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The dynamic changes of RNA N6-methyladenosine (m 6 A) during cancer progression participate in various cellular processes. However, less is known about a possible direct connection between upstream regulator and m 6 A modification, and therefore affects oncogenic progression. Here, we have identified that a key enzyme in N4-acetylcytidine (ac4C) acetylation NAT10 is highly expressed in human osteosarcoma tissues, and its knockdown enhanced m 6 A contents and significantly suppressed osteosarcoma cell growth, migration and invasion. Further results revealed that NAT10 silence inhibits mRNA stability and translation of m 6 A reader protein YTHDC1, and displayed an increase in glucose uptake, a decrease in lactate production and pyruvate content. YTHDC1 recognizes differential m 6 A sites on key enzymes of glycolysis phosphofructokinase (PFKM) and lactate dehydrogenase A (LDHA) mRNAs, which suppress glycolysis pathway by increasing mRNA stability of them in an m 6 A methylation-dependent manner. YTHDC1 partially abrogated the inhibitory effect caused by NAT10 knockdown in tumor models in vivo, lentiviral overexpression of YTHDC1 partially restored the reduced stability of YTHDC1 caused by lentiviral depleting NAT10 at the cellular level. Altogether, we found ac4C driven RNA m 6 A modification can positively regulate the glycolysis of cancer cells and reveals a previously unrecognized signaling axis of NAT10/ac4C-YTHDC1/m 6 A-LDHA/PFKM in osteosarcoma. Video Abstract., (© 2024. The Author(s).)

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., (© 2022. The Author(s).)

The reprogramming of energy metabolism is one of the hallmarks of cancer and is crucial for tumor progression. Altered aerobic glycolysis is a well-known characteristic of cancer cell metabolism. In the present study, the expression profiles of key metabolic genes (HK2, PFKM, and PKM2) were assessed in the breast cancer cohort of Pakistan using quantitative polymerase chain reaction (qPCR) and IHC. Expression patterns were correlated with molecular subtypes and clinical parameters in the patients. A significant upregulation of key glycolytic genes was observed in tumor samples in comparison to their adjacent controls (p < 0.0001). The expression of the studied glycolytic genes was significantly increased in late clinical stages, positive nodal involvement, and distant metastasis (p < 0.05). HK2 and PKM2 were found to be upregulated in luminal B, whereas PFKM was overexpressed in the luminal A subtype of breast cancer. The genes were positively correlated with the proliferation marker Ki67 (p < 0.001). Moreover, moderate positive linear correlations between HK2 and PKM2 (r = 0.476), HK2 and PFKM (r = 0.473), and PKM2 and PFKM (r = 0.501) were also observed (p < 0.01). These findings validate that the key regulatory genes in glycolysis can serve as potential biomarkers and/or molecular targets for breast cancer management. However, the clinical significance of these molecules needs to be further validated through in vitro and in vivo experiments.

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., Competing Interests: The authors declared no conflict of interest., (Copyright © 2021 Linlin Gao et al.)

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., Competing Interests: Declaration of interests V.K.M. is a paid scientific advisor to 5AM Ventures and Janssen Pharmaceuticals. O.S.S. is a paid consultant for Proteinaceous. R.S. holds equity in BlueBird Bio. G.W.Y. is co-founder, member of the Board of Directors, on the scientific advisory board, equity holder, and paid consultant for Locanabio and Eclipse Bioinnovations. G.W.Y. is a visiting professor at the National University of Singapore. G.W.Y.’s interest(s) have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. A.A.J. and V.K.M. are co-inventors on a US provisional patent application related to the work in this manuscript. The authors declare no other competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.

Dental pulp stem cells (DPSCs) can differentiate into diverse cell lineages, including odontogenic cells that are responsible for dentin formation, which is important in pulp repair and tooth regeneration. While glycolysis plays a central role in various cellular activities in both physiological and pathological conditions, its role and regulation in odontogenic differentiation are unknown. Here, we show that aerobic glycolysis is induced during odontoblastic differentiation from human DPSCs. Importantly, we demonstrate that during odontoblastic differentiation, protein expression levels of phosphofructokinase 1 muscle isoform (PFKM) and PFK2, but not other glycolytic enzymes, are mainly upregulated by AKT activation, resulting in increased total PFK enzyme activity. Increased PFK activity is essential to enhance aerobic glycolysis, which plays an important role in the odontoblastic differentiation of human DPSCs. These findings underscore that PFK activation-induced aerobic glycolysis accompanies, and participates in, human DPSCs differentiation into odontogenic lineage, and could play a role in the regulation of dental pulp repair., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Phosphofructokinase-M (PFKM) is a key enzyme in glycolysis. The expression and activity of PFKM is closely related to the occurrence and development of malignant tumors, but its role in the regulation of renal cell carcinoma (RCC) is still unknown. We found that the expression of PFKM was lower in RCC tumor tissue than in adjacent normal tissues, and that low expression of PFKM was related to the poor overall survival of RCC patients. In addition, our results showed that FOXO3 mediated PFKM inhibited the growth, migration and invasion of RCC cells, suggesting that PFKM is a protective factor for RCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interest statement: The Sponsor declares a conflict of interest. A.V.S. is a coinventor on the patent for growth hormone-releasing hormone analogs, assigned to the University of Miami, Miami, FL, and the Veterans Affairs Medical Center, Miami, FL. The other authors declare no conflict of interest., (Copyright © 2020 the Author(s). Published by PNAS.)

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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Insulin secretion from pancreatic islet β-cells occurs in a pulsatile fashion, with a typical period of ∼5 min. The basis of this pulsatility in mouse islets has been investigated for more than four decades, and the various theories have been described as either qualitative or mathematical models. In many cases the models differ in their mechanisms for rhythmogenesis, as well as other less important details. In this Perspective, we describe two main classes of models: those in which oscillations in the intracellular Ca 2+ concentration drive oscillations in metabolism, and those in which intrinsic metabolic oscillations drive oscillations in Ca 2+ concentration and electrical activity. We then discuss nine canonical experimental findings that provide key insights into the mechanism of islet oscillations and list the models that can account for each finding. Finally, we describe a new model that integrates features from multiple earlier models and is thus called the Integrated Oscillator Model. In this model, intracellular Ca 2+ acts on the glycolytic pathway in the generation of oscillations, and it is thus a hybrid of the two main classes of models. It alone among models proposed to date can explain all nine key experimental findings, and it serves as a good starting point for future studies of pulsatile insulin secretion from human islets., (© 2018 by the American Diabetes Association.)

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., (© 2017 Webb et al.)

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.

Background: Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1 (-/-) mouse (iMSBmal1 (-/-))., Results: Progressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1 (-/-) mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1 (-/-) muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1 (-/-) muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle., Conclusions: These data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.

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.

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.

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.

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).

Aim: Endurance and resistance training (ET and RT, respectively) in older subjects have been proven beneficial against metabolic or cardiovascular disorders and against sarcopaenia respectively. Like ET, RT may also increase muscle oxidative capacities. In addition, it could be questioned whether RT, similarly to ET, is able to increase muscle energetic stores such as intra-myocellular lipids (IMCL) and glycogen contents. To evaluate a possible ET- and RT-induced parallel increase in oxidative capacity and energetic stores, active elderly men (72 ± 2 years) were submitted to a 14-week training programme (three times week(-1) ) combining lower body endurance and upper body resistance., Methods: Muscle samples were collected in ET vastus lateralis (VLat) and RT deltoid (Del) muscles before and after training. IMCL and glycogen contents were assessed by histochemistry (Oil Red O and periodic acid-Schiff staining, respectively) and by biochemical assay for glycogen. Citrate synthase (CS, marker of mitochondrial citric acid cycle), β-hydroxyacyl coenzyme A dehydrogenase (β-HAD, beta-oxidation) and phosphofructokinase (PFK, glycolytic pathway) activities were determined and so was the capillary interface index (LC/PF)., Results: Both training regimens significantly increased CS and LC/PF in ET-VLat and RT-Del. IMCL content and β-HAD activity increased (P < 0.05) only in ET-VLat, whereas PFK activity increased (P < 0.05) only in RT-Del. Glycogen content was not significantly altered in response to training in both muscles., Conclusion: Unlike RT, which induced an increase in PFK, ET is able to increase IMCL content and β-oxidation capacity in active elderly men, even though both training may improve CS activity and LC/PF., (© 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.)

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., (Copyright © 2011 Wiley Periodicals, Inc.)

Longissimus muscle samples from the pig genotypes Duroc (Du), Pietrain (MHS homozygote negative (PiNN), positive (PiPP)) and a Duroc-Pietrain crossbreed (DuPi) were analyzed. The PiPP samples showed a faster pH drop and higher electrical conductivity, drip loss and lightness values. Before slaughter the concentrations of the adenine nucleotides were comparable between the genotypes, but 40 min after slaughter (p.m.) the ATP concentrations decreased and IMP increased, to a higher extent in the PiPP pigs. The nucleotide values of the 12 h p.m. samples were again comparable. Activities of glycogen phosporylase (GP), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were nearly similar before slaughter. Forty minutes after slaughter the LDH activities increased in all pigs and the PFK activities in all genotypes but not in the PiPP. GP results were rather inconsistent indicating an earlier activation of this enzyme. The study showed that the reduced meat quality in the PiPP pigs is accompanied with rapid ATP degradation and accelerated enzyme activation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Human cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved., Methodology/principal Findings: In the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux., Conclusions/significance: Posttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.

Muscle glycogen synthase (GYS1) is the rate-limiting enzyme in glycogen synthesis, and its activity is regulated by the phosphorylation states of certain amino acid residues encoded by the GYS1 gene. In the present study, the authors molecularly characterized the full-length equine GYS1 (eGYS1) cDNA and found that it contains a less common polyadenylation signal (AATACA). An amino acid alignment with other mammalian GYS1 showed that the phosphorylation sites in eGYS1 are completely conserved. Genomic DNA analysis revealed that the equine-specific substitutions (Glu 16 Asp and Ala 252 Thr) were completely conserved among six equine species. The tissue expression profiles of eGYS1, equine type II hexokinase (eHKII) and muscle-type phosphofructokinase (ePFKM) were determined by real-time PCR and western blot analysis. The mRNA expression level of eGYS1 was significantly higher in the cervical muscle as compared to other tissues. The cervical muscle and heart tissue samples contained a broad range of eGYS1 protein bands that appeared to reflect multiple phosphorylation states. eHKII was predominately expressed only in the cervical muscle; unlike its expression in other mammals, eHKII was not substantially expressed in the insulin-responsive heart or adipose tissue of horse. The expression level of ePFKM mRNA was significantly higher in the heart than in the cervical muscle, which differs from the PFKM expression pattern of other mammals. These tissue expression profiles are fundamental for the understanding of equine glucose metabolism.

Phosphofructokinase is a sophisticated allosteric enzyme that is fundamental for the control of glycolysis. The structure of the bacterial enzyme is well characterized. However, little is known about the structural organization of the more complex enzyme from mammals. We have obtained the structure of human muscle phosphofructokinase in the presence of fructose 6-phosphate at a resolution of 1.8 nm by electron microscopy (EM). Particles of the tetrameric enzyme corresponded to an elongated molecule (14.5 × 9 nm) arranged into 2 dimeric subdomains. Image analysis and 3-dimensional reconstruction showed the presence of a prominent channel in one of the dimers but not in the opposite one, revealing that they are in greatly different conformations. Fitting of bacterial structures into the EM model suggested disruption of the fructose 6-phosphate catalytic and the fructose 2,6-bisphophate allosteric sites in the cavity-containing dimer. Therefore, the reported structure might have major implications for the function of mammalian phosphofructokinase.

Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory mechanism that is modulated by a large variety of metabolites, drugs, and intracellular proteins. To date, the mammalian enzyme structure has not yet been resolved. However, it is known that PFK undergoes an intricate oligomerization process, shifting among monomers, dimers, tetramers, and more complex oligomeric structures. The equilibrium between PFK dimers and tetramers is directly correlated with the enzyme regulation, because the dimer exhibits very low catalytic activity, whereas the tetramer is fully active. Several PFK ligands modulate the enzyme, favoring the formation of its dimers or tetramers. The present review integrates recent findings regarding the regulatory aspects of muscle type PFK and discusses their relation to the control of metabolism.

We hypothesized that a season of ice hockey would result in extensive remodeling of muscle. Tissue sampled from the vastus lateralis of 15 players (age = 20.6 ± 0.4 years; mean ± SE) prior to (PRE) and following (POST) a season was used to characterize specific adaptations. Measurement of representative metabolic pathway enzymes indicated higher maximal activities in POST than in PRE (p < 0.05) for succinic dehydrogenase (3.26 ± 0.31 vs. 3.91 ± 0.11 mol mg protein(-1) min(-1)), citrate synthase (7.26 ± 0.70 vs. 8.70 ± 0.55 mol mg protein(-1) min(-1)), and phosphofructokinase (12.8 ± 1.3 vs. 14.4 ± 0.96 mol mg protein(-1) min(-1)) only. The season resulted in an increase in Na+-K+-ATPase concentration (253 ± 6.3 vs. 265 ± 6.0 pmol g(-1) wet weight), a decrease (p < 0.05) in maximal activity of the sarcoplasmic reticulum Ca2+-ATPase (107 ± 4.2 micromol g protein(-1) min(-1) vs. 92.0 ± 4.6 micromol g protein(-1) min(-1)), and no change in the distribution (%) of fibre types. A smaller (p < 0.05) cross-sectional area (CSA) for both type I (-11.7%) and type IIA (-18.2%) fibres and a higher (p < 0.05) capillary count/CSA for type I (+17.9%) and type IIA (+17.2%) were also found over the season. No changes were found in peak oxygen consumption (51.4 ± 1.2 mL kg(-1) min(-1) vs. 52.3 ± 1.3 mL kg(-1) min(-1)). The results suggest, based on the alterations in oxidative and perfusion potentials and muscle mass, that the dominant adaptations are in support of oxidative metabolism, which occurs at the expense of fibre CSA and possibly force-generating potential.

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4, HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4, HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells., (2010 Elsevier Inc. All rights reserved.)

Synthesis of triacylglycerol requires the glucose-derived glycerol component, and glucose uptake has been viewed as the rate-limiting step in glucose metabolism in adipocytes. Furthermore, adipose tissue contains all three isoforms of the glycolytic enzyme phosphofructokinase (PFK). We here report that mice deficient in the muscle isoform PFK-M have greatly reduced fat stores. Mice with disrupted activity of the PFK-M distal promoter were obtained from Lexicon Pharmaceuticals, developed from OmniBank OST#56064. Intra-abdominal fat was measured by magnetic resonance imaging of the methylene proton signal. Lipogenesis from labeled glucose was measured in isolated adipocytes. Lipolysis (glycerol and free fatty acid release) was measured in perifused adipocytes. Intra-abdominal fat in PFK-M-deficient female mice (5-10 months old) was 17 +/- 3% of that of wild-type littermates (n = 4; P < 0.02). Epididymal fat weight in 15 animals (7-9.5 months) was 34 +/- 4% of control littermate (P < 0.002), with 10-30% lower body weight. Basal and insulin-stimulated lipogenesis in PFK-M-deficient epididymal adipocytes was 40% of the rates in cells from heterozygous littermates (n = 3; P < 0.05). The rate of isoproterenol-stimulated lipolysis in wild-type adipocytes declined approximately 10% after 1 h and 50% after 2 h; in PFK-M-deficient cells it declined much more rapidly, 50% in 1 h and 90% in 2 h, and lipolytic oscillations appeared to be damped (n = 4). These results indicate an important role for PFK-M in adipose metabolism. This may be related to the ability of this isoform to generate glycolytic oscillations, because such oscillations may enhance the production of the triacylglycerol precursor alpha-glycerophosphate.

Mammalian sperm require ATP for motility, and most of it is generated by glycolysis. The glycolytic enzymes segregate to the principal piece region of the flagellum, where some are bound tightly to a novel cytoskeletal structure defining this region, the fibrous sheath (FS), and others are easily extracted with detergents. One of the latter is the spermatogenic cell-specific variant isozyme of hexokinase type 1 (HK1S), characterized by an N-terminal 24-amino acid spermatogenic cell-specific region (SSR). Yeast two-hybrid screens carried out using the SSR as bait determined that HK1S is tethered to muscle-type phosphofructokinase (PFKM) in the principal piece region. This led to the identification of four testis-specific Pfkm splice variants, one that overlapped a variant reported previously (Pfkm&#95;v1) and three that were novel (Pfkm&#95;v2, Pfkm&#95;v3, and Pfkm&#95;v4). They differ from Pfkm transcripts found in somatic cells by encoding a novel 67-amino acid N-terminal extension, the testis-specific region (TSR), producing a spermatogenic cell-specific PFKM variant isozyme (PFKMS). An antiserum generated to the TSR demonstrated that PFKMS is present in the principal piece and is insoluble in 1% Triton X-100 detergent. In subsequent yeast two-hybrid screens, the TSR was found to interact with glutathione S-transferase mu class 5 (GSTM5), identified previously as a spermatogenic cell-specific component of the FS. These results demonstrated that HK1S is tethered in the principal piece region by PFKMS, which in turn is bound tightly to GSTM5 in the FS.

In muscle tissue the protein caveolin-3 forms caveolae--flask-shaped invaginations localized on the cytoplasmic surface of the sarcolemmal membrane. Caveolae have a key role in the maintenance of plasma membrane integrity and in the processes of vesicular trafficking and signal transduction. Mutations in the caveolin-3 gene lead to skeletal muscle pathology through multiple pathogenetic mechanisms. Indeed, caveolin-3 deficiency is associated to sarcolemmal membrane alterations, disorganization of skeletal muscle T-tubule network and disruption of distinct cell-signaling pathways. To date, there have been 30 caveolin-3 mutations identified in the human population. Caveolin-3 defects lead to four distinct skeletal muscle disease phenotypes: limb girdle muscular dystrophy, rippling muscle disease, distal myopathy, and hyperCKemia. In addition, one caveolin-3 mutant has been described in a case of hypertrophic cardiomyopathy. Many patients show an overlap of these symptoms and the same mutation can be linked to different clinical phenotypes. This variability can be related to additional genetic or environmental factors. This review will address caveolin-3 biological functions in muscle cells and will describe the muscle and heart disease phenotypes associated with caveolin-3 mutations.

The present study examined muscle adaptations and alterations in work capacity in endurance-trained runners as a result of a reduced amount of training combined with speed endurance training. For a 6- to 9-wk period, 17 runners were assigned to either a speed endurance group with a 25% reduction in the amount of training but including speed endurance training consisting of six to twelve 30-s sprint runs 3-4 times/wk (SET group n = 12) or a control group (n = 5), which continued the endurance training ( approximately 55 km/wk). For the SET group, the expression of the muscle Na(+)-K(+) pump alpha(2)-subunit was 68% higher (P < 0.05) and the plasma K(+) level was reduced (P < 0.05) during repeated intense running after 9 wk. Performance in a 30-s sprint test and the first of the supramaximal exhaustive runs was improved (P < 0.05) by 7% and 36%, respectively, after the speed endurance training period. In the SET group, maximal O(2) uptake was unaltered, but the 3-km (3,000-m) time was reduced (P < 0.05) from 10.4 +/- 0.1 to 10.1 +/- 0.1 min and the 10-km (10,000-m) time was improved from 37.3 +/- 0.4 to 36.3 +/- 0.4 min (means +/- SE). Muscle protein expression and performance remained unaltered in the control group. The present data suggest that both short- and long-term exercise performances can be improved with a reduction in training volume if speed endurance training is performed and that the Na(+)-K(+) pump plays a role in the control of K(+) homeostasis and in the development of fatigue during repeated high-intensity exercise.

The objective of this study was to determine the effects of oral administration of sodium citrate (CIT) or acetate (ACE) to pigs on blood parameters, postmortem glycolysis, pH decline, and quality attributes of pork. Previous studies have shown that CIT has the potential to inhibit phosphofructokinase (PFK), a key enzyme in postmortem muscle glycolysis. In Exp. 1, CIT, ACE, or water was orally administered (0.75 g/kg of BW) to 24 pigs. After a 30-min rest, pigs were exercised, and blood samples were taken at 45 and 75 min after oral treatment. Citrate and ACE tended (P = 0.08) to increase blood pH and increased (P = 0.02) bicarbonate levels immediately after exercise. After a 30-min rest, blood pH of pigs administered ACE tended (P = 0.09) to remain higher, whereas blood pH of CIT-treated pigs was similar to that of control pigs. Bicarbonate levels in ACE- and CIT-treated pigs were still greater (P < 0.05) than those of control pigs at 75 min after oral treatment. In Exp. 2, 30 pigs were administered CIT, ACE, or water 45 min before stunning (electric plus captive bolt). Antemortem treatments had no effect (P > 0.10) on muscle pH or postmortem concentrations of the glycolytic metabolites of glucose-6 phosphate, fructose-6 phosphate, fructose-1,6 bisphosphate, glyceraldehyde-3 phosphate, dihydroxyacetone phosphate, or lactate. Minor, but inconsistent, differences in quality attributes were found in LM chops, and no differences in quality attributes were found between control and CIT- or ACE-treated pigs for inside and outside semimembranosus muscles (P > 0.10). There was no significant inhibition of the PFK enzyme by orally administered CIT or ACE; however, the PFK glycolytic metabolite data analysis indicated that PFK was a main regulatory enzyme in postmortem muscle.

Objective: To determine insulin sensitivity, proportions of muscle fiber types, and activities of glycogenolytic and glycolytic enzymes in Belgians with and without polysaccharide storage myopathy (PSSM)., Animals: 10 Quarter Horses (QHs) and 103 Belgians in which PSSM status had been determined., Procedures: To determine insulin sensitivity, a hyperinsulinemic euglycemic clamp (HEC) technique was used in 5 Belgians with PSSM and 5 Belgians without PSSM. Insulin was infused i.v. at 3 mU/min/kg for 3 hours, and concentrations of blood glucose and plasma insulin were determined throughout. An i.v. infusion of glucose was administered to maintain blood glucose concentration at 100 mg/dL. Activities of glycogenolytic and glycolytic enzymes were assessed in snap-frozen biopsy specimens of gluteus medius muscle obtained from 4 Belgians with PSSM and 5 Belgians without PSSM. Percentages of type 1, 2a, and 2b muscle fibers were determined via evaluation of >or= 250 muscle fibers in biopsy specimens obtained from each Belgian used in the aforementioned studies and from 10 QHs (5 with PSSM and 5 without PSSM)., Results: Belgians with and without PSSM were not significantly different with respect to whole-body insulin sensitivity, muscle activities of glycogenolytic and glycolytic enzymes, or proportions of muscle fiber types. However, Belgians had an increased proportion of type 2a and decreased proportion of type 2b muscle fibers, compared with proportions in QHs, regardless of PSSM status., Conclusions and Clinical Relevance: PSSM in Belgians may be attributable to excessive glycogen synthesis rather than decreased glycogen utilization or enhanced glucose uptake into muscle cells.

A substrate cycle composed of phosphofructo 1-kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca(2+)]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH-nitroblue tetrazolium-linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose- and glucose-6-P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co-regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca(2+) signals that can be important for sperm development and function.

Two phosphofructokinase (PFK) chimeras were constructed by exchanging the N- and C-terminal halves of the mammalian M- and C-type isozymes, to investigate the contribution of each terminus to the catalytic site and the fructose-2,6-P(2)/fructose-1,6-P(2) allosteric site. The homogeneously-purified chimeric enzymes organized into tetramers, and exhibited kinetic properties for fructose-6-P and MgATP similar to those of the native enzyme that furnished the N-terminal domain in each case, whereas their fructose-2,6-P(2) activatory characteristics coincided with those of the isozyme that provided the C-terminal half. This reflected the role of each domain in the formation of the corresponding binding site. Grafting the N-terminus of PFK-M onto the C-terminus of the fructose-1,6-P(2) insensitive PFK-C restored transduction of this signal to the catalytic site, which significance is also discussed.

This study aimed to determine the impact of short-term normobaric hypoxia on physiology and performance in highly trained athletes. Twelve (7 male and 5 female) athletes were randomly assigned into two groups and spent 8 h per night for two consecutive nights a week over 3 weeks under either short-term normobaric hypoxia (simulating 3636 m altitude, inspired O2=13%) or in normobaric normoxia in a single-blind study. Following a 3 week washout period, athletes were then exposed to the other condition. Athletes were tested for maximal oxygen consumption and time to exhaustion on an electromagnetically braked cycle ergometer before and after each treatment in addition to being tested for anaerobic performance (Wingate test) on a modified Monark cycle ergometer. Blood samples were taken throughout the experiment and vastus lateralis muscle biopsies were taken before and after each treatment. Increases in red blood cell count, haematocrit, haemoglobin, platelet number and erythropoietin concentration were observed following short-term normobaric hypoxia. Except for a modest decrease in phosphofructokinase activity following short-term normobaric hypoxia, no changes were observed in muscle enzyme activities, buffer capacity, capillary density or morphology. No performance measures were changed following short-term normobaric hypoxia or normobaric normoxia. Although short-term normobaric hypoxia exposure increased levels of a number of haematological parameters, this was not associated with improved aerobic or anaerobic performance in highly trained athletes.

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.

The present study investigates the effects of power training on mechanical efficiency (ME) in jumping. Twenty-three subjects, including ten controls, volunteered for the study. The experimental group trained twice a week for 15 weeks performing various jumping exercises such as drop jumps, hurdle jumps, hopping and bouncing. In the maximal jumping test, the take-off velocity increased from 2.56 (0.24) m.s(-1) to 2.77 (0.18) m.s(-1) ( P<0.05). In the submaximal jumping of 50% of the maximum, energy expenditure decreased from 660 (110) to 502 (68) J.kg(-1).min(-1) ( P<0.001) while, simultaneously, ME increased from 37.2 (8.4)% to 47.4 (8.2)% ( P<0.001). Some muscle enzyme activities of the gastrocnemius muscle increased during the training period: citrate synthase from 35 (8) to 39 (7) micromol.g(-1) dry mass.min(-1) ( P<0.05) and beta-hydroxyacyl CoA dehydrogenase from 21 (4) to 23 (5) micromol.g(-1) dry mass.min(-1) ( P<0.05), whereas no significant changes were observed in phosphofructokinase and lactate dehydrogenase. In the control group, no changes in ME or in enzyme activities were observed. In conclusion, the enhanced performance capability of 8% in maximal jumping as a result of power training was characterized by decreased energy expenditure of 24%. Thus, the increased neuromuscular performance, joint control strategy, and intermuscular coordination (primary factors), together with improved aerobic capacity (secondary factor), may result in reduced oxygen demands and increased ME.

The associations between skeletal muscle morphological and metabolic properties and the changes in body composition and metabolic rates in response to long-term overfeeding were investigated in 24 healthy young male identical twins (12 pairs). The proportions of muscle fiber types (type I, type IIA, and type IIB) and the activities of creatine kinase (CK), oxoglutarate dehydrogenase (OGDH), and phosphofructokinase (PFK) were determined from biopsies of the vastus lateralis before and after the overfeeding protocol. Body weight, fat mass (FM), fat-free mass (FFM), percent body fat (%FAT), resting metabolic rate (RMR), and thermic effect of a standardized meal (TEM) were also measured before and after 100 days of overfeeding. Type I muscle fiber proportions correlated inversely with the changes of FM and %FAT (r = -0.43, P =.035; r = -0.49, P =.01), and type IIA positively with the same overfeeding-induced changes (r = 0.43, P =.035; r = 0.47, P =.021). Baseline CK and PFK activities correlated negatively with the changes of RMR (r = -0.49, P =.017; r = -0.53, P =.01). OGDH activity at baseline correlated negatively with the changes of FM (r = -0.47, P = 0.02) but the ratio of PFK/OGDH correlated positively with the change of FM (r = 0.46, P =.02). We conclude that overfeeding induced a lower gain of FM in individuals with higher proportions of type I fiber, lower proportions of type IIA fiber, and higher OGDH activities at baseline. CK and PFK activities at baseline were associated with an attenuated increase in RMR when challenged by overfeeding. The significant correlations range from 0.43 to 0.53, and account for 18% to 28% of the variance in the response to overfeeding. The results suggest that an elevated skeletal muscle oxidative capacity plays a protective role in the response to long-term positive energy balance., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.