Silencing of PFKFB3 protects podocytes against high glucose­induced injury by inducing autophagy.

Diabetic nephropathy (DN) is a diabetic complication that threatens the health of patients with diabetes. In addition, podocyte injury can lead to the occurrence of DN. The protein 6­phosphofructo­2­kinase/fructose­2,6-biphosphatase 3 (PFKFB3) may be associated with diabetes; however, the effects of PFKFB3 knockdown by small interfering (si)RNA on the growth of podocytes remains unknown. To investigate the mechanism by which PFKFB3 mediates podocyte injury, MPC5 mouse podocyte cells were treated with high­glucose (HG), and cell viability and apoptosis were examined by Cell Counting Kit­8 assay and flow cytometry, respectively. In addition, the expression of autophagy­related proteins were measured using western blot analysis and immunofluorescence staining. Cell migration was investigated using a Transwell assay and phalloidin staining was performed to observe the cytoskeleton. The results revealed that silencing of PFKFB3 significantly promoted MPC5 cell viability and inhibited apoptosis. In addition, the migration of the MPC5 cells was notably downregulated by siPFKFB3. Moreover, PFKFB3 silencing notably reversed the HG­induced decrease in oxygen consumption rate, and the HG­induced increase in extracellular acidification rate was rescued by PFKFB3 siRNA. Furthermore, silencing of PFKFB3 induced autophagy in HG­treated podocytes through inactivating phosphorylated (p­)mTOR, p­AMPKα, LC3 and sirtuin 1, and activating p62. In conclusion, silencing of PFKFB3 may protect podocytes from HG­induced injury by inducing autophagy. Therefore, PFKFB3 may serve as a potential target for treatment of DN.

The Role of HIF1α-PFKFB3 Pathway in Diabetic Retinopathy.

Diabetic retinopathy (DR) is the leading cause of blindness for adults in developed countries. Both microvasculopathy and neurodegeneration are implicated in mechanisms of DR development, with neuronal impairment preceding microvascular abnormalities, which is often underappreciated in the clinic. Most current therapeutic strategies, including anti-vascular endothelial growth factor (anti-VEGF)-antibodies, aim at treating the advanced stages (diabetic macular edema and proliferative diabetic retinopathy) and fail to target the neuronal deterioration. Hence, new therapeutic approach(es) intended to address both vascular and neuronal impairment are urgently needed. The hypoxia-inducible factor 1α (HIF1α)-6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) pathway is critically implicated in the islet pathology of diabetes. Recent evidence highlighted the pathway relevance for pathologic angiogenesis and neurodegeneration, two key aspects in DR. PFKFB3 is key to the sprouting angiogenesis, along with VEGF, by determining the endothelial tip-cell competition. Also, PFKFB3-driven glycolysis compromises the antioxidative capacity of neurons leading to neuronal loss and reactive gliosis. Therefore, the HIF1α-PFKFB3 signaling pathway is unique as being a pervasive pathological component across multiple cell types in the retina in the early as well as late stages of DR. A metabolic point-of-intervention based on HIF1α-PFKFB3 targeting thus deserves further consideration in DR.

PFKFB2 regulates glycolysis and proliferation in pancreatic cancer cells.

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

Indole Alleviates Diet-Induced Hepatic Steatosis and Inflammation in a Manner Involving Myeloid Cell 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3.

BACKGROUND AND AIMS: Indole is a microbiota metabolite that exerts anti-inflammatory responses. However, the relevance of indole to human non-alcoholic fatty liver disease (NAFLD) is not clear. It also remains largely unknown whether and how indole acts to protect against NAFLD. The present study sought to examine the association between the circulating levels of indole and liver fat content in human subjects and explore the mechanisms underlying indole actions in mice with diet-induced NAFLD. APPROACH AND RESULTS: In a cohort of 137 subjects, the circulating levels of indole were reversely correlated with body mass index. In addition, the circulating levels of indole in obese subjects were significantly lower than those in lean subjects and were accompanied with increased liver fat content. At the whole-animal level, treatment of high-fat diet (HFD)-fed C57BL/6J mice with indole caused significant decreases in the severity of hepatic steatosis and inflammation. In cultured cells, indole treatment stimulated the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a master regulatory gene of glycolysis, and suppressed macrophage proinflammatory activation in a PFKFB3-dependent manner. Moreover, myeloid cell-specific PFKFB3 disruption exacerbated the severity of HFD-induced hepatic steatosis and inflammation and blunted the effect of indole on alleviating diet-induced NAFLD phenotype. CONCLUSIONS: Taken together, our results demonstrate that indole is relevant to human NAFLD and capable of alleviating diet-induced NAFLD phenotypes in mice in a myeloid cell PFKFB3-dependent manner. Therefore, indole mimetic and/or macrophage-specific PFKFB3 activation may be the viable preventive and/or therapeutic approaches for inflammation-associated diseases including NAFLD.

PFKFB3-mediated endothelial glycolysis promotes pulmonary hypertension.

Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3)-mediated endothelial glycolysis plays a critical role in the development of PH. Heterozygous global deficiency of Pfkfb3 protected mice from developing hypoxia-induced PH, and administration of the PFKFB3 inhibitor 3PO almost completely prevented PH in rats treated with Sugen 5416/hypoxia, indicating a causative role of PFKFB3 in the development of PH. Immunostaining of lung sections and Western blot with isolated lung endothelial cells showed a dramatic increase in PFKFB3 expression and activity in pulmonary endothelial cells of rodents and humans with PH. We generated mice that were constitutively or inducibly deficient in endothelial Pfkfb3 and found that these mice were incapable of developing PH or showed slowed PH progression. Compared with control mice, endothelial Pfkfb3-knockout mice exhibited less severity of vascular smooth muscle cell proliferation, endothelial inflammation, and leukocyte recruitment in the lungs. In the absence of PFKFB3, lung endothelial cells from rodents and humans with PH produced lower levels of growth factors (such as PDGFB and FGF2) and proinflammatory factors (such as CXCL12 and IL1ß). This is mechanistically linked to decreased levels of HIF2A in lung ECs following PFKFB3 knockdown. Taken together, these results suggest that targeting PFKFB3 is a promising strategy for the treatment of PH.

The Glycolytic Enzyme PFKFB3 Controls TNF-α-Induced Endothelial Proinflammatory Responses.

Endothelial cells play an important role in health and a variety of diseases. Recent evidences show that endothelial cells rely on glycolysis rather than on oxidative phosphorylation to generate energy to support cellular functions such as angiogenesis. However, the effect of endothelial glycolysis on vascular inflammation remains little known. Here, we investigate the role of key glycolytic enzyme PFKFB3 in tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory responses. siRNAs were used to knockdown the expression of PFKFB3. In some experiments, PFKFB3 inhibitors were also used. TNF-α at 20 ng/ml was added to confluent endothelial cells for different time period of stimulation. PFKFB3 expression was examined by RT-PCR and western blotting. Cytokine antibody panel membranes were employed to detect different cytokines/chemokines in culture supernatant of endothelial cells. The determination of monocyte adhesion to endothelial cells after TNF-α treatment was conducted using THP-1 cells. The monocyte attraction was performed using Transwell filters. For further mechanisms, NF-κB-p65 localization was examined by immunofluorescence. Expression of total IκB, phospho-IκB, phospho-NF-κB-p65, and Ikkß was detected by western blotting. DNA-binding activity of NF-κB was assessed using electrophoretic mobility shift assay. We found that TNF-α increased endothelial PFKFB3 expression. Knockdown of PFKFB3 almost blocked all TNF-α-induced release of the proinflammatory cytokines/chemokines (MCP-1, IL-8, CXCL1, GMCSF, RANTES, TNF-α) and ICAM-1. PFKFB3 knockdown also significantly inhibited TNF-α-induced monocyte adhesion and transmigration. Furthermore, inhibition of PFKFB3 inhibited TNF-α-induced Ikkß phosphorylation, IκBα phosphorylation and degradation, NF-κB-p65 phosphorylation, nuclear translocation, and DNA-binding activity. Thus, our results demonstrate that glycolytic enzyme PFKFB3 plays a critical role in TNF-α-induced endothelial inflammation.

Analysis of Malignant Melanoma Cell Lines Exposed to Hypoxia Reveals the Importance of PFKFB4 Overexpression for Disease Progression.

BACKGROUND/AIM: Most melanomas develop in hypoxic conditions. Since hypoxia via HIF-1 induces glycolysis, a process essential for malignant melanoma growth/survival, the goal of this study was to analyze the influence of hypoxia on the expression of HIF-1 target genes involved in glucose metabolism. MATERIALS AND METHODS: The response of melanoma cell lines to hypoxic conditions was analyzed by RT-PCR and western blotting. A Kaplan-Meier survival analysis for patients with high and low expression level of PFKFB4 was performed. Further analysis of patients' data was performed using the R/Bioconductor environment. RESULTS: Induction of PFKFB4 gene expression can be considered a crucial mechanism behind glycolysis enhancement in hypoxic melanoma cells. Analysis of a publicly available database revealed that high PFKFB4 expression contributes to poor prognosis of melanoma patients. CONCLUSION: Currently available anti-melanoma therapeutic strategies may significantly benefit from agents targeting PFKFB4 activity.

Non-canonical roles of PFKFB3 in regulation of cell cycle through binding to CDK4.

There is growing interest in studying the molecular mechanisms of crosstalk between cancer metabolism and the cell cycle. 6-phosphate fructose-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a well-known glycolytic activator that plays an important role in tumorigenesis. We investigated whether PFKFB3 was directly involved in oncogenic signaling networks. Mass Spectrometry showed that PFKFB3 interacts with cyclin-dependent kinase (CDK) 4, which controls the transition from G1 phase to S phase of the cell cycle. Further analysis indicated that lysine 147 was a key site for the binding of PFKBFB3 to CDK4. PFKFB3 binding resulted in the accumulation of CDK4 protein by inhibiting ubiquitin proteasome degradation mediated by the heat shock protein 90-Cdc37-CDK4 complex. The proteasome-dependent degradation of CDK4 was accelerated by disrupting the interaction of PFKFB3 with CDK4 by mutating lysine (147) to alanine. Blocking PFKFB3-CDK4 interaction improved the therapeutic effect of FDA-approved CDK4 inhibitor palbociclib on breast cancer. These findings suggest that PFKFB3 is a hub for coordinating cell cycle and glucose metabolism. Combined targeting of PFKFB3 and CDK4 may be new strategy for breast cancer treatment.

TIGAR cooperated with glycolysis to inhibit the apoptosis of leukemia cells and associated with poor prognosis in patients with cytogenetically normal acute myeloid leukemia.

BACKGROUND: Cancer cells show increased glycolysis and take advantage of this metabolic pathway to generate ATP. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects tumor cells from intracellular reactive oxygen species (ROS)-associated apoptosis. However, the function of TIGAR in glycolysis and survival of acute myeloid leukemia cells remains unclear. METHODS: We analyzed TIGAR expression in cytogenetically normal (CN-) AML patients and the correlations with clinical and biological parameters. In vivo and in vitro, we tested whether glycolysis may induce TIGAR expression and evaluated the combination effect of glycolysis inhibitor and TIGAR knockdown on human leukemia cell proliferation. RESULTS: High TIGAR expression was an independent predictor of poor survival and high incidence of relapse in adult patients with CN-AML. TIGAR also showed high expression in multiple human leukemia cell lines and knockdown of TIGAR activated glycolysis through PFKFB3 upregulation in human leukemia cells. Knockdown of TIGAR inhibited the proliferation of human leukemia cells and sensitized leukemia cells to glycolysis inhibitor both in vitro and in vivo. Furthermore, TIGAR knockdown in combination with glycolysis inhibitor 2-DG led leukemia cells to apoptosis. In addition, the p53 activator Nutlin-3α showed a significant combinational effect with TIGAR knockdown in leukemia cells. However, TIGAR expression and its anti-apoptotic effects were uncoupled from overexpression of exogenous p53 in leukemia cells. CONCLUSIONS: TIGAR might be a predictor of poor survival and high incidence of relapse in AML patients, and the combination of TIGAR inhibitors with anti-glycolytic agents may be novel therapies for the future clinical use in AML patients.

miR-26b inhibits proliferation, migration, invasion and apoptosis induction via the downregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 driven glycolysis in osteosarcoma cells.

MicroRNAs (miRNAs) are differentially expressed and play crucial roles in cancer development and progression. Elevated glycolysis provides survival advantage and metastatic phenotype. Emerging evidence indicates that glycolysis in cancers can be regulated by miRNAs. In the present study, the role of miR-26b in the proliferation, invasion and glycolytic phenotype of osteosarcoma (OS) cells was investigated. miR-26b was reported to be downregulated in OS tissues, however, the effect of miR-26b on OS has not been distinctly evaluated. The present study therefore investigated the miR-26b sensitivity mechanism in OS. To determine the role of miR-26, we reinstated its expression in the U2OS OS cell line through transfection with miR-26b mimics and examined the effects on cell proliferation, migration, invasion, cell cycle progression and glycolytic parameters. The computational prediction tool was employed to identify the molecular target of miR-26b and was confirmed experimentally. Restoration of miR-26b expression inhibited cell proliferation, migration and invasion, arrested cell cycle progression, and induced cell apoptosis accompanied by the downregulation of glycolytic phenotype. Moreover, the binding site for miR-26b was predicted in the 3'UTR of gene 6-phosphofructo-2-kinase/fructose­2,6-bisphosphatase-3 (PFKFB3), suggesting a role for miR-26b in metabolic alteration in OS cells. Further studies showed that overexpression of miR-26b repressed PFKFB3 mRNA and protein levels followed by modulation of the expression of glycolytic components (LDHA, GLUT-1) and markers of invasion and cell cycle such as MMP-9, MMP-2, cyclin D1 and p27. Collectively, the data suggested the tumor suppressive role of miR-26b which functions by targeting the glycolytic metabolism in OS cells, and providing a possible therapeutic strategy for OS patients by targeting miRNA expression.

Endothelial PFKFB3 plays a critical role in angiogenesis.

OBJECTIVE: Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3) is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo. APPROACH AND RESULTS: Under hypoxic conditions or after treatment with angiogenic factors, endothelial PFKFB3 was upregulated both in vitro and in vivo. The knockdown or overexpression of PFKFB3 suppressed or accelerated endothelial proliferation and migration in vitro, respectively. Neonatal mice from a model of oxygen-induced retinopathy showed suppressed neovascular growth in the retina when endothelial PFKFB3 was genetically deleted or when the mice were treated with a PFKFB3 inhibitor. In addition, tumors implanted in mice deficient in endothelial PFKFB3 grew more slowly and were provided with less blood flow. A lower level of phosphorylated protein kinase B was observed in PFKFB3-knockdown endothelial cells, which was accompanied by a decrease in intracellular lactate. The addition of lactate to PFKFB3-knockdown cells rescued the suppression of endothelial proliferation and migration. CONCLUSIONS: The blockade or deletion of endothelial PFKFB3 decreases angiogenesis both in vitro and in vivo. Thus, PFKFB3 is a promising target for the reduction of endothelial glycolysis and its related pathological angiogenesis.

Substrate fate in activated macrophages: a comparison between innate, classic, and alternative activation.

Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differential activations result in the expression of specific sets of genes involved in the release of pro- or anti-inflammatory stimuli. In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2-(13)C(2)]glucose tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-gamma or TLR-2, -3, -4, and -9 results in similar flux distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to Hif-1alpha activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for Hif-1alpha, the switch of PFK2 isoenzymes still occurs in LPS/IFN-gamma-activated macrophages, suggesting that this pathway regulates ubiquitous PFK2 expression through Hif-1alpha-independent mechanisms.

Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of both GKRP and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic beta-cells.

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

Niche-specific regulation of central metabolic pathways in a fungal pathogen.

To establish an infection, the pathogen Candida albicans must assimilate carbon and grow in its mammalian host. This fungus assimilates six-carbon compounds via the glycolytic pathway, and two-carbon compounds via the glyoxylate cycle and gluconeogenesis. We address a paradox regarding the roles of these central metabolic pathways in C. albicans pathogenesis: the glyoxylate cycle is apparently required for virulence although glyoxylate cycle genes are repressed by glucose at concentrations present in the bloodstream. Using GFP fusions, we confirm that glyoxylate cycle and gluconeogenic genes in C. albicans are repressed by physiologically relevant concentrations of glucose, and show that these genes are inactive in the majority of fungal cells infecting the mouse kidney. However, these pathways are induced following phagocytosis by macrophages or neutrophils. In contrast, glycolytic genes are not induced following phagocytosis and are expressed in infected kidney. Mutations in all three pathways attenuate the virulence of this fungus, highlighting the importance of central carbon metabolism for the establishment of C. albicans infections. We conclude that C. albicans displays a metabolic program whereby the glyoxylate cycle and gluconeogenesis are activated early, when the pathogen is phagocytosed by host cells, while the subsequent progression of systemic disease is dependent upon glycolysis.

Targeted disruption of inducible 6-phosphofructo-2-kinase results in embryonic lethality.

Inducible 6-phosphofructo-2-kinase (iPFK-2; PFKFB3) produces fructose-2,6-bisphosphate (F2,6BP), which is a potent allosteric activator of 6-phosphofructo-1-kinase (PFK-1), the rate-limiting step in glycolysis. iPFK-2 functions as an activator of anaerobic glycolysis within the hypoxic microenvironment of growing tumors. The early embryo is challenged similarly since the process of vasculogenesis does not begin until after embryonic day 7. We hypothesized that iPFK-2 expression is essential for the survival of the growing embryo. First, we cloned the mouse homolog of iPFK2 and found that it is abundantly expressed in cortical neurons, epithelial cells, and secretory cells of the choroid plexus, pancreas, and adrenal gland of the adult mouse. Using gene targeting, we then disrupted exons 3-7 of the mouse iPFK2 gene, which encode the substrate binding site. No full-term homozygous iPFK-2(-/-) progeny were produced from 11 F7 iPFK-2(+/-) crosses and no homozygous iPFK-2(-/-) embryos were detected after 8 days of embryogenesis.

Identification and characterization of the hypoxia-responsive element of the human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K, identical to PFKFB3) is expressed in a variety of cells and tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, primary blood mononuclear cells and cancer cells. We observed previously that the enhancer region of the HP2K gene, which has been identified in the 5'-flanking region between -1265 and -1329, could respond to serum stimulation following the transfection of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. The HP2K enhancer region also contains two copies of the hypoxia-inducible factor-1 (HIF-1) binding motif (5'-ACGTG-3'). In this study we performed characterization of the HP2K gene expression in response to hypoxic conditions. Both electrophoretic mobility shift and co-transfection assays of the HP2K promoter-luciferase reporter with HIF-1 expression vectors indicated that HIF-1 binds to the hypoxia-responsive element (HRE) of HP2K, thereby upregulating its gene expression. In addition, we demonstrated using site-directed mutagenesis that a complete tandem repeat of the HIF-1 binding motif with a 4-bp interruption is required for full induction of HP2K expression (up to 22-fold) under hypoxic conditions, and that this response is much stronger than that of the erythropoietin (EPO) gene. These results suggest that the sequence 5'-ACGTGNNNNACGTG-3' in the HP2K enhancer is the authentic HRE consensus motif that mediates increased transcription, under hypoxic conditions, via HIF-1.

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis.

Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.

The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase.

The activation of monocytes involves a stimulation of glycolysis, release of potent inflammatory mediators, and alterations in gene expression. All of these processes are known to be further increased under hypoxic conditions. The activated monocytes express inducible 6-phosphofructo-2-kinase (iPFK-2), which synthesizes fructose 2,6-bisphosphate, a stimulator of glycolysis. During ischemia, AMP-activated protein kinase (AMPK) activates the homologous heart 6-phosphofructo-2-kinase isoform by phosphorylating its Ser-466. Here, we studied the involvement of AMPK and iPFK-2 in the stimulation of glycolysis in activated monocytes under hypoxia. iPFK-2 was phosphorylated on the homologous serine (Ser-461) and activated by AMPK in vitro. The activation of human monocytes by lipopolysaccharide induced iPFK-2 expression and increased fructose 2,6-bisphosphate content and glycolysis. The incubation of activated monocytes with oligomycin, an inhibitor of oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, fructose 2,6-bisphosphate content, and glycolysis. In cultured human embryonic kidney 293 cells, the expression of a dominant-negative AMPK prevented both the activation and phosphorylation of co-transfected iPFK-2 by oligomycin. It is concluded that the stimulation of glycolysis by hypoxia in activated monocytes requires the phosphorylation and activation of iPFK-2 by AMPK.