RESUMO
BACKGROUND: The fetal cortical neuroepithelium is a mosaic of distinct progenitor populations that elaborate diverse cellular fates. Ethanol induces apoptosis and interferes with the survival of differentiating neurons. However, we know little about ethanol's effects on neuronal progenitors. We therefore exposed neurosphere cultures from fetal rat cerebral cortex, to varying ethanol concentrations, to examine the impact of ethanol on stem cell fate. RESULTS: Ethanol promoted cell cycle progression, increased neurosphere number and increased diversity in neurosphere size, without inducing apoptosis. Unlike controls, dissociated cortical progenitors exposed to ethanol exhibited morphological evidence for asymmetric cell division, and cells derived from ethanol pre-treated neurospheres exhibited decreased proliferation capacity. Ethanol significantly reduced the numbers of cells expressing the stem cell markers CD117, CD133, Sca-1 and ABCG2, without decreasing nestin expression. Furthermore, ethanol-induced neurosphere proliferation was not accompanied by a commensurate increase in telomerase activity. Finally, cells derived from ethanol-pretreated neurospheres exhibited decreased differentiation in response to retinoic acid. CONCLUSION: The reduction in stem cell number along with a transient ethanol-driven increase in cell proliferation, suggests that ethanol promotes stem to blast cell maturation, ultimately depleting the reserve proliferation capacity of neuroepithelial cells. However, the lack of a concomitant change in telomerase activity suggests that neuroepithelial maturation is accompanied by an increased potential for genomic instability. Finally, the cellular phenotype that emerges from ethanol pre-treated, stem cell depleted neurospheres is refractory to additional differentiation stimuli, suggesting that ethanol exposure ablates or delays subsequent neuronal differentiation.
Assuntos
Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Etanol/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Células Neuroepiteliais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Feminino , Feto , Regeneração Nervosa/fisiologia , Células Neuroepiteliais/citologia , Células Neuroepiteliais/fisiologia , Gravidez , Ratos , Ratos Sprague-Dawley , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of long-chain fatty acids (LCFA) for oxidation and for peroxisome proliferator-activated receptor alpha (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced beta-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. During standard diet, mitochondrial HMG CoA synthase mRNA was selectively reduced in L-FABP null liver. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism, whereas L-FABP can activate ketogenic gene expression in fed mice. Thus, the mechanisms whereby L-FABP affects fatty acid oxidation may vary with physiological condition.
Assuntos
Proteínas de Transporte/metabolismo , Jejum/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/metabolismo , Animais , Proteínas de Transporte/genética , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/química , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Cetonas/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Oxirredução , Ácido Palmítico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. We recently showed that ethanol depletes reserve cerebral cortical stem cell capacity. Moreover, proliferating neuroepithelial cells exposed to ethanol were resistant to subsequent retinoic acid-induced differentiation. Emerging evidence suggests that cytokines play a crucial growth-promoting role in the developing neural tube. METHODS: We cultured murine cortical neurosphere cultures in control or ethanol-supplemented mitogenic medium, to mimic alcohol exposure during the period of neuroepithelial proliferation. Cultures were then treated with a step-wise mitogen-withdrawal, integrin-activation model to mimic subsequent phases of neuronal migration and early differentiation. We examined the impact of alcohol exposure during neurogenesis on the secretion of inflammatory and growth-promoting cytokines. RESULTS: Cortical neurosphere cultures exhibit increasingly complex differentiation phenotypes in response to step-wise mitogen-withdrawal and laminin exposure. Some inflammation-modulating cytokines were secreted independent of differentiation state. However, chemotactic cytokines were specifically secreted at high levels, as a function of differentiation stage. monocyte chemotactic protein-1, vascular endothelial growth factor-A, and interleukin (IL)-10 were coordinately decreased during differentiation compared with neuroepithelial proliferation, while granulocyte macrophage-colony stimulating factor (GM-CSF) was induced during differentiation, compared with the neuroepithelial proliferation period. Ethanol exposure during the period of neuroepithelial proliferation prevented the early differentiation-induced increase in GM-CSF while inducing differentiation-associated increase in IL-12 secretion. CONCLUSION: Embryonic cerebral cortical neuroepithelial-derived precursors secrete high levels of several angiogenic and neural-growth-promoting cytokines as they differentiate into neurons. Our data collectively suggest that ethanol exposure during the period of neuroepithelial proliferation significantly disrupts cytokine signals that are required for the support of emerging neurovascular networks, and the maintenance of neural stem cell beds.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Depressores do Sistema Nervoso Central/toxicidade , Córtex Cerebral/citologia , Citocinas/metabolismo , Etanol/toxicidade , Sistema Nervoso/embriologia , Células Neuroepiteliais/citologia , Proteínas Angiogênicas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso/efeitos dos fármacos , Células Neuroepiteliais/efeitos dos fármacos , Gravidez , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of approximately 75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver.