scholarly journals Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver

1975 ◽  
Vol 150 (1) ◽  
pp. 51-58 ◽  
Author(s):  
F J Moreno ◽  
L Sánchez-Urrutia ◽  
J M Medina ◽  
F Sánchez-Medina ◽  
F Mayor
Keyword(s):  
Cyclic Amp ◽  
Nicotinic Acid ◽  
Rat Liver ◽  
Hormonal Regulation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Actinomycin D ◽  
De Novo ◽  
Glycogen Depletion ◽  
Perfusion Medium ◽  
Low Concentrations

1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably < the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.

scholarly journals Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element.

1990 ◽  
Vol 10 (7) ◽  
pp. 3357-3364 ◽  
Author(s):  
P G Quinn ◽  
D K Granner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Nuclear Factors ◽  
Dependent Protein

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

scholarly journals The effects of glucagon and insulin on adenosine 3′:5′-cyclic monophosphate concentrations in an organ culture of mature rat liver

1973 ◽  
Vol 132 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Kenneth Siddle ◽  
Barbara Kane-Maguire ◽  
Anthony K. Campbell
Keyword(s):  
Cyclic Amp ◽  
Organ Culture ◽  
Rat Liver ◽  
Incubation Medium ◽  
Radioactive Tracer ◽  
Maximal Increase ◽  
Low Concentrations ◽  
Glucagon And Insulin ◽  
Millipore Filters ◽  
Glucagon Concentration

1. A modified radioimmunoassay for cyclic AMP was developed from the method of Steiner et al. (1969). Cyclic [3H]AMP was used as the radioactive tracer. Free and antibody-bound nucleotides were separated by adsorption of protein to Millipore filters. The assay was used to measure amounts of cyclic AMP down to 0.1pmol in 50μl. 2. The effect of glucagon on cyclic AMP content in pieces of mature rat liver maintained for 6 days in organ culture was studied. 3. Cyclic AMP content in the tissue reached a maximum in 5–15min and then decreased. This may have been partly due to an inhibitor of glucagon action formed in the tissue. Small amounts of cyclic AMP were released into the incubation medium. 4. The maximal increase in cyclic AMP content produced by glucagon decreased over 6 days in culture. However, liver pieces cultured for 2 and 6 days were more sensitive to low concentrations of glucagon than were fresh liver pieces. Glucagon concentrations for half-maximal effects were approx. 1μm and 0.05μm for fresh liver and 2-day cultured liver respectively. 5. Insulin (3.5μm) lowered the cyclic AMP content by 30% in the presence of a submaximal glucagon concentration in liver cultured for 2 days. No effect of insulin was demonstrated on fresh liver pieces. 6. Insulin and glucagon were rapidly destroyed by fresh liver pieces.

Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element

1990 ◽  
Vol 10 (7) ◽  
pp. 3357-3364
Author(s):  
P G Quinn ◽  
D K Granner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Nuclear Factors ◽  
Dependent Protein

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.

scholarly journals Effect of muscular exercise and glycogen depletion on rat liver and kidney phosphoenolpyruvate carboxykinase

FEBS Letters ◽  
1971 ◽  
Vol 19 (2) ◽  
pp. 128-130 ◽  
Author(s):  
F. Sanchez-Medina ◽  
L. Sanchez-Urrutia ◽  
J.M. Medina ◽  
F. Mayor
Keyword(s):  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Muscular Exercise ◽  
Glycogen Depletion ◽  
Liver And Kidney

scholarly journals Effect of 3-mercaptopicolinic acid on gluconeogenesis and gluconeogenic metabolite concentrations in the isolated perfused rat liver

1975 ◽  
Vol 150 (1) ◽  
pp. 137-139 ◽  
Author(s):  
M N Goodman
Keyword(s):  
Rat Liver ◽  
Metabolic Regulation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver ◽  
Ideal Agent ◽  
Low Concentrations ◽  
Metabolite Concentrations

3-Mercaptopicolinic acid inhibited gluconeogenesis from lactate and alanine, but not dihydroxyacetone, in the perfused rat liver. Hepatic metabolite concentrations suggested that gluconeogenesis was inhibited at phosphoenolpyruvate carboxykinase. The compound is very effective at low concentrations, and seems an ideal agent for use in studying metabolic regulation involving gluconeogenesis and anaplerotic mechanisms.

scholarly journals Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver

1986 ◽  
Vol 234 (2) ◽  
pp. 325-334 ◽  
Author(s):  
N J Pyne ◽  
M E Cooper ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Gmp ◽  
Integral Membrane Protein ◽  
Soluble Enzyme ◽  
Cyclic Amp Phosphodiesterase ◽  
Apparent Homogeneity ◽  
Low Concentrations ◽  
Considerable Homology ◽  
Hydrolysis Of

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a ‘particulate enzyme’ found as an integral membrane protein associated with the plasma membrane, and a ‘soluble’ enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.

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