Àurea Navarro-Sabaté

PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate

Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated gluconeogenesis in the liver. This discovery has led to the realization that this compound plays a significant role in directing carbohydrate fluxes in all eukaryotes. Biophysical studies of the enzyme that both synthesizes and degrades this biofactor have yielded insight into its molecular enzymology. Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential in the treatment of diabetes.

PFKFB3 gene silencing decreases glycolysis, induces cell‐cycle delay and inhibits anchorage‐independent growth in HeLa cells

The high rate of glycolysis despite the presence of oxygen in tumor cells (Warburg effect) suggests an important role for this process in cell division. The glycolytic rate is dependent on the cellular concentration of fructose 2,6‐bisphosphate (Fru‐2,6‐P2), which, in turn, is controlled by the bifunctional enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2). The ubiquitous PFK‐2 isoenzyme (uPFK‐2, alternatively named UBI2K5 or ACG) coded by the pfkfb3 gene is induced by different stimuli (serum, progesterone, insulin, hypoxia, etc.) and has the highest kinase/phosphatase activity ratio amongst all PFK‐2 isoenzymes discovered to date, which is consistent with its role as a powerful activator of glycolysis. uPFK‐2 is expressed in brain, placenta, transformed cells and proliferating cells. In the present work, we analyze the impact of small interfering RNA (siRNA)‐induced silencing of uPFK‐2 on the inhibition of cell proliferation. HeLa cells treated with uPFK‐2 siRNA showed a decrease in uPFK‐2 RNA levels measured at 24 h. uPFK‐2 protein levels were severely depleted at 48–72 h when compared with cells treated with an unrelated siRNA, correlating with decreased glycolytic activity, Fru‐2,6‐P2, lactate and ATP concentrations. These metabolic changes led to reduced viability, cell‐cycle delay and an increase in the population of apoptotic cells. Moreover, uPFK‐2 suppression inhibited anchorage‐independent growth. The results obtained highlight the importance of uPFK‐2 on the regulation of glycolysis, on cell viability and proliferation and also on anchorage‐independent growth. These data underscore the potential for uPFK‐2 as an effective tumor therapeutic target.

Cooperation of Adenosine with Macrophage Toll-4 Receptor Agonists Leads to Increased Glycolytic Flux through the Enhanced Expression of PFKFB3 Gene

Macrophages activated through Toll receptor triggering increase the expression of the A2A and A2B adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A2A and A2B receptors, we demonstrate that the A2A receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A2B receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1α is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A2 receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages.

TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells

BACKGROUND AND PURPOSE The TP53 induced glycolysis and apoptosis regulator (TIGAR) functions to lower fructose-2,6-bisphosphate (Fru-2,6-P(2)) levels in cells, consequently decreasing glycolysis and leading to the scavenging of reactive oxygen species (ROS), which correlate with a higher resistance to cell death. The decrease in intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic lesions. Given these good prospects of TIGAR for metabolic regulation and p53-response modulation, we analyzed the effects of TIGAR knockdown in U87MG and T98G glioblastoma-derived cell lines. METHODS/RESULTS After TIGAR-knockdown in glioblastoma cell lines, different metabolic parameters were assayed, showing an increase in Fru-2,6-P(2), lactate and ROS levels, with a concomitant decrease in reduced glutathione (GSH) levels. In addition, cell growth was inhibited without evidence of apoptotic or autophagic cell death. In contrast, a clear senescent phenotype was observed. We also found that TIGAR protein levels were increased shortly after irradiation. In addition, avoiding radiotherapy-triggered TIGAR induction by gene silencing resulted in the loss of capacity of glioblastoma cells to form colonies in culture and the delay of DNA repair mechanisms, based in γ-H2AX foci, leading cells to undergo morphological changes compatible with a senescent phenotype. Thus, the results obtained raised the possibility to consider TIGAR as a therapeutic target to increase radiotherapy effects. CONCLUSION TIGAR abrogation provides a novel adjunctive therapeutic strategy against glial tumors by increasing radiation-induced cell impairment, thus allowing the use of lower radiotherapeutic doses.

Insulin induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells.

Fructose 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 microM 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.

Regulation of ubiquitous 6‐phosphofructo‐2‐kinase by the ubiquitin‐proteasome proteolytic pathway during myogenic C2C12 cell differentiation

6‐Phosphofructo‐2‐kinase catalyzes the synthesis and degradation of fructose 2,6‐bisphosphate, activator of phosphofructokinase‐1 and inhibitor of fructose 1,6‐bisphosphatase. These properties confer to this bifunctional enzyme a key role in the control of glycolysis and gluconeogenesis. Several mammalian isozymes generated by alternative splicing from four genes, designated pfkfb1–4, have been identified. The results presented in this study demonstrate the expression of the pfkfb3 gene in C2C12 cells and its downregulation during myogenic cell differentiation. We also show that the decrease of ubiquitous 6‐phosphofructo‐2‐kinase isozyme levels, product of pfkfb3 gene, is due to its enhanced degradation through the ubiquitin‐proteasome proteolytic pathway.

Akt-dependent Activation of the Heart 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFKFB2) Isoenzyme by Amino Acids

Background: Metabolites may activate signal transduction pathways that regulate cell metabolism. Results: Amino acids activate Fru-2,6-P2 synthesis through Akt-dependent phosphorylation of a specific PFKFB2 isoform. Conclusion: Amino acids regulate Fru-2,6-P2 metabolism via Akt signaling. Significance: This study shows how signaling and metabolism are inextricably linked. Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked.

Pfkfb3 is transcriptionally upregulated in diabetic mouse liver through proliferative signals.

The ubiquitous isoform of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (uPFK‐2), a product of the Pfkfb3 gene, plays a crucial role in the control of glycolytic flux. In this study, we demonstrate that Pfkfb3 gene expression is increased in streptozotocin‐induced diabetic mouse liver. The Pfkfb3/‐3566 promoter construct linked to the luciferase reporter gene was delivered to the liver via hydrodynamic gene transfer. This promoter was upregulated in streptozotocin‐induced diabetic mouse liver compared with transfected healthy cohorts. In addition, increases were observed in Pfkfb3 mRNA and uPFK‐2 protein levels, and intrahepatic fructose‐2,6‐bisphosphate concentration. During streptozotocin‐induced diabetes, phosphorylation of both p38 mitogen‐activated protein kinase and Akt was detected, together with the overexpression of the proliferative markers cyclin D and E2F. These findings indicate that uPFK‐2 induction is coupled to enhanced hepatocyte proliferation in streptozotocin‐induced diabetic mouse liver. Expression decreased when hepatocytes were treated with either rapamycin or LY 294002. This shows that uPFK‐2 regulation is phosphoinositide 3‐kinase–Akt–mammalian target of rapamycin dependent. These results indicate that fructose‐2,6‐bisphosphate is essential to the maintenance of the glycolytic flux necessary for providing energy and biosynthetic precursors to dividing cells.

TP53-inducible Glycolysis and Apoptosis Regulator (TIGAR) Metabolically Reprograms Carcinoma and Stromal Cells in Breast Cancer.

A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer.

Mediators of rat ischemic hepatic preconditioning after cold preservation identified by microarray analysis

Hepatic ischemia‐reperfusion injury associated with liver transplantation is an as yet unresolved problem in clinical practice. Preconditioning protects the liver against the deleterious effects of ischemia, although the mechanism underlying this preconditioning is still unclear. To profile gene expression patterns involved in hepatic ischemic preconditioning, we analyzed the changes in gene expression in rat livers by DNA microarray analysis. Approximately 116 genes were found to have altered gene expression after 8 hours of cold ischemia. Moreover, the expression of 218 genes was modified by classic preconditioning followed by the same ischemia process. Given the importance of the effects of ischemic preconditioning (IP) in minimizing the liver damage induced by sustained ischemia before reperfusion, this study analyzed the putative genes involved in the beneficial role of IP in liver grafts undergoing cold ischemia before its implantation in the recipient (IP+I). Great differences were found in the gene expression pattern of ischemic preconditioning + long cold ischemia (IP+I) group when compared with the long cold ischemia alone condition (I), which could explain the protective regulatory mechanisms that take place after preconditioning. Twenty‐six genes that were downregulated in cold ischemia were found upregulated after preconditioning preceding a long cold ischemia period. These would be genes activated or maintained by preconditioning. Heat shock protein genes and 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase are among the most markedly induced transcripts. Liver Transpl. 12:1615–1625, 2006.