scholarly journals Effect of thyroid state on cyclic AMP-mediated induction of hepatic phosphoenolpyruvate carboxykinase

1985 ◽  
Vol 226 (1) ◽  
pp. 67-73 ◽  
Author(s):  
W Höppner ◽  
W Süssmuth ◽  
H J Seitz
Keyword(s):  
Cyclic Amp ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Close Correlation ◽  
Perfused Liver ◽  
Thyroid State ◽  
Fed State ◽  
Isolated Perfused Liver ◽  
Translatable Mrna

Hepatic phosphoenolpyruvate carboxykinase (PEPCK) is significantly increased in the hyperthyroid starved rat, and moderately decreased in the hypothyroid starved rat. As tri-iodothyronine by itself has only a small and sustained effect on the induction of this enzyme, as was previously shown in the isolated perfused organ, the effect of hypo- and hyper-thyroidism on the increase in cytosolic PEPCK provoked by dibutyryl cyclic AMP (Bt2cAMP) was investigated in vivo and in the isolated perfused liver. Compared with euthyroid fed controls, in hypothyroid fed rats Bt2cAMP provoked in 2 h only a small increase in translatable mRNA coding for PEPCK. In contrast, in hyperthyroid animals PEPCK mRNA as measured by translation in vitro was already increased in the fed state, and further enhanced by Bt2cAMP injection to values as in euthyroid controls. Under all thyroid states a close correlation between PEPCK mRNA activity and PEPCK synthesis was observed. In the isolated perfused liver from the hyperthyroid fed rat, the increase in PEPCK provoked by Bt2cAMP or Bt2cAMP + isobutylmethylxanthine was considerably enhanced compared with those obtained in livers of hypothyroid rats. Also, adrenaline provoked a stimulated induction of PEPCK in hyperthyroid rats compared with hypothyroid rats. To summarize, our data indicate that the primary action of thyroid hormones on the synthesis of hepatic cytosolic PEPCK is to accelerate the cyclic AMP- or adrenaline-induction of the enzyme, acting primarily at a pretranslational level.

Hepatotrophic effects of insulin on glucose, glycogen, and adenine nucleotides in hepatocytes isolated from fed adult rats

1980 ◽  
Vol 58 (10) ◽  
pp. 1004-1011 ◽  
Author(s):  
Khursheed N. Jeejeebhoy ◽  
Joseph Ho ◽  
Rajni Mehra ◽  
Alan Bruce-Robertson
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Close Correlation ◽  
Adult Rats ◽  
Fed State ◽  
Adenine Nucleotide Pool ◽  
Intracellular Glucose

In vivo observations have suggested that there is an hepatotrophic effect of insulin. By contrast, subsequent in vitro work, using the isolated perfused liver system, showed no effect or indeterminate effects of insulin on the transport of glucose into the hepatocyte. However because this system may not have endured long enough to show such an influence we explored the transport of glucose using a 48-h suspension culture of hepatocytes isolated from young adult fed rats, the suspension being infused continuously with insulin at a rate approximating the maximum entering portal blood in the fed state. (In a separate study phloridzin was added after 2 h of incubation.) DNA, intracellular glucose and its inward transport, glycogen, and the adenine nucleotides were measured at intervals. By comparison with control or untreated cells, insulin-treated cells showed significantly more DNA and intracellular glucose, and the differences were abolished by phloridzin. Glucose transport rates fell to low values in untreated controls and still lower with insulin plus phloridzin. but the initial rate was maintained to the end (48 h) by insulin alone. Results for glycogen were similar to those for intracellular glucose. There was a close correlation (r = 0.96) between these two. The total adenine nucleotide pool and the concentration of ATP were maintained for about 24 h and fell to half their initial values by 48 h. Insulin had increased these concentrations significantly by 6 h. Although concentrations of ADP and AMP decreased gradually in all groups of cells, insulin enhanced the level of ADP by 12 h but had no measurable effect on that of AMP. The energy charge increased slightly throughout incubation but more so (by 6 h) in the presence of insulin. In conclusion the data support the concept that in the longer term (> 12 h) insulin in the portal circulation maintains the characteristic free permeability of the hepatocyte to glucose and this permits a variety of effects related to glucose entry into the hepatocyte.

Effect of Adrenocortical and Hypophyseal Hormones on Uptake of Fat by Isolated Perfused Liver

1958 ◽  
Vol 195 (3) ◽  
pp. 678-680 ◽  
Author(s):  
Murray Heimberg ◽  
H. C. Meng ◽  
D. Bradley
Keyword(s):  
Anterior Pituitary ◽  
Pituitary Hormones ◽  
Perfused Liver ◽  
Rapid Rate ◽  
Perfused Rat Liver ◽  
Perfusion Fluid ◽  
Fed State ◽  
Fat Uptake

The effect of adrenocortical and anterior pituitary hormones on the uptake of neutral fat by the perfused rat liver has been investigated. It was observed that adrenalectomy increased the rate of fat uptake. Cortisone treatment of adrenalectomized animals reduced the rate of fat uptake to that of the normal fed state. Cortisone and hydrocortisone administered in vivo, or added to the perfusion fluid in vitro, reduced the rapid rate of fat uptake resulting from 48 hours of fasting. Hydrocortisone administered to normal fed rats did not depress the rate of fat uptake. The administration of crude anterior pituitary extracts to the normal fed animal significantly increased fat uptake. Hypophysectomy increased the rate of fat uptake. Administration of growth hormone to the normal fed animal 3–8 hours prior to killing, or added to perfusion fluid in vitro, increased the rate of fat uptake.

Influence of hepatic taurine concentration on bile acid conjugation with taurine.

1977 ◽  
Vol 232 (1) ◽  
pp. E75 ◽  
Author(s):  
W G Hardison ◽  
J H Proffitt
Keyword(s):  
Bile Acid ◽  
Cholic Acid ◽  
Sodium Cholate ◽  
Total Bile Acid ◽  
Perfused Liver ◽  
Taurine Concentration ◽  
Isolated Perfused Liver ◽  
Liver Homogenates

In vitro, addition of taurine to liver homogenates increases the proportion of cholic acid conjugated with taurine. In the present study, the relation between hepatic taurine concentration and the proportion of infused sodium cholate conjugated with taurine was studied in the whole organ. The isolated perfused liver was studied to eliminate possible transfer of taurine to or from the large extrahepatic poosl present in vivo. During cholate infusion, the proportion of taurocholate excreted in bile decreased, and the proportion of glycocholate increased in a complementary fashion. Infusion of taurine with cholate prevented these changes. Hepatic taurine concentration, calculated from measured hepatic taurine concentrations before and at the end of cholate infusion, fell. Fall in proportion of total bile acid excreted as taurocholate was most rapid at low hepatic taurine concentrations between about 1.4 and 0.65 mumol/g liver. Hepatic taurine concentrations is a major determinant of the proportion of bile acid conjugated with taurine.

scholarly journals Metabolic fates of diethylstilboestrol sulphates in the rat

1977 ◽  
Vol 164 (2) ◽  
pp. 423-430 ◽  
Author(s):  
P A Barford ◽  
A H Olavesen ◽  
C G Curtis ◽  
G M Powell
Keyword(s):  
Rat Liver ◽  
Whole Body ◽  
Perfused Liver ◽  
Isolated Perfused Liver

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the rat. Both of the esters were desulphated to some extent in vivo. In addition, significant amounts of radioactivity appeared in the bile as diethylstilboestrol mono[35S]sulphate monoglucuronide. The percentage of the dose appearing in bile as the diconjugate was substantially greater in experiments with diethylstilboestrol mono[35S]sulphate than with diethylstilboestrol di[35S]sulphate. Whole-body radioautography and studies with isolated perfused liver confirmed the liver as the major metabolic organ for both esters. When the metabolite diethylstilboestrol mono[35S]sulphate monoglucuronide isolated from the bile was reinjected, it was excreted in the bile unchanged. Studies in vitro demonstrated that both esters were substrates for arylsulphatase C with Km values in the range 52-76 micrometer. The metabolic fates and modes of excretion of the esters are discussed in relation to the enzyme complement of rat liver.

In vitro Lipolysis as a Tool for the Establishment of IVIVC for Lipid-Based Drug Delivery Systems

2019 ◽  
Vol 16 (8) ◽  
pp. 688-697
Author(s):  
Ravinder Verma ◽  
Deepak Kaushik
Keyword(s):  
Drug Delivery ◽  
Ph Value ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Rapid Screening ◽  
Resonance Spectroscopy ◽  
Fed State ◽  
In Vitro Lipolysis ◽  
Ph Stat

: In vitro lipolysis has emerged as a powerful tool in the development of in vitro in vivo correlation for Lipid-based Drug Delivery System (LbDDS). In vitro lipolysis possesses the ability to mimic the assimilation of LbDDS in the human biological system. The digestion medium for in vitro lipolysis commonly contains an aqueous buffer media, bile salts, phospholipids and sodium chloride. The concentrations of these compounds are defined by the physiological conditions prevailing in the fasted or fed state. The pH of the medium is monitored by a pH-sensitive electrode connected to a computercontrolled pH-stat device capable of maintaining a predefined pH value via titration with sodium hydroxide. Copenhagen, Monash and Jerusalem are used as different models for in vitro lipolysis studies. The most common approach used in evaluating the kinetics of lipolysis of emulsion-based encapsulation systems is the pH-stat titration technique. This is widely used in both the nutritional and the pharmacological research fields as a rapid screening tool. Analytical tools for the assessment of in vitro lipolysis include HPLC, GC, HPTLC, SEM, Cryo TEM, Electron paramagnetic resonance spectroscopy, Raman spectroscopy and Nanoparticle Tracking Analysis (NTA) for the characterization of the lipids and colloidal phases after digestion of lipids. Various researches have been carried out for the establishment of IVIVC by using in vitro lipolysis models. The current publication also presents an updated review of various researches in the field of in vitro lipolysis.

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.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230

1992 ◽  
Vol 12 (4) ◽  
pp. 1507-1514
Author(s):  
C L Denis ◽  
S C Fontaine ◽  
D Chase ◽  
B E Kemp ◽  
L T Bemis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Growth Conditions ◽  
Dependent Protein Kinase ◽  
Inactive Form ◽  
Dependent Protein ◽  
Kinase Phosphorylation

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

scholarly journals Properties of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesized in hepatoma cells in the presence of amino acid analogues

1975 ◽  
Vol 146 (3) ◽  
pp. 585-593 ◽  
Author(s):  
S E Knowles ◽  
J M Gunn ◽  
L Reshef ◽  
R W Hanson ◽  
F J Ballard
Keyword(s):  
Amino Acid ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Hepatoma Cells ◽  
Dibutyryl Cyclic Amp ◽  
Enzyme Degradation ◽  
Amino Acid Analogues ◽  
Enzyme Molecules ◽  
Natural Amino Acids

1. Phosphoenolpyruvate carboxykinase (GTP) was induced by a combination of dibutyryl cyclic AMP, theophyline and dexamethasone in Reuber H35 hepatoma cells under conditions where an amino acid in the medium was replaced by an appropriate analogue. 2. With canavanine replacing arginine or with 5-fluorotryptophan or 6-fluorotryptophan replacing tryptophan the induced enzyme had a lower catalytic activity-relative to antibody reactivity. 3. These aberrant enzyme molecules were heat-labile in vitro. 4. Measurements of enzyme degradation in vivo indicated that the canavanine-containing enzyme and the 6-fluorotryptophan-containing enzyme were degraded more rapidly than the enzyme containing all natural amino acids.

scholarly journals Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485 ◽  
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

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