RESUMO
Reports that ataxia telangiectasia mutated (ATM) is required for full activation of Akt raise the hypothesis that ATM plays a role in insulin-like growth factor 1 (IGF-1) signalling through the Akt/mammalian target of rapamycin (mTOR) pathway. Differentiated C2C12 cells harbouring either ATM-targeting short hairpin RNA (shRNA) or non-targeting shRNA and myotubes from a C2C12 lineage previously exposed to empty vector lentivirus were incubated in the presence or absence of 10 nm IGF-1 followed by Western blot analysis. Parallel experiments were performed in isolated soleus muscles from mice expressing only one functional ATM allele (ATM(+/-)) compared with muscles from wild-type (ATM(+/+)) mice. Insulin-like growth factor 1 increased phosphorylation of Akt S473, Akt T308 and p70 S6 kinase (S6K) in myotubes expressing non-targeting shRNA and in empty vector controls, but the IGF-1 effects were significantly reduced in myotubes with shRNA-mediated ATM knockdown. Likewise, IGF-1-stimulated phosphorylation of Akt S473, Akt T308, mTOR and S6K was lower in isolated soleus muscles from ATM(+/-) mice compared with muscles from ATM(+/+) mice. The ATM inhibitor KU55933 prevented stimulation of S6K phosphorylation in C2C12 myotubes exposed to IGF-1, suggesting that decreased IGF-1 action is not limited to chronic conditions of decreased ATM function. Stimulation of insulin receptor substrate 1 tyrosine 612 phosphorylation by IGF-1 was unaffected by ATM deficiency, though IGF-1 phosphatidylinositol 3-kinase activity tended to be lower in muscle from ATM haploinsufficient mice compared with wild-type muscle. The data suggest that ATM is a modulator of IGF-1 signalling downstream of insulin receptor substrate 1 in skeletal muscle.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/enzimologia , Mioblastos Esqueléticos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Vetores Genéticos , Genótipo , Haploinsuficiência , Proteínas Substratos do Receptor de Insulina/metabolismo , Lentivirus/genética , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Músculo Esquelético/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Fenótipo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pironas/farmacologia , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Transfecção , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
AIMS: There are reports that ataxia telangiectasia mutated (ATM) can activate the AMP-activated protein kinase (AMPK) and also Akt, two kinases that play integral parts in cardioprotection and metabolic function. We hypothesized that chloroquine and resveratrol, both known ATM activators, would also activate AMPK and Akt. MAIN METHODS: Phosphorylation of AMPK and Akt was assessed after C2C12 myotubes were exposed to chloroquine or resveratrol. Additional experiments were done in cells expressing shRNA against ATM or in the presence of the ATM inhibitor KU55933. The effects of chloroquine on intracellular calcium were assessed with the fluorescent probe Calcium Green-1 AM. KEY FINDINGS: 0.5 mM chloroquine increased AMPK phosphorylation by nearly 4-fold (P<0.05), and 0.25 mM chloroquine roughly doubled Akt phosphorylation (P<0.05). Chloroquine also increased autophosphorylation of ATM by ~50% (P<0.05). Resveratrol (0.15 mM) increased AMPK phosphorylation about three-fold (P<0.05) but in contrast to chloroquine sharply decreased Akt phosphorylation. Chloroquine increased AMPK and Akt phosphorylation in myotubes expressing shRNA against ATM that reduced ATM protein levels by about 90%. Likewise, chloroquine-stimulated phosphorylation of AMPK and Akt and resveratrol-stimulated phosphorylation of AMPK were not altered by inhibition of ATM. Chloroquine decreased intracellular calcium by >50% concomitant with a decrease in glucose transport. SIGNIFICANCE: These ATM-independent effects of chloroquine on AMPK and Akt and the additional effect to decrease intracellular calcium are likely to partially underlie the positive metabolic effects of chloroquine that have been reported in the literature.
RESUMO
Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4'-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3beta-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.
Assuntos
Flavonoides/biossíntese , Glycine max/enzimologia , Liases Intramoleculares/metabolismo , Isoflavonas/biossíntese , Sequência de Aminoácidos , Catálise , Clonagem Molecular , DNA de Plantas/genética , Ativação Enzimática , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Genes de Plantas , Engenharia Genética , Liases Intramoleculares/classificação , Liases Intramoleculares/genética , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Glycine max/genéticaRESUMO
5-epi-Aristolochene dihydroxylase (EAH) catalyzes unique stereo- and regiospecific hydroxylations of a bicyclic sesquiterpene hydrocarbon to generate capsidiol. To define functional and mechanistic features of the EAH enzyme, the utility of a coupled assay using readily available sesquiterpene synthases and microsomes from yeast overexpressing the EAH enzyme was determined. Capsidiol and deoxycapsidiol biosyntheses were readily measured in coupled assays consisting of 5-epi-aristolochene synthase and EAH as determined by the incorporation of radiolabeled farnesyl diphosphate into thin-layer chromatography-isolated products and verified by gas chromatography-mass spectrometry analysis. The assays were dependent on the amounts of synthase and hydroxylase protein added, the incubation times, and the presence of nicotinamide adenine dinucleotide phosphate. The utility of this coupled assay was extended by examining the relative efficiency of the EAH enzyme to catalyze hydroxylations of different sesquiterpene skeletons generated by other terpene synthases.