Palmitate inhibits liver glycolysis. Involvement of fructose 2,6-bisphosphate in the glucose/fatty acid cycle.
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ABSTRACT:
SUBMITTER: Hue L
PROVIDER: S-EPMC1149034 | biostudies-other | 1988 Apr
REPOSITORIES: biostudies-other
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Project description:The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS(483)VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.
| S-EPMC165633 | biostudies-literature
Project description:Incubation of isolated rat hepatocytes from fasted rats with 0-6 mM-glucose caused an increase in [fructose 2,6-bisphosphate] (0.2 to about 5 nmol/g) without net lactate production. A release of 3H2O from [3-3H]glucose was, however, detectable, indicating that phosphofructokinase was active and that cycling occurred between fructose 6-phosphate and fructose 1,6-bisphosphate. A relationship between [fructose 2,6-bisphosphate] and lactate production was observed when hepatocytes were incubated with [glucose] greater than 6 mM. Incubation with glucose caused a dose-dependent increase in [hexose 6-phosphates]. The maximal capacity of liver cytosolic proteins to bind fructose 2,6-bisphosphate was 15 nmol/g, with affinity constants of 5 X 10(6) and 0.5 X 10(6) M-1. One can calculate that, at 5 microM, more than 90% of fructose 2,6-bisphosphate is bound to cytosolic proteins. In livers of non-anaesthetized fasted mice, the activation of glycogen synthase was more sensitive to glucose injection than was the increase in [fructose 2,6-bisphosphate], whereas the opposite situation was observed in livers of fed mice. Glucose injection caused no change in the activity of liver phosphofructokinase-2 and decreased the [hexose 6-phosphates] in livers of fed mice.
| S-EPMC1144513 | biostudies-other
Project description:The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.
| S-EPMC1147977 | biostudies-other
Project description:1. Incubation of hepatocytes from fed or starved rats with increasing glucose concentrations caused a stimulation of lactate production, which was further increased under anaerobic conditions. 2. When glycolysis was stimulated by anoxia, [fructose 2,6-bis-phosphate] was decreased, indicating that this ester could not be responsible for the onset of anaerobic glycolysis. In addition, the effect of glucose in increasing [fructose 2,6-bisphosphate] under aerobic conditions was greatly impaired in anoxic hepatocytes. [Fructose 2,6-bisphosphate] was also diminished in ischaemic liver, skeletal muscle and heart. 3. The following changes in metabolite concentration were observed in anaerobic hepatocytes: AMP, ADP, lactate and L-glycerol 3-phosphate were increased; ATP, citrate and pyruvate were decreased: phosphoenolpyruvate and hexose 6-phosphates were little affected. Concentrations of adenine nucleotides were, however, little changed by anoxia when hepatocytes from fed rats were incubated with 50 mM-glucose. 4. The activity of ATP:fructose 6-phosphate 2-phosphotransferase was not affected by anoxia but decreased by cyclic AMP. 5. The role of fructose 2,6-bisphosphate in the regulation of glycolysis is discussed.
| S-EPMC1158592 | biostudies-other
Project description:Recombinant-derived human interleukin 1 (IL1) alpha and beta and interferon gamma (IFN-gamma) each produced similar increases in rheumatoid synovial cell (RSC) glycolysis, as judged by increased values for glucose uptake, lactate production and cellular fructose 2,6-bisphosphate [Fru(2,6)P2]. Measurement of Fru(2,6)P2 proved to be the most sensitive parameter for an assessment of glycolysis: IL1 alpha, IL1 beta and IFN-gamma all produced a 3-6-fold increase in this metabolite whereas tumour necrosis factor (TNF alpha) was far less effective. Prostaglandin E production was stimulated predominantly by IL1 alpha and IL1 beta rather than by IFN-gamma or TNF alpha. When combinations of cytokines were examined the addition of IFN-gamma with either IL1 alpha, IL1 beta or murine IL1 produced a synergistic increase in cellular Fru(2,6)P2. The three forms of IL1 increased Fru(2,6)P2 via the same pathway, whereas IFN-gamma acted via a different mechanism. The increase in Fru(2,6)P2 in subcultured RSC produced by addition of medium from a primary culture exceeded the maximal effects of any of the single cytokines studied, suggesting the presence of a mixture of cytokines in the primary RSC culture medium.
| S-EPMC1148822 | biostudies-other
Project description:The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser(45) → His substantially fills the central cavity of pFBPase, and the triple mutation Ser(45) → His, Thr(46) → Arg, and Leu(186) → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.
| S-EPMC3961670 | biostudies-literature
Project description:The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.
| S-EPMC5544942 | biostudies-literature
Project description:Fructose-2,6-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates 6-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities. PFKFB4 is over-expressed in human cancers, induced by hypoxia and required for survival and growth of several cancer cell lines. Although PFKFB4 appears to be a rational target for anti-neoplastic drug development, it is not clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-fold greater than its phosphatase activity, siRNA and genomic deletion of PFKFB4 decrease F2,6BP, PFKFB4 over-expression increases F2,6BP and selective PFKFB4 inhibition in vivo markedly reduces F2,6BP, glucose uptake and ATP. Last, we find that PFKFB4 is required for cancer cell survival during the metabolic response to hypoxia, presumably to enable glycolytic production of ATP when the electron transport chain is not fully operational. Taken together, our data indicate that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP. We predict that pharmacological disruption of the PFKFB4 kinase domain may have clinical utility for the treatment of human cancers.
| S-EPMC4196155 | biostudies-literature
Project description:Mammalian phosphofructokinase originated by duplication, fusion, and divergence of a primitive prokaryotic gene, with the duplicated fructose 6-phosphate catalytic site in the C-terminal half becoming an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared across the interface between subunits aligned in an antiparallel orientation, the N-terminal half of one subunit facing the C-terminal half of the other. The composition of these binding sites and the way in which subunits interact to form the dimer within the tetrameric enzyme have been reexamined by systematic point mutations to alanine of key amino acid residues of human muscle phosphofructokinase. We found that residues His-199, His-298, Arg-201, and Arg-292 contribute to the catalytic site and not to the allosteric site, because their mutation decreased the affinity for fructose 6-phosphate without affecting the activation by fructose 2,6-bisphosphate or its binding affinity. In contrast, residues Arg-566, Arg-655, and His-661 were critical components of the fructose bisphosphate allosteric site, because their mutation strongly reduced the action and affinity of the activator, with no alteration of substrate binding to the active site. Our results suggest that mammalian phosphofructokinase subunits associate with the N-terminal halves facing each other to form the two catalytic sites/dimer and the C-terminal halves forming the allosteric sites. Additionally, mutation of certain residues eliminated activation by fructose 1,6-bisphosphate, but not its binding, with little effect on activation by fructose 2,6-bisphosphate, indicating a divergence in the signal transduction route despite their binding to the same site.
| S-EPMC2666562 | biostudies-literature
Project description:Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=72.39, b=127.71, c=157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.
| S-EPMC3169401 | biostudies-literature