RESUMO
Nonalcoholic fatty liver disease (NAFLD) is a major health burden in the aging society with an urging medical need for a better understanding of the underlying mechanisms. Mitochondrial fatty acid oxidation and mitochondrial-derived reactive oxygen species (ROS) are considered critical in the development of hepatic steatosis, the hallmark of NAFLD. Our study addressed in C57BL/6J mice the effect of high fat diet feeding and age on liver mitochondria at an early stage of NAFLD development. We therefore analyzed functional characteristics of hepatic mitochondria and associated alterations in the mitochondrial proteome in response to high fat feeding in adolescent, young adult, and middle-aged mice. Susceptibility to diet-induced obesity increased with age. Young adult and middle-aged mice developed fatty liver, but not adolescent mice. Fat accumulation was negatively correlated with an age-related reduction in mitochondrial mass and aggravated by a reduced capacity of fatty acid oxidation in high fat-fed mice. Irrespective of age, high fat diet increased ROS production in hepatic mitochondria associated with a balanced nuclear factor erythroid-derived 2 like 2 (NFE2L2) dependent antioxidative response, most likely triggered by reduced tethering of NFE2L2 to mitochondrial phosphoglycerate mutase 5. Age indirectly influenced mitochondrial function by reducing mitochondrial mass, thus exacerbating diet-induced fat accumulation. Therefore, consideration of age in metabolic studies must be emphasized.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Ingestão de Energia/fisiologia , Fígado Gorduroso/fisiopatologia , Fígado/metabolismo , Mitocôndrias/metabolismo , Fatores Etários , Animais , Estudos Transversais , Ácidos Graxos/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/veterinária , Metabolismo dos Lipídeos , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Oxirredução , Fosfoproteínas Fosfatases/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismoRESUMO
The intestinal peptide transporter PEPT1 provides bulk quantities of amino acids to epithelial cells. PEPT1 is a high-capacity and low-affinity solute carrier of the SLC15 family found in apical membranes of enterocytes in small intestine and distal colon. Surprisingly, murine PEPT1 (mPEPT1) has an apparent molecular mass of â¼95 kDa in the small intestine but â¼105 kDa in the large intestine. Here we describe studies on mPEPT1 protein glycosylation and how glycans affect transport function. Putative N-glycosylation sites of mPEPT1 were altered by site-directed mutagenesis followed by expression in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515, and N532 by glutamine (Q) resulted in a decrease of the mPEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation-deficient transporters to be functional with comparable expression levels in oocyte membranes. Strikingly, the mutant protein with N50Q exhibited a twofold decreased affinity for Gly-Sar but a 2.5-fold rise in the maximal inward currents compared with the wild-type protein. Elevated maximal transport currents were also recorded for cefadroxil and tri-l-alanine. Tracer flux studies performed with [(14)C]-Gly-Sar confirmed the reduction in substrate affinity and showed twofold increased maximal transport rates for the N50Q transporter. Elimination of individual N-glycosylation sites did not alter membrane expression in oocytes or overall transport characteristics except for the mutant protein N50Q. Because transporter surface density was not altered in N50Q, removal of the glycan at this location appears to accelerate the substrate turnover rate.
Assuntos
Simportadores/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Glicosilação , Camundongos , Mutagênese Sítio-Dirigida , Transportador 1 de Peptídeos , XenopusRESUMO
The intestinal peptide transporter PEPT-1 plays an important role in development, growth, reproduction, and stress tolerance in Caenorhabditis elegans, as revealed by the severe phenotype of the pept-1-deficient strain. The reduced number of offspring and increased stress resistance were shown to result from changes in the insulin/IGF-signaling cascade. To further elucidate the regulatory network behind the phenotypic alterations in PEPT1-deficient animals, a quantitative proteome analysis combined with transcriptome profiling was applied. Various target genes of XBP-1, the major mediator of the unfolded protein response, were found to be downregulated at the mRNA and protein levels, accompanied by a reduction of spliced xbp-1 mRNA. Proteome analysis also revealed a markedly reduced content of numerous ribosomal proteins. This was associated with a reduction in the protein synthesis rate in pept-1 C. elegans, a process that is strictly regulated by the TOR (target of rapamycine) complex, the cellular sensor for free amino acids. These data argue for a central role of PEPT-1 in cellular amino acid homeostasis. In PEPT-1 deficiency, amino acid levels dropped systematically, leading to alterations in protein synthesis and in the IRE-1/XBP-1 pathway.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Aminoácidos/análise , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Western Blotting , Caenorhabditis elegans , Proteínas de Transporte/genética , Primers do DNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/metabolismo , Proteômica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/metabolismo , Resposta a Proteínas não Dobradas/genéticaRESUMO
Selenium is an essential trace element and mediates its functions via various selenoproteins such as glutathione peroxidases or thioredoxin reductases. A suboptimal selenium supply causes metabolic disturbances and is associated with an increased risk to develop different disorders, including cancer or cardiovascular diseases. This study aimed to assess the impact of a suboptimal selenium status on the hepatic metabolome of male mice analyzed by a targeted liquid chromatography/tandem mass spectrometry and a method based on non-targeted gas chromatography hyphenated with mass spectrometry. Feeding animals a diet with about half of the recommended selenium content supplied as selenomethionine caused liver glutathione peroxidase and thioredoxin reductase activities to decline and lipid peroxidation to increase. Serum T3 thyroid hormone concentration also declined via a reduced hepatic deiodinase activity. Metabolite profiling revealed predominantly changes in cysteine and carbon-1 metabolism as well as in selected lipid subclasses. In particular the concentrations of palmitoylcarnitines and oleoylcarnitines (C18:1 and C16:1) and various phosphatidylcholine species containing saturated fatty acids were elevated. Increased taurine levels suggested an enhanced cysteine flux through the salvage pathway whereas increased homocysteine levels appeared to be a consequence of a massive down-regulation of cystathionine ß lyase (cystathionine ß synthase) and a reduced flux through the transsulfuration pathway. The findings demonstrate that a suboptimal selenium status causes alterations in lipid and carbon-1 metabolism in mouse liver. These changes may contribute to the development of diseases associated with a suboptimal selenium status.
Assuntos
Fígado/metabolismo , Selênio/metabolismo , Animais , Carnitina/análogos & derivados , Carnitina/metabolismo , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Glicogênio/metabolismo , Peroxidação de Lipídeos , Masculino , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de Massas em Tandem , Tri-Iodotironina/metabolismoRESUMO
Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.
Assuntos
Transportador de Glucose Tipo 2/metabolismo , Glucose/metabolismo , Mucosa Intestinal/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo , Animais , Glicemia/metabolismo , Glucose/farmacologia , Transportador de Glucose Tipo 2/deficiência , Incretinas/sangue , Incretinas/metabolismo , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Absorção Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Camundongos , Transportador 1 de Glucose-Sódio/deficiênciaRESUMO
BACKGROUND: PEPT1 is a rheogenic transport protein in the apical membrane of intestinal epithelial cells capable of transporting essentially all possible di- and tripeptides that are generated from the luminal protein breakdown. In addition, several anticancer, antimicrobial and antiviral drugs are taken up from the intestinal lumen via PEPT1 and therefore PEPT1 is a target for efficient drug delivery via prodrug approaches. Thus, understanding PEPT1 gene regulation is not only of importance for dietary adaptation but also for drug treatment. METHODS: In silico analysis of the Pept1 promoter was performed using MatInspector. Pept1 promoter constructs were generated and cotransfected with an Nrf2 expression plasmid. Caco-2 cells were stimulated with Nrf2 inducers followed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Biological relevance was investigated using western blot analysis and transport activity assays. RESULTS: Reporter gene assays showed transcriptional activation of the Pept1 promoter in response to Nrf2 overexpression. EMSA as well as ChIP analysis validated Nrf2 binding to the ARE located closest to the start codon (Pept1-ARE1). Induction of the Nrf2 pathway resulted in increased endogenous PEPT1 protein abundance as well as transport activity. Moreover, we demonstrate that also the induction of autophagy by MG132 resulted in elevated Nrf2 binding to Pept1-ARE1 and increased PEPT1 protein expression. CONCLUSION: In summary, we identified a biologically active Nrf2 binding site within the Pept1 promoter which links Pept1 to the cellular defense program activated by Nrf2. GENERAL SIGNIFICANCE: This study identifies Pept1 as an inducible target gene of the Nrf2 pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fator 2 Relacionado a NF-E2/fisiologia , Simportadores/genética , Autofagia , Sítios de Ligação , Células CACO-2 , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Transportador 1 de Peptídeos , Regiões Promotoras GenéticasRESUMO
The development of the nematode Caenorhabditis elegans is a highly dynamic process. Although various studies have assessed global transcriptome changes, information on the dynamics of the proteome during ontogenesis is not available. We metabolically labeled C. elegans by using ¹5N ammonium chloride as a precursor in Escherichia coli feeding bacteria grown in minimal media as a new cost-effective technique. Quantitative proteome analysis was performed by LC-MS/MS in animals harvested at different times during ontogenesis. We identified and quantified 245 proteins at all larval stages in two independent replicates. Between larval stages (20 and 40 h after hatching) 61 were found to change significantly in level. Among those ribosomal proteins, aminoacyl tRNA synthetases and enzymes of energy metabolism increased in abundance, while extracellular matrix proteins and muscle proteins dominated groups displaying reduced levels. Moreover, changes observed for selected proteins such as VIT-6 and SOD-1 matched with previously published findings confirming the validity of our approach. The metabolic labeling technique applied seems well suited to assess changes in the proteome changes of C. elegans in a quantitative manner during larval development. The data set generated provides the basis for further exploitation of the role of individual proteins or protein clusters during ontogenesis.
Assuntos
Caenorhabditis elegans/metabolismo , Isótopos de Nitrogênio/análise , Proteoma/metabolismo , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Marcação por Isótopo/métodos , Larva , Isótopos de Nitrogênio/metabolismo , Mapas de Interação de Proteínas , Proteoma/análise , Proteômica/métodos , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/metabolismo , Espectrometria de Massas em TandemRESUMO
Like most microorganisms, the yeast Saccharomyces cerevisiae is prototrophic for riboflavin (vitamin B2). Riboflavin auxotrophic mutants with deletions in any of the RIB genes frequently segregate colonies with improved growth. We demonstrate by reporter assays and Western blots that these suppressor mutants overexpress the plasma-membrane riboflavin transporter MCH5. Frequently, this overexpression is mediated by the transcription factor Put3, which also regulates the proline catabolic genes PUT1 and PUT2. The increased expression of MCH5 may increase the concentrations of FAD, which is the coenzyme required for the activity of proline oxidase, encoded by PUT1. Thus, Put3 regulates proline oxidase activity by synchronizing the biosynthesis of the apoenzyme and the coenzyme FAD. Put3 is known to bind to the promoters of PUT1 and PUT2 constitutively, and we demonstrate by gel-shift assays that it also binds to the promoter of MCH5. Put3-mediated transcriptional activation requires proline as an inducer. We find that the increased activity of Put3 in one of the suppressor mutants is caused by increased intracellular levels of proline. Alternative PUT3-dependent and -independent mechanisms might operate in other suppressed strains.