RESUMO
Fructose consumption in humans and animals has been linked to enhanced de novo lipogenesis, dyslipidemia, and insulin resistance. Hereditary deficiency of ketohexokinase (KHK), the first enzymatic step in fructose metabolism, leads to essential fructosuria in humans, characterized by elevated levels of blood and urinary fructose following fructose ingestion but is otherwise clinically benign. To address whether KHK deficiency is associated with altered glucose and lipid metabolism, a Khk knockout (KO) mouse line was generated and characterized. NMR spectroscopic analysis of plasma following ingestion of [6-13C] fructose revealed striking differences in biomarkers of fructose metabolism. Significantly elevated urine and plasma 13C-fructose levels were observed in Khk KO vs. wild-type (WT) control mice, as was reduced conversion of 13C-fructose into plasma 13C-glucose and 13C-lactate. In addition, the observation of significant levels of fructose-6-phosphate in skeletal muscle tissue of Khk KO, but not WT, mice suggests a potential mechanism, whereby fructose is metabolized via muscle hexokinase in the absence of KHK. Khk KO mice on a standard chow diet displayed no metabolic abnormalities with respect to ambient glucose, glucose tolerance, body weight, food intake, and circulating trigylcerides, ß-hydroxybutyrate, and lactate. When placed on a high-fat and high-fructose (HF/HFruc) diet, Khk KO mice had markedly reduced liver weight, triglyceride levels, and insulin levels. Together, these results suggest that Khk KO mice may serve as a good model for essential fructosuria in humans and that inhibition of KHK offers the potential to protect from diet-induced hepatic steatosis and insulin resistance.
Assuntos
Dieta , Frutoquinases/deficiência , Erros Inatos do Metabolismo da Frutose/genética , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal/genética , Dieta Hiperlipídica , Ingestão de Alimentos/genética , Frutoquinases/genética , Frutoquinases/metabolismo , Erros Inatos do Metabolismo da Frutose/metabolismo , Frutosefosfatos/sangue , Intolerância à Glucose/genética , Resistência à Insulina , Metabolismo dos Lipídeos/genética , Camundongos KnockoutRESUMO
Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2NI) and R531G (AMPKγ2RG), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2NI or AMPKγ2RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2NI or AMPKγ2RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2NI and AMPKγ2RG, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2NI or AMPKγ2RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2WT mice, AMPKγ2NI and AMPKγ2RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2RG but not AMPKγ2NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2NI and AMPKγ2RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Mutação , Insuficiência Renal/genética , Síndrome de Wolff-Parkinson-White/genética , Animais , Apoptose , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Inflamação/genética , Inflamação/patologia , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Insuficiência Renal/patologia , Síndrome de Wolff-Parkinson-White/patologiaRESUMO
Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.
Assuntos
Colesterol/sangue , Pirazóis/farmacologia , Receptores de Glucagon/antagonistas & inibidores , beta-Alanina/análogos & derivados , Animais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Humanos , Hipercolesterolemia/induzido quimicamente , Concentração Inibidora 50 , Absorção Intestinal , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pirazóis/efeitos adversos , beta-Alanina/efeitos adversos , beta-Alanina/farmacologiaRESUMO
H atom produced in the thermal decomposition of CH(3)OH highly diluted in Ar (0.48-10 ppm) was monitored behind reflected shock waves by atomic resonance absorption spectrometry (ARAS) at fixed temperatures (and pressures), that is, 1660 (1.73 atm), 1760 (2.34 atm), 1860 (2.04 atm), 1950 (2.18 atm), and 2050 K (1.76 atm) (+/-10 K, respectively). High sensitivity for the H atom has been attained by signal averaging of the ARAS signals down to the concentrations of approximately 1 x 10(11) atoms/cm(3) and enables us to determine the branching fraction for the direct H atom production channel, CH(3)OH --> CH(2)OH + H (channel 1c ) in a mixture of 1 ppm CH(3)OH. Channel 1c is confirmed to be minor, that is, branching fraction for channel 1c is expressed by Log(k(1c)/k(1)) = (- 2.88 +/- 1.88) x 10(3)/T - (0.23 +/- 1.02), which corresponds to k(1c)/k(1) < 0.03 for the present temperature range. By using 0.48 and 1.0 ppm CH(3)OH with (100-1000) ppm H(2), the total decomposition rate k(1) for CH(3)OH --> products is measured from the time dependence of H atom, where the radical products of main channels 1a and 1b , that is, OH, CH(3), and CH(2), were converted rapidly into H atoms. The experimental result is summarized as Log(k(1)/cm(3)molecule(-1)s(-1)) = (-12.82 +/- 0.71) x 10(3)/T - (8.5 +/- 0.38). A theoretical study based on ab initio/TST calculations with high accuracy has been conducted for the reaction: (3)CH(2) + H(2) --> CH(3) + H (reaction 3 ). The rate is given by k(3)/cm(3)molecule(-1) s(-1) = (7.32 x 10(-19))T(2.3) exp (-3699/T). This result is used for numerical simulations to evaluate k(1). Present experimental results on the thermal decomposition rate of CH(3)OH are found to be consistent with previous works. It is also found that time dependence of [H] observed in the 10 ppm CH(3)OH in Ar can be reproduced very well by kinetic simulations by using a reaction mechanism composed of 36 elementary reactions.
RESUMO
Physical activity promotes metabolic and cardiovascular health benefits that derive in part from the transcriptional responses to exercise that occur within skeletal muscle and other organs. There is interest in discovering a pharmacologic exercise mimetic that could imbue wellness and alleviate disease burden. However, the molecular physiology by which exercise signals the transcriptional response is highly complex, making it challenging to identify a single target for pharmacological mimicry. The current studies evaluated the transcriptome responses in skeletal muscle, heart, liver, and white and brown adipose to novel small molecule activators of AMPK (pan-activators for all AMPK isoforms) compared to that of exercise. A striking level of congruence between exercise and pharmacological AMPK activation was observed across the induced transcriptome of these five tissues. However, differences in acute metabolic response between exercise and pharmacologic AMPK activation were observed, notably for acute glycogen balances and related to the energy expenditure induced by exercise but not pharmacologic AMPK activation. Nevertheless, intervention with repeated daily administration of short-acting activation of AMPK was found to mitigate hyperglycemia and hyperinsulinemia in four rodent models of metabolic disease and without the cardiac glycogen accretion noted with sustained pharmacologic AMPK activation. These findings affirm that activation of AMPK is a key node governing exercise mediated transcription and is an attractive target as an exercise mimetic.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Animais , Metabolismo Energético , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Homeostase , Camundongos Endogâmicos C57BL , Oxirredução , Condicionamento Físico AnimalRESUMO
5'-Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of mammalian energy homeostasis and has been implicated in mediating many of the beneficial effects of exercise and weight loss including lipid and glucose trafficking. As such, the enzyme has long been of interest as a target for the treatment of Type 2 Diabetes Mellitus. We describe the optimization of ß1-selective, liver-targeted AMPK activators and their evolution into systemic pan-activators capable of acutely lowering glucose in mouse models. Identifying surrogates for the key acid moiety in early generation compounds proved essential in improving ß2-activation and in balancing improvements in plasma unbound fraction while avoiding liver sequestration.
RESUMO
5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cardiomegalia/induzido quimicamente , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Imidazóis/farmacologia , Piridinas/farmacologia , Animais , Benzimidazóis , Glicemia/efeitos dos fármacos , Jejum , Glicogênio/metabolismo , Hipoglicemia/induzido quimicamente , Imidazóis/efeitos adversos , Imidazóis/química , Insulina/farmacologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Piridinas/efeitos adversos , Piridinas/químicaRESUMO
Novel dihydropyrrolopyrazole-substituted benzimidazoles were synthesized and evaluated in vitro as inhibitors of transforming growth factor-beta type I receptor (TGF-beta RI), TGF-beta RII, and mixed lineage kinase-7 (MLK-7). These compounds were found to be potent TGF-beta RI inhibitors and selective versus TGF-beta RII and MLK-7 kinases. Benzimidazole derivative 8b was active in an in vivo target (TGF-beta RI) inhibition assay.
Assuntos
Benzimidazóis/síntese química , MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/síntese química , Pirróis/síntese química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Células Cultivadas , Humanos , Camundongos , Camundongos Nus , Vison , Estrutura Terciária de Proteína , Pirazóis/química , Pirazóis/farmacologia , Pirróis/química , Pirróis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alpha actinin (ACTN) has emerged as a multitasking protein, whose roles range from bundling actin filaments to functioning as a versatile protein interaction platform for proteins involved in structural or signaling aspects. We report here that ACTN2, one of the four ACTN isoforms, may shuttle between the cytoplasm and nucleus where the nuclear exportation takes place in a CRM1-dependent manner. The majority of ACTN2 was found to localize in the cytoplasm and exhibit a lower stability which was demonstrated using either mutants carrying mutated nuclear receptor binding motif or inhibitors against the ubiquitin- and calpain-dependent degradation pathways. Horse serum induced differentiation of C2C12 cells also caused the redistribution of nuclear ACTN2 to the cytoplasm, which subcellular compartment the ACTN2 behaves as an unstable protein. Our data indicated that the model in which ACTN2 functions as a multi-talented coregulator may be controlled by the differential protein stability modulated via nucleo-cytoplasmic trafficking in C2C12 cells.