Liver Phosphofructokinase Research Articles

High fat high fructose diet induces mild oxidative stress and reorganizes intermediary metabolism in male mouse liver: Alpha-ketoglutarate effects

BackgroundDiets rich in fats and/or carbohydrates are used to study obesity and related metabolic complications. We studied the effects of a high fat high fructose diet (HFFD) on intermediary metabolism and the development of oxidative stress in mouse liver and tested the ability of alpha-ketoglutarate to prevent HFFD-induced changes. MethodsMale mice were fed a standard diet (10% kcal fat) or HFFD (45% kcal fat, 15% kcal fructose) with or without addition of 1% alpha-ketoglutarate (AKG) in drinking water for 8 weeks. ResultsThe HFFD had no effect on body mass but activated fructolysis and glycolysis and induced inflammation and oxidative stress with a concomitant increase in activity of antioxidant enzymes in the mouse liver. HFFD-fed mice also showed lower mRNA levels of pyruvate dehydrogenase kinase 4 (PDK4) and slightly increased intensity of mitochondrial respiration in liver compared to mice on the standard diet. No significant effects of HFFD on transcription of PDK2 and PGC1α, a peroxisome proliferator-activated receptor co-activator-1α, or protein levels of p-AMPK, an active form of AMP-activated protein kinase, were found. The addition of AKG to HFFD decreased oxidized glutathione levels, did not affect levels of lipid peroxides and PDK4 transcripts but increased activities of hexokinase and phosphofructokinase in mouse liver. ConclusionsSupplementation with AKG had weak modulating effects on HFFD-induced oxidative stress and changes in energetics in mouse liver. General significanceOur research expands the understanding of diet-induced metabolic switching and elucidates further roles of alpha-ketoglutarate as a metabolic regulator.

Every-Other-Day Feeding Decreases Glycolytic and Mitochondrial Energy-Producing Potentials in the Brain and Liver of Young Mice.

Intermittent fasting is used to reduce body mass in obese adult humans and animals. However, information on the impact of one type of intermittent fasting (IF) called every-other-day feeding (EODF) on young animals is scarce. In this study, 1-month-old mice of both sexes were subjected to a 4-week regimen of EODF using age-matched counterparts fed ad libitum as controls. At the end of EODF exposure, experimental male and female mice weighed 14 and 13% less than the control counterparts. The EODF regimen resulted in lower liver levels of glycogen, glucose, and lactate, but did not affect lactate level in mouse cerebral cortex of both sexes. Activities of key glycolytic enzymes (hexokinase, phosphofructokinase, and pyruvate kinase) in liver of experimental mice were lower than those in controls. In the cerebral cortex, only hexokinase and pyruvate kinase activities were lower than in controls, but phosphofructokinase activity was not affected in IF females and was higher in IF males as compared with ad libitum fed males. Mitochondria isolated from liver of IF mice had lower respiratory control ratios, but those from the cortex had the same values as control animals. The concentration of β-hydroxybutyrate and the activity of β-hydroxybutyrate dehydrogenase were lower in the IF mouse liver, but not changed or enhanced in the IF cerebral cortex. Thus, animal responses to IF do not depend significantly on sex and are directed to decrease energy metabolism to save resources, and the effects are more pronounced in the liver than in the brain.

Scutellariae Radix and Coptidis Rhizoma Improve Glucose and Lipid Metabolism in T2DM Rats via Regulation of the Metabolic Profiling and MAPK/PI3K/Akt Signaling Pathway.

Aim Scutellariae Radix (SR) and Coptidis Rhizoma (CR) have often been combined to cure type 2 diabetes mellitus (T2DM) in the clinical practice for over thousands of years, but their compatibility mechanism is not clear. Mitogen-activated protein kinase (MAPK) signaling pathway has been suggested to play a critical role during the process of inflammation, insulin resistance, and T2DM. This study was designed to investigate their compatibility effects on T2DM rats and explore the underlying mechanisms by analyzing the metabolic profiling and MAPK/PI3K/Akt signaling pathway. Methods The compatibility effects of SR and CR were evaluated with T2DM rats induced by a high-fat diet (HFD) along with a low dose of streptozocin (STZ). Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to discover potential biomarkers. The levels of pro-inflammatory cytokines; biochemical indexes in serum, and the activities of key enzymes related to glycometabolism in liver were assessed by ELISA kits. qPCR was applied to examine mRNA levels of key targets in MAPK and insulin signaling pathways. Protein expressions of p65; p-p65; phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K); phosphorylated-PI3K (p-PI3K); protein kinase B (Akt); phosphorylated Akt (p-Akt) and glucose transporter 2 (Glut2) in liver were investigated by Western blot analysis. Results Remarkably, hyperglycaemia, dyslipidemia, inflammation, and insulin resistance in T2DM were ameliorated after oral administration of SR and CR, particularly their combined extracts. The effects of SR, CR, low dose of combined extracts (LSC) and high dose of combined extracts (HSC) on pro-inflammatory cytokine transcription in T2DM rats showed that the MAPK pathway might account for the phenomenon with down-regulation of MAPK (P38 mitogen-activated protein kinases (P38), extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK)) mRNA, and protein reduction in p-P65. While mRNA levels of key targets such as insulin receptor substrate 1 (IRS1), PI3K, Akt2, and Glut2 in the insulin signaling pathway were notably up-modulated, phosphorylations of PI3K, Akt, and expression of Glut2 were markedly enhanced. Moreover, the increased activities of phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), glucose 6-phosphatase (G6Pase), and glycogen phosphorylase (GP) were highly reduced and the decreased activities of glucokinase (GK), phosphofructokinase (PFK), pyruvate kinase (PK), and glycogen synthase (GS) in liver were notably increased after treatment. Further investigation indicated that the metabolic profiles of plasma and urine were clearly improved in T2DM rats. Fourteen potential biomarkers (nine in plasma and five in urine) were identified. After intervention, these biomarkers returned to normal level to some extent. Conclusion The results showed that SR, CR, and combined extract groups were normalized. The effects of combined extracts were more remarkable than single herb treatment. Additionally, this study also showed that the metabonomics method is a promising tool to unravel how traditional Chinese medicines work.

Kinetic Characterization of Human Liver Phosphofructokinase

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate in an ATP dependent reaction. This reaction represents the first committed step of the glycolytic pathway and as such, plays an important role in metabolism. In liver, this regulation is especially interesting, as hepatocytes can alternatively undergo gluconeogenesis or glycolysis. While PFKs from the livers of several mammalian species have been characterized, human liver PFK has not been thoroughly examined. The gene encoding human liver PFK was cloned into an expression vector utilizing the tac promoter. Human liver PFK was expressed in an Escherichia coli cell line, RL257a, which contains no native PFK. The protein has been purified to a specific activity of 105 U/mg through a combination of heat denaturation, ammonium sulfate precipitation, anion exchange chromatography, and gel filtration chromatography. Human liver PFK is inhibited by ATP with a coupling free energy (ΔGax) of 3.0 kcal/mol, as compared to rat liver PFK (ΔGax 4.1 kcal/mol), a well characterized mammalian PFK. Additionally, human liver PFK demonstrates a 10-fold increase in F6P affinity when the pH is increased as demonstrated by the dissociation constants at pH 7 (Kia 0.26) and pH 9 (Kia 0.032). Ammonium sulfate activates the enzyme (ΔGax −1.9 kcal/mol), while citrate inhibits the enzyme (ΔGax 0.89 kcal/mol). This work seeks to quantify the kinetic and allosteric behaviors of human liver PFK and compare the magnitude of the effects between rat and human liver PFKs and is supported by the NIH, Grant #GM033216.

Use of 2-Photon Fluorescence Correlation Spectroscopy to Investigate Rat Liver Phosphofructokinase Self-Association

Phosphofructokinase (PFK) catalyzes the first committed step of glycolysis. The smallest active oligomer of PFK from rat liver (RLPFK) is a tetramer, however previous studies have demonstrated that at a physiological enzyme concentrations, fructose 6-phosphate (F6P) stabilizes species much larger than a tetramer. Using a Weber linkage argument, we propose that a highly associated species would demonstrate a higher affinity for F6P. 2-photon fluorescence correlation spectroscopy was used to quantify the oligomeric state of RLPFK in the presence of either F6P or MgATP. 11.6μM RLPFK labeled with fluorescein isothiocyanate, initially in the presence of F6P, was diluted to various concentrations from 116 nM to 1.5 nM into buffer with various concentrations, ranging from 3.2 mM to 0.2 mM, of either F6P and observed for 4 hours after dilution. In the presence of MgATP, 116nM RLPFK exists as a stable species with an apparent diffusion coefficient of 24μm2/sec, consistent with the value predicted for a tetramer. Diluting RLPFK to a concentration of 58nM or below in the presence of MgATP resulted in some dissociation into species smaller than the tetramer. The tetramer was stabilized by high [MgATP]. Dilution of RLPFK to 116nM in the presence of F6P gave an apparent diffusion coefficient of 8μM2/sec indicating a species much larger than a tetramer. This large species was stable over four hours in the presence of 3.2 to 0.8 mM F6P. At F6P concentrations of 0.4 mM and lower, RLPFK slowly dissociated into smaller species with an apparent diffusion coefficient of 16μM2/sec. The rate and extent of dissociation was dependent on both [RLPFK] and [F6P]. At low concentration of RLPFK and F6P the enzyme dissociates into species smaller than a tetramer. Funding: NIH-GM33216, NIH-CBI and Welch-A1543.

Metabolic and molecular responses in Nile tilapia, Oreochromis niloticus during short and prolonged hypoxia

The strictly aquatic breathing Nile tilapia, Oreochromis niloticus is an extremely hypoxia-tolerant fish. To augment our understanding of the effects of hypoxia on anaerobic glycolysis in the Nile tilapia, we studied the effect of short-term for 1 day (trial 1) and long-term for 30 days (trial 2) hypoxia on a selected glycolytic enzymes activity and mRNA expression in liver and white muscle. The hypoxic oxygen concentrations used in the two trials were 2, 1, and 0.5 mg O2 L−1 for comparison with a control normoxic group 8 mg O2 L−1. The activity of phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) in liver and white muscle except liver LDH decreased in trial 1 and increased in trial 2. Assessments of mRNA levels in trial 1 revealed that PFK was downregulated and LDH was upregulated in liver and white muscle, while PK fluctuated between upregulation in liver and downregulation in white muscle. Meanwhile, PK and LDH were upregulated while PFK was similar to control values in both tissues in trial 2. Comet assay results demonstrated an increase in DNA damage that was directly proportional to increasing hypoxic concentrations. This damage was more pronounced in trial 1. This suggests that the Nile tilapia cope better with long-term hypoxic conditions, possibly as an adaptive response.

Kinetic Characterization of Human Liver Phosphofructokinase

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate in an ATP dependent reaction. This reaction represents the first committed step of the glycolytic pathway and as such, plays an important role in metabolism. In liver, this regulation is especially interesting, as hepatocytes can alternatively undergo gluconeogenesis or glycolysis. While PFKs from the livers of several mammalian species have been characterized, human liver PFK has not been thoroughly examined. The gene encoding human liver PFK was cloned into an expression vector utilizing the tac promoter. Human liver PFK was expressed in an Escherichia coli cell line, RL257a, which contains no native PFK. The protein has been purified to a specific activity of 105 U/mg through a combination of heat denaturation, ammonium sulfate precipitation, anion exchange chromatography, and gel filtration chromatography. Human liver PFK is inhibited by ATP with a coupling free energy (ΔGax) of 3.0 kcal/mol, as compared to rat liver PFK (ΔGax 4.1 kcal/mol), a well characterized mammalian PFK. Additionally, human liver PFK demonstrates a 10-fold increase in F6P affinity when the pH is increased as demonstrated by the dissociation constants at pH 7 (Kia 0.26) and pH 9 (Kia 0.032). Ammonium sulfate activates the enzyme (ΔGax −1.9 kcal/mol), while citrate inhibits the enzyme (ΔGax 0.89 kcal/mol). This work seeks to quantify the kinetic and allosteric behaviors of human liver PFK and compare the magnitude of the effects between rat and human liver PFKs and is supported by the NIH, Grant #GM033216.

Use of 2-Photon Fluorescence Correlation Spectroscopy and Electron Microscopy to Ellucidate the Dependence of RLPFK Self Association on Ligand Concentration

Phosphofructokinase (PFK) catalyzes the first committed step of glycolysis. Allosteric regulation of PFK makes its activity, and thus glycolysis, sensitive to intercellular metabolic conditions. Regulation of PFK from liver is complex because of the liver's responsibility to maintain glucose homeostasis, which means that glycolysis and gluconeogenesis must be regulated reciprocally. Allosteric regulation of PFK activity is achieved by modifying the binding affinity of its substrate, fructose 6-phosphate (Fru-6-P). However, in vitro studies performed on rat liver PFK (RLPFK) suggest that at physiological concentrations of Fru-6-P RLPFK activity is negligible even in the presence of known allosteric activators. Additional mechanisms must exist to account for the activity required to support flux through glycolysis. The smallest active oligomer of RLPFK is a tetramer, however fluorescence polarization studies have previously demonstrated that at a physiological enzyme concentration both Fru-6-P and other activators stabilize species much larger than a tetramer. A Weber linkage argument predicts that the highly associated species would demonstrate a higher affinity for Fru-6-P resulting in activation. RLPFK exists as a tetramer in the dilute enzyme concentrations necessary for in vitro activity assays whereas, at physiological concentrations, RLPFK can be highly associated. 2-photon fluorescence correlation spectroscopy (FCS) and electron microscopy have been used to quantify the oligomeric state of RLPFK at high and low enzyme concentrations. FCS performed on alexa-488 labeled RLPFK suggests a complex self-association behavior in the presence of Fru-6-P. Electron microscopy indicates that, in the presence of Fru-6-P, rat liver PFK can form long fibrils that consist of up to 36 PFK tetramers extending over 200 nm in length. These effects are counteracted by MgATP, a known allosteric inhibitor of the enzyme. Funding: NIH-GM33216, NIH-CBI and Welch-A1543

Application of three-photon excitation FCS to the study of protein oligomerization.

Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three-photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in the presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three-photon excitation process was verified from the slope of the log–log plot of intensity against laser power.

Effects of Roux-en-Y gastric bypass and ileal transposition surgeries on glucose and lipid metabolism in skeletal muscle and liver

BackgroundRoux-en Y gastric bypass (RYGB) and ileal transposition (IT) surgeries produce weight loss and improve diabetic control; however, the mechanisms of glycemic improvements are largely unknown. Because skeletal muscle and liver play a key role in glucose homeostasis, we compared the effects of RYGB and IT surgeries on key molecules of glucose and lipid metabolism in muscle and liver. MethodsSprague-Dawley rats were subjected to RYGB, IT, or sham surgeries; sham-animals were ad-lib fed or pair-fed to RYGB rats (n = 7-9/group). At 8 weeks postoperatively, blood samples were collected for glucagon-like peptide-1 (GLP-1) and insulin analyses by ELISA. Leg muscle and liver tissues were analyzed for mRNA (RT-qPCR) and/or protein abundance (immuno blotting) of important molecules of glucose and lipid metabolism [glucose transporter-4 (GLUT-4), hexokinase, phosphofructokinase (PFK), adenosine monophosphate activated protein kinase-α (AMPKα), cytochrome C oxidase-IV (COX-IV), citrate synthase, carnitine palmitoyl transferase-1 (CPT-1), medium-chain acyl-CoA dehydrogenase (MCAD), peroxisome proliferator-activated receptor gamma co-activator 1 α (PGC-1 α), PGC-1-related coactivator (PRC), uncoupling protein-3 (UCP-3)]. ResultsPlasma GLP-1 concentrations were increased comparably with RYGB and IT. RYGB and IT increased muscle GLUT-4 protein content, muscle hexokinase mRNA, and liver PFK mRNA. IT increased muscle AMPKα and COX-IV protein content and liver citrate synthase activity. IT increased muscle CPT-1, MCAD and PRC mRNA, whereas RYGB increased UCP-3 mRNA in muscle and liver, and PGC-1 α mRNA in liver. ConclusionThe data suggest that RYGB and IT surgeries lead to enhanced GLP-1 secretion and produce similar stimulatory effects on important molecules of glucose metabolism but differential effects on key molecules of lipid oxidation in muscle and liver.

Inhibition of Rat Muscle and Liver Phosphofructokinases by High Doses of Ethanol

Activities of both rat muscle and liver phosphofructokinases are significantly inhibited after a single ethanol intake in the dose of 2.5 g per kg of body weight. This inhibitory effect is indirect, since ethanol in concentration (50 mM) close to that established after 2.5 g per kg of body weight intake cannot decrease their activities in vitro. Inhibition of liver phosphofructokinase activity after the 5.0 g per kg ethanol intake may be direct, since liver phosphofructokinase activity decreases in vitro when ethanol is added to supernatants of rat liver tissue in 100 mM concentration. According to the results of molecular docking, ethanol at high concentrations can be bound by adenine-binding pocket of the allosteric ADP-binding site of liver phosphofructokinase (Asp543, Phe308, Phe538, and Phe671) and its activation by ADP can be blocked by C2H5OH molecule. Direct inhibition of muscle phosphofructokinase activity, probably due to the binding of ethanol to the similar ADP-binding site, is possible when the concentration of ethanol (500 mM) is much higher than the level which can be established in living cells. So, inhibition of muscle phosphofructokinase activity after a single 5.0 g per kg intake is indirect and probably linked with the inhibition of the enzyme by elevated citrate and phosphoenolpyruvate levels.

Expression Analysis of Glycerol Synthesis–Related Liver Transcripts in Rainbow Smelt (Osmerus mordax) Exposed to a Controlled Decrease in Temperature

Rainbow smelt (Osmerus mordax) accumulate high glycerol levels to avoid freezing at subzero temperatures. Glyceroneogenesis is activated by low temperature and occurs in liver via a branch in glycolysis and gluconeogenesis. In this study, carbohydrate and liver transcript levels of 21 genes potentially associated with glycerol production were assessed during a controlled warm to cold transition. Smelt were held at 8°C (warm smelt; non-glycerol accumulating) or subjected to a controlled decrease in water temperature from 8° to 0°C (cold smelt; glycerol accumulating) and sampled at the end of the temperature decrease and 1 mo later. In cold smelt compared with warm smelt, liver glycogen levels were lower and phosphoglucomutase transcript levels were higher. Plasma glycerol levels were higher and increased over time in cold smelt; in cold smelt, liver phosphofructokinase and pyruvate dehydrogenase kinase transcript levels increased over time. These findings imply that glycerol production is being fueled by glycogen degradation and inhibition of pyruvate oxidation serves to channel metabolic flux toward glycerol as opposed to complete glycolysis. Plasma glucose and liver glucose-6-phosphatase transcript levels were higher. Lipoprotein lipase transcript levels were higher, suggesting enhanced lipid breakdown to fuel energy metabolism. Glutamine synthetase transcript levels were higher, perhaps to store nitrogen for biosynthesis in spring.

Modulation of phosphofructokinase (PFK) from Setaria cervi, a bovine filarial parasite, by different effectors and its interaction with some antifilarials

BackgroundPhosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11, PFK) is of primary importance in the regulation of glycolytic flux. This enzyme has been extensively studied from mammalian sources but relatively less attention has been paid towards its characterization from filarial parasites. Furthermore, the information about the response of filarial PFK towards the anthelmintics/antifilarial compounds is lacking. In view of these facts, PFK from Setaria cervi, a bovine filarial parasite having similarity with that of human filarial worms, was isolated, purified and characterized.ResultsThe S. cervi PFK was cytosolic in nature. The adult parasites (both female and male) contained more enzyme activity than the microfilarial (Mf) stage of S. cervi, which exhibited only 20% of total activity. The S. cervi PFK could be modulated by different nucleotides and the response of enzyme to these nucleotides was dependent on the concentrations of substrates (F-6-P and ATP). The enzyme possessed wide specificity towards utilization of the nucleotides as phosphate group donors. S. cervi PFK showed the presence of thiol group(s) at the active site of the enzyme, which could be protected from inhibitory action of para-chloromercuribenzoate (p-CMB) up to about 76% by pretreatment with cysteine or β-ME. The sensitivity of PFK from S. cervi towards antifilarials/anthelmintics was comparatively higher than that of mammalian PFK. With suramin, the Ki value for rat liver PFK was 40 times higher than PFK from S. cervi.ConclusionsThe results indicate that the activity of filarial PFK may be modified by different effectors (such as nucleotides, thiol group reactants and anthelmintics) in filarial worms depending on the presence of varying concentrations of substrates (F-6-P and ATP) in the cellular milieu. It may possess thiol group at its active site responsible for catalysis. Relatively, 40 times higher sensitivity of filarial PFK towards suramin as compared to the analogous enzyme from the mammalian system indicates that this enzyme could be exploited as a potential chemotherapeutic target against filariasis.

Enzymatic and mitochondrial responses to 5 months of aerial exposure in the slender lungfish Protopterus dolloi

Mitochondrial respiration and activities of key metabolic enzymes from liver and white skeletal muscle were compared between control aquatic slender lungfish Protopterus dolloi, and those exposed to air for 5 months. Activities of citrate synthase, glycogen phosphorylase, phosphofructokinase and pyruvate kinase in liver were not affected by air‐exposure. In muscle, air‐exposure reduced citrate synthase and pyruvate kinase activities (relative to tissue wet mass) by 63 and 50%, respectively. Liver carnitine palmitoyl transferase activity (relative to mitochondrial protein) decreased by half following air‐exposure, but there was no change in muscle. In mitochondria isolated from muscle, state 3 and state 4 respiration were reduced by 74 and 89%, respectively following air‐exposure, but liver mitochondria were not affected. In liver, air‐exposure increased activities of ornithine‐urea cycle enzymes including glutamine synthase, carbamoyl‐phosphate synthase III and arginase, by 1·9‐ to 4·2‐fold. Carbamoyl‐phosphate synthase III activity could not be detected in muscle, indicating that urea is not synthesized in this tissue. These data suggest that skeletal muscle metabolism is downregulated in air‐exposure, conserving energy and protein during a period when the animals cannot forage. In contrast, ATP production capacities in the liver are maintained, and this may permit expensive urea biosynthesis to continue during aerial exposure.

Metabolic adaptation of the fetal and postnatal ovine heart: regulatory role of hypoxia-inducible factors and nuclear respiratory factor-1.

Numerous metabolic adaptations occur in the heart after birth. Important transcription factors that regulate expression of the glycolytic and mitochondrial oxidative genes are hypoxia-inducible factors (HIF-1alpha and -2alpha) and nuclear respiratory factor-1 (NRF-1). The goal of this study was to examine expression of HIF-1alpha, HIF-2alpha, and NRF-1 and the genes they regulate in pre- and postnatal myocardium. Ovine right and left ventricular myocardium was obtained at four time points: 95 and 140 d gestation (term = 145 d) and 7 d and 8 wk postnatally. Steady-state mRNA and protein levels of HIF-1alpha and NRF-1 and protein levels of HIF-2alpha were measured along with mRNA of HIF-1alpha-regulated genes (aldolase A, alpha- and beta-enolase, lactate dehydrogenase A, liver and muscle phosphofructokinase) and NRF-1-regulated genes (cytochrome c, Va subunit of cytochrome oxidase, and carnitine palmitoyltransferase I ). HIF-1alpha protein was present in fetal myocardium but dropped below detectable levels at 7 d postnatally. HIF-2alpha protein levels were similar at the four time points. Steady-state mRNA levels of alpha-enolase, lactate dehydrogenase A, and liver phosphofructokinase declined significantly postnatally. Aldolase A, beta-enolase, and muscle phosphofructokinase mRNA levels increased postnatally. Steady-state mRNA and protein levels of NRF-1 decreased postnatally in contrast to the postnatal increases in cytochrome c, subunit Va of cytochrome oxidase, and carnitine palmitoyltransferase I mRNA levels. The in vivo postnatal regulation of enzymes encoding glycolytic and mitochondrial enzymes is complex. As transactivation response elements for the genes encoding metabolic enzymes continue to be characterized, studies using the fetal-to-postnatal metabolic transition of the heart will continue to help define the in vivo role of these transcription factors.

Metabolic Adaptation of the Fetal and Postnatal Ovine Heart: Regulatory Role of Hypoxia-Inducible Factors and Nuclear Respiratory Factor-1

Numerous metabolic adaptations occur in the heart after birth. Important transcription factors that regulate expression of the glycolytic and mitochondrial oxidative genes are hypoxia-inducible factors (HIF-1α and -2α) and nuclear respiratory factor-1 (NRF-1). The goal of this study was to examine expression of HIF-1α, HIF-2α, and NRF-1 and the genes they regulate in pre- and postnatal myocardium. Ovine right and left ventricular myocardium was obtained at four time points: 95 and 140 d gestation (term = 145 d) and 7 d and 8 wk postnatally. Steady-state mRNA and protein levels of HIF-1α and NRF-1 and protein levels of HIF-2α were measured along with mRNA of HIF-1α-regulated genes (aldolase A, α- and β-enolase, lactate dehydrogenase A, liver and muscle phosphofructokinase) and NRF-1-regulated genes (cytochrome c, Va subunit of cytochrome oxidase, and carnitine palmitoyltransferase I ). HIF-1α protein was present in fetal myocardium but dropped below detectable levels at 7 d postnatally. HIF-2α protein levels were similar at the four time points. Steady-state mRNA levels of α-enolase, lactate dehydrogenase A, and liver phosphofructokinase declined significantly postnatally. Aldolase A, β-enolase, and muscle phosphofructokinase mRNA levels increased postnatally. Steady-state mRNA and protein levels of NRF-1 decreased postnatally in contrast to the postnatal increases in cytochrome c, subunit Va of cytochrome oxidase, and carnitine palmitoyltransferase I mRNA levels. The in vivo postnatal regulation of enzymes encoding glycolytic and mitochondrial enzymes is complex. As transactivation response elements for the genes encoding metabolic enzymes continue to be characterized, studies using the fetal-to-postnatal metabolic transition of the heart will continue to help define the in vivo role of these transcription factors.

Effects of anoxia exposure and aerobic recovery on metabolic enzyme activities in the freshwater turtleTrachemys scripta elegans

The effects of anoxic submergence (20 h at 7°C in nitrogen-bubbled water) and subsequent aerobic recovery (24 h at 7°C) on the maximal activities of 21 metabolic enzymes were assessed in liver, kidney, heart, brain, and red and white skeletal muscle of an anoxia-tolerant freshwater turtle, the red-eared slider, Trachemys scripta elegans. Anoxia exposure affected the activities of only a few enzymes; for example, it reduced the activity of phosphofructokinase in liver and brain, hexokinase in kidney, glycerol-3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase in heart, glutamate dehydrogenase and serine dehydratase in brain, and 3-hydroxyacyl-CoA dehydrogenase in red muscle. During aerobic recovery, activities of most of these enzymes rebounded and activities of 10 others that were not affected by anoxia rose during recovery. Anoxia-induced changes in selected enzymes appear to meet very specific needs such as glycolytic-rate depression, regulation of glycolytic versus gluconeogenic flux in liver, or alterations in amino acid neurotransmitter levels in brain. Overall, the data demonstrate that the enzymatic make-up of turtle organs undergoes very few changes during anoxia exposure and recovery, which shows that the constitutive activities of enzymes are well designed to meet the metabolic demands of anoxic excursions.

Modification of liver cytosol enzyme activities promoted in vitro by reduced sulfur species generated from cystine with γ-cystathionase

Liver cytosolic γ-cystathionase catalyzes the generation of reduced sulfur species, referred to as `bound sulfur,' in the presence of cystine. Incubating a rat liver cytosol fraction in the presence of cystine or oxidized glutathione inactivated certain cytosolic enzyme activities. The activities of cytosolic phosphofructokinase (PFK) and pyruvate kinase rapidly decreased at pH 7.4 during incubation with a lower concentration of cystine than during incubation with oxidized glutathione. Hexokinase and 11 other enzymes in the system were affected minimally or not at all. Adding dithiothreitol to the system reactivated the modified enzymes. Inactivated PFK activity could also be recovered when reduced glutathione or NADPH was added to the cytosol fraction. In these reconstitution systems, purified rat liver PFK was directly inactivated with cystine trisulfide (one of the low molecular types of bound sulfur), but not by cystine (below 0.1 mM). Purified PFK was also inactivated by incubation with cystine plus γ-cystathionase freshly prepared from cytosol. This was not observed, however, when γ-cystathionase was pretreated with a specific inhibitor, d,l-propargylglycine. The cystine-dependent inactivation of PFK observed in liver cytosol is shown to be caused mainly by the reaction between bound sulfur and the enzyme, but not by the direct thiol/disulfide exchange. Thus, in vitro modification of the cytosolic enzymes by bound sulfur generated from cystine with γ-cystathionase has high potency and relatively specific.

Rabbit Liver Phosphofructokinase: Rapid Purification and Phosphorylation Site Identification

Liver phosphofructokinase can be selectively precipitated by the addition of protamine sulfate to a heat-treated crude extract and redissolved, giving nearly full recovery of catalytic activity in combination with a 67-fold increase in specific activity. We have incorporated protamine sulfate precipitation into a five step purification procedure that can be completed in one day—giving 47% recovery of electrophoretically homogeneous liver phosphofructokinase having a specific activity of 50 units/mg. The radio-labeled fragment isolated from a CNBr digest of liver phosphofructokinase that has undergonein vitrophosphorylation catalyzed by the cAMP-dependent protein kinase has the sequence AEYVSGELEHVTRRSLS.

Fibroblast Growth Factor-1 Induces Phosphofructokinase, Fatty Acid Synthase and Ca2+-ATPase mRNA Expression in NIH 3T3 Cells

Polypeptide growth factors act in part by inducing the expression of specific proteins that perform functions critical to cell cycle progression. We have used a differential display technique to identify genes that are expressed at higher levels following fibroblast growth factor (FGF)-1 (acidic FGF) stimulation of quiescent murine NIH 313 fibroblasts. Three such genes - liver (B-type) phosphofructokinase (PFK), fatty acid synthase (FAS) and sarco(endo)plasmic reticulum Ca2+-ATPase type 2 (SERCA2) - are described in this report. The level of FAS and SERCA2 mRNA expression is increased rapidly after FGF-1 addition; in contrast, PFK mRNA is induced with kinetics more typical of delayed-early genes. These results indicate that enhanced expression of the PFK, FAS and SERCA2 proteins may be important for FGF-1-stimulated cell proliferation.