RESUMO
A tight hormonal control of energy homeostasis is of pivotal relevance for animals. Recent evidence suggests an involvement of the nuclear receptor NR1i3 (CAR). Fasting induces CAR by largely unknown mechanisms and CAR-deficient mice are defective in fasting adaptation. In rat hepatocytes CAR was induced by WY14643, a PPARalpha-agonist. A DR1 motif in the CAR promoter was necessary and sufficient for this control. The PPARalpha-dependent increase in CAR potentiated the phenobarbital-induced transcription of the prototypical CAR-dependent gene CYP2B1. Since free fatty acids are natural ligands for PPARalpha, a fasting-induced increase in free fatty acids might induce CAR. In accordance with this hypothesis, CAR induction by fasting was abrogated in PPARalpha-deficient mice.
Assuntos
Adaptação Fisiológica , Regulação da Expressão Gênica , PPAR alfa/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Inanição/genética , Fatores de Transcrição/genética , Animais , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B1/metabolismo , Metabolismo Energético , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , PPAR alfa/agonistas , Regiões Promotoras Genéticas , Pirimidinas/farmacologia , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/metabolismo , Inanição/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Two isoforms of the rat prostaglandin E(2) receptor, rEP3alpha-R and rEP3beta-R, differ only in their C-terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3alpha-ST341-349A-R) were stably expressed in HEK293 cells. All rEP3-R receptors showed a similar ligand-binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. Repeated exposure of cells expressing the rEP3alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi-response as well as plasma membrane localization of the rEP3beta-R and the rEP3alpha-ST341-349A-R were not affected by prior agonist-stimulation. Agonist-stimulation of HEK293-rEP3alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3beta-R was not phosphorylated and the rEP3alpha-ST341-349A-R was phosphorylated only weakly. These results led to the hypothesis that agonist-induced desensitization of the rEP3alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341-349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization.
Assuntos
AMP Cíclico/metabolismo , Receptores de Prostaglandina E/metabolismo , Serina/metabolismo , Treonina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP3 , Serina/genética , Treonina/genética , TransfecçãoRESUMO
Reduced expression of the INDY (I'm not dead yet) tricarboxylate carrier increased the life span in different species by mechanisms akin to caloric restriction. Mammalian INDY homolog (mIndy, SLC13A5) gene expression seems to be regulated by hormonal and/or nutritional factors. The underlying mechanisms are still unknown. The current study revealed that mIndy expression and [(14)C]-citrate uptake was induced by physiological concentrations of glucagon via a cAMP-dependent and cAMP-responsive element-binding protein (CREB)-dependent mechanism in primary rat hepatocytes. The promoter sequence of mIndy located upstream of the most frequent transcription start site was determined by 5'-rapid amplification of cDNA ends. In silico analysis identified a CREB-binding site within this promoter fragment of mIndy. Functional relevance for the CREB-binding site was demonstrated with reporter gene constructs that were induced by CREB activation when under the control of a fragment of a wild-type promoter, whereas promoter activity was lost after site-directed mutagenesis of the CREB-binding site. Moreover, CREB binding to this promoter element was confirmed by chromatin immunoprecipitation in rat liver. In vivo studies revealed that mIndy was induced in livers of fasted as well as in high-fat-diet-streptozotocin diabetic rats, in which CREB is constitutively activated. mIndy induction was completely prevented when CREB was depleted in these rats by antisense oligonucleotides. Together, these data suggest that mIndy is a CREB-dependent glucagon target gene that is induced in fasting and in type 2 diabetes. Increased mIndy expression might contribute to the metabolic consequences of diabetes in the liver.