RESUMO
Glucokinase activators (GKAs) have been developed as blood glucose lowering drugs for type 2 diabetes. Despite good short-term efficacy, several GKAs showed a decline in efficacy chronically during clinical trials. The underlying mechanisms remain incompletely understood. We tested the hypothesis that deficiency in the liver glucokinase regulatory protein (GKRP) as occurs with common human GCKR variants affects chronic GKA efficacy. We used a Gckr-P446L mouse model for the GCKR exonic rs1260326 (P446L) variant and the Gckr-del/wt mouse to model transcriptional deficiency to test for chronic efficacy of the GKA, AZD1656 in GKRP-deficient states. In the Gckr-P446L mouse, the blood glucose lowering efficacy of AZD1656 (3â¯mg/kg body wt) after 2â¯weeks was independent of genotype. However after 19â¯weeks, efficacy was maintained in wild-type but declined in the LL genotype, in conjunction with raised hepatic glucokinase activity and without raised liver lipids. Sustained blood glucose lowering efficacy in wild-type mice was associated with qualitatively similar but more modest changes in the liver transcriptome compared with the P446L genotype, consistent with GKA therapy representing a more modest glucokinase excess than the P446L genotype. Chronic treatment with AZD1656 in the Gckr-del/wt mouse was associated with raised liver triglyceride and hepatocyte microvesicular steatosis. The results show that in mouse models of liver GKRP deficiency in conjunction with functional liver glucokinase excess as occurs in association with common human GCKR variants, GKRP-deficiency predisposes to declining efficacy of the GKA in lowering blood glucose and to GKA induced elevation in liver lipids.
RESUMO
OBJECTIVE: Stable isotope studies have shown that hepatic de novo lipogenesis (DNL) plays an important role in the pathogenesis of intrahepatic lipid (IHL) deposition. Furthermore, previous research has demonstrated that fructose 1-phosphate (F1P) not only serves as a substrate for DNL, but also acts as a signalling metabolite that stimulates DNL from glucose. The aim of this study was to elucidate the mediators of F1P-stimulated DNL, with special focus on two key regulators of intrahepatic glucose metabolism, i.e., glucokinase regulatory protein (GKRP) and carbohydrate response element binding protein (ChREBP). METHODS: Aldolase B deficient mice (Aldob-/-), characterized by hepatocellular F1P accumulation, enhanced DNL, and hepatic steatosis, were either crossed with GKRP deficient mice (Gckr-/-) or treated with short hairpin RNAs directed against hepatic ChREBP. RESULTS: Aldob-/- mice showed higher rates of de novo palmitate synthesis from glucose when compared to wildtype mice (p < 0.001). Gckr knockout reduced de novo palmitate synthesis in Aldob-/- mice (p = 0.017), without affecting the hepatic mRNA expression of enzymes involved in DNL. In contrast, hepatic ChREBP knockdown normalized the hepatic mRNA expression levels of enzymes involved in DNL and reduced fractional DNL in Aldob-/- mice (p < 0.05). Of interest, despite downregulation of DNL in response to Gckr and ChREBP attenuation, no reduction in intrahepatic triglyceride levels was observed. CONCLUSIONS: Both GKRP and ChREBP mediate F1P-stimulated DNL in aldolase B deficient mice. Further studies are needed to unravel the role of GKRP and hepatic ChREBP in regulating IHL accumulation in aldolase B deficiency.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Frutose-Bifosfato Aldolase , Lipogênese , Fígado , Camundongos Knockout , Triglicerídeos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Camundongos , Fígado/metabolismo , Triglicerídeos/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Frutose-Bifosfato Aldolase/genética , Masculino , Camundongos Endogâmicos C57BL , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Glucose/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de TransporteRESUMO
OBJECTIVES: The Glucokinase Regulatory Protein GKRP, encoded by GCKR, enables acute regulation of liver glucokinase to support metabolic demand. The common human GCKR rs1260326:Pro446 > Leu variant within a large linkage disequilibrium region associates with pleiotropic traits including lower Type 2 diabetes risk and raised blood triglycerides and cholesterol. Whether the GCKR-P446 > L substitution is causal to the raised lipids is unknown. We determined whether mouse GKRP phenocopies the human GKRP:P446 > L substitution and studied a GKRP:P446L knockin mouse to identify physiological consequences to P446 > L. METHODS: GKRP-deficient hepatocytes were transfected with adenoviral vectors for human or mouse GKRP:446 P or 446 L for cellular comprehensive analysis including transcriptomics consequent to P446 > L. Physiological traits in the diet-challenged P446L mouse were compared with pleiotropic associations at the human rs1260326 locus. Transcriptomics was compared in P446L mouse liver with hepatocytes overexpressing glucokinase or GKRP:446 P/L. RESULTS: 1. P446 > L substitution in mouse or human GKRP similarly compromises protein expressivity of GKRP:446 L, nuclear sequestration of glucokinase and counter-regulation of gene expression. 2. The P446L knockin mouse has lower liver glucokinase and GKRP protein similar to human liver homozygous for rs1260326-446 L. 3. The diet-challenged P446L mouse has lower blood glucose, raised blood cholesterol and altered hepatic cholesterol homeostasis consistent with relative glucokinase-to-GKRP excess, but not raised blood triglycerides. CONCLUSIONS: Mouse GKRP phenocopies the human GKRP:P446 > L substitution despite the higher affinity for glucokinase of human GKRP. The diet-challenged P446L mouse replicates several traits found in association with the rs1260326 locus on chromosome 2 including raised blood cholesterol, lower blood glucose and lower liver glucokinase and GKRP protein but not raised blood triglycerides.
Assuntos
Glicemia , Diabetes Mellitus Tipo 2 , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinase/metabolismo , Fígado/metabolismo , Triglicerídeos/metabolismoRESUMO
The Carbohydrate response element binding protein, ChREBP encoded by the MLXIPL gene, is a transcription factor that is expressed at high levels in the liver and has a prominent function during consumption of high-carbohydrate diets. ChREBP is activated by raised cellular levels of phosphate ester intermediates of glycolysis, gluconeogenesis and the pentose phosphate pathway. Its target genes include a wide range of enzymes and regulatory proteins, including G6pc, Gckr, Pklr, Prkaa1,2, and enzymes of lipogenesis. ChREBP activation cumulatively promotes increased disposal of phosphate ester intermediates to glucose, via glucose 6-phosphatase or to pyruvate via glycolysis with further metabolism by lipogenesis. Dietary fructose is metabolized in both the intestine and the liver and is more lipogenic than glucose. It also induces greater elevation in phosphate ester intermediates than glucose, and at high concentrations causes transient depletion of inorganic phosphate, compromised ATP homeostasis and degradation of adenine nucleotides to uric acid. ChREBP deficiency predisposes to fructose intolerance and compromised cellular phosphate ester and ATP homeostasis and thereby markedly aggravates the changes in metabolite levels caused by dietary fructose. The recent evidence that high fructose intake causes more severe hepatocyte damage in ChREBP-deficient models confirms the crucial protective role for ChREBP in maintaining intracellular phosphate homeostasis. The improved ATP homeostasis in hepatocytes isolated from mice after chronic activation of ChREBP with a glucokinase activator supports the role of ChREBP in the control of intracellular homeostasis. It is hypothesized that drugs that activate ChREBP confer a protective role in the liver particularly in compromised metabolic states.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Fígado/metabolismo , Doenças Metabólicas/prevenção & controle , Substâncias Protetoras/metabolismo , Elementos de Resposta , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Gluconeogênese , Glicólise , Humanos , Lipogênese , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologiaRESUMO
The ionic liquid 1-octyl-3-methylimidazolium (M8OI) has been found in the environment and identified as a hazard for triggering the liver disease primary biliary cholangitis (PBC). Given limited toxicity data for M8OI and other structurally-related ionic liquids, target organs for M8OI toxicity were examined. Adult male C57Bl6 mice were acutely exposed to 0-10 mg/kg body weight M8OI via 2 intraperitoneal injections (time zero and 18 h) and effects examined at 24 h. At termination, tissue histopathology, serum and urinary endpoints were examined. No overt pathological changes were observed in the heart and brain. In contrast, focal and mild to multifocal and moderate degeneration with a general trend for an increase in severity with increased dose was observed in the kidney. These changes were accompanied by a dose-dependent increased expression of Kim1 in kidney tissue, marked elevations in urinary Kim1 protein and a dose-dependent increase in serum creatinine. Hepatic changes were limited to a significant dose-dependent loss of hepatic glycogen and a mild but significant increase in portal tract inflammatory recruitment and/or fibroblastic proliferation accompanied by a focal fibrotic change. Cultured mouse tissue slices reflected these in vivo effects in that dose-dependent injury was observed in kidney slices but not in the liver. Kidney slices accumulated higher levels of M8OI than liver slices (e.g. at 10 µM, greater than 4 fold) and liver slices where markedly more active in the metabolism of M8OI. These data indicate that the kidney is a target organ for the toxic effects of M8OI accompanied by mild cholangiopathic changes in the liver after intraperitoneal administration.
Assuntos
Substâncias Perigosas/toxicidade , Líquidos Iônicos/toxicidade , Rim/efeitos dos fármacos , Testes de Toxicidade , Animais , Íons/farmacologia , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
AIM: To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis. METHODS: C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge. RESULTS: Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-ß), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-ß mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2. CONCLUSIONS: Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-ß and both catalytic subunits of AMP-activated protein kinase.
Assuntos
Glucoquinase , Fígado , Trifosfato de Adenosina , Animais , Proteínas de Transporte/genética , Glucoquinase/genética , Glucoquinase/metabolismo , Glucose , Glucose-6-Fosfato , Hepatócitos/metabolismo , Homeostase , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Elementos de RespostaRESUMO
Metformin therapy lowers blood glucose in type 2 diabetes by targeting various pathways including hepatic gluconeogenesis. Despite widespread clinical use of metformin the molecular mechanisms by which it inhibits gluconeogenesis either acutely through allosteric and covalent mechanisms or chronically through changes in gene expression remain debated. Proposed mechanisms include: inhibition of Complex 1; activation of AMPK; and mechanisms independent of both Complex 1 inhibition and AMPK. The activation of AMPK by metformin could be consequent to Complex 1 inhibition and raised AMP through the canonical adenine nucleotide pathway or alternatively by activation of the lysosomal AMPK pool by other mechanisms involving the aldolase substrate fructose 1,6-bisphosphate or perturbations in the lysosomal membrane. Here we review current interpretations of the effects of metformin on hepatic intermediates of the gluconeogenic and glycolytic pathway and the candidate mechanistic links to regulation of gluconeogenesis. In conditions of either glucose excess or gluconeogenic substrate excess, metformin lowers hexose monophosphates by mechanisms that are independent of AMPK-activation and most likely mediated by allosteric activation of phosphofructokinase-1 and/or inhibition of fructose bisphosphatase-1. The metabolite changes caused by metformin may also have a prominent role in counteracting G6pc gene regulation in conditions of compromised intracellular homeostasis.
Assuntos
Gluconeogênese , Hipoglicemiantes/farmacologia , Fígado/metabolismo , Metformina/farmacologia , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Glicólise , Humanos , Fígado/efeitos dos fármacosRESUMO
The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.
Assuntos
Glucose-6-Fosfato/metabolismo , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Trifosfato de Adenosina/metabolismo , Animais , Di-Hidroxiacetona/farmacologia , Gluconeogênese/efeitos dos fármacos , Glucose/farmacologia , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Masculino , Metformina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfofrutoquinase-1/antagonistas & inibidores , Fosfofrutoquinase-1/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Rotenona/farmacologiaRESUMO
Several C-ß-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(ß-d-glucopyranosyl)-imidazoles and 2-aryl-4-(ß-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 µM; 13b = 4.58 µM). Ten C-ß-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a-c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30-60 µM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form.
Assuntos
Azóis/farmacologia , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicogenólise/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Animais , Azóis/química , Células CACO-2 , Desenho de Fármacos , Inibidores Enzimáticos/química , Glicogênio Fosforilase/metabolismo , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Coelhos , Relação Estrutura-AtividadeRESUMO
The mechanisms by which metformin (dimethylbiguanide) inhibits hepatic gluconeogenesis at concentrations relevant for type 2 diabetes therapy remain debated. Two proposed mechanisms are 1) inhibition of mitochondrial Complex 1 with consequent compromised ATP and AMP homeostasis or 2) inhibition of mitochondrial glycerophosphate dehydrogenase (mGPDH) and thereby attenuated transfer of reducing equivalents from the cytoplasm to mitochondria, resulting in a raised lactate/pyruvate ratio and redox-dependent inhibition of gluconeogenesis from reduced but not oxidized substrates. Here, we show that metformin has a biphasic effect on the mitochondrial NADH/NAD redox state in mouse hepatocytes. A low cell dose of metformin (therapeutic equivalent: <2 nmol/mg) caused a more oxidized mitochondrial NADH/NAD state and an increase in lactate/pyruvate ratio, whereas a higher metformin dose (≥5 nmol/mg) caused a more reduced mitochondrial NADH/NAD state similar to Complex 1 inhibition by rotenone. The low metformin dose inhibited gluconeogenesis from both oxidized (dihydroxyacetone) and reduced (xylitol) substrates by preferential partitioning of substrate toward glycolysis by a redox-independent mechanism that is best explained by allosteric regulation at phosphofructokinase-1 (PFK1) and/or fructose 1,6-bisphosphatase (FBP1) in association with a decrease in cell glycerol 3-phosphate, an inhibitor of PFK1, rather than by inhibition of transfer of reducing equivalents. We conclude that at a low pharmacological load, the metformin effects on the lactate/pyruvate ratio and glucose production are explained by attenuation of transmitochondrial electrogenic transport mechanisms with consequent compromised malate-aspartate shuttle and changes in allosteric effectors of PFK1 and FBP1.
Assuntos
Gluconeogênese/efeitos dos fármacos , Hepatócitos/metabolismo , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Mitocôndrias Hepáticas/metabolismo , NAD/metabolismo , Animais , Ácido Aspártico/metabolismo , Células Cultivadas , Frutose-Bifosfatase/metabolismo , Glucose/metabolismo , Glicólise , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Ácido Láctico/metabolismo , Malatos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Oxirredução , Fosfofrutoquinase-1/metabolismo , Ratos , Ratos WistarRESUMO
Recent environmental sampling around a landfill site in the UK demonstrated that unidentified xenoestrogens were present at higher levels than control sites; that these xenoestrogens were capable of super-activating (resisting ligand-dependent antagonism) the murine variant 2â¯ERß and that the ionic liquid 1-octyl-3-methylimidazolium chloride (M8OI) was present in some samples. To determine whether M8OI was a contributor to the xenoestrogen pool in the soils, activation of human estrogen receptors by M8OI was examined. M8OI activated the human ERα in MCF7 cells in a dose-response manner. These effects were inhibited by the ER antagonist ICI182780; occurred in the absence of any metabolism of M8OI and were confirmed on examination of ER-dependent induction of trefoil factor 1 mRNA in MCF7 cells. M8OI also super-activated the murine variant 2â¯ERß in a murine hepatopancreatobiliary cell line. The human ERß was not activated by M8OI when expressed in HEK293â¯cells. These data demonstrate that M8OI is a xenoestrogen capable of activating the human ERα and super-activating the murine variant 2â¯ERß.
Assuntos
Receptor alfa de Estrogênio/agonistas , Imidazóis/farmacologia , Líquidos Iônicos/farmacologia , Animais , Células Cultivadas , Receptor alfa de Estrogênio/metabolismo , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Imidazóis/química , Imidazóis/metabolismo , Líquidos Iônicos/química , Líquidos Iônicos/metabolismo , Células MCF-7 , Camundongos , Estrutura MolecularRESUMO
BACKGROUND: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method. RESULTS: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors. CONCLUSION: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.
Assuntos
DNA de Cadeia Simples , Edição de Genes/métodos , Marcação de Genes , Camundongos/genética , Alelos , Animais , Sistemas CRISPR-Cas , Mutação , Reprodutibilidade dos TestesRESUMO
AIM: Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti-hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials. MATERIALS AND METHODS: We used rat hepatocytes to test the hypothesis that GKAs raise hepatocyte glucose 6-phosphate (G6P, the glucokinase product) and down-stream metabolites with consequent repression of the liver glucokinase gene ( Gck). We compared a GKA with metformin, the most widely prescribed drug for T2D. RESULTS: Treatment of hepatocytes with 25 mM glucose raised cell G6P, concomitantly with Gck repression and induction of G6pc (glucose 6-phosphatase) and Pklr (pyruvate kinase). A GKA mimicked high glucose by raising G6P and fructose-2,6-bisphosphate, a regulatory metabolite, causing a left-shift in glucose responsiveness on gene regulation. Fructose, like the GKA, repressed Gck but modestly induced G6pc. 2-Deoxyglucose, which is phosphorylated by glucokinase but not further metabolized caused Gck repression but not G6pc induction, implicating the glucokinase product in Gck repression. Metformin counteracted the effect of high glucose on the elevated G6P and fructose 2,6-bisphosphate and on Gck repression, recruitment of Mlx-ChREBP to the G6pc and Pklr promoters and induction of these genes. CONCLUSIONS: Elevation in hepatocyte G6P and downstream metabolites, with consequent liver Gck repression, is a potential contributing mechanism to the loss of GKA efficacy during chronic therapy. Cell metformin loads within the therapeutic range attenuate the effect of high glucose on G6P and on glucose-regulated gene expression.
Assuntos
Ativadores de Enzimas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucoquinase/metabolismo , Hepatócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Tiazóis/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Cultivadas , Dieta Ocidental/efeitos adversos , Frutose/administração & dosagem , Frutose/efeitos adversos , Frutosedifosfatos/metabolismo , Glucoquinase/antagonistas & inibidores , Glucoquinase/química , Glucoquinase/genética , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Glucose-6-Fosfato/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Masculino , Camundongos Endogâmicos C3H , Sobrepeso/enzimologia , Sobrepeso/metabolismo , Sobrepeso/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/química , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Ratos WistarRESUMO
Liver glucose metabolism is dependent on glucokinase activity. Glucokinase expression is transcriptionally regulated by hormones and metabolites of glucose, and glucokinase activity is dependent on reversible binding of glucokinase to a specific inhibitor protein, glucokinase regulatory protein (GKRP), and to other binding proteins such as 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK2/FBP2), which functions as an activator. Glucokinase is inhibited in the postabsorptive state by sequestration in the nucleus bound to GKRP, and it is activated postprandially by portal hyperglycemia and fructose through dissociation from GKRP, translocation to the cytoplasm, and binding to PFK2/FBP2. Glucagon dissociates this interaction, promoting translocation back to the nucleus. In humans, changes in glucokinase expression and activity are associated with poorly controlled type 2 diabetes and with nonalcoholic fatty liver disease, and a common variant of GKRP with altered binding affinity for glucokinase is associated with increased blood and liver lipids and other metabolic traits that implicate a role for GKRP in maintaining intrahepatic metabolite homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucagon/metabolismo , Glucoquinase/metabolismo , Fígado/enzimologia , Modelos Biológicos , Fosfofrutoquinase-2/metabolismo , Animais , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinase/antagonistas & inibidores , Glucoquinase/química , Glucoquinase/genética , Homeostase , Humanos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Período Pós-Prandial , Transporte ProteicoRESUMO
Type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) are associated with elevated hepatic glucose production and fatty acid synthesis (de novo lipogenesis (DNL)). High carbohydrate diets also increase hepatic glucose production and lipogenesis. The carbohydrate-response element-binding protein (ChREBP, encoded by MLXIPL) is a transcription factor with a major role in the hepatic response to excess dietary carbohydrate. Because its target genes include pyruvate kinase (PKLR) and enzymes of lipogenesis, it is regarded as a key regulator for conversion of dietary carbohydrate to lipid for energy storage. An alternative hypothesis for ChREBP function is to maintain hepatic ATP homeostasis by restraining the elevation of phosphate ester intermediates in response to elevated glucose. This is supported by the following evidence: (i) A key stimulus for ChREBP activation and induction of its target genes is elevation of phosphate esters; (ii) target genes of ChREBP include key negative regulators of the hexose phosphate ester pool (GCKR, G6PC, SLC37A4) and triose phosphate pool (PKLR); (iii) ChREBP knock-down models have elevated hepatic hexose phosphates and triose phosphates and compromised ATP phosphorylation potential; (iv) gene defects in G6PC and SLC37A4 and common variants of MLXIPL, GCKR and PKLR in man are associated with elevated hepatic uric acid production (a marker of ATP depletion) or raised plasma uric acid levels. It is proposed that compromised hepatic phosphate homeostasis is a contributing factor to the elevated hepatic glucose production and lipogenesis that associate with type 2 diabetes, NAFLD and excess carbohydrate in the diet.
RESUMO
Liver glycogen is synthesized after a meal in response to an increase in blood glucose concentration in the portal vein and endocrine and neuroendocrine signals, and is degraded to glucose between meals to maintain blood glucose homeostasis. Glycogen degradation and synthesis during the diurnal cycle are mediated by changes in the activities of phosphorylase and glycogen synthase. Phosphorylase is regulated by phosphorylation of serine-14. Only the phosphorylated form of liver phosphorylase (GPa) is catalytically active. Interconversion between GPa and GPb (unphosphorylated) is dependent on the activities of phosphorylase kinase and of phosphorylase phosphatase. The latter comprises protein phosphatase-1 in conjunction with a glycogen-targeting protein (G-subunit) of the PPP1R3 family. At least two of six G-subunits (GL and PTG) expressed in liver are involved in GPa dephosphorylation. GPa to GPb interconversion is dependent on the conformational state of phosphorylase which can be relaxed (R) or tense (T) depending on the concentrations of allosteric effectors such as glucose, glucose 6-phosphate and adenine nucleotides and on the acetylation state of lysine residues. The G-subunit, GL, encoded by PPP1R3B gene is expressed at high levels in liver and can function as a phosphorylase phosphatase and a synthase phosphatase and has an allosteric binding site for GPa at the C-terminus which inhibits synthase phosphatase activity. GPa to GPb conversion is a major upstream event in the regulation of glycogen synthesis by glucose, its downstream metabolites and extracellular signals such as insulin and neurotransmitters.
Assuntos
Glicogênio Fosforilase/metabolismo , Glicogênio Hepático/metabolismo , Fígado/metabolismo , Glucose/metabolismo , Humanos , Fosforilação/fisiologiaRESUMO
The concept of activation of glucokinase (encoded by the Gck gene) as a potential therapy for type 2 diabetes has been explored by several pharmaceutical companies. Small-molecule Gck activators (GKAs) were found to be effective at increasing glucose disposal by hepatocytes and lowering blood glucose in animal models of diabetes during acute or chronic exposure and in human type 2 diabetes during short-term exposure. However, several clinical trials of GKAs were discontinued because of declining efficacy during chronic exposure or other issues. In some cases, declining efficacy was associated with an increase in plasma triglycerides. Accordingly, increased hepatic triglyceride production or steatosis was inferred as the likely cause for declining efficacy. However, other mechanisms of tachyphylaxis need to be considered. For example, elevated glucose concentration causes induction of glucose 6-phosphatase (G6pc) and repression of Gck in hepatocytes. This is best explained as an adaptative mechanism to maintain intracellular phosphometabolite homeostasis. Enhancement of G6pc induction and Gck repression by GKAs because of perturbed phosphometabolite homeostasis could explain the decline in GKA efficacy during chronic exposure. Progress in understanding the mechanisms of intracellular phosphometabolite homeostasis is crucial for development of better drug therapies and appropriate dietary intervention for type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativadores de Enzimas/farmacologia , Glucoquinase/efeitos dos fármacos , Glucoquinase/metabolismo , Hipoglicemiantes/farmacologia , Animais , Ativadores de Enzimas/uso terapêutico , Humanos , Hipoglicemiantes/uso terapêutico , Patentes como AssuntoRESUMO
Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 N-acyl-ß-d-glucopyranosylamines putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a 'consensus scoring' approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants' values, in vitro, ranged from 5 to 377µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Glucosamina/análogos & derivados , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosamina/síntese química , Glucosamina/química , Glucosamina/farmacologia , Glicogênio Fosforilase Hepática/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Glucagon/farmacologia , Glucoquinase/metabolismo , Hepatócitos/metabolismo , Fosfofrutoquinase-2/metabolismo , Transporte Proteico/efeitos dos fármacos , Animais , Glicemia/metabolismo , Western Blotting , Proteínas de Transporte/genética , Células Cultivadas , Imunofluorescência , Fármacos Gastrointestinais/farmacologia , Glucoquinase/genética , Glicólise/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fosfofrutoquinase-2/genética , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AR42J-B-13 (B-13) cells form hepatocyte-like (B-13/H) cells in response to glucocorticoid treatment. To establish its utility in toxicity and genotoxicity screening, cytochrome P450 (CYP) induction, susceptibility to toxins, and transporter gene expression were examined. Conversion to B-13/H cells resulted in expression of male-specific CYP2C11 and sensitivity to methapyrilene. B-13/H cells constitutively expressed CYP1A, induced expression in response to an aryl hydrocarbon receptor agonist, and activated benzo[α]pyrene to a DNA-damaging species. Functional CYP1A2 was not expressed due to deletions in the Cyp1a2 gene. A B-13 cell line stably expressing the human CYP1A2 was therefore engineered (B-13(-TR/h1A2)) and the derived B-13/H cells expressed metabolically functional CYP1A2. Treatment with the cooked food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine resulted in a dose-dependent increase in DNA damage. B-13/H cells expressed constitutive androstane receptor (CAR) and induced CYP2B1 mRNA levels in response to classical CAR activators. However, translation to functional CYP2B1 protein was low and increased minimally by CAR activator treatment. B-13/H cells expressed high levels of pregnane X-receptor (PXR) and induced CYP3A1 in response to classical PXR activators. CYP3A genes were inducible, functional, and activated aflatoxin B1 to a DNA-damaging species. All 23 major hepatic transporters were induced when B-13 cells were converted to B-13/H cells, although in many cases, levels remained below those present in adult rat liver. However, bile salt export pump, Abcb1b, multidrug resistance-associated protein, and breast cancer resistance protein transporters were functional in B-13/H cells. These data demonstrate that the B-13 cell generates hepatocyte-like cells with functional drug metabolism and transporter activities, which can alone--or in a humanized form--be used to screen for hepatotoxic and genotoxic endpoints in vitro.