RESUMEN
Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.
Asunto(s)
Fosfatasas de Especificidad Dual/genética , Glucógeno Sintasa/metabolismo , Glucógeno/biosíntesis , Resistencia a la Insulina/genética , Obesidad/patología , Envejecimiento/genética , Animales , Fosfatasas de Especificidad Dual/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucógeno/genética , Glucógeno Sintasa/genética , Humanos , Insulina/genética , Insulina/metabolismo , Hígado/enzimología , Hígado/metabolismo , Hígado/patología , Ratones , Obesidad/etiología , Obesidad/genética , Fosforilación , Proteínas Tirosina Fosfatasas no ReceptorasRESUMEN
OBJECTIVE: Nicotine, the main addictive ingredient in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Considerable genetic variation in the CYP2A6 gene results in large variation in the rates of nicotine metabolism, which in turn alters smoking behaviours (e.g. amount of cigarettes smoked, risk for dependence and success in smoking cessation). The aim of this study was to identify and characterize novel variants in CYP2A6. MATERIALS AND METHODS: The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced and seven novel variants were identified [CYP2A6*39 (V68M), CYP2A6*40 (I149M), CYP2A6*41 (R265Q), CYP2A6*42 (I268T), CYP2A6*43 (T303I), CYP2A6*44 (E390K), CYP2A6*44 (L462P)]. Variants were introduced into a bi-cistronic cDNA expression construct containing CYP2A6 and P450 oxidoreductase and assessed for protein expression, enzymatic activity and stability as evaluated using western blotting and nicotine metabolism. Genotyping assays were developed and allelic frequencies were assessed in 534 African Americans. RESULTS: The variants showed significantly lower protein expression (P<0.001) when compared with the wild-type as well as reduced metabolism of nicotine to cotinine when controlling for cDNA expression using P450 oxidoreductase (P<0.001). The variants also showed reduced stability at 37°C. Allelic frequencies ranged from 0.1 to 0.6% with a collective genotype frequency of 3.2%; the impact in vitro correlated significantly with in-vivo activity (R(2)=0.40-0.48, P<0.05). Together, those with a novel variant had significantly lower nicotine metabolism in vivo than those without genetic variants (P<0.01). CONCLUSION: Here, we identified a number of novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 genetic variability.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Negro o Afroamericano/genética , Nicotina/metabolismo , Citocromo P-450 CYP2A6 , Estabilidad de Enzimas , Evolución Molecular , Frecuencia de los Genes , Variación Genética , Genotipo , Humanos , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
Lafora disease is the most common teenage-onset neurodegenerative disease, the main teenage-onset form of progressive myoclonus epilepsy (PME), and one of the severest epilepsies. Pathologically, a starch-like compound, polyglucosan, accumulates in neuronal cell bodies and overtakes neuronal small processes, mainly dendrites. Polyglucosan formation is catalyzed by glycogen synthase, which is activated through dephosphorylation by glycogen-associated protein phosphatase-1 (PP1). Here we remove PTG, one of the proteins that target PP1 to glycogen, from mice with Lafora disease. This results in near-complete disappearance of polyglucosans and in resolution of neurodegeneration and myoclonic epilepsy. This work discloses an entryway to treating this fatal epilepsy and potentially other glycogen storage diseases.
Asunto(s)
Glucanos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedad de Lafora/fisiopatología , Animales , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Glucanos/análisis , Glucógeno Sintasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Enfermedad de Lafora/genética , Ratones , Ratones NoqueadosRESUMEN
The harlequin (hq)/Big Blue mouse is a novel model of premature ageing distinguished by a patchy coat, early-onset neurodegeneration, stress-induced heart disease and a mutation detection assay applicable to individual tissues. The hq mutation causes down-regulation of apoptosis-inducing factor and an elevation of reactive oxygen species (ROS). Neural tissues have elevated mutant frequency and early-onset degeneration. This is the first examination of mutations and histology in the skin of hq disease mice. The frequency and pattern of cII mutations in skin from adult hq disease and wild-type (WT) mice 15 days after a single intraperitoneal (i.p.) injection of paraquat (PQ; 10 mg/kg) or vehicle control (VC) were determined to assess spontaneous mutagenesis and sensitivity to an exogenous ROS-inducing mutagen. Skin of hq disease mice shows elevated levels of ROS (P < 0.001) and reduced numbers of hair follicles and associated epidermal cells (P < 0.001) compared to WT control. Acute PQ exposure did not produce detectable skin histopathology. Spontaneous and PQ-induced mutation frequency is elevated in hq skin (P = 0.03 and P = 0.01, respectively) compared to VC-treated WT mice. Despite elevated mutation frequency, mutation patterns were unaltered. Acute PQ exposure resulted in a 1.6-fold increase in mutation frequency in WT mice compared to the level of spontaneous mutations but no significant impact on mutation frequency in hq disease mice. Increased mutation frequency in skin of hq disease mice may be relevant to mechanisms underlying the patchy coat and useful as a biomarker in tests of antioxidant efficacy in preventing the hq disease phenotype. Unaltered mutation patterns with hq disease are consistent with the multiple mutation types associated with ROS. Acute PQ exposure had only subtle effects in WT mice and reduced mitochondrial complex I activity and elevated antioxidant enzyme activity in hq disease mice may lead to PQ resistance.