RESUMEN

Intestinal barrier dysfunction usually occurred in acute pancreatitis (AP) but the mechanism remains unclear. In this study, RNA sequencing of ileum in L-arginine-induced AP mice demonstrated that phosphoenolpyruvate kinase 1 (Pck1) was significantly up-regulated. Increased Pck1 expression in intestinal epithelial cells (IECs) was further validated in ileum of AP mice and duodenum of AP patients. In AP mice, level of Pck1 was positively correlated with pancreatic and ileal histopathological scores, serum amylase activity, and intestinal permeability (serum diamine oxidase (DAO), D-lactate, and endotoxin). In AP patients, level of Pck1 had a positive correlation with Ranson scores, white blood cell count and C-reactive protein. Inhibition of Pck1 by 3-Mercaptopicolinic acid hydrochloride (3-MPA) alleviated pancreatic and ileal injuries in AP mice. AP + 3-MPA mice showed improved intestinal permeability, including less epithelial apoptosis, increased tight junction proteins (TJPs) expression, decreased serum DAO, D-lactate, endotoxin, and FITC-Dextran levels, and reduced bacteria translocation. Lysozyme secreted by Paneth cells and mucin2 (MUC2) secretion in goblet cells were also partly restored in AP + 3-MPA mice. Meanwhile, inhibition of Pck1 improved intestinal immune response during AP, including elevation of M2/M1 macrophages ratio and secretory immunoglobulin A (sIgA) and reduction in neutrophils infiltration. In vitro, administration of 3-MPA dramatically ameliorated inflammation and injuries of epithelial cells in enteroids treated by LPS. In conclusion, inhibition of Pck1 in IECs might alleviate AP via modulating intestinal homeostasis.

Asunto(s)

Células Epiteliales , Mucosa Intestinal , Pancreatitis , Fosfoenolpiruvato Carboxiquinasa (GTP) , Animales , Ratones , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Homeostasis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Ratones Endogámicos C57BL , Pancreatitis/metabolismo , Pancreatitis/patología , Pancreatitis/tratamiento farmacológico , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ácidos Picolínicos/farmacología

RESUMEN

Suppression of excessive hepatic gluconeogenesis is an effective strategy for controlling hyperglycemia in type 2 diabetes (T2D). In the present study, we screened our compounds library to discover the active molecules inhibiting gluconeogenesis in primary mouse hepatocytes. We found that SL010110 (5-((4-allyl-2-methoxyphenoxy) methyl) furan-2-carboxylic acid) potently inhibited gluconeogenesis with 3 µM and 10 µM leading to a reduction of 45.5% and 67.5%, respectively. Moreover, SL010110 caused suppression of gluconeogenesis resulted from downregulating the protein level of phosphoenolpyruvate carboxykinase 1 (PEPCK1), but not from affecting the gene expressions of PEPCK, glucose-6-phosphatase, and fructose-1,6-bisphosphatase. Furthermore, SL010110 increased PEPCK1 acetylation, and promoted PEPCK1 ubiquitination and degradation. SL010110 activated p300 acetyltransferase activity in primary mouse hepatocytes. The enhanced PEPCK1 acetylation and suppressed gluconeogenesis caused by SL010110 were blocked by C646, a histone acetyltransferase p300 inhibitor, suggested that SL010110 inhibited gluconeogenesis by activating p300. SL010110 decreased NAD+/NADH ratio, inhibited SIRT2 activity, and further promoted p300 acetyltransferase activation and PEPCK1 acetylation. These effects were blocked by NMN, an NAD+ precursor, suggested that SL010110 inhibited gluconeogenesis by inhibiting SIRT2, activating p300, and subsequently promoting PEPCK1 acetylation. In type 2 diabetic ob/ob mice, single oral dose of SL010110 (100 mg/kg) suppressed gluconeogenesis accompanied by the suppressed hepatic SIRT2 activity, increased p300 activity, enhanced PEPCK1 acetylation and degradation. Chronic oral administration of SL010110 (15 or 50 mg/kg) significantly reduced the blood glucose levels in ob/ob and db/db mice. This study reveals that SL010110 is a lead compound with a distinct mechanism of suppressing gluconeogenesis via SIRT2-p300-mediated PEPCK1 degradation and potent anti-hyperglycemic activity for the treatment of T2D.

Asunto(s)

Diabetes Mellitus Experimental/metabolismo , Gluconeogénesis/efectos de los fármacos , Glucosa/metabolismo , Hipoglucemiantes/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Sirtuina 2/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Gluconeogénesis/fisiología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Ratones Transgénicos , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Proteolisis/efectos de los fármacos , Sirtuina 2/antagonistas & inhibidores

RESUMEN

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.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Desarrollo de Músculos/efectos de los fármacos , Fosfoenolpiruvato Carboxiquinasa (ATP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Ácidos Picolínicos/farmacología , Animales , Biomarcadores , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Gluconeogénesis/genética , Isoenzimas , Ratones , Células Musculares , Regiones Promotoras Genéticas , ARN Mensajero/genética , Serina/biosíntesis

RESUMEN

Phosphoenolpyruvate carboxykinase (PEPCK) has traditionally been characterized for its role in the first committed step of gluconeogenesis. The current understanding of PEPCK's metabolic role has recently expanded to include it serving as a general mediator of tricarboxylic acid cycle flux. Selective inhibition of PEPCK in vivo and in vitro has been achieved with 3-mercaptopicolinic acid (MPA) (Ki ∼ 8 µM), whose mechanism of inhibition has been elucidated only recently. On the basis of crystallographic and mechanistic data of various inhibitors of PEPCK, MPA was used as the initial chemical scaffold to create a potentially more selective inhibitor, 3-[(carboxymethyl)thio]picolinic acid (CMP), which has been characterized both structurally and kinetically here. These data demonstrate that CMP acts as a competitive inhibitor at the OAA/PEP binding site, with its picolinic acid moiety coordinating directly with the M1 metal in the active site (Ki ∼ 29-55 µM). The extended carboxy tail occupies a secondary binding cleft that was previously shown could be occupied by sulfoacetate (Ki ∼ 82 µM) and for the first time demonstrates the simultaneous occupation of both OAA/PEP subsites by a single molecular structure. By occupying both the OAA/PEP binding subsites simultaneously, CMP and similar molecules can potentially be used as a starting point for the creation of additional selective inhibitors of PEPCK.

Asunto(s)

Inhibidores Enzimáticos/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/química , Ácidos Picolínicos/química , Animales , Cristalografía/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Ácidos Picolínicos/farmacología , Estructura Secundaria de Proteína , Ratas

RESUMEN

Dysregulated hepatic gluconeogenesis is a hallmark of insulin resistance and type 2 diabetes mellitus (T2DM). Although existing drugs have been proven to improve gluconeogenesis, achieving this objective with functional food is of interest, especially using conjugated linoleic acid (CLA) found in dairy products. Both cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) isomers of CLA were tested in human (HepG2) and rat (H4IIE) hepatocytes for their potential effects on gluconeogenesis. The hepatocytes exposed for 24 h with 20 µM of c9,t11-CLA had attenuated the gluconeogenesis in both HepG2 and H4IIE by 62.5% and 80.1%, respectively. In contrast, t10,c12-CLA had no effect. Of note, in HepG2 cells, the exposure of c9,t11-CLA decreased the transcription of gluconeogenic enzymes, cytosolic phosphoenolpyruvate carboxykinase (PCK1) by 87.7%, and glucose-6-phosphatase catalytic subunit (G6PC) by 38.0%, while t10,c12-CLA increased the expression of G6PC, suggesting the isomer-specific effects of CLA on hepatic glucose production. In HepG2, the peroxisome proliferator-activated receptor (PPAR) agonist, rosiglitazone, reduced the glucose production by 72.9%. However, co-administration of c9,t11-CLA and rosiglitazone neither exacerbated nor attenuated the efficacy of rosiglitazone to inhibit glucose production; meanwhile, t10,c12-CLA abrogated the efficacy of rosiglitazone. Paradoxically, PPARγ antagonist GW 9662 also led to 70.2% reduction of glucose production and near undetectable PCK1 expression by abrogating CLA actions. Together, while the precise mechanisms by which CLA isomers modulate hepatic gluconeogenesis directly or via PPAR warrant further investigation, our findings establish that c9,t11-CLA suppresses gluconeogenesis by decreasing PEPCK on hepatocytes.

Asunto(s)

Glucosa/biosíntesis , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Ácidos Linoleicos Conjugados/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Animales , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Células Hep G2 , Hepatocitos/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ácidos Linoleicos Conjugados/química , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estereoisomerismo , Relación Estructura-Actividad

RESUMEN

Gluconeogenesis is a major source of hyperglycemia in patients with type 2 diabetes mellitus (T2DM), thus targeting gluconeogenesis to suppress glucose production is a promising strategy for anti-T2DM drug discovery. In our preliminary in vitro studies, we found that a small-molecule (E)-3-(2-(quinoline-4-yl)vinyl)-1H-indol-6-ol (QVO) inhibited the hepatic glucose production (HGP) in primary hepatocytes. We further revealed that QVO suppressed hepatic gluconeogenesis involving calmodulin-dependent protein kinase kinase ß- and liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathways as well as AMPK-independent mitochondrial function-related signaling pathway. To evaluate QVO's anti-T2DM activity in vivo, which was impeded by the complicated synthesis route of QVO with a low yield, we designed and synthesized 4-[2-(1H-indol-3-yl)vinyl]quinoline (IVQ) as a prodrug with easier synthesis route and higher yield. IVQ did not inhibit the HGP in primary hepatocytes in vitro. Pharmacokinetic studies demonstrated that IVQ was quickly converted to QVO in mice and rats following administration. In both db/db and ob/ob mice, oral administration of IVQ hydrochloride (IVQ-HCl) (23 and 46 mg/kg every day, for 5 weeks) ameliorated hyperglycemia, and suppressed hepatic gluconeogenesis and activated AMPK signaling pathway in the liver tissues. Furthermore, IVQ caused neither cardiovascular system dysfunction nor genotoxicity. The good druggability of IVQ has highlighted its potential in the treatment of T2DM and the prodrug design for anti-T2DM drug development.

Asunto(s)

Diabetes Mellitus Tipo 2/tratamiento farmacológico , Gluconeogénesis/efectos de los fármacos , Hipoglucemiantes/uso terapéutico , Indoles/uso terapéutico , Profármacos/uso terapéutico , Quinolinas/uso terapéutico , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Activadores de Enzimas/uso terapéutico , Activadores de Enzimas/toxicidad , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/toxicidad , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Hepatocitos/efectos de los fármacos , Hipoglucemiantes/toxicidad , Indoles/toxicidad , Hígado/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Profármacos/toxicidad , Quinolinas/toxicidad , Transducción de Señal/efectos de los fármacos

RESUMEN

Heat stress (HS) leads to increased lipid storage and expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) in pig adipocytes. However, the importance of PCK1 activation and lipid storage in the adaptive response to HS is unknown. Therefore, in vitro experiments were conducted to investigate the effect of PCK1 inhibition with 3-mercaptopicolinic acid (3MPA) on lipid storage and adipocyte response during HS. In vitro culture of adipocytes under HS (41.0 °C) increased (P < 0.05) triacylglycerol accumulation compared with control (37.0 °C). HS increased (P < 0.05) reactive oxygen species level and 3MPA further upregulated (P < 0.05) its level. Heat shock protein 70 (HSP70) gene expression was induced (P < 0.05) by HS compared to control, and PCK1 inhibition with 3MPA attenuated (P < 0.05) its induction by HS. The endoplasmic reticulum (ER) stress markers, C/EBP homologous protein (CHOP) was also upregulated by HS and 3MPA further upregulated (P < 0.05) CHOP mRNA level. These results suggest that with inhibition of PCK1 during HS, in vitro cultured adipocytes were less able to induce adaptive responses such as upregulation of HSP70 and triglycerides, and this exacerbated ER stress during HS. Thus, PCK1 may function to alleviate ER stress that occurs during HS.

Asunto(s)

Adaptación Fisiológica , Respuesta al Choque Térmico , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Porcinos/fisiología , Adipocitos/fisiología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Células Cultivadas , Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Masculino , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Ácidos Picolínicos/farmacología , ARN Mensajero/metabolismo , Porcinos/genética , Triglicéridos/metabolismo

RESUMEN

CD8+ memory T (Tm) cells are fundamental for protective immunity against infections and cancers 1-5 . Metabolic activities are crucial in controlling memory T-cell homeostasis, but mechanisms linking metabolic signals to memory formation and survival remain elusive. Here we show that CD8+ Tm cells markedly upregulate cytosolic phosphoenolpyruvate carboxykinase (Pck1), the hub molecule regulating glycolysis, tricarboxylic acid cycle and gluconeogenesis, to increase glycogenesis via gluconeogenesis. The resultant glycogen is then channelled to glycogenolysis to generate glucose-6-phosphate and the subsequent pentose phosphate pathway (PPP) that generates abundant NADPH, ensuring high levels of reduced glutathione in Tm cells. Abrogation of Pck1-glycogen-PPP decreases GSH/GSSG ratios and increases levels of reactive oxygen species (ROS), leading to impairment of CD8+ Tm formation and maintenance. Importantly, this metabolic regulatory mechanism could be readily translated into more efficient T-cell immunotherapy in mouse tumour models.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Glucógeno/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Melanoma Experimental/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Neoplasias Cutáneas/genética , Ácido 3-Mercaptopropiónico/farmacología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/genética , Ciclo del Ácido Cítrico/inmunología , Inhibidores Enzimáticos/farmacología , Femenino , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Gluconeogénesis/inmunología , Glucosa/inmunología , Glucógeno/inmunología , Glucólisis/efectos de los fármacos , Glucólisis/genética , Glucólisis/inmunología , Homeostasis/inmunología , Memoria Inmunológica , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/inmunología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NADP/inmunología , NADP/metabolismo , Vía de Pentosa Fosfato/efectos de los fármacos , Vía de Pentosa Fosfato/genética , Vía de Pentosa Fosfato/inmunología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/inmunología , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo

RESUMEN

There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 µM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 µM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C.

Asunto(s)

Inhibidores Enzimáticos/química , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/química , Ácidos Picolínicos/química , Animales , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Proteínas Recombinantes/química , Porcinos

RESUMEN

For almost 40 years, it has been known that tryptophan metabolites and picolinic acid analogues act as inhibitors of gluconeogenesis. Early studies observed that 3-mercaptopicolinic acid (MPA) was a potent hypoglycemic agent via inhibition of glucose synthesis through the specific inhibition of phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenesis pathway. Despite prior kinetic investigation, the mechanism of the inhibition by MPA is unclear. To clarify the mechanism of inhibition exerted by MPA on PEPCK, we have undertaken structural and kinetic studies. The kinetic data in concert with crystallographic structures of PEPCK in complex with MPA and the substrates for the reaction illustrate that PEPCK is inhibited by the binding of MPA at two discrete binding sites: one acting in a competitive fashion with PEP/OAA (∼10 µM) and the other acting at a previously unidentified allosteric site (Ki ∼ 150 µM). The structural studies suggest that binding of MPA to the allosteric pocket stabilizes an altered conformation of the nucleotide-binding site that in turn reduces the affinity of the enzyme for the nucleotide.

Asunto(s)

Regulación Alostérica/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipoglucemiantes/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ácidos Picolínicos/farmacología , Animales , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Fosfoenolpiruvato Carboxiquinasa (GTP)/química , Ratas

RESUMEN

The aim of this study was to elucidate the mechanisms involved in the inhibition of renal gluconeogenesis occurring under conditions of lowered activity of NADPH oxidase (Nox), the enzyme considered to be one of the main sources of reactive oxygen species in kidneys. The in vitro experiments were performed on primary cultures of rat renal proximal tubules, with the use of apocynin, a selective Nox inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a potent superoxide radical scavenger. In the in vivo experiments, Zucker diabetic fatty (ZDF) rats, a well established model of diabetes type 2, were treated with apocynin solution in drinking water. The main in vitro findings are the following: (1) both apocynin and TEMPOL attenuate the rate of gluconeogenesis, inhibiting the step catalyzed by phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the process; (2) in the presence of the above-noted compounds the expression of PEPCK and the phosphorylation of transcription factor CREB and ERK1/2 kinases are lowered; (3) both U0126 (MEK inhibitor) and 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione (ERK inhibitor) diminish the rate of glucose synthesis via mechanisms similar to those of apocynin and TEMPOL. The observed apocynin in vivo effects include: (1) slight attenuation of hyperglycemia; (2) inhibition of renal gluconeogenesis; (3) a decrease in renal PEPCK activity and content. In view of the results summarized above, it can be concluded that: (1) the lowered activity of the ERK1/2 pathway is of importance for the inhibition of renal gluconeogenesis found under conditions of lowered superoxide radical production by Nox; (2) the mechanism of this phenomenon includes decreased PEPCK expression, resulting from diminished activity of transcription factor CREB; (3) apocynin-evoked inhibition of renal gluconeogenesis contributes to the hypoglycemic action of this compound observed in diabetic animals. Thus, the study has delivered some new insights into the recently discussed issue of the usefulness of Nox inhibition as a potential antidiabetic strategy.

Asunto(s)

Diabetes Mellitus Experimental/tratamiento farmacológico , Gluconeogénesis/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , NADPH Oxidasas/metabolismo , Acetofenonas/farmacología , Animales , Antioxidantes/farmacología , Butadienos/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Óxidos N-Cíclicos/farmacología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Regulación de la Expresión Génica , Gluconeogénesis/genética , Hipoglucemiantes/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Nitrilos/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Zucker , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Marcadores de Spin , Tiazolidinedionas/farmacología

RESUMEN

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism.

Asunto(s)

Neoplasias/genética , Neoplasias/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Factor de Transcripción Activador 4/metabolismo , Aminoácidos/metabolismo , Animales , Estrés del Retículo Endoplásmico , Femenino , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Ratones , Mitocondrias/enzimología , Modelos Biológicos , Células 3T3 NIH , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismo

RESUMEN

The plasticizer di-(2-ethylhexyl) phthalate (DEHP) is used to add flexibility to polyvinylchloride polymers and as a component of numerous consumer and medical products. DEHP and its metabolites have been detected in amniotic fluid and umbilical cord blood, suggesting fetal exposure. In the present study, we used an in utero exposure model in which pregnant rat dams were exposed to 1- to 300-mg DEHP/kg·d from gestational day 14 until birth. We previously reported that this window of exposure to environmentally relevant doses of DEHP resulted in reduced levels of serum testosterone and aldosterone in adult male offspring and that the effects on aldosterone were sustained in elderly rats and resulted in decreased blood pressure. Here, we characterized the long-term effects of in utero DEHP exposure by performing global gene expression analysis of prepubertal (postnatal d 21) and adult (postnatal d 60) adrenal glands. We found that the peroxisome proliferator-activated receptor and lipid metabolism pathways were affected by DEHP exposure. Expression of 2 other DEHP targets, hormone-sensitive lipase and phosphoenolpyruvate carboxykinase 1 (Pck1), correlated with reduced aldosterone levels and may account for the inhibitory effect of DEHP on adrenal steroid formation. The angiotensin II and potassium pathways were up-regulated in response to DEHP. In addition, the potassium intermediate/small conductance calcium-activated channel Kcnn2 and 2-pore-domain potassium channel Knck5 were identified as DEHP targets. Based on this gene expression analysis, we measured fatty acid-binding protein 4 and phosphoenolpyruvate carboxykinase 1 in sera from control and DEHP-exposed rats and identified both proteins as putative serum biomarkers of in utero DEHP exposure. These results shed light on molecular targets that mediate DEHP long-term effects and, in doing so, provide means by which to assess past DEHP exposure.

Asunto(s)

Glándulas Suprarrenales/efectos de los fármacos , Dietilhexil Ftalato/toxicidad , Disruptores Endocrinos/toxicidad , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Receptores Activados del Proliferador del Peroxisoma/antagonistas & inhibidores , Plastificantes/toxicidad , Efectos Tardíos de la Exposición Prenatal , Glándulas Suprarrenales/crecimiento & desarrollo , Glándulas Suprarrenales/metabolismo , Aldosterona/sangre , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Dietilhexil Ftalato/administración & dosificación , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Disruptores Endocrinos/administración & dosificación , Femenino , Hipotensión/inducido químicamente , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/sangre , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/sangre , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Plastificantes/administración & dosificación , Embarazo , Ratas , Ratas Sprague-Dawley , Esterol Esterasa/antagonistas & inhibidores , Esterol Esterasa/genética , Esterol Esterasa/metabolismo , Testosterona/sangre

RESUMEN

Jazf1 is a 27 kDa nuclear protein containing three putative zinc finger motifs that is associated with diabetes mellitus and prostate cancer; however, little is known about the role that this gene plays in regulation of metabolism. Recent evidence indicates that Jazf1 transcription factors bind to the nuclear orphan receptor TR4. This receptor regulates PEPCK, the key enzyme involved in gluconeogenesis. To elucidate Jazf1's role in metabolism, we fed a 60% fat diet for up to 15 weeks. In Jazf1 overexpression mice, weight gain was found to be significantly decreased. The expression of Jazf1 in the liver also suppressed lipid accumulation and decreased droplet size. These results suggest that Jazf1 plays a critical role in the regulation of lipid homeostasis. Finally, Jazf1 may provide a new therapeutic target in the management of obesity and diabetes.

Asunto(s)

Proteínas Portadoras/genética , Dieta Alta en Grasa , Metabolismo de los Lípidos/genética , Proteínas Nucleares/genética , Aumento de Peso/genética , Animales , Glucemia/análisis , Proteínas Co-Represoras , Proteínas de Unión al ADN , Prueba de Tolerancia a la Glucosa , Homeostasis , Insulina/fisiología , Ratones , Ratones Transgénicos , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

As an enzyme acting at the junction of gluconeogenic pathway, phosphoenolpyruvate carboxykinase (PEPCK) controls substrate flow from Krebs cycle toward glucose production. Therefore, it would be advantageous to design effective inhibitors to inactivate PEPCK in diabetes mellitus and other abnormalities caused by insulin resistance. Such inhibitors may compensate the metabolic consequences of ex-activity of PEPCK at these conditions. Understanding the mechanism by which inhibitors exert their effect on enzyme activity is of great interest for designing stronger inhibitors. In the present work, molecular dynamic simulations were used to study enzyme-inhibitor interactions. Our results indicate that inhibitors of PEPCK with their short chains interact with enzyme active site through non-covalent interactions of electrostatic and hydrogen bond nature. The data also show that inhibitors neither reach a stable state in their binding site nor make static complex with the enzyme active site. Instead, they interact with functional groups of active site residues in a dynamic fashion. In this way, oxalate and sulfoacetate carrying two negative groups of higher charge density and optimum spacing from each other, show more dynamic behavior (lower stability in their binding site) and more inhibitory effects than other inhibitors used (phosphonoformate, phosphoglycolate and 3-phosphonopropionate).

Asunto(s)

Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/química , Ácidos Acíclicos/metabolismo , Animales , Dominio Catalítico , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Ratas

RESUMEN

Hepatic gluconeogenesis is crucial for glucose homeostasis. Although sirtuin 1 (Sirt1) is implicated in the regulation of gluconeogenesis in the liver, the effects of other histone deacetylases (HDAC) on gluconeogenesis are unclear. The aim of this study was to identify the role of class I HDACs in hepatic gluconeogenesis. In HepG2 cells and the liver of mice, the expressions of phosphoenol pyruvate carboxykinase (PEPCK) and hepatocyte nuclear factor 4α (HNF4α) were significantly decreased by treatment with a newly designed class I HDAC inhibitor, Ky-2. SiRNA knockdown of HDAC1 expression, but not of HDAC2 or HDAC3, in HepG2 cells decreased PEPCK and HNF4α expression. In HepG2 cells, insulin-stimulated phosphorylation of Akt and forkhead box O 1 (FoxO1) was increased by Ky-2. Pyruvate tolerance tests in Ky-2-treated high-fat-diet (HFD)-fed mice showed a marked reduction in blood glucose compared with vehicle-treated HFD mice. These data suggest that class I HDACs increase HNF4α protein expression and the transcriptional activity of FoxO1, followed by the induction of PEPCK mRNA expression and gluconeogenesis in liver.

Asunto(s)

Gluconeogénesis , Glucosa/metabolismo , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Hígado/metabolismo , Péptidos Cíclicos/farmacología , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Línea Celular Tumoral , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Factor Nuclear 4 del Hepatocito/antagonistas & inhibidores , Histona Desacetilasa 1/genética , Humanos , Insulina/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosforilación , ARN Interferente Pequeño/genética , Transcripción Genética

RESUMEN

Excessive hepatic glucose production through the gluconeogenesis pathway is partially responsible for the elevated glucose levels observed in patients with type 2 diabetes mellitus (T2DM). The forkhead transcription factor forkhead box O1 (Foxo1) plays a crucial role in mediating the effect of insulin on hepatic gluconeogenesis. Here, using a db/db mouse model, we demonstrate the effectiveness of Foxo1 inhibitor, an orally active small-molecule compound, as a therapeutic drug for treating T2DM. Using mass spectrometric affinity screening, we discovered a series of compounds that bind to Foxo1, identifying among them the compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856), which potently inhibits human Foxo1 transactivation and reduces glucose production through the inhibition of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase mRNA levels in a rat hepatic cell line. Oral administration of AS1842856 to diabetic db/db mice led to a drastic decrease in fasting plasma glucose level via the inhibition of hepatic gluconeogenic genes, whereas administration to normal mice had no effect on the fasting plasma glucose level. Treatment with AS1842856 also suppressed an increase in plasma glucose level caused by pyruvate injection in both normal and db/db mice. Taken together, these findings indicate that the Foxo1 inhibitor represents a new class of drugs for use in treating T2DM.

Asunto(s)

Factores de Transcripción Forkhead/antagonistas & inhibidores , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/farmacología , Quinolonas/farmacología , Animales , Línea Celular Tumoral , Ayuno , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Glucosa/biosíntesis , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Glucosa-6-Fosfatasa/genética , Humanos , Hiperglucemia/metabolismo , Hipoglucemiantes/uso terapéutico , Masculino , Espectrometría de Masas , Ratones , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Ácido Pirúvico/farmacología , Quinolonas/uso terapéutico , ARN Mensajero/antagonistas & inhibidores , Ratas , Relación Estructura-Actividad , Activación Transcripcional

RESUMEN

The mechanisms of molecular recognition of phosphoenolpyruvate (PEP) and oxaloacetate (OAA) by cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) were investigated by the systematic evaluation of a variety of PEP and OAA analogues as potential reversible inhibitors of the enzyme against PEP. The molecules that inhibit the enzyme in a competitive fashion were found to fall into two general classes. Those molecules that mimic the binding geometry of PEP, namely phosphoglycolate and 3-phosphonopropionate, are found to bind weakly (millimolar Ki values). In contrast, those competitive inhibitors that mimic the binding of OAA (oxalate and phosphonoformate) coordinate directly to the active site manganese ion and bind an order of magnitude more tightly (micromolar Ki values). The competitive inhibitor sulfoacetate is found to be an outlier of these two classes, binding in a hybrid fashion utilizing modes of recognition of both PEP and OAA in order to achieve a micromolar inhibition constant in the absence of direct coordination to the active site metal. The kinetic studies in combination with the structural characterization of the five aforementioned competitive inhibitors demonstrate the molecular requirements for high affinity binding of molecules to the active site of the enzyme. These features include cis-planar carbonyl groups that are required for coordination to the active site metal, a bridging electron rich atom at the position corresponding to the C2 methylene group of OAA to facilitate interactions with R405, a carboxylate or sulfonate moiety at a position corresponding to the C1 carboxylate of OAA, and the edge-on aromatic interaction between a carboxylate and Y235.

Asunto(s)

Citosol/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Sitios de Unión , Cristalización , Citosol/enzimología , Inhibidores Enzimáticos/química , Cinética , Modelos Moleculares , Estructura Molecular , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo

RESUMEN

The structures of the rat cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) reported in the PEPCK-Mn2+, -Mn2+-oxaloacetic acid (OAA), -Mn2+-OAA-Mn2+-guanosine-5'-diphosphate (GDP), and -Mn2+-Mn2+-guanosine-5'-tri-phosphate (GTP) complexes provide insight into the mechanism of phosphoryl transfer and decarboxylation mediated by this enzyme. OAA is observed to bind in a number of different orientations coordinating directly to the active site metal. The Mn2+-OAA and Mn2+-OAA-Mn2+GDP structures illustrate inner-sphere coordination of OAA to the manganese ion through the displacement of two of the three water molecules coordinated to the metal in the holo-enzyme by the C3 and C4 carbonyl oxygens. In the PEPCK-Mn2+-OAA complex, an alternate bound conformation of OAA is present. In this conformation, in addition to the previous interactions, the C1 carboxylate is directly coordinated to the active site Mn2+, displacing all of the waters coordinated to the metal in the holo-enzyme. In the PEPCK-Mn2+-GTP structure, the same water molecule displaced by the C1 carboxylate of OAA is displaced by one of the gamma-phosphate oxygens of the triphosphate nucleotide. The structures are consistent with a mechanism of direct in-line phosphoryl transfer, supported by the observed stereochemistry of the reaction. In the catalytically competent binding mode, the C1 carboxylate of OAA is sandwiched between R87 and R405 in an environment that would serve to facilitate decarboxylation. In the reverse reaction, these two arginines would form the CO2 binding site. Comparison of the Mn2+-OAA-Mn2+GDP and Mn2+-Mn2+GTP structures illustrates a marked difference in the bound conformations of the nucleotide substrates in which the GTP nucleotide is bound in a high-energy state resulting from the eclipsing of all three of the phosphoryl groups along the triphosphate chain. This contrasts a previously determined structure of PEPCK in complex with a triphosphate nucleotide analogue in which the analogue mirrors the conformation of GDP as opposed to GTP. Last, the structures illustrate a correlation between conformational changes in the P-loop, the nucleotide binding site, and the active site lid that are important for catalysis.

Asunto(s)

Citosol/enzimología , Transferencia de Energía , Modelos Químicos , Oxaloacetatos/química , Oxaloacetatos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/química , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Cationes Bivalentes/química , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , Cristalografía por Rayos X , Descarboxilación , Inhibidores Enzimáticos , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/aislamiento & purificación , Humanos , Cinética , Sustancias Macromoleculares/metabolismo , Manganeso/química , Manganeso/metabolismo , Manganeso/farmacología , Modelos Moleculares , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/aislamiento & purificación , Fosforilación , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación

RESUMEN

New modifications on the C-8 4-aminobenzyl unit of the previously reported 3-alkyl-1,8-dibenzylxanthine inhibitors of cPEPCK are presented. The most active compound reported here is the 5-chloro-1,3-dimethyl-1H-pyrazole-4-sulfonic acid amide derivative 2 with an IC(50) of 0.29+/-0.08 microM. An X-ray analysis of a heteroaromatic sulfonamide is presented showing a new pi-pi interaction.

Asunto(s)

Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Fosfoenolpiruvato Carboxiquinasa (GTP)/antagonistas & inhibidores , Xantinas/farmacología , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Xantinas/química