Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation.

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering.

Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates - glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An in vitro synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T. maritima) were overexpressed in E. coli BL21(DE3) and purified by simple heat precipitation. The optimal pH and temperature of one-pot biosynthesis were 7.2 and 70°C, respectively. The optimal enzyme ratios of αGP, PGM, PGI and PFK were 2:2:1:2 in terms of units. Via step-wise addition of new substrates, up to 125 ± 4.6mM FDP was synthesized after 7-h reaction. This de novo ATP-free enzymatic pathway comprised of all hyperthermophilic enzymes could drastically decrease the manufacturing costs of FDP and its derivatives GA3P and DHAP, better than those catalyzed by ATP-regeneration cascade biocatalysis, the use of mesophilic enzymes, whole cell lysates, and microbial cell factories.

Fructose-2,6-bisphosphate synthesis by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is required for the glycolytic response to hypoxia and tumor growth.

Fructose-2,6-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates 6-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities. PFKFB4 is over-expressed in human cancers, induced by hypoxia and required for survival and growth of several cancer cell lines. Although PFKFB4 appears to be a rational target for anti-neoplastic drug development, it is not clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-fold greater than its phosphatase activity, siRNA and genomic deletion of PFKFB4 decrease F2,6BP, PFKFB4 over-expression increases F2,6BP and selective PFKFB4 inhibition in vivo markedly reduces F2,6BP, glucose uptake and ATP. Last, we find that PFKFB4 is required for cancer cell survival during the metabolic response to hypoxia, presumably to enable glycolytic production of ATP when the electron transport chain is not fully operational. Taken together, our data indicate that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP. We predict that pharmacological disruption of the PFKFB4 kinase domain may have clinical utility for the treatment of human cancers.

Viral hepatitis infection and insulin resistance: a review of the pathophysiological mechanisms.

Viral hepatitis is a common cause of morbidity in Mexico. Insulin resistance (IR) is related to the liver damage caused by some viral infections, especially chronic infections. Chronic viral infection is an important risk factor for the development of type 2 diabetes mellitus, disease that is currently among the 10 main causes of morbidity and the most common cause of mortality. Although several studies have reported an association between IR and hepatitis B virus or hepatitis C virus (HCV) infection, the pathophysiology has been studied thoroughly only for the association between IR and HCV infection. It is thought that HCV infection causes direct damage through the action of the core proteins, which induces an inflammatory state characterized by secretion of proinflammatory cytokines that interfere with normal insulin signaling and disturb glucose, lipid and protein metabolism. This review summarizes the mechanisms by which viral infection is thought to induce IR.

Viral hepatitis infection and insulin resistance: a review of the pathophysiological mechanisms / Hepatitis virales y resistencia a la insulina: revisión de los mecanismos fisiopatológicos

Viral hepatitis is a common cause of morbidity in Mexico. Insulin resistance (IR) is related to the liver damage caused by some viral infections, especially chronic infections. Chronic viral infection is an important risk factor for the development of type 2 diabetes mellitus, disease that is currently among the 10 main causes of morbidity and the most common cause of mortality. Although several studies have reported an association between IR and hepatitis B virus or hepatitis C virus (HCV) infection, the pathophysiology has been studied thoroughly only for the association between IR and HCV infection. It is thought that HCV infection causes direct damage through the action of the core proteins, which induces an inflammatory state characterized by secretion of proinflammatory cytokines that interfere with normal insulin signaling and disturb glucose, lipid and protein metabolism. This review summarizes the mechanisms by which viral infection is thought to induce IR.

Las hepatitis virales son una causa común de morbilidad en México. La resistencia a la insulina (RI) ha sido relacionada con el daño hepático causado por infecciones virales crónicas, haciendo de ellas un factor de riesgo para el desarrollo de diabetes mellitus tipo 2, problema de salud que se encuentra entre las primeras 10 causas de morbilidad y es la primera de mortalidad. Aunque varios estudios han reportado una asociación entre la RI y la infección con virus de la hepatitis B y virus de la hepatitis C, sólo con el último se ha estudiado su fisiopatología. Se ha sugerido que produce daño directo a través de proteínas de su núcleo e induce un estado inflamatorio que interfiere con la señalización normal de insulina, resultando en una alteración del metabolismo de glucosa, lípidos y proteínas. Esta revisión resume los mecanismos por los que se sugiere que estas infecciones inducen RI.

Effect of corosolic acid on gluconeogenesis in rat liver.

Corosolic acid (CRA), an active component of Banaba leaves (Lagerstroemia speciosa L.), decreases blood glucose in diabetic animals and humans. In this study, we investigated the mechanism of action of CRA on gluconeogenesis in rat liver. CRA (20-100 microM) dose-dependently decreased gluconeogenesis in perfused liver and in isolated hepatocytes. Fructose-2,6-bisphosphate (F-2,6-BP), a gluconeogenic intermediate, plays a critical role in hepatic glucose output by regulating gluconeogenesis and glycolysis in the liver. CRA increased the production of F-2,6-BP along with a decrease in intracellular levels of cAMP both in the presence and in the absence of forskolin in isolated hepatocytes. While a cAMP-dependent protein kinase (PKA) inhibitor inhibited hepatic gluconeogenesis, the drug did not intensify the inhibitory effect of CRA on hepatic gluconeogenesis in isolated hepatocytes. These results indicate that CRA inhibits gluconeogenesis by increasing the production of F-2,6-BP by lowering the cAMP level and inhibiting PKA activity in isolated hepatocytes. Furthermore, CRA increased glucokinase activity in isolated hepatocytes without affecting glucose-6-phosphatase activity, suggesting the promotion of glycolysis. These effects on hepatic glucose metabolism may underlie the various anti-diabetic actions of CRA.

Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate.

Heat-treated Escherichia coli producing Thermus polyphosphate kinase regenerated ATP by using exogenous polyphosphate. This recombinant could be used as a platform to produce valuable compounds in combination with thermostable phosphorylating or energy-requiring enzymes. In this work, we demonstrated the production of fructose 1,6-diphosphate from fructose and polyphosphate.

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli.

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.

Effects of brazilin on the production of fructose-2,6-bisphosphate in rat hepatocytes.

Increased hepatic glucose output is one of the major mechanisms of hyperglycemia in diabetic patients. Fructose-2,6-bisphosphate (F-2,6-BP), a gluconeogenic intermediate, plays a critical role in hepatic glucose output by regulating gluconeogenesis and glycolysis in the liver. Brazilin, an active component of sappan wood (Caesalpinia sappan), decreases blood glucose in diabetic animals. In this study, the effect of brazilin on gluconeogenic intermediate production and enzyme activity were examined to investigate the hypoglycemic mechanism of brazilin. Brazilin increased the production of F-2,6-BP in hepatocytes by elevating intracellular levels of fructose-6-phosphate (F-6-P) and hexose-6-phosphate (H-6-P). Brazilin was also found to significantly increase the activity of 6-phosphofructo-2-kinase (PFK-2) and pyruvate kinase in glucagon-treated hepatocytes. However, glucose-6-phosphatase activity was not affected by brazilin. This data suggests that brazilin inhibits hepatic gluconeogenesis by elevating the F-2,6-BP level in hepatocytes, possibly by elevating cellular F-6-P/H-6-P levels and PFK-2 activity. Increased pyruvate kinase activity may also play a role in the anti-gluconeogenic action of brazilin.

Itaconate reduces visceral fat by inhibiting fructose 2,6-bisphosphate synthesis in rat liver.

OBJECTIVE: Itaconate is an analog of phosphoenolpyruvate, which is an inhibitor of fructose-6-phosphate 2-kinase (F6P2Kinase), an enzyme that synthesizes fructose 2,6-bisphosphate (F26BP). Carbohydrates ingested are preferentially used for glycogen synthesis in the liver and muscles, and excess carbohydrates are metabolized by glycolysis in the liver and used for fatty acid synthesis. We hypothesized that itaconate is incorporated into liver cells and suppresses fat synthesis by inhibiting liver glycolysis at the step of phosphofructokinase, which is activated by F26BP. METHODS: Rats were allowed to eat ad libitum for 3 wk or, in separate experiments, to limit food intake by pair feeding. One group was given drinking water (control group) and the other group was given a 10 g/L itaconate solution (itaconate group). We measured body weight gain, visceral fat accumulation, and F6P2Kinase activity. RESULTS: Body weight gain in the itaconate group was lower than that in the control group (P < 0.05). In the dietary-controlled rats, there was no difference in body weight increase between groups, but visceral fat content (P < 0.01), plasma free fatty acid, and triacylglycerol levels (P < 0.05) were lower in the itaconate group than in the control group. Further, itaconate decreased the F26BP level (P < 0.05) in vivo and partly inhibited rat liver-type F6P2Kinase in vitro. CONCLUSIONS: These results indicate that itaconate, which is a decarboxylate and resembles phosphoenolpyruvate, is incorporated into liver cells and suppresses glycolysis by decreasing the level of F26BP, resulting in decreased visceral fat.

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit.

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.

Central modulatory neurons control fuel selection in flight muscle of migratory locust.

Insect flight is one of the most intense and energy-demanding physiological activities. High carbohydrate oxidation rates are necessary for take-off, but, to spare the limited carbohydrate reserves, long-distance flyers, such as locusts, soon switch to lipid as the main fuel. We demonstrate that before a flight, locust muscles are metabolically poised for take-off by the release of octopamine from central modulatory dorsal unpaired median (DUM) neurons, which increases the levels of the potent glycolytic activator fructose 2,6-bisphosphate in flight muscle. Because DUM neurons innervating the flight muscles are active during rest but selectively inhibited during flight, they stimulate carbohydrate catabolism during take-off but tend to decrease muscle glycolysis during prolonged flight. cAMP-dependent protein kinase A is necessary but not sufficient for signal transduction, suggesting parallel control via a calcium-dependent pathway. Locust flight is the first reported instance of a direct and specific involvement of neuronal activity in the control of muscle glycolysis in working muscle during exercise.

Lepidimoic acid increases fructose 2,6-bisphosphate in Amaranthus seedlings.

This study examines the influence of the growth promoter, lepidimoic acid, on the level of an important cytosolic signal metabolite, fructose 2,6-bisphosphate (Fru-2,6-P2), which can activate pyrophosphatedependent:phosphofructokinase (PFP, EC 2.7.1.90), and on glycolytic metabolism in Amaranthus caudatus seedlings. Fru-2,6-P2 concentrations were respectively increased by approximately 2-, 3- and 4-fold when the seedlings were treated with 0.3, 3 and 30 mM lepidimoic acid. Exogenous lepidimoic acid also affected levels of glycolytic intermediates in the seedlings. The increase in fructose 1,6-bisphosphate and decreases in fructose 6-phosphate and glucose 6-phosphate were found in response to the elevated concentration of lepidimoic acid. These results suggest that lepidimoic acid may affect glycolytic metabolism in the Amaranthus seedlings by increasing the activity of PFP due to increasing level of Fru-2,6-P2.

The proinflammatory mediator macrophage migration inhibitory factor induces glucose catabolism in muscle.

Severe infection or tissue invasion can provoke a catabolic response, leading to severe metabolic derangement, cachexia, and even death. Macrophage migration inhibitory factor (MIF) is an important regulator of the host response to infection. Released by various immune cells and by the anterior pituitary gland, MIF plays a critical role in the systemic inflammatory response by counterregulating the inhibitory effect of glucocorticoids on immune-cell activation and proinflammatory cytokine production. We describe herein an unexpected role for MIF in the regulation of glycolysis. The addition of MIF to differentiated L6 rat myotubes increased synthesis of fructose 2,6-bisphosphate (F2,6BP), a positive allosteric regulator of glycolysis. Increased expression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) enhanced F2,6BP production and, consequently, cellular lactate production. The catabolic effect of TNF-alpha on myotubes was mediated by MIF, which served as an autocrine stimulus for F2, 6BP production. TNF-alpha administered to mice decreased serum glucose levels and increased muscle F2,6BP levels; pretreatment with a neutralizing anti-MIF mAb completely inhibited these effects. Anti-MIF also prevented hypoglycemia and increased muscle F2,6BP levels in TNF-alpha-knockout mice that were administered LPS, supporting the intrinsic contribution of MIF to these inflammation-induced metabolic changes. Taken together with the recent finding that MIF is a positive, autocrine stimulator of insulin release, these data suggest an important role for MIF in the control of host glucose disposal and carbohydrate metabolism.

Apoptosis induced by growth factor withdrawal in fibroblasts overproducing fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate is a potent endogenous stimulator of glycolysis. A high aerobic glycolytic rate often correlates with increased cell proliferation. To investigate this relationship, we have produced clonal cell lines of Rat-1 fibroblasts that stably express transgenes coding for 6-phosphofructo-2-kinase, which catalyzes the synthesis of fructose 2,6-bisphosphate, or for fructose 2,6-bisphosphatase, which catalyzes its degradation. While serum deprivation in culture reduced the growth rate of control cells, it caused apoptosis in cells overproducing fructose 2,6-bisphosphate. Apoptosis was inhibited by 5-amino-4-imidazolecarboxamide riboside, suggesting that 5'-AMP-activated protein kinase interferes with this phenomenon.

Stimulation of fructose 1,6-bisphosphate production in melanoma cells by alpha-melanocyte-stimulating hormone-- 31P/13C-NMR and 32P-labeling studies.

In a 31P-NMR spectroscopic study of cultured M2R mouse melanoma cells, we previously demonstrated the acute stimulation of three peaks in the phosphomonoester region of the spectrum by [Ahx4, DPhe7]alpha-melanotropin (concomitant with an increase in cellular adenosine 3',5'-phosphate (cAMP) and a decrease in ATP [Degani, H., DeJordy, J. O. & Salomon, Y. (1991) Proc. Natl Acad. Sci. USA 88, 1506-1510]. Chemical identification of these metabolites was performed in this study using 32P metabolic labeling and polyethyleneimine-cellulose thin layer chromatography in combination with 31P-NMR and 13C-NMR spectroscopic methods. Two of the stimulated signals were identified as P1 and P6 of fructose 1,6-bisphosphate (FruP2) and their mode of regulation by alpha-melanotropin was examined. The FruP2 response to alpha-melanotropin coincided in time and dose with a rise in cAMP and a decrease in levels of ATP, while elevation of cAMP by forskolin alone did not increase FruP2. The stimulatory effect of alpha-melanotropin was not associated with a change in the overall rate of glycolysis, suggesting that FruP2 levels were not rate limiting in this process. The data suggest the presence of a previously unknown response of M2R melanoma cells to alpha-melanotropin, which coincides in time with enhanced cAMP accumulation but is not mediated by cAMP and may relate to the control of FruP2 in a non glycolytic context.

Anomeric specificity of rat hepatic 6-phosphofructo-2-kinase: an NMR study.

The anomeric specificity of 6-phosphofructo-2-kinase for D-fructose-6-phosphate was determined by nuclear magnetic spectroscopy. A mutant 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (His258-Ala) was used to minimize degradation of fructose-2,6-bisphosphate by the bisphosphatase activity. The 1H NMR spectrum of the fructose-2,6-bisphosphate formed from the reaction was identical in the spectral region (3.5 to 4.0 ppm) to that reported for D-fructose-2,6-bisphosphate by Voll et al. (7). The integration of this region accounted for the 7 nonexchangeable protons of the furanose form of fructose. The measured coupling constants and the chemical shifts were identical to those of commercially prepared D-fructose-2,6-bisphosphate. The long range (through 4-bond: P-2, O-2, C-2, C-3, and H-3) coupling between P-2 and H-3, 4JH-3, P-2, was found to be 1.06 Hz and provides strong evidence for the beta-anomer. Additionally, failure to find a similar coupling to the H-la peak ruled out the possibility of existence of the alpha-anomer. These results indicate that only beta-D-fructose-2,6-bisphosphate was synthesized via the 6-phosphofructo-2-kinase reaction. It was concluded that 6-phosphofructo-2-kinase has an absolute stereo specificity for the beta-anomer of D-fructose-6-phosphate.

Glucose-stimulated synthesis of fructose 2,6-bisphosphate in rat liver. Dephosphorylation of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase and activation by a sugar phosphate.

The effect of glucose on hepatic fructose (Fru) 2,6-P2 in starved rats was investigated. When livers were perfused with high glucose (40 mM), hexose-P in the liver increased immediately reaching the maximum within in 2 min, but Fru 2,6-P2 after a lag period of 4 min increased linearly. The activation of Fru 6-P,2-kinase and inactivation of Fru 2,6-Pase also showed a similar lag period. Determination of the phosphate contents of the bifunctional enzyme after 10 min of glucose perfusion revealed that 90% of the enzyme was in the dephospho form while only 10% of the control liver enzyme was dephosphorylated. Comparison of crude extracts of liver perfused with either high glucose or normal glucose (5.6 mM) showed that high glucose livers contained 50% higher protein phosphatase activity, which dephosphorylated the bifunctional enzyme. Subcellular fractionation of the extract showed that activation of the protein phosphatase occurred in the cytosol. Desalting of the cytosolic fraction resulted in a 50% loss of the protein phosphatase activity. The low molecular weight activator in the cytosol was isolated, and by various chemical and enzymatic methods it was identified as xylulose 5-P. The activation of protein phosphatase by xylulose 5-P showed a highly sigmoidal saturation curve. The rate of formation of xylulose 5-P in the perfused liver showed a lag period of approximately 2 min, and after 4 min its concentration reached 10 microM, the minimum concentration necessary for the activation of the protein phosphatase. We conclude that the mechanism of glucose-induced Fru 2,6-P2 synthesis was not due to increased Fru 6-P as generally thought but occurred as a result of dephosphorylation of Fru 6-P,2-kinase:Fru 2,6-Pase. Moreover, the dephosphorylation was enhanced by increased xylulose 5-P, which activated a specific protein phosphatase. The results suggest a mechanism for coordinated regulation of glycolysis and the pentose shunt pathway that is mediated by xylulose 5-P.

Correlation between fructose 2,6-bisphosphate and lactate production in skeletal muscle.

The epinephrine-induced production of lactate in nonexercising muscles may be due in part to allosteric activation of 6-phosphofructo-1-kinase by fructose 2,6-bisphosphate (F-2,6-P2). To determine if a correlation exists between F-2,6-P2 and lactate production in skeletal muscle, isolated rat hindlimbs were perfused for 30 min with a medium containing epinephrine at concentrations varying between 1.7 +/- 0.5 and 72.4 +/- 4.2 nM. In comparison to control values, hindlimbs perfused with 72.4 +/- 4.2 nM epinephrine had a two- to threefold increase in F-2,6-P2 and a fourfold increase in muscle lactate production. Hindlimb lactate production was highly correlated to gastrocnemius adenosine 3',5'-cyclic monophosphate (r = 0.80), fructose 6-phosphate (r = 0.87), and F-2,6-P2 (r = 0.81). The adenosine 3',5'-cyclic monophosphate-mediated increase in glycogenolysis with consequent increase in fructose 6-phosphate (substrate for 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase) is likely important for induction of lactate production by inactive muscle. The high correlation between muscle F-2,6-P2 and muscle lactate production at varying concentrations of epinephrine supports the hypothesis that the epinephrine-induced activation of glycolysis and lactate production in nonexercising muscle is mediated in part by increases in F-2,6-P2 levels.

CS-045, a new oral antidiabetic agent, stimulates fructose-2,6-bisphosphate production in rat hepatocytes.

Fructose-2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key enzyme in glycolysis. We previously revealed that sulfonylureas stimulate fructose-2,6-bisphosphate production in the rat liver by activating 6-phosphofructo-2-kinase. In the present study, we show that CS-045, a new antidiabetic agent, activated 6-phosphofructo-2-kinase and raised fructose-2,6-bisphosphate levels in dispersed rat hepatocytes. This action was time- and dose-dependent. Ten micromolar CS-045 raised the fructose-2,6-bisphosphate content linearly to the submaximal level in 20 min. Dose dependency was observed in the range of 1-30 microM. Thirty micromolar CS-045 completely reversed the inhibitory effect of 0.1 nM glucagon on fructose-2,6-bisphosphate production. CS-045 activated 6-phosphofructo-2-kinase by decreasing the Km value for the substrate (fructose-6-phosphate) without affecting the Vmax. The combination of suboptimal doses of CS-045 and tolbutamide increased fructose-2,6-bisphosphate content more than that induced by each agent alone. These results indicate that CS-045 may reduce plasma glucose by facilitating glycolysis in the liver.