RESUMEN
AIM: By acting as both insulin sensitizers and lipid-lowering agents, dual-acting peroxisome proliferator-activated receptors alpha/gamma (PPARalpha/gamma) agonists may be used to improve glucose tolerance in type 2 diabetic patients without inducing adiposity and body weight gain. Here, in an animal model of obesity and insulin resistance, the metabolic response to cevoglitazar, a dual PPARalpha/gamma, was characterized using a combination of in vivo and ex vivo magnetic resonance methodologies and compared to treatment effects of fenofibrate, a PPARalpha agonist, and pioglitazone, a PPARgamma agonist. METHODS: Four groups of fatty Zucker rats: (i) Vehicle; (ii) fenofibrate 150 mg/kg; (iii) pioglitazone 30 mg/kg; and (iv) cevoglitazar 5 mg/kg were investigated before and after treatment. Animals were fed a fat-enriched (54% kcal fat) diet for 6 weeks, 2 weeks high of fat-exposure alone followed by a 4-week dosing period. RESULTS AND CONCLUSIONS: Cevoglitazar was as effective as pioglitazone at improving glucose tolerance. However, unlike pioglitazone, both fenofibrate and cevoglitazar reduced BW gain and adiposity, independent of food intake. All three treatment regimens normalized intramyocellular lipids. Metabolic profiling showed that in the muscle cevoglitazar improves the lipid profile via both PPARalpha- and PPARgamma-mediated mechanisms. Pioglitazone reduced hepatic lipid accumulation, while cevoglitazar and fenofibrate reduced hepatic lipid concentration below baseline levels (p < 0.05). Metabolic profiling showed that in the liver, cevoglitazar functions largely through PPARalpha agonism resulting in increased beta-oxidation. Cevoglitazar only induced small changes to the lipid composition of visceral fat. In subcutaneous fat, however, cevoglitazar induced changes similar to those observed with fenofibrate suggesting export of fatty acids from this depot.
Asunto(s)
Grasa Abdominal/efectos de los fármacos , Adiposidad/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Fenofibrato/farmacología , Hipoglucemiantes/farmacología , Hipolipemiantes/farmacología , Animales , Grasas de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Resistencia a la Insulina , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Músculo Esquelético/química , Obesidad/metabolismo , PPAR alfa/agonistas , PPAR gamma/agonistas , Pioglitazona , Ratas , Ratas Zucker , Tiazolidinedionas/farmacologíaRESUMEN
Nateglinide, a D-phenylalanine derivative, belongs to a new group of insulinotropic agents with rapid onset and short duration of action. These agents have been developed to reduce the risk of hypoglycaemia associated with pharmacological control and to decrease the likelihood of pancreatic beta-cell exhaustion. Nateglinide mediates the release of insulin from beta-cells by binding to the sulphonylurea receptors, which leads to the closure of ATP-sensitive K(+) channels. Increasing evidence from receptor binding, mechanistic and in vitro and in vivo insulin studies indicate unique pharmacodynamic and pharmacokinetic properties with nateglinide that are distinct from those of sulphonylureas. The time required by nateglinide to close beta-cell K(ATP) channels is comparable to that of glyburide but threefold and fivefold faster than repaglinide and glimepiride, respectively. Furthermore, its effects are rapidly reversed with an off-rate at the K(ATP) channel twice as fast as that of glyburide and glimepiride and five times faster than repaglinide. This results in a rapid and short insulin response characteristic of the physiological pattern of post-mealtime insulin release. Internalisation into beta-cells is not required for the action of nateglinide. Given that the kinetic profile of the agent is associated with selective enhancement of early-phase insulin secretion, nateglinide is expected to minimise post-meal hyperglycaemia with minimal propensity for hypoglycaemia.
Asunto(s)
Ciclohexanos/farmacología , Hipoglucemiantes/farmacología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Canales de Potasio de Rectificación Interna , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/farmacología , Ciclohexanos/metabolismo , Ciclohexanos/farmacocinética , Humanos , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacocinética , Insulina/metabolismo , Secreción de Insulina , Activación del Canal Iónico/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cinética , Nateglinida , Fenilalanina/metabolismo , Fenilalanina/farmacocinética , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , Receptores de Droga/metabolismo , Receptores de SulfonilureasRESUMEN
Hyperglycemia of diabetes is caused in part by perturbation of hepatic glucose metabolism. Hepatic glucokinase (GK) is an important regulator of glucose storage and disposal in the liver. GK levels are lowered in patients with maturity-onset diabetes of the young and in some diabetic animal models. Here, we explored the adenoviral vector-mediated overexpression of GK in a diet-induced murine model of type 2 diabetes as a treatment for diabetes. Diabetic mice were treated by intravenous administration with an E1/E2a/E3-deleted adenoviral vector encoding human hepatic GK (Av3hGK). Two weeks posttreatment, the Av3hGK-treated diabetic mice displayed normalized fasting blood glucose levels (95 +/- 4.8 mg/dl; P < 0.001) when compared with Av3Null (135 +/- 5.9 mg/dl), an analogous vector lacking a transgene, and vehicle-treated diabetic mice (134 +/- 8 mg/dl). GK treatment also resulted in lowered insulin levels (632 +/- 399 pg/ml; P < 0.01) compared with the control groups (Av3Null, 1,803 +/- 291 pg/ml; vehicle, 1,861 +/- 392 pg/ml), and the glucose tolerance of the Av3hGK-treated diabetic mice was normalized. No significant increase in plasma or hepatic triglycerides, or plasma free fatty acids was observed in the Av3hGK-treated mice. These data suggest that overexpression of GK may have a therapeutic potential for the treatment of type 2 diabetes.
Asunto(s)
Diabetes Mellitus/genética , Expresión Génica/fisiología , Glucoquinasa/genética , Adenoviridae/genética , Animales , Glucemia/análisis , Diabetes Mellitus/fisiopatología , Ingestión de Alimentos , Ayuno/sangre , Técnicas de Transferencia de Gen , Vectores Genéticos , Glucoquinasa/metabolismo , Glucógeno/metabolismo , Humanos , Insulina/sangre , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Triglicéridos/metabolismoRESUMEN
The enzyme glucokinase (GK) plays a central role in glucose homeostasis. Hepatic GK activity is acutely controlled by the action of the GK regulatory protein (GKRP). In vitro evidence suggests that GKRP reversibly binds to GK and inhibits its activity; however, less is known about the in vivo function of GKRP. To further explore the physiological role of GKRP in vivo, we used an E1/E2a/E3-deficient adenoviral vector containing the cDNA encoding human GKRP (Av3hGKRP). High fat diet-induced diabetic mice were administered Av3hGKRP or a control vector lacking a transgene (Av3Null). Surprisingly, the Av3hGKRP-treated mice showed a significant improvement in glucose tolerance and had lower fasting blood glucose levels than Av3Null-treated mice. A coincident decrease in insulin levels indicated that the Av3hGKRP-treated mice had sharply improved insulin sensitivity. These mice also exhibited lower leptin levels, reduced body weight, and decreased liver GK activity. In vitro experiments indicated that GKRP was able to increase both GK protein and enzymatic activity levels, suggesting that another role for GKRP is to stabilize and/or protect GK. These data are the first to indicate the ability of GKRP to treat type 2 diabetes and therefore have significant implications for future therapies of this disease.
Asunto(s)
Proteínas Portadoras , Diabetes Mellitus Tipo 2/terapia , Terapia Genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Virus del Sarcoma Aviar/genética , Glucemia/metabolismo , Peso Corporal , Células Cultivadas , Diabetes Mellitus Tipo 2/etiología , Grasas de la Dieta/efectos adversos , Ayuno , Vectores Genéticos , Glucoquinasa/antagonistas & inhibidores , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/terapia , Prueba de Tolerancia a la Glucosa , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Hígado/fisiología , Glucógeno Hepático/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Ratas , Ratas Sprague-Dawley , Virus 40 de los Simios/genética , Transfección , Células Tumorales CultivadasRESUMEN
Nateglinide (A-4166) is an amino acid derivative with insulinotrophic action in clinical development for treatment of type 2 diabetes. The aim of this study was to determine whether nateglinide's interaction at the K(ATP) channel/sulfonylurea receptor underlies its more rapid onset and shorter duration of action in animal models. Binding studies were carried out with membranes prepared from RIN-m5F cells and HEK-293 cells expressing recombinant human sulfonylurea receptor 1 (SUR1). The relative order for displacement of [(3)H]glibenclamide in competitive binding experiments with RIN-m5F cell membranes was glibenclamide > glimepiride > repaglinide > glipizide > nateglinide > L-nateglinide > tolbutamide. The results with HEK-293/recombinant human SUR1 cells were similar with the exception that glipizide was more potent than repaglinide. Neither nateglinide nor repaglinide had any effect on the dissociation kinetics for [(3)H]glibenclamide, consistent with both compounds competitively binding to the glibenclamide-binding site on SUR1. Finally, the inability to measure [(3)H]nateglinide binding suggests that nateglinide dissociates rapidly from SUR1. Direct interaction of nateglinide with K(ATP) channels in rat pancreatic beta-cells was investigated with the patch-clamp method. The relative potency for inhibition of the K(ATP) channel was repaglinide > glibenclamide > nateglinide. Kinetics of the inhibitory effect on K(ATP) current showed that the onset of inhibition by nateglinide was comparable to glibenclamide but more rapid than that of repaglinide. The time for reversal of channel inhibition by nateglinide was also faster than with glibenclamide and repaglinide. These results suggest that the unique characteristics of nateglinide are largely the result of its interaction at the K(ATP) channel.
Asunto(s)
Carbamatos/farmacología , Ciclohexanos/farmacología , Hipoglucemiantes/farmacología , Islotes Pancreáticos/metabolismo , Proteínas de la Membrana , Fenilalanina/análogos & derivados , Piperidinas/farmacología , Canales de Potasio/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae , Compuestos de Sulfonilurea/farmacología , Transportadoras de Casetes de Unión a ATP , Animales , Unión Competitiva/efectos de los fármacos , Carbamatos/farmacocinética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Separación Celular , Células Cultivadas , Glucosa/metabolismo , Gliburida/farmacología , Glicosiltransferasas , Humanos , Hipoglucemia/sangre , Hipoglucemia/inducido químicamente , Técnicas In Vitro , Insulina/metabolismo , Canales KATP , Cinética , Masculino , Nateglinida , Técnicas de Placa-Clamp , Fenilalanina/farmacología , Piperidinas/farmacocinética , Bloqueadores de los Canales de Potasio , Canales de Potasio de Rectificación Interna , Ratas , Ratas Sprague-Dawley , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Compuestos de Sulfonilurea/farmacocinéticaRESUMEN
Lipoprotein(a) [Lp(a)] is associated with atherosclerosis and with disease processes involving thrombosis. Lp(a) contains apoprotein (a) [apo(a)], which has a sequence highly homologous to plasminogen. Hence, Lp(a) binds directly to extracellular matrix, cellular plasminogen receptors and fibrin(ogen) and competes for the binding of plasminogen to these regulatory surfaces. These interactions may contribute to the proatherothrombogenic consequences of high Lp(a) levels. These interactions are mediated by lysine binding sites (LBS). Therefore, we examined the role of apo(a) kringle IV-10 [the only apo(a) kringle demonstrated to exhibit lysine binding activity in the intact lipoprotein] in the interaction of Lp(a) with these regulatory molecules. We have compared directly apo(a) KIV-10 with plasminogen K4 to examine whether these highly structurally homologous kringle modules are also functionally homologous. Futhermore, because the plasminogen K5-protease domain (K5-PD) binds directly to fibrin, we have also examined the ability of this plasminogen fragment to inhibit the interaction of Lp(a) with these regulatory molecules and with extracellular matrix. Apo(a) KIV-10 competed effectively for the binding of 125I-Lp(a) to these surfaces but was less effective than either intact Lp(a), plasminogen K4 or plasminogen. Plasminogen KS-PD was a better competitor than apo(a) KIV-10 for 125I-Lp(a) binding to the representative extracellular matrix, Matrigel, and to plasmin-treated fibrinogen. In contrast, plasminogen K5-PD did not compete for the interaction of Lp(a) with cells, although it effectively competed for plasminogen binding. These results suggest that Lp(a) recognizes sites in all of the regulatory molecules that are also recognized by apo(a) KIV-10 and that Lp(a) recognizes sites in extracellular matrix and in plasmin-modified fibrinogen that also are recognized by plasminogen K5-PD. Thus, the interaction of Lp(a) with cells is clearly distinct from that with extracellular matrix and with plasmin-treated fibrinogen and the recognition sites within Lp(a) and plasminogen for these regulatory molecules are not identical.
Asunto(s)
Apolipoproteínas A/metabolismo , Kringles , Lipoproteína(a)/metabolismo , Plasminógeno/metabolismo , Apolipoproteínas A/química , Coagulación Sanguínea , Fibrinógeno/química , Fibrinógeno/metabolismo , Humanos , Lipoproteína(a)/química , Plasminógeno/química , Unión ProteicaRESUMEN
Kringles are protein modules found within a wide variety of fibrinolytic and coagulation-related proteins that show binding affinity for lysine, lysine analogs and for fibrin. We report here the crystal structures of apolipoprotein(a) kringle IV37 (apo(a) K4(37)) in its free state and in separate complexes with two omega-amino acids, 6-aminohexanoic acid (6AHA) and p-aminomethylbenzoic acid (PAMBA). The structures of the unliganded form and of both complexes have been determined and refined by restrained least-squares methods to about 2.0 angstrom. The overall kringle architecture is essentially identical with that determined in other kringles but it shows some small significant structural changes in the lysine binding site. Ther is virtually no difference in conformation between the unliganded and complexed forms, suggesting that apo(a) K4(37) does not undergo any conformational rearrangement upon binding. The 6AHA molecule binds to apo(a) K4(37) in a completely different way from that observed with the kringle 4 of plasminogen (PGK4). Its amino group makes an ion pair interaction with the two aspartate residues (Asp55/Asp57) of the anionic center and its carboxylate group faces out into the solvent making water-mediated contacts with the protein. The mode of binding of PAMBA resembles more that decribed for 6AHA when bound to PGK4. The PAMBA molecule is bound by ion pair interactions with the two aspartate residues (Asp55/Asp57) and with Arg71 from the cationic center and by van der Waals contacts. The relative importance of the cationic center from kringles for binding zwitterionic ligands is discussed.
Asunto(s)
Ácido Aminocaproico/metabolismo , Apolipoproteínas A/química , Kringles , para-Aminobenzoatos , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/metabolismo , Secuencia de Aminoácidos , Ácido Aminocaproico/química , Apolipoproteínas A/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Ligandos , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Plasminógeno/química , Plasminógeno/metabolismo , Unión Proteica , Alineación de SecuenciaRESUMEN
A portion of kringle IV37 (KIV37) of apolipoprotein (a), (apo(a)), was polymerase chain reaction-cloned from human liver cDNA. The protein product of this clone was expressed in Escherichia coli as a poly histidine fusion protein. Based on recovery of purified fusion apo(a) KIV37 protein expression levels were estimated to be 10 mg/g of E. coli cell paste. Mass spectral analysis showed the molecular mass of fusion apo(a) KIV37 to be 12,260 +/- 1 daltons. Almost all fusion apo(a) KIV37 was expressed as inclusion bodies and had to be refolded. Fusion apo(a) KIV37 was isolated from the inclusion bodies and purified by lysine-Sepharose affinity chromatography by eluting with 0.2 M epsilon-aminocaproic acid. The fusion protein was treated with thrombin to yield a homogeneous, functional apo(a) KIV37 domain composed of 92 amino acids having a molecular mass of 10,510 +/- 1 daltons. N-terminal protein sequencing and amino acid analysis have confirmed the sequence and composition of apo(a) KIV37. The molar extinction coefficient, epsilon, for apo(a) KIV37 was determined to be 3.1 x 10(4) M-1 cm-1, and the pI was measured to be 6.7 +/- 0.1. In addition, the dissociation constants, Kd, for a series of 11 lysine analogs have been determined by measuring the change in intrinsic fluorescence of apo(a) KIV37 upon saturable binding with these compounds. Kd values ranged from 4.2 +/- 0.9 microM for trans-4-(aminomethyl)cyclohexanecarboxylic acid to 4.6 +/- 0.4 mM for L-arginine. Apo(a) KIV37 binds to plasmin-treated fibrinogen with an EC50 value of 14 +/- 1.2 microM and prevents the binding of Lp(a) to plasmin-treated fibrinogen with an IC50 value of 16 +/- 6 microM. Lp(a) binds to the plasmin-treated fibrinogen surface with an EC50 value of approximately 1.0 +/- 0.3 nM. These studies demonstrate that apo(a) KIV37 can be expressed at high levels, refolded properly, and used as a fully functional lysine-binding domain. In addition, these results also demonstrate that apo(a) KIV37 provides the major interaction of Lp(a) with fibrinogen. One additional weak binding site in Lp(a) is adequate to describe overall Lp(a) binding to fibrinogen.
Asunto(s)
Apolipoproteínas A/genética , Kringles/genética , Lisina/metabolismo , Fragmentos de Péptidos/genética , Apolipoproteínas A/biosíntesis , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Fibrinógeno/efectos de los fármacos , Fibrinógeno/metabolismo , Fibrinolisina/farmacología , Humanos , Lisina/análogos & derivados , Datos de Secuencia Molecular , Fragmentos de Péptidos/biosíntesis , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Yeast squalene synthetase which has been truncated by 24 amino acids at the C-terminus has been overexpressed in Escherichia coli and constitutes approximately 20% of the total soluble cell protein. For the first time, milligram quantities of this essential enzyme in the cholesterol biosynthetic pathway have been purified to near homogeneity by ammonium sulfate precipitation and Mono Q anion-exchange chromatography so that the steady-state rate constants could be measured. A combination of 10% methanol, 10% glycerol, 30 mM octyl-beta-D-glucopyranoside, 0.4% Brij-58, and 1 mM dithiothreitol in 25 mM sodium phosphate, pH 7.4, was essential for the stability and maximal enzyme activity of the near homogeneous enzyme. Kinetic analysis indicated a Km for farnesyl pyrophosphate of 2.5 microM, suggesting fairly tight binding of farnesyl pyrophosphate to truncated yeast squalene synthetase. The turnover number, kcat, for the conversion of farnesyl pyrophosphate to squalene was 0.53 s-1, and the apparent second order rate constant, kcat/Km, was 2.1 x 10(5) M-1 s-1, indicating a relatively slow conversion of farnesyl pyrophosphate to squalene and a low specificity constant for this enzyme. In addition, Km for NADPH and NADH was 0.5 and 3.6 mM, respectively. Moreover, truncated yeast squalene synthetase shows a preference for NADPH over NADH as reflected in the sevenfold higher kcat/Km value for NADPH similar to that for the native enzyme.
Asunto(s)
Farnesil Difosfato Farnesil Transferasa/aislamiento & purificación , Farnesil Difosfato Farnesil Transferasa/metabolismo , Saccharomyces cerevisiae/enzimología , Cromatografía por Intercambio Iónico , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Farnesil Difosfato Farnesil Transferasa/biosíntesis , Immunoblotting , Cinética , Peso Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.