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1.
J Clin Endocrinol Metab ; 97(9): 3261-9, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22802091

RESUMEN

CONTEXT: Conflicting data exist on mitochondrial function and physical activity in type 2 diabetes mellitus (T2DM) development. OBJECTIVE: The aim was to assess mitochondrial function at different stages during T2DM development in combination with physical exercise in longstanding T2DM patients. DESIGN AND METHODS: We performed cross-sectional analysis of skeletal muscle from 12 prediabetic 11 longstanding T2DM male subjects and 12 male controls matched by age and body mass index. INTERVENTION: One-year intrasubject controlled supervised exercise training intervention was done in longstanding T2DM patients. MAIN OUTCOME MEASUREMENTS: Extensive ex vivo analyses of mitochondrial quality, quantity, and function were collected and combined with global gene expression analysis and in vivo ATP production capacity after 1 yr of training. RESULTS: Mitochondrial density, complex I activity, and the expression of Krebs cycle and oxidative phosphorylation system-related genes were lower in longstanding T2DM subjects but not in prediabetic subjects compared with controls. This indicated a reduced capacity to generate ATP in longstanding T2DM patients only. Gene expression analysis in prediabetic subjects suggested a switch from carbohydrate toward lipid as an energy source. One year of exercise training raised in vivo skeletal muscle ATP production capacity by 21 ± 2% with an increased trend in mitochondrial density and complex I activity. In addition, expression levels of ß-oxidation, Krebs cycle, and oxidative phosphorylation system-related genes were higher after exercise training. CONCLUSIONS: Mitochondrial dysfunction is apparent only in inactive longstanding T2DM patients, which suggests that mitochondrial function and insulin resistance do not depend on each other. Prolonged exercise training can, at least partly, reverse the mitochondrial impairments associated with the longstanding diabetic state.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Mitocondrias Musculares/fisiología , Miopatías Mitocondriales/metabolismo , Miopatías Mitocondriales/terapia , Actividad Motora/fisiología , Músculo Esquelético/metabolismo , Adenosina Trifosfato/biosíntesis , Anciano , Presión Sanguínea/fisiología , Composición Corporal/fisiología , Índice de Masa Corporal , Ciclo del Ácido Cítrico/genética , Ciclo del Ácido Cítrico/fisiología , Diabetes Mellitus Tipo 2/terapia , Progresión de la Enfermedad , Femenino , Expresión Génica/fisiología , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/metabolismo , Fosforilación Oxidativa , Aptitud Física/fisiología , Estado Prediabético/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa
2.
Mol Genet Metab ; 106(1): 73-82, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22405600

RESUMEN

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). This enzyme is required for the degradation of dermatan sulfate. In its absence, dermatan sulfate accumulates in cells and is excreted in large quantities in urine. Specific therapeutic intervention is available; however, accurate and timely diagnosis is crucial for maximal benefit. To better understand the current practices for diagnosis and to establish diagnostic guidelines, an international MPS VI laboratory diagnostics scientific summit was held in February of 2011 in Miami, Florida. The various steps in the diagnosis of MPS VI were discussed including urinary glycosaminoglycan (uGAG) analysis, enzyme activity analysis, and molecular analysis. The following conclusions were reached. Dilute urine samples pose a significant problem for uGAG analysis and MPS VI patients can be missed by quantitative uGAG testing alone as dermatan sulfate may not always be excreted in large quantities. Enzyme activity analysis is universally acknowledged as a key component of diagnosis; however, several caveats must be considered and the appropriate use of reference enzymes is essential. Molecular analysis supports enzyme activity test results and is essential for carrier testing, subsequent genetic counseling, and prenatal testing. Overall the expert panel recommends caution in the use of uGAG screening alone to rule out or confirm the diagnosis of MPS VI and acknowledges enzyme activity analysis as a critical component of diagnosis. Measurement of another sulfatase enzyme to exclude multiple sulfatase deficiency was recommended prior to the initiation of therapy. When feasible, the use of molecular testing as part of the diagnosis is encouraged. A diagnostic algorithm for MPS VI is provided.


Asunto(s)
Glicosaminoglicanos/orina , Mucopolisacaridosis VI/diagnóstico , N-Acetilgalactosamina-4-Sulfatasa , Cerebrósido Sulfatasa/sangre , Cerebrósido Sulfatasa/orina , Pruebas con Sangre Seca , Humanos , Mucopolisacaridosis VI/enzimología , N-Acetilgalactosamina-4-Sulfatasa/sangre , N-Acetilgalactosamina-4-Sulfatasa/genética , N-Acetilgalactosamina-4-Sulfatasa/orina
3.
J Med Genet ; 49(1): 10-5, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22114105

RESUMEN

BACKGROUND: Mitochondrial disorders are associated with abnormalities of the oxidative phosphorylation (OXPHOS) system and cause significant morbidity and mortality in the population. The extensive clinical and genetic heterogeneity of these disorders due to a broad variety of mutations in several hundreds of candidate genes, encoded by either the mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), impedes a straightforward genetic diagnosis. A new disease gene is presented here, identified in a single Kurdish patient born from consanguineous parents with neonatally fatal Leigh syndrome and complex I deficiency. METHODS AND RESULTS: Using homozygosity mapping and subsequent positional candidate gene analysis, a total region of 255.8 Mb containing 136 possible mitochondrial genes was identified. A pathogenic mutation was found in the complex I subunit encoding the NDUFA9 gene, changing a highly conserved arginine at position 321 to proline. This is the first disease-causing mutation ever reported for NDUFA9. Complex I activity was restored in fibroblasts of the patient by lentiviral transduction with wild type but not mutant NDUFA9, confirming that the mutation causes the complex I deficiency and related disease. CONCLUSIONS: The data show that homozygosity mapping and candidate gene analysis remain an efficient way to detect mutations even in small consanguineous pedigrees with OXPHOS deficiency, especially when the enzyme deficiency in fibroblasts allows appropriate candidate gene selection and functional complementation.


Asunto(s)
Complejo I de Transporte de Electrón/genética , Enfermedad de Leigh/diagnóstico , Enfermedad de Leigh/genética , Mutación Missense , Secuencia de Aminoácidos , Células Cultivadas , Consanguinidad , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/metabolismo , Resultado Fatal , Estudios de Asociación Genética , Homocigoto , Humanos , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Datos de Secuencia Molecular , Neuroimagen
4.
Mol Genet Metab ; 104(3): 273-8, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21843962

RESUMEN

Earlier research on ten horses suffering from the frequently fatal disorder atypical myopathy showed that MADD (multiple acyl-CoA dehydrogenase deficiency) is the biochemical derangement behind atypical myopathy. From five horses that died as a result of this disease and seven healthy control horses, urine and plasma were collected ante mortem and muscle biopsies were obtained immediately post-mortem (2 patients and 7 control horses), to analyse creatine, purine and carbohydrate metabolism as well as oxidative phosphorylation. In patients, the mean creatine concentration in urine was increased 17-fold and the concentration of uric acid approximately 4-fold, compared to controls. The highest degree of depletion of glycogen was observed in the patient with the most severe myopathy clinically. In this patient, glycolysis was more active than in the other patients and controls, which may explain this depletion. One patient demonstrated very low phosphoglycerate mutase (PGAM) activity, less than 10% of reference values. Most respiratory chain complex activity in patients was 20-30% lower than in control horses, complex II activity was 42% lower than normal, and one patient had severely decrease ATP-synthase activity, more than 60% lower than in control horses. General markers for myopathic damage are creatine kinase (CK) and lactic acid in plasma, and creatine and uric acid in urine. To obtain more information about the cause of the myopathy analysis of carbohydrate, lipid and protein metabolism as well as oxidative phosphorylation is advised. This study expands the diagnostic possibilities of equine myopathies.


Asunto(s)
Creatina , Enfermedades de los Caballos/metabolismo , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/complicaciones , Fosforilación Oxidativa , Fosfoglicerato Mutasa/deficiencia , Condicionamiento Físico Animal , Rabdomiólisis/metabolismo , Animales , Aspartato Aminotransferasas/sangre , Secuencia de Bases , Creatina/orina , Creatina Quinasa/sangre , Cartilla de ADN/genética , Femenino , Caballos , L-Lactato Deshidrogenasa/sangre , Malonatos/orina , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Datos de Secuencia Molecular , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/sangre , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/orina , Fosfoglicerato Mutasa/genética , Rabdomiólisis/etiología , Análisis de Secuencia de ADN , Succinatos/orina , Ácido Úrico/orina
5.
J Med Genet ; 47(8): 507-12, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19542079

RESUMEN

BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.


Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Enfermedad de Leigh/enzimología , Enfermedad de Leigh/genética , Metiltransferasas/genética , Proteínas Mitocondriales/genética , Mutación/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Secuencia de Bases , Preescolar , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/genética , Familia , Femenino , Homocigoto , Humanos , Enfermedad de Leigh/diagnóstico por imagen , Enfermedad de Leigh/metabolismo , Leucocitos Mononucleares/enzimología , Imagen por Resonancia Magnética , Masculino , Metiltransferasas/química , Proteínas Mitocondriales/química , Datos de Secuencia Molecular , Marruecos , Linaje , Tomografía Computarizada por Rayos X , Adulto Joven
6.
J Med Genet ; 44(4): e74, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17400793

RESUMEN

BACKGROUND: Detection of mutations in the mitochondrial DNA (mtDNA) is usually limited to common mutations and the transfer RNA genes. However, mutations in other mtDNA regions can be an important cause of oxidative phosphorylation (OXPHOS) disease as well. OBJECTIVE: To investigate whether regions in the mtDNA are preferentially mutated in patients with OXPHOS disease. METHODS: Screening of the mtDNA for heteroplasmic mutations was performed by denaturing high-performance liquid chromatography analysis of 116 patients with OXPHOS disease but without the common mtDNA mutations. RESULTS: An mtDNA sequence variant was detected in 15 patients, 5 of which were present in the ND5 gene. One sequence variant was new and three were known, one of which was found twice. The novel sequence variant m.13511A-->T occurred in a patient with a Leigh-like syndrome. The known mutation m.13513G-->A, associated with mitochondrial encephalomyopathy lactic acidosis and stroke-like syndrome (MELAS) and MELAS/Leigh/Leber hereditary optic neuropathy overlap syndrome, was found in a relatively low percentage in two patients from two different families, one with a MELAS/Leigh phenotype and one with a MELAS/chronic progressive external ophthalmoplegia phenotype. The known mutation m.13042G-->A, detected previously in a patient with a MELAS/myoclonic epilepsy, ragged red fibres phenotype and in a family with a prevalent ocular phenotype, was now found in a patient with a Leigh-like phenotype. The sequence variant m.12622G-->A was reported once in a control database as a polymorphism, but is reported in this paper as heteroplasmic in three brothers, all with infantile encephalopathy (Leigh syndrome) fatal within the first 15 days of life. Therefore, a causal relationship between the presence of this sequence variant and the onset of mitochondrial disease cannot be entirely excluded at this moment. CONCLUSIONS: Mutation screening of the ND5 gene is advised for routine diagnostics of patients with OXPHOS disease, especially for those with MELAS- and Leigh-like syndrome with a complex I deficiency.


Asunto(s)
Complejo I de Transporte de Electrón/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , Secuencia de Aminoácidos , Animales , Encéfalo/anomalías , Niño , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , ADN Mitocondrial/genética , Enfermedades en Gemelos , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/fisiología , Resultado Fatal , Femenino , Pruebas Genéticas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Recién Nacido , Enfermedad de Leigh/genética , Síndrome MELAS/genética , Masculino , Mitocondrias Musculares/enzimología , Proteínas Mitocondriales/química , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/fisiología , Datos de Secuencia Molecular , Mutación Missense , Fenotipo , Subunidades de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido
7.
Acta Physiol (Oxf) ; 187(1-2): 295-303, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16734766

RESUMEN

AIM: The role of high cholesterol-containing microdomains in the signal transduction cascade leading to the activation of volume-regulated anion channels (VRACs) was studied. METHODS: Osmotic cell swelling-induced efflux of 125I- was determined in human epithelial Intestine 407 cells and in skin fibroblasts obtained from healthy controls or Niemann-Pick type C (NPC) patients. Cellular cholesterol content was modulated by pre-incubation with 2-hydroxypropyl-beta-cyclodextrin in the presence of acceptor lipid vesicles. RESULTS: Osmotic cell swelling of human Intestine 407 cells leads to the rapid activation of a compensatory anion conductance. Treatment of the cells with cyclodextrin enhanced the response to submaximal hypotonic stimulation by approx. twofold, but did not further increase the efflux elicited by a saturating stimulus. In contrast, the volume-sensitive anion efflux was markedly inhibited when cholesterol-loaded cyclodextrin was used. Potentiation of the response by cholesterol depletion was maintained in caveolin-1 deficient Caco-2 colonocytes as well as in sphingomyelinase-treated Intestine 407 cells, indicating that cholesterol-rich microdomains are not crucially involved. However, treatment of the cells with progesterone, an inhibitor of NPC1-dependent endosomal cholesterol trafficking, not only markedly reduced the hypotonicity-provoked anion efflux, but also prevented its potentiation by cyclodextrin. In addition, the volume-sensitive anion efflux from human NPC skin fibroblasts was significantly smaller when compared with control fibroblasts. CONCLUSIONS: The results support a model of regulatory volume decrease involving recruitment of volume-sensitive anion channels from intracellular compartments to the plasma membrane.


Asunto(s)
Canales de Cloruro/metabolismo , Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina , Transporte Biológico , Estudios de Casos y Controles , Caveolina 1/farmacología , Línea Celular , Membrana Celular/metabolismo , Tamaño de la Célula , Células Cultivadas , Excipientes/farmacología , Fibroblastos/metabolismo , Humanos , Soluciones Hipotónicas , Membrana Dobles de Lípidos , Progesterona/farmacología , Estructura Terciaria de Proteína , Esfingomielina Fosfodiesterasa/farmacología , beta-Ciclodextrinas/farmacología
8.
J Inherit Metab Dis ; 28(5): 733-41, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16151905

RESUMEN

6-Hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMUPC) was shown to be a specific substrate for the determination of acid (lysosomal) sphingomyelinase (ASM; gene SMPD1). Fibroblasts (n = 27) and leukocytes (n = 8) from both the A and B types of Niemann-Pick disease showed < 6% and < 10% of mean normal ASM activity, respectively. Niemann-Pick A or B patients bearing the Q292K mutation had apparently normal ASM activity with our new artificial substrate. These patients with false-normal sphingomyelinase activity, however, could readily be detected by determining the extent of inhibition of enzymatic hydrolysis of the artificial substrate HMU-PC by an unlabelled natural substrate, in particular lysosphingomyelin. This approach is generally applicable. Our novel assay for ASM combines the ease of a rapid and robust enzyme assay using a fluorogenic substrate with the specificity of an ASM assay using a natural substrate. Such assays are obviously more convenient to the diagnostic laboratory, since radiolabelled substrates are not required.


Asunto(s)
Análisis Químico de la Sangre/métodos , Química Clínica/métodos , Fluorometría/métodos , Enfermedades de Niemann-Pick/diagnóstico , Esfingomielina Fosfodiesterasa/química , Ceramidas/química , Pruebas Enzimáticas Clínicas , Diagnóstico Diferencial , Relación Dosis-Respuesta a Droga , Fibroblastos/enzimología , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica , Hexosaminidasas/química , Humanos , Hidrólisis , Leucocitos/enzimología , Leucocitos/metabolismo , Mutación , Enfermedades de Niemann-Pick/enzimología , Éteres Fosfolípidos/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Unión Proteica , Reproducibilidad de los Resultados , Piel/metabolismo , Esfingomielinas/química , Esfingosina/análogos & derivados , Esfingosina/química , Especificidad por Sustrato , Factores de Tiempo
9.
Mol Hum Reprod ; 11(3): 223-8, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15709156

RESUMEN

A family presented with three affected children with Leigh syndrome, a progressive neurodegenerative disorder. Analysis of the OXPHOS complexes in muscle of two affected patients showed an increase in activity of pyruvate dehydrogenase and a decrease of complex V activity. Mutation analysis revealed the T9176C mutation in the mtATPase 6 gene (OMIM 516060) and the mutation load was above 90% in the patients. Unaffected maternal relatives were tested for carrier-ship and one of them, with a mutation load of 55% in blood, was pregnant with her first child. The possibility of prenatal diagnosis was evaluated. The main problem was the lack of data on genotype-phenotype associations for the T9176C mutation and on variation of the mutation percentage in tissues and in time. Therefore, multiple tissues of affected and unaffected carriers were analysed. Eventually, prenatal diagnosis was offered with understanding by the couple that there could be considerable uncertainty in the interpretation of the results. Prenatal diagnosis was carried out twice on cultured and uncultured chorion villi and amniotic fluid cells. The result was a mutation percentage just below the assumed threshold of expression (90%). The couple decided to continue the pregnancy and an apparently healthy child was born with an as yet unclear prognosis. This is the first prenatal diagnosis for a carrier of the T9176C mutation. Prenatal diagnosis for this mutation is technically reliable, but the prognostic predictions are not straightforward.


Asunto(s)
ADN Mitocondrial/genética , Enfermedad de Leigh/diagnóstico , ATPasas de Translocación de Protón Mitocondriales/genética , Diagnóstico Prenatal , Niño , Análisis Mutacional de ADN , Femenino , Humanos , Enfermedad de Leigh/genética , Masculino , Músculo Esquelético/enzimología , Linaje , Fenotipo , Mutación Puntual , Embarazo , Complejo Piruvato Deshidrogenasa/análisis
10.
Biochim Biophys Acta ; 1393(1): 137-42, 1998 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-9714777

RESUMEN

Dihydroxyacetonephosphate acyltransferase (DHAP-acyltransferase) and alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) are the first two enzymes involved in the biosynthesis of ether phospholipids. Both peroxisomal enzymes have recently been purified to homogeneity and their molecular weights under denaturing conditions were reported. To determine the in situ functional size of both enzymes, radiation inactivation experiments were performed. Alkyl-DHAP synthase showed single exponential decays, both when enzymatic activity and when immunoreactive protein levels were measured, from which target sizes of 79+/-2 kDa and 78+/-4 kDa, respectively, were calculated. DHAP-acyltransferase activity increased at lower doses and decayed upon further irradiation with an apparent target size of 62+/-7 kDa. We conclude from these data that the functional unit sizes for both enzymes in situ are represented by their single polypeptide chains.


Asunto(s)
Aciltransferasas/química , Transferasas Alquil y Aril/química , Hígado/enzimología , Proteínas de la Membrana/química , Análisis por Activación , Animales , Cobayas , Microcuerpos/enzimología , Peso Molecular
11.
Biochem J ; 330 ( Pt 2): 765-9, 1998 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-9480888

RESUMEN

Lipoprotein lipase (LPL) purified from bovine milk showed variable abilities to stimulate the binding of low density lipoprotein (LDL) to J774 macrophages. The presence of a 37 kDa protein in the LPL sample seemed to be of importance for its stimulatory capacity. In order to investigate this, we isolated LPL from bovine milk via heparin Sepharose chromatography using a continuous salt gradient. Fractions containing the 37 kDa protein (as shown by SDS/PAGE under reducing conditions) eluted first from the column, followed by the 56 kDa LPL protein. The LPL enzymatic activity co-eluted with the 56 kDa protein, whereas the amount of 37 kDa protein fully paralleled the stimulatory effect on the binding of LDL to J774 cells. Samples not containing the 37 kDa protein were far less effective in stimulating the binding. Western blotting using a monoclonal antibody 5D2 against amino acids 396-405 in the carboxy-terminal domain of LPL, showed that the 37 kDa protein may be the C-terminal domain of LPL, presumably generated by proteolytic degradation of the mature LPL protein by milk proteases during its isolation. Furthermore, the functional mass of LPL for stimulation of the binding of LDL, as determined by radiation inactivation, was shown to be 30.9+/-1.8 kDa. We therefore suggest that cleavage of LPL at protease-sensitive sites causes a conformational change, generating an LPL protein which is more effective in mediating the binding and uptake of lipoproteins by cells.


Asunto(s)
Lipoproteína Lipasa/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Animales , Western Blotting , Bovinos , Células Cultivadas , Cromatografía en Gel , Humanos , Ratones , Leche/enzimología , Peso Molecular
12.
Biochem J ; 330 ( Pt 2): 785-9, 1998 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-9480891

RESUMEN

Hepatic lipase (HL) plays a key role in the metabolism of several lipoproteins. Metabolically active HL is bound in liver parenchymal cells to specific binding sites. We studied the nature of the HL binding in rat liver. Rat livers were perfused with heparin, which lead to a loss of 80% of the HL binding capacity of the liver. The heparin-containing perfusates possessed HL binding capacity, determined by slot-blot assay. The perfusates were loaded on to a heparin-Sepharose column and eluted with a linear salt gradient (0.2-1 M). HL binding activity, assessed by a slot-blot binding assay, eluted both at 0.3 M and at 0.8 M NaCl. A 0.5 M NaCl eluate was used to further characterize the HL binding activity. In this fraction the major protein had a molecular mass of 70 kDa. The fraction showed saturable HL binding in a solid-phase binding assay. Cross-linking of the 0.5 M NaCl fraction to 125I-labelled HL yielded a complex of 130 kDa, suggesting the cross-linking of the 57 kDa 125I-labelled HL to a protein of about 73 kDa. We concluded that heparin releases a protein of about 73 kDa from rat liver, which associates with HL. This protein may represent the HL binding site in liver.


Asunto(s)
Heparina/farmacología , Lipoproteína Lipasa/metabolismo , Hígado/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Sitios de Unión , Cromatografía de Afinidad , Hígado/efectos de los fármacos , Masculino , Peso Molecular , Ratas , Ratas Wistar
13.
Atherosclerosis ; 141 Suppl 1: S31-4, 1998 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9888639

RESUMEN

The pivotal role of heparin-releasable lipases in the clearing of postprandial lipids is known for a long time. The hydrolysis of triglycerides under influence of lipoprotein lipase is among the first recognised and well defined processes in postprandial lipid metabolism. More recently, also hepatic lipase has been implicated in the clearing of postprandial lipoproteins. Lipoprotein lipase as well as hepatic lipase are also involved in the metabolism of several other lipoproteins. However, their capacity is limited. This may lead to interaction of different metabolic processes and competition for the available lipase by different lipoproteins. Indeed, it is generally accepted that the exaggerated postprandial response in subjects with hypertriglyceridemia is at least partially due to competition between endogenous (VLDL) and exogenous (chylomicrons) lipoproteins. Similar mechanisms may also take place in the liver where hepatic lipase plays a role in the metabolism of several lipoproteins. In this short review, the roles of lipoprotein lipase and hepatic lipase in postprandial lipoprotein metabolism are discussed in relation(s) to their suggested function in the metabolism of different lipoproteins.


Asunto(s)
Metabolismo de los Lípidos , Lipoproteína Lipasa/metabolismo , Hígado/metabolismo , Animales , Humanos , Lipasa/metabolismo , Lipoproteínas/metabolismo , Periodo Posprandial
14.
Biochem J ; 321 ( Pt 2): 425-30, 1997 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-9020876

RESUMEN

Hepatic lipase (HL) is thought to be located at the vascular endothelium in the liver. However, it has also been implicated in the binding and internalization of chylomicron remnants in the parenchymal cells. In view of this apparent discrepancy between localization and function, we re-investigated the localization of HL in rat liver using biochemical and immunohistochemical techniques. The binding of HL to endothelial cells was studied in primary cultures of rat liver endothelial cells. Endothelial cells bound HL in a saturable manner with high affinity. However, the binding capacity accounted for at most 1% of the total HL activity present in the whole liver. These results contrasted with earlier studies, in which non-parenchymal cell (NPC) preparations had been found to bind HL with a high capacity. To study HL binding to the different components of the NPC preparations, we separated endothelial cells, Kupffer cells and blebs by counterflow elutriation. Kupffer cells and endothelial cells showed a relatively low HL-binding capacity. In contrast, the blebs, representing parenchymal-cell-derived material, had a high HL-binding capacity (33 m-units/mg of protein) and accounted for more than 80% of the total HL binding in the NPC preparation. In contrast with endothelial and Kupffer cells, the HL-binding capacity of parenchymal cells could account for almost all the HL activity found in the whole liver. These data strongly suggest that HL binding occurs at parenchymal liver cells. To confirm this conclusion in situ, we studied HL localization by immunocytochemical techniques. Using immunofluorescence, we confirmed the sinusoidal localization of HL. Immunoelectron microscopy demonstrated that virtually all HL was located at the microvilli of parenchymal liver cells, with a minor amount at the endothelium. We conclude that, in rat liver, HL is localized at the microvilli of parenchymal cells.


Asunto(s)
Lipasa/metabolismo , Hígado/enzimología , Hígado/ultraestructura , Animales , Células Cultivadas , Endotelio/citología , Endotelio/enzimología , Endotelio/ultraestructura , Inmunohistoquímica , Lipasa/ultraestructura , Hígado/citología , Masculino , Microscopía Inmunoelectrónica , Microvellosidades/enzimología , Microvellosidades/ultraestructura , Unión Proteica , Ratas , Ratas Wistar
15.
Biochem J ; 318 ( Pt 2): 463-7, 1996 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-8809034

RESUMEN

Lipoprotein lipase (LPL) is functionally active only as a dimer. It is also generally assumed that the highly homologous hepatic lipase functions as a dimer, but no clear evidence has been presented. A hepatic lipase-like activity, also indicated as L-type lipase, is present in adrenal and ovary tissues. This enzyme is thought to originate from the liver and to be identical to hepatic lipase. We determined the functional molecular mass of hepatic lipase in rat liver, adrenal gland and ovary by radiation inactivation, a method for determining the functional size of a protein without the need of prior purification. Samples were exposed to ionizing radiation at -135 degrees C. Hepatic lipase activity in liver homogenate showed a single exponential decay. The functional molecular mass was calculated to be 63 +/- 10 kDa. Hepatic lipase activity in adrenal homogenate was found to have a functional molecular mass of 117 +/- 16 kDa. The functional molecular masses of the lipases partially purified from rat liver perfusate, adrenal homogenate or ovarian homogenate showed the same pattern, a target mass for the liver enzyme of 56 +/- 6 kDa and a target mass of 117 +/- 14 kDa for the enzyme from adrenal gland or ovary. In Western blot analysis the mass of the structural units of hepatic lipase in liver was 57 kDa and in adrenal and ovary tissue 51 kDa. We conclude that the functional unit of hepatic lipase in the liver is a monomer. The enzyme in adrenal gland and ovary is different from the liver and the functional unit may be a dimer.


Asunto(s)
Glándulas Suprarrenales/enzimología , Lipasa/química , Hígado/enzimología , Ovario/enzimología , Animales , Western Blotting , Relación Dosis-Respuesta en la Radiación , Electroforesis en Gel de Poliacrilamida , Femenino , Cinética , Lipasa/antagonistas & inhibidores , Lipasa/efectos de la radiación , Masculino , Peso Molecular , Especificidad de Órganos , Ratas , Ratas Wistar
16.
Biochem J ; 302 ( Pt 3): 717-22, 1994 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-7945195

RESUMEN

The binding of hepatic lipase to rat liver was studied in an ex vivo perfusion model. The livers were perfused with media containing partially purified rat hepatic lipase or bovine milk lipoprotein lipase. The activity of the enzymes was determined in the perfusion media before and after passage through the liver. During perfusion with a hepatic-lipase-containing medium the lipase activity in the medium did not change, indicating that there was no net binding of lipase by the liver. In contrast, more than 80% of the lipoprotein lipase was removed from the medium. This lipoprotein lipase activity could be recovered into the perfusion medium completely by heparin perfusion of the liver. If livers, first depleted of hepatic lipase by heparin, were subsequent perfused with a hepatic-lipase-containing medium, 90 +/- 24 m-units of the lipase activity was bound per g of liver (up to 1000 m-units/total liver). However, heparin treatment of the liver decreases the ability of the liver to re-bind hepatic lipase by 80%. Perfusion of rat livers with 0.3 M NaCl released 60% of the lipase activity into the medium. Upon subsequent perfusion of these livers with hepatic-lipase-containing media, 541 +/- 164 m-units of hepatic lipase could be bound per g of liver (up to 5000 m-units/total liver). The binding of hepatic lipase was also studied in livers of corticotropin (ACTH)-pre-treated rats. In these rats also, hepatic lipase bound only to livers which had been pre-perfused with heparin or 0.3 M NaCl. After heparin pre-perfusion, 88 +/- 12 m-units of hepatic lipase could be bound per g of liver, similar to that with livers of control rats not treated with ACTH. After prior salt perfusion, however, the capacity of the livers of ACTH-pre-treated rats to bind hepatic lipase was 212 +/- 60 m-units/g of liver. This is less than in livers of control rats (541 +/- 164 m-units/g of liver). These results indicate that in rat liver the binding of hepatic lipase is heterogeneous in character and consists of heparin-resistant and heparin-sensitive components. The hepatic-lipase binding capacity of the liver is saturable and fully utilized under various conditions. The heparin-sensitive binding capacity is lowered in ACTH-treated rats, whereas the heparin-resistant binding is unaffected. We postulate that the functional hepatic lipase activity can be regulated by changes in the binding capacity of the liver.


Asunto(s)
Lipasa/metabolismo , Lipoproteína Lipasa/metabolismo , Hígado/enzimología , Hormona Adrenocorticotrópica/farmacología , Animales , Anticuerpos Monoclonales , Sitios de Unión , Bovinos , Ensayo de Inmunoadsorción Enzimática , Heparina/farmacología , Lipasa/aislamiento & purificación , Lipoproteína Lipasa/inmunología , Hígado/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Cloruro de Sodio/farmacología
17.
Mol Cell Biochem ; 116(1-2): 171-9, 1992 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-1480146

RESUMEN

The vascular endothelium can be regarded as a widely distributed organ, interposed between the intravascular and extravascular spaces, with a pluripotent function in the regulation of capillary diameter, vascular homeostasis, lipoprotein metabolism and the vascular response to injury. In the basal physiological state these processes provide a non-thrombotic, non-inflammatory vascular lining preventing uncontrolled inflammation and coagulation. Endothelial cells respond to potential harmful conditions (mechanical stress, anoxia, ischemia and oxidative stress) and a variety of hormones and vasoactive mediators by inducing coagulation and production of inflammatory mediators through the production of 'bioactive' lipids. Although the number of studies in isolated myocardial endothelial cells is limited, from the presumed metabolic analogy with endothelial cells isolated (and cultured) from other organs, one may conclude that the bioactive lipids include oxygenated arachidonate metabolites (eicosanoids) and the platelet activating factor (1--O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF). All aspects of lipid metabolism, related to the production of eicosanoids and PAF, are present within myocardial endothelial cells. There is uptake and incorporation of fatty acids by endothelial cells and liberation from endogenous triacylglycerol and (membrane) phospholipid stores by (phospho)lipases. Endothelial cells oxidize fatty acids in a carnitine-dependent, mitochondrial, pathway. Endothelial cells actively interact with high density lipoprotein (HDL) and low density lipoprotein (LDL) leading to uptake of cholesterol(esters) that undergo intracellular hydrolysis, and re-esterification to phospho- and neutral lipids, and leaving the LDL-particle modified in a way that makes them bind to the scavenger receptor on macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Endocardio/metabolismo , Metabolismo de los Lípidos , Animales , Transporte Biológico , Colesterol/metabolismo , Eicosanoides/biosíntesis , Endocardio/citología , Humanos , Lipólisis , Lipoproteínas/metabolismo , Lípidos de la Membrana/metabolismo , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Oxidación-Reducción , Fosfolípidos/metabolismo , Factor de Activación Plaquetaria/biosíntesis , Triglicéridos/metabolismo
18.
Biochim Biophys Acta ; 1055(2): 189-92, 1990 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-2146976

RESUMEN

The importance of the glucose/fatty acid cycle in the control of cardiac lipolysis is emphasized by the following observations. Addition of the glycogen debranching inhibitor deoxynojirimycin or an O2-vehicle, fluorocarbon F-43, to media perfusing paced, lipid-enriched, Langendorff hearts lower cardiac lactate and glycerol 3-phosphate levels together with inhibition of glucagon-stimulated glycerol (and lactate) release. The absence of fluorocarbon during perfusion of 5 Hz paced langendorff hearts probably results in limited tissue oxygenation, resulting in glycogenolysis and lipolysis. The results indicate hormonal control of cardiac lipolysis by glyco(geno)lysis.


Asunto(s)
Glucógeno/metabolismo , Glucólisis , Lipólisis , Miocardio/metabolismo , 1-Desoxinojirimicina , Animales , Glucagón/farmacología , Glucosamina/análogos & derivados , Glucosamina/farmacología , Glicerol/metabolismo , Glucólisis/efectos de los fármacos , Hipoxia , Técnicas In Vitro , Lactatos/metabolismo , Lipólisis/efectos de los fármacos , Ratas
19.
Biochem J ; 268(2): 487-92, 1990 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-2163615

RESUMEN

Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.


Asunto(s)
Aciltransferasas/metabolismo , Enfermedad Coronaria/enzimología , Diabetes Mellitus/enzimología , Glucagón/farmacología , Miocardio/enzimología , Fosfatidato Fosfatasa/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Transporte Biológico , Enfermedad Coronaria/complicaciones , Enfermedad Coronaria/tratamiento farmacológico , Citosol/efectos de los fármacos , Citosol/enzimología , Complicaciones de la Diabetes , Diabetes Mellitus/tratamiento farmacológico , Diacilglicerol O-Acetiltransferasa , Ácidos Grasos/metabolismo , Técnicas In Vitro , Cinética , Magnesio/farmacología , Masculino , Microsomas/enzimología , Ácidos Oléicos/farmacología , Ratas
20.
Basic Res Cardiol ; 85(2): 153-63, 1990.
Artículo en Inglés | MEDLINE | ID: mdl-2350329

RESUMEN

The hormonal regulation and enzymatic basis of endogenous lipolysis in heart are not yet completely elucidated. The lysosomal fraction from rat heart appeared to be markedly enriched in triglycerides and a significant reduction in triglycerides in this fraction was found after prolonged perfusion or stimulation of lipolysis with glucagon. The enhanced rate of lipolysis, measured as glycerol release from the isolated perfused rat heart, was abolished 10-15 min after continuous glucagon administration. Omission of glucagon for another 60 min restored the ability of glucagon to stimulate lipolysis, indicating the limited availability of endogenous triglycerides and the presence of a transfer-system for triglycerides from a non-metabolically active pool to a metabolically active pool. The enhanced lipolysis induced by low-flow ischemia was found to be inhibited by the lysosomotropic agent methylamine (5 mM). Methylamine-perfusion during low-flow ischemia was accompanied by an increased recovery of myocardial triglycerides in the lysosomal fraction. The possible role of lysosome-like particles in myocardial triglyceride homeostasis was further investigated by studying the kinetics of uptake and degradation of labeled triglycerides by membrane-particles recovered in the subcellular fraction enriched with lysosomal marker enzymes. It appeared that isolated lysosomal membranes take up added triglycerides at an average rate of 30 nmoles/min/g protein. The bulk of these triglycerides taken up is stored whereas 20% is degraded to diglycerides and free fatty acids. More than 90% of the free fatty acids formed were released from the lysosomes into the supernatant. The uptake and degradation of triglyceride-filled liposomes by isolated myocardial lysosomes was inhibited during incubation with methylamine (5 mM). On the other hand, a lowering of pH during in vitro incubation increased the rate of uptake and degradation of added triglycerides by isolated lysosomes. These results indicate that lysosomes or lysosome-like particles are involved in the enhanced lipolysis during myocardial ischemia.


Asunto(s)
Circulación Coronaria/fisiología , Enfermedad Coronaria/fisiopatología , Lisosomas/fisiología , Miocardio/metabolismo , Triglicéridos/metabolismo , Equilibrio Ácido-Base/fisiología , Adenosina Monofosfato/metabolismo , Animales , Calcio/metabolismo , Glicerol/metabolismo , Lipólisis/fisiología , Masculino , Metilaminas/farmacología , Reperfusión Miocárdica , Ratas , Ratas Endogámicas
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