RESUMEN
BACKGROUND: Glucokinase (GK) is a metabolic enzyme encoded by the GCK gene and expressed in glucose-sensitive tissues, principally pancreatic islets cell and hepatocytes. The GK protein acts in pancreatic islets as a "glucose sensor" that couples fluctuations in the blood glucose concentration to changes in cellular function and insulin secretion. GCK and GK have proposed importance in the development and progression of diabetes mellitus and are potential therapeutic targets for diabetes treatment. The study was undertaken to determine the nucleotide sequence of feline pancreatic GK cDNA, predict the amino acid sequence and structure of the feline GK protein, and perform comparative bioinformatic analysis of feline cDNA and protein. Routine PCR techniques were used with cDNA from feline pancreas. Clones were assembled to obtain the full length cDNA. Protein prediction and modeling were performed using bioinformatic tools. RESULTS: Full-length feline pancreatic GK cDNA contains a 1398 nucleotide coding sequence with high identity to other pancreatic GK cDNAs. The deduced 465 amino acid feline protein has 15 amino acid substitutions not found in other mammalian GK proteins but maintains high structural homology with human GK. Feline pancreatic GK is highly conserved at nucleotide and protein levels. Residues crucial for substrate binding and catalysis are completely conserved in the feline protein. CONCLUSION: Molecular analysis predicts that feline pancreatic GK functions similarly to other mammalian GK proteins.
Asunto(s)
Gatos/metabolismo , Clonación Molecular , Regulación Enzimológica de la Expresión Génica , Glucoquinasa/metabolismo , Islotes Pancreáticos/enzimología , Secuencia de Aminoácidos , Animales , Glucoquinasa/genética , Islotes Pancreáticos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
In most mammals, glucokinase (GK) acts as a hepatic "glucose sensor" that permits hepatic metabolism to respond appropriately to changes in plasma glucose concentrations. GK activity is potently regulated by the glucokinase regulatory protein (GKRP), which is encoded by the GCKR gene. GKRP binds GK in the nucleus and inhibits its activity. GK becomes active when it is released from GKRP and translocates to the cytosol. Low glucokinase (GK) activity is reported to be a principal feature of feline hepatic carbohydrate metabolism but the molecular pathways that regulate GK activity are not known. This study examined the hypothesis that species-specific differences in GKRP expression parallel the low GK activity observed in feline liver. Hepatic GKRP expression was examined using RT-PCR, immunoblot, and confocal immunomicroscopy. The results show that the GCKR gene is present in the feline genome but GCKR mRNA and the GKRP protein were absent in feline liver. The lack of GKRP expression in feline liver indicates that the low GK activity cannot be the result of GKRP-mediated inhibition of the GK enzyme. However, the absence of the permissive effects of GCKR expression on GK expression and activity may contribute to reduced GK enzyme activity in feline liver. The study results show that the cat is a natural model for GCKR knockout and may be useful to study regulation of GCKR expression and its role in hepatic glucose-sensing and carbohydrate metabolism.
Asunto(s)
Proteínas Portadoras/biosíntesis , Gatos/metabolismo , Glucoquinasa/metabolismo , Hígado/metabolismo , Animales , Proteínas Portadoras/genética , Hepatocitos/enzimología , Hepatocitos/metabolismo , Immunoblotting/veterinaria , Hígado/enzimología , Microscopía Confocal/veterinaria , ARN/química , ARN/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinariaRESUMEN
Ketohexokinase (KHK) metabolizes dietary fructose and is an important regulator of hepatic glucose metabolism. The veterinary literature contains conflicting data regarding the role of KHK in feline fructose metabolism. The study objectives were to determine tissue expression of KHK mRNA and protein in cats, with special emphasis on hepatic expression. KHK mRNA and protein expression were determined using routine RT-PCR and immunoblot techniques. KHK mRNA was detected in feline liver, pancreas, spleen and striated muscle but not in lung. The partial sequence of feline KHK mRNA obtained was highly similar to known KHK mRNA sequences. Immunoblot studies confirmed KHK protein expression in the feline liver. The tissue distribution of KHK mRNA in cats is similar to KHK expression in other species. KHK mRNA and protein expression in feline liver is consistent with previous reports of hepatic fructokinase activity in this species.