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1.
J Lipid Res ; 54(5): 1335-45, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23359610

RESUMO

Chronic ethanol consumption is a prominent cause of liver disease worldwide. Dysregulation of an important lipid uptake and trafficking gene, liver-fatty acid binding protein (L-FABP), may contribute to alterations in lipid homeostasis during early-stage alcoholic liver. We have reported the detrimental effects of ethanol on the expression of L-FABP and hypothesize this may deleteriously impact metabolic networks regulating fatty acids. Male wild-type (WT) and L-FABP(-/-) mice were fed a modified Lieber-DeCarli liquid diet for six weeks. To assess the response to chronic ethanol ingestion, standard biochemical indicators for alcoholic liver disease (ALD) and oxidative stress were measured. Ethanol ingestion resulted in attenuation of hepatic triglyceride accumulation and elevation of cholesterol in L-FABP(-/-) mice. Lipidomics analysis validated multiple alterations in hepatic lipids resulting from ethanol treatment. Increased immunohistochemical staining for the reactive aldehydes 4-hydroxynonenal and malondialdehyde were observed in WT mice ingesting ethanol; however, L-FABP(-/-) mice displayed prominent protein adducts in liver sections evaluated from pair-fed and ethanol-fed mice. Likewise, alterations in glutathione, thiobarbituric acid reactive substances (TBARS), 8-isoprostanes, and protein carbonyl content all indicated L-FABP(-/-) mice exhibit high sustained oxidative stress in the liver. These data establish that L-FABP is an indirect antioxidant protein essential for sequestering FFA and that its impairment could contribute to in the pathogenesis of ALD.


Assuntos
Etanol/toxicidade , Proteínas de Ligação a Ácido Graxo/metabolismo , Hepatopatias Alcoólicas/genética , Estresse Oxidativo/efeitos dos fármacos , Animais , Colesterol/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Ácidos Graxos/metabolismo , Predisposição Genética para Doença , Humanos , Lipídeos/análise , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/genética
2.
Hum Genomics ; 5(3): 170-91, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21504868

RESUMO

Fatty acid-binding proteins (FABPs) are members of the intracellular lipid-binding protein (iLBP) family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20) fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied.


Assuntos
Evolução Molecular , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Família Multigênica , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma Humano , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Especificidade da Espécie
3.
Chem Res Toxicol ; 25(5): 1012-21, 2012 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-22502949

RESUMO

Hepatic oxidative stress and subsequent lipid peroxidation are well-recognized consequences of sustained ethanol consumption. The covalent adduction of nucleophilic amino acid side-chains by lipid electrophiles is significantly increased in patients with alcoholic liver disease (ALD); a global assessment of in vivo protein targets and the consequences of these modifications, however, has not been conducted. In this article, we describe the identification of novel protein targets for covalent adduction in a 6-week murine model for ALD. Ethanol-fed mice displayed a 2-fold increase in hepatic TBARS, while immunohistochemical analysis for the reactive aldehydes 4-hydroxynonenal (4-HNE), 4-oxononenal (4-ONE), acrolein (ACR), and malondialdehyde (MDA) revealed a marked increase in the staining of modified proteins in the ethanol-treated mice. Increased protein carbonyl content was confirmed utilizing subcellular fractionation of liver homogenates followed by biotin-tagging through hydrazide chemistry, where approximately a 2-fold increase in modified proteins was observed in microsomal and cytosolic fractions. To determine targets of protein carbonylation, a secondary hydrazide method coupled to a highly sensitive 2-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS or MuDPIT) technique was utilized. Our results have identified 414 protein targets for modification by reactive aldehydes in ALD. The presence of novel in vivo sites of protein modification by 4-HNE (2), 4-ONE (4) and ACR (2) was also confirmed in our data set. While the precise impact of protein carbonylation in ALD remains unknown, a bioinformatic analysis of the data set has revealed key pathways associated with disease progression, including fatty acid metabolism, drug metabolism, oxidative phosphorylation, and the TCA cycle. These data suggest a major role for aldehyde adduction in the pathogenesis of ALD.


Assuntos
Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Fígado/metabolismo , Fígado/patologia , Carbonilação Proteica , Proteínas/metabolismo , Acroleína/análise , Aldeídos/análise , Animais , Etanol/efeitos adversos , Etanol/metabolismo , Ácidos Graxos/metabolismo , Masculino , Malondialdeído/análise , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/análise , Proteômica , Espectrometria de Massas em Tandem , Substâncias Reativas com Ácido Tiobarbitúrico/análise
4.
J Proteome Res ; 10(4): 1837-47, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21319786

RESUMO

Alcoholic liver disease (ALD) is a prominent cause of morbidity and mortality in the United States. Alterations in protein folding occur in numerous disease states, including ALD. The endoplasmic reticulum (ER) is the primary site of post-translational modifications (PTM) within the cell. Glycosylation, the most abundant PTM, affects protein stability, structure, localization, and activity. Decreases in hepatic glycosylation machinery have been observed in rodent models of ALD, but specific protein targets have not been identified. Utilizing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, glycoproteins were identified in hepatic microsomal fractions from control and ethanol-fed mice. This study reports for the first time a global decrease in ER glycosylation. Additionally, the identification of 30 glycoproteins within this fraction elucidates pathway-specific alterations in ALD impaired glycosylation. Among the identified proteins, triacylglycerol hydrolase (TGH) is positively affected by glycosylation, showing increased activity following the addition of sugar moieties. Impaired TGH activity is associated with increased cellular storage of lipids and provides a potential mechanism for the observed pathologies associated with ALD.


Assuntos
Retículo Endoplasmático/metabolismo , Etanol/metabolismo , Glicoproteínas/análise , Fígado/química , Fígado/citologia , Animais , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional/métodos , Etanol/administração & dosagem , Glicoproteínas/genética , Glicosilação , Humanos , Fígado/patologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/química , Estresse Oxidativo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
5.
Mol Pharmacol ; 79(6): 941-52, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21415306

RESUMO

The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is proposed to be an important factor in the etiology of alcoholic liver disease. To understand the effects of 4-HNE on homeostatic signaling pathways in hepatocytes, cellular models consisting of the human hepatocellular carcinoma cell line (HepG2) and primary rat hepatocytes were evaluated. Treatment of both HepG2 cells and primary hepatocytes with subcytotoxic concentrations of 4-HNE resulted in the activation of Akt within 30 min as demonstrated by increased phosphorylation of residues Ser473 and Thr308. Quantification and subsequent immunocytochemistry of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)[rsqb] resulted in a 6-fold increase in total PtdIns(3,4,5)P(3) and increased immunostaining at the plasma membrane after 4-HNE treatment. Cotreatment of HepG2 cells with 4-HNE and the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) or the protein phosphatase 2A (PP2A) inhibitor okadaic acid revealed that the mechanism of activation of Akt is PI3K-dependent and PP2A-independent. Using biotin hydrazide detection, it was established that the incubation of HepG2 cells with 4-HNE resulted in increased carbonylation of the lipid phosphatase known as "phosphatase and tensin homolog deleted on chromosome 10" (PTEN), a key regulator of Akt activation. Activity assays both in HepG2 cells and recombinant PTEN revealed a decrease in PTEN lipid phosphatase activity after 4-HNE application. Mass spectral analysis of 4-HNE-treated recombinant PTEN detected a single 4-HNE adduct. Subsequent analysis of Akt dependent physiological consequences of 4-HNE in HepG2 cells revealed significant increases in the accumulation of neutral lipids. These results provide a potential mechanism of Akt activation and cellular consequences of 4-HNE in hepatocytes.


Assuntos
Aldeídos/farmacologia , Hepatócitos/efeitos dos fármacos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Primers do DNA , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Hepatócitos/enzimologia , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/antagonistas & inibidores
6.
Chem Res Toxicol ; 24(5): 651-62, 2011 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-21449565

RESUMO

4-Hydroxynonenal (4-HNE) is an endogenous product of lipid peroxidation known to play a role in cellular signaling through protein modification and is a major precursor for protein carbonyl adducts found in alcoholic liver disease (ALD). In the present study, a greater than 2-fold increase in protein carbonylation of sirtuin 3 (SIRT3), a mitochondrial class III histone deacetylase, is reported in liver mitochondrial extracts of ethanol-consuming mice. The consequence of this in vivo carbonylation on SIRT3 deacetylase activity is unknown. Interestingly, mitochondrial protein hyperacetylation was observed in a time-dependent increase in a model of chronic ethanol consumption; however, the underlying mechanisms for this remain unknown. Tandem mass spectrometry was used to identify and characterize the in vitro covalent modification of rSIRT3 by 4-HNE at Cys(280), a critical zinc-binding residue, and the resulting inhibition of rSIRT3 activity via pathophysiologically relevant concentrations of 4-HNE. Computational-based molecular modeling simulations indicate that 4-HNE modification alters the conformation of the zinc-binding domain inducing minor changes within the active site, resulting in the allosteric inhibition of SIRT3 activity. These conformational data are supported by the calculated binding energies derived from molecular docking studies suggesting the substrate peptide of acetyl-CoA synthetase 2 (AceCS2-K(ac)) and display a greater affinity for native SIRT3 as compared with the 4-HNE adducted protein. The results of this study characterize altered mitochondrial protein acetylation in a mouse model of chronic ethanol ingestion and thiol-specific allosteric inhibition of rSIRT3 resulting from 4-HNE adduction.


Assuntos
Aldeídos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Sirtuína 3/metabolismo , Compostos de Sulfidrila/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Etanol/metabolismo , Humanos , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , Carbonilação Proteica , Alinhamento de Sequência
7.
Mol Immunol ; 72: 9-18, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26922040

RESUMO

BACKGROUND: Ethanol feeding in mice activates complement via C1q binding to apoptotic cells in the liver; complement contributes to ethanol-induced inflammation and injury. Despite the critical role of C1q in ethanol-induced injury, the mechanism by which ethanol activates C1q remains poorly understood. Secretory IgM (sIgM), traditionally considered to act as an anti-microbial, also has critical housekeeping functions, facilitating clearance of apoptotic cells, at least in part through activation of C1q. Therefore, we hypothesized that (1) ethanol-induced apoptosis in the liver recruits sIgM, facilitating the activation of C1q and complement and (2) C1INH (C1 esterase inhibitor), which inhibits C1 functional activity, prevents complement activation and decreases ethanol-induced liver injury. METHODS: Female C57BL/6 wild-type, C1qa(-/-), BID(-/-) and sIgM(-/-) mice were fed ethanol containing liquid diets or pair-fed control diets. C1INH or vehicle was given via tail vein injection to ethanol- or pair-fed wild-type mice at 24 and 48h prior to euthanasia. RESULTS: Ethanol exposure increased apoptosis in the liver, as well as the accumulation of IgM in the liver. In the early stages of ethanol feeding, C1q co-localized with IgM in the peri-sinusoidal space of the liver and accumulation of IgM and C3b was dependent on ethanol-induced BID-dependent apoptosis. sIgM(-/-) mice were protected from both ethanol-induced activation of complement and early ethanol-induced liver injury when compared to wild-type mice. Treatment with C1INH also decreased hepatic C3b deposition and ethanol-induced injury. CONCLUSION: These data indicate that sIgM contributes to activation of complement and ethanol-induced increases in inflammatory cytokine expression and hepatocyte injury in the early stages of ethanol-induced liver injury.


Assuntos
Apoptose , Ativação do Complemento , Complemento C1q/imunologia , Imunoglobulina M/imunologia , Hepatopatias Alcoólicas/imunologia , Animais , Proteínas Inativadoras do Complemento 1/farmacologia , Proteína Inibidora do Complemento C1 , Complemento C1q/antagonistas & inibidores , Citocinas/biossíntese , Feminino , Hepatócitos/imunologia , Hepatopatias Alcoólicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Solubilidade
8.
Free Radic Biol Med ; 73: 411-20, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24924946

RESUMO

Pathogenesis in alcoholic liver disease (ALD) is complicated and multifactorial but clearly involves oxidative stress and inflammation. Currently, conflicting reports exist regarding the role of endoplasmic reticulum (ER) stress in the etiology of ALD. The glucose-regulated protein 78 (GRP78) is the ER homolog of HSP70 and plays a critical role in the cellular response to ER stress by serving as a chaperone assisting protein folding and by regulating the signaling of the unfolded protein response (UPR). Comprising three functional domains, an ATPase, a peptide-binding, and a lid domain, GRP78 folds nascent polypeptides via the substrate-binding domain. Earlier work has indicated that the ATPase function of GRP78 is intrinsically linked and essential to its chaperone activity. Previous work in our laboratory has indicated that GRP78 and the UPR are not induced in a mouse model of ALD but that GRP78 is adducted by the lipid electrophiles 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) in vivo. As impairment of GRP78 has the potential to contribute to pathogenesis in ALD, we investigated the functional consequences of aldehyde adduction on GRP78 function. Identification of 4-HNE and 4-ONE target residues in purified human GRP78 revealed a marked propensity for Lys and His adduction within the ATPase domain and a relative paucity of adduct formation within the peptide-binding domain. Consistent with these findings, we observed a concomitant dose-dependent decrease in ATP-binding and ATPase activity without any discernible impairment of chaperone function. Collectively, our data indicate that ATPase activity is not essential for GRP78-mediated chaperone activity and is consistent with the hypothesis that ER stress does not play a primary initiating role in the early stages of ALD.


Assuntos
Adenosina Trifosfatases/metabolismo , Aldeídos/química , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Choque Térmico/metabolismo , Hepatopatias Alcoólicas/patologia , Aldeídos/metabolismo , Sequência de Aminoácidos , Animais , Simulação por Computador , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Humanos , Inflamação/patologia , Hepatopatias Alcoólicas/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estresse Oxidativo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Resposta a Proteínas não Dobradas
9.
J Nutr Biochem ; 24(8): 1436-45, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23465594

RESUMO

OBJECTIVE: The objective of the study was to examine the interaction of moderate and high dietary fat and ethanol with respect to formation of steatosis and regulation of the AMP-activated protein kinase (AMPK) pathway in a mouse model of chronic ethanol consumption. METHODS: Male C57BL/6J mice were pair-fed a modified Lieber-DeCarli diet composed of either moderate fat [30% fat-derived calories (MF)] or high fat [45% fat-derived calories (HF)] combined with increasing concentrations of ethanol (2%-6%) for 6 weeks. RESULTS: Chronic ethanol consumption resulted in significant increases in plasma alanine aminotransferase in MF (1.84-fold) and HF mice (2.33-fold), yet liver triglycerides only increased significantly in the HF model (1.62-fold). Ethanol addition significantly increased plasma adiponectin under conditions of MF but not HF. In combination with MF, the addition of ethanol significantly decreased total and hepatic pThr(172)AMPKα and acetyl CoA Carboxylase (ACC). HF plus ethanol decreased pSer(108)AMPKß, yet a marked 1.5-fold increase in pThr(172)AMPKα occurred. No change was evident in pSer(79)ACC under conditions of ethanol and HF ingestion. In both models, nuclear levels of sterol response element binding protein 1c and carbohydrate response element binding protein were decreased. Surprisingly, MF plus ethanol significantly elevated protein expression of medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and very long chain acyl-CoA dehydrogenase but did not significantly affect mRNA expression of other proteins involved in ß-oxidation and fatty acid synthesis. HF plus ethanol significantly reduced mRNA expression of both stearoyl CoA desaturase 1 and fatty acid elongase 5, but did not have an effect on MCAD or LCAD. CONCLUSION: These data suggest that, when co-ingested with ethanol, dietary fat differentially contributes to dysregulation of adiponectin-dependent activation of the AMPK pathway in the liver of mice.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Gorduras na Dieta/administração & dosagem , Etanol/efeitos adversos , Fígado Gorduroso/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Adiponectina/sangue , Alanina Transaminase/sangue , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Modelos Animais de Doenças , Fígado Gorduroso/induzido quimicamente , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triglicerídeos/sangue
10.
PLoS One ; 7(6): e38459, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22701647

RESUMO

4-Hydroxynonenal (4-HNE) is a reactive α,ß-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd(1) = 0.395 µM and Kd(2) = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a contributing factor in the pathogenesis of ALD.


Assuntos
Aldeídos/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatopatias Alcoólicas/fisiopatologia , Modelos Moleculares , Conformação Proteica , Naftalenossulfonato de Anilina/metabolismo , Animais , Clonagem Molecular , Primers do DNA/genética , Eletroforese em Gel Bidimensional , Etanol/efeitos adversos , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Imunoprecipitação , Hepatopatias Alcoólicas/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinação
11.
J Toxicol ; 2012: 207594, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22829816

RESUMO

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

12.
Chem Biol Interact ; 192(1-2): 107-12, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-21354120

RESUMO

OBJECTIVES: Oxidative stress is one component of alcoholic liver disease (ALD) that is manifested in the peroxidation of cellular lipids producing the electrophile, 4-hydroxynonenal (4-HNE). This electrophile is proposed to modify essential cellular proteins resulting in loss of protein function and cellular homeostasis. Studies were initiated to identify hepatic proteins that are targets of 4-HNE modification and determine their relationship with progression of the early stages of ALD. METHODS: Rat and mouse models were developed using the Lieber-DeCarli diet to simulate early stages of ALD consisting of fatty liver (steatosis) and hepatocellular injury indicated by a 1.5-2-fold elevation of plasma ALT activity. Liver samples obtained from control and ethanol treated animals were subjected to two-dimensional electrophoresis and immunoblotting using polyclonal antibodies generated against 4-HNE epitopes for detection of proteins modified by 4-HNE. Following identification of 4-HNE adducted proteins, the respective recombinant proteins modified with physiologic concentrations of 4-HNE were evaluated to determine the functional consequences of 4-HNE modification. RESULTS: One group of proteins identified included Hsp70, Hsp90 and protein disulfide isomerase (PDI), all of which are involved in protein folding or processing are targets of adduction. In vitro assays indicated significant impairment of the protein activities following modification with physiologically relevant concentrations of 4-HNE. Liver fatty acid binding protein, L-FABP, was also identified as a target and additional studies revealed that the levels of this protein were significantly decreased because of chronic ethanol ingestion. Erk1/2 was identified as a target for modification and subsequently determined to have impaired activity. CONCLUSIONS: Inhibition of Hsp70, Hsp90 and PDI function could be involved in initiation of the early phases of ER stress contributing to stimulation and accumulation of hepatic lipids. Likewise, impairment of L-FABP activity could also disrupt lipid transport also contributing to steatosis. The modification and inhibition of Erk1/2 by 4-HNE may also contribute to the decreased hepatocellular proliferation associated with ALD. Collectively, these results provide new information concerning the mechanisms whereby the modification of hepatic proteins by 4-HNE contributes to ALD.


Assuntos
Peroxidação de Lipídeos , Hepatopatias Alcoólicas/metabolismo , Fígado/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley
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