RESUMEN
BACKGROUND: Lipoprotein lipase (LPL) is important for lipid deposition in adipose tissue (AT) and responds rapidly to changes in the nutritional state. Animal experiments indicate that short-term regulation of LPL is mainly post-translational. Different processing of LPL in different AT depots may play a role in the distribution of lipids in the body. MATERIALS AND METHODS: Lipoprotein lipase mRNA, mass and activity were measured in pieces of omental adipose tissue (OAT) and subcutaneous adipose tissue (SAT) from 15 subjects undergoing gastrointestinal surgery (four male and 11 female subjects, mean age 54 +/- 5 years, BMI 28 +/- 2 kg m(-2)). RESULTS: Lipoprotein lipase activity was higher in OAT than in SAT (18 +/- 2.1 compared with 12 +/- 1.6 mU g(-1), P < 0.01), whereas LPL mass was lower in OAT than in SAT (100 +/- 9 compared with 137 +/- 16 mU g(-1), P < 0.05). Consequently, the specific LPL activity (ratio of activity over mass) was approximately twofold greater in OAT compared with SAT. There was correlation between LPL mRNA and LPL activity in SAT (P < 0.05) and a similar tendency in OAT (P = 0.08). There were strong correlations (P < 0.01) for mRNA abundance as well as for LPL activity between the two depots. In contrast there was no correlation between the LPL mass and LPL mRNA or activity in any of the depots. CONCLUSIONS: These results indicate that long-term regulation, as reflected in the mRNA abundance, is similar in the two types of adipose tissue. The displayed activity reflects the mRNA abundance and the fraction of newly synthesized LPL molecules which the post-translational mechanism allows to become/remain active. This fraction was on average twofold greater in OAT compared with SAT.
Asunto(s)
Tejido Adiposo/enzimología , Lipoproteína Lipasa/metabolismo , Epiplón/enzimología , Abdomen/cirugía , Antropometría , Biopsia , Femenino , Expresión Génica , Humanos , Lípidos/sangre , Lipoproteína Lipasa/biosíntesis , Lipoproteína Lipasa/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Grasa Subcutánea/enzimologíaRESUMEN
With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA-poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA--poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA--poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.
Asunto(s)
ADN/administración & dosificación , Galactosa/metabolismo , Polilisina/administración & dosificación , Células 3T3 , Animales , Apolipoproteína A-I/genética , Arteriosclerosis/genética , Arteriosclerosis/terapia , Cloranfenicol O-Acetiltransferasa/genética , Genes Reporteros , Terapia Genética , Humanos , Técnicas In Vitro , Operón Lac , Hígado/metabolismo , Ratones , Polilisina/metabolismo , Ratas , Células Tumorales CultivadasAsunto(s)
Regulación Enzimológica de la Expresión Génica , Glucuronidasa/genética , Lisosomas/enzimología , Testosterona/fisiología , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucuronidasa/metabolismo , Riñón/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Orquiectomía , Especificidad de Órganos , Pene/enzimología , Glándulas Salivales/enzimología , Testosterona/farmacologíaRESUMEN
To study structure-function relationships in low density lipoprotein receptor (LDLR), a key protein in human cholesterol metabolism, it is reasonable to operate with separate protein domains. To obtain highly purified functionally active LDLR ligand-binding domain, we have cloned the corresponding LDLR cDNA fragment in two expression plasmid vectors of Escherichia coli. We have developed methods to purify fusion and practically individual recombinant proteins and characterized the obtained products biochemically. Antibodies raised against fused with beta-galactosidase and individual recombinant protein have been shown to be efficient in identification of LDLR protein in crude lysates of human fibroblasts (cell line HT-1080).