miR-21-5p/Tiam1-mediated glycolysis reprogramming drives breast cancer progression via enhancing PFKL stabilization.

T lymphoma invasion and metastasis 1 (Tiam1) as a tumor-associated gene specifically activates Rho-like GTPases Rac1 and implicates in the invasive phenotype of many cancers. Altering the glycolytic pathway is foreseen as a sound approach to trigger cancer regression. However, the mechanism of Tiam1 in breast cancer (BC) glycolysis reprogramming remains to be clarified. Here, we reported the Tiam1 high expression and prognostic significance in BC. In vitro and in vivo experimental assays identified the functional role of Tiam1 in promoting BC cell proliferation, metastasis and glycolysis reprogramming. Mechanistically, we showed for the first time that Tiam1 could interact with the crucial glycolytic enzyme phosphofructokinase, liver type (PFKL) and promote the evolution of BC in a PFKL-dependent manner. Moreover, miR-21-5p was found to exacerbate the BC proliferation and aggression by targeting Tiam1. Altogether, our study highlights the critical role of Tiam1 in BC development and that the miR-21-5p/Tiam1/PFKL signaling pathway may serve as a target for new anti-BC therapeutic strategies.

Relocation of phosphofructokinases within epithelial cells is a novel event preceding breast cancer recurrence that accurately predicts patient outcomes.

Although recurrent cancers can become life threatening, little is known about the intracellular events required for cancer recurrences. Due to this lack of mechanistic information, there is no test to predict cancer recurrences or nonrecurrences during early stages of disease. In this retrospective study, we use ductal carcinoma in situ of the breast as a framework to better understand the mechanism of cancer recurrences using patient outcomes as the physiological observable. Conventional pathology slides were labeled with anti-phosphofructokinase type L (PFKL) and anti-phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) reagents. PFKL and PFKFB4 were found in ductal epithelial cell nucleoli from DCIS samples of women who did not experience a cancer recurrence. In contrast, PFKL and PFKFB4 may be found near the plasma membrane in samples from patients who will develop recurrent cancer. With the use of machine learning to predict patient outcomes, holdout studies of individual patient micrographs for the three biomarkers PFKL, PFKFB4, and phosphorylated glucose transporter 1 (GLUT1) demonstrated 38.6% true negatives, 49.5% true positives, 11.9% false positives, and 0% false negatives (n = 101). A subpopulation of recurrent samples demonstrated PFKL, PFKFB4, and phosphorylated GLUT1 accumulation at the apical surface of epithelial cells, suggesting that carbohydrates can be harvested from the ducts' luminal spaces as an energy source. We suggest that PFK isotype patterns are metabolic switches representing key mechanistic steps of recurrences. Furthermore, PFK enzyme patterns within epithelial cells contribute to an accurate diagnostic test to classify DCIS patients as high or low recurrence risk.

Selective activation of PFKL suppresses the phagocytic oxidative burst.

In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.

Polymorphisms in Glycolysis-Related Genes Are Associated with Clinical Outcomes of Paclitaxel-Cisplatin Chemotherapy in Non-Small Cell Lung Cancer.

OBJECTIVE: This study was conducted to investigate whether polymorphisms in glycolysis-related genes are associated with clinical outcomes of patients with advanced-stage non-small cell lung cancer (NSCLC) undergoing chemotherapy. METHODS: A total of 377 patients with NSCLC were enrolled. Sixty-five single-nucleotide polymorphisms in 26 genes involved in the glycolytic pathway were evaluated. The associations of the variants with the chemotherapy response and overall survival (OS) were analyzed. RESULTS: Among the 65 variants investigated, PFKL rs2073436C>G and GPI rs7248411C>G significantly correlated with clinical outcomes after chemotherapy in multivariate analyses. PFKL rs2073436C>G was significantly associated with both a worse response to chemotherapy (adjusted odds ratio [aOR] = 0.64, 95% CI = 0.45-0.90, p = 0.01) and a worse OS (adjusted hazard ratio [aHR] = 1.35, 95% CI = 1.14-1.61, p = 0.001). GPI rs7248411C>G was significantly associated with both a better chemotherapy response (aOR = 1.58, 95% CI = 1.07-2.23, p = 0.02) and a better OS (aHR = 0.80, 95% CI = 0.66-0.98, p = 0.03). When stratified by tumor histology, PFKL rs2073436C>G was significantly associated with OS only in squamous cell carcinoma, whereas GPI rs7248411C>G exhibited a significant association with the chemotherapy response and OS only in adenocarcinoma. CONCLUSION: This result suggests that the PFKL rs2073436C>G and GPI rs7248411C>G are useful for predicting the clinical outcome of first-line paclitaxel-cisplatin chemotherapy in NSCLC.

A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma.

Abnormal expression of the E3 ubiquitin ligase A20 has been found in some malignant cancers, including hepatocellular carcinoma (HCC). Here, we discovered that A20 is an E3 ubiquitin ligase for phosphofructokinase, liver type (PFKL) in HCC A20 interacts with PFKL and promotes its degradation, therefore inhibiting glycolysis in HCC cell lines. Downregulation of A20 in HCC cells promotes proliferation, migration, and glycolysis, all of which can be inhibited by targeting PFKL with RNA interference. Importantly, A20 is downregulated in advanced HCC tissues and inversely correlated with PFKL expression. Thus, our findings establish A20 as a critical regulator of glycolysis and reveal a novel mechanism for A20 in tumor suppression and PFKL regulation. Given that an increased level of glycolysis is linked with HCC, this study also identifies potential therapeutic targets for HCC treatment.

Integration of flux measurements to resolve changes in anabolic and catabolic metabolism in cardiac myocytes.

Although ancillary pathways of glucose metabolism are critical for synthesizing cellular building blocks and modulating stress responses, how they are regulated remains unclear. In the present study, we used radiometric glycolysis assays, [13C6]-glucose isotope tracing, and extracellular flux analysis to understand how phosphofructokinase (PFK)-mediated changes in glycolysis regulate glucose carbon partitioning into catabolic and anabolic pathways. Expression of kinase-deficient or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat neonatal cardiomyocytes co-ordinately regulated glycolytic rate and lactate production. Nevertheless, in all groups, >40% of glucose consumed by the cells was unaccounted for via catabolism to pyruvate, which suggests entry of glucose carbons into ancillary pathways branching from metabolites formed in the preparatory phase of glycolysis. Analysis of 13C fractional enrichment patterns suggests that PFK activity regulates glucose carbon incorporation directly into the ribose and the glycerol moieties of purines and phospholipids, respectively. Pyrimidines, UDP-N-acetylhexosamine, and the fatty acyl chains of phosphatidylinositol and triglycerides showed lower 13C incorporation under conditions of high PFK activity; the isotopologue 13C enrichment pattern of each metabolite indicated limitations in mitochondria-engendered aspartate, acetyl CoA and fatty acids. Consistent with this notion, high glycolytic rate diminished mitochondrial activity and the coupling of glycolysis to glucose oxidation. These findings suggest that a major portion of intracellular glucose in cardiac myocytes is apportioned for ancillary biosynthetic reactions and that PFK co-ordinates the activities of the pentose phosphate, hexosamine biosynthetic, and glycerolipid synthesis pathways by directly modulating glycolytic intermediate entry into auxiliary glucose metabolism pathways and by indirectly regulating mitochondrial cataplerosis.

The glycolytic enzyme phosphofructokinase-1 assembles into filaments.

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.

Identification of a multienzyme complex for glucose metabolism in living cells.

Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells.

Testicular lactate content is compromised in men with Klinefelter Syndrome.

Klinefelter syndrome (KS) is the most common genetic cause of human infertility, but the mechanism(s) responsible for its phenotype remain largely unknown. KS is associated with alterations in body composition and with a higher risk of developing metabolic diseases. We therefore hypothesized that KS men seeking fertility treatment possess an altered testicular metabolism profile that may hamper the nutritional support of spermatogenesis. Testicular biopsies from control (46, XY) (n = 6) and KS (47, XXY) (n = 6) individuals were collected and analyzed by proton high-resolution magic-angle spinning nuclear magnetic resonance spectroscopy. The mRNA and protein expression of crucial glycolysis-associated enzymes and transporters were evaluated in parallel by quantitative PCR and Western blot, respectively. Our data revealed altered regulation of glucose transporters (GLUT1 and GLUT3); phosphofructokinase 1, liver isoform (PFKL); and lactate dehydrogenase A (LDHA) expression in the testis of KS patients. Moreover, we detected a severe reduction in lactate and creatine accumulation within testicular tissue from KS men. The aberrant levels of the biomarkers detected in testicular biopsies of KS men may therefore be associated with the infertility phenotypes presented by these men. Mol. Reprod. Dev. 83: 208-216, 2016. © 2015 Wiley Periodicals, Inc.

Hypoglycemic effect of deoxynojirimycin-polysaccharide on high fat diet and streptozotocin-induced diabetic mice via regulation of hepatic glucose metabolism.

Type 2 diabetes mellitus (T2DM) is currently considered a worldwide epidemic and finding effective therapeutic strategies against this disease is highly important. A deoxynojirimycin-polysaccharide mixture (DPM) has previously been shown to exert hypoglycemic effects on alloxan- or streptozotocin (STZ)-induced diabetic mice. The purpose of the present study was to evaluate the therapeutic effects and underlying mechanism(s) of DPM on T2DM induced by high fat diet following low-dose STZ treatment in mice. After daily oral treatment of diabetic mice with DPM (150 mg/kg b.w.) for 90 d, significant decline in blood glucose, pyruvate, triglyceride (TG), aspartate transaminase (AST), alanine transaminase (ALT), creatinine (Cr), lipid peroxide (LPO) and malondialdehyde (MDA) levels as well as evident increases in high density lipoprotein (HDL-c) and hepatic glycogen concentrations were observed. In the first stage, in which DPM was administered for 60 d, blood insulin levels did not undergo significant change but a significant decrease in the HOMA-IR index was detected. By contrast, the HOMA-IR index increased significantly in T2MD controls. In the second stage, in which DPM treatment was continued for another 30 d, insulin levels significantly increased in DPM-treated mice in comparison with T2DM controls. These results indicate that insulin resistance in the pre-diabetic period and the dysfunction of pancreatic ß-cells are ameliorated by DPM treatment. DPM also down-regulated protein levels of insulin receptor (IR) and gluconeogenic enzymes (pyruvate carboxylase, fructose-1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in peripheral tissues (liver and/or muscle), but enhanced the expressions of insulin in pancreas, lipoprotein lipase (LPL) and glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylase E1) in the liver. Furthermore, deoxynojirimycin (DNJ) and polysaccharide (P) were found to increase proliferation of hepatic LO-2 cells and scavenging of radicals in vitro. These results support the results of our biochemical analyses and underscore possible mechanisms underlying the protective effects of DPM on STZ-induced damage to the pancreas and the liver. Taken together, our findings suggest that DPM may be developed as an antihyperglycemic agent for the treatment of diabetes mellitus.

Phosphofructokinase 1 glycosylation regulates cell growth and metabolism.

Cancer cells must satisfy the metabolic demands of rapid cell growth within a continually changing microenvironment. We demonstrated that the dynamic posttranslational modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a key metabolic regulator of glucose metabolism. O-GlcNAcylation was induced at serine 529 of phosphofructokinase 1 (PFK1) in response to hypoxia. Glycosylation inhibited PFK1 activity and redirected glucose flux through the pentose phosphate pathway, thereby conferring a selective growth advantage on cancer cells. Blocking glycosylation of PFK1 at serine 529 reduced cancer cell proliferation in vitro and impaired tumor formation in vivo. These studies reveal a previously uncharacterized mechanism for the regulation of metabolic pathways in cancer and a possible target for therapeutic intervention.

Expression analysis of glycerol synthesis-related liver transcripts in rainbow smelt (Osmerus mordax) exposed to a controlled decrease in temperature.

Rainbow smelt (Osmerus mordax) accumulate high glycerol levels to avoid freezing at subzero temperatures. Glyceroneogenesis is activated by low temperature and occurs in liver via a branch in glycolysis and gluconeogenesis. In this study, carbohydrate and liver transcript levels of 21 genes potentially associated with glycerol production were assessed during a controlled warm to cold transition. Smelt were held at 8°C (warm smelt; non-glycerol accumulating) or subjected to a controlled decrease in water temperature from 8° to 0°C (cold smelt; glycerol accumulating) and sampled at the end of the temperature decrease and 1 mo later. In cold smelt compared with warm smelt, liver glycogen levels were lower and phosphoglucomutase transcript levels were higher. Plasma glycerol levels were higher and increased over time in cold smelt; in cold smelt, liver phosphofructokinase and pyruvate dehydrogenase kinase transcript levels increased over time. These findings imply that glycerol production is being fueled by glycogen degradation and inhibition of pyruvate oxidation serves to channel metabolic flux toward glycerol as opposed to complete glycolysis. Plasma glucose and liver glucose-6-phosphatase transcript levels were higher. Lipoprotein lipase transcript levels were higher, suggesting enhanced lipid breakdown to fuel energy metabolism. Glutamine synthetase transcript levels were higher, perhaps to store nitrogen for biosynthesis in spring.

Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells.

Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.

Beneficiary effect of Tinospora cordifolia against high-fructose diet induced abnormalities in carbohydrate and lipid metabolism in Wistar rats.

High intake of dietary fructose has been shown to exert a number of adverse metabolic eff ects in humans and experimental animals. The present study was designed to investigate the eff ect of the aqueous extract of Tinospora cordifolia stem (TCAE) on the adverse eff ects of fructose loading toward carbohydrate and lipid metabolism in rats. Adult male Wistar rats of body weight around 200 g were divided into four groups, two of which were fed with starch diet and the other two with high fructose (66 %) diet. Plant extract of TC (400 mg/kg/day) was administered orally to each group of the starch fed rats and the highfructose fed rats. At the end of 60 days of experimental period, biochemical parameters related to carbohydrate and lipid metabolism were assayed. Hyperglycemia, hyperinsulinemia, hypertriglyceridemia, insulin resistance, and elevated levels of hepatic total lipids, cholesterol, triglycerides, and free fatty acids (p < 0.05) observed in fructose-fed rats were completely prevented with TCAE treatment. Alterations in the activities of enzymes of glucose metabolism (hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, and glucose-6-phosphate dehydrogenase) and lipid metabolism (fatty acid synthetase, lipoprotein lipase, and malic enzyme) as observed in the high fructose-fed rats were prevented with TCAE administration. In conclusion, our fi ndings indicate improvement of glucose and lipid metabolism in high-fructose fed rats by treatment with Tinospora cordifolia, and suggest that the plant can be used as an adjuvant for the prevention and/or management of insulin resistance and disorders related to it.

Effect of guava (Psidium guajava Linn.) leaf soluble solids on glucose metabolism in type 2 diabetic rats.

This study investigated the effect of aqueous and ethanol soluble solid extracts of guava (Psidium guajava Linn.) leaves on hypoglycemia and glucose metabolism in type 2 diabetic rats. Low-dose streptozotocin (STZ) and nicotinamide were injected into Sprague-Dawley (SD) rats to induce type 2 diabetes. Acute and long-term feeding tests were carried out, and an oral glucose tolerance test (OGTT) to follow the changes in plasma glucose and insulin levels was performed to evaluate the antihyperglycemic effect of guava leaf extracts in diabetic rats.The results of acute and long-term feeding tests showed a significant reduction in the blood sugar level in diabetic rats fed with either the aqueous or ethanol extract of guava leaves (p < 0.05). Long-term administration of guava leaf extracts increased the plasma insulin level and glucose utilization in diabetic rats. The results also indicated that the activities of hepatic hexokinase, phosphofructokinase and glucose-6-phosphate dehydrogenase in diabetic rats fed with aqueous extracts were higher than in the normal diabetic group (p < 0.05). On the other hand, diabetic rats treated with the ethanol extract raised the activities of hepatic hexokinase and glucose-6-phosphate dehydrogenase (p < 0.05) only. The experiments provided evidence to support the antihyperglycemic effect of guava leaf extract and the health function of guava leaves against type 2 diabetes.

Purification and kinetic characterization of 6-phosphofructo-1-kinase from the liver of gilthead sea bream (Sparus aurata).

6-phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.

Two types of phosphofructokinase-1 differentially regulate the glycolytic pathway in insulin-stimulated chicken skeletal muscle.

To elucidate the precise regulation of glucose homeostasis in chicken skeletal muscle, expression of muscle- and liver-type phosphofructokinase-1 (EC:2.7.1.11, PFK-M, PFK-L) was characterized in the insulin-stimulated state by Real-Time PCR. Firstly, chicken PFK-M and PFK-L full-length cDNA sequences were identified. The deduced amino acid sequences were 81.6% and 86.5% identical with human PFK-M and PFK-L, respectively. In pectoralis superficialis (PS) muscle and extensor digitorum longus (EDL), PFK-M mRNA levels were unchanged following insulin stimulation. Surprisingly, although mammalian PFK-L has been reported to be expressed in liver, kidney and brain, chicken PFK-L was not detected in liver and kidney, however, strong expression was detected in skeletal muscle and brain by Northern blot analysis. However, using PCR, PFK-L mRNA was detected in liver. Taken together, chicken PFK-L mRNA expression was at a very low level, below the detection limit of Northern blot analysis. Chicken PFK-L mRNA levels were increased 200% in PS muscle but decreased by 40% in EDL following insulin stimulation. These results suggest that two types of PFK regulate the glycolytic pathway in the insulin-stimulated state and, therefore, that glucose metabolism in chicken skeletal muscle may be regulated in a very different manner compared to mammals.