RESUMEN
The affinity of a synthetic polymer nanoparticle (NP) to a target biomacromolecule is determined by the association and dissociation rate constants (kon, koff) of the interaction. The individual rates and their sensitivity to local environmental influences are important factors for the on-demand capture and release a target biomacromolecule. Positively charged NPs for small interfering RNA (siRNA) delivery is a case in point. The knockdown efficacy of siRNA can be strongly influenced by the binding kinetics to the NP. Here, we show that kon and koff of siRNA to NPs can be individually engineered by tuning the chemical structure and composition of the NP. N-Isopropylacrylamide-based NPs functionalized with hydrophobic and amine monomers were used. koff decreased by increasing the amount of amine groups in the NP, whereas kon did not change. Importantly, NPs showing a low koff at pH 5.5 together with a high koff at pH 7.4 showed high knockdown efficiency when NP/siRNA complexes were packaged in lipid nanoparticles. These results provide direct evidence for the premise that the efficacy of an siRNA delivery vector is linked with the strong affinity to the siRNA in the endosome and low affinity in the cytoplasm.
Asunto(s)
Técnicas de Transferencia de Gen , Nanopartículas/química , ARN Interferente Pequeño/metabolismo , Acrilamidas/química , Animales , Línea Celular Tumoral , Citoplasma/metabolismo , Endosomas/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Ratones , ARN Interferente Pequeño/genética , Polímeros de Estímulo Receptivo/químicaRESUMEN
It is very effective to combine physiological examination (resistive index by renal Doppler) and laboratory testing (estimated glomerular filtration rate and albuminuria) when analyzing diabetic nephropathy associated with metabolic syndrome. In particular, the resistive index value is important for increasing diagnostic capabilities. We expect renal blood flow measurement using Sonazoid to increase accuracy.
Asunto(s)
Técnicas de Laboratorio Clínico , Nefropatías Diabéticas/diagnóstico , Síndrome Metabólico/complicaciones , Circulación Renal , Nefropatías Diabéticas/complicaciones , Nefropatías Diabéticas/fisiopatología , Compuestos Férricos , Humanos , Hierro , Óxidos , Ultrasonografía Doppler , Resistencia VascularRESUMEN
This study identifies a novel organic anion transporter Oat9 expressed in mouse liver and kidney. Two variants were detected by screening a mouse liver cDNA library; these varients consist of 1815 (designated Oat9S) and 2165 (Oat9L) base pairs which encode 443 and 551 amino acid proteins, respectively. Oat9S has a predicted structure containing eight transmembrane domains (TMD); whereas, Oat9L possesses twelve TMD. Oat9 mRNA expression was detected in kidney and liver. This transporter was located at the apical side of the late portion of proximal tubules and at the sinusoidal side of hepatocytes. When expressed in Xenopus oocytes, Oat9S mediated the transport of L-carnitine (Km = 2.9 microM), a representative zwitterion, as well as cimetidine (Km = 16.1 microM) and salicylic acid (Km = 175.5 microM), while Oat9L did not show any transport activity. Oat9S-mediated L-carnitine uptake was inhibited by D-carnitine, acetylcarnitine, octanoylcarnitine, betaine, and other organic compounds, suggesting that quaternary ammonium cation bulkiness and relative hydrophobicity are important factors for Oat9S-substrate interactions. Among OATs, Oat9S appears to be the first member to mediate the transport of carnitine and possesses eight TMD. Overall, these new results provide added insight into the structure-activity relationship comprising the organic ion-permeation pathway.
Asunto(s)
Carnitina/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Transportadores de Anión Orgánico/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Ratones , Datos de Secuencia Molecular , Oocitos/metabolismo , Transportadores de Anión Orgánico/análisis , Transportadores de Anión Orgánico/clasificación , Transportadores de Anión Orgánico/genética , Alineación de Secuencia , Xenopus laevisRESUMEN
A 76 year-old woman was admitted to our hospital because of pyrexia and fatigue. One year earlier, she was diagnosed as nephrotic syndrome(NS) caused by focal segmental glomerulosclerosis and immunosuppressive therapy was started with marked amelioration of proteinuria. Thereafter, her renal function worsened, but only supportive treatment was continued. After admission, a cerebrospinal fluid (CSF) examination revealed Cryptococcus neoformans (C. neoformans) by india ink staining and a subsequent CSF culture confirmed C. neoformans infection. Accordingly, we made the diagnosis of cryptococcal meningitis and immediately started multiple anti fungal drugs with dosage modification according to her impaired renal function. Immunosuppressive therapy for NS was temporarily terminated. The inflammatory signs and symptoms soon were markedly improved, but the anti cryptococcal antibody titer in the serum and CSF remained high. Immunosuppressive therapy was started again at a low dosage because urinary protein had increased again. One hundred and eight days from admission, she was discharged with a regimen of multiple anti fungal drugs. Proteinuria and renal insufficiency was almost stable during hospitalization. Most fungal infection develops in patients in an immunosuppressive state induced by immunosuppressive drugs, HIV infection and so on. Patients with NS are frequently in an immunosuppressive state because of urinary loss of immunoglobulins and the use of immunosuppressive drugs. Therefore, it should be remembered that patients with NS are at a high risk of suffering from fungal infection.
Asunto(s)
Inmunosupresores/uso terapéutico , Meningitis Criptocócica/etiología , Síndrome Nefrótico/tratamiento farmacológico , Insuficiencia Renal/tratamiento farmacológico , Anciano , Anfotericina B/administración & dosificación , Antifúngicos/administración & dosificación , Cryptococcus neoformans/aislamiento & purificación , Ciclosporina/administración & dosificación , Esquema de Medicación , Femenino , Fluconazol/administración & dosificación , Humanos , Meningitis Criptocócica/tratamiento farmacológico , Síndrome Nefrótico/complicaciones , Prednisolona/administración & dosificación , Insuficiencia Renal/complicacionesRESUMEN
Many of macromolecular toxins induce cell death by directly interacting with cells or induction of inflammatory cytokines. Abiotic polymer ligands (PLs) composed of functional monomers are able to bind and neutralize toxins in vivo and are of great interest for efficient antidotes. However, little has been reported about recognition and neutralization of target molecules in the bloodstream because of readily elimination from the bloodstream. Here, we report a rational design of PLs-decorated lipid nanoparticles (PL-NPs) for neutralizing a target toxin in vivo. PL that decorated on the NPs would cooperatively interacts with target biomacromolecules since the lipid molecules in NPs have a high degree of freedom. In the present study, N-isopropylacrylamide based PLs interacting with histones, major mediators of sepsis, were synthesized. Affinity between PL-NPs and histones depends on monomer composition and polymer length. The optimized PL-NP showed little affinity for plasma proteins. The PL-NPs inhibited the toxicity of histones both in vitro and in vivo, suggesting that PLs on the NPs cooperatively bound to histones and neutralized their toxicity. In addition, circulation time of optimized PL was significantly prolonged by the modification onto NPs. These results provide a platform for designing antidote nanoparticles neutralizing toxic biomacromolecules.
Asunto(s)
Antídotos/administración & dosificación , Histonas/toxicidad , Nanopartículas/administración & dosificación , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diseño de Fármacos , Ligandos , Lípidos/administración & dosificación , Masculino , Ratones Endogámicos BALB C , Polímeros/administración & dosificaciónRESUMEN
The effect of metformin or rosiglitazone monotherapy versus placebo on insulin signaling and gene expression in skeletal muscle of patients with newly diagnosed type 2 diabetes was determined. A euglycemic-hyperinsulinemic clamp, combined with skeletal muscle biopsies and glucose uptake measurements over rested and exercised muscle, was performed before and after 26 weeks of metformin (n = 9), rosiglitazone (n = 10), or placebo (n = 11) treatment. Insulin-mediated whole-body and leg muscle glucose uptake was enhanced 36 and 32%, respectively, after rosiglitazone (P < 0.01) but not after metformin or placebo treatment. Insulin increased insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation, IRS-1-associated phosphatidylinositol (PI) 3-kinase activity, and phosphorylation of Akt Ser473 and AS160, a newly described Akt substrate that plays a role in GLUT4 exocytosis, approximately 2.3 fold before treatment. These insulin signaling parameters were unaltered after metformin, rosiglitazone, or placebo treatment. Expression of selected genes involved in glucose and fatty acid metabolism in skeletal muscle was unchanged between the treatment groups. Low-intensity acute exercise increased insulin-mediated glucose uptake but was without effect on insulin signaling. In conclusion, the insulin-sensitizing effects of rosiglitazone are independent of enhanced signaling of IRS-1/PI 3-kinase/Akt/AS160 in patients with newly diagnosed type 2 diabetes.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucosa/metabolismo , Hipoglucemiantes/uso terapéutico , Insulina/fisiología , Metformina/uso terapéutico , Tiazolidinedionas/uso terapéutico , Glucemia/efectos de los fármacos , Péptido C/sangre , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Ácidos Grasos no Esterificados/sangre , Regulación de la Expresión Génica , Humanos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , RosiglitazonaRESUMEN
Small interfering RNA (siRNA) has the potential to be a candidate as a cure for intractable diseases. However, an appropriate vector is required for siRNA delivery because of the low transfection efficiency of siRNA without a vector and its easy degradation in vivo. Here, we report a simple, only one step, and efficient method for siRNA encapsulation into a lipidic nanocarrier by freeze-thawing: siRNA was entrapped between the lipid layers of multi-layer liposomes by freeze-thawing of lipoplexes composed of polycation liposomes (PCLs) and siRNA. siRNA-holding capacity to the PCL was increased by repeating freeze-thaw of the lipoplex up to 5cycles. Although siRNA in the conventional lipoplex was degraded after incubation in 90% fetal bovine serum for 72h, siRNA in the frozen and thawed lipoplex was not degraded. Interestingly, we found that the lipoplex formed a "packed multi-layer" structure after the freeze-thawing of "single-layer" PCL and siRNA complex, suggesting that siRNA exists between the lipid layers working as a binder. The frozen and thawed lipoplex showed significantly higher knockdown efficacy compared with the conventional lipoplex. In addition, PEGylated freeze-thawed lipoplexes delivered a higher amount of siRNA to a tumor in vivo compared with the PEGylated conventional ones. These results provide an attractive strategy for "one-step" encapsulation of siRNA into liposomes by freeze-thawing.
Asunto(s)
Liposomas/química , Poliaminas/química , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular Tumoral , Congelación , Lípidos/química , Masculino , Ratones , Ratones Endogámicos BALB C , Polielectrolitos , Polietilenglicoles/química , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , TransfecciónRESUMEN
Tanis is a recently described protein reported to be a putative receptor for serum amyloid A and found to be dysregulated with diabetes in the Israeli sand rat Psamommys obesus. Tanis has also been identified as a selenoprotein, one of the first two identified membrane selenoproteins. We determined mRNA expression of the human homologue of Tanis, SelS/AD-015, in skeletal muscle and adipose tissue biopsies obtained from 10 type 2 diabetic patients and 11 age- and weight-matched healthy subjects. Expression of Tanis/SelS mRNA in skeletal muscle and adipose tissue biopsies was similar between diabetic and control subjects. A subset of subjects underwent a euglycemic-hyperinsulinemic clamp, and adipose tissue expression of Tanis/SelS was determined after in vivo insulin stimulation. Adipose tissue Tanis/SelS mRNA expression was unchanged after insulin infusion in control subjects, whereas Tanis/SelS mRNA increased in seven of eight subjects following insulin stimulation in diabetic subjects. Skeletal muscle and adipose tissue Tanis/SelS mRNA expression were positively correlated with plasma serum amyloid A. In conclusion, there is a strong trend toward upregulation of Tanis/SelS following insulin infusion in adipose tissue from type 2 diabetic subjects. Moreover, the positive relationship between Tanis mRNA and the acute-phase protein serum amyloid A suggests an interaction between innate immune system responses and Tanis expression in muscle and adipose tissue.
Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Esquelético/metabolismo , Proteína Amiloide A Sérica/metabolismo , Estudios de Casos y Controles , Técnica de Clampeo de la Glucosa , Humanos , Insulina/farmacología , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , ARN Mensajero/metabolismo , Selenoproteínas , Regulación hacia ArribaRESUMEN
Although it is known that diabetic nephropathy is accelerated by hypertension, the mechanisms involved in this process are not clear. In this study we aimed to clarify these mechanisms using male Wistar fatty rats (WFR) as a type 2 diabetic model and male Wistar lean rats (WLR) as a control. Each group was fed a normal or high sodium diet from the age of 6 to 14 weeks. We determined the blood pressure and urinary albumin excretion (UAE). At the end of the study, the expressions of mitogen-activated protein kinases (MAPK) and transforming growth factor-beta1 (TGF-beta1) were examined in the isolated glomeruli by Western blot analysis, and the number of glomerular lesions was determined by conventional histology. High sodium load caused hypertension and a marked increase in UAE in the WFR but not in the WLR. Glomerular volume was increased in the hypertensive WFR. There was no difference among the four groups in the expression of c-Jun-NH2-terminal kinase (JNK). In contrast, the expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and its upstream regulator, MAPK/ERK kinase 1 (MEK1), were augmented in the hypertensive WFR. Expression of p38 MAPK was increased in the normotensive WFR, and further enhanced in the hypertensive WFR. Moreover, administration of high sodium load to WFR augmented the expression of TGF-beta1. In conclusion, systemic hypertension in WFR accelerates the diabetic nephropathy in type 2 diabetes via MEK-ERK and p38 MAPK cascades. TGF-beta1 is also involved in this mechanism.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/patología , Hipertensión/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Albuminuria/metabolismo , Animales , Western Blotting , Peso Corporal/fisiología , Creatinina/orina , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/metabolismo , Hemodinámica/fisiología , Hipertensión/enzimología , Hipertensión/metabolismo , Riñón/patología , Glomérulos Renales/patología , Masculino , Tamaño de los Órganos/fisiología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Ratas Wistar , Sodio/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
A 54-year-old man was found to have hypertension at age 32, and a diagnosis of Werner's Syndrome was made at age 36 when he was examined for hyperlipidemia. Diabetes mellitus was found at age 42. Proteinuria appeared at age 49, and microscopic hematuria was seen at age 50. At age 51, serum creatinin level began to rise and atrophy of bilateral kidneys was observed by abdominal CT. There after, the renal function gradually worsened. At age 53, the serum creatinin level rose to 8.3 mg/dl, and systemic edema as well as loss of appetite appeared, resulting in the initiation of hemodialysis. In Werner's syndrome, though arteriosclerosis arises frequently, case reports with chronic renal failure are extremely rare. To investigate the cause of the renal dysfunction, renal biopsy was performed and the samples were histologically examined, revealing the presence of hypertensive glomerular changes. It is, thus, conceivable that hypertension had played a major role in the progression of renal failure in this case.
Asunto(s)
Fallo Renal Crónico/terapia , Diálisis Renal , Síndrome de Werner/complicaciones , Humanos , Fallo Renal Crónico/etiología , Masculino , Persona de Mediana EdadAsunto(s)
Antagonistas Adrenérgicos beta/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Ensayos Clínicos como Asunto , Hipertensión/complicaciones , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/etiología , Albuminuria/tratamiento farmacológico , Albuminuria/etiología , Albuminuria/prevención & control , Antagonistas de Receptores de Angiotensina , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/prevención & control , Quimioterapia Combinada , Humanos , Hipertensión/tratamiento farmacológico , Enfermedades Renales/prevención & controlRESUMEN
BACKGROUND/AIMS: The present study evaluated the clinical efficacy and pharmacokinetics of microemulsion cyclosporine A (ME-CyA) with modification from postprandial to preprandial administration in adult patients with refractory nephrotic syndrome. METHODS: We investigated 19 patients with refractory nephrotic syndrome who had been switched from the postprandial administration of ME-CyA to preprandial administration. The pharmacokinetics of ME-CyA were also evaluated before and 6 months after switching from postprandial to preprandial administration by serial measurement of the blood CyA concentration in 10 patients. RESULTS: This study showed that 16 of 19 patients (84%) displayed an improvement in their clinical condition or continued to maintain remission after switching from post- to preprandial administration. In particular among 14 patients with minimal change nephrotic syndrome (MCNS) in this study, 13 patients maintained or achieved remission under preprandial ME-CyA administration. Only three of 10 patients with postprandial administration showed a peak concentration> 500 ng/ml within 1-2 h after administration, while with preprandial administration, nine of 10 patients showed this good absorption profiles. This effectiveness of preprandial administration seems to be dependent on the improved pharmacokinetics with the increase of area under the curve from 0-4 h (AUC(0-4)) and peak concentration. There were no statistical differences in the mean daily doses of ME-CyA between both administration periods. No ME-CyA-induced nephrotoxicity or other harmful events were encountered throughout the study. CONCLUSION: The preprandial administration of ME-CyA results in a good pharmacokinetic profile and is useful for management of refractory nephrotic syndrome in adults, particularly in patients with MCNS.
Asunto(s)
Ciclosporina/administración & dosificación , Ciclosporina/farmacocinética , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacocinética , Absorción Intestinal , Nefrosis Lipoidea/tratamiento farmacológico , Administración Oral , Adolescente , Adulto , Anciano , Ciclosporina/sangre , Emulsiones , Humanos , Inmunosupresores/sangre , Persona de Mediana Edad , Periodo Posprandial , Adulto JovenRESUMEN
Insulin stimulates Na(+),K(+)-ATPase activity and induces translocation of Na(+),K(+)-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na(+),K(+)-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na(+),K(+)-ATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Na(+),K(+)-ATPase alpha-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive (86)Rb(+) uptake and [(3)H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunits. Insulin-stimulated Na(+),K(+)-ATPase activation, phosphorylation, and translocation of alpha-subunits to the plasma membrane were abolished by 20 microm PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nm wortmannin) and protein kinase C (10 microm GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of alpha(1)- and alpha(2)-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate alpha-subunit of purified human Na(+),K(+)-ATPase in vitro. In conclusion, insulin stimulates Na(+),K(+)-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the alpha-subunits by ERK1/2 mitogen-activated protein kinase.
Asunto(s)
Insulina/farmacología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Músculo Esquelético/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Biotinilación , Células Cultivadas , Activación Enzimática , Humanos , Proteína Quinasa 3 Activada por Mitógenos , Ouabaína/metabolismo , Fosforilación , Proteína Quinasa C/fisiología , Subunidades de Proteína , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
The gene of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase gives rise to several splice variants. We hypothesized that the expression of p85alpha splice variants may be altered in skeletal muscle from subjects with type 2 diabetes mellitus. Skeletal muscle biopsies were obtained from nine type 2 diabetic and eight healthy men, matched for age, body mass index (BMI) and physical fitness. PI 3-kinase activity in skeletal muscle following in vitro insulin stimulation was reduced in subjects with type 2 diabetes. p85alpha mRNA was elevated fourfold in type 2 diabetic as compared to healthy control subjects ( P<0.05). p85alpha mRNA abundance was positively correlated with plasma insulin concentration ( P<0.01) and serum glucose concentration ( P<0.01). Despite this, protein levels of p85alpha, p55alpha, and the novel human p50alpha were not altered in type 2 diabetic subjects. Thus, although gene expression of full-length p85alpha is increased in skeletal muscle from type 2 diabetics, this is not reflected by increased protein levels. Therefore, defects in PI 3-kinase activity are likely due to impaired activation of the enzyme rather than changes in protein expression of the isoforms of the regulatory subunit.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Expresión Génica , Músculo Esquelético/fisiopatología , Fosfatidilinositol 3-Quinasas/genética , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/metabolismoRESUMEN
We investigated whether insulin resistance in patients with chronic heart failure (CHF) is associated with impaired insulin signalling in skeletal muscle and whether exercise training would lead to an improvement in insulin signalling, concomitant with enhanced insulin action. Fourteen men with CHF due to idiopathic dilated cardiomyopathy, with mild-to-moderate limitation of physical activity and a left-ventricular ejection fraction of less than 45 %, were studied before and after either a 5 month exercise training programme (n = 7) or standard care (n = 7). Seven healthy men participated as controls. Whole-body insulin-stimulated glucose uptake was determined by the euglycaemic hyperinsulinaemic clamp technique and skeletal muscle biopsy samples were obtained before and after the insulin infusion for insulin signalling measurements. Insulin-stimulated glucose uptake was 20 % lower in CHF patients versus healthy subjects. Physiological hyperinsulinaemia increased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 by approximately 2.5-fold, IRS-1-associated phosphatidylinositol 3-kinase (PI-3-kinase) activity by approximately 2-fold and Akt (protein kinase B) phosphorylation by approximately 3-fold, with similar responses between healthy subjects and CHF patients. Insulin-mediated glucose uptake was not altered in patients after standard care, whereas exercise training elicited a 25 % increase in glucose uptake. Neither standard care nor exercise training altered insulin-stimulated tyrosine phosphorylation of IRS-1, IRS-1-associated PI-3-kinase activity or Akt phosphorylation. In conclusion, the CHF patients demonstrated impaired insulin-stimulated glucose uptake, despite normal signal transduction in skeletal muscle at the level of IRS-1, PI-3-kinase and Akt. Of clinical relevance is the finding that exercise training improves glucose uptake. However, these changes in insulin action after exercise training appear to be independent of enhanced insulin signalling at the level of IRS-1, PI-3-kinase or Akt.
Asunto(s)
Gasto Cardíaco Bajo/fisiopatología , Resistencia a la Insulina , Insulina/metabolismo , Músculo Esquelético/fisiopatología , Transducción de Señal , Gasto Cardíaco Bajo/etiología , Cardiomiopatía Dilatada/complicaciones , Estudios de Casos y Controles , Enfermedad Crónica , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/irrigación sanguínea , Educación y Entrenamiento Físico , Flujo Sanguíneo Regional , Volumen SistólicoRESUMEN
l-Carnitine is an essential component of mitochondrial fatty acid beta-oxidation and plays a pivotal role in the maturation of spermatozoa within the male reproductive tract. Epididymal plasma contains the highest levels of l-carnitine found in the human body, and initiation of sperm motility occurs in parallel to l-carnitine increase in the epididymal lumen. Using a specific carrier, epididymal epithelium secretes l-carnitine into the lumen by an active transport mechanism; however, the structure-activity relationship comprising the carnitine-permeation pathway is poorly understood. We discovered a novel carnitine transporter (CT2) specifically located in human testis. Analyzing the primary structure of CT2 revealed that it is phylogenetically located between the organic cation transporter (OCT/OCTN) and anion transporter (OAT) families. Hence, CT2 represents a novel transporter family. When expressed in Xenopus oocytes, CT2 mediates the high affinity transport of l-carnitine but does not accept mainstream OCT/OCTN cationic or OAT anionic substrates. We synthesized and tested various carnitine-related compounds and investigated the physicochemical properties of substrate recognition by semi-empirical computational chemistry. The data suggest that the quaternary ammonium cation bulkiness and relative hydrophobicity be the most important factors that trigger CT2-substrate interactions. Immunohistochemistry showed that the CT2 protein is located in the luminal membrane of epididymal epithelium and within the Sertoli cells of the testis. The identification of CT2 represents an interesting evolutionary link between OCT/OCTNs and OATs, as well as provides us with an important insight into the maturation of human spermatozoa.