Activation of glycolysis by insulin with a sequential increase of the 6-phosphofructo-2-kinase activity, fructose-2,6-bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes

The involvement of 6-phosphofructo-2-kinase, fructose 2,6-bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin-dependent short-term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short-term influence of insulin on these parameters was dependent on the insulin concentration used for the long-term culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as 'insulin cells' and 'control cells', respectively. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two-fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6-phosphofructo-2-kinase remained constant. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6-phosphofructo-2-kinase directly after the medium change. However, both parameters increased during a 1-2-h incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value. Addition of insulin to control cells led to a 5-8-fold stimulation of glycolysis but only after a 30-90-min lag phase. During this lag period insulin strongly increased sequentially the 6-phosphofructo-2-kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin-stimulated glycolysis. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin-independently, led to an immediate increase in glycolysis without a lag phase. It is concluded that in this insulin-sensitive cell system: the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin-dependent stimulation of glycolysis in insulin cells; activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level. The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion.     

Lithium's effects on rat liver glucose metabolism in vivo

Oral administration of lithium carbonate to fed-healthy rats strongly decreased liver glycogen content, despite the simultaneous activation of glycogen synthase and the inactivation of glycogen phosphorylase. The effect seemed to be related to a decrease in glucose 6-phosphate concentration and to a decrease in glucokinase activity. Moreover, in these animals lithium markedly decreased liver fructose 2,6-bisphosphate, which could be a consequence of the fall in glucose 6-phosphate and of the inactivation of 6-phosphofructo-2-kinase. Liver pyruvate kinase activity and blood insulin also decreased after lithium administration. Lower doses of lithium carbonate had less intense effects. Lithium administration to starved-healthy and fed-streptozotocin-diabetic rats caused a slight increase in blood insulin, which was simultaneous with increases in liver glycogen, glucose 6-phosphate, and fructose 2, 6-phosphate. Glucokinase, 6-phosphofructo-2-kinase, and pyruvate kinase activities also increased after lithium administration in starved-healthy and fed-diabetic rats. Lithium treatment activated glycogen synthase and inactivated glycogen phosphorylase in a manner similar to that observed in fed-healthy rats. Glycemia was not modified in any group of animals. These results indicate that lithium acts on liver glycogen metabolism in vivo in at least two different ways: one related to changes in insulinemia, and the other related to the direct action of lithium on the activity of some key enzymes of liver glucose metabolism.     

Fructose 2,6-bisphosphate and insulin stimulation of glycolysis in 3T3-L1 adipocytes

1. Insulin is able to stimulate lactate production and to enhance fructose 2,6-bisphosphate (Fru-2,6-P2) content in 3T3-L1 adipocytes. 2. Phorbol 12-myristate 13-acetate is more efficacious than insulin in rising Fru-2,6-P2 content and less effective in the stimulation of glycolysis. 3. 3T3-L1 adipocyte 6-phosphofructo-l-kinase appears to be very sensitive to exogenous Fru-2,6-P2. 4. Insulin treatment does not affect the maximum activity of 6-phosphofructo-1-kinase whereas it markedly increases the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The role of Fru-2,6-P2 in the insulin induced enhancement of glycolytic flux is discussed.     

Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6-bisphosphate and of glycolytic intermediates in Trypanosoma brucei

Upon differential centrifugation of cell-free extracts of Trypanosoma brucei, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6-phosphofructo-2-kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycerol 3-phosphate. The Km of fructose-2,6-bisphosphatase was 7 microM; this enzyme was inhibited by fructose 1,6-bisphosphate (Ki = 10 microM) and, less potently, by fructose 6-phosphate, phosphoenolpyruvate and glycerol 3-phosphate. Melarsen oxide inhibited 6-phosphofructo-2-kinase (Ki less than 1 microM) and fructose-2,6-bisphosphatase (Ki = 2 microM) much more potently than pyruvate kinase (Ki greater than 100 microM). The intracellular concentrations of fructose 2,6-bisphosphate and hexose 6-phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6-bisphosphate, ATP, hexose 6-phosphate and fructose 1,6-bisphosphate. These studies indicate that the concentration of fructose 2,6-bisphosphate is mainly controlled by the concentration of the substrates of 6-phosphofructo-2-kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6-bisphosphate on pyruvate kinase. At micromolar concentrations, melarsen oxide blocked almost completely the formation of fructose 2,6-bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6-phosphates. At higher concentrations (3-10 microM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolypyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.     

Changes in key regulatory enzymes of hepatic carbohydrate metabolism after glucose loading of starved rats

When glucose was given to starved rats there was an increase in both 6-phosphofructo 2-kinase and pyruvate kinase activity and a decrease in fructose 2,6-bisphosphatase activity 30 min and 60 min later. These changes were accompanied by an increase in glycogen deposition and by modest, but significant increases in fructose 2,6-bisphosphate levels at the same time. Metabolite measurements indicated that flux through 6-phosphofructo 1-kinase and pyruvate kinase were increased. These results suggest that although glycogen deposition may occur via the gluconeogenic pathway, glycolysis is activated at the same time by changes in the phosphorylation state of key regulatory enzymes as well as by the small rise in fructose 2,6-bisphosphate.     

Insulin-like effects of vanadate on glucokinase activity and fructose 2,6-bisphosphate levels in the liver of diabetic rats

Streptozotocin diabetic rats showed more than a 4-fold increase in blood glucose levels, whereas hepatic glycogen, fructose 2,6-bisphosphate concentration, and 6-phosphofructo-2-kinase activity were decreased. The "total" 6-phosphofructo-2-kinase and the "active" (nonphosphorylated) form of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Vanadate administration for a 2-week period restored the altered values in the diabetic rats without modifying significantly in the control animals any of the parameters studied. Glucokinase activity was essentially lacking in the diabetic animals, and vanadate treatment restored the activity to about 65% of its control value, a good correlation between the recovery of the enzyme and the blood glucose level being observed. These results show an insulin-like effect of vanadate in the whole animal and suggest that insulin and vanadate possess similar actions on hepatic intracellular events.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

Vanadate counteracts glucagon effects in isolated rat hepatocytes

The incubation of isolated rat hepatocytes with vanadate increased the concentration of fructose 2,6-bisphosphate without modifying 6-phosphofructo-2-kinase activity. Vanadate also reverted and prevented the decrease of fructose 2,6-bisphosphate levels, of the "active" form of the 6-phosphofructo 2-kinase and of the pyruvate kinase activity ratio produced by glucagon, by probably counteracting the increase in cyclic AMP concentration.     

Perturbation of glucose flux in the liver by decreasing F26P2 levels causes hepatic insulin resistance and hyperglycemia

Hepatic insulin resistance is one of the characteristics of type 2 diabetes and contributes to the development of hyperglycemia. How changes in hepatic glucose flux lead to insulin resistance is not clearly defined. We determined the effects of decreasing the levels of hepatic fructose 2,6-bisphosphate (F26P(2)), a key regulator of glucose metabolism, on hepatic glucose flux in the normal 129J mice. Upon adenoviral overexpression of a kinase activity-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme that determines F26P(2) level, hepatic F26P(2) levels were decreased twofold compared with those of control virus-treated mice in basal state. In addition, under hyperinsulinemic conditions, hepatic F26P(2) levels were much lower than those of the control. The decrease in F26P(2) leads to the elevation of basal and insulin-suppressed hepatic glucose production. Also, the efficiency of insulin to suppress hepatic glucose production was decreased (63.3 vs. 95.5% suppression of the control). At the molecular level, a decrease in insulin-stimulated Akt phosphorylation was consistent with hepatic insulin resistance. In the low hepatic F26P(2) states, increases in both gluconeogenesis and glycogenolysis in the liver are responsible for elevations of hepatic glucose production and thereby contribute to the development of hyperglycemia. Additionally, the increased hepatic gluconeogenesis was associated with the elevated mRNA levels of peroxisome proliferator-activated receptor-gamma coactivator-1alpha and phosphoenolpyruvate carboxykinase. This study provides the first in vivo demonstration showing that decreasing hepatic F26P(2) levels leads to increased gluconeogenesis in the liver. Taken together, the present study demonstrates that perturbation of glucose flux in the liver plays a predominant role in the development of a diabetic phenotype, as characterized by hepatic insulin resistance.     

Fructose 2,6-bisphosphate in isolated foetal hepatocytes

Fru 2,6-P2 was present in isolated foetal hepatocytes at a concentration of 1.6 nmol per g cells. When foetal hepatocytes were exposed to glucagon no changes were observed either in the concentration of Fru 2,6-P2 and lactate release or in the activities of 6-phosphofructo-2-kinase and pyruvate kinase. Incubation of purified 6-phosphofructo-2-kinase with the catalytic subunit of protein kinase did not change the enzyme activity. The inhibition by sn-glycerol 3-phosphate was much lower for the foetal than for adult enzyme. These results suggest that an isoenzyme of 6-phosphofructo-2-kinase in foetal hepatocytes different from that of adult hepatocytes may be present.     

The mechanism by which glucose increases fructose 2,6-bisphosphate concentration in Saccharomyces cerevisiae. A cyclic-AMP-dependent activation of phosphofructokinase 2

When glucose was added to a suspension of Saccharomyces cerevisiae in stationary phase, it caused a transient increase in the concentration of cyclic AMP and a more persistent increase in the concentration of hexose 6-phosphate and of fructose 2,6-bisphosphate. These effects of glucose on cyclic AMP and fructose 2,6-bisphosphate but not that on hexose 6-phosphate were greatly decreased in the presence of 0.15 mM acridine orange or when a temperature-sensitive mutant deficient in adenylate cyclase was used at the restrictive temperature. Incubation of the cells in the presence of dinitrophenol and in the absence of glucose increased the concentration of both cyclic AMP and fructose 2,6-bisphosphate, but with a minimal change in that of hexose 6-phosphate. Glucose induced also in less than 3 min a severalfold increase in the activity of 6-phosphofructo-2-kinase and this effect was counteracted by the presence of acridine orange. When a cell-free extract of yeast in the stationary phase was incubated with ATP-Mg and cyclic AMP, there was a 10-fold activation of 6-phosphofructo-2-kinase. Finally, the latter enzyme was purified 150-fold and its activity could then be increased about 10-fold upon incubation with ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase. This activation resulted from a 4.3-fold increase in V and a 2-fold decrease in Km. Both forms of the enzyme were inhibited by sn-glycerol 3-phosphate. From these results it is concluded that the effect of glucose in increasing the concentration of fructose 2,6-bisphosphate in S. cerevisiae is mediated by the successive activation of adenylate cyclase and of cyclic-AMP-dependent protein kinase and by the phosphorylation of 6-phosphofructo-2-kinase by the latter enzyme. In deep contrast with what is known of the liver enzyme, yeast 6-phosphofructo-2-kinase is activated by phosphorylation instead of being inactivated.     

Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes.

The ability of glucagon and of adrenaline to affect the concentration of fructose 2,6-bisphosphate in isolated hepatocytes was re-investigated because of important discrepancies existing in the literature. We were unable to detect a significant difference in the sensitivity of the hepatocytes with regard to the effect of glucagon to initiate the interconversion of phosphorylase, pyruvate kinase, 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase, and also to cause the disappearance of fructose 2,6-bisphosphate. In contrast, we have observed differences in the time-course of these various changes, since the interconversions of phosphorylase and of pyruvate kinase were at least twice as fast as those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. When measured in a cell-free system in the presence of MgATP, the cyclic AMP-dependent interconversion of pyruvate kinase was 5-10-fold more rapid than those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. These data indicate that 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase are relatively poor substrates for cyclic AMP-dependent protein kinase; they also support the hypothesis that the two catalytic activities belong to a single protein. Adrenaline had only a slight effect on the several parameters under investigation, except for the activation of phosphorylase. In the absence of Ca2+ ions from the incubation medium, however, adrenaline had an effect similar to that of glucagon.     

Regulation of fructose 2,6-bisphosphate concentration in white adipose tissue.

Injection of insulin to fed rats diminished the concentration of fructose 2,6-bisphosphate in white adipose tissue. Incubation of epididymal fat-pads or adipocytes with insulin stimulated lactate release and sugar detritiation and also decreased fructose 2,6-bisphosphate concentration. Such a decrease was, however, not observed in fat-pads from starved or alloxan-diabetic rats. Incubation of adipocytes from fed rats with various concentrations of glucose or fructose led to a dose-dependent rise in fructose 2,6-bisphosphate which correlated with lactate output and detritiation of 3-3H-labelled sugar. In adipocytes from fed rats, palmitate stimulated the detritiation of [3-3H]glucose without affecting lactate production and fructose 2,6-bisphosphate concentration. Incubation of epididymal fat-pads from fed rats in the presence of antimycin stimulated lactate output but decreased fructose 2,6-bisphosphate concentration. Changes in lipolytic rates brought about by noradrenaline, insulin, adenosine and corticotropin in adipocytes from fed rats were not related to changes in fructose 2,6-bisphosphate or to rates of lactate output. In fed rats, the activity of 6-phosphofructo-2-kinase was not changed after treatment of adipocytes with insulin, noradrenaline or adenosine. It is suggested that the decrease in fructose 2,6-bisphosphate concentration observed after insulin treatment can be explained by the increase in sn-glycerol 3-phosphate, an inhibitor of 6-phosphofructo-2-kinase.     

Effect of benzoate on the metabolism of fructose 2,6-bisphosphate in yeast

When benzoate (2 mM, pH 3.5) was added together with glucose (0.1 M) to a suspension of Saccharomyces cerevisiae in the stationary phase, it caused a relative increase in the concentration of glucose 6-phosphate and fructose 6-phosphate and a decrease in the concentration of fructose 1,6-bisphosphate. These effects are in confirmation of similar observations made by Krebs et al. [Biochem. J. 214, 657-663 (1983)] and are indicative of an inhibition of 6-phosphofructo-1-kinase. Benzoate also caused an about fourfold relative decrease in the concentration of fructose 2,6-bisphosphate, an increase in that of cyclic AMP with no change in that of ATP. It also greatly decreased the activation of 6-phosphofructo-2-kinase, but not that of trehalase, both of which normally occur upon addition of glucose to a yeast suspension. When added 10 min after glucose, benzoate caused a rapid (within 2-3 min) decrease in fructose 2,6-bisphosphate concentration and in 6-phosphofructo-2-kinase activity. In the presence of benzoate, there was also a parallel decrease in the concentration of fructose 2,6-bisphosphate and in the rate of ethanol production when the external pH was dropped from 5.0 to 2.5, with minimal change in the concentration of ATP. Purified 6-phosphofructo-2-kinase was inhibited by benzoate and also by an acid pH. Experiments with cell-free extracts did not provide an explanation for the rapid disappearance of fructose-2,6-bisphosphate or the inactivation of 6-phosphofructo-2-kinase in yeast upon addition of benzoate.     

Insulin-mediated regulation of heart atrial and ventricular 6-phosphofructo-1-kinase

Atrial 6-phosphofructo-1-kinase activity from the hearts of diabetic rats was decreased by 50%, but ventricular 6-phosphofructo-1-kinase activity was found not to be insulin-sensitive. This decrease in atrial 6-phosphofructo-1-kinase activity during diabetes was characterized by diminished levels of all three types of 6-phosphofructo-1-kinase subunits. As shown by immunological titration and column chromatography, the population of native 6-phosphofructo-1-kinase isozymes in the ventricles was not measurably affected during insulin deprivation. However, the atrial isozyme population in diabetic rat heart appeared to contain, on a relative basis, higher levels of the isozymic forms containing the L-type subunit. Measurement of the levels of this subunit indicated that in diabetic atria it was less affected than the other subunits. In the ventricles, insulin deficiency did not promote significant losses of fructose-2,6-P2; but, in diabetic rats, the atrial levels of this activator were decreased by 80% and subsequently restored by insulin treatment. These data suggest that any insulin-mediated effects on ventricular 6-phosphofructo-1-kinase activity and resultant effects on ventricular glycolysis do not appear to be exerted through changes in enzyme concentration, but probably through changes in modulators other than fructose-2,6-P2. In contrast to the ventricles, it appears that insulin exerts its effects on atrial 6-phosphofructo-1-kinase activity and, in part, influences atrial glycolysis through alteration of fructose-2,6-P2 levels, enzyme concentration, and isozymic content.     

Yeast 6-phosphofructo-2-kinase: sequence and mutant.

We have reported yeast 6-phosphofructo-2-kinase (EC 2.7.1.105) as having a ca. 96-kDa subunit size, as well as isolation of its structural gene, PFK26. Sequencing now shows an open reading frame of 827 amino acids and 93.5 kDa. The deduced amino acid sequence has 42% identity with the 55-kDa subunit of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver with extra material at both ends. Although the yeast sequence is especially similar to the liver one in its bisphosphatase domain, the essential His-258 of the liver enzyme is, in yeast, a serine, which may explain the apparent lack of bisphosphatase activity. Also, the yeast enzyme known to be activated via protein kinase A, has a putative phosphorylation site near its C-terminus and lacks the N-terminal phosphorylation sequence involved in inhibition of the liver enzyme. In a chromosomal null mutant strain, pfk26::LEU2, activity was marginal and the protein was not detectable as antigen. The mutant strain grew well on glucose and contained a near-normal level of fructose 2,6-P2. But in its growth on pyruvate, by contrast with the wild-type strain, no fructose 2,6-P2 was detectable, and it did not form after glucose addition in the presence of cycloheximide either. Such resting cells, however, metabolized glucose at the normal high rate. Glucose addition to the pfk26 mutant strain in the absence of cycloheximide, on the other hand, caused a ca. 10% normal rate of fructose 2,6-P2 accumulation, presumably employing a glucose-inducible second enzyme. Using strains also lacking 6-phosphofructo-1-kinase, affinity chromatography revealed the second enzyme as a minor peak amounting to 6% of 6-phosphofructo-2-kinase activity in a PFK26 strain and as the sole peak, in similar amount, in a pfk26 mutant strain.     

Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was phosphorylated by cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Treatment of the 32P-labeled enzyme with thermolysin removed all of the radioactivity from the enzyme core and produced a single labeled peptide. The phosphopeptide was purified by ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography. The sequence of the 12-amino acid peptide was found to be Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser(P)-Ser-Ile-Pro-Gln. Correlation of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated. The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of the native bifunctional enzyme was compared with that of other rat liver protein substrates. The Km for 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (10 microM) was less than that for rat liver pyruvate kinase (39 microM), fructose-1,6-bisphosphatase (222 microM), and 6- phosphofructose -1-kinase (230 microM). Comparison of the initial rate of phosphorylation of a number of protein substrates of the cyclic AMP-dependent protein kinase revealed that only skeletal muscle phosphorylase kinase was phosphorylated more rapidly than the bifunctional enzyme. Skeletal muscle glycogen synthase, heart regulatory subunit of cyclic AMP-dependent protein kinase, and liver pyruvate kinase were phosphorylated at rates nearly equal to that of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, while phosphorylation of fructose-1,6-bisphosphatase and 6-phosphofructo-1-kinase was barely detectable. Phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was not catalyzed by any other protein kinase tested. These results are consistent with a primary role of the cyclic AMP-dependent protein kinase in regulation of the enzyme in intact liver.     

Involvement of PI 3-kinase and activated ERK in facilitating insulin-stimulated triacylglycerol synthesis in hepatocytes

Triacylglycerol synthesis was studied in hepatocytes isolated from fasted/refed rats by EDTA perfusion. Insulin induced a 1.5-fold increase in glucose incorporation into triacylglycerol. Insulin-stimulated triacylglycerol synthesis and insulin-stimulated protein kinase B/Akt activity were inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY 294002, and the mitogen-activated protein kinase kinase inhibitor PD 98059. Inhibition of p70 ribosomal protein-S6 kinase with rapamycin was without effect. Insulin-stimulated pyruvate dehydrogenase activity was abolished by phosphatidylinositol 3-kinase inhibitors. No effect of insulin on acetyl CoA carboxylase activity was observed.     

Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae.

The influence of 6-phosphofructo-1-kinase on glycolytic flux in the yeast Saccharomyces cerevisiae was assessed by measuring the effects of enzyme overexpression on glucose consumption, ethanol production, and glycolytic intermediate levels under aerobic and anaerobic conditions. Enzyme overexpression had no effect on glycolytic flux under anaerobic conditions, but under aerobic conditions, it increased glycolytic flux up to the anaerobic level. The Pasteur effect was thus abolished in these cells. The increased glycolytic flux was accompanied by a compensatory decrease in flux in oxidative phosphorylation. The concentrations of the enzyme substrates showed only small or insignificant changes. These data imply that the enzyme has a low flux control coefficient for glycolysis. However, in cells overexpressing the enzyme, there was a compensatory decrease in 6-phosphofructo-2-kinase activity which was accompanied by a corresponding decrease in fructose 2,6-bisphosphate concentration. Measurements in vitro showed that the decrease in the concentration of this positive allosteric effector of 6-phosphofructo-1-kinase could significantly lower its specific activity in the cell and that this could compensate for the increased enzyme concentration in the overproducer.