreased in human umbilical vein endothelial cells after treatment with uremic porcine serum or carboxymethyllysine-modified albumin, an advanced glycation end product (AGE) known to induce endothelial dysfunction. AGE-mediated suppression of KLF2 is dependent on activation of the receptor for AGE, as measured by small interfering RNA knockdown of the receptor for AGE. Furthermore, KLF2 suppression promotes endothelial dysfunction, because adenoviral overexpression of KLF2 inhibits reactive oxygen species production and leukocyte adhesion in human umbilical vein endothelial cells. In addition, the application of hemodynamic shear stress, prolonged serum dialysis, or treatment with the receptor for AGE antagonist azeliragon (TTP488) is sufficient to prevent KLF2 suppression in vitro. To decipher the mechanism by which uremic AGEs suppress KLF2 expression, we assessed the role of the receptor for AGE in activation of nuclear factor-κB signaling, a hallmark of endothelial cell activation. Using a constitutively active form of IκBα, we show that translocation of p65 to the nucleus is necessary for KLF2 suppression after treatment with uremic AGEs. CONCLUSIONS: These data identify KLF2 suppression as a consequence of the uremic milieu, which may exacerbate endothelial dysfunction and resultant cardiovascular disease.
Assuntos
Proteínas Sanguíneas/metabolismo , Produtos Finais de Glicação Avançada/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Insuficiência Renal Crônica/sangue , Soroalbumina Bovina/toxicidade , Uremia/sangue , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/agonistas , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Diálise Renal , Insuficiência Renal Crônica/terapia , Sus scrofa , Fator de Transcrição RelA/metabolismo , Uremia/terapiaRESUMO
Brown adipose tissue (BAT) is a specialized thermogenic organ in mammals. The ability of BAT mitochondria to generate heat in response to cold-challenge to maintain core body temperature is essential for organismal survival. While cold activated BAT mitochondrial biogenesis is recognized as critical for thermogenic adaptation, the contribution of mitochondrial quality control to this process remains unclear. Here, we show mitophagy is required for brown adipocyte mitochondrial homeostasis during thermogenic adaptation. Mitophagy is significantly increased in BAT from cold-challenged mice (4 °C) and in ß-agonist treated brown adipocytes. Blockade of mitophagy compromises brown adipocytes mitochondrial oxidative phosphorylation (OX-PHOS) capacity, as well as BAT mitochondrial integrity. Mechanistically, cold-challenge induction of BAT mitophagy is UCP1-dependent. Furthermore, our results indicate that mitophagy coordinates with mitochondrial biogenesis, maintaining activated BAT mitochondrial homeostasis. Collectively, our in vivo and in vitro findings identify mitophagy as critical for brown adipocyte mitochondrial homeostasis during cold adaptation.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo Marrom/fisiologia , Hipotermia/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Termogênese , Proteína Desacopladora 1/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Temperatura Baixa , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biogênese de Organelas , Fosforilação Oxidativa , Proteína Desacopladora 1/genéticaRESUMO
Neutrophils are the most abundant white blood cells in circulation and are key components of the innate immune response. Clinical and experimental studies support an important role for the neutrophils in a broad spectrum of acute and chronic inflammatory conditions. However, our understanding of nodal points that control neutrophil activation remains incompletely understood. Over the past decade, studies have linked members of the Kruppel-like family of transcription factors (KLFs) to myeloid cell differentiation and function. Here we show that KLF4 is a critical transcriptional regulator of neutrophil biology. KLF4-deficient neutrophils exhibited impaired responses to inflammatory stimulation ex vivo, including reduced production of cytokines and reactive oxygen species, impaired degranulation, and impaired bacterial killing and clearance. Consequently, mice bearing myeloid-specific conditional KLF4 deficiency (K4-cKO) exhibited enhanced susceptibility to bacterial infection but resistance to lipopolysaccharide-induced septic shock and experimental autoimmune encephalomyelitis. Finally, mechanistic studies revealed that the defects in KLF4-deficient neutrophils likely resulted from the defective Toll-like receptor 4-NF-κB signaling. Collectively, these findings identify KLF4 as a novel transcriptional regulator of neutrophil activation.
RESUMO
Adipose tissue stores energy in the form of triglycerides. The ability to regulate triglyceride synthesis and breakdown based on nutrient status (e.g., fed versus fasted) is critical for physiological homeostasis and dysregulation of this process can contribute to metabolic disease. Whereas much is known about hormonal control of this cycle, transcriptional regulation is not well understood. Here, we show that the transcription factor Kruppel-like factor 15 (KLF15) is critical for the control of adipocyte lipid turnover. Mice lacking Klf15 in adipose tissue (AK15KO) display decreased adiposity and are protected from diet-induced obesity. Mechanistic studies suggest that adipose KLF15 regulates key genes of triglyceride synthesis and inhibits lipolytic action, thereby promoting lipid storage in an insulin-dependent manner. Finally, AK15KO mice demonstrate accelerated lipolysis and altered systemic energetics (e.g., locomotion, ketogenesis) during fasting conditions. Our study identifies adipose KLF15 as an essential regulator of adipocyte lipid metabolism and systemic energy balance.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Proteínas de Ligação a DNA/genética , Glucose/metabolismo , Lipogênese/genética , Lipólise/genética , Fatores de Transcrição/genética , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/deficiência , Jejum/fisiologia , Regulação da Expressão Gênica , Glucose/farmacologia , Humanos , Insulina/metabolismo , Insulina/farmacologia , Fatores de Transcrição Kruppel-Like , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais , Fatores de Transcrição/deficiência , Triglicerídeos/metabolismoRESUMO
Maintenance of vascular integrity in the adult animal is needed for survival, and it is critically dependent on the endothelial lining, which controls barrier function, blood fluidity, and flow dynamics. However, nodal regulators that coordinate endothelial identity and function in the adult animal remain poorly characterized. Here, we show that endothelial KLF2 and KLF4 control a large segment of the endothelial transcriptome, thereby affecting virtually all key endothelial functions. Inducible endothelial-specific deletion of Klf2 and/or Klf4 reveals that a single allele of either gene is sufficient for survival, but absence of both (EC-DKO) results in acute death from myocardial infarction, heart failure, and stroke. EC-DKO animals exhibit profound compromise in vascular integrity and profound dysregulation of the coagulation system. Collectively, these studies establish an absolute requirement for KLF2/4 for maintenance of endothelial and vascular integrity in the adult animal.
Assuntos
Coagulação Sanguínea/genética , Permeabilidade Capilar/genética , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Animais , Transtornos da Coagulação Sanguínea/genética , Insuficiência Cardíaca/genética , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Knockout , Infarto do Miocárdio/genética , Acidente Vascular Cerebral/genéticaRESUMO
Loss of protein and organelle quality control secondary to reduced autophagy is a hallmark of aging. However, the physiologic and molecular regulation of autophagy in long-lived organisms remains incompletely understood. Here we show that the Kruppel-like family of transcription factors are important regulators of autophagy and healthspan in C. elegans, and also modulate mammalian vascular age-associated phenotypes. Kruppel-like family of transcription factor deficiency attenuates autophagy and lifespan extension across mechanistically distinct longevity nematode models. Conversely, Kruppel-like family of transcription factor overexpression extends nematode lifespan in an autophagy-dependent manner. Furthermore, we show the mammalian vascular factor Kruppel-like family of transcription factor 4 has a conserved role in augmenting autophagy and improving vessel function in aged mice. Kruppel-like family of transcription factor 4 expression also decreases with age in human vascular endothelium. Thus, Kruppel-like family of transcription factors constitute a transcriptional regulatory point for the modulation of autophagy and longevity in C. elegans with conserved effects in the murine vasculature and potential implications for mammalian vascular aging.KLF family transcription factors (KLFs) regulate many cellular processes, including proliferation, survival and stress responses. Here, the authors position KLFs as important regulators of autophagy and lifespan in C. elegans, a role that may extend to the modulation of age-associated vascular phenotypes in mammals.