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.

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.

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.