Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 59
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Cell Mol Gastroenterol Hepatol ; 7(4): 763-781, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30831319

RESUMO

BACKGROUND & AIMS: Obesity promotes the development of nonalcoholic fatty liver diseases (NAFLDs), yet not all obese patients develop NAFLD. The underlying causes for this discrepancy remain elusive. LPGAT1 is an acyltransferase that catalyzes the remodeling of phosphatidylglycerol (PG), a mitochondrial phospholipid implicated in various metabolic diseases. Here, we investigated the role of LPGAT1 in regulating the onset of diet-induced obesity and its related hepatosteatosis because polymorphisms of the LPGAT1 gene promoter were strongly associated with susceptibility to obesity in Pima Indians. METHODS: Mice with whole-body knockout of LPGAT1 were generated to investigate the role of PG remodeling in NAFLD. RESULTS: LPGAT1 deficiency protected mice from diet-induced obesity, but led to hepatopathy, insulin resistance, and NAFLD as a consequence of oxidative stress, mitochondrial DNA depletion, and mitochondrial dysfunction. CONCLUSIONS: This study identified an unexpected role of PG remodeling in obesity, linking mitochondrial dysfunction to NAFLD.

2.
Aging Cell ; 18(3): e12941, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30838774

RESUMO

Cardiolipin (CL) is a mitochondrial signature phospholipid that is required for membrane structure, respiration, dynamics, and mitophagy. Oxidative damage of CL by reactive oxygen species is implicated in the pathogenesis of Parkinson's disease (PD), but the underlying cause remains elusive. This work investigated the role of ALCAT1, an acyltransferase that catalyzes pathological remodeling of CL in various aging-related diseases, in a mouse model of PD induced by 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine (MPTP). We show that MPTP treatment caused oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain. In contrast, ablation of the ALCAT1 gene or pharmacological inhibition of ALCAT1 prevented MPTP-induced neurotoxicity, apoptosis, and motor deficits. ALCAT1 deficiency also mitigated mitochondrial dysfunction by modulating DRP1 translocation to the mitochondria. Moreover, pharmacological inhibition of ALCAT1 significantly improved mitophagy by promoting the recruitment of Parkin to dysfunctional mitochondria. Finally, ALCAT1 expression was upregulated by MPTP and by α-synucleinopathy, a key hallmark of PD, whereas ALCAT1 deficiency prevented α-synuclein oligomerization and S-129 phosphorylation, implicating a key role of ALCAT1 in the etiology of mouse models of PD. Together, these findings identify ALCAT1 as a novel drug target for the treatment of PD.

3.
Medicine (Baltimore) ; 97(30): e11438, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30045265

RESUMO

This study is to characterize the transcription factor expression profiles for the peripheral CD4 T-cell subsets, and analyze its associations with the clinical measures of the hepatitis B virus (HBV) infection.Totally 275 subjects were included. The expression levels of transcription factors (T-bet, GATA-3, Foxp3, RORγt, and Bcl-6) in the peripheral blood mononuclear cells (PBMCs) were determined by the real-time fluorimetry quantitative PCR (FQ-PCR).Lowest expression levels of all these transcription factors were observed for the HBsAb(-) group, which were higher in the HBsAb(+) and RHB groups. The T-bet/GATA-3 ratios in the CHB and RHB groups were significantly lower than the HBsAb(-) group, whereas the RORγt/Foxp3 ratios in the AHB and RHB groups were significantly higher than the CHB and HBsAb(+) groups. Furthermore, the RORγt mRNA expression levels were significantly different among groups with different disease severities or with different alanine aminotransferase (ALT) levels. The asymptomatic carrier (AsC) group and the group with ALT ≤ 40 had the highest express level. The mRNA expression levels of T-bet, GATA-3, Foxp3, and RORγt varied along with the aspartate aminotransferase (AST) levels, with AST ≤ 40 having the highest expression levels. In addition, significant differences were observed in the transcription factor expression levels between the group with the serum HBV DNA load of (1.000-9.999) × 10 copies/mL and other groups.Expression profile of critical transcription factors for peripheral CD4 T-cell subsets may indicate clinical outcomes of HBV infection.


Assuntos
Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Linfócitos T CD4-Positivos/patologia , Fatores de Transcrição Forkhead/genética , Hepatite B , Leucócitos Mononucleares , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Adulto , Feminino , Hepatite B/sangue , Hepatite B/diagnóstico , Hepatite B/genética , Vírus da Hepatite B/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Fatores de Transcrição/genética
4.
Endocrinology ; 159(8): 3036-3047, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29873699

RESUMO

Rho GDP-dissociation inhibitor (GDIα) inhibits glucose-stimulated insulin secretion (GSIS) in part by locking Rho GTPases in an inactive GDP-bound form. The onset of GSIS causes phosphorylation of GDIα at Ser174, a critical inhibitory site for GDIα, leading to the release of Rho GTPases and their subsequent activation. However, the kinase regulator(s) that catalyzes the phosphorylation of GDIα in islet ß cells remains elusive. We propose that SAD-A, a member of AMP-activated protein kinase-related kinases that promotes GSIS as an effector kinase for incretin signaling, interacts with and inhibits GDIα through phosphorylation of Ser174 during the onset GSIS from islet ß cells. Coimmunoprecipitation and phosphorylation analyses were carried out to identify the physical interaction and phosphorylation site of GDIα by SAD-A in the context of GSIS from INS-1 ß cells and primary islets. We identified GDIα directly binds to SAD-A kinase domain and phosphorylated by SAD-A on Ser174, leading to dissociation of Rho GTPases from GDIα complexes. Accordingly, overexpression of SAD-A significantly stimulated GDIα phosphorylation at Ser174 in response to GSIS, which is dramatically potentiated by glucagonlike peptide-1, an incretin hormone. Conversely, SAD-A deficiency, which is mediated by short hairpin RNA transfection in INS-1 cells, significantly attenuated endogenous GDIα phosphorylation at Ser174. Consequently, coexpression of SAD-A completely prevented the inhibitory effect of GDIα on insulin secretion in islets. In summary, glucose and incretin stimulate insulin secretion through the phosphorylation of GDIα at Ser174 by SAD-A, which leads to the activation of Rho GTPases, culminating in insulin exocytosis.

5.
J Med Virol ; 90(5): 926-935, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29350417

RESUMO

Hepatic fibrosis is a frequent feature of chronic hepatitis C virus (HCV) infection. Some evidence has suggested the potential role of silent information regulator 1 (SIRT1) in organ fibrosis. The aim of this study was to investigate the effect of HCV core protein on expression of SIRT1 of liver sinusoidal endothelial cell (LSEC) and function of LSEC. LSECs were co-cultured with HepG2 cells or HepG2 cells expressing HCV core protein and LSECs cultured alone were used as controls. After co-culture, the activity and expression levels of mRNA and protein of SIRT1 in LSEC were detected by a SIRT1 fluorometric assay kit, real time-PCR (RT-PCR), Western blot, respectively. The levels of adiponectin receptor 2 (AdipoR2), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by Western blot. Cluster of differentiation 31 (CD31), CD14, and von Willebrand factor (vWf) of LSECs was performed by flow cytometry. The level of reactive oxygen species (ROS) was assayed. Malondialdehyde (MDA), superoxide dismutase (SOD), adiponectin, nitric oxide (NO), and endothelin-1 (ET-1) levels in the co-culture supernatant were measured. The co-culture supernatant was then used to cultivate LX-2 cells. The levels of α-smooth muscle actin (ASMA) and transforming growth factor-ß1 (TGF-ß1) protein in LX-2 cells were measured by Western blot. Compared with LSEC co-cultured with HepG2 cells group, in LSEC co-cultured with HepG2-core cells group, the activity and expression level of mRNA and protein of SIRT1 reduced; the level of adiponectin reduced and the expression level of AdipoR2 protein decreased; ROS levels increased; the expression level of eNOS, VEGF protein decreased; and the expression level of CD14 decreased; the expression level of vWf and CD31 increased; NO and SOD levels decreased; whereas ET-1 and MDA levels increased; the levels of ASMA and TGF-ß1 protein in LX-2 cells increased. SIRT1 activator improved the above-mentioned changes. HCV core protein may down-regulate the activity and the expression of SIRT1 of LSEC, then decreasing synthesis of adiponectin and the expression of AdipoR2, thus inducing contraction of LSEC and hepatic sinusoidal capillarization and increasing oxidative stress, ultimately cause hepatic stellate cell (HSC) activation. Treatment with SIRT1 activator restored the function of LSEC and inhibited the activation of HSC.

6.
Nat Commun ; 8: 15986, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28656979

RESUMO

Excessive activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including cardiovascular and Alzheimer's disease. Here we show that microtubule-affinity regulating kinase 4 (MARK4) binds to NLRP3 and drives it to the microtubule-organizing centre, enabling the formation of one large inflammasome speck complex within a single cell. MARK4 knockdown or knockout, or disruption of MARK4-NLRP3 interaction, impairs NLRP3 spatial arrangement and limits inflammasome activation. Our results demonstrate how an evolutionarily conserved protein involved in the regulation of microtubule dynamics orchestrates NLRP3 inflammasome activation by controlling its transport to optimal activation sites, and identify a targetable function for MARK4 in the control of innate immunity.

7.
Nat Commun ; 8: 14824, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28327662

RESUMO

Hepatic de novo lipogenesis (DNL) converts carbohydrates into triglycerides and is known to influence systemic lipid homoeostasis. Here, we demonstrate that the zinc finger protein Zbtb20 is required for DNL. Mice lacking Zbtb20 in the liver exhibit hypolipidemia and reduced levels of liver triglycerides, along with impaired hepatic lipogenesis. The expression of genes involved in glycolysis and DNL, including that of two ChREBP isoforms, is decreased in livers of knockout mice. Zbtb20 binds to and enhances the activity of the ChREBP-α promoter, suggesting that altered metabolic gene expression is mainly driven by ChREBP. In addition, ChREBP-ß overexpression largely restores hepatic expression of genes involved in glucose and lipid metabolism, and increases plasma and liver triglyceride levels in knockout mice. Finally, we show that Zbtb20 ablation protects from diet-induced liver steatosis and improves hepatic insulin resistance. We suggest ZBTB20 is an essential regulator of hepatic lipogenesis and may be a therapeutic target for the treatment of fatty liver disease.


Assuntos
Lipogênese , Fígado/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carboidratos/química , Núcleo Celular/metabolismo , Carboidratos da Dieta , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Deleção de Genes , Regulação da Expressão Gênica , Glucose/metabolismo , Glicólise , Homeostase , Humanos , Resistência à Insulina , Lipogênese/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Transporte Proteico , Fatores de Transcrição/deficiência , Transcrição Genética , Triglicerídeos/sangue , Triglicerídeos/metabolismo
8.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(1): 114-129, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27502688

RESUMO

Autophagy is an evolutionarily conserved mechanism that maintains nutrient homeostasis by degrading protein aggregates and damaged organelles. Autophagy is reduced in aging, which is implicated in the pathogenesis of aging-related diseases, including cancers, obesity, type 2 diabetes, cardiovascular diseases, and neurodegenerative diseases. Mitochondria-derived phospholipids cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol are critical throughout the autophagic process, from initiation and phagophore formation to elongation and fusion with endolysosomal vesicles. Cardiolipin is also required for mitochondrial fusion and fission, an important step in isolating dysfunctional mitochondria for mitophagy. Furthermore, genetic screen in yeast has identified a surprising role for cardiolipin in regulating lysosomal function. Phosphatidylethanolamine plays a pivotal role in supporting the autophagic process, including autophagosome elongation as part of lipidated Atg8/LC3. An emerging role for phosphatidylglycerol in AMPK and mTORC1 signaling as well as mitochondrial fission may provide the first glimpse into the function of phosphatidylglycerol apart from being a precursor for cardiolipin. This review examines the effects of manipulating phospholipids on autophagy and mitophagy in health and diseases, as well as current limitations in the field. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.


Assuntos
Autofagia/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosfolipídeos/metabolismo , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Humanos , Degradação Mitocondrial/fisiologia , Dinâmica Mitocondrial/fisiologia
9.
Nat Commun ; 7: 11121, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27079169

RESUMO

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.


Assuntos
Linhagem da Célula/genética , Lactotrofos/metabolismo , Adeno-Hipófise/metabolismo , Fatores de Transcrição/genética , Animais , Western Blotting , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Hipopituitarismo/genética , Hipopituitarismo/metabolismo , Hipotálamo/embriologia , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Lactotrofos/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Adeno-Hipófise/embriologia , Adeno-Hipófise/crescimento & desenvolvimento , Prolactina/genética , Prolactina/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo
10.
Sci Rep ; 6: 20453, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26857140

RESUMO

Bif-1 is a membrane-curvature inducing protein that is implicated in the regulation of autophagy and tumorigenesis. Here, we report that Bif-1 plays a critical role in regulating lipid catabolism to control the size of lipid droplets and prevent the development of obesity and insulin resistance upon aging or dietary challenge. Our data show that Bif-1 deficiency promotes the expansion of adipose tissue mass without altering food intake or physical activities. While Bif-1 is dispensable for adipose tissue development, its deficiency reduces the basal rate of adipose tissue lipolysis and results in adipocyte hypertrophy upon aging. The importance of Bif-1 in lipid turnover is not limited to adipose tissue since fasting and refeeding-induced lipid droplet clearance is also attenuated by Bif-1 loss in the liver. Interestingly, obesity induced by a high fat-diet or Bif-1 deficiency downregulates the expression of proteins involved in the autophagy-lysosomal pathway, including Atg9a and Lamp1 in the adipose tissue. These findings thus identify Bif-1 as a novel regulator of lipid homeostasis to prevent the pathogenesis of obesity and its associated metabolic complications.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Tecido Adiposo , Resistência à Insulina/genética , Gotículas Lipídicas , Metabolismo dos Lipídeos/genética , Obesidade , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
11.
Sci Rep ; 6: 20438, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26830324

RESUMO

The recombination efficiency and cell specificity of Cre driver lines are critical for exploring pancreatic ß cell biology with the Cre/LoxP approach. Some commonly used Cre lines are based on the short Ins2 promoter fragment and show recombination activity in hypothalamic neurons; however, whether this stems from endogenous Ins2 promoter activity remains controversial. In this study, we generated Ins2-Cre knockin mice with a targeted insertion of IRES-Cre at the Ins2 locus and demonstrated with a cell lineage tracing study that the Ins2 gene is not transcriptionally active in the hypothalamus. The Ins2-Cre driver line displayed robust Cre expression and activity in pancreatic ß cells without significant alterations in insulin expression. In the brain, Cre activity was mainly restricted to the choroid plexus, without significant recombination detected in the hippocampus or hypothalamus by the LacZ or fluorescent tdTomato reporters. Furthermore, Ins2-Cre mice exhibited normal glucose tolerance and insulin secretion upon glucose stimulation in vivo. In conclusion, this Ins2-Cre driver line allowed high-fidelity detection of endogenous Ins2 promoter activity in vivo, and the negative activity in the hypothalamus demonstrated that this system is a promising alternative tool for studying ß cell biology.


Assuntos
Técnicas de Introdução de Genes , Loci Gênicos , Hipotálamo/citologia , Hipotálamo/metabolismo , Insulina/genética , Integrases/genética , Integrases/metabolismo , Neurônios/metabolismo , Animais , Ativação Enzimática , Ordem dos Genes , Marcação de Genes , Vetores Genéticos/genética , Glucose/metabolismo , Recombinação Homóloga , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo
12.
Diabetologia ; 59(2): 316-24, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26564177

RESUMO

AIMS/HYPOTHESIS: 'Glucotoxicity' is a term used to convey the negative effect of hyperglycaemia on beta cell function; however, the underlying molecular mechanisms that impair insulin secretion and gene expression are poorly defined. Our objective was to define the role of transcription factor v-ets avian erythroblastosis virus E26 oncogene homologue 1 (Ets-1) in beta cell glucotoxicity. METHODS: Primary islets and Min6 cells were exposed to high glucose and Ets-1 expression was measured. Recombinant adenovirus and transgenic mice were used to upregulate Ets-1 expression in beta cells in vitro and in vivo, and insulin secretion was assessed. The binding activity of H3/H4 histone on the Ets-1 promoter, and that of forkhead box (FOX)A2, FOXO1 and Ets-1 on the Pdx-1 promoter was measured by chromatin immunoprecipitation and quantitative real-time PCR assay. RESULTS: High glucose induced upregulation of Ets-1 expression and hyperacetylation of histone H3 and H4 at the Ets-1 gene promoter in beta cells. Ets-1 overexpression dramatically suppressed insulin secretion and biosynthesis both in vivo and in vitro. Besides, Ets-1 overexpression increased the activity of FOXO1 but decreased that of FOXA2 binding to the pancreatic and duodenal homeobox 1 (PDX-1) homology region 2 (PH2), resulting in inhibition of Pdx-1 promoter activity and downregulation of PDX-1 expression and activity. In addition, high glucose promoted the interaction of Ets-1 and FOXO1, and the activity of Ets-1 binding to the Pdx-1 promoter. Importantly, PDX-1 overexpression reversed the defect in pancreatic beta cells induced by Ets-1 excess, while knockdown of Ets-1 prevented hyperglycaemia-induced dysfunction of pancreatic beta cells. CONCLUSIONS/INTERPRETATION: Our observations suggest that Ets-1 links glucotoxicity to pancreatic beta cell dysfunction through inhibiting PDX-1 expression in type 2 diabetes.


Assuntos
Diabetes Mellitus Experimental/genética , Proteínas de Homeodomínio/genética , Hiperglicemia/genética , Células Secretoras de Insulina/fisiologia , Proteína Proto-Oncogênica c-ets-1/fisiologia , Transativadores/genética , Animais , Glicemia/fisiologia , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Hiperglicemia/sangue , Hiperglicemia/fisiopatologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Transativadores/metabolismo
13.
Biol Trace Elem Res ; 171(2): 419-426, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26463750

RESUMO

Selenium (Se) mainly performs its function through Se-containing proteins. Selenoprotein W (SelW), one member of the selenoprotein family, plays important roles in the normal function of the heart. To investigate the possible relationship between Se and SelW for the regulation of oxidative damage in chicken embryo myocardial cells, we treated myocardial cells with Se and H2O2. Then, the levels of lactate dehydrogenase (LDH) and 3,4-methylenedioxyamphetamine in the culture media, levels of SelW, inflammatory genes NF-κB, tumor necrosis factor (TNF)-α, p53, and the cell cycle were analyzed. Furthermore, the correlation between SelW and the levels of these factors was determined. The results indicated that Se treatment increased the expression of SelW (P < 0.05) and caused a downregulation of p53, NF-κB, and TNF-α (P < 0.05). In contrast, H2O2 increased the expression of p53, NF-κB, TNF-α, and LDH (P < 0.05) and induced early cell apoptosis, which was alleviated by treatment with Se. In addition, SelW had a positive correlation with the levels of inflammatory genes investigated. Taken together, our findings suggested that SelW is sensitive to Se levels and oxidative stress, and may play a role in the protective function of Se against oxidative damage and inflammation in chicken myocardial cells.


Assuntos
Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo , Selênio/farmacologia , Selenoproteína W/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Selênio/administração & dosagem , Relação Estrutura-Atividade
14.
Autophagy ; 11(4): 643-52, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25919711

RESUMO

Tafazzin (TAZ) is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of TAZ cause Barth syndrome, which is characterized by mitochondrial dysfunction and dilated cardiomyopathy, leading to premature death. However, the molecular mechanisms underlying the cause of mitochondrial dysfunction in Barth syndrome remain poorly understood. Here we investigated the role of TAZ in regulating mitochondrial function and mitophagy. Using primary mouse embryonic fibroblasts (MEFs) with doxycycline-inducible knockdown of Taz, we showed that TAZ deficiency in MEFs caused defective mitophagosome biogenesis, but not other autophagic processes. Consistent with a key role of mitophagy in mitochondria quality control, TAZ deficiency in MEFs also led to impaired oxidative phosphorylation and severe oxidative stress. Together, these findings provide key insights on mitochondrial dysfunction in Barth syndrome, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for this lethal condition.


Assuntos
Autofagia/fisiologia , Cardiolipinas/metabolismo , Degradação Mitocondrial/fisiologia , Fatores de Transcrição/metabolismo , Animais , Autofagia/genética , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Humanos , Camundongos , Mitocôndrias/genética , Mutação/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
15.
Curr Drug Targets ; 16(12): 1372-80, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25706109

RESUMO

Accumulation of toxic lipids is the most common etiology of insulin resistance in type 2 diabetes and associated metabolic disorders such as obesity and non-alcoholic fatty liver disease. Understanding of the underlying mechanisms has revealed various opportunities to target key regulators in lipid metabolic pathways for the treatment of metabolic diseases. Here, we review the discovery and development of potential anti-diabetic drugs with primary effects on cellular targets leading to reductions of intracellular lipids in key organs. We will particularly focus on AMPK, SIRT1, PGC-1α, SREBP-1c, ChREBP, ACC, PPARs and HSPs which either stimulate in fatty acid oxidation (energy expenditure) or inhibit de novo lipogenesis.


Assuntos
Descoberta de Drogas/métodos , Hipoglicemiantes/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Hipoglicemiantes/uso terapêutico , Resistência à Insulina
16.
Hepatology ; 61(2): 486-96, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25203315

RESUMO

UNLABELLED: Defective autophagy is implicated in the pathogenesis of nonalcoholic fatty liver diseases (NAFLD) through poorly defined mechanisms. Cardiolipin is a mitochondrial phospholipid required for bioenergetics and mitophagy from yeast to mammals. Here we investigated a role for ALCAT1 in the development of NAFLD. ALCAT1 is a lysocardiolipin acyltransferase that catalyzes pathological cardiolipin remodeling in several aging-related diseases. We show that the onset of diet-induced NAFLD caused autophagic arrest in hepatocytes, leading to oxidative stress, mitochondrial dysfunction, and insulin resistance. In contrast, targeted deletion of ALCAT1 in mice prevented the onset of NAFLD. ALCAT1 deficiency also restored mitophagy, mitochondrial architecture, mitochondrial DNA (mtDNA) fidelity, and oxidative phosphorylation. In support of a causative role of the enzyme in a mitochondrial etiology of the disease, hepatic ALCAT1 expression was significantly up-regulated in mouse models of NAFLD. CONCLUSION: Forced expression of ALCAT1 in primary hepatocytes led to multiple defects that are highly reminiscent of NAFLD, including steatosis, defective autophagy, and mitochondrial dysfunction, linking pathological cardiolipin remodeling by ALCAT1 to the pathogenesis of NAFLD.


Assuntos
Aciltransferases/metabolismo , Degradação Mitocondrial , Hepatopatia Gordurosa não Alcoólica/etiologia , Animais , Autofagia , Fibrose , Hepatócitos/fisiologia , Lipogênese , Fígado/patologia , Masculino , Camundongos Knockout , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo
17.
J Biol Chem ; 289(52): 36284-302, 2014 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-25391657

RESUMO

The calcium-permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full-length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of forkhead box transcription factor 3a (FOXO3a) and superoxide dismutase 2. Cells expressing the dominant negative short isoform (TRPM2-S) had reduced FOXO3a and superoxide dismutase 2 levels, reduced calcium influx in response to oxidative stress, and enhanced reactive oxygen species, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared with tumors expressing TRPM2-L. Expression of hypoxia-inducible factor (HIF)-1/2α was significantly reduced in TRPM2-S-expressing tumor cells as was expression of target proteins regulated by HIF-1/2α including those involved in glycolysis (lactate dehydrogenase A and enolase 2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (cytochrome oxidase 4.1/4.2 in complex IV). The reduction in HIF-1/2α was mediated through both significantly reduced HIF-1/2α mRNA levels and increased levels of von Hippel-Lindau E3 ligase in TRPM2-S-expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S-expressing cells after doxorubicin treatment was rescued by gain of HIF-1 or -2α function. These data suggest that TRPM2 activity is important for tumor growth and for cell viability and survival following doxorubicin treatment and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2α, mitochondrial function, and mitophagy.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neuroblastoma/metabolismo , Canais de Cátion TRPM/fisiologia , Glândulas Suprarrenais/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Doxorrubicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Potencial da Membrana Mitocondrial , Potenciais da Membrana , Camundongos Nus , Transplante de Neoplasias , Neuroblastoma/patologia , Isoformas de Proteínas/fisiologia , Transporte Proteico , Carga Tumoral
18.
J Biol Chem ; 289(47): 33044-53, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25315780

RESUMO

CGI-58 is a lipid droplet-associated protein that, when mutated, causes Chanarin-Dorfman syndrome in humans, which is characterized by excessive storage of triglyceride in various tissues. However, the molecular mechanisms underlying the defect remain elusive. CGI-58 was previously reported to catalyze the resynthesis of phosphatidic acid as a lysophosphatidic acid acyltransferase. In addition to triglyceride, phosphatidic acid is also used a substrate for the synthesis of various mitochondrial phospholipids. In this report, we investigated the propensity of CGI-58 in the remodeling of various phospholipids. We found that the recombinant CGI-58 overexpressed in mammalian cells or purified from Sf9 insect cells catalyzed efficiently the reacylation of lysophosphatidylglycerol to phosphatidylglycerol (PG), which requires acyl-CoA as the acyl donor. In contrast, the recombinant CGI-58 was devoid of acyltransferase activity toward other lysophospholipids. Accordingly, overexpression and knockdown of CGI-58 adversely affected the endogenous PG level in C2C12 cells. PG is a substrate for the synthesis of cardiolipin, which is required for mitochondrial oxidative phosphorylation and mitophagy. Consequently, overexpression and knockdown of CGI-58 adversely affected autophagy and mitophagy in C2C12 cells. In support for a key role of CGI-58 in mitophagy, overexpression of CGI-58 significantly stimulated mitochondrial fission and translocation of PINK1 to mitochondria, key steps involved in mitophagy. Furthermore, overexpression of CGI-58 promoted mitophagic initiation through activation of 5'-AMP-activated protein kinase and inhibition of mTORC1 mammalian target of rapamycin complex 1 signaling, the positive and negative regulators of autophagy, respectively. Together, these findings identified novel molecular mechanisms by which CGI-58 regulates lipid homeostasis, because defective autophagy is implicated in dyslipidemia and fatty liver diseases.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Autofagia , Lisofosfolipídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Aciltransferases/genética , Animais , Western Blotting , Células COS , Linhagem Celular , Cercopithecus aethiops , Células HEK293 , Homeostase , Humanos , Cinética , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Alvo Mecanístico do Complexo 1 de Rapamicina , Microscopia Confocal , Microssomos/metabolismo , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Fosfatidilgliceróis/metabolismo , Interferência de RNA , Células Sf9 , Especificidade por Substrato , Serina-Treonina Quinases TOR/metabolismo
19.
Int J Infect Dis ; 23: 75-81, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24704332

RESUMO

BACKGROUND: Insulin resistance is highly prevalent in patients with chronic hepatitis C (CHC) and to some extent accounts for fibrosis and reducing viral eradication. Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with insulin resistance and steatosis. We investigated the role of the endocannabinoid system in glucose metabolism disorders induced by hepatitis C virus (HCV) replication. METHODS: Human hepatic stellate cells (HSC; LX-2 cells) were co-cultured with Huh-7.5 cells or Huh-7.5 cells harboring HCV replicon (replicon cells). Endocannabinoid levels were then measured by liquid chromatography/mass spectrometry. The expression of CB1R and its downstream glucose metabolism genes in hepatocytes were determined by real-time PCR and Western blot. Glucose uptake by hepatocytes and glucose production were measured. Glucose metabolism tests and measurements of HCV RNA levels and nonstructural protein 5A (NS5A) levels were taken after treatment with CB1R agonist arachidonyl-2-chloroethanolamide (ACEA) or antagonist AM251. RESULTS: Compared to the co-culture with Huh-7.5 cells, the level of 2-arachidonoylglycerol (2-AG) and the CB1R mRNA and protein levels increased in the co-culture of LX-2 cells with replicon cells. The activation of CB1R decreased AMP-activated protein kinase (AMPK) phosphorylation, inhibited cell surface expression of glucose transporter 2 (GLUT2), and suppressed cellular glucose uptake; furthermore, it increased cyclic AMP response element-binding protein H (CREBH), then up-regulated phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes and down-regulated the glucokinase (GK) gene, thus promoting glucose production. Interferon treatment restored the aforementioned changes. CB1R antagonist improved glucose metabolism disorders by an increase in glucose uptake and a decrease in glucose production, and inhibited HCV replication. CONCLUSIONS: HCV replication may not only increase the 2-AG content, but may also up-regulate the expression of CB1R of hepatocytes, then change the expression profile of glucose metabolism-related genes, thereby causing glucose metabolism disorders of hepatocytes and promoting HCV replication. Treatment with CB1R antagonist improved glucose metabolism disorders and inhibited viral genome replication.


Assuntos
Endocanabinoides/metabolismo , Transtornos do Metabolismo de Glucose/virologia , Hepacivirus/isolamento & purificação , Hepatócitos/virologia , Receptor CB1 de Canabinoide/metabolismo , Replicação Viral , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Linhagem Celular , Sobrevivência Celular , Técnicas de Cocultura , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genoma Viral , Transtornos do Metabolismo de Glucose/patologia , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Glicerídeos/metabolismo , Hepacivirus/fisiologia , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/virologia , Hepatite C Crônica/patologia , Hepatócitos/metabolismo , Humanos , Fosforilação , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/metabolismo , Pirazóis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Transdução de Sinais , Regulação para Cima
20.
J Biol Chem ; 289(15): 10909-18, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24573674

RESUMO

Acyl-CoA:monoacylglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze the two consecutive steps in the synthesis of triacylglycerol, a key process required for dietary fat absorption into the enterocytes of the small intestine. In this report, we investigated the tendency of MGAT2 to form an enzyme complex with DGAT1 and DGAT2 in intact cells. We demonstrated that in addition to the 38-kDa monomer of the MGAT2 enzyme predicted by its peptide sequence, a 76-kDa moiety was detected in SDS-PAGE without reducing agent and heat inactivation. The 76-kDa MGAT2 moiety was greatly enhanced by treatment with a cross-linking reagent in intact cells. Additionally, the cross-linking reagent dose-dependently yielded a band corresponding to the tetramer (152 kDa) in SDS-PAGE, suggesting that the MGAT2 enzyme primarily functions as a homotetrameric protein and as a tetrameric protein. Likewise, DGAT1 also forms a homodimer under nondenaturing conditions. When co-expressed in COS-7 cells, MGAT2 heterodimerized with DGAT1 without treatment with a cross-linking reagent. MGAT2 also co-eluted with DGAT1 on a gel filtration column, suggesting that the two enzymes form a complex in intact cells. In contrast, MGAT2 did not heterodimerize with DGAT2 when co-expressed in COS-7 cells, despite high sequence homology between the two enzymes. Furthermore, systematic deletion analysis demonstrates that N-terminal amino acids 35-80 of DGAT1, but not a signal peptide at the N terminus of MGAT2, is required for the heterodimerization. Finally, co-expression of MGAT2 with DGAT1 significantly increased lipogenesis in COS-7 cells, indicating the functional importance of the dimerization.


Assuntos
Diacilglicerol O-Aciltransferase/metabolismo , Metabolismo dos Lipídeos , N-Acetilglucosaminiltransferases/metabolismo , Absorção , Sequência de Aminoácidos , Animais , Células COS , Cercopithecus aethiops , Cromatografia em Gel , Reagentes para Ligações Cruzadas/química , Diabetes Mellitus/metabolismo , Gorduras na Dieta/metabolismo , Deleção de Genes , Humanos , Lipogênese , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA