Bone marrow mesenchymal stem cell-derived exosomal miR-21a-5p alleviates renal fibrosis by attenuating glycolysis by targeting PFKM.

Renal fibrosis is a common pathological feature and outcome of almost all chronic kidney diseases, and it is characterized by metabolic reprogramming toward aerobic glycolysis. Mesenchymal stem cell-derived exosomes (MSC-Exos) have been proposed as a promising therapeutic approach for renal fibrosis. In this study, we investigated the effect of MSC-Exos on glycolysis and the underlying mechanisms. We demonstrated that MSC-Exos significantly ameliorated unilateral ureter obstruction (UUO)-induced renal fibrosis by inhibiting glycolysis in tubular epithelial cells (TECs). miRNA sequencing showed that miR-21a-5p was highly enriched in MSC-Exos. Mechanistically, miR-21a-5p repressed the expression of phosphofructokinase muscle isoform (PFKM), a rate-limiting enzyme of glycolysis, thereby attenuating glycolysis in TECs. Additionally, knockdown of miR-21a-5p abolished the renoprotective effect of MSC-Exos. These findings revealed a novel role for MSC-Exos in the suppression of glycolysis, providing a new insight into the treatment of renal fibrosis.

Association of PFKM gene polymorphisms and susceptibility to cryptorchidism in a Chinese Han population.

BACKGROUND: Cryptorchidism is one of the most common congenital anomalies in newborn boys. There are various risk factors that have been verified to have relationship with cryptorchidism, including exogenous and genetic, but the pathogenesis of cryptorchidism remains unclear. PFKM gene is a critical gene encodes for a regulatory enzyme, which limits the rate in the pathway of glycolysis. We assumed that cryptorchidism risk may associated with PFKM gene single-nucleotide polymorphisms (SNPs). Thus we selected three tag SNPs in the PFKM gene and aimed to investigate the possible association between PFKM gene polymorphisms and cryptorchidism risk. METHODS: The SNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. 140 cases and 227 controls were enrolled in this study, including 105 unilateral cryptorchidism and 35 bilateral cases. The testis position was decided by the higher one in bilateral cases. RESULTS: The frequency of allele G of SNP rs2228500 is increased in cryptorchidism patients compared to that in controls (p < 0.05). Genotypic frequencies of rs2228500 are associated with the susceptibility of cryptorchidism in the codominant model (p < 0.05). And compared with G/G genotype in the dominant model, notable decreased frequencies of A carriers (A/G-A/A genotypes) were observed in cryptorchidism patients (p = 0.0069, OR = 1.80, 95% CI 1.17-2.75). CONCLUSIONS: This research first revealed that PFKM gene polymorphisms were associated with cryptorchidism in a Chinese Han population. We have offered primary evidence that the G allele and the G/G genotype of rs2228500 SNP in the PFKM gene are more frequent in patients with cryptorchidism than healthy controls.

MicroRNA-383-5p Regulates Oxidative Stress in Mice with Acute Myocardial Infarction through the AMPK Signaling Pathway via PFKM.

OBJECTIVE: The purpose of this study is to explore the regulating role of microRNA-383-5p (miR-383-5p) in oxidative stress after acute myocardial infarction (AMI) through AMPK pathway via phosphofructokinase muscle-type (PFKM). METHODS: We established the AMI model, and the model mice were injected with miR-383-5p agomir to study the effect of miR-383-5p in AMPK signaling pathways. The target gene for miR-383-5p was reported to be PFKM, so we hypothesized that overexpression of miR-383-5p inhibits activation of the AMPK signaling pathway. RESULTS: In this research, we found that overexpression of miR-383-5p decreases myocardial oxidative stress, myocardial apoptosis, the expression level of PFKM malondialdehyde (MDA), and reactive oxygen species (ROS) in the myocardial tissues after AMI, and finally, AMI-induced cardiac systolic and diastolic function could be improved. CONCLUSION: This study demonstrated that miR-383-5p could reduce the oxidative stress after AMI through AMPK signaling pathway by targeting PFKM.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

ZEB1 enhances Warburg effect to facilitate tumorigenesis and metastasis of HCC by transcriptionally activating PFKM.

Metabolic reprogramming, especially Warburg effect, is a key event in tumor initiation and progression. ZEB1 plays a vital role in metastasis of various cancers. We previously found that ZEB1 was excessively expressed in hepatocellular carcinoma (HCC) and its high expression was closely correlated with metastasis and recurrence of HCC. We want to know whether glycolytic enzymes are regulated by ZEB1 and contribute to carcinogenesis and metastasis of HCC. Methods: To explore whether ZEB1 could enhance glycolysis in HCC, we knocked down ZEB1 by short hairpin RNA (shRNA) in MHCC-97H and HCC-LM3 cells and performed glucose uptake, lactate production, ECAR and OCR assays. To investigate how ZEB1 enhances glycolysis, the protein levels of glycolytic enzymes were detected in the same cell lines using Western blot. The regulatory effect of ZEB1 on PFKM mRNA level was confirmed by RT-qPCR, luciferase report assay and ChIP assay. In order to assess the role of ZEB1-PFKM axis in cell proliferation, cell counting and CCK-8 assays were performed in MHCC-97H and HCC-LM3 cell lines knocked down for ZEB1 and further re-expressed for either ZEB1 or PFKM or not. To explored whether the ZEB1-PFKM axis also functions in HCC cell migration, invasion and metastasis, the same MHCC-97H and HCC-LM3 cell lines were performed for wound healing assays, transwell assays and colony formation assays, meanwhile, MHCC-97H cell lines were performed for orthotopic liver transplantation assays. Finally, the expression of ZEB1 and PFKM were examined in human liver cancer specimens and non-tumorous liver tissues using immunohistochemical and Western blot. Results: We found that ZEB1 transcriptionally upregulates the expression of the muscle isoform of phosphofructokinase-1 (PFKM), a rate-limiting enzyme in glycolysis. Intriguingly, a non-classic ZEB1-binding sequence in the promoter region of PFKM was identified through which ZEB1 directly activates the transcription of PFKM. Silencing of ZEB1 in MHCC-97H and HCC-LM3 cell leads to impaired PFKM expression, glycolysis, proliferation and invasion, and such impairments are rescued by exogenous expression of PFKM. Importantly, in-situ HCC xenograft assays and studies from TCGA database demonstrate that ZEB1-PFKM axis is crucial for carcinogenesis and metastasis of HCC. Conclusions: Our study reveals a novel mechanism of ZEB1 in promoting HCC by activating the transcription of PFKM, establishing the direct link of ZEB1 to the promotion of glycolysis and Warburg effect and suggesting that inhibition of ZEB1 transcriptional activity toward PFKM may be a potential therapeutic strategy for HCC.

miRNA biomarkers for predicting overall survival outcomes for head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a malignant tumor of the upper aerodigestive tract. The loss and gain of miRNA function promote cancer development through various mechanisms. RNA sequencing (RNA-seq) and miRNAs sequencing data from the Cancer Genome Atlas (TCGA) was used to show the dysfunctional miRNAs microenvironment and to provide useful biomarkers for miRNAs therapy. Seven miRNAs were found to be independent prognostic factors of HNSCC patients in the training cohort. A total of 60 target genes for these miRNAs were predicted. Nine target genes (CDCA4, CXCL14, FLNC, KLF7, NBEAL2, P4HA1, PFKM, PFN2 and SEPPINE1) were correlated with patient's overall survival (OS) outcomes. We identified novel miRNAs markers for the prognosis of head and neck squamous cell carcinoma.

Splice variant of growth hormone-releasing hormone receptor drives esophageal squamous cell carcinoma conferring a therapeutic target.

The extrahypothalamic growth hormone-releasing hormone (GHRH) and its cognate receptors (GHRH-Rs) and splice variants are expressed in a variety of cancers. It has been shown that the pituitary type of GHRH-R (pGHRH-R) mediates the inhibition of tumor growth induced by GHRH-R antagonists. However, GHRH-R antagonists can also suppress some cancers that do not express pGHRH-R, yet the underlying mechanisms have not been determined. Here, using human esophageal squamous cell carcinoma (ESCC) as a model, we were able to reveal that SV1, a known splice variant of GHRH-R, is responsible for the inhibition induced by GHRH-R antagonist MIA-602. We demonstrated that GHRH-R splice variant 1 (SV1) is a hypoxia-driven promoter of tumor progression. Hypoxia-elevated SV1 activates a key glycolytic enzyme, muscle-type phosphofructokinase (PFKM), through the nuclear factor kappa B (NF-κB) pathway, which enhances glycolytic metabolism and promotes progression of ESCC. The malignant actions induced by the SV1-NF-κB-PFKM pathway could be reversed by MIA-602. Altogether, our studies demonstrate a mechanism by which GHRH-R antagonists target SV1. Our findings suggest that SV1 is a hypoxia-induced oncogenic promoter which can be an alternative target of GHRH-R antagonists.

A uniparental isodisomy event introducing homozygous pathogenic variants drives a multisystem metabolic disorder.

Uniparental isodisomy (UPiD) is a rare genetic event that occurs when two identical copies of a single chromosome are inherited from one parent. Here we report a patient with a severe, multisystem metabolic disorder who inherited two copies of Chromosome 12 from her father. He was a heterozygous carrier of a variant in the muscle-specific enzyme 6-phosphofructokinase (PFKM) gene and of a truncating variant in the pseudouridine synthase 1 (PUS1) gene (both on Chromosome 12), resulting in a homozygous state of these mutations in his daughter. The PFKM gene functions in glycolysis and is linked to Tarui syndrome. The PUS1 gene functions in mitochondrial tRNA processing and is linked to myopathy, lactic acidosis, and sideroblastic anemia (MLASA). Analysis of human dermal fibroblasts, which do not express PFKM, revealed a loss of PUS1 mRNA and PUS1 protein only in the patient cells compared to healthy controls. The patient cells also revealed a reduction of the mitochondrial-encoded protein MTCO1, whereas levels of the nuclear-encoded SDHA remained unchanged, suggesting a specific impairment of mitochondrial translation. Further destabilization of these cells is suggested by the altered levels of BAX, BCL-2, and TP53 proteins, alterations that become augmented upon exposure of the cells to DNA damage. The results illustrate the efficacy of UPiD events to reveal rare pathogenic variants in human disease and demonstrate how these events can lead to cellular destabilization.

Glycolytic reprogramming in cancer cells: PKM2 dimer predominance induced by pulsatile PFK-1 activity.

The glycolytic enzyme pyruvate kinase M2 (PKM2) exists in both catalytically inactive dimeric and active tetrameric forms. In cancer cells, PKM2 dimer predominance contributes to tumor growth by triggering glycolytic reprogramming. However, the mechanism that promotes PKM2 dimer predominance over tetramer in cancer cells remains elusive. Here, we show that pulsatile phosphofructokinase (PFK-1) activity results in PKM2 dimer predominance. Mathematical simulations predict that pulsatile PFK-1 activity prevents the formation of PKM2 tetramer even under high levels of fructose-1,6-bisphosphate (FBP), a PKM2 tetramer-promoting metabolite produced by PFK-1. We experimentally confirm these predictions at the single-molecule level by providing evidence for pulsatile PFK-1 activity-induced synchronized dissociation of PKM2 tetramers and the subsequent accumulation of PKM2 dimers under high levels of FBP in HeLa cells. Moreover, we show that pulsatile PFK-1 activity-induced PKM2 dimer predominance also controls cell proliferation. Thus, our study reveals the significance of pulsatile PFK-1 activity in cancer cell metabolism.

Kallikrein-related peptidase 6 can cleave human-muscle-type 6-phosphofructo-1-kinase into highly active shorter fragments.

PURPOSE: Cancer cells consume more glucose than normal human cells and convert most glucose into lactate. It has been proposed that deregulated glycolysis is triggered by the posttranslational modification of 85 kDa muscle-type 6-phosphofructo-1-kinase (PFK-M) which is cleaved by a specific protease to form shorter, highly active, feedback-inhibition-resistant PFK-M fragments. PRINCIPAL RESULTS: To find the protease involved in PFK-M modification, analyses of the protease target sites on the human PFK-M enzyme yielding 45-47 kDa fragments were performed in silico. The results suggested that an enzyme in the kallikrein (KLK) family may be involved. Kallikreins can be self-activated in the cytosol and are often overexpressed in cancer cells. After incubating the internally quenched FRET peptide with a sequence characteristic of the target site, along with the active KLK6, the cleavage of the peptide was observed. The ability of KLK6 to cleave native PFK-M and form highly active citrate-resistant 45 kDa fragments was further confirmed by enzymatic tests and SDS-PAGE. A role of KLK6 in the posttranslational modification of native PFK-M was ultimately confirmed in vivo. A yeast strain that encoded native human PFK-M as the only PFK1 enzyme was additionally transformed with proKLK6 or KLK6 genes under the control of an inducible promoter. The transformants growth rate was found to increase after the induction of proKLK6 gene expression as compared to the strain with the native PFK-M enzyme. CONCLUSION: KLK6 may be the key protease involved in the modification of PFK-M and trigger deregulated glycolytic flux in cancer cells.

Differential expression of genes related to glucose metabolism in domesticated pigs and wild boar.

Glucose metabolism is a basic biological process that shows substantial variation within and between species. Using pig as a model organism, we investigated differences in glucose metabolic genes in seven tissues from domesticated pigs (Rongchang pig and Tibetan pig, meanwhile, the Tibetan pig just as a special case of the domesticated pig under plateau condition) and wild boar. We found large differences in the expression of genes involved in multiple aspects of glucose metabolism, including genes associated with glucose transport, gluconeogenesis, and glycolysis. In addition, we identified microRNAs (miRNAs) that may be involved in the divergence of glucose metabolism in pig. A combined analysis of mRNA and miRNA expression indicated that some miRNA:mRNA pairs showed ab facto function in it. Our results provide a valuable resource for further determination of miRNA regulatory roles in pig glucose metabolism and reveal the divergence of glucose metabolism in pigs under domestication.

Genetic polymorphisms in glycolytic pathway are associated with the prognosis of patients with early stage non-small cell lung cancer.

This study was conducted to investigate whether polymorphisms of genes involved in glycolysis are associated with the prognosis of patients with non-small cell lung cancer (NSCLC) after surgical resection. Forty-four single nucleotide polymorphisms (SNPs) of 17 genes in glycolytic pathway were investigated in a total of 782 patients with NSCLC who underwent curative surgical resection. The association of the SNPs with overall survival (OS) and disease free survival (DFS) were analyzed. Among the 44 SNPs investigated, four SNPs (ENO1 rs2274971A > G, PFKM rs11168417C > T, PFKP rs1132173C > T, PDK2 rs3785921G > A) were significantly associated with survival outcomes in multivariate analyses. When stratified by tumor histology, three SNPs (ENO1 rs2274971A > G, PFKM rs11168417C > T, and PDK2 rs3785921G > A) were significantly associated with OS and/or DFS only in squamous cell carcinoma, whereas PFKP rs1132173C > T exhibited a significant association with survival outcomes only in adenocarcinoma. When the four SNPs were combined, OS and DFS decreased as the number of bad genotypes increased (Ptrend = 8 × 10-4 and 3 × 10-5, respectively). Promoter assays showed that ENO1 rs2274971G allele had significantly higher promoter activity compared to the rs2274971A allele. The four SNPs, especially ENO1 rs2274971A > G, may be useful for the prediction of prognosis in patients with surgically resected NSCLC.

Molecular characterization, expression profile, and association study with meat quality traits of porcine PFKM gene.

Glycolytic potential is a hot aspect to meat quality research in recent years. Phosphofructokinase, muscle type (PFKM), is a key regulatory enzyme used to catalyze the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in glycolysis. The present study was designed to investigate the association of PFKM SNP and meat quality traits in pigs. In this study, the 2,864-bp full-length cDNA sequence of the porcine PFKM gene was obtained, which contained 30 bp of 5' UTR, 2,343 bp of coding region, and 491 bp of 3' UTR. The porcine PFKM mRNA was predominantly expressed in skeletal muscle and heart. One single nucleotide polymorphism (SNP) T129C in exon 13 of PFKM gene was detected, with its allele frequencies significantly different between Chinese indigenous pig breed and Western pig breeds. The SNP was significantly associated with meat color value (m. biceps femoris), meat marbling (m. longissimus dorsi), meat marbling (m. biceps femoris), intramuscular fat (m. longissimus dorsi) (P<0.01), and water moisture (m. longissimus dorsi) in the Large White×Meishan F2 population. These results laid a foundation for further investigations on the detailed physiological function of porcine PFKM gene.

A genome-wide association study of early-onset breast cancer identifies PFKM as a novel breast cancer gene and supports a common genetic spectrum for breast cancer at any age.

Early-onset breast cancer (EOBC) causes substantial loss of life and productivity, creating a major burden among women worldwide. We analyzed 1,265,548 Hapmap3 single-nucleotide polymorphisms (SNP) among a discovery set of 3,523 EOBC incident cases and 2,702 population control women ages ≤ 51 years. The SNPs with smallest P values were examined in a replication set of 3,470 EOBC cases and 5,475 control women. We also tested EOBC association with 19,684 genes by annotating each gene with putative functional SNPs, and then combining their P values to obtain a gene-based P value. We examined the gene with smallest P value for replication in 1,145 breast cancer cases and 1,142 control women. The combined discovery and replication sets identified 72 new SNPs associated with EOBC (P < 4 × 10(-8)) located in six genomic regions previously reported to contain SNPs associated largely with later-onset breast cancer (LOBC). SNP rs2229882 and 10 other SNPs on chromosome 5q11.2 remained associated (P < 6 × 10(-4)) after adjustment for the strongest published SNPs in the region. Thirty-two of the 82 currently known LOBC SNPs were associated with EOBC (P < 0.05). Low power is likely responsible for the remaining 50 unassociated known LOBC SNPs. The gene-based analysis identified an association between breast cancer and the phosphofructokinase-muscle (PFKM) gene on chromosome 12q13.11 that met the genome-wide gene-based threshold of 2.5 × 10(-6). In conclusion, EOBC and LOBC seem to have similar genetic etiologies; the 5q11.2 region may contain multiple distinct breast cancer loci; and the PFKM gene region is worthy of further investigation. These findings should enhance our understanding of the etiology of breast cancer.

Expression of phosphofructokinase in skeletal muscle is influenced by genetic variation and associated with insulin sensitivity.

Using an integrative approach in which genetic variation, gene expression, and clinical phenotypes are assessed in relevant tissues may help functionally characterize the contribution of genetics to disease susceptibility. We sought to identify genetic variation influencing skeletal muscle gene expression (expression quantitative trait loci [eQTLs]) as well as expression associated with measures of insulin sensitivity. We investigated associations of 3,799,401 genetic variants in expression of >7,000 genes from three cohorts (n = 104). We identified 287 genes with cis-acting eQTLs (false discovery rate [FDR] <5%; P < 1.96 × 10(-5)) and 49 expression-insulin sensitivity phenotype associations (i.e., fasting insulin, homeostasis model assessment-insulin resistance, and BMI) (FDR <5%; P = 1.34 × 10(-4)). One of these associations, fasting insulin/phosphofructokinase (PFKM), overlaps with an eQTL. Furthermore, the expression of PFKM, a rate-limiting enzyme in glycolysis, was nominally associated with glucose uptake in skeletal muscle (P = 0.026; n = 42) and overexpressed (Bonferroni-corrected P = 0.03) in skeletal muscle of patients with T2D (n = 102) compared with normoglycemic controls (n = 87). The PFKM eQTL (rs4547172; P = 7.69 × 10(-6)) was nominally associated with glucose uptake, glucose oxidation rate, intramuscular triglyceride content, and metabolic flexibility (P = 0.016-0.048; n = 178). We explored eQTL results using published data from genome-wide association studies (DIAGRAM and MAGIC), and a proxy for the PFKM eQTL (rs11168327; r(2) = 0.75) was nominally associated with T2D (DIAGRAM P = 2.7 × 10(-3)). Taken together, our analysis highlights PFKM as a potential regulator of skeletal muscle insulin sensitivity.

[Metabolic myopathies]. / Miopatías metabólicas.

AIM: To review the metabolic myopathies manifested only by crisis of myalgias, cramps and rigidity of the muscles with decreased voluntary contractions and normal inter crisis neurologic examination in children and adolescents. DEVELOPMENT: These metabolic myopathies are autosomic recessive inherited enzymatic deficiencies of the carbohydrates and lipids metabolisms. The end result is a reduction of intra muscle adenosine triphosphate, mainly through mitochondrial oxidative phosphorylation, with decrease of available energy for muscle contraction. The one secondary to carbohydrates intra muscle metabolism disorders are triggered by high intensity brief (< 10 min) exercises. Those secondary to fatty acids metabolism disorders are triggered by low intensity prolonged (> 10 min) exercises. The conditions in the first group in order of decreasing frequency are the deficiencies of myophosforilase (GSD V), muscle phosphofructokinase (GSD VII), phosphoglycerate mutase 1 (GSD X) and beta enolase (GSD XIII). The conditions in the second group in order of decreasing frequency are the deficiencies of carnitine palmitoyl transferase II and very long chain acyl CoA dehydrogenase. CONCLUSIONS: The differential characteristics of patients in each group and within each group will allow to make the initial presumptive clinical diagnosis in the majority and then to order only the necessary tests to achieve the final diagnosis. Treatment during the crisis includes hydration, glucose and alkalinization of urine if myoglobin in blood and urine are elevated. Prevention includes avoiding exercise which may induce the crisis and fasting. The prognosis is good with the exception of rare cases of acute renal failure due to hipermyoglobinemia because of severe rabdomyolisis.

TITLE: Miopatias metabolicas.Objetivo. Revisar las miopatias metabolicas manifestadas solamente por crisis de mialgias, calambres y rigidez musculares con dificultad para contraer los musculos afectados y el examen neurologico normal entre las crisis en niños y adolescentes. Desarrollo. Estas miopatias metabolicas se deben a deficits enzimaticos heredados en forma autosomica recesiva del metabolismo de los carbohidratos y lipidos. El resultado final es una reduccion del trifosfato de adenosina principalmente a traves de la fosforilacion oxidativa mitocondrial con disminucion de la energia disponible para la contraccion muscular. Las secundarias a trastornos del metabolismo de los carbohidratos se producen por ejercicios de alta intensidad y breves (< 10 min) y las secundarias a trastornos de los lipidos, por ejercicios de baja intensidad y prolongados (> 10 min). Los deficits enzimaticos en el primer grupo son de miofosforilasa (glucogenosis V), fosfofructocinasa muscular (glucogenosis VII), fosfoglicerato mutasa 1 (glucogenosis X) y beta enolasa (glucogenosis XIII), y en el segundo, de carnitina palmitol transferasa tipo II y de acil-CoA deshidrogenasa de cadena muy larga. Conclusiones. Las caracteristicas diferenciales de los pacientes en cada grupo y dentro de cada grupo permitiran el diagnostico clinico presuntivo inicial en la mayoria y solicitar solamente los examenes necesarios para corroborar el diagnostico. El tratamiento de las crisis consiste en hidratacion, glucosa y alcalinizacion de la orina. Las medidas preventivas son evitar el tipo de ejercicio que induce las crisis y el ayuno. No existe cura o tratamiento especifico. El pronostico es bueno con la excepcion de casos raros de insuficiencia renal aguda debido a la elevacion sanguinea de la mioglobina producto de una rabdomiolisis grave.

Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.

Glycolysis is the initial step of glucose catabolism and is up-regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H(2)O(2)-treated myotubes, we show up-regulation of the rate-limiting glycolytic enzyme muscle-type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P<0.05). Using microRNA profiling, we identify miR-320a as a regulator of PFKm expression. Reduced miR-320a levels (to ∼50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR-320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR-320a appears to regulate oxidative stress-induced PFKm expression, and reduced miR-320a allows greater induction of glycolysis in response to H(2)O(2) treatment. We show that this microRNA-mediated regulation occurs through PFKm's 3' untranslated region and that Ets proteins are involved in the regulation of PFKm via miR-320a. These findings suggest that oxidative stress-responsive microRNA-320a may regulate glycolysis broadly within nature.

Functional linkage of adenine nucleotide binding sites in mammalian muscle 6-phosphofructokinase.

6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).

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.