RESUMO
Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.
Assuntos
Hipoglicemiantes/metabolismo , Microssomos Hepáticos/metabolismo , Fenformin/análogos & derivados , Fenformin/metabolismo , Acetatos/química , Animais , Soluções Tampão , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Feminino , Concentração de Íons de Hidrogênio , Hipoglicemiantes/análise , Hipoglicemiantes/química , Espectrometria de Massas , Metformina/análise , Metanol/química , Microssomos Hepáticos/enzimologia , Sistemas On-Line/instrumentação , Concentração Osmolar , Fenformin/análise , Fenformin/química , Ratos , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
In the present work, we demonstrate the presence of a glucose inhibitory effect on the phenobarbital-mediated induction of the delta-aminolevulinate synthase mRNA in normal rat hepatocytes, consistent with the results obtained with the delta-aminolevulinate synthase activity previously reported. This "glucose effect" can be prevented by adding cAMP, adenylate cyclase activators, or a phosphodiesterase inhibitor. Delta-Aminolevulinate synthase mRNA half-life is not modified in the presence of phenobarbital or glucose. When the same experiments are performed using diabetic cells, no glucose effect is observed, even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study suggest that glucose decreases delta-aminolevulinate synthase biosynthesis by acting at a pretranslational step. Assuming that the glucose effect operates by a repression mechanism exerted by metabolites derived from or related to glucose, the present results may reflect a derangement in the formation of these metabolites as a result of the abnormal metabolism operating in the diabetic state.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Diabetes Mellitus Experimental/enzimologia , Glucose/farmacologia , Fígado/efeitos dos fármacos , Fenobarbital/toxicidade , Porfirias/induzido quimicamente , 1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 5-Aminolevulinato Sintetase/genética , Adenilil Ciclases/metabolismo , Animais , Glicemia/fisiologia , Bucladesina/farmacologia , AMP Cíclico/farmacologia , Diabetes Mellitus Experimental/sangue , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Heme/biossíntese , Imunidade Inata , Fígado/enzimologia , Masculino , Fenobarbital/antagonistas & inibidores , Fenobarbital/farmacologia , Porfirias/etiologia , Ratos , Sistemas do Segundo Mensageiro/fisiologia , EstreptozocinaRESUMO
We examined the mechanism underlying the effect of cAMP on delta-aminolevulinate synthase mRNA biosynthesis in isolated hepatocytes from normal and experimental diabetic rats. We have demonstrated that the potentiation by dibutyryl cAMP of the phenobarbital-mediated induction of delta-aminolevulinate synthase enzyme activity, observed in our previously reported studies, reflects an increased amount of its mRNA. The inducing effect exerted by phenobarbital on the biosynthesis of delta-aminolevulinate synthase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response to the increased level of endogenous cAMP in diabetic hepatocytes is apparently sufficient for a maximum activation of the cAMP-dependent protein kinase. The present results suggest that in rat liver dibutyryl cAMP modulates delta-aminolevulinate synthase mRNA biosynthesis by acting predominantly, if not exclusively, at the level of gene transcription.
Assuntos
5-Aminolevulinato Sintetase/genética , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulação Enzimológica da Expressão Gênica , Fígado/metabolismo , Fenobarbital/farmacologia , 5-Aminolevulinato Sintetase/biossíntese , Animais , Sequência de Bases , Northern Blotting , Bucladesina/farmacologia , Relação Dose-Resposta a Droga , Meia-Vida , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
The induction of ferrochelatase activity by phenobarbital and its potentiation by dibutyryl cAMP assayed in normal rat hepatocytes are associated with increased activity of ferrochelatase mRNA. Glucose inhibits this stimulatory effect. This inhibition can be reversed with increasing concentrations of dibutyryl cAMP. The inducing effect exerted by phenobarbital on the activity of ferrochelatase mRNA in diabetic hepatocytes is greater than that observed in normal cells. This enhanced response in diabetic rat hepatocytes is neither potentiated by adding dibutyryl cAMP nor repressed by glucose. The absence of a glucose effect persists even when the endogenous cAMP content is lowered to normal levels. The results obtained in this study are consistent with those reported in other published studies of ferrochelatase activity. This adds more experimental evidence to support the concept that ferrochelatase is inducible. The results obtained suggest that ferrochelatase is more susceptible to induction with phenobarbital in diabetic rat hepatocytes than in normal rat hepatocytes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Ferroquelatase/biossíntese , Regulação da Expressão Gênica , Fígado/metabolismo , RNA Mensageiro/biossíntese , Animais , Bucladesina/farmacologia , Indução Enzimática/efeitos dos fármacos , Glucose/farmacologia , Masculino , Fenobarbital/farmacologia , Ratos , Ratos EndogâmicosRESUMO
The present work demonstrates that phenformin exerted an inducing effect on delta-aminolevulinic acid synthase (ALA-S) and ferrochelatase activities and on cytochrome P-450 content in isolated hepatocytes from rats with experimental diabetes. Similar results were obtained with respect to ALA-S activity and cytochrome P-450 content when chlorpropamide was used. The inducing effect exerted by allylisopropylacetamide (AIA) on ALA-S and ferrochelatase activities in diabetic hepatic cells was markedly greater than that observed in normal hepatocytes. This stimulatory response was not enhanced by adding dibutyryl cyclic AMP (cAMP). When phenformin was added to isolated rat hepatocytes of normal rats, induction of ALA-S and ferrochelatase activities and cytochrome P-450 content was observed only in the presence of added dibutyryl cAMP. Addition of chlorpropamide to this in vitro system did not exert an inducing effect on the same enzymes even in the presence of dibutyryl cAMP. The present results add more experimental evidence about the lability of the heme pathway of diabetic hepatocytes.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , AMP Cíclico/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Diabetes Mellitus Experimental/enzimologia , Ferroquelatase/biossíntese , Liases/biossíntese , Fenformin/farmacologia , Alilisopropilacetamida/farmacologia , Animais , Bucladesina/farmacologia , Clorpropamida/farmacologia , Indução Enzimática/efeitos dos fármacos , Técnicas In Vitro , Chumbo/farmacologia , Fígado/enzimologia , Masculino , RatosRESUMO
In the present work we demonstrate that insulin decreases the phenobarbital-induced activities of delta-aminolevulinic acid synthase and ferrochelatase in isolated hepatocytes from normal and experimental-diabetic rats. Insulin concentrations required to produce significant inhibition in diabetic hepatocytes were higher than in normal cells. Under similar experimental conditions, insulin decreased the basal activities of delta-aminolevulinic acid synthase and ferrochelatase in hepatocytes from normal rats; no inhibitory effect was observed on the basal activity of delta-aminolevulinic acid synthase in hepatocytes from diabetic rats. Cytochrome P-450 content of both normal and diabetic cells was not affected by insulin in absence or presence of phenobarbital. The inhibitory action of insulin was exerted even when effective concentrations of glucagon, dexamethasone, or 8-(p-chlorophenylthio)-cAMP were present.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Heme/biossíntese , Insulina/fisiologia , Fígado/enzimologia , 5-Aminolevulinato Sintetase/metabolismo , Animais , AMP Cíclico/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Ferroquelatase/metabolismo , Glucagon/fisiologia , Técnicas In Vitro , Fígado/efeitos dos fármacos , Masculino , Fenobarbital/farmacologia , RatosRESUMO
In the present work we have been able to demonstrate the existence of some interrelationship between intracellular level of cAMP content and phenobarbital induction of delta-aminolevulinic acid synthase, ferrochelatase, and cytochrome P-450 biosynthesis in isolated rat hepatocytes. The increase of the level of intracellular cAMP produced by activators of adenylate cyclase, inhibitors of phosphodiesterase, or added cyclic nucleotides is reflected by an increase of the phenobarbital induction effect. The greater induction observed in hepatocytes of diabetic rats may be due to a higher level of the intracellular cAMP. The lack of potentiation of added cAMP in diabetic cells is mainly due to the fact that the maximum induction that could be attained is already achieved by the effect of the preexisting high level of the endogenous cAMP.
Assuntos
AMP Cíclico/fisiologia , Diabetes Mellitus Experimental/metabolismo , Heme/biossíntese , Fígado/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/análogos & derivados , Cicloeximida/farmacologia , Sistema Enzimático do Citocromo P-450/fisiologia , Dactinomicina/farmacologia , Ferroquelatase/metabolismo , Técnicas In Vitro , Fígado/citologia , Hepatopatias/etiologia , Masculino , Fenobarbital/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Porfirias/etiologia , RatosRESUMO
Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40,000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240,000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h, but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 microM for the metal and 30.3 microM for mesoporphyrin, and for cobaltochelatase activity, 27 and 45.5 microM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.