RESUMEN
Mitochondrial DNA m.3243A > G mutation causes mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and its associated multi-organ disorders, including diabetes. To clarify associations between m.3243A > G organ heteroplasmy and clinical phenotypes, including the age at death, we combined genetic and pathological examinations from seven unreported and 36 literature cases of autopsied subjects. Clinical characteristics of subjects were as follows: male, 13; female, 28; unknown, 2; the age at death, 36.9 ± 20.2 [4-82] years; BMI, 16.0 ± 2.9 [13.0-22.3]; diabetes, N = 21 (49%), diabetes onset age 38.6 ± 14.2 years; deafness, N = 27 (63%); stroke-like episodes (StLEp), N = 25 (58%); congestive heart failure (CHF), N = 15 (35%); CHF onset age, 51.3 ± 14.5 years. Causes of death (N = 32) were as follows: cardiac, N = 13 (41%); infection, N = 8 (25%); StLEp, N = 4 (13%); gastrointestinal, N = 4 (13%); renal, N = 2 (6%); hepatic, N = 1 (2%). High and low heteroplasmies were confirmed in non-regenerative and regenerative organs, respectively. Heteroplasmy of the liver, spleen, leukocytes, and kidney for all subjects was significantly associated with the age at death. Furthermore, the age at death was related to juvenile-onset (any m.3243A > G-related symptoms appeared before 20) and stroke-like episodes. Multiple linear regression analysis with the age at death as an objective variable showed the significant contribution of liver heteroplasty and juvenile-onset to the age at death. m.3243A > G organ heteroplasmy levels, particularly hepatic heteroplasmy, are significantly associated with the age at death in deceased cases.
Asunto(s)
Diabetes Mellitus , Síndrome MELAS , Accidente Cerebrovascular , Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , Preescolar , Niño , Adolescente , Adulto Joven , Anciano de 80 o más Años , Heteroplasmia , ADN Mitocondrial/genética , Mutación , Accidente Cerebrovascular/complicaciones , Hígado/patología , Síndrome MELAS/genéticaRESUMEN
BACKGROUND: Phenotype of autosomal recessive hypercholesterolaemia (ARH), a rare lipid disorder, is known to be milder than that of homozygous familial hypercholesterolaemia (FH) with LDL receptor gene mutation. However, few data exist regarding the functional differences in ARH and FH particularly in terms of remnant-like particles' (RLP) metabolism. MATERIALS AND METHODS: Blood sampling was performed up to 6h after OFTT cream loading (50 g/body surface area) with 2-h intervals in a single ARH proband, four heterozygous FH patients with LDL receptor gene mutation and four normal controls. Plasma lipoprotein and RLP fraction were determined by HPLC system. The area under curve (AUC) of each lipoprotein including RLP fractions was evaluated. RESULTS: The AUC of TG, RLP cholesterol (RLP-C) and RLP triglyceride (RLP-TG) levels of heterozygous FH subjects was significantly higher than those of controls (466±71 mg/dL×h vs. 303±111 mg/dL×h, P<0·05; 35±7 mg/dL×h vs. 21±8 mg/dL×h, P<0·05; 124±57 mg/dL×h vs. 51±13 mg/dL×h, P<0·05, respectively). Under these conditions, those values of ARH were close to those of controls (310 mg/dL×h, 22 mg/dL×h, 23 mg/dL×h, respectively). CONCLUSION: These data demonstrate that unlike in FH, RLP clearance is preserved in ARH. The preservation of post-prandial RLP clearance may contribute to the mild phenotype of ARH compared with FH.
Asunto(s)
Colesterol/metabolismo , Hiperlipoproteinemia Tipo II/metabolismo , Lipoproteínas/metabolismo , Triglicéridos/metabolismo , Área Bajo la Curva , Ensayo de Inmunoadsorción Enzimática , Femenino , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Periodo Posprandial/fisiologíaRESUMEN
Prebeta1-HDL, a putative discoid-shaped high-density lipoprotein (HDL) is known to participate in the retrieval of cholesterol from peripheral tissues. In this study, to clarify potential sources of this lipoprotein, we conducted heparin injection on four Japanese volunteer men and found that serum triglyceride (TG) level decreased in parallel with the increase in serum nonesterified fatty acids and plasma lipoprotein lipase (LPL) protein mass after heparin injection. Plasma prebeta1-HDL showed considerable increases at 15 min after the heparin injection in all of the subjects. In contrast, serum HDL-C levels did not change. Gel filtration with fast protein liquid chromatography system (FPLC) study on lipoprotein profile revealed that in post-heparin plasma, low-density lipoprotein and alphaHDL fractions did not change, whereas there was a considerable decrease in very low-density lipoprotein (VLDL) fraction and an increase in prebeta1-HDL fraction when compared with those in pre-heparin plasma. We also conducted in vitro analysis on whether prebeta1-HDL was produced during VLDL lipolysis by LPL. One hundred microliters of VLDL extracted from pooled serum by ultracentrifugation was incubated with purified bovine milk LPL at 37 degrees C for 0-120 min. Prebeta1-HDL concentration increased in a dose dependent manner with increased concentration of added LPL in the reaction mixture and with increased incubation time, indicating that prebeta1-HDL was produced during lipolysis of VLDL by LPL. Taken these in vivo and in vitro analysis together, we suggest that lipolysis of VLDL particle by LPL is an important source for formation of prebeta1-HDL.
Asunto(s)
Lipoproteínas de Alta Densidad Pre-beta/biosíntesis , Lipólisis , Triglicéridos/metabolismo , Adulto , Animales , Bovinos , Heparina/administración & dosificación , Lipoproteínas de Alta Densidad Pre-beta/sangre , Humanos , Masculino , Persona de Mediana Edad , Leche/metabolismo , Triglicéridos/sangreRESUMEN
In an attempt to clarify the role of the type 2 corticotropin-releasing hormone (CRH) receptor (CRHR-2) in the brain in activation of the hypothalamic-pituitary-adrenocortical axis, we conducted experiments using male Wistar rats. First, an injection of urocortin-2 (7.5 microg) into the lateral ventricle resulted in transient increases in CRH heteronuclear RNA (hnRNA) in parvocellular paraventricular nucleus (PVN) and in plasma adrenocorticotropic hormone (ACTH), whereas sustained increases in arginine vasopressin (AVP) hnRNA and c-fos mRNA in the parvocellular PVN were observed as compared with vehicle treatment. Pretreatment with the selective CRHR-2 antagonist antisauvagine-30 (20 microg) into the lateral ventricle 15 min prior to agonist injection attenuated the stimulatory effects of urocortin-2 on the above-mentioned hypothalamic-pituitary-adrenal axis variables. These effects were similar or rather more potent than those induced by pretreatment with 50 microg of alpha-helical CRH. Second, we found longer-lasting increases in CRH and AVP hnRNA and c-fos mRNA in parvocellular PVN and in plasma ACTH following central administration of urocortin-3 (7.5 microg) than following urocortin-2. Pretreatment with antisauvagine-30 antagonized the effects of urocortin-3 on the above-mentioned variables. Finally, central administration of antisauvagine-30 as well as alpha-helical CRH profoundly attenuated restraint-stress-induced increases in AVP hnRNA. However, alpha-helical CRH, but not antisauvagine-30, attenuated restraint-stress-induced increases in CRH hnRNA in the PVN. Both antagonists transiently attenuated stress responses of c-fos mRNA in PVN and plasma ACTH. These results indicate that there is a CRHR-2-mediated mechanism in the brain that stimulates CRH- and AVP-producing neurons in the PVN which results in the promotion of plasma ACTH secretion.
Asunto(s)
Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Hormona Liberadora de Corticotropina/fisiología , Animales , Hormona Liberadora de Corticotropina/metabolismo , Masculino , Ratones , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas , Ratas Wistar , Receptores de Hormona Liberadora de Corticotropina/agonistas , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidoresRESUMEN
Previous studies have demonstrated that various types of stress increase type 1 corticotropin-releasing hormone (CRH) receptor (currently abbreviated to CRF1 receptor) mRNA in the hypothalamic paraventricular nucleus (PVN) of rats, but not mice. This study investigated whether different sensitivities of glucocorticoid-mediated negative feedback effects can explain this species difference in stress-induced PVN CRF1 receptor mRNA expression. First, the CRF1 receptor mRNA level in the PVN of CRH knockout (KO) mice during acute restraint stress was compared with that in wild-type (WT) mice. Consistent with previous findings, WT mice showed no induction of CRF1 receptor mRNA in the PVN following acute restraint, regardless of normal hypothalamic-pituitary-adrenocortical responses. In contrast, CRF1 receptor mRNA in the PVN of CRH KO mice was increased following 2 h of restraint. Since the response of tyrosine hydroxylase (TH) mRNA in the locus coeruleus (LC) to restraint was similar between CRH KO and WT mice, it is unlikely that enhanced noradrenergic input into the PVN was responsible for the CRF1 receptor mRNA induction in CRH KO mice. Second, to determine whether CRH KO per se or a low corticosterone response to stress is required to induce CRF1 receptor mRNA expression in the PVN in mice, the response of adrenalectomized WT mice was examined. Acute restraint increased the CRF1 receptor mRNA level in the PVN of adrenalectomized WT mice, similar to the case for CRH KO mice. TH mRNA in the LC showed similar increases in sham and adrenalectomized WT mice. These results indicate that PVN CRF1 receptor mRNA is much more sensitive to glucocorticoid-mediated negative feedback in mice than in rats, such that a normal increase in plasma corticosterone during stress can mask CRF1 receptor mRNA induction in the PVN of mice.
Asunto(s)
Hormona Liberadora de Corticotropina/deficiencia , Núcleo Hipotalámico Paraventricular/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Restricción Física/métodos , Estrés Fisiológico/metabolismo , Adrenalectomía/métodos , Animales , Corticosterona/sangre , Hormona Liberadora de Corticotropina/genética , Regulación de la Expresión Génica/fisiología , Hibridación in Situ/métodos , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Receptores de Hormona Liberadora de Corticotropina/genéticaRESUMEN
Single stress induces long-lasting changes in the hypothalamo-pituitary--adrenal (HPA) axis of adult animals. Selective oxytocin (OXT) receptor antagonist was administrated into the cerebroventricle of male rats to test its influence on plasma adrenocorticotropic hormone (ACTH) responses induced by immobilization stress. The ACTH level is significantly higher than the control level (P<0.05) up to 6 days after single stress. Although the OXT antagonist did not change the plasma ACTH level at the end of single stress (P=0.59), the antagonist significantly decreased the ACTH concentration at the end of repeated (3 days) stress and 2 days after single stress (P<0.05) compared with controls. The results suggest that endogenous brain OXT enhances the long-lasting but not immediate HPA axis response to stress.
Asunto(s)
Hormona Adrenocorticotrópica/sangre , Sistema Hipotálamo-Hipofisario/metabolismo , Oxitocina/análogos & derivados , Oxitocina/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Oxitocina/antagonistas & inhibidores , Estrés Fisiológico/metabolismo , Regulación hacia Arriba/fisiología , Hormona Adrenocorticotrópica/metabolismo , Animales , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Inyecciones Intraventriculares , Masculino , Oxitocina/farmacología , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Oxitocina/metabolismo , Estrés Fisiológico/fisiopatología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUNDS: Familial hypercholesterolemia (FH) is an autosomal dominant disease characterized by hypercholesterolemia, tendon xanthomas, and premature coronary heart disease. FH is caused by mutations of "FH genes," which include the LDL-receptor (LDLR), apolipoprotein B-100 (APOB) or proprotein convertase subtilisin/kexin type 9 (PCSK9). We evaluated the usefulness of FH gene analysis for diagnosing homozygous FH (homo-FH), particularly in cases caused by gain-of-function (g-o-f) mutations in PCSK9 (PCSK9 E32K). OBJECTIVES: To evaluate the frequency of homo-FH caused by PCSK9 E32K compared with FH due to other genetic causes and to report the phenotypic features of homo-FH caused by PCSK9 E32K. METHODS: Genomic DNA was prepared from white blood cells, and LDLR and PCSK9 mutations were identified using the Invader assay method. RESULTS: Of the 1055 hetero-FH patients, 62 patients (5.9%) carried the PCSK9 E32K mutation, while in the 82 alleles of 41 homo-FH patients, 13 (15.9%) had double mutations of LDLR allele and PCSK9 E32K mutation. Mean plasma total cholesterol (TC) (9.93 ± 2.95 mmol/L, mean ± SD) in true homo-FH cases with PCSK9 E32K or double hetero-FH cases with PCSK9 E32K and LDLR mutations were significantly lower than those in true homo-FH (18.06 ± 4.96 mmol/L) and compound heterozygous cases with LDLR mutations (14.84 ± 1.62 mmol/L). Mean plasma TC concentrations in the 59 hetero-FH cases with PCSK9 E32K (7.21 ± 1.55 mmol/L) were significantly lower than those (8.94 ± 1.53 mmol/L) in the hetero-FH by LDLR mutations. CONCLUSIONS: FH caused by PCSK9 g-o-f mutations is relatively common in Japan and causes a mild type of homo- and hetero-FH compared with FH caused by LDLR mutations.
Asunto(s)
Hiperlipoproteinemia Tipo II/genética , Proproteína Convertasas/genética , Serina Endopeptidasas/genética , Alelos , Sustitución de Aminoácidos , Pueblo Asiatico/genética , Colesterol/sangre , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Genes Dominantes , Heterogeneidad Genética , Genotipo , Homocigoto , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/epidemiología , Japón , Masculino , Mutación Missense , Linaje , Fenotipo , Mutación Puntual , Proproteína Convertasa 9 , Proproteína Convertasas/fisiología , Receptores de LDL/genética , Serina Endopeptidasas/fisiología , Triglicéridos/sangreRESUMEN
BACKGROUND: The half of hyperalphalipoproteinemia (HALP) in Japan is caused by CETP gene mutations. Other than two prevalent mutations (D442G and Intron 14 splicing donor site +1G>A), some rare CETP mutations are found in Japanese HALP subjects. METHODS: CETP gene analysis of genomic DNA from subjects was performed by restriction fragment length polymorphism (RFLP) and sequencing analysis. Mutations which were suspected to cause a splicing defect or a protein secretion defect were investigated in COS-1 cells transfected with a CETP minigene construct or a cDNA expression vector. RESULTS: Each of three subjects was identified as a carrier of CETP gene mutation of a compound heterozygote of c.653_654delGGinsAAAC and Intron 14 splicing donor site +1G>A, a heterozygote of c.658G>A or a homozygote of L261R. The c.658G>A mutation was located at the last nucleotide of exon 7, and it was confirmed to cause splicing abnormality revealed by the CETP minigene analysis. The L261R CETP was not secreted to conditioned media of the cells. CONCLUSIONS: Three novel CETP gene mutations are responsible for HALP by CETP deficiency. It is predicted that there are more rare CETP gene mutations in Japanese, and these multiple rare mutations alone or a combination with each of prevalent mutations is responsible for mild-to-moderate or marked HALP, respectively.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/genética , Hiperlipoproteinemias/genética , Anciano , Empalme Alternativo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Femenino , Humanos , Hiperlipoproteinemias/sangre , Hiperlipoproteinemias/epidemiología , Japón/epidemiología , Persona de Mediana Edad , Mutación , Linaje , Polimorfismo de Longitud del Fragmento de Restricción/genética , Análisis de Secuencia de ADNRESUMEN
Aggressive low-density lipoprotein (LDL) cholesterol-lowering therapy is important for high-risk patients. However, sparse data exist on the impact of combined aggressive LDL cholesterol-lowering therapy in familial hypercholesterolemia (FH), particularly on side effects to changes in plasma coenzyme Q10 and proprotein convertase subtilisin/kexin type 9 levels. We enrolled 17 Japanese patients with heterozygous FH (12 men, 63.9 ± 7.4 years old) with single LDL receptor gene mutations in a prospective open randomized study. Permitted maximum doses of rosuvastatin (20 mg/day), ezetimibe (10 mg/day), and granulated colestimide (3.62 g/day) were introduced sequentially. Serum levels of LDL cholesterol decreased significantly by -66.4% (p <0.001) and 44% of participants achieved LDL cholesterol levels <100 mg/dl. There were no serious side effects or abnormal laboratory data that would have required the protocol to have been terminated except for 1 patient with myalgia. Coadministration of ezetimibe and granulated colestimide further lowered serum LDL cholesterol more than rosuvastatin alone without changing plasma coenzyme Q10 and proprotein convertase subtilisin/kexin type 9 levels. In conclusion, adequate introduction of this aggressive cholesterol-lowering regimen can improve the lipid profile of FH.
Asunto(s)
Azetidinas/administración & dosificación , LDL-Colesterol/sangre , Epiclorhidrina/administración & dosificación , Fluorobencenos/administración & dosificación , Hiperlipoproteinemia Tipo II/tratamiento farmacológico , Imidazoles/administración & dosificación , Pirimidinas/administración & dosificación , Resinas Sintéticas/administración & dosificación , Sulfonamidas/administración & dosificación , Anciano , Anciano de 80 o más Años , Resinas de Intercambio Aniónico , Anticolesterolemiantes/administración & dosificación , LDL-Colesterol/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Ezetimiba , Femenino , Estudios de Seguimiento , Heterocigoto , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/genética , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Rosuvastatina Cálcica , Resultado del TratamientoRESUMEN
BACKGROUND: Autosomal recessive hypercholesterolemia (ARH) exhibits different responsiveness to statins compared with that in homozygous familial hypercholesterolemia (FH). However, few data exist regarding lipoprotein metabolism of ARH. Therefore, we aimed to clarify lipoprotein metabolism, especially the remnant lipoprotein fractions of ARH before and after statin therapy. METHODS AND RESULTS: We performed a lipoprotein kinetic study in an ARH patient and 7 normal control subjects, using stable isotope methodology (10 mg/kg of [(2)H(3)]-leucine). These studies were performed at baseline and after the 20 mg daily dose of atorvastatin. Tracer/tracee ratio of apolipoprotein B (apoB) was determined by gas chromatography/mass spectrometry and fractional catabolic rates (FCR) were determined by multicompartmental modeling, including remnant lipoprotein fractions. FCR of low-density lipoprotein (LDL) apoB of ARH was significantly lower than those of control subjects (0.109 versus 0.450±0.122 1/day). In contrast, the direct removal of very-low-density lipoprotein remnant was significantly greater in ARH than those in control subjects (47.5 versus 2±2%). Interestingly, FCR of LDL apoB in ARH dramatically increased to 0.464 1/day, accompanying reduction of LDL cholesterol levels from 8.63 to 4.22 mmol/L after treatment with atorvastatin of 20 mg/d for 3 months. CONCLUSIONS: These results demonstrate that ARH exhibits decreased LDL clearance associated with decreased FCR of LDL apoB and increased clearance for very-low-density lipoprotein remnant. We suggest that increased clearance of remnant lipoprotein fractions could contribute to the great responsiveness to statins, providing new insights into the lipoprotein metabolism of ARH and the novel pharmacological target for LDLRAP1.
Asunto(s)
Hipercolesterolemia/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Adulto , Anciano , Anticolesterolemiantes/uso terapéutico , Apolipoproteínas B/metabolismo , Atorvastatina , Cromatografía de Gases y Espectrometría de Masas , Ácidos Heptanoicos/uso terapéutico , Humanos , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/patología , Marcaje Isotópico , Cinética , Masculino , Persona de Mediana Edad , Linaje , Pirroles/uso terapéuticoRESUMEN
The therapeutic efficacy of interferon (IFN) for the hepatitis C is closely related with mutations in interferon sensitivity determining region or V3 domain of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A). However, the relationship between alanine aminotransferase (ALT) normalization and the NS5A variability remains unclear. To clarify these features of NS5A, we examined the genetic variability of the patients' NS5A from 33 HCV genotype 1b-infected patients: 11 sustained virological response (SVR), 11 end-of-treatment response (ETR) with normal ALT (<40 IU/L), and 11 non-response (NR) with abnormal ALT (>40 IU/L) after IFN treatment for >24 weeks. The amino acid in position 2378 (followed by HCV-J prototype strain) with alanine (A2378) before IFN treatment was frequent in both SVR and ETR after IFN treatment, whereas that with threonine (T2378) was significant in NR. Moreover, substitution of threonine for alanine in HCV subgenomic replicon showed a 3- to 4-fold reduction of IFN transactivation and replication even in the presence of IFN, suggesting an IFN-resistant phenotype. These observations suggest that a single amino acid in position 2378 of NS5A plays important roles for both ALT normalization and IFN response in HCV-1b infected patients.
Asunto(s)
ADN Viral/análisis , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Interferón-alfa/administración & dosificación , Proteínas no Estructurales Virales , Adulto , Anciano , Alanina/genética , Alanina Transaminasa/sangre , Análisis Mutacional de ADN , Farmacorresistencia Viral/genética , Femenino , Estudios de Seguimiento , Hepacivirus/patogenicidad , Hepatitis C/sangre , Hepatitis C/fisiopatología , Humanos , Interferón-alfa/efectos adversos , Masculino , Persona de Mediana Edad , Mutagénesis Sitio-Dirigida , Mutación/genética , Transgenes/genética , Resultado del Tratamiento , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
BACKGROUND: We elucidated the difference between the effects of bezafibrate and atorvastatin in hypertriglyceridemia with apoE2/2 and 3/3. METHODS: An open randomized crossover study consisted of a 4-week treatment period with bezafibrate (400 mg daily) or atorvastatin (10 mg daily) and a 4-week wash-out period. RESULTS: Bezafibrate significantly decreased serum concentrations of triglyceride (apoE2/2, E3/3: -49.2%, -39.0%) and significantly increased high-density lipoprotein (HDL) cholesterol (+28.5%, +26.1%) in both apoE phenotypes but did not change serum concentrations of low-density lipoprotein (LDL) cholesterol. Atorvastatin significantly decreased serum concentrations of LDL cholesterol (-34.0%, -30.0%) and triglyceride (-27.6%, -25.8%) in both apoE phenotypes but did not change HDL cholesterol concentrations. Changes in cholesterol in lipoprotein subfractions were not different between apoE2/2 and E3/3. Bezafibrate changed cholesterol distribution from small- to large-sized LDL and from large- to small-sized HDL. On the other hand, atorvastatin decreased cholesterol in all apoB-containing lipoprotein subfractions but did not change any of the HDL subfractions. CONCLUSION: Bezafibrate and atorvastatin improve atherogenic dyslipidemia in considerably different ways. Extrapolating from the present data, we presume that the combination of these drugs may contribute to reduce LDL-C/HDL-C ratio effectively as well as lowering concentrations of serum triglyceride.
Asunto(s)
Bezafibrato/farmacología , Ácidos Heptanoicos/farmacología , Hiperlipoproteinemia Tipo III/sangre , Hipolipemiantes/farmacología , Lipoproteínas/sangre , Pirroles/farmacología , Anciano , Apolipoproteína E2/sangre , Apolipoproteína E2/genética , Apolipoproteína E3/sangre , Apolipoproteína E3/genética , Atorvastatina , Bezafibrato/efectos adversos , Bezafibrato/uso terapéutico , Cromatografía Líquida de Alta Presión , Estudios Cruzados , Femenino , Ácidos Heptanoicos/efectos adversos , Ácidos Heptanoicos/uso terapéutico , Humanos , Hiperlipoproteinemia Tipo III/tratamiento farmacológico , Hiperlipoproteinemia Tipo III/genética , Hipolipemiantes/efectos adversos , Hipolipemiantes/uso terapéutico , Masculino , Persona de Mediana Edad , Fenotipo , Pirroles/efectos adversos , Pirroles/uso terapéuticoRESUMEN
AIM: Familial hypercholesterolemia (FH) is caused by mutations of FH genes, i.e. LDL-receptor (LDLR), PCSK9 and apolipoprotein B (ApoB) gene. We evaluated the usefulness of DNA analysis for the diagnosis of homozygous FH (homo-FH), and studied the frequency of FH in the Hokuriku district of Japan. METHODS: Twenty-five homo-FH patients were recruited. LDLR mutations were identified using the Invader assay method. Mutations in PCSK9 were detected by PCR-SSCP followed by direct sequence analysis. RESULTS: We confirmed 15 true homozygotes and 10 compound heterozygotes for LDLR mutations. Three types of double heterozygotes for LDLR and PCSK9 were found. No FH patients due to ApoB mutations were found. The incidences of homo-FH and hetero-FH in the Hokuriku district were 1/171,167 and 1/208, respectively. CONCLUSIONS: Our observations underlined the value of FH gene analysis in diagnosing homo-FH and confirmed extraordinarily high frequency of FH in the Hokuriku district of Japan.
Asunto(s)
Apolipoproteínas B/genética , Pueblo Asiatico/genética , Análisis Mutacional de ADN , Hiperlipoproteinemia Tipo II/genética , Mutación , Receptores de LDL/genética , Serina Endopeptidasas/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Homocigoto , Humanos , Hiperlipoproteinemia Tipo II/etnología , Incidencia , Lactante , Japón/epidemiología , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Proproteína Convertasa 9 , Proproteína Convertasas , Medición de Riesgo , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND: Autosomal recessive hypercholesterolemia (ARH) is an extremely rare inherited hypercholesterolemia, the cause of which is mutations in low-density lipoprotein (LDL) receptor adaptor protein 1 (LDLRAP1) gene. METHODS: A total of 146 heterozygous familial hypercholesterolemic (FH) patients with a mutation in LDLR gene were screened for genes encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLRAP1. RESULTS: Among the 146 subjects, we identified a 79-year-old Japanese female with double mutations in LDLR gene (c.2431A>T) and LDLRAP1 gene (c.606dup). Two other relatives with double mutations in those genes in her family were also identified. Although the proband exhibited massive Achilles tendon xanthoma and coronary and aortic valvular disease, serum LDL-C level of subjects with double mutations was similar with that of subjects with single LDLR mutation (284.0±43.5 versus 265.1 ± 57.4 mg/dl). CONCLUSION: Additional mutation in LDLRAP1 may account for severer phenotype in terms of xanthoma and atherosclerotic cardiovascular disease in FH patients.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Heterocigoto , Hiperlipoproteinemia Tipo II/genética , Mutación , Receptores de LDL/genética , Tendón Calcáneo/patología , Adulto , Anciano , Biomarcadores/sangre , Enfermedades Cardiovasculares/genética , Proteínas de Transferencia de Ésteres de Colesterol/genética , LDL-Colesterol/sangre , Análisis Mutacional de ADN , Femenino , Predisposición Genética a la Enfermedad , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/complicaciones , Japón , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Proproteína Convertasa 9 , Proproteína Convertasas/genética , Serina Endopeptidasas/genética , Índice de Severidad de la Enfermedad , Xantomatosis/genética , Xantomatosis/patologíaRESUMEN
BACKGROUND: Bezafibrate and fenofibrate show different binding properties against peroxisome proliferator-activated receptor subtypes, which could cause different clinical effects on circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and on various metabolic markers. METHODS: An open, randomized, four-phased crossover study using 400 mg of bezafibrate or 200mg of fenofibrate was performed. Study subjects were 14 dyslipidemia with impaired glucose tolerance or type 2 diabetes mellitus (61 ± 16 years, body mass index (BMI) 26 ± 3 kg/m², total cholesterol (TC) 219 ± 53 mg/dL, triglyceride (TG) 183 ± 83 mg/dL, high-density lipoprotein-cholesterol (HDL-C) 46 ± 8 mg/dL, fasting plasma glucose 133 ± 31 mg/dL and HbA1c 6.2 ± 0.8%). Subjects were given either bezafibrate or fenofibrate for 8 weeks, discontinued for 4 weeks and then switched to the other fibrate for 8 weeks. Circulating PCSK9 levels and other metabolic parameters, including adiponectin, leptin and urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured at 0, 8, 12 and 20 weeks. RESULTS: Plasma PCSK9 concentrations were significantly increased (+39.7% for bezafibrate and +66.8% for fenofibrate, p<0.001) in all patients except for one subject when treated with bezafibrate. Both bezafibrate and fenofibrate caused reductions in TG (-38.3%, p<0.001 vs. -32.9%, p<0.01) and increases in HDL-C (+18.0%, p<0.001 vs. +11.7%, p<0.001). Fenofibrate significantly reduced serum cholesterol levels (TC, -11.2%, p<0.01; non-HDL-C, -17.3%, p<0.01; apolipoprotein B, -15.1%, p<0.01), whereas bezafibrate significantly improved glucose tolerance (insulin, -17.0%, p<0.05) and metabolic markers (γ-GTP, -38.9%, p<0.01; adiponectin, +15.4%, p<0.05; urine 8-OHdG/Cre, -9.5%, p<0.05). CONCLUSION: Both bezafibrate and fenofibrate increased plasma PCSK9 concentrations. The addition of a PCSK9 inhibitor to each fibrate therapy may achieve beneficial cholesterol lowering along with desirable effects of respective fibrates.
Asunto(s)
Adipoquinas/metabolismo , Bezafibrato/administración & dosificación , Fenofibrato/administración & dosificación , Proproteína Convertasas/genética , Serina Endopeptidasas/genética , 8-Hidroxi-2'-Desoxicoguanosina , Adiponectina/sangre , Adulto , Anciano , Índice de Masa Corporal , Colesterol/metabolismo , HDL-Colesterol/sangre , Estudios Cruzados , Desoxiguanosina/análogos & derivados , Desoxiguanosina/farmacología , Diabetes Mellitus Tipo 2/sangre , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Leptina/sangre , Masculino , Persona de Mediana Edad , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Proproteína Convertasa 9 , Factores de Tiempo , Triglicéridos/metabolismoRESUMEN
OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol trafficking by mediating degradation of cell-surface LDL receptors (LDLR). Gain-of-function PCSK9 mutations are known to increase plasma LDL-C levels. We attempted to find gain-of-function PCSK9 mutations in Japanese subjects and determine the frequency and impacts of these mutations, especially on circulating PCSK9 and LDL-C levels. METHODS: PCR-SSCP followed by direct sequence analysis was performed for all 12 exons and intronic junctions of the PCSK9 in 55 subjects with clinically diagnosed familial hypercholesterolaemia (clinical-FH), who were confirmed to have no LDLR mutations. Among the mutations detected, PCSK9 E32K was likely to be a gain-of-function mutation, and screening was performed by PCR-RFLP in clinical-FH and general Japanese controls. The levels of PCSK9 in plasma from subjects and in media of HepG2 cells transfected with PCSK9 constructs were measured by ELISA. RESULTS: We detected 7 PCSK9 variants, including E32K. The frequency of PCSK9 E32K in clinical-FH (6.42%) was significantly higher than that in controls (1.71%). Three cases representing homozygous FH phenotypes were double heterozygous for PCSK9 E32K and LDLR C183S, C292X or K790X. Two cases were true homozygous for PCSK9 E32K; to our knowledge, these are the first true homozygotes for gain-of-function PCSK9 mutations reported to date. The PCSK9 E32K mutant had over 30% increased levels of PCSK9 in plasma from the subjects and in media of transiently transfected HepG2 cells as compared with those in controls. Furthermore, LDL-C levels in the PCSK9 E32K true homozygotes and heterozygotes were 2.10- and 1.47-fold higher than those in controls with comparable circulating PCSK9 levels, respectively, suggesting enhanced function of PCSK9 E32K. CONCLUSIONS: We found 2 true homozygotes for PCSK9 E32K and 3 double heterozygotes for PCSK9 E32K and LDLR mutations associated with autosomal dominant hypercholesterolaemia. This study provided evidence that PCSK9 E32K significantly affects LDL-C levels via increased mass and function of PCSK9, and could exacerbate the clinical phenotypes of patients carrying LDLR mutations.
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Hiperlipoproteinemia Tipo II/genética , Serina Endopeptidasas/genética , Adulto , Pueblo Asiatico/genética , LDL-Colesterol/sangre , Ensayo de Inmunoadsorción Enzimática , Exones , Heterocigoto , Homocigoto , Humanos , Intrones , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Proproteína Convertasa 9 , Proproteína Convertasas , Serina Endopeptidasas/sangre , Serina Endopeptidasas/fisiologíaRESUMEN
We have previously reported reduced glucocorticoid receptor (GR) mRNA levels in the hippocampus and hypothalamic paraventricular nucleus (PVN) during repeated immobilization, which is potentially associated with persistent activation of the hypothalamic-pituitary-adrenocortical axis. We used in situ hybridization and Western blot to examine the transcriptional regulation of the GR gene, GR nuclear translocation, and expression of cytosolic heat shock protein 90 (hsp90), a chaperone protein essential for GR nuclear translocation, in the hippocampus, PVN, and anterior pituitary (AP) during single immobilization (sIMO) and the final immobilization on d 7 after daily IMO for 6 days (rIMO). As with GR mRNA, GR heteronuclear RNA levels decreased in the hippocampus and PVN and increased in the AP during sIMO and rIMO, indicating that the GR mRNA levels in these regions were regulated at the transcriptional level. In both sIMO and rIMO, nuclear GR levels were significantly increased in the hippocampus, medial basal hypothalamus (MBH), and AP. However, GR nuclear translocation was reduced in the hippocampus, unchanged in the MBH, and enhanced in the AP during rIMO, as compared with sIMO. Cytosolic hsp90 expression was unchanged in the hippocampus and MBH, whereas it significantly increased in the AP at 30 min during rIMO but not during sIMO. These results suggest that the site-specific changes in GR nuclear translocation during sIMO vs. rIMO are partially linked to hsp90 responses to immobilization. The reduced nuclear translocation of GR in the hippocampus during rIMO may reflect decreased glucocorticoid-mediated negative feedback on the hypothalamic-pituitary-adrenocortical axis.
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Núcleo Celular/metabolismo , Inmovilización/métodos , Receptores de Glucocorticoides/genética , Estrés Psicológico/fisiopatología , Transporte Activo de Núcleo Celular , Hormona Adrenocorticotrópica/sangre , Animales , Western Blotting , Corticosterona/sangre , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Hibridación in Situ , Masculino , Núcleo Hipotalámico Paraventricular/metabolismo , Adenohipófisis/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Receptores de Glucocorticoides/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
BACKGROUND: The objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method. METHODS: Our method comprised the following 2 features: first, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay. RESULTS: Based on the genetic mutation, the FH subjects were divided into 2 groups, K790X, (n=20) and P664L, (n=5), and their LDLR activities was measured by this method, which was found to be 55.3+/-8.9% and 63.9+/-13.8%, respectively, of that of the control group (n=15). In comparison, the LDLR activity was 86.1+/-11.6% (K790X) and 73.3+/-6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differentiated with our method than with the conventional method (paired t-test, p<0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4+/-13.2% (K790X) and 76.9+/-17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1+/-9.7% (K790X) and 70.3+/-26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used. CONCLUSION: These results demonstrate that our new method is applicable for measuring LDLR activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.
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Análisis Químico de la Sangre/métodos , Antígenos CD28/inmunología , Complejo CD3/inmunología , Hiperlipoproteinemia Tipo II/sangre , Linfocitos/metabolismo , Receptores de LDL/metabolismo , Alelos , Animales , Anticuerpos/inmunología , Femenino , Heparina/metabolismo , Heterocigoto , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/patología , Lipoproteínas/deficiencia , Masculino , Persona de Mediana Edad , Mutación , Receptores de LDL/sangre , Sensibilidad y EspecificidadRESUMEN
Hepatitis C virus (HCV) proteins appear to play an important role in IFN-resistance, but the molecular mechanism remains unclear. To clarify the mechanism in HCV replicon RNA harboring Huh-7 cells (Huh-9-13), we isolated cellular clones with impaired IFNalpha-sensitivity. Huh-9-13 was cultured for approximately 2 months in the presence of IFNalpha, and 4 IFNalpha-resistant cell clones showing significant resistances were obtained. When total RNA from clones was introduced into Huh-7 cells, the transfected cells also exhibited IFNalpha-resistance. Although no common mutations were present, mutations in NS3 and NS5A regions were accumulated. Transactivation of IFNalpha and IFNalpha-stimulated Stat-1 phosphorylation were reduced, and the elimination of HCV replicon RNA from the clones restored the IFNalpha signaling. These results suggest that the mutations in the HCV replicon RNA, at least in part, cause an inhibition of IFN signaling and are important for acquisition of IFNalpha resistance in Huh-9-13.
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Genes Virales/genética , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Interferón-alfa/farmacología , Proteínas no Estructurales Virales/genética , Línea Celular Tumoral , Células Clonales , Farmacorresistencia Viral , Humanos , Mutación , Fosforilación , ARN Viral/genética , Replicón/genética , Factor de Transcripción STAT1/metabolismo , TransfecciónRESUMEN
We have previously reported that repeated immobilization produces persistent activation of the hypothalamic-pituitary-adrenocortical axis in rats. In an attempt to assess whether any adaptational responses occur at the pituitary level, we examined the detailed time courses of proopiomelanocortin (POMC) gene transcription in the anterior pituitary (AP) in comparison with those of corticotropin-releasing hormone (CRH) gene transcription in the hypothalamic paraventricular nucleus (PVN) during single and repeated immobilization using both intronic and exonic probes. During single immobilization, there was a robust and rapid increase in both CRH heteronuclear RNA (hnRNA) in the PVN and POMC hnRNA in the AP, together with a slower increase in CRH mRNA, but no significant increase in POMC mRNA. Single immobilization also caused significant increases in the plasma concentrations of both ACTH and corticosterone. Daily immobilization for 6 days increased the basal levels of CRH hnRNA and CRH mRNA in the PVN and POMC mRNA in the AP. Both CRH hnRNA and POMC hnRNA responded rapidly to a final episode of acute immobilization on day 7, whereas the peak values of CRH hnRNA and POMC hnRNA after 15 min of the final stress were smaller than those during single immobilization. In contrast to single stress, CRH mRNA did not change significantly, whereas POMC mRNA robustly increased after the final immobilization on day 7. Plasma ACTH increased to a similar degree to single stress, but its initial increase at 5 min was significantly higher than that during single immobilization. The increase in the plasma corticosterone concentration was higher during final immobilization than during single stress. These results suggest that, in response to the hypothalamic drive during repeated immobilization stress, pituitary corticotrophs are capable of upregulating the basal and stress-induced POMC mRNA levels via increased efficiency of the posttranscriptional processing of the hnRNA and/or increased mRNA stability.