Insulin induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells

AbstractFructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 μM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide

Liver Glucokinase and Lipid Metabolism

Control of energy metabolism is crucial for optimal functioning of organs and tissues. Amongst all nutrients, glucose is the principal energy source for most cells and, therefore, minimum blood glucose levels must be guaranteed. Alterations in glycaemia can lead to hyperglycaemic states (producing protein glycosylation and toxicity in glucose-sensitive cells) or hypoglycaemic states (that can affect brain function), both harmful. Therefore, mechanisms must exist to keep glycaemia in a narrow physiological range (4-8 mM) independently of the nutritional state. To achieve control of blood glucose levels, our body has a complex, interorgan signaling system using nutrients (glucose, lipids, amino acids), hormones (insulin, glucagon, ghrelin, etc.) and the autonomic nervous system. In response to these signals, organs and tissues (mainly intestine, endocrine pancreas, liver, skeletal muscle, adipose tissue, brain and adrenal glands) adapt their function to energetic requirements. The liver plays a pivotal role in the maintenance of glucose homeostasis by continuously adapting its metabolism to energetic needs. In the fed state, when blood glucose levels are high and there is insulin, liver takes-up part glucose to replenish glycogen stores. Besides, when glucose stores are full, the liver has the capacity to synthesize lipids de novo from glucose for-long term energy storage. Lipids are packaged in very low-density lipoprotein (VLDL) particles and then transported to the adipose tissue. Conversely during starvation, when glycaemia falls and glucagon increases, the liver produces glucose to maintain circulating glucose levels by breaking down glycogen stores or by synthesizing glucose de novo through gluconeogenesis. Gluconeogenesis, as an energy-consuming pathway, is linked to -oxidation of fatty acids (fuel supplier pathway)..

Liver GlucokinaseA456V Induces Potent Hypoglycemia without Dyslipidemia through a Paradoxical Induction of the Catalytic Subunit of Glucose-6-Phosphatase

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GKA456V, described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulation in vivo. GKA456V was overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affected in vivo, demonstrating impaired GKRP regulation. GKA456V overexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GKA456V-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy

Glycosylation defects, offset by PEPCK-M, drive entosis in breast carcinoma cells

On glucose restriction, epithelial cells can undergo entosis, a cell-in-cell cannibalistic process, to allow considerable withstanding to this metabolic stress. Thus, we hypothesized that reduced protein glycosylation might participate in the activation of this cell survival pathway. Glucose deprivation promoted entosis in an MCF7 breast carcinoma model, as evaluated by direct inspection under the microscope, or revealed by a shift to apoptosis + necrosis in cells undergoing entosis treated with a Rho-GTPase kinase inhibitor (ROCKi). In this context, curbing protein glycosylation defects with N-acetyl-glucosamine partially rescued entosis, whereas limiting glycosylation in the presence of glucose with tunicamycin or NGI-1, but not with other unrelated ER-stress inducers such as thapsigargin or amino-acid limitation, stimulated entosis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is upregulated by glucose deprivation, thereby enhancing cell survival. Therefore, we presumed that PEPCK-M could play a role in this process by offsetting key metabolites into glycosyl moieties using alternative substrates. PEPCK-M inhibition using iPEPCK-2 promoted entosis in the absence of glucose, whereas its overexpression inhibited entosis. PEPCK-M inhibition had a direct role on total protein glycosylation as determined by Concanavalin A binding, and the specific ratio of fully glycosylated LAMP1 or E-cadherin. The content of metabolites, and the fluxes from C-13-glutamine label into glycolytic intermediates up to glucose-6-phosphate, and ribose- and ribulose-5-phosphate, was dependent on PEPCK-M content as measured by GC/MS. All in all, we demonstrate for the first time that protein glycosylation defects precede and initiate the entosis process and implicates PEPCK-M in this survival program to dampen the consequences of glucose deprivation. These results have broad implications to our understanding of tumor metabolism and treatment strategies

Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2+

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology

PEPCK-M recoups tumor cell anabolic potential in a PKC-ζ-dependent manner

Background: Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M; PCK2) is expressed in all cancer types examined and in neuroprogenitor cells. The gene is upregulated by amino acid limitation and ER-stress in an ATF4- dependent manner, and its activity modulates the PEP/Ca2+ signaling axis, providing clear arguments for a functional relationship with metabolic adaptations for cell survival. Despite its potential relevance to cancer metabolism, the mechanisms responsible for its pro-survival activity have not been completely elucidated. Methods: [U-13C]glutamine and [U-13C]glucose labeling of glycolytic and TCA cycle intermediates and their anabolic end-products was evaluated quantitatively using LC/MS and GC/MS in conditions of abundant glucose and glucose limitation in loss-of-function (shRNA) and gain-of-function (lentiviral constitutive overexpression) HeLa cervix carcinoma cell models. Cell viability was assessed in conjunction with various glucose concentrations and in xenografts in vivo. Results: PEPCK-M levels linearly correlated with [U-13C]glutamine label abundance in most glycolytic and TCA cycle intermediate pools under nutritional stress. In particular, serine, glycine, and proline metabolism, and the anabolic potential of the cell, were sensitive to PEPCK-M activity. Therefore, cell viability defects could be rescued by supplementing with an excess of those amino acids. PEPCK-M silenced or inhibited cells in the presence of abundant glucose showed limited growth secondary to TCA cycle blockade and increased ROS. In limiting glucose conditions, downregulation of PKC-ζ tumor suppressor has been shown to enhance survival. Consistently, HeLa cells also sustained a survival advantage when PKC-ζ tumor suppressor was downregulated using shRNA, but this advantage was abolished in the absence of PEPCK-M, as its inhibition restores cell growth to control levels. The relationship between these two pathways is also highlighted by the anti-correlation observed between PEPCK-M and PKC-ζ protein levels in all clones tested, suggesting co-regulation in the absence of glucose. Finally, PEPCK-M loss negatively impacted on anchorage-independent colony formation and xenograft growth in vivo. Conclusions: All in all, our data suggest that PEPCK-M might participate in the mechanisms to regulate proteostasis in the anabolic and stalling phases of tumor growth. We provide molecular clues into the clinical relevance of PEPCK-M as a mechanism of evasion of cancer cells in conditions of nutrient stress

Molecular structure and biodegradation kinetics of Linear Alkylbenzene Sulphonates in sea water.

The present paper describes the results of the application of the biodegradation test proposed by the United States Environmental Protection Agency (USEPA) “Biodegradability in sea water” Office of Prevention, Pesticides, and Toxic Substances (OPPTS) 835.3160, to Linear Alkylbenzene Sulphonate (LAS), the synthetic surfactant with the highest consumption volume on a world-wide basis. High performance liquid chromatography (HPLC) has been employed for the separation and quantification of the different homologues and isomers of the surfactant. Water from the Bay of Cádiz (South–West of the Iberian peninsula) has been used as test medium. The results indicate how both lag and t50 time shows a significant linear relationship with the length of the alkyl chain of the homologue; the effect of this is that the homologues of longer chain length not only begin to degrade first but also degrade at a faster rate. Regarding the isomeric composition, it is observed that as the percentage of biodegradation increases, there is an increase in the proportion of internal isomers, in comparison with the isomeric relationships of the original test substanc

Impulsividad y conciencia del problema predicen la adherencia terapéutica y el abandono del tratamiento en el trastorno por juego de azar

Este estudio investiga el valor predictivo de la impulsividad como rasgo (evaluada con la escala de conducta impulsiva UPPS-P) y de covariados relevantes (variables sociodemográficas, severidad del juego de azar, estado de ánimo disfórico, otras conductas adictivas e inteligencia no verbal), con respecto al abandono del tratamiento y los niveles de cumplimiento de las prescripciones terapéuticas en pacientes con trastorno por juego de azar. Sesenta y seis pacientes con este trastorno, participantes del proyecto G-Brain, fueron evaluados inicialmente en impulsividad rasgo y en los covariados mencionados. Dicha evaluación se realizó durante los seis primeros meses desde el inicio de su tratamiento. En el seguimiento realizado a los 6 meses, 24 pacientes habían abandonado (grupo ABD) y 42 continuaban el tratamiento (grupo NABD). Los análisis multivariados con las subescalas de impulsividad mostraron diferencias prospectivas entre ambos grupos. Aparentemente, estas diferencias son atribuibles a las dimensiones afectivas de impulsividad (urgencias positiva y negativa). Entre ambas dimensiones, solo la urgencia positiva fue un predictor independiente de un ligero incremento en la probabilidad de abandono. Dentro del grupo NABD, un mayor grado de adherencia terapéutica vino predicho, de manera independiente, tanto por una baja búsqueda de sensaciones como por una mayor conciencia de los problemas vinculados al juego. Estos resultados sugieren que los rasgos de impulsividad de origen afectivo son predictores de abandono del tratamiento en pacientes con trastorno por juego. La conciencia de problemas asociados al juego de azar y una baja búsqueda de sensaciones predisponen a una mayor adherencia a las prescripciones terapéuticas

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background: Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required. Methodology/Principal Findings: We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/ progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients. Conclusions/Significance: Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signatur

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required.We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients.Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature