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How phosphate levels are detected in mammals is unknown. The bone-derived hormone fibroblast growth factor 23 (FGF23) lowers blood phosphate levels by reducing kidney phosphate reabsorption and 1,25(OH)2D production, but phosphate does not directly stimulate bone FGF23 expression. Using PET scanning and LC-MS, we found that phosphate increases kidney-specific glycolysis and synthesis of glycerol-3-phosphate (G-3-P), which then circulates to bone to trigger FGF23 production. Further, we found that G-3-P dehydrogenase 1 (Gpd1), a cytosolic enzyme that synthesizes G-3-P and oxidizes NADH to NAD+, is required for phosphate-stimulated G-3-P and FGF23 production and prevention of hyperphosphatemia. In proximal tubule cells, we found that phosphate availability is substrate-limiting for glycolysis and G-3-P production and that increased glycolysis and Gpd1 activity are coupled through cytosolic NAD+ recycling. Finally, we show that the type II sodium-dependent phosphate cotransporter Npt2a, which is primarily expressed in the proximal tubule, conferred kidney specificity to phosphate-stimulated G-3-P production. Importantly, exogenous G-3-P stimulated FGF23 production when Npt2a or Gpd1 were absent, confirming that it was the key circulating factor downstream of glycolytic phosphate sensing in the kidney. Together, these findings place glycolysis at the nexus of mineral and energy metabolism and identify a kidney-bone feedback loop that controls phosphate homeostasis., Introduction Phosphate is essential for life, with a fundamental role in bone mineralization, cell signaling, synthesis of lipids and nucleic acids, and energy metabolism. Bacteria and yeast sense inorganic phosphate [...]

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial [F.sub.0][F.sub.1]-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or [alpha]-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of [F.sub.o][F.sub.1]-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized [HIF1.sub.[alpha]], leading to transcriptional activation of glycolysis. Cardiacspecific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives [F.sub.o][F.sub.1]-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart., Introduction Mitochondrial [F.sub.o][F.sub.1]-ATP synthase, or complex V, is composed of a soluble catalytic [F.sub.1] region (main subunit composition [[alpha].sub.3][[beta].sub.3][gamma]) in the mitochondrial matrix and a membrane-embedded [F.sub.o] region (1). The [...]

The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including L-serine, L-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1[alpha] in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect., Introduction Cardiac hypertrophy is an initial response of the heart to hemodynamic overload, including high blood pressure, termed pressure overload (PO). Long-term cardiac PO also often elicits other pathological effects [...]

Glycolysis, or its variations, is a fundamental metabolic pathway in life that functions in almost all organisms to decompose external or intracellular sugars. The pathway involves the partial oxidation and [...]

Beta adrenergic receptor blocking drugs ([beta]-blockers) are used chronically in many cardiovascular diseases such as hypertension, ischemic heart disease, arrhythmia, and heart failure. Beneficial effects are associated with the inhibition of symphathetic nervous system hyperactivity, reduction of heart rate, and remodeling by blocking the mitogenic activity of catecholamines. A possible effect of [beta]-blockers on substrate metabolism has also been suggested. The direct effects of [beta]-blockers on mouse C2C12 cells were investigated in this study. C2C12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and differentiated into myotubes in the same medium that contained 1% FBS. Palmitic acid oxidation and glycolysis were measured by using [9,10-[sup.3]H]palmitate and [5-[sup.3]H]glucose, respectively. The amount of [sup.3][H.sup.2]O was measured as an indicator of substrate usage. Carvedilol (100 [micro]mol/L) inhibited palmitate oxidation and increased glycolysis by nearly 50%. Prazosin altered substrate metabolism in a similar fashion as carvedilol, whereas propranolol or bisoprolol were devoid of metabolic effects. When added to mimic sympathetic activation, epinephrine stimulated glycolysis but did not alter fatty acid oxidation. Based on these results, carvedilol appears to have direct effects on substrate metabolism that are related to the blockade of [alpha]l adrenergic receptors. Key words: carvedilol, bisoprolol, [beta][beta]-blockers, C2C12, fatty acid oxidation, glycolysis. Les medicaments qui bloquent les recepteurs beta-adrenergiques ([beta]- bloquants) sont utilises de facon chronique pour controler plusieurs maladies cardiovasculaires comme l'hypertension, les maladies cardiaques ischemiques, l'arythmie et l'insuffisance cardiaque. Leurs effets benefiques sont associes a l'inhibition de l'hyperactivite du systeme nerveux sympathique, a la reduction du rythme cardiaque et au remodelage, en bloquant l'activite mitogene des catecholamines. Un effet possible des [beta]-bloquants sur le metabolisme energetique a aussi ete suggere. Les effets directs des [beta]-bloquants sur les cellules de souris C2C12 ont ete examines dans cette etude. Les cellules C2C12 ont ete cultivees dans du milieu DMEM contenant 10 % de serum de veau foetal (SVF) et ont ete differenciees en myotubes dans le meme milieu contenant 1 % de SVF. L'oxydation de l'acide palmitique et la glycolyse ont ete mesures a l'aide de [9,10-[sup.3]H]palmitate et de [5-[sup.3]H]glucose, respectivement. La quantite de [sup.3][H.sub.2]O pris comme indicateur de l'utilisation des substrats a ete mesuree. Le carvedilol (100 [micro]mol/L) inhibait l'oxydation du palmitate et augmentait la glycolyse de presque 50 %. Le prazosine modifiait le metabolisme energetique de la meme facon que le carvedilol alors que le propanolol et le bisoprolol etaient depourvus d'effets metaboliques. Lorsqu'ajoutee pour mimer l'activation sympathique, l'epinephrine stimulait la glycolyse mais ne modifiait pas l'oxydation des acides gras. Selon ces resultats, le carvedilol semble exercer des effets directs sur le metabolisme energetique qui sont relies au blocage des recepteurs [alpha]l adrenergiques. Mots-cles : carvedilol, bisoprolol, [beta]-bloquants, C2C12, oxydation des acides gras, glycolyse. [Traduit par la Redaction], Introduction Beta adrenergic receptor antagonists ([beta]-blockers) are the mainstay in the treatment of many cardiovascular diseases such as hypertension, ischemic heart disease, arrhythmia, and heart failure. In addition, they are [...]

Aminoglycolysis and simultaneous hydrolysis-aminoglycolysis of poly(ethylene terephthalate) (PET) wastes by diethanolamine (DEA) was attempted in the presence of xylene, with the aim of obtaining intermediates containing both hydroxyl and carboxyl end groups, which could be employed in polyurethane surface coatings. Aminoglycolysis and simultaneous hydrolysis-aminoglycolysis reactions were carried out with the catalysis of zinc acetate (ZnAc) at high temperatures and relatively high pressures. Depolymerization products were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimeter (DSC). To prepare polyurethane surface coating films, the depolymerization products were reacted with 1,4 butanediol (BDO) and toluene diisocyanate (TDI) in the presence of dibutiltin dilaurate (DBTDL) catalyst and pyrogallol inhibitor. After the films prepared, the physical properties such as hardness, drying degree, adhesion, and impact resistance of these films were investigated. The intermediates yielded from medium to very hard coatings with excellent adhesion and having medium to very good drying degree. POLYM. ENG. SCI., 51:746-754, 2011. © 2011 Society of Plastics Engineers, INTRODUCTION The chemical recycling of poly(ethylene terephthalate) (PET) is carried out with the aim of obtaining either its monomers such as terephthalic acid (TPA), ethylene glycol (EG), and bis(2-hydroxyethyl terephthalate) [...]

Amyloid-[beta] (A[beta]) plaque deposition can precede the clinical manifestations of dementia of the Alzheimer type (DAT) by many years and can be associated with changes in brain metabolism. Both the All plaque deposition and the changes in metabolism appear to be concentrated in the brain's default-mode network. In contrast to prior studies of brain metabolism which viewed brain metabolism from a unitary perspective that equated glucose utilization with oxygen consumption, we here report on regional glucose use apart from that entering oxidative phosphorylation (so-called 'aerobic glycolysis'). Using PET, we found that the spatial distribution of aerobic glycolysis in normal young adults correlates spatially with A[beta] deposition in individuals with DAT and cognitively normal participants with elevated A[beta], suggesting a possible link between regional aerobic glycolysis in young adulthood and later development of Alzheimer pathology. Alzheimer's disease | default mode network | positron emission tomography doi/ 10.1073/pnas.1010461107

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A. angiogenesis; progenitor cells; repair doi: 10.1152/ajplung.00274.2009.

Hypoxia-inducible factor (HIF)-l[alpha] is a key regulator of anaerobic energy metabolism. We asked the following question: Does the breakdown of microtubular structures influence glycolysis in hypoxic cardiomyocytes by regulating HIF-l[alpha] Neonatal rat cardiomyocytes were cultured under hypoxic conditions, while microtubule-stabilizing (pacfitaxel) and -depolymerizing (colchicine) agents were used to change microtubular structure. Models of high microtubule-associated protein 4 (MAP4) expression and RNA interference of microtubulin expression were established. Microtubular structural changes and intracellular HIF-l[alpha] protein distribution were observed with laser confocal scanning microscopy. Content of key glycolytic enzymes, viability, and energy content of cardiomyocytes were determined by colorimetry and high-performance liquid chromatography. HIF-l[alpha] protein content and mRNA expression were determined by Western blotting and real-time PCR, respectively. Low doses of microtubule-stabilizing agent (10 [micro]mol/1 paclitaxel) and enhanced expression of MAP4 stabilized the reticular microtubular structures in hypoxic cardiomyocytes, increased the content of key glycolytic enzymes, ameliorated energy supply and enhanced cell viability, and upregulated HIF-1[alpha] protein expression and endonuclear aggregation. In contrast, the microtubule-depolymerizing agent (10 [micro]mol/1 colchicine) or reduced microtubulin expression had adverse affects on the same parameters, in particular, HIF-l[alpha] protein content and endonuclear aggregation. We conclude that microtubular structural changes influence glycolysis in the early stages of hypoxia in cardiomyocytes by regulating HIF-1[alpha] content. Stabilizing microtubular structures increases endonuclear and total HIF-l[alpha] expression, content of key glycolytic enzymes, and energy supply. These findings provide potential therapeutic targets for ameliorating cell energy metabolism during early myocardial hypoxia. pacfitaxel; colchicine; microtubule-associated protein 4; interference RNA; PCR; immunofluorescence; glycolytic enzymes; ATP; ADP; pyruvate kinase; hexokinase; phosphofructokinase; lactate; [alpha]-microtubulin doi: 10.1152/ajpheart.01039.2009.

The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [[1-.sup.13]C]- and [[3-.sup.13]C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme. doi: 10.1128/JB.01281-09

Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism. doi: 10.1128/JB.00959-09

Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdhl with PFKFB3--either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1--or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/ C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer. aerobic glycolysis | cell cycle | PFKFB3 | cancer www.pnas.org/cgi/doi/10.1073/pnas.0913668107

In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed 'the Warburg effect.' Aerobic glycolysis is an inefficient way to generate adenosine 5'-triphosphate (ATP), however, and the advantage it confers to cancer cells has been unclear. Here we propose that the metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass (e.g., nucleotides, amino acids, and lipids) needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production. A better understanding of the mechanistic links between cellular metabolism and growth control may ultimately lead to better treatments for human cancer.

In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glyco-protein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.

The glucose analogue 2-deoxy-D-glucose (2-DG) restrains growth of normal and malignant cells, prolongs the lifespan of C. elegans, and is widely used as a glycolytic inhibitor to study metabolic activity with regard to cancer, neurodegeneration, calorie restriction, and aging. Here, we report that separating glycolysis and the pentose phosphate pathway highly increases cellular tolerance to 2-DG. This finding indicates that 2-DG does not block cell growth solely by preventing glucose catabolism. In addition, 2-DG provoked similar concentration changes of sugar-phosphate intermediates in wild-type and 2-DG-resistant yeast strains and in human primary fibroblasts. Finally, a genome-wide analysis revealed 19 2-DG-resistant yeast knockouts of genes implicated in carbohydrate metabolism and mitochondrial homeostasis, as well as ribosome biogenesis, mRNA decay, transcriptional regulation, and cell cycle. Thus, processes beyond the metabolic block are essential for the biological properties of 2-DG. cell growth inhibition | glycolysis | off-target effect | pentose phosphate pathway | carbohydrate metabolism

ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation. glycosome | PEX14 | regulation of glycolysis | systems biology | Trypanosoma brucei

At concentrations around [10.sup.-9] M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed [10.sup.-10] M. We investigated whether physiological concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon doseresponse curves emerged. One was an expected increase in left ventricular pressure (LVP) occurring between [10.sup.-9.5] and [10.sup.-8] M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration ([10.sup.-8] M) were blocked by the AC inhibitor NKY80 (20 [micro]M). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels ([10.sup.-11] to [10.sup.-10] M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon ([10.sup.-10] M) also increased glucose oxidation and suppressed palmitate oxidation, mimicking known effects of insulin, without altering ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon ([10.sup.-10] M) or insulin (4 x [10.sup.-10] M) were abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (10 [micro]M) but not significantly affected by NKY80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3K, and these effects were also abolished by LY-294002. The results are consistent with the hypothesis that physiological levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3K and not AC/cAMP. insulin; glycolysis; phosphoinositide 3-kinase; cyclic adenosine monophosphate

The insulin-responsive glucose transporter 4 (GLUT4) plays a key role in glucose uptake and metabolism in insulin target tissues. Being a rate-limiting step in glucose metabolism, the expression and function of the GLUT4 isoform has been extensively studied and found to be tightly regulated at both mRNA and protein levels. Adaptation to states of enhanced metabolic demand is associated with increased glucose metabolism and GLUT4 gene expression, whereas states of insulin resistance such as type 2 diabetes mellitus (DM2), obesity, and aging are associated with impaired regulation of GLUT4 gene expression and function. The present review focuses on the interplay among hormonal, nutritional, and transcription factors in the regulation of GLUT4 transcription in health and sickness. adipose; diabetes; gene expression; muscle; obesity; peroxisome proliferator-activated receptor-[gamma]; forkhead box O1

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-l,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. Key words: pork, glycolysis, citrate, acetate, meat quality

The Rapoport--Luebering glycolytic bypass comprises evolutionar-ily conserved reactions that generate and dephosphorylate 2,3bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyosteli-um, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase, Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mipp 1 expression in Dictyostelium, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37[degrees]C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4[degrees]C, revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered. 2,3-BPG | erythrocyte | glycolysis

In this study, we have investigated the hypothesis that an exercise protocol designed to repeatedly induce a large dependence on carbohydrate and large increases in glycolytic flux rate would result in rapid increases in the principal glucose and lactate transporters in working muscle, glucose transporter (GLUT)-4 and monocarboxylate transporter (MCT)4, respectively, and in activity of hexokinase (Hex), the enzyme used to phosphorylate glucose. Transporter abundance and Hex activity were assessed in homogenates by Western blotting and quantitative chemiluminescence and fluorometric techniques, respectively, in samples of tissue obtained from the vastus lateralis in 12 untrained volunteers [peak aerobic power ([Vo.sub.2peak]) = 44.3 -+ 2.3 ml*[kg.sup.-1]*[min.sup.-1]] before cycle exercise at repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). The 16 repetitions of the exercise were performed for 6 min at ~90% [Vo.sub.2peak], once per hour. Compared with R1, GLUT-4 increased (P < 0.05) by 28% at R2 and remained elevated (P < 0.05) at R9 and R16. For MCT-4, increases (P < 0.05) of 24% were first observed at R9 and persisted at R16. No changes were observed in GLUT-1 and MCT-1 or in Hex activity. The ~17- to 24-fold increase (P < 0.05) in muscle lactate observed at R1 and R2 was reduced (P < 0.05) to an 11-fold increase at R9 and R16. It is concluded that an exercise protocol designed to strain muscle carbohydrate reserves and to result in large increases in lactic acid results in a rapid upregulation of both GLUT-4 and MCT-4. contractile activity; muscle; glucose transporters; lactate transporters; glycolysis

Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-l,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro [sup.31]p NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.

SgrS is a 227-nt small RNA that is expressed in Escherichia coil during glucose-phosphate stress, a condition associated with intracellular accumulation of glucose-6-phosphate caused by disruption of glycolytic flux. Under stress conditions, SgrS negatively regulates translation and stability of the ptsG mRNA, encoding the major glucose transporter, by means of a base pairing-dependent mechanism requiring the RNA chaperone Hfq. SgrS activity mitigates the effects of glucose-phosphate stress, and the present study has elucidated a function of SgrS that is proposed to contribute to the stress response. The 5' end of SgrS, upstream of the nucleotides involved in base pairing with the ptsG mRNA, contains a 43-aa ORF, sgrT, that is conserved in most species that contain SgrS-like small RNAs. The sgrT gene is translated in E. coil under conditions of glucose-phosphate stress. Analysis of alleles that separate the base pairing function of SgrS from the sgrT coding sequence revealed that either of these functions alone are sufficient for previously characterized SgrS phenotypes. SgrS-dependent down-regulation of ptsG mRNA stability does not require SgrT and SgrT by itself has no effect on ptsG mRNA stability. Cells expressing sgrT alone had a defect in glucose uptake even though they had nearly wild-type levels of PtsG ([IICB.sup.Glc]). Together, these data suggest that SgrS represents a previously unrecognized paradigm for small RNA (sRNA) regulators as a bifunctional RNA that encodes physiologically redundant but mechanistically distinct functions contributing to the same stress response. riboregulation | RNA stability | small proteins I phosphoeno|pyruvate phosphotransferase system | glycolytic flux

Phosphofructokinase is a key enzyme of glycolysis that exists as homoand heterotetramers of three subunit isoforms: muscle, liver, and C type. Mice with a disrupting tag inserted near the distal promoter of the phosphofructokinase-M gene showed tissue-dependent differences in loss of that isoform: 99% in brain and 95-98% in islets, but only 50-75% in skeletal muscle and little if any loss in heart. This correlated with the continued presence of proximal transcripts specifically in muscle tissues. These data strongly support the proposed two-promoter system of the gene, with ubiquitous use of the distal promoter and additional use of the proximal promoter selectively in muscle. Interestingly, the mice were glucose intolerant and had somewhat elevated fasting and fed blood glucose levels; however, they did not have an abnormal insulin tolerance test, consistent with the less pronounced loss of phosphofructokinase-M in muscle. Isolated pefifused islets showed about 50% decreased glucosestimulated insulin secretion and reduced amplitude and regularity of secretory oscillations. Oscillations in cytoplasmic free [Ca.sup.2+] and the rise in the ATP/ADP ratio appeared normal. Secretory oscillations still occurred in the presence of diazoxide and high KC1, indicating an oscillation mechanism not requiring dynamic [Ca.sup.2+] changes. The results suggest the importance of phosphofructokinase-M for insulin secretion, although glucoldnase is the overall rate-limiting glucose sensor. Whether the [Ca.sup.2+] oscillations and residual insulin oscillations in this mouse model are due to the residual 2-5% phosphofructoldnase-M or to other phosphofunctokinase isoforms present in islets or involve another metabolic oscillator remains to be determined. OmniBank; calcium; pancreatic islets; adenosine 5'-triphosphate/ adenosine 5'-diphosphate ratio

The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh, encoding NADH dehydrogenase II, and cyoABCDE, encoding the cytochrome bo-type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system.

The role of glycolytically generated ATP in [Ca.sup.2+]/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular [Ca.sup.2+] signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular [Ca.sup.2+] concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + [beta]-hydroxybutyrate ([beta]-HB), or iodoacetic acid (IAA) caused an increase of intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [[[Ca.sup.2+]].sub.i]. The rise of [[[Ca.sup.2+]].sub.i] was characterized by a transient spike followed by a small sustained plateau of elevated [[[Ca.sup.2+]].sub.i]. In the absence of extracellular [Ca.sup.2+] 2-DG caused an increase in [[[Ca.sup.2+]].sub.i], suggesting that inhibition of glycolysis directly triggered release of [Ca.sup.2+] from intracellular endoplasmic reticulum (ER) [Ca.sup.2+] stores. The inositol-1,4,5-trisphosphate receptor ([IP.sub.3]R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced [Ca.sup.2+] response. [Ca.sup.2+] release was initiated in peripheral cytoplasmic processes from which activation propagated as a [[[Ca.sup.2+]].sub.i] wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [[[Ca.sup.2+]].sub.i]. Propagating [[[Ca.sup.2+]].sub.i] waves were preceded by [[[Ca.sup.2+]].sub.i] oscillations and small, highly localized elevations of [[[Ca.sup.2+]].sub.i] ([Ca.sup.2+] puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced [Ca.sup.2+] response, and vice versa during inhibition of CaMKII 2-DG-induced [Ca.sup.2+] release was attenuated. Similar results were obtained with pyr + [beta]-HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular [Mg.sup.2+] concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER [Ca.sup.2+] release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the [IP.sub.3]R. [Ca.sup.2+]/calmodulin-dependent kinase II; glycolysis; calcium regulation doi:10.1152/ajpcell.00543.2006

Fifty years ago, Kornberg and Krebs established the glyoxylate cycle as the pathway for the synthesis of cell constituents from [C.sub.2]-units. However, since then, many bacteria have been described that do not contain isocitrate lyase, the key enzyme of this pathway. Here, a pathway termed the ethylmalonyl-CoA pathway operating in such organisms is described. Isotopically labeled acetate and bicarbonate were transformed to ethylmalonyl-CoA by cell extracts of acetate-grown, isocitrate lyase-negative Rhodobacter sphaeroides as determined by NMR spectroscopy. Crotonyl-CoA carboxylase/reductase, catalyzing crotonyl-CoA + C[O.sub.2] + NADPH [right arrow] ethylmalonyl-[CoA.sup.-] + [NADP.sup.+] was identified as the key enzyme of the ethylmalonyl-CoA pathway. The reductive carboxylation of an enoyl-thioester is a unique biochemical reaction, unprecedented in biology. The enzyme from R. sphaeroides was heterologously produced in Escherichia coli and characterized. Crotonyl-CoA carboxylase/reductase (or its gene) can be used as a marker for the presence of the ethylmalonyl-CoA pathway, which functions not only in acetyl-CoA assimilation. In Streptomyces sp., it may also supply precursors (ethylmalonyl-CoA) for antibiotic biosynthesis. For methylotrophic bacteria such as Methylobacterium extorquens, extension of the serine cycle with reactions of the ethylmalonyl-CoA pathway leads to a simplified scheme for isocitrate lyase-independent [C.sub.1] assimilation. acetyl-CoA assimilation | glyoxylate cycle | methylotrophy | polyketide | serine cycle