Cyclic AMP induced inhibition of pyruvate kinase flux in the intact liver cell.

The rate of pyruvate kinase flux in the intact cell is estimated by a new procedure, involving trapping of ¹⁴C from NaH¹⁴CO₃ in a large pyruvate + lactate pool, and calculation of the specific activity of phosphoenol pyruvate. With high concentrations of pyruvate as substrate for isolated rat liver cells, cyclic AMP (0.1 mM) depresses pyruvate kinase flux by about 45%, in addition to inhibiting both glucose and lactate formation. The inhibition of pyruvate kinase may cause an inhibition of hydrogen translocation from the mitochondria to the cytosol.     

Epinephrine-stimulated phosphorylation of pyruvate kinase in hepatocytes

Incubation of rat liver parenchymal cells with 10^?5m epinephrine or norepinephrine resulted in a rapid incorporation of ³²P into pyruvate kinase. Inclusion of α-adrenergic blocking agents (phenoxybenzamine or phentolamine) in the hepatocyte incubation medium prior to addition of epinephrine suppressed the subsequent phosphorylation of pyruvate kinase. On the other hand, inclusion of the β-adrenergic antagonist, propranolol, in the hepatocyte incubation medium prior to addition of epinephrine did not suppress the epinephrine-elicited phosphorylation of pyruvate kinase. Exogenous addition of either cyclic AMP or cyclic GMP to the hepatocyte incubation medium also resulted in increased phosphorylation of pyruvate kinase. To investigate whether the same amino acid residue(s) of liver pyruvate kinase was being phosphorylated in each instance, ³²P-labeled pyruvate kinase was isolated from hepatocytes after incubation in the presence or absence of either glucagon or epinephrine. In addition, purified liver pyruvate kinase was phosphorylated in vitro with a rat liver cyclic AMP-dependent protein kinase. Each ³²P-labeled pyruvate kinase was then subjected to tryptic digestion, two-dimensional thin-layer peptide mapping, and autoradiography. Each ³²P-labeled pyruvate kinase sample yielded 44 to 48 tryptic peptides upon staining with ninhydrin and 4 peptides that contain ³²P as detected by autoradiography. Furthermore, the same 4 peptides of pyruvate kinase were radiolabeled in each instance. Thus phosphorylation of pyruvate kinase in vitro with [γ-³²P]ATP or upon addition of either glucagon or epinephrine to hepatocytes incubated with ³²P_i resulted in phosphorylation of the same amino acid residues.     

Cyclic AMP-dependent phosphorylation of erythrocyte variant pyruvate kinase

Cyclic AMP-dependent phosphorylation of a variant erythrocyte pyruvate kinase (PK; EC 2.7.1.40) was studied. This variant PK shows a faster electrophoretic mobility than the normal enzyme. The decreased enzyme activity observed in this variant is associated with a quantitative decrease of enzyme protein. Other parameters are within normal ranges. The partially purified variant PK is phosphorylated with a subsequent increase of k0.5s (phosphoenolpyruvate) similar to the normal control, suggesting that the structural abnormality of the variant enzyme has no influence on the phosphorylation-deactivation mechanism. On the other hand, the variant PK in the erythrocyte was less extensively phosphorylated than PK in normal erythrocytes. This may be the result of abnormal metabolism in the patient's red cells, including increased 2,3-diphosphoglycerate and decreased adenosine triphosphate levels.     

Cyclic AMP-dependent inactivation of human liver pyruvate kinase.

Human liver pyruvate kinase is rapidly (within 2 min) inactivated by incubation of a human liver supernatant with cyclic AMP, when measured at suboptimal substrate concentrations. Half-maximal inactivation is reached with 0.04 μM cyclic AMP. The apparent K_0.5 for phosphoenolpyruvate shifts from 0.5 mM to 1.1 mM by incubation with cyclic AMP. It is concluded that cyclic AMP-dependent protein kinase may catalyze the phosphorylation of human liver pyruvate kinase $\text{in vivo}$.     

Epidermal growth factor stimulates the phosphorylation of pyruvate kinase in freshly isolated rat hepatocytes.

Epidermal growth factor (EGF) has previously been shown to stimulate gluconeogenesis in rat liver by decreasing the activity of pyruvate kinase [(1988) Biochem. J. 255, 361-364]. Here we investigate the mechanism underlying the inactivation of the enzyme. EGF was found to increase the incorporation of phosphate into pyruvate kinase, with maximal phosphorylation achieved only after 10 min in the presence of the growth factor. The increase in phosphorylation was not additive with that caused by cyclic AMP. Phosphoamino acid analysis of pyruvate kinase isolated from cells treated with EGF indicated that EGF increases phosphorylation solely on serine residues. The exact site of EGF-mediated phosphorylation has yet to be identified.     

Cyclic AMP stimulated protein kinase activity within the secretory vesicle fraction of rat islets.

The isolated rat islet secretory vesicle fraction (24,000xg) was incubated with gamma [³²P ATP] and the TCA-insoluble material was pelleted. The pellet was acid hydrolyzed, lyophilized, redissolved, then subjected to two-dimensional chromatographic separation. Two labeled compounds were identified, i.e., phosphoserine and inorganic phosphorus. With the addition of cyclic AMP (3.5 × 10^?6M), there was a 235% increase in phosphoserine radioactivity (P<.01) within endogenous protein subunits. When histones were added to the incubation media, the addition of cyclic AMP resulted in a threefold increase in phosphoserine radioactivity in the TCA-insoluble material (P<.01). A comparison was made of cyclic AMP-stimulated protein kinase activity in the homogenate and various islet subcellular fractions. Cyclic AMP-stimulated protein kinase activity is associated with the 24,000xg (secretory vesicle) fraction.     

Glucagon-induced phosphorylation of pyruvate kinase (type L) in rat liver slices.

The effect of glucagon on the phosphorylation of pyruvate kinase in ³²P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10^?7 M glucagon, the incorporation of [³²P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10^?9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.     

Cyclic AMP promotes neuronal survival by phosphorylation of glycogen synthase kinase 3beta

Agents that elevate intracellular cyclic AMP (cAMP) levels promote neuronal survival in a manner independent of neurotrophic factors. Inhibitors of phosphatidylinositol 3 kinase and dominant-inactive mutants of the protein kinase Akt do not block the survival effects of cAMP, suggesting that another signaling pathway is involved. In this report, we demonstrate that elevation of intracellular cAMP levels in rat cerebellar granule neurons leads to phosphorylation and inhibition of glycogen synthase kinase 3beta (GSK-3beta). The increased phosphorylation of GSK-3beta by protein kinase A (PKA) occurs at serine 9, the same site phosphorylated by Akt. Purified PKA is able to phosphorylate recombinant GSK-3beta in vitro. Inhibitors of GSK-3 block apoptosis in these neurons, and transfection of neurons with a GSK-3beta mutant that cannot be phosphorylated interferes with the prosurvival effects of cAMP. These data suggest that activated PKA directly phosphorylates GSK-3beta and inhibits its apoptotic activity in neurons.     

Cyclic AMP and free fatty acids in the longer-term regulation of pyruvate dehydrogenase kinase in rat soleus muscle.

Starvation increased pyruvate dehydrogenase (PDH) kinase activity in extracts of freshly excised rat soleus 2.2-fold (from 0.6 min-1 in fed rats to 1.31 min-1 in 48-h-starved rats). In fed rats, activities were unchanged following 24 h of culture in medium 199, but increased 2.1-fold on 24 h of culture with 50 microM dibutyryl cAMP plus 1 mM n-octanoate and 1.6-1.7-fold with either agent alone. Approx. 70% of the increase in PDH kinase induced by starvation was lost following 24 h of culture in medium 199; the loss was prevented by 50 microM dibutyryl cAMP plus 1 mM n-octanoate. cAMP concentrations in fresh soleus muscle were 1 nmol/g (fed rats) and 1.6 nmol/g (starved rats). After 20-60 min of culture the fed-starved difference disappeared and [cAMP] fell to 0.4 nmol/g. Calcitonin-gene-related peptide (CGRP) increased cAMP 3-fold; the increase was maintained throughout 24 h of culture, but was readily reversed at 30 min or 24 h of culture by 60-min incubation with CGRP-free medium. Starvation of the rat (48 h) had no effect on the sensitivity of soleus towards the [cAMP]-increasing effect of CGRP. It is concluded that culture may reverse effects of starvation on PDH kinase activity by lowering cAMP and by removal from the in vivo effects of circulating free fatty acids; and that starvation and CGRP had no detectable long-term effects on the cAMP system in soleus muscle.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose. Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

Abstract. From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose.
Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Cyclic AMP independence of Escherichia coli protein phosphorylation

The effect of cyclic AMP on protein phosphorylation was analyzed comparatively in two strains of E.coli differing in their capacity to synthesize this nucleotide, one of them lacking the adenylate cyclase activity. The results obtained from both in vivo and in vitro experiments concurred in showing that the bacterial protein kinase activity is cAMP-independent.     

Membrane-associated pyruvate kinase in developing guinea-pig liver.

During analysis of pyruvate kinase distribution in developing guinea-pig liver it was observed that a substantial proportion of the activity remained associated with the microsomal membrane fraction ('microsomes'). Although some of this could be removed by washing with sucrose, the majority required detergent treatment for liberation, and even then at least one-half remained attached to the microsomes. Estimates of the contribution of this fraction to total cell pyruvate kinase activity indicated that it was more than 50% of the total, and this is likely to be an underestimate because of the continued latency of the enzyme even in the presence of detergent. The susceptibility of the microsomal enzyme, whether released by detergent or sucrose washing, to inactivation by Triton X-100 suggested it to be different from the cytosolic enzyme, which was stable under such conditions. (The microsomal enzyme required the presence of additional protein, such as bovine serum albumin, to maintain stability.) This view was confirmed by DEAE-cellulose chromatography and particularly isoelectric focusing, where the microsomal enzyme was shown to consist of at least four forms, which were distinctly different from those in the cytosol. Those data and the kinetic properties of the four forms in the membrane fraction indicate that the microsomal pyruvate kinase could consist of four counterparts to the cytosolic isoenzyme forms. These results are discussed in relation to the two possible explanations for the phenomenon (not mutually exclusive): that the more hydrophobic membrane forms are precursors of the cytosolic enzyme and that they may be part of functional glycolytic pathway in the microsomes of developing liver.     

Modulation of the phosphorylation state of rat liver pyruvate kinase by allosteric effectors and insulin.

The regulation of pyruvate kinase in isolated hepatocytes from fasted rats was studied where the intracellular level of fructose 1,6-bisphosphate was elevated 5-fold by the addition of 5 mM dihydroxyacetone. In this case, flux through pyruvate kinase was increased. The increase in flux correlated with an elevation in fructose bisphosphate levels but not with P-enolpyruvate levels which were unchanged. Pyruvate kinase was activated and its affinity for P-enolpyruvate was increased 7-fold in hepatocyte homogenates. Precipitation of the enzyme from homogenates with ammonium sulfate removed fructose 1,6-bisphosphate and activation was no longer observed. These results indicate that flux through and activity of pyruvate kinase can be controlled by the intracellular level of fructose 1,6-bisphosphate. The effect of elevated fructose 1,6-bisphosphate levels on the ability of glucagon to inactivate pyruvate kinase was also studied where only covalent enzyme modification is observed. Inactivation by maximally effective hormone concentrations was unaffected by elevated levels of fructose 1,6-bisphosphate, but the half-maximally effective concentration was increased from 0.3 to 0.8 nM. Activation of the cyclic AMP-dependent protein kinase by 0.3 nM glucagon was unaffected, but the initial rate of pyruvate kinase inactivation was suppressed. These results suggest that alterations in the level of fructose 1,6-bisphosphate can affect the ability of physiological concentrations of glucagon to inactivate pyruvate kinase by opposing phosphorylation of the enzyme. Consistent with this view was the finding that physiological concentrations of fructose 1,6-bisphosphate inhibited in vitro phosphorylation of purified pyruvate kinase. Inactivation of pyruvate kinase by 0.3 nM glucagon or 1 microM phenylephrine was also suppressed by 10 nM insulin. Insulin did not act by increasing fructose 1,6-bisphosphate levels. The antagonism to glucagon correlated well with the ability of insulin to suppress activation of the cyclic AMP-dependent protein kinase. However, no such correlation was observed with phenylephrine in the absence or presence of insulin. Thus, insulin can enhance pyruvate kinase activity by both cyclic AMP-dependent and independent mechanisms.     

Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase

In the red cell incubated with ortho-[32P] phosphate, CaCl₂ and calcium ionophore A 23187, phosphorylation of erythrocyte pyruvate kinase was demonstrated using the double antibody technique and autoradiography. Phosphorylation was inhibited by calmodulin inhibitors, trifluoperazine or ZnCl₂. In the presence of purified erythrocyte calmodulin, CaCl₂ and [γ-³²P] ATP, the partially purified erythrocyte pyruvate kinase containing cytozol protein kinases was phosphorylated. This was also inhibited by trifluoperazine or ZnCl₂. From these results, it was concluded that erythrocyte pyruvate kinase is phosphorylated by a calcium-calmodulin dependent process.     

Turnover of liver pyruvate kinase

The relative rates of synthesis and degradation for liver pyruvate kinase have been determined in rats fed standard lab chow, fasted, and refed a high-carbohydrate-low-protein diet. Relative rates of synthesis and apparent rates of degradation were determined by pulse-labeling the enzyme in vivo with l-[4,5-³H]leucine and by measuring the incorporation of radioactivity into liver pyruvate kinase after quantitative precipitation of the enzyme with anti-liver pyruvate kinase immunoglobulin. The relative rate of synthesis decreased approximately 75% upon fasting and then increased 20- to 30-fold upon refeeding the high-carbohydrate diet. The apparent half-lives for liver pyruvate kinase in fasted, control, and refed animals are very similar (55, 59, and 47 h, respectively). Thus, the nutritional alterations in the levels of liver pyruvate kinase seem to result primarily from alterations in the rate of enzyme synthesis.     

Differential phosphorylation of MAP-2 stimulated by calcium-calmodulin and cyclic AMP.

Comparison of cyclic AMP- and calcium-dependent phosphorylation in rat brain cytosol reveals MAP-2 to be a common endogenous substrate. Examination of limited protease digestion patterns indicates that the two kinases phosphorylate MAP-2 at distinct sites and suggests that such phosphorylation may have differential effects on MAP-2 function.     

Glucagon regulation of pyruvate kinase in relation to phosphenol pyruvate substrate cycling in isolated rat hepatocytes.

The rate of flux through pyruvate kinase in isolated rat hepatocytes has been estimated by a new procedure involving direct spectrophotometric measurement of pyruvate production by liver cells suspended in an oxygenated medium containing lactate dehydrogenase and NADH. For the substrates, glucose, dihydroxyacetone, fructose, propionate and galactose only the rate of pyruvate production from glucose and galactose was inhibited by the addition of 1 μM-glucagon. These results imply that glucagon mediates glycolytic flux at a point in the pathway preceding the point of entry of fructose and dihydroxyacetone and not at pyruvate kinase.