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.

scholarly journals Labeling of the releasable adenine nucleotides of washed human platelets

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 89-99 ◽  
Author(s):  
HJ Reimers ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Human Platelets ◽  
Transport Processes ◽  
Adenylate Energy Charge ◽  
Prolonged Incubation ◽  
Atp Pool ◽  
Adenine Nucleotide Pool

Abstract In rabbit platelets, the metabolically active ATP pool equilibrates with the releasable ATP pool within 1 day. The studies showing this have now been extended to human platelets. Human platelets labeled with 14C-adenosine or 14C-adenine were incubated for up to 10 hr in vitro at 37 degrees C. After 10 hr, about 12% of the total platelet 14C-ATP and 14C-ADP had become releasable with thrombin (4.2 units/ml). Lysis of platelets did not occur, since less than 1% of the platelet-bound 51Cr from platelets labeled with this radioisotope appeared in the ambient fluid upon thrombin treatment. The 14C-ATP/14C-ADP ratio of the released adenine nucleotides (7.6) was similar to the 14C-ATP/14C-ADP ratio of the nonreleasable adenine nucleotides (7.1) 2 hr after the labeling with 14C-adenosine. However, upon prolonged incubation (10 hr) in vitro, the 14C-ATP/14C-ADP ratio of the releasable adenine nucleotides decreased to 2.7. The adenylate energy charge and the 14C- ATP/14C-ADP ratio of the metabolic adenine nucleotide pool did not change significantly during the time of observation. The 14C-ATP content of the platelets decreased by less than 1% hr of incubation at 37 degrees C. These observations are interpreted to mean that the 14C is transferred from the metabolically active, nonreleasable adenine nucleotide pool of human platelets into the releasable adenine nucleotide pool as ATP and is partially hydrolyzed there to yield ADP. The transfer of ATP across the storage organelle membrane of platelets may be similar to transport processes in the chromaffin cells of the adrenal medulla and may represent a general phenomenon in cells that possess storage organelles containing adenine nucleotides.

scholarly journals Labeling of the releasable adenine nucleotides of washed human platelets

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 89-99 ◽  
Author(s):  
HJ Reimers ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Human Platelets ◽  
Transport Processes ◽  
Adenylate Energy Charge ◽  
Prolonged Incubation ◽  
Atp Pool ◽  
Adenine Nucleotide Pool

In rabbit platelets, the metabolically active ATP pool equilibrates with the releasable ATP pool within 1 day. The studies showing this have now been extended to human platelets. Human platelets labeled with 14C-adenosine or 14C-adenine were incubated for up to 10 hr in vitro at 37 degrees C. After 10 hr, about 12% of the total platelet 14C-ATP and 14C-ADP had become releasable with thrombin (4.2 units/ml). Lysis of platelets did not occur, since less than 1% of the platelet-bound 51Cr from platelets labeled with this radioisotope appeared in the ambient fluid upon thrombin treatment. The 14C-ATP/14C-ADP ratio of the released adenine nucleotides (7.6) was similar to the 14C-ATP/14C-ADP ratio of the nonreleasable adenine nucleotides (7.1) 2 hr after the labeling with 14C-adenosine. However, upon prolonged incubation (10 hr) in vitro, the 14C-ATP/14C-ADP ratio of the releasable adenine nucleotides decreased to 2.7. The adenylate energy charge and the 14C- ATP/14C-ADP ratio of the metabolic adenine nucleotide pool did not change significantly during the time of observation. The 14C-ATP content of the platelets decreased by less than 1% hr of incubation at 37 degrees C. These observations are interpreted to mean that the 14C is transferred from the metabolically active, nonreleasable adenine nucleotide pool of human platelets into the releasable adenine nucleotide pool as ATP and is partially hydrolyzed there to yield ADP. The transfer of ATP across the storage organelle membrane of platelets may be similar to transport processes in the chromaffin cells of the adrenal medulla and may represent a general phenomenon in cells that possess storage organelles containing adenine nucleotides.

In vivo control of phosphofructokinase: system models suggest new experimental protocols

1989 ◽  
Vol 257 (4) ◽  
pp. R878-R888 ◽  
Author(s):  
R. J. Connett
Keyword(s):  
Adenine Nucleotide ◽  
Energy State ◽  
Adenine Nucleotides ◽  
Experimental Studies ◽  
Energy System ◽  
Absolute Size ◽  
Steady State Kinetics ◽  
Adenine Nucleotide Pool

There is still uncertainty as to how much control of in vivo rates of glycolysis by phosphofructokinase (PFK) depends on cytosolic phosphate energy state. Three models of PFK kinetics incorporating sensitivity to pH, adenine nucleotides, and inorganic phosphate (Pi) were embedded in the physiological "phosphate energy system" of creatine-containing tissues [Connett, R.J. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988]. Effects of changes in phosphate energy state and total adenine nucleotide and phosphate pools on steady-state kinetics were examined. Analyses mimicking in vitro experiments indicated no activity at the pH and [ATP] of working muscles. When tested using the coordinated changes in Pi and adenine nucleotides expected in vivo, all models showed reasonable activity. Control was dominated by [Pi] in the normal physiological range of energy states. The almost linear response to phosphate energy state, measured by creatine charge (phosphocreatine/total creatine), is insensitive to the absolute size of the adenine nucleotide pool. A step to almost full activation occurred when phosphocreatine buffering of [ATP] was exceeded. Several experimental studies are suggested.

scholarly journals The role of leptin in the regulation of TSH secretion in the fed state: in vivo and in vitro studies

2002 ◽  
Vol 174 (1) ◽  
pp. 121-125 ◽  
Author(s):  
TM Ortiga-Carvalho ◽  
KJ Oliveira ◽  
BA Soares ◽  
CC Pazos-Moura
Keyword(s):  
Fold Increase ◽  
Adult Rats ◽  
Fed State ◽  
Rat Pituitary ◽  
Pituitary Thyroid Axis ◽  
Tsh Secretion ◽  
Thyroid Axis

Leptin has been shown to stimulate the hypothalamus-pituitary-thyroid axis in fasting rodents; however, its role in thyroid axis regulation under physiological conditions is still under investigation. Here it was investigated in freely fed rats whether leptin modulates thyrotroph function in vivo and whether leptin has direct pituitary effects on TSH release. Since leptin is produced in the pituitary, the possibility was also investigated that leptin may be a local regulator of TSH release. TSH was measured by specific RIA. Freely fed adult rats 2 h after being injected with a single s.c. injection of 8 microg leptin/100 g body weight showed a 2-fold increase in serum TSH (P<0.05). Hemi-pituitary explants incubated with 10(-9) and 10(-7) M leptin for 2 h showed a reduced TSH release of 40 and 50% respectively (P<0.05). Conversely, incubation of hemi-pituitary explants with antiserum against leptin, aiming to block the action of locally produced leptin, resulted in higher TSH release (45%, P<0.05). In conclusion, also in the fed state, leptin has an acute stimulatory effect on TSH release in vivo, acting probably at the hypothalamus. However, the direct pituitary effect of leptin is inhibitory and data also provide evidence that in the rat pituitary leptin may act as an autocrine/paracrine inhibitor of TSH release.

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.

scholarly journals Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1980 ◽  
Vol 188 (3) ◽  
pp. 913-920 ◽  
Author(s):  
Georges Van Den Berghe ◽  
Françoise Bontemps ◽  
Henri-Géry Hers
Keyword(s):  
High Speed ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Amp Deaminase ◽  
Purine Nucleotides ◽  
Isolated Rat Hepatocytes ◽  
Adenine Nucleotide Pool ◽  
Isolated Rat

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [14C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [14C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.

Bicarbonate versus Lactate Buffer in Peritoneal Dialysis Solutions: The Beneficial Effect on Rbc Metabolism

1996 ◽  
Vol 16 (5) ◽  
pp. 511-518 ◽  
Author(s):  
Umberto Buoncristiani ◽  
Francesco Galli ◽  
Simona Rovidati ◽  
M. Cristina Albertini ◽  
Carla Covarelli ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Adenine Nucleotides ◽  
Polyacrylamide Gel Electrophoresis ◽  
Energy Charge ◽  
Sodium Dodecyl ◽  
Reducing Power ◽  
Significant Drop ◽  
Bicarbonate Buffer

Objective Using the erythrocyte as a model for other kinds of cells not directly exposed to peritoneal dialysis (PD) solutions, we investigated the tolerance of the cell metabolism to lactate and bicarbonate buffers Design We studied, in vivo (in two groups of 5 PD patients each) and in vitro, the Embden-Meyerh of pathway (EMP) because it represents a potential target for the unphysiological effects of lactate or bicarbonate buffers. The EMP is the main glucose-utilizing route in the red blood cell (RBC), producing energy and reducing power. Methods The enzymatic activities of the key steps in the glycolytic pathway and the energy charge (EC), determined by the levels of phosphorylated adenine nucleotides, were investigated spectrophotometrically and by high performance liquid chromatography (HPLC) in two groups of patients undergoing lactate (L-group) and bicarbonate (B-group) PD, respectively. The in vitro effects of both bicarbonate and lactate buffers on some EMP enzyme activities and energy production were determined. Cellular pH (pHi) was also investigated. Results The B-group showed an EC value near the control levels, while in the L-group a significantly lower EC value was observed (t-test: p < 0.05 vs both B-group and controls). The key enzymes in the EMP, and in particular hexokinase, were higher in the L versus B-group (p < 0.03 for the comparison of the Hk mean values). As demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the bound form of glyceraldehyde-3-phosphate dehydrogenase (G-3-PD), an inactive form of this EMP enzyme, was significantly higher in the Lgroup with respect to the B-group (p< 0.004). In thein vitro experiments, high lactate concentrations acutely inhibited the key enzymatic steps of glycolysis, producing a significant decrease in glucose consumption and adenosine triphosphate production. These effects were not observed when bicarbonate was used in the incubations. Bothin vivoandin vitrolactate, but not bicarbonate, induce a significant drop in pHi (p < 0.05). Decreased levels of pHi like those observed in the lactate-incubated RBC were demonstrated to be able to inhibit G-3-PD activity (25±2%) here used as an indicator of the actual decrease in pH. Conclusion This study provides evidence for a damaging action of lactate with respect to bicarbonate buffer on the RBC metabolism. This condition was demonstrated observing a cell energy depletion, which coincidesin vitro with an acute EMP impairment; the lactate accumulation together with the consequent lowering of pHi seem to be responsible for this effect, which was not observed when bicarbonate was used instead of lactate.

Energy status of the rapidly paced canine myocardium in congestive heart failure

1992 ◽  
Vol 73 (6) ◽  
pp. 2363-2367 ◽  
Author(s):  
C. Montgomery ◽  
N. Hamilton ◽  
C. D. Ianuzzo
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Lactate Concentration ◽  
Energy Status ◽  
Glycogen Depletion ◽  
Myocardial Layers ◽  
Adenine Nucleotide Pool

Rapid ventricular pacing (RVP) is used as an experimental model of congestive heart failure (CHF). The purpose of this study was to determine the energy status of the dog myocardium after the development of CHF via chronic RVP. The myocardium had a significantly lower (P < 0.05) energy charge (EC) during CHF (0.63 +/- 0.01) than in sham-operated controls (0.82 +/- 0.02). This was due to significant differences in concentrations in ATP (-48%), ADP (29%), and AMP (275%) in the RVP group. However, the total adenine nucleotide pool was not different between groups. Myocardial lactate concentration was also similar. Glycogen was significantly lower (P < 0.05) by 20% at peak CHF. The adenine nucleotides were similar among the different myocardial layers (endo-, mid-, and epicardium). The administration of enalapril (an inhibitor of angiotension-converting enzyme) to decrease vascular resistance had no effect on the myocardial energy status of CHF dogs. These findings suggest that the lower EC in CHF animals is not the result of subendocardial ischemia. Also, lower EC is not associated with endogenous glycogen depletion or increased lactate concentration. The energy status of the myocardium in RVP-induced CHF is unlike that seen in ischemia-induced heart failure. This suggests that CHF in RVP is not vascular in origin.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029 ◽  
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Abstract Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

Sign in / Sign up

Export Citation Format

Share Document