RESUMO
From the 95% aqueous ethanol extract of Murraya microphylla, five pairs of new carbazole alkaloid enantiomers, (+/-)-microphylines N-R (1a/1b-5a/5b), were isolated, together with 20 known carbazole alkaloids. The structures of the new compounds were determined by the HRMS and NMR spectroscopic data, along with the calculated electronic circular dichroism (ECD) and Mo2(AcO)4-induced CD data. The known compound (+)-mahanine (21) showed significant cytotoxicities against Du145, HepG2, HeLa, and HCT-116 cell lines, and its possible mechanism was deduced to target on phosphoenolpyruvate carboxykinase 2 (PCK2) protein via surface plasmon resonance (SPR) and molecular docking.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Murraya/química , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Alcaloides/química , Alcaloides/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Carbazóis/química , Carbazóis/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Relação Estrutura-AtividadeRESUMO
Phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme with a cytosolic (Pck1/PEPCK-C) and mitochondrial (Pck2/PEPCK-M) isoform. Here we investigate the effect of 3-mercaptopicolinic acid (3-MPA), a PEPCK inhibitor, on C2C12 muscle cells. We report that Pck2 mRNA is 50-5000-fold higher than Pck1 during C2C12 myogenesis, indicating Pck2 is the predominant PEPCK isoform. C2C12 cell proliferation was inhibited in a dose-dependent manner following 48 h 3-MPA treatment (0.01-1 mM). C2C12 myogenic differentiation was significantly induced following 3-MPA treatment (0.25, 0.5, 1 mM) from day 0 of differentiation, demonstrated by increased creatine kinase activity, fusion index and myotube diameter; likewise, the myosin heavy chain (MyHC)-IIB isoform (encoded by Myh4) is an indicator of hypertrophy, and both porcine MYH4-promoter activity and endogenous Myh4 mRNA were also significantly induced. High doses (0.5 and/or 1 mM) of 3-MPA reduced mRNA expression of Pck2 and genes associated with serine biosynthesis (Phosphoglycerate dehydrogenase, Phgdh; phosphoserine aminotransferase-1, Psat1) following treatment from days 0 and 4. To conclude, as Pck2/PEPCK-M is the predominant isoform in C2C12 cells, we postulate that 3-MPA promoted myogenic differentiation through the inhibition of PEPCK-M. However, we were unable to confirm that 3-MPA inhibited PEPCK-M enzyme activity as 3-MPA interfered with the PEPCK enzyme assay, particularly at 0.5 and 1 mM.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Ácidos Picolínicos/farmacologia , Animais , Biomarcadores , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/genética , Isoenzimas , Camundongos , Células Musculares , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Serina/biossínteseRESUMO
BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate. The mitochondrial isozyme, PEPCK-M is highly expressed in cancer cells, where it plays a role in nutrient stress response. To date, pharmacological strategies to target this pathway have not been pursued. METHODS: A compound embodying a 3-alkyl-1,8-dibenzylxanthine nucleus (iPEPCK-2), was synthesized and successfully probed in silico on a PEPCK-M structural model. Potency and target engagement in vitro and in vivo were evaluated by kinetic and cellular thermal shift assays (CETSA). The compound and its target were validated in tumor growth models in vitro and in murine xenografts. RESULTS: Cross-inhibitory capacity and increased potency as compared to 3-MPA were confirmed in vitro and in vivo. Treatment with iPEPCK-2 inhibited cell growth and survival, especially in poor-nutrient environment, consistent with an impact on colony formation in soft agar. Finally, daily administration of the PEPCK-M inhibitor successfully inhibited tumor growth in two murine xenograft models as compared to vehicle, without weight loss, or any sign of apparent toxicity. CONCLUSION: We conclude that iPEPCK-2 is a compelling anticancer drug targeting PEPCK-M, a hallmark gene product involved in metabolic adaptations of the tumor.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Animais , Biomarcadores Tumorais/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Estrutura Secundária de Proteína , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Diabetes mellitus is a medical condition characterized by the body's loss of control over blood sugar. The frequency of diagnosed cases and consequential increases in medical costs makes it a rapidly growing chronic disease that threatens human health worldwide. In addition, its unnerving statistical projections are perilous to both the economy of the nation and man's life expectancy. Type-I and type-II diabetes are the two clinical forms of diabetes mellitus. Type-II diabetes mellitus (T2DM) is illustrated by the abnormality of glucose homeostasis in the body, resulting in hyperglycemia. Although significant research attention has been devoted to the development of diabetes regimens, which demonstrates success in lowering blood glucose levels, their efficacies are unsustainable due to undesirable side effects such as weight gain and hypoglycemia. Over the years, heterocyclic scaffolds have been the basis of anti-diabetic chemotherapies; hence, in this review we consolidate the use of bioactive scaffolds, which have been evaluated for their biological response as inhibitors against their respective anti-diabetic molecular targets over the past five years (2012-2017). Our investigation reveals a diverse target set which includes; protein tyrosine phosphatase 1â¯B (PTP1B), dipeptidly peptidase-4 (DPP-4), free fatty acid receptors 1 (FFAR1), G protein-coupled receptors (GPCR), peroxisome proliferator activated receptor-γ (PPARγ), sodium glucose co-transporter-2 (SGLT2), α-glucosidase, aldose reductase, glycogen phosphorylase (GP), fructose-1,6-bisphosphatase (FBPase), glucagon receptor (GCGr) and phosphoenolpyruvate carboxykinase (PEPCK). This review offers a medium on which future drug design and development toward diabetes management may be modelled (i.e. optimization via structural derivatization), as many of the drug candidates highlighted show promise as an effective anti-diabetic chemotherapy.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Dipeptidil Peptidase 4/metabolismo , Inibidores Enzimáticos/química , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/metabolismo , Humanos , Hipoglicemiantes/química , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-GlicoseRESUMO
BACKGROUND: Increased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells. METHODS: 3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). RESULTS: 3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPß), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control. CONCLUSION: These results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity.
Assuntos
Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Camundongos , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Phosphoenolpyruvate carboxykinase (PEPCK) is involved in glycolysis in the cestode parasite, Raillietina echinobothrida; whereas, it executes a gluconeogenic role in its host, Gallus domesticus. Because of its differing primary function in the cestode parasite and its host, this enzyme is regarded as a plausible anthelmintic target. Hence, the biological significance of PEPCK in the parasite was analysed using siRNA against PEPCK from R. echinobothrida (RePEPCK). In order to find out the functional differences between RePEPCK and GdPEPCK (PEPCK from its host, G. domesticus), PEPCK genes from both sources were cloned, over-expressed, characterized, and some properties of the purified enzymes were compared. RePEPCK and GdPEPCK showed a standard Michaelis-Menten kinetics with K mapp of 46.9 and 22.9 µ m, respectively, for phosphoenolpyruvate and K mapp of 15.4 µ m for oxaloacetate in GdPEPCK decarboxylation reaction. Here, we report antagonist behaviours of recombinant PEPCKs derived from the parasite and its host. In search of possible modulators for PEPCK, few phytoestrogens were examined on the purified enzymes and their inhibitory constants were determined and discussed. This study stresses the potential of these findings to validate PEPCK as the anthelmintic drug target for parasitism management.
Assuntos
Cestoides/enzimologia , Infecções por Cestoides/veterinária , Galinhas/parasitologia , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fitoestrógenos/farmacologia , Doenças das Aves Domésticas/parasitologia , Animais , Cestoides/genética , Infecções por Cestoides/parasitologia , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Cinética , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , RNA Interferente Pequeno , Proteínas RecombinantesRESUMO
SCOPE: Functional foods can be used alone or in combination with existing therapies in preventing and treating type 2 diabetes (T2D). Trans-2,3,5,4'-tetrahydroxystilbene 2-O-ß-glucopyranoside (trans-THSG), a dominant bioactive compound from Polygonum multiflorum (PM)-a popular medicinal food in Asia, has attracted increasing research interests due to its strong antioxidant activity. The content of naturally occurring cis-THSG (cis-2,3,5,4'-tetrahydroxystilbene 2-O-ß-glucopyranoside) was very low in PM root, but was prepared in this study by mimicking the traditional process of PM. The anti-diabetic effects of trans- and cis-THSG were evaluated in T2D to search for more efficacious food ingredient(s). METHODS AND RESULTS: Trans-THSG was chromatographically purified from PM roots and cis-THSG was prepared with our innovative process via exposure of trans-THSG to UV-light. The anti-diabetic effects of both THSGs were tested with HFD-induced male CF-1 diabetic mice. Cis-THSG was found more effective than trans-THSG in hypoglycemic effect and in ameliorating glucose intolerance and insulin resistance. In HepG2 cells, cis-THSG also demonstrated more potent activity than trans-THSG in suppressing transcription of phosphoenopyruvate carboxykinase (PEPCK). CONCLUSION: Cis-THSG can be an enriched bioactive ingredient in PM roots from post-processing and is significantly more effective against hyperglycemia than trans-THSG. One of the effective pathways was through inhibition of PEPCK.
Assuntos
Fallopia multiflora/química , Glucosídeos/química , Glucosídeos/farmacologia , Hipoglicemiantes/farmacologia , Estilbenos/química , Estilbenos/farmacologia , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células Hep G2 , Humanos , Hipoglicemiantes/química , Resistência à Insulina , Masculino , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/genéticaRESUMO
This study investigated the metabolic requirements for neuronal progenitor maintenance in vitro and in vivo by examining the metabolic adaptations that support neuronal progenitors and neural stem cells (NSCs) in their undifferentiated state. We demonstrate that neuronal progenitors are strictly dependent on lactate metabolism, while glucose induces their neuronal differentiation. Lactate signaling is not by itself capable of maintaining the progenitor phenotype. The consequences of lactate metabolism include increased mitochondrial and oxidative metabolism, with a strict reliance on cataplerosis through the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) pathway to support anabolic functions, such as the production of extracellular matrix. In vivo, lactate maintains/induces populations of postnatal neuronal progenitors/NSCs in a PEPCK-M-dependent manner. Taken together, our data demonstrate that, lactate alone or together with other physical/biochemical cues maintain NSCs/progenitors with a metabolic signature that is classically found in tissues with high anabolic capacity.
Assuntos
Ácido Láctico/metabolismo , Células-Tronco Neurais/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Bromodesoxiuridina , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Glucose/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidoresRESUMO
Aging involves progressive loss of cellular function and integrity, presumably caused by accumulated stochastic damage to cells. Alterations in energy metabolism contribute to aging, but how energy metabolism changes with age, how these changes affect aging, and whether they can be modified to modulate aging remain unclear. In locomotory muscle of post-fertile Caenorhabditis elegans, we identified a progressive decrease in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), a longevity-associated metabolic enzyme, and a reciprocal increase in glycolytic pyruvate kinase (PK) that were necessary and sufficient to limit lifespan. Decline in PEPCK-C with age also led to loss of cellular function and integrity including muscle activity, and cellular senescence. Genetic and pharmacologic interventions of PEPCK-C, muscle activity, and AMPK signaling demonstrate that declines in PEPCK-C and muscle function with age interacted to limit reproductive life and lifespan via disrupted energy homeostasis. Quantifications of metabolic flux show that reciprocal changes in PEPCK-C and PK with age shunted energy metabolism toward glycolysis, reducing mitochondrial bioenergetics. Last, calorie restriction countered changes in PEPCK-C and PK with age to elicit anti-aging effects via TOR inhibition. Thus, a programmed metabolic event involving PEPCK-C and PK is a determinant of aging that can be modified to modulate aging.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicólise , Dinâmica Mitocondrial , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Piruvato Quinase/metabolismo , Envelhecimento , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Restrição Calórica , Citosol/enzimologia , Citosol/metabolismo , Citosol/ultraestrutura , Metabolismo Energético , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Interferência de RNA , Análise de SobrevidaRESUMO
Cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) is a critical enzyme involved in gluconeogenesis, glyceroneogenesis and cataplerosis. cPEPCK converts oxaloacetic acid (OAA) into phosphoenol pyruvate (PEP) in the presence of GTP. cPEPCK is known to be associated with type 2 diabetes. Genistein is an isoflavone compound that shows anti-diabetic and anti-obesitic properties. Experimental studies have shown a decrease in the blood glucose level in the presence of genistein by lowering the functional activity of cPEPCK, an enzyme of gluconeogenesis. Using computational techniques such as molecular modeling, molecular docking, molecular dynamics simulation and binding free energy calculations, we identified cPEPCK as a direct target of genistein. We studied the molecular interactions of genistein with three possible conformations of cPEPCK-unbound cPEPCK (u_cPEPCK), GTP bound cPEPCK (GTP_cPEPCK) and GDP bound cPEPCK (GDP_cPEPCK). Binding of genistein was also compared with an already known cPEPCK inhibitor. We analyzed the interactions of genistein with cPEPCK enzyme and compared them with its natural substrate (OAA), product (PEP) and known inhibitor (3-MPA). Our results demonstrate that genistein uses the mechanism of mixed inhibition to block the functional activity of cPEPCK and thus can serve as a potential anti-diabetic and anti-obesity drug candidate. We also identified an extended binding site in the catalytic cleft of cPEPCK which is used by 3-MPA to inhibit cPEPCK non-competitively. We demonstrate that extended binding site of cPEPCK can further be exploited for designing new drugs against cPEPCK.
Assuntos
Genisteína/química , Modelos Moleculares , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/química , Sítios de Ligação , Catálise , Genisteína/metabolismo , Humanos , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Ligação ProteicaRESUMO
Class IIa histone deacetylases (HDACs), such as HDAC4, HDAC5, and HDAC7, provide critical mechanisms for regulating glucose homeostasis. Here we report that HDAC9, another class IIa HDAC, regulates hepatic gluconeogenesis via deacetylation of a Forkhead box O (FoxO) family transcription factor, FoxO1, together with HDAC3. Specifically, HDAC9 expression can be strongly induced upon hepatitis C virus (HCV) infection. HCV-induced HDAC9 upregulation enhances gluconeogenesis by promoting the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, indicating a major role for HDAC9 in the development of HCV-associated exaggerated gluconeogenic responses. Moreover, HDAC9 expression levels and gluconeogenic activities were elevated in livers from HCV-infected patients and persistent HCV-infected mice, emphasizing the clinical relevance of these results. Our results suggest HDAC9 is involved in glucose metabolism, HCV-induced abnormal glucose homeostasis, and type 2 diabetes.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Gluconeogênese , Hepatite C Crônica/metabolismo , Histona Desacetilases/metabolismo , Resistência à Insulina , Fígado/metabolismo , Proteínas Repressoras/metabolismo , Acetilação , Animais , Biópsia por Agulha Fina , Linhagem Celular Tumoral , Indução Enzimática , Feminino , Proteína Forkhead Box O1 , Hepatite C Crônica/sangue , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Histona Desacetilases/genética , Humanos , Fígado/patologia , Fígado/virologia , Masculino , Camundongos Transgênicos , Ocludina/antagonistas & inibidores , Ocludina/genética , Ocludina/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Viral/antagonistas & inibidores , RNA Viral/sangue , RNA Viral/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Tetraspanina 28/antagonistas & inibidores , Tetraspanina 28/genética , Tetraspanina 28/metabolismoRESUMO
Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.
Assuntos
Benzamidas/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/química , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Benzamidas/química , Cristalografia por Raios X , Regulação da Expressão Gênica , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Isoxazóis/antagonistas & inibidores , Isoxazóis/farmacologia , Masculino , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Cultura Primária de Células , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores X de Retinoides/agonistas , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Transdução de SinaisRESUMO
Pyruvate kinase (PK; EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are essential regulatory enzymes of glucose oxidation in helminths, the PK/PEPCK branch point being the first divergent step between carbohydrate catabolism of the parasites and their hosts. Recently, PEPCK from the cestode parasite, Raillietina echinobothrida, has been purified and characterized. In order to find out the differential kinetics, if any, at PK/PEPCK branch point in the parasite, in this study, we purified and characterized the parasite PK and compared it with the parasite PEPCK. The purified PK displayed standard Michaelis-Menten kinetics with Kmapp of 77.8 µM for its substrate PEP, whereas the Kmapp was 46.9 µM for PEPCK. PEP exhibited differential kinetics at PK/PEPCK branch point of the parasite and behaved as a homotropic effector for PEPCK, but not for PK. The inhibitory constant (Ki) for genistein and daidzein (phytochemicals from Flemingia vestita) was determined and discussed. From these results, we hypothesize that PK/PEPCK branch point is a probable site for anthelmintic action.
Assuntos
Anticestoides/química , Cestoides/enzimologia , Inibidores Enzimáticos/química , Fabaceae/química , Fosfoenolpiruvato Carboxiquinase (ATP)/química , Extratos Vegetais/química , Piruvato Quinase/química , Animais , Cestoides/química , Cestoides/efeitos dos fármacos , Genisteína/química , Isoflavonas/química , Cinética , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/isolamento & purificaçãoRESUMO
OBJECTIVE: Chromium is an essential mineral that is thought to be necessary for normal glucose homeostasis. Numerous studies give evidence that chromium picolinate can modulate blood glucose and insulin resistance. The main ingredient of Tianmai Xiaoke (TMXK) Tablet is chromium picolinate. In China, TMXK Tablet is used to treat type 2 diabetes. This study investigated the effect of TMXK on glucose metabolism in diabetic rats to explore possible underlying molecular mechanisms for its action. METHODS: Diabetes was induced in rats by feeding a high-fat diet and subcutaneously injection with a single dose of streptozotocin (50 mg/kg, tail vein). One week after streptozotocin-injection, model rats were divided into diabetic group, low dose of TMXK group and high dose of TMXK group. Eight normal rats were used as normal control. After 8 weeks of treatment, skeletal muscle was obtained and was analyzed using Roche NimbleGen mRNA array and quantitative polymerase chain reaction (qPCR). Fasting blood glucose, oral glucose tolerance test and homeostasis model assessment of insulin resistance (HOMA-IR) index were also measured. RESULTS: The authors found that the administration of TMXK Tablet can reduce the fasting blood glucose and fasting insulin level and HOMA-IR index. The authors also found that 2 223 genes from skeletal muscle of the high-dose TMXK group had significant changes in expression (1 752 increased, 471 decreased). Based on Kyoto encyclopedia of genes and genomes pathway analysis, the most three significant pathways were "insulin signaling pathway", "glycolysis/gluconeogenesis" and "citrate cycle (TCA)". qPCR showed that relative levels of forkhead box O3 (FoxO3), phosphoenolpyruvate carboxykinase 2 (Pck2), and protein tyrosine phosphatase 1B (Ptp1b) were significantly decreased in the high-dose TMXK group, while v-akt murine thymoma viral oncogene homolog 1 (Akt1) and insulin receptor substrate 2 (Irs2) were increased. CONCLUSION: Our data show that TMXK Tablet reduces fasting glucose level and improves insulin resistance in diabetic rats. The mechanism may be linked to the inactivation of PTP1B and PCK enzymes, or through intracellular pathways, such as the insulin signaling pathway.
Assuntos
Glicemia/análise , Cromo/administração & dosagem , Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina/fisiologia , Medicina Tradicional Chinesa , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Resistência à Insulina , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , ComprimidosRESUMO
The effect of univalent cations on activity of key enzymes of C2-metabolism has been investigated in the producer of biosurfactants, Acinetibacter calcoaceticus IMV B-7241 grown on ethanol. It was established that potassium cations are inhibitors of pyroquinolinequinone-dependent alcohol- and acetaldehyde dehydrogenases, the enzymes of biosynthesis of surface-active aminolipids (NADP-dependent glutamate dehydrogenase) and glycolipids (phosphoenopyruvate (PhEP)-carboxikinase), while ammonium cations are activators of these enzymes and PhEP-carboxylase. A decrease of potassium cations concentration in the cultivation medium to 1 mM and increase of the content of amine nitrogen to 10 mM as a result of potassium nitrate substitution by equimolar, as to nitrogen, urea concentration were accompanied by the increase of activity of enzymes of ethanol metabolism and SAS biosynthesis, as well as by the 2-fold increase of conditional concentration of the biosurfactants.
Assuntos
Acinetobacter calcoaceticus/efeitos dos fármacos , Acinetobacter calcoaceticus/enzimologia , Amônia/farmacologia , Potássio/farmacologia , Tensoativos/metabolismo , Acinetobacter calcoaceticus/crescimento & desenvolvimento , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Amônia/metabolismo , Cátions Monovalentes , Meios de Cultura , Ativação Enzimática/efeitos dos fármacos , Etanol/metabolismo , Desidrogenase de Glutamato (NADP+)/antagonistas & inibidores , Desidrogenase de Glutamato (NADP+)/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxilase/antagonistas & inibidores , Fosfoenolpiruvato Carboxilase/metabolismo , Potássio/metabolismo , Especificidade por Substrato , Ureia/metabolismoRESUMO
The interactions between oxaloacetic (OAA) and phosphoenolpyruvic carboxykinase (PEPCK) binding pocket in the presence and absence of hydrazine were carried out using quantum chemical calculations, based on the two-layered ONIOM (ONIOM2) approach. The complexes were partially optimized by ONIOM2 (B3LYP/6-31G(d):PM6) method while the interaction energies between OAA and individual residues surrounding the pocket were performed at the MP2/6-31G(d,p) level of theory. The calculated interaction energies (INT) indicated that Arg87, Gly237, Ser286, and Arg405 are key residues for binding to OAA with the INT values of -1.93, -2.06, -2.47, and -3.16 kcal mol(-1), respectively. The interactions are mainly due to the formation of hydrogen bonding interactions with OAA. Moreover, using ONIOM2 (B3LYP/6-31G(d):PM6) applied on the PEPCKHS complex, two proton transfers were observed; first, the proton was transferred from the carboxylic group of OAA to hydrazine while the second one was from Asp311 to Lys244. Such reactions cause the generation of binding strength of OAA to the pocket via electrostatic interaction. The orientations of Lys243, Lys244, His264, Asp311, Phe333, and Arg405 were greatly deviated after hydrazine incorporation. These indicate that hydrazine plays an important role in terms of not only changing the conformation of the binding pocket, but is also tightly bound to OAA resulting in its conformation change in the pocket. The understanding of such interaction can be useful for the design of hydrazine-based inhibitor for antichachexia agents.
Assuntos
Inibidores Enzimáticos/química , Hidrazinas/química , Ácido Oxaloacético/química , Fosfoenolpiruvato Carboxiquinase (ATP)/química , Prótons , Motivos de Aminoácidos , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Ligação Proteica , Estrutura Terciária de Proteína , Teoria Quântica , Eletricidade Estática , TermodinâmicaRESUMO
Adiponectin and its receptors are inversely related to the degree of obesity and have been identified as potential therapeutic targets for the treatment of obesity. In this study, we evaluated the effect of hydrodynamic delivery of adiponectin and/or its receptor 2 (adipoR2) genes on controlling the development of obesity and insulin resistance in AKR/J mice fed a high-fat diet. An increase in adiponectin and adipoR2 gene expression by hydrodynamic gene delivery prevented diet-induced weight gain, reduced fat accumulation in liver and adipose tissue, and improved insulin sensitivity. Beneficial effects were seen with reduced gluconeogenesis in the liver and lipogenesis in the liver, white adipose tissue and skeletal muscle. Real-time PCR analysis demonstrated overexpression of adiponectin and adipoR2 significantly suppressed transcription of phosphoenolpyruvate carboxykinase (pepck), glucose-6-phosphatase (g6pase), stearoyl CoA desaturase 1 (scd-1) and fatty acid synthase (fas) gene. Inhibition effects were mediated by activating the AMP-activated protein kinase (AMPK). These results prove that elevation of adiponectin and/or adipoR2 expression via gene transfer is an effective approach in managing obesity epidemics.
Assuntos
Adiponectina/genética , Terapia Genética , Obesidade/genética , Obesidade/terapia , Receptores de Adiponectina/genética , Adiponectina/administração & dosagem , Animais , Dieta Hiperlipídica , Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Transferência de Genes , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/metabolismo , Humanos , Hidrodinâmica , Insulina/genética , Insulina/metabolismo , Resistência à Insulina/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Camundongos , Obesidade/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Receptores de Adiponectina/administração & dosagem , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/metabolismoRESUMO
Sodium arsenite has been demonstrated to alter the expression of genes associated with glucose homeostasis in tissues involved in the pathogenesis of type 2 diabetes; however, the underlying molecular mechanism has not been fully elucidated yet. In this study, we report that the sodium arsenite-induced gene expression of the small heterodimer partner (SHP; NR0B2), an atypical orphan nuclear receptor, regulates the expression of hepatic gluconeogenic genes. Sodium arsenite augments hepatic SHP mRNA levels in an AMP-activated protein kinase (AMPK)-dependent manner. Sodium arsenite activated AMPK and was shown to perturb cellular ATP levels. The arsenite-induced SHP mRNA level was blocked by adenoviral overexpression of dominant negative AMPK (Ad-dnAMPKalpha) or by the AMPK inhibitor compound C in hepatic cell lines. We demonstrated the dose-dependent induction of SHP mRNA levels by sodium arsenite and repressed the forskolin/dexamethasone-induced gene expression of the key hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Ad-dnAMPKalpha blocked the repressive effects of arsenite-induced SHP on PEPCK and G6Pase. Sodium arsenite inhibited the promoter activity of PEPCK and G6Pase, and this repression was abolished by small interfering (si)RNA SHP treatments. The knockdown of SHP expression by oligonucleotide siRNA SHP or adenoviral siRNA SHP released the sodium arsenite-mediated repression of forskolin/dexamethasone-stimulated PEPCK and G6Pase gene expression in a variety of hepatic cell lines. Results from our study suggest that sodium arsenite induces SHP via AMPK to inhibit the expression of hepatic gluconeogenic genes and also provide us with a novel molecular mechanism of arsenite-mediated regulation of hepatic glucose homeostasis.
Assuntos
Arsenitos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Compostos de Sódio/farmacologia , Proteínas Quinases Ativadas por AMP , Trifosfato de Adenosina/metabolismo , Animais , Northern Blotting , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Gluconeogênese/efeitos dos fármacos , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/biossíntese , Glucose-6-Fosfatase/genética , Humanos , Fígado/enzimologia , Masculino , Complexos Multienzimáticos/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
During the evolution of angiosperms, C4 phosphoenolpyruvate carboxylases have evolved several times independently from ancestral non-photosynthetic isoforms. They show distinct kinetic and regulatory properties when compared with the C3 isozymes. To identify the evolutionary alterations which are responsible for C4-specific properties, particularly the increased tolerance towards the allosteric inhibitor L-malate, the photosynthetic phosphoenolpyruvate carboxylase of Flaveria trinervia Mohr C4 and its ortholog from the closely related C3 plant Flaveria pringlei Gand. were examined using reciprocal enzyme chimeras. The main determinants for a high tolerance towards L-malate were located in the C-terminal region of the C4 enzyme. The effect of interchanging the region between amino acids 296 and 437 was strongly dependent upon the activation of the enzyme by glucose-6-phosphate. This confirms earlier observations that this region is important for the regulation of the enzyme by glucose-6-phosphate and that it harbours determinants for the different response of the C3 and the C4 enzyme towards this allosteric activator. In addition, it was possible to demonstrate that the only C4-specific amino acid, a serine in the C-terminal part of the enzyme, is not involved in conferring an increased L-malate tolerance to the C4 enzyme.
Assuntos
Evolução Molecular , Flaveria/enzimologia , Flaveria/genética , Malatos/farmacologia , Fosfoenolpiruvato Carboxiquinase (ATP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Sequência de Aminoácidos , Inibidores Enzimáticos , Regulação da Expressão Gênica de Plantas/fisiologia , Glucose-6-Fosfato , Dados de Sequência Molecular , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
This study focuses on the alterations suffered by the serotoninergic and kinurenergic routes of tryptophan (TRP) metabolism in liver, and their relation with gluconeogenic phosphoenolpyruvate-carboxykinase (PEPCK) blockage in experimental acute porphyria. This porphyria was induced in rats by a combined treatment of 2-allyl-2-isopropylacetamide (100, 250, 500 mg/kg bw) and 3,5-dietoxicarbonil 1,4-dihydrocollidine (constant 50 mg/kg bw dose). Results showed a marked dose-dependent increase of all TRP pyrrolase (TRPp) forms, active (holo, total) and inactive (apo), and a decrease in the degree of enzyme saturation by heme. Increases for holo, total, and apo-TRPp were 90, 150, and 230%, respectively, at the highest dose assayed (H). The treatment also impaired the serotoninergic route of TRP metabolism in liver, causing a decrease in serotonin level (H, 38%), and a concomitant enhancement in TRP content (H, 23%). The porphyrinogenic treatment promoted a blockage in PEPCK activity (H, 30%). This occurred in correlation to the development of porphyria, to TRPp alterations and to the production of hepatic microsomal thiobarbituric acid reactive substances. Porphyria was estimated through increases in 5-aminolevulinic acid-synthase (ALA-S) activity, ALA and porphobilinogen contents, and a decrease in ferrochelatase activity. Thus, the TRP kynurenine route was augmented whereas the serotoninergic route was reduced. PEPCK blockage could be partly attributed to quinolinate generated from TRP by the increase of TRPp activity, which would be due to the effect of porphyrinogenic drugs on TRP. The contribution of ROS to PEPCK blockage is analyzed. Likewise, the implication of these results in the control of porphyrias by glucose is discussed.