RESUMO
The peroxisome proliferator-activated receptor-γ (PPARγ) improves whole-body insulin sensitivity by regulating the adipogenic and metabolic functions of mature adipocytes. We have previously demonstrated that an active splice variant of X-box binding protein 1 (XBP1s) enhances PPARγ expression during adipogenesis. In this study, we investigated the role of XBP1s, particularly with respect to PPARγ, in the mechanisms underlying insulin sensitivity in mature adipocytes. Insulin was able to stimulate XBP1s generation by activating inositol-requiring enzyme 1 (IRE1) α and was also able to increase its transcriptional activity by inducing nuclear translocation. XBP1s also upregulated the levels of phosphorylated IRS1 and AKT, demonstrating a positive feedback regulatory mechanism linking insulin and XBP1s. XBP1s enhanced the expression of fibroblast growth factor 21 and, in turn, increased PPARγ activity, translocation of GLUT4 to the cell surface, and glucose uptake rate in adipocytes. In addition, XBP1s abolished palmitate-induced insulin resistance in adipocytes by increasing adiponectin secretion, repressing the secretion of pro-inflammatory adipokines such as leptin, monocyte chemoattractant protein 1, and tumor necrosis factor α, and decreasing fatty acid release. These findings provide a novel mechanism by which XBP1s stimulate insulin sensitivity in adipocytes through fibroblast growth factor 21 induction and PPARγ activation.
Assuntos
Adipócitos/metabolismo , Endorribonucleases/metabolismo , Glucose/metabolismo , Insulina/farmacologia , PPAR gama/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Resistência à Insulina , Camundongos , Modelos Biológicos , Ácido Palmítico , Transporte Proteico/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/genéticaRESUMO
In obese patients, free fatty acids ectopically accumulated in non-adipose tissues cause cell death. Saturated fatty acids are more deleterious to non-adipose cells, and supplementation with monounsaturated fatty acids has been proposed to rescue cells from saturated fatty acid-induced cytotoxicity; however, the mechanisms are not well understood. To understand the cytoprotective role of monounsaturated fatty acids in lipotoxic cell death of macrophages, we investigated the antagonizing effect of oleate and the underlying mechanisms in palmitate-treated RAW264.7 cells. Palmitate strongly induced apoptosis in macrophages by increasing CD36 expression, which was identified to mediate both endoplasmic reticulum stress and the generation of reactive oxygen species. Co-treatment with oleate significantly reduced CD36 expression and its downstream signaling pathways of apoptosis in palmitate-treated cells. These findings provide a novel mechanism by which oleate protects macrophages from palmitate-induced lipotoxicity.
Assuntos
Apoptose/efeitos dos fármacos , Antígenos CD36/biossíntese , Regulação para Baixo/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Ácido Oleico/farmacologia , Palmitatos/farmacologia , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-AtividadeRESUMO
Wnt10b, an endogenous inhibitor of adipogenesis, maintains preadipocytes in an undifferentiated state by suppressing adipogenic transcription factors. We have previously demonstrated that Wnt10b transcription during adipogenesis is negatively regulated by X-box-binding protein 1 (XBP1), an important transcription factor of the unfolded protein response. In this report, we demonstrate that XBP1s can directly induce the transcription of microRNA-148a, which in turn mediates the silencing of Wnt10b mRNA during adipogenic differentiation of 3T3-L1 cells. Stability of Wnt10b mRNA was found to be significantly increased by knockdown of XBP1s. Using computational algorithms, a set of microRNAs was predicted to bind Wnt10b mRNA, of which microRNA-148a was selected as a potential target for XBP1s. Our results revealed that microRNA-148a could bind to the 3'UTR of Wnt10b mRNA. Its ectopic expression significantly suppressed both Wnt10b expression and ß-catenin activity. When we altered the expression of XBP1 in 3T3-L1 cells, microRNA-148a levels changed accordingly. A potential XBP1 response element was found in the promoter region of microRNA-148a, and XBP1s directly bound to this response element as shown by point mutation analysis and chromatin immunoprecipitation assay. In addition, a microRNA-148a mimic significantly restored adipogenic potential in XBP1-deficient 3T3-L1 cells. These findings provide the first evidence that XBP1s can regulate Wnt10b by a post-transcriptional mechanism through directly inducing microRNA-148a.
Assuntos
Adipogenia/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Inativação Gênica , MicroRNAs/genética , Interferência de RNA , Fatores de Transcrição/genética , Proteínas Wnt/genética , Regiões 3' não Traduzidas , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Sítios de Ligação , Humanos , Camundongos , Estabilidade de RNA , Fatores de Transcrição de Fator Regulador X , Transcrição Gênica , Via de Sinalização Wnt , Proteína 1 de Ligação a X-BoxRESUMO
X-box binding protein 1 (XBP1), a transcription factor of the unfolded protein response, plays various roles in many biological processes. We examined its pro-adipogenic activity and target genes during adipogenic differentiation in wild-type and genetically modified 3T3-L1 cells. Signalling pathways that contribute to Xbp1 mRNA splicing, and the correlation of the transcriptionally active XBP1 isoform (XBP1s) level with body mass index and the level of peroxisome proliferator-activated receptor γ2 (PPARγ2) in human adipose tissues were also examined. The mRNA and nuclear protein expression levels of XBP1s increased immediately following hormonal induction of adipogenesis, reaching a peak at 6 h. Results from cDNA microarray and gene expression analyses using genetically modified cells indicated that PPARγ2 was a principal target of XBP1s. The XBP1s-specific binding motif, which is distinct from the CCAAT/enhancer-binding protein α binding site, was identified in the PPARγ2 promoter by site-directed mutagenesis. Fetal bovine serum, insulin, 3-isobutyl-1-methylxanthine and dexamethasone contributed independently to Xbp1 mRNA splicing. In human subcutaneous adipose tissues, the levels of both Xbp1s and Pparγ2 mRNA increased proportionally with body mass index, and there was a significant positive correlation between the two genes. These data suggest for the first time that positive regulation of PPARγ2 is a principal mechanism of XBP1s-mediated adipogenesis in 3T3-L1 cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , PPAR gama/metabolismo , Fatores de Transcrição/fisiologia , Ativação Transcricional , Células 3T3-L1 , Adipogenia , Adulto , Animais , Sequência de Bases , Sítios de Ligação , Índice de Massa Corporal , Feminino , Expressão Gênica , Humanos , Camundongos , Pessoa de Meia-Idade , PPAR gama/genética , Regiões Promotoras Genéticas , Ligação Proteica , Splicing de RNA , Fatores de Transcrição de Fator Regulador X , Gordura Subcutânea , Proteína 1 de Ligação a X-Box , Adulto JovemRESUMO
Differentiation of preadipocytes into adipocytes is controlled by various transcription factors. Recently, the pro-adipogenic function of XBP1, a transcription factor upregulated by endoplasmic reticulum stress, has been reported. In this study, we demonstrated that XBP1 suppresses the expression of Wnt10b, an anti-adipogenic Wnt, during the differentiation of 3T3-L1 preadipocytes. The expression pattern of XBP1 was reciprocal to that of Wnt10b during the early stage of adipogenesis. The intracellular protein levels of ß-catenin were negatively regulated by XBP1. Direct binding of XBP1 to the Wnt10b promoter and the subsequent decrease of the ß-catenin signalling pathway represent a novel adipogenic differentiation mechanism.
Assuntos
Adipogenia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Inativação Gênica , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Células 3T3-L1 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Wnt/genética , Via de Sinalização Wnt , Proteína 1 de Ligação a X-Box , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Adipocytes are continuously stimulated by proinflammatory cytokines such as TNFα, which cause adipocyte dysfunction by facilitating the inflammatory response. Although miR-130 was reported to be an important regulator of adipogenesis by targeting PPARγ mRNA, little is known about the mechanisms regulating miR-130 expression during the proinflammatory response. Here, we examined miR-130 levels in white adipose tissue (WAT) from high-fat diet (HFD) mice and TNFα-stimulated adipocytes. Primary transcripts of miR-130 were increased after TNFα stimulation, indicating that induction of miR-130 during the pro-inflammatory response is regulated by a transcriptional event. A chromatin immunoprecipitation assay showed that p65 binding to the promoter regions of miR-130 was enhanced after TNFα treatment. Taken together, our findings suggest that induction of miR-130 by TNFα is responsible for adipocyte dysfunction.
RESUMO
Adipocytes are continuously stimulated by proinflammatory cytokines such as TNFα, which cause adipocyte dysfunction by facilitating the inflammatory response. Although miR-130 was reported to be an important regulator of adipogenesis by targeting PPARγ mRNA, little is known about the mechanisms regulating miR-130 expression during the proinflammatory response. Here, we examined miR-130 levels in white adipose tissue (WAT) from high-fat diet (HFD) mice and TNFα-stimulated adipocytes. Primary transcripts of miR-130 were increased after TNFα stimulation, indicating that induction of miR-130 during the pro-inflammatory response is regulated by a transcriptional event. A chromatin immunoprecipitation assay showed that p65 binding to the promoter regions of miR-130 was enhanced after TNFα treatment. Taken together, our findings suggest that induction of miR-130 by TNFα is responsible for adipocyte dysfunction.
Assuntos
Adipócitos/metabolismo , Inflamação/metabolismo , MicroRNAs/metabolismo , Obesidade/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Dieta Hiperlipídica/efeitos adversos , Inflamação/etiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células NIH 3T3 , Obesidade/complicações , Obesidade/etiologia , PPAR gama/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Brown adipose tissue is specialized to burn lipids for thermogenesis and energy expenditure. Second-generation antipsychotics (SGA) are the most commonly used drugs for schizophrenia with several advantages over first-line drugs, however, it can cause clinically-significant weight gain. To reveal the involvement of brown adipocytes in SGA-induced weight gain, we compared the effect of clozapine, quetiapine, and ziprasidone, SGA with different propensities to induce weight gain, on the differentiation and the expression of brown fat-specific markers, lipogenic genes and adipokines in a mouse brown preadipocyte cell line. On Oil Red-O staining, the differentiation was inhibited almost completely by clozapine (40 µM) and partially by quetiapine (30 µM). Clozapine significantly down-regulated the brown adipogenesis markers PRDM16, C/EBPß, PPARγ2, UCP-1, PGC-1α, and Cidea in dose- and time-dependent manners, whereas quetiapine suppressed PRDM16, PPARγ 2, and UCP-1 much weakly than clozapine. Clozapine also significantly inhibited the mRNA expressions of lipogenic genes ACC, SCD1, GLUT4, aP2, and CD36 as well as adipokines such as resistin, leptin, and adiponectin. In contrast, quetiapine suppressed only resistin and leptin but not those of lipogenic genes and adiponectin. Ziprasidone (10 µM) did not alter the differentiation as well as the gene expression patterns. Our results suggest for the first time that the inhibition of brown adipogenesis may be a possible mechanism to explain weight gain induced by clozapine and quetiapine.
Assuntos
Antipsicóticos , Clozapina , Dibenzotiazepinas , Piperazinas , Tiazóis , Aumento de Peso/efeitos dos fármacos , Adipócitos Marrons/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Adipocinas/metabolismo , Animais , Antipsicóticos/administração & dosagem , Antipsicóticos/efeitos adversos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clozapina/administração & dosagem , Clozapina/efeitos adversos , Dibenzotiazepinas/administração & dosagem , Dibenzotiazepinas/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Piperazinas/administração & dosagem , Piperazinas/efeitos adversos , Fumarato de Quetiapina , Esquizofrenia/tratamento farmacológico , Tiazóis/administração & dosagem , Tiazóis/efeitos adversosRESUMO
When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2alpha phosphorylation. Transcription of two downstream targets of eIF2alpha, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Embrionárias/citologia , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular , Meios de Cultura/farmacologia , Proteínas de Ligação a DNA , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dobramento de Proteína , Ratos , Células EstromaisRESUMO
This study aimed to investigate the effect of serum albumin at admission, measured within 24 h after stroke onset, on the functional outcome in ischaemic stroke patients. The medical records of 76 first-ever hemiplegic ischaemic stroke patients were reviewed. Collected data included age, sex, initial stroke severity, cerebrovascular risk factors, lesion-related variables, aetiologic subtype of stroke and serum albumin at admission. The functional outcome was measured by functional independence measure (FIM) and modified Barthel index (MBI). Serum albumin at admission and initial National Institutes of Health Stroke Scale (NIHSS) score were correlated with the functional outcome, respectively. Serum albumin at admission was an independent predictor of MBI gain on multiple regression analysis. Serum albumin at admission would be a useful predictor of the functional outcome and trials for the correction of hypoalbuminaemia from the acute stage would be helpful to decrease the risk of poor outcome in ischaemic stroke patients.
Assuntos
Isquemia Encefálica/diagnóstico , Albumina Sérica/metabolismo , Acidente Vascular Cerebral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Biomarcadores/sangue , Análise Química do Sangue/métodos , Isquemia Encefálica/sangue , Isquemia Encefálica/fisiopatologia , Avaliação da Deficiência , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estado Nutricional/fisiologia , Valor Preditivo dos Testes , Prognóstico , Análise de Regressão , Albumina Sérica/análise , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/fisiopatologia , Adulto JovemRESUMO
BACKGROUND/AIMS: Apoptosis contributes to cyclosporine (CsA)-induced renal cell death. This study tested the effects of CsA-induced endoplasmic reticulum (ER) stress on apoptotic cell death in an experimental model of chronic CsA nephropathy. METHODS: CsA (15 mg/kg per day) was given to rats for 7 or 28 days. The ER stress response was evaluated with BiP expression, and the proapoptotic response was assessed with CHOP and caspase 12 expression. ER structure was evaluated by transmission electron microscopy, and apoptotic cell death was detected with TUNEL staining. RESULTS: Short-term treatment of CsA for 7 days activated both the ER stress response (induction of BiP mRNA and protein) and the proapoptotic response (upregulation of caspase 12 and CHOP mRNAs and proteins). However, long-term treatment with CsA for 28 days decreased BiP and further increased CHOP. The imbalance between the two responses coincided with the timing of the appearance of apoptotic cell death and the disruption of the ER structure. CONCLUSION: Prolonged CsA-induced ER stress causes apoptotic cell death by depleting molecular chaperones and activating the proapoptotic pathway.
Assuntos
Apoptose , Morte Celular , Ciclosporina , Retículo Endoplasmático/efeitos dos fármacos , Nefropatias/induzido quimicamente , Animais , Northern Blotting , Caspase 12/análise , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Nefropatias/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/análiseRESUMO
Brain/kidney (B/K) protein is a novel double C2-like-domain protein that is highly expressed in rat brain and kidney, but its cellular localization and functional role in the kidney are still undetermined. We examined the cellular localization of B/K protein in the rat kidney under normal and ischemic conditions. Ischemia-reperfusion (I/R) injury was induced by clamping both renal arteries for 45 min, and animals were killed at 1 and 6 h and 1, 2, 3, 5, 7, 14, and 28 days after the reperfusion. Kidney tissues were processed for immunohistochemistry and immunoblot analyses using rabbit anti-B/K polyclonal antibodies. In control kidneys, B/K protein was expressed primarily in distal tubules including the thick ascending limb, distal convoluted and connecting tubules, and collecting duct. Notably, B/K protein was also expressed in the straight portion (S3 segment), but not in the S1 or S2, of proximal tubules, and podocytes of the glomerulus. In rat kidneys with I/R injury, expression of B/K protein was differentially regulated according to the anatomic location. In distal tubules, overall expression of B/K protein was markedly decreased. On the other hand, I/R injury significantly increased B/K protein expression in the S3 segment of the outer medulla as well as in the rat proximal tubular epithelial cell line NRK-52E in vitro. Furthermore, B/K protein was strongly expressed in many exfoliated cells in the lumen and urine. These findings suggest that B/K protein is closely associated with cell death in proximal tubules, which are vulnerable to I/R injury in the kidney.