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.

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.

Characterization of the inhibition of pyruvate kinase caused by phenylalanine and phenylpyruvate in rat brain cortex.

Pyruvate kinase plays a crucial role on the glycolytic pathway, the main route that provides energy for brain functioning. In the present study, we investigated the kinetics of the inhibition of pyruvate kinase provoked by phenylalanine and its main metabolite, phenylpyruvate, in mitochondria-free cerebral cortex homogenate from 22-day-old Wistar rats. We found that phenylalanine and phenylpyruvate inhibit PK activity by competition with the enzyme substrates ADP and phosphoenolpyruvate. We also investigated the interaction between phenylalanine and phenylpyruvate, and the kinetics of alanine prevention of the inhibitory action of phenylalanine and phenylpyruvate on pyruvate kinase activity. We observed that alanine per se had no effect on PK activity but prevented the inhibitory action of phenylalanine and phenylpyruvate by competition. The data suggest that phenylalanine, phenylpyruvate, and alanine act on a common site in the enzyme, probably an allosteric one. It is possible that inhibition of brain PK activity may be related to the reduction of glucose metabolism observed in the brain of phenylketonuric patients and may be one of the mechanisms responsible for the neurological dysfunction found in these patients. Further studies, however, are necessary to evaluate the benefit of carbohydrate and alanine supplementation to the diet of phenylketonuric patients.

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).

A highly efficient cell-free protein synthesis system from Escherichia coli.

We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture. First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter. Second, the use of a condensed 30000 x g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis. This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract. For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids. This improved cell-free protein synthesis system is superior in productivity to any other cell-free systems reported so far, including the continuous-flow cell-free system.

Suppression of chemotactic activity of neutrophils in hyperosmotic conditions comparable to the renal medulla: partial preservation by phosphoenolpyruvate.

Chemotaxis is one of the most important functions of the polymorphonuclear leukocyte (PMN). In the host defense against pyelonephritis, the renal medulla is a site of interaction between bacteria and PMNs. At this site the osmotic pressure is elevated due to a high concentration of NaCl and urea. We evaluated the in vitro chemotactic activity of PMNs under the hyperosmolar conditions created by high concentrations of NaCl and urea. This activity was suppressed by the stimulation of opsonized zymosan and formyl-methionyl-leucyl-phenylalanine. The inhibition of chemotaxis was partially preserved by phosphoenolpyruvic acid (PEP), a precursor of adenosine triphosphate (ATP), in hyperosmolar NaCl but not in urea. The intracellular content of ATP was increased by supplementing the hyperosmolar NaCl with PEP. These observations suggest that inhibition of the chemotactic activity of PMNs is due to differing mechanisms for each NaCl and urea, and that PEP may protect the PMNs against hyperosmolar NaCl by maintaining ATP content.

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.

Interrelated effects of nucleoside mono- and triphosphates and phosphoenolpyruvate on rat hearts subjected to cardioplegic arrest at normothermia.

Supplementation with phosphoenolpyruvate (PEP) and ATP was previously found to enhance the protective effect of potassium cardioplegia on rat hearts subjected to extensive ischemic trauma (30 min at 37 degrees C) in the paracorporeal rat heart model. In the present experiments, the ischemia time was reduced to 20 min (37 degrees C). Ventricular work after ischemia was best in control rats with potassium cardioplegia only. Supplementing the cardioplegic solution with PEP and ATP (group I) resulted in significantly reduced postischemic ventricular work and increased efflux of the creatine kinase isoenzyme MB (CK-MB). The same result was obtained when adenosine monophosphate (AMP) was added to the supplementation (group II). When guanosine monophosphate (GMP) was added instead of AMP (group III), the negative effects of PEP and ATP in the cardioplegic solution were partly abolished. Plain potassium cardioplegia, without additives, nevertheless gave the best results. There were no significant intergroup differences in the myocardial content of adenine nucleotides. The results contrasted with those in the previous study of more protracted ischemic trauma (30 min at 37 degrees C) and possible explanations are discussed.

Enhanced protection of rat hearts during ischemia by phosphoenolpyruvate and ATP in cardioplegia.

The paracorporeal rat heart model was used to investigate the extent of myocardial protection by potassium cardioplegia supplemented with phosphoenolpyruvate (PEP) (14.4 mM) and ATP (0.067 mM) singly or in combination. Rat hearts were subjected to 30 minutes of ischemia at 37 degrees. They were subsequently reperfused for 40 minutes during which time the left ventricular isovolumic work was measured and blood samples were taken for creatine kinase isoenzyme MB (CK-MB) analysis. At the end of each experiment the hearts were freeze-clamped for later analyses of high energy phosphate compounds. Supplementation with PEP and ATP (Group I) and with only ATP (Group II) showed a significantly better left ventricular isovolumic work and a significantly higher adenylate charge potential (ACP). Supplementation with only PEP (Group III) resulted in significantly better left ventricular isovolumic work than the control group but significantly lower than groups I and II. There were no significant differences between the groups in regard to the CK-MB efflux. Supplementation with PEP and ATP in combination did not show any positive effect at 40 minutes of reperfusion over and above that which was achieved with ATP only.

Changes in adenosine triphosphate, 2,3 diphosphoglycerate, and P50 of dog blood following transfusion of autologous red cells pretreated with phosphoenolpyruvate in vitro.

Red cells treated with phosphoenolpyruvate (PEP) in vitro were reinfused into the donor dogs and were monitored for changes in adenosine triphosphate (ATP), 2, 3 diphosphoglycerate (2,3 DPG), and P50. ATP and 2,3 DPG concentrations increased to 116 and 143 percent of control, respectively, when these cells were incubated with 60 mM PEP for 90 minutes at 37 degrees C. The oxygen dissociation curve shifted to the right, and P50 increased from 24.5 to 30.6 torr as a result of the PEP treatment. When one-half of the circulating red cell volume was treated with PEP and reinfused into the animal, the red cell 2,3 DPG increased to 120 percent of pretransfusion values. The 2,3 DPG level remained elevated during the following day and returned to near pretransfusion levels on the third day. The P50 of the circulating blood paralleled the variations in the red cell 2,3 DPG level, and the capacity for oxygen delivery was calculated to be raised by 13 to 38 percent for a period of 24 hours. In contrast, elevated red cell ATP returned to control values immediately after transfusion. In vivo viability, i.e., 24-hour survival and one-half disappearance time, of the cells pretreated with PEP were determined by a single-isotope technique using 51Cr. The results showed that PEP treatment did not injure the red cells. In addition, there was neither acute toxicity nor a deleterious hemodynamic effect when large amounts of PEP were administered intravenously. These results suggest that PEP could be used clinically to improve the capacity of the circulating red cells to deliver oxygen to the tissues.

Reconstitution of regulatory properties of adenylate cyclase in Escherichia coli extracts.

The inhibition of adenylate cyclase activity of Escherichia coli by methyl alpha-glucoside has been demonstrated in intact or in permeable cells but not in cell-free extracts. In intact or permeable cells, this inhibition is demonstrable only in strains expressing the genes for proteins of the phosphoenolpyruvate:glycose phosphotransferase system (PTS); in permeable cells, the inhibition also requires potassium phosphate. Using homogeneous proteins of the PTS, we have reconstituted in cell-free extracts many of the features of the regulated form of adenylate cyclase: (i) In the absence of K2HPO4, permeable cells have lower adenylate cyclase activity than extracts; addition of homogeneous PTS proteins to the extracts brings adenylate cyclase activity close to the level observed in permeable cells. (ii) The low activity observed in permeable cells is stimulated by potassium phosphate; this stimulation is also observed in extracts supplemented with PTS proteins and phosphoenolpyruvate. (iii) In permeable cells, potassium phosphate-stimulated adenylate cyclase activity is inhibited by methyl alpha-glucoside or pyruvate; extracts behaved similarly when supplemented with PTS proteins, K2HPO4, and phosphoenolpyruvate. Thus, the regulated form of adenylate cyclase has been reconstituted in cell-free extracts by addition of homogeneous PTS proteins.

Metabolic and functional effects of phosphoenolpyruvate and adenosine triphosphate on rat hearts subjected to global ischemia.

The effect of phosphoenolpyruvate (PEP) and adenosine triphosphate (ATP) administered during ischemia was investigated, using the paracorporeal rat heart model. During 15 min of global ischemia the hearts were perfused twice with PEP and ATP (supplemented) or with NaCl only (non-supplemented). In hearts that were freeze-clamped after the ischemic period (group A), the myocardial content of high-energy phosphates showed only minor differences between hearts with and without supplemention. In the supplemented hearts there was increased myocardial content of pyruvate and, to some extent, of lactate, indicating that PEP was metabolized to pyruvate and partly to lactate. In groups B and C the hearts were reperfused for 40 min before freeze-clamping. The non-supplemented hearts showed higher energy content and better left ventricular performance and, concomitantly, the creatine kinase isoenzyme MB (CK-MB) efflux was less than for the supplemented hearts. A clear inverse relationship between left ventricular performance and CK-MB efflux indicated that CK-MB served as a marker of cell integrity. These results contrasted with previus finding of an unequivocally positive effect from PEP and ATP administration. Possible explanations are discussed.

Myocardial pyruvate, lactate, and orthophosphate contents under different postischemic conditions. A study in a paracorporeal rat heart model.

The myocardial contents of pyruvate, lactate and orthophosphate after ischemia were investigated in a paracorporeal rat heart model under different conditions. The arterial blood was supplemented with phosphoenolpyruvate (PEP) and adenosine triphosphate early during reperfusion of excised hearts subjected to 15 min of complete global ischemia at 37 degrees C. The increase in myocardial pyruvate was significant after 4 min of reperfusion compared with the content in non-supplemented hearts subjected to the same ischemic trauma. Such dynamic changes were not observed for lactate and orthophosphate under corresponding conditions. The distinct increase in myocardial pyruvate content on supplementation with PEP and adenosine triphosphate was most probably due to an adenosine triphosphate-mediated PEP translocation into myocardial cells, with rapid metabolization of translocated PEP into adenosine triphosphate (in the presence of cellular adenosine diphosphate) and pyruvate. The pyruvate and lactate relationship varied, depending on the postischemic conditions. The postischemic myocardial orthophosphate content was stable, with only minor fluctuations. This possibility to supply the postischemic myocardium with substrate for immediate intracellular energy production is of clinical interest and merits further studies.

Acid-citrate-dextrose-phosphoenolpyruvate medium as a rejuvenant for blood storage.

Stored, depleted RBC were rejuvenated with respect to their levels of adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), and P50 by acid-citrate-dextrose perservatives containing phosphoenolpyruvate (PEP) without sucrose. The restorations of P50 and 2,3-DPG were dependent on the phosphoenolpyruvate concentration. Erythrocyte P50 and 2,3-DPG, even after treatment with these preservatives, decreased with increasing storage period, but the P50 and 2,3-DPG of five-week-old blood were still higher than the corresponding values of fresh blood. ATP concentration was also increased by treating stored blood with preservatives containing phosphoenolpyruvate, but the elevated ATP of five-week-old blood was only about 50 percent of fresh blood. The ATP level could not be raised further by increasing phosphoenolpyruvate concentration but was improved by supplementation with adenine and nucleosides. Incubation of stored blood with 15 mM phosphoenolpyruvate was sufficient to restore ATP, 2,3-DPG and P50 of three-week-old blood to nearly normal. The results of these studies indicate that sucrose is not necessary for PEP to be effective as a preservative additive.

Myocardial energy restoration of ischemic damage by administration of phosphoenolpyruvate during reperfusion. A study in a paracorporeal rat heart model.

The myocardial energy restoration of ischemic damage by administration of phosphoenolpyruvate during reperfusion was investigated in a paracorporeal heart model modified in the rat. Excised hearts were subjected to 15 min of complete global ischemia at ischemia at 37 degrees C before pulsatile blood reperfusion for 30 min. One group (n = 7) was supplemented with 67 mumol of phosphoenolpyruvate and 0.67 mumol of adenosine triphosphate dissolved in 10 ml of saline and another with plain saline (nonsupplemented) during reperfusion. All hearts were freeze-clamped after 30 min of reperfusion and subjected to energy content analysis. The adenylate charge potential was 0.91 +/- 0.01 (mean +/- SD) for the supplemented and 0.85 +/- 0.06 (mean +/- SD) for the nonsupplemented group. This difference was significant (p less than 0.02). Concomitantly the outflow of creatine kinase was less for the supplemented group. The translocation of phosphoenolpyruvate into the myocardial cells seems possible by blood supplementation of a lower dose of adenosine triphosphate.