RESUMO
Adipose-specific inactivation of both AP-1 and CCAAT-enhancer-binding protein (C/EBP) families of B-ZIP transcription factors in transgenic mice causes severe lipoatrophy. To evaluate whether inactivation of only C/EBP members was critical for lipoatrophy, A-C/EBP, a dominant-negative protein that specifically inhibits the DNA binding of the C/EBP members, was expressed in adipose tissue. For the first 2 weeks after birth, aP2-A-C/EBP mice had no white adipose tissue (WAT), drastically reduced brown adipose tissue (BAT), and exhibited marked hepatic steatosis, hyperinsulinemia, and hyperlipidemia. However, WAT appeared during the third week, coinciding with significantly improved metabolic functioning. In adults, BAT remained reduced, causing cold intolerance. At 30 weeks, the aP2-A-C/EBP mice had only 35% reduced WAT, with clear morphological signs of lipodystrophy in subcutaneous fat. Circulating leptin and adiponectin levels were less than the wild-type levels, and these mice exhibited impaired triglyceride clearance. Insulin resistance, glucose intolerance, and reduced free fatty acid release in response to ß3-adrenergic agonist suggest improper functioning of the residual WAT. Gene expression analysis of inguinal WAT identified reduced mRNA levels of several enzymes involved in fatty acid synthesis and glucose metabolism that are known C/EBPα transcriptional targets. There were increased levels for genes involved in inflammation and muscle differentiation. However, when dermal fibroblasts from aP2-A-C/EBP mice were differentiated into adipocytes in tissue culture, muscle markers were elevated more than the inflammatory markers. These results demonstrate that the C/EBP family is essential for adipose tissue development during the early postnatal period, the regulation of glucose and lipid homeostasis in adults, and the suppression of the muscle lineage.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Lipodistrofia/etiologia , Lipodistrofia/metabolismo , Proteínas/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Western Blotting , Composição Corporal/genética , Composição Corporal/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Calorimetria Indireta , Células Cultivadas , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Proteínas de Ligação a Ácido Graxo/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Lipodistrofia/genética , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Proteínas/genética , Triglicerídeos/metabolismoRESUMO
Cellular levels of RNAs containing transposable elements increase in response to various stresses. Polymerase III (Pol III)-dependent transcripts of transposons are different from transposon-containing RNAs generated by read-through Pol II-dependent transcription. Until now, Pol III transcripts were detected by primer extension followed by time-consuming gel electrophoresis. In this paper, we describe a more sensitive PCR-based method for the selective detection of Pol III-transcribed RNAs. The method is based on the difference in sequences at the 5' ends of the Pol II- and Pol III-dependent transcripts. We employed this method to quantify Pol III transcripts of transposon B1 in rodent cells and revealed that their levels are affected by UV irradiation. We therefore conclude that the abundance of the Pol III-transcribed fraction of cellular RNA may serve as marker of stress response and can be conveniently quantified by the method described.
Assuntos
DNA Polimerase III/metabolismo , Elementos de DNA Transponíveis/genética , Perfilação da Expressão Gênica/métodos , Hepatócitos/química , Reação em Cadeia da Polimerase/métodos , RNA/biossíntese , Estresse Fisiológico/genética , Raios Ultravioleta/efeitos adversos , Animais , Núcleo Celular/metabolismo , Células Cultivadas/metabolismo , Células Cultivadas/efeitos da radiação , Elementos de DNA Transponíveis/efeitos da radiação , Hepatócitos/enzimologia , Queratinócitos/enzimologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Camundongos , RNA/genética , Ratos , Elementos Nucleotídeos Curtos e Dispersos/genéticaAssuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/classificação , Fatores de Transcrição/química , Fatores de Transcrição/classificação , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Dimerização , Humanos , Zíper de Leucina , Dados de Sequência Molecular , Ligação Proteica , TermodinâmicaRESUMO
The beta2-neuronal nicotinic acetylcholine receptor gene (CHRNB2) is a logical candidate for influencing smoking behavior and nicotine dependence. We discovered six single nucleotide polymorphisms (SNPs) in the CHRNB2 gene by surveying 15.4 kb of genomic sequence including a previously undescribed 3' untranslated region that extends 4.0 kb downstream of the coding region. One of the SNPs causes an amino acid substitution in exon 5, one occurs in the promoter region, one changes an intronic base, and three occur in the 3' untranslated region. The ethnically dependent allele frequencies and the marker-to-marker linkage disequilibrium patterns of five of these polymorphisms were determined. The SNPs were assayed in 743 individuals for whom information on smoking history and lifelong nicotine dependence was available. No significant associations of the individual markers or their haplotypes to smoking behavior or level of nicotine dependence were found.