RESUMO
Arachidonic acid (AA), a polyunsaturated fatty acid, is involved in the modulation of neuronal excitability in the brain. Arachidonate lipoxygenase 3 (ALOXE3), a critical enzyme in the AA metabolic pathway, catalyzes the derivate of AA into hepoxilins. However, the expression pattern of ALOXE3 and its role in the brain has not been described until now. Here we showed that the levels of Aloxe3 mRNA and protein kept increasing since birth and reached the highest level at postnatal day 30 in the mouse hippocampus and temporal cortex. Histomorphological analyses indicated that ALOXE3 was enriched in adult hippocampus, somatosensory cortex and striatum. The distribution was restricted to the neurites of function-specific subregions, such as mossy fibre connecting hilus and CA3 neurons, termini of Schaffer collateral projections, and the layers III and IV of somatosensory cortex. The spatiotemporal expression pattern of ALOXE3 suggests its potential role in the modulation of neural excitability and seizure susceptibility. In fact, decreased expression of ALOXE3 and elevated concentration of AA in the hippocampus was found after status epilepticus (SE) induced by pilocarpine. Local overexpression of ALOXE3 via adeno-associated virus gene transfer restored the elevated AA level induced by SE, alleviated seizure severities by increasing the latencies to myclonic switch, clonic convulsions and tonic hindlimb extensions, and decreased the mortality rate in the pilocarpine-induced SE model. These results suggest that the expression of ALOXE3 is a crucial regulator of AA metabolism in brain, and potentially acts as a regulator of neural excitability, thereby controlling brain development and seizure susceptibility.
Assuntos
Convulsões , Estado Epiléptico , Animais , Encéfalo/metabolismo , Hipocampo/metabolismo , Camundongos , Pilocarpina , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , Estado Epiléptico/induzido quimicamenteRESUMO
Valproate (VPA), a widely-used antiepileptic drug, is a selective inhibitor of histone deacetylase (HDAC) that play important roles in epigenetic regulation. The patient with different diseases receiving this drug tend to exhibit weight gain and abnormal metabolic phenotypes, but the underlying mechanisms remain largely unknown. Here we show that VPA increases the Fto mRNA and protein expression in mouse hypothalamic GT1-7 cells. Interestingly, VPA promotes histone H3/H4 acetylation and the FTO expression which could be reversed by C646, an inhibitor for histone acetyltransferase. Furthermore, VPA weakens the FTO's binding and enhances the binding of transcription factor TAF1 to the Fto promoter, and C646 leads to reverse effect of the VPA, suggesting an involvement of the dynamic of histone H3/H4 acetylation in the regulation of FTO expression. In addition, the mice exhibit an increase in the food intake and body weight at the beginning of 2-week treatment with VPA. Simultaneously, in the hypothalamus of the VPA-treated mice, the FTO expression is upregulated and the H3/H4 acetylation is increased; further the FTO's binding to the Fto promoter is decreased and the TAF1's binding to the promoter is enhanced, suggesting that VPA promotes the assembly of the basal transcriptional machinery of the Fto gene. Finally, the inhibitor C646 could restore the effects of VPA on FTO expression, H3/H4 acetylation, body weight, and food intake; and loss of FTO could reverse the VPA-induced increase of body weight and food intake. Taken together, this study suggests an involvement of VPA in the epigenetic upregulation of hypothalamic FTO expression that is potentially associated with the VPA-induced weight gain.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/biossíntese , Epigênese Genética/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Ácido Valproico/farmacologia , Aumento de Peso/efeitos dos fármacos , Animais , Anticonvulsivantes/farmacologia , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Epigênese Genética/fisiologia , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Aumento de Peso/fisiologiaRESUMO
Nicotine, a major addictive component in tobacco, plays an important role in the changes of body weight upon smoking and its cessation. Here we showed that nicotine-treated mice exhibited weight loss and nicotine withdrawal led to weight gain. Using TMT-based proteomic analysis, we obtained the different hypothalamic protein profiles in response to nicotine and its withdrawal. A total of ~5000 proteins were identified from the hypothalamus with 50 altered proteins upon 28-day nicotine treatment and 28 altered proteins upon 15-day nicotine withdrawal. Of the altered proteins, CASP3, LCMT2, GRIN2D, CCNT2, FADS3 and MRPS18B were inversely changed in response to nicotine and withdrawal, coincidence with the change of body weight. Of them, CASP3, LCMT2, GRIN2D and CCNT2 were found to be associated with several GO terms and KEGG pathways linking with cell apoptosis, neurotransmission and metabolism. Further Western blot and RT-qPCR analyses confirmed that the levels of the 4 proteins CASP3, LCMT2, GRIN2D and CCNT2, instead of their mRNA transcripts, altered in response to nicotine and withdrawal. Thus this study provides nicotine- and withdrawal-induced hypothalamic protein profiles and suggests potential roles of these altered proteins in the change of body weight. SIGNIFICANCE: Cigarette smoking is one of important factors harming human health. Most smokers tend to have lower body weights and smoking cessation often lead to overweight or obesity, which is an important reason for smokers to insist on smoking. It is known that nicotine, a critical component in tobacco, is associated with the alteration in body weight by affecting hypothalamic function. Through TMT-based proteomic analysis, this study identified differential hypothalamic protein profiles in response to nicotine treatment and its withdrawal, and 4 nicotine- and withdrawal-induced contrary proteins CASP3, LCMT2, GRIN2D and CCNT2 are involved in several enriched GO terms and KEGG pathways, which are associated with cell apoptosis, neurotransmission and metabolism. Our study may provide novel targets for further investigation of the molecular mechanisms of nicotine- and withdrawal-induced alteration in body weight.