HIF-1 is a transcription factor that controls a widespread range of genes in metazoan organisms in response to hypoxia and is composed of α and β subunits. In shrimp, phosphofructokinase (PFK) and ...fructose bisphosphatase (FBP) are up-regulated in hypoxia. We hypothesized that HIF-1 is involved in the regulation of PFK and FBP genes in shrimp hepatopancreas under hypoxia. Long double stranded RNA (dsRNA) intramuscular injection was utilized to silence simultaneously both HIF-1 subunits, and then, we measured the relative expression of PFK and FBP, as well as their corresponding enzymatic activities in hypoxic shrimp hepatopancreas. The results indicated that HIF-1 participates in the up-regulation of PFK transcripts under short-term hypoxia since the induction caused by hypoxia (~1.6 and ~4.2-fold after 3 and 48h, respectively) is significantly reduced in the dsRNA animals treated. Moreover, PFK activity was significantly ~2.8-fold augmented after 3h in hypoxia alongside to an ~1.9-fold increment in lactate. However, when animals were dsRNA treated, both were significantly reduced. On the other hand, FBP transcripts were ~5.3-fold up-regulated in long-term hypoxic conditions (48h). HIF-1 is involved in this process since FBP transcripts were not induced by hypoxia when HIF-1 was silenced. Conversely, the FBP activity was not affected by hypoxia, which suggests its possible regulation at post-translational level. Taken together, these results position HIF-1 as a prime transcription factor in coordinating glucose metabolism through the PFK and FBP genes among others, in shrimp under low oxygen environments.
Tuberculosis (TB) remains one of the major health concerns worldwide.
(Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key ...metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While
gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.
Glucagon-like peptide-1 signalling impacts glucose homeostasis and appetite thereby indirectly affecting substrate availability at the whole-body level. The incretin canonically produces an ...insulinotropic effect, thereby lowering blood glucose levels by promoting the uptake and inhibiting the production of the sugar by peripheral tissues. Likewise, GLP-1 signalling within the central nervous system reduces the appetite and food intake, whereas its gastric effect delays the absorption of nutrients, thus improving glycaemic control and reducing the risk of postprandial hyperglycaemia. We review the molecular aspects of the GLP-1 signalling, focusing on its impact on intracellular energy metabolism. Whilst the incretin exerts its effects predominantly via a Gs receptor, which decodes the incretin signal into the elevation of intracellular cAMP levels, the downstream signalling cascades within the cell, acting on fast and slow timescales, resulting in an enhancement or an attenuation of glucose catabolism, respectively.
•Glucagon-like peptide-1 signalling impacts glucose metabolism within target cells.•Molecular aspects of this signalling depend on the dynamic of GLP-1 stimulus and isoforms of key metabolic enzymes expressed.•The consequences can differ significantly from tissue to tissue, however, the acute effect stimulates glucose catabolism, whereas the chronic effect is linked with an attenuation of glycolytic flux.
We examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhibited ...PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine residues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indirectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhibition of glucose consumption in tissues.
In some archaea, the phosphorylation of glucose and fructose 6‐phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the ...hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis. Assay of the enzyme activity with different sugars, metals and nucleotides allows us to conclude that the enzyme is able to phosphorylate both fructose 6P and glucose in the presence of ADP and a divalent metal cation. Kinetic characterization of the enzyme revealed complex regulation by the free Mg2+ concentration and AMP, with the latter appearing to be a key metabolite. To determine whether this enzyme could have a role in gluconeogenesis, we evaluated the reversibility of both reactions and found that glucokinase activity is reversible, whereas phosphofructokinase activity is not. To determine the important residues for glucose and fructose 6P binding, we modeled the bifunctional phosphofructokinase/glucokinase enzyme from M. maripaludis and its interactions with both sugar substrates using protein–ligand docking. Comparison of the active site of the phosphofructokinase/glucokinase enzyme from M. maripaludis with the structural models constructed for all the homology sequences present in the order Methanococcales shows that all of the ADP‐dependent kinases from this order would be able to phosphorylate glucose and fructose 6P, which rules out the current annotation of these enzymes as specific phosphofructokinases.
Database
Model data are available in the Protein Model Data Base under accession numbers PM0079106, PM0079107, PM0079108, PM0079109, PM0079110, PM0079111, PM0079112, PM0079113, PM0079114, PM0079115 and PM0079116
Here we demonstrated that besides the enzyme from Methanocaldococcus jannaschii all the enzymes from the Methanococcales group would be able to phosphoryated glucose and fructose‐6‐phosphate, Characterization of the Methanococcus maripaludis enzyme show a complex regulation by free Mg2+ and AMP, not recognized previously, and reversibility of the GK activity which could be responsible for glucose formation in this archaeon.
The main hallmark of myocardial substrate metabolism in cardiac hypertrophy or heart failure is a shift from fatty acid oxidation to greater reliance on glycolysis. However, the close correlation ...between glycolysis and fatty acid oxidation and underlying mechanism by which causes cardiac pathological remodelling remain unclear. We confirm that KLF7 simultaneously targets the rate-limiting enzyme of glycolysis, phosphofructokinase-1, liver, and long-chain acyl-CoA dehydrogenase, a key enzyme for fatty acid oxidation. Cardiac-specific knockout and overexpression KLF7 induce adult concentric hypertrophy and infant eccentric hypertrophy by regulating glycolysis and fatty acid oxidation fluxes in male mice, respectively. Furthermore, cardiac-specific knockdown phosphofructokinase-1, liver or overexpression long-chain acyl-CoA dehydrogenase partially rescues the cardiac hypertrophy in adult male KLF7 deficient mice. Here we show that the KLF7/PFKL/ACADL axis is a critical regulatory mechanism and may provide insight into viable therapeutic concepts aimed at the modulation of cardiac metabolic balance in hypertrophied and failing heart.
Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the ...substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.
Fresh meat quality development is influenced by pH decline that results from muscle glycolyzing energy substrates postmortem. The exact reason why glycolysis stops in the presence of residual ...glycogen remains unclear. We hypothesized that a critical glycolytic enzyme loses activity near the ultimate pH of meat. Porcine longissimus muscle samples were subjected to an in vitro system that mimics postmortem anaerobic metabolism at buffered pH values (7.0, 6.5, 6.0, 5.5 or 5.0). At pH7.0, 6.5, and 6.0, glycogenolysis and glycolysis proceeded normally while pH5.5 stopped lactate formation. Additional experimentation indicated that phosphofructokinase lost activity at pH5.5 while all other glycolytic enzymes remained active. A similar inactivation of phosphofructokinase was observed when using chicken and beef muscle. Elevated temperature hastened pH decline and phosphofructokinase activity loss. Thus, pH inactivates phosphofructokinase and arrests postmortem glycolysis, which may explain the similar ultimate pH across meat of different species.
•We reestablish the Scopes buffer system as a tool for postmortem metabolism research.•Phosphofructokinase (PFK) is pH inactivated postmortem.•PFK partially explains the consistency in ultimate pH in meat of different species.•Rapid glycolysis allows more substrate to pass PFK which may lower ultimate pH.•An updated model of the factors controlling postmortem metabolism is proposed.
The objective of this study was to analyze the differences in the proteins in non-capacitated and capacitated boar sperm and to identify the functions of the differential proteins and key ...capacitation proteins of boar sperm before and after capacitation. Transwell chambers were used to separate capacitated sperm proteins using a unique polycarbonate membrane. Meanwhile, isotopic tags for relative and absolute quantification combined with LC‒MS/MS analysis were used for quantitative determination of differential proteins. Through the comparative analysis of different databases, 475 different proteins were identified in non-capacitated sperm and capacitated sperm, of which 303 were significantly upregulated and 172 were significantly downregulated. These differentially-expressed proteins are mainly involved in redox processes, cell biosynthesis processes and cell aromatic compound metabolism biological processes. They also participate in the signaling pathways of phosphorylation, ketone synthesis and degradation, most of which interact to varying degrees. Among these differentially-expressed proteins, phosphofructokinase attracted our attention as a potential capacitated protein. We further verified that phosphofructokinase can promote boar sperm capacitation by immunoblotting.
•Transwell chamber was used to separate sperm proteins by 0.4 μm polycarbonate membrane.•There are major changes in proteins responsible for energy metabolism and plasma membrane stability during sperm capacitation.•Phosphofructokinase is a potential capacitation-promoting factor.
PDF
