Wheat germ peptide improves glucose metabolism and insulin resistance in HepG2 hepatocytes via regulating SOCS3/IRS1/Akt pathway.

Evidence has demonstrated that oxidative stress plays a crucial role in regulating cellular glucose metabolism. In previous studies, wheat germ peptide (WGP) was found to effectively mitigate oxidative stress induced by high glucose. Based on the information provided, we hypothesized that WGP could exhibit antihyperglycemic and anti-insulin-resistant effects in cells. The insulin-resistant cell model was established by insulin stimulation. The glucose consumption, glycogen content, and the activities of hexokinase and pyruvate kinase following WGP treatment were measured. The protein expression of SOCS3, phosphorylated insulin receptor substrate-1 (p-IRS1), IRS1, phosphorylated protein kinase B (p-Akt), Akt, glucose transporter 2 (GLUT2), phosphorylated GSK 3ß, GSK 3ß, FOXO1, G6P, and phosphoenolpyruvate carboxykinase were assessed by western blot analysis. Our results demonstrated that WGP treatment increased cellular glucose consumption and glycogen synthesis and enhanced hexokinase and pyruvate kinase activities. Additionally, WGP treatment was observed to cause a significant reduction in the expression of SOCS3, FOXO1, G6P, and phosphoenolpyruvate carboxykinase, as well as in the ratio of p-IRS1/IRS1. Conversely, the expression of GLUT2 and the ratios of p-Akt/Akt and p-GSK3ß/GSK3ß were upregulated by WGP. These findings suggested that WGP can activate the SOCS3/IRS1/Akt signaling pathway, thus promoting the phosphorylation of GSK-3ß and increasing the expression of FOXO1 and GLUT2, which contribute to enhancing glycogen synthesis, inhibiting gluconeogenesis, and promoting glucose transport in insulin-resistant HepG2 cells.

KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

Glycolytic metabolite phosphoenolpyruvate protects host from viral infection through promoting AATK expression.

Viral infections can result in metabolism rewiring of host cells, which in turn affects the viral lifecycle. Phosphoenolpyruvate (PEP), a metabolic intermediate in the glycolytic pathway, plays important roles in several biological processes including anti-tumor T cell immunity. However, whether PEP might participate in modulating viral infection remains largely unknown. Here, we demonstrate that PEP generally inhibits viral replication via upregulation of apoptosis-associated tyrosine kinase (AATK) expression. Targeted metabolomic analyses have shown that the intracellular level of PEP was increased upon viral infection. PEP treatment significantly restricted viral infection and hence declined subsequent inflammatory response both in vitro and in vivo. Besides, PEP took inhibitory effect on the stage of viral replication and also decreased the mortality of mice with viral infection. Mechanistically, PEP significantly promoted the expression of AATK. Knockdown of AATK led to enhanced viral replication and consequent increased levels of cytokines. Moreover, AATK deficiency disabled the antiviral effect of PEP. Together, our study reveals a previously unknown role of PEP in broadly inhibiting viral replication by promoting AATK expression, highlighting the potential application of activation or upregulation of the PEP-AATK axis in controlling viral infections.

Phosphoenolpyruvate induces endothelial dysfunction and cell senescence through stimulation of metabolic reprogramming.

Endothelial dysfunction is a key early link in the pathogenesis of atherosclerosis, and the accumulation of senescent vascular endothelial cells causes endothelial dysfunction. Phosphoenolpyruvate (PEP), which is a high-energy glycolytic intermediate, protects against ischemia-reperfusion injury in isolated rat lung, heart, and liver tissue by quickly providing ATP. However, it was reported that serum PEP concentrations are 13-fold higher in healthy elderly compare to the young. Unlike that of other cell types, the energy required for the physiological function of endothelial cells is mainly derived from glycolysis. Recently, it is unclear whether circulating accumulation of PEP affects endothelial cell function. In this study, we found for the first time that 50-250 µM of PEP significantly promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs) through increased expression of vascular endothelial adhesion factor 1 (VCAM1) and intercellular adhesion factor 1 (ICAM1) in HUVECs. Meanwhile, 50-250 µM of PEP decreased the expression of endothelial nitric oxide synthase (eNOS) and cellular level of nitric oxide (NO) in HUVECs. Moreover, PEP increased levels of ROS, enhanced the numbers of SA-ß-Gal-positive cells and upregulated the expression of cell cycle inhibitors such as p21, p16 and the phosphorylation level of p53 on Ser15, and the expression of proinflammatory factors including TNF-α, IL-1ß, IL-6, IL-8, IL-18 and MCP-1 in HUVECs. Furthermore, PEP increased both oxygen consumption rate (OCR) and glycolysis rate, and was accompanied by reduced NAD+/NADH ratios and enhanced phosphorylation levels of AMPKα (Thr172), p38 MAPK (T180/Y182) and NF-κB p65 (Ser536) in HUVECs. Notably, PEP had no significant effect on hepG2 cells. In conclusion, these results demonstrated that PEP induced dysfunction and senescence in vascular endothelial cells through stimulation of metabolic reprogramming.

The antidiabetic effects of Bifidobacterium longum subsp. longum BL21 through regulating gut microbiota structure in type 2 diabetic mice.

Bifidobacterium longum subsp. longum BL21 (BL21) possesses hypoglycemic activity, but its anti-diabetic mechanism has rarely been illustrated. In the present work, the effects of BL21 on type 2 diabetes mellitus (T2DM) were investigated in diabetic mice induced via a high-fat diet combined with streptozotocin (STZ). Our data indicated that BL21 at a dose of 109 CFU per day significantly lowered the levels of fasting blood glucose and alleviated insulin resistance in diabetic mice. Meanwhile, BL21 enhanced the anti-oxidative capacity, increased the hepatic glycogen content, and significantly decreased the gene expression levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the livers of diabetic mice. Endotoxemia-related inflammation and impaired intestinal barrier function in diabetic mice were also improved using BL21. More importantly, the disturbance of intestinal flora was regulated by BL21, including increased levels of the genera Akkermansia, Alloprevotella, Bacteroides, and Alistipes and decreased levels of Lachnospiraceae_NK4A136_group, Mucispirillum, and Odoribacter. Collectively, the amelioration of T2DM via BL21 supplementation might be partially attributed to regulation of the parameters related to glucose metabolism and the modulation of gut microbiota. Therefore, BL21 could be a potential functional food for ameliorating T2DM.

Organic chromium derived from the chelation of Ganoderma lucidum polysaccharide and chromium (III) alleviates metabolic syndromes and intestinal microbiota dysbiosis induced by high-fat and high-fructose diet.

Organic chromium is of great interest and has become an important chromium supplement resource in recent years because of its low toxicity and easy absorption. In our previous study, we synthesized a novel organic chromium [GLP-Cr] through the chelation of Ganoderma lucidum polysaccharide and chromium (III). The purpose of this study was to investigate the beneficial effects of GLP-Cr on the improvement of metabolic syndromes (MetS) in mice fed with a high-fat and high-fructose diet (HFHFD) and its mechanism of action. The results indicated that oral administration of GLP-Cr inhibited the excessive exaltation of body weight, glucose tolerance, fasting blood glucose and lipid levels, hepatic total cholesterol (TC), triglyceride (TG) levels caused by HFHFD. Besides, 16S rRNA amplicon sequencing showed that GLP-Cr intervention evidently ameliorated intestinal microbiota dysbiosis by changing the proportions of some intestinal microbial phylotypes. In addition, correlation network-based analysis indicated that the key intestinal microbial phylotypes were closely related to biochemical parameters associated with MetS under GLP-Cr intervention. Liver metabolomics analysis suggested that GLP-Cr intervention significantly regulated the levels of some biomarkers involved in alpha-linolenic acid metabolism, fatty acid biosynthesis, steroid hormone biosynthesis, glycerophospholipid metabolism, glycerolipid metabolism, steroid hormone biosynthesis, primary bile acid biosynthesis, and so on. Moreover, GLP-Cr intervention regulated liver mRNA levels of key genes associated with glucose and lipid metabolism. The mRNA level of glucose transporter type 4 (Glut4) was markedly increased by GLP-Cr intervention, and the mRNA levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6Pase) in the liver were significantly decreased. Meanwhile, GLP-Cr intervention significantly decreased hepatic mRNA levels of cluster of differentiation 36 (Cd36), acetyl-CoA carboxylase 1 (Acc1) and sterol regulatory element binding protein-1c (Srebp-1c), indicating that GLP-Cr intervention inhibited the excessive accumulation of free fatty acids in the liver. These findings suggest that the prevention of hyperglycemia and dyslipidemia by GLP-Cr may be closely related to the regulation of gut microbial composition and hepatic metabolic pathways, thus GLP-Cr can be serving as a functional component in the prevention of MetS.

Metabolite Regulatory Interactions Control Plant Respiratory Metabolism via Target of Rapamycin (TOR) Kinase Activation.

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O2 consumption rate (RN) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour RN measurements following leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids) identified phosphoenolpyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf RN Using metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling RN Many amino acids, as well as Glc analogs, were found to potently inhibit the RN stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro- and Ala-stimulated RN were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway. Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2.

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.

Ixodes scapularis Tick Cells Control Anaplasma phagocytophilum Infection by Increasing the Synthesis of Phosphoenolpyruvate from Tyrosine.

The obligate intracellular pathogen, Anaplasma phagocytophilum, is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host-pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis, the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum. The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate.

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP(+)/NBT/PMS (ß-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H37Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H37Ra PGI was 226 µg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H37Ra PGI inhibition by PEP.

Allosteric regulation of Lactobacillus plantarum xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp).

UNLABELLED: Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657-663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCE: Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains.

Phosphoenolpyruvate administration protects ischemia-reperfusion injury in isolated rabbit lungs.

Phosphoenolpyruvate (PEP) is an intermediate metabolite of the glycolytic pathway and an in vivo high-energy phosphate compound. We have examined the protective effects of PEP on ischemia-reperfusion lung injury in isolated rabbits lungs perfused with a physiological salt solution. The lungs were divided into three treatment groups: (1) ischemia-reperfusion (IR), (2) ischemia-reperfusion with PEP treatment (PEP-IR), in which 1 mM PEP was pre-administered into the perfusate during the stable period, and (3) ventilation-perfusion continued without interruption (Cont). In the IR and PEP-IR groups, ventilation-perfusion was discontinued for about 60 min after a 30-min stable period and then restarted. The capillary filtration coefficients (K fc) and pyruvate concentration in the perfusate were determined immediately before ischemia and 30 and 60 min after reperfusion. The left lungs were dried at the end of the experiment to calculate the tissue wet-to-dry weight ratio (W/D). The K fc values after reperfusion were significantly higher in the IR group than in the other two groups. Pyruvate concentrations were significantly higher at three time-points in the PEP-IR group than in the other two groups. The W/D was significantly higher in the IR group than in the other two groups. Based on these results, we conclude that the administration of PEP prior to lung ischemia alleviates lung ischemia-reperfusion injury.

Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase - The importance of accommodating the active site water.

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9±0.4µM. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated.

Allosteric regulation in phosphofructokinase from the extreme thermophile Thermus thermophilus.

An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and activated by ADP by modifying the affinity exhibited for the substrate fructose 6-phosphate (Fru-6-P) in a manner analogous to other prokaryotic PFKs. However, TtPFK binds both of these allosteric ligands significantly more tightly than other bacterial PFKs while effecting a substantially more modest extent of inhibition or activation at 25 °C, reinforcing the principle that binding affinity and effectiveness can be both independent and uncorrelated to one another. These properties have allowed us to establish rigorously that PEP only inhibits by antagonizing the binding of Fru-6-P and not by influencing turnover, a conclusion that requires kcat to be determined under conditions in which both inhibitor and substrate are saturating simultaneously. In addition, the temperature dependence of the allosteric effects on Fru-6-P binding indicate that the coupling free energies are entropy-dominated, as observed previously for PFK from Bacillus stearothermophilus but not for PFK from Escherichia coli , supporting the hypothesis that entropy-dominated allosteric effects may be a characteristic of enzymes derived from thermostable organisms. For such enzymes, the root cause of the allosteric effect may not be easily discerned from static structural information such as that obtained from X-ray crystallography.

Phosphoenolpyruvic acid, an intermediary metabolite of glycolysis, as a potential cytoprotectant and anti-oxidant in HeLa cells.

This study examined the cytoprotective and anti-oxidative properties of phosphoenolpyruvic acid (PEP), a glycolysis metabolite with a high-energy phosphate group. PEP (0.1-10 mM) significantly attenuated the decrease in cell viability induced by hydrogen peroxide (H(2)O(2)) in HeLa cells in a dose-dependent manner. PEP also inhibited the decrease in calcein-acetomethoxy-stained cells and the increase in propidium iodide-stained cells that were induced by H(2)O(2). The H(2)O(2)-stimulated increase in intracellular reactive oxygen species was significantly reduced by PEP. PEP also demonstrated scavenging potential against hydroxyl radicals, as assessed by the electron paramagnetic resonance method. In addition, PEP demonstrated scavenging potential against the 1,1-diphenyl-2-picrylhydrazyl radical, a representative artificial radical, although the potential is very weak. PEP (10 mM) slightly inhibited the decrease in cellular ATP content induced by H(2)O(2), but did not show any effects at low doses (0.1, 1 mM). PEP (0.1-10 mM) also attenuated the cell injury but not the decrease in intracellular ATP content, induced by 2-deoxy-D-glucose, a glycolysis inhibitor. These results indicate that PEP exerts cytoprotective effects and has anti-oxidative potential, although the precise cytoprotective mechanisms are not fully elucidated. We suggest that PEP is a functional carbohydrate metabolite with cytoprotective and anti-oxidative activity, and is potentially useful as a therapeutic agent against diseases that involve the oxidative stress.

Identification of allosteric-activating drug leads for human liver pyruvate kinase.

There is a growing appreciation of the beneficial attributes of allosteric drugs. However, the development of this special class of drugs has in large part been via serendipitous findings from high-throughput screens of drug libraries. Limited success at deliberately identifying allosteric drugs may be due to a focus on enzyme inhibitors, a parallel to the historic focus on competitive inhibitors. In contrast to inhibition, activation of an enzyme by a small molecule can only occur through a limited number of mechanisms, mainly allosteric regulation. Activation of human liver pyruvate kinase (hL-PYK) in an effort to create a glycolytic/gluconeogenic futile cycle is one potential mechanism to counteract hyperglycemia. Using hL-PYK, we demonstrate the potential of drug library screens to identify allosteric-activator drug leads.

Phosphoenolpyruvate and Mg2+ binding to pyruvate kinase monitored by infrared spectroscopy.

Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg(2+) binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg(2+) binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-(13)C(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO(3)(2-) stretching vibrations); and 967 cm(-1) (symmetric PO(3)(2-) stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm(-1). The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na(+) exhibited the same band positions as in the presence of K(+), indicating very similar interaction strengths between ligand and protein in both cases.

The quaternary structure of pyruvate kinase type 1 from Escherichia coli at low nanomolar concentrations.

Pyruvate kinase (PK) is the key control point of glycolysis-the biochemical pathway central to energy metabolism and the production of precursors used in biosynthesis. PK type 1 from Escherichia coli (Ec-PK1) is activated by both fructose-1,6-bisphosphate (FBP) and its substrate, phosphoenol pyruvate (PEP). To date, it has not been possible to determine whether the enzyme is tetrameric at the low concentrations (i.e. low nM range) used to study the steady-state kinetics, or assess whether its allosteric effectors alter the oligomeric state of the enzyme at these concentrations. Employing the new technique of analytical ultracentrifugation with fluorescence detection we have, for the first time, shown that the K(D)(4-2) for Ec-PK1 is in the subnanomolar range, well below the concentrations used in kinetic studies. In addition, we show that, unlike some other PK isoenzymes, the modulation of oligomeric state by the allosteric effectors FBP and PEP does not occur at a concentration of 10 nM or above.

Diffusion restrictions surrounding mitochondria: a mathematical model of heart muscle fibers.

Several experiments on permeabilized heart muscle fibers suggest the existence of diffusion restrictions grouping mitochondria and surrounding ATPases. The specific causes of these restrictions are not known, but intracellular structures are speculated to act as diffusion barriers. In this work, we assume that diffusion restrictions are induced by sarcoplasmic reticulum (SR), cytoskeleton proteins localized near SR, and crowding of cytosolic proteins. The aim of this work was to test whether such localization of diffusion restrictions would be consistent with the available experimental data and evaluate the extent of the restrictions. For that, a three-dimensional finite-element model was composed with the geometry based on mitochondrial and SR structural organization. Diffusion restrictions induced by SR and cytoskeleton proteins were varied with other model parameters to fit the set of experimental data obtained on permeabilized rat heart muscle fibers. There are many sets of model parameters that were able to reproduce all experiments considered in this work. However, in all the sets, <5-6% of the surface formed by SR and associated cytoskeleton proteins is permeable to metabolites. Such a low level of permeability indicates that the proteins should play a dominant part in formation of the diffusion restrictions.

Phosphoenolpyruvate-dependent inhibition of collagen biosynthesis, alpha2beta1 integrin and IGF-I receptor signaling in cultured fibroblasts.

The mechanism of collagen biosynthesis regulation is not fully understood. The finding that prolidase plays an important role in collagen biosynthesis and phosphoenolpyruvate inhibits prolidase activity "in vitro" led to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with millimolar concentrations (1-4 mM) of phosphoenolpyruvate monopotassium salt (PEP) for 24 h. It was found that PEP-dependent decrease in prolidase activity and expression was accompanied by parallel decrease in collagen biosynthesis. However, the experiments with inhibitor of PEP production, 3-mercaptopicolinate revealed no direct correlation between collagen biosynthesis and prolidase activity and expression. Since insulin-like growth factor (IGF-I) is the most potent stimulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by beta(1) integrin signaling, the effect of PEP on IGF-I receptor (IGF-IR) and beta(1) integrin receptor expressions were evaluated. It was found that the exposure of the cells to 4 mM PEP contributed to a decrease in IGF-IR and beta(1) integrin receptor expressions. The data suggest that PEP-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts may undergo through depression of alpha(2)beta(1) integrin and IGF-IR signaling. The hypothetical mechanism of the role of prolidase in IGF-IR, beta(1) integrin receptor expressions, and clinical significance of the process are discussed.