Exogenous, but not endogenous, cyclic GMP reduces hepatic pyruvate kinase activity

We investigated the effects of exogenous cyclic GMP and stimulants of endogenous cyclic GMP accumulation on L-form (hepatic) pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) activity in isolated rat hepatocytes. Exogenous cyclic GMP (200 muM) reduced pyruvate kinase activity, but was less potent than exogenous cyclic AMP (50 muM) (Ki congruent to 120 muM vs. 30 muM, respectively), had a slower onset of action (1.0 vs. 0.3 min, respectively) and a less rapid maximal effect (5.0 vs. 1.0 min, respectively). Similar results were noted with dibutyryl cyclic GMP or dibutyryl cyclic AMP. 1.0 muM acetylcholine increased cyclic GMP concentrations in isolated hepatocytes from 233 +/- 16 to 447 +/- 3 pmol/g cell protein (P less than 0.001), but did not alter pyruvate kinase activity. Similar results were noted with carbamylcholine, NaN3 or acetylcholine plus eserine sulfate. The results suggest a differential effect of exogenous vs. endogenous cyclic GMP on L-form pyruvate kinase activity, and question the physiological relevance of observations with exogenous cyclic GMP in this system.     

Production of cyclic AMP from extracellular ATP by intact LM cells.

Intact LM cells, a line of cultured mouse fibroblasts, exhibited an adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence of exogenous [alpha-32P]ATP which was 20--30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E1 stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an adenylate cyclase activity which is accessible to extracellular ATP.     

Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Adenylate cyclase and phosphodiesterase activities in rat hepatocytes cultured in the presence and absence of dexamethasone

The effects of glucagon and dexamethasone on the activities of the enzymes involved in cyclic adenosine 3':5'-monophosphate (cyclic AMP) metabolism in primary monolayer cell cultures of adult rat hepatocytes were examined. Short-term experiments indicated that the magnitude of the cultured cells' response to glucagon, as measured by production of cyclic AMP, was essentially the same as that for freshly isolated hepatocytes. However, the time course of this response was markedly different. Although the activity of adenylate cyclase is maintained throughout the culture period at a level similar to that of the freshly isolated hepatocytes, the activity of both low and high Km forms of phosphodiesterase decreases rapidly with length of time in vitro. This is reflected by an increase in cyclic AMP produced in response to glucagon and theophylline by cells of different ages. Dexamethasone caused an increased loss of phosphodiesterase activity, as well as increased cyclic AMP accumulation in the presence or absence of theophylline. Various agents failed to restore the lost phosphodiesterase activity. These results may indicate that phosphodiesterase activity is more sensitive to the inevitable inadequacies of the in vitro environment of cultured hepatocytes than adenylate cyclase. It was also found that a modification of the method of Seglen (1) for the preparation of isolated hepatocytes yielded cells that had less phosphodiesterase activity than those prepared by the method of Berry and Friend (2).     

P2-purinergic control of liver glycogenolysis.

Purinergic agonists cause a dose-dependent activation of glycogen phosphorylase in isolated rat hepatocytes. Half-maximally effective concentrations are 5 X 10(-7)M for ATP, 2 X 10(-6)M for ADP, and about 5 X 10(-5) M for AMP and adenosine. This potency series indicates the presence of P2-purinergic receptors. The mode of action of ATP appears to be identical with that of the Ca2+-dependent glycogenolytic hormones angiotensin, vasopressin and alpha 1-adrenergic agonists. (1) They all require Ca2+ for phosphorylase activation; (2) they do not increase cyclic AMP levels; (3) they are susceptible to heterologous desensitization by vasopressin and phenylephrine; (4) they lower cyclic AMP concentrations in hepatocytes stimulated by glucagon, most probably mediated by an enhanced phosphodiesterase activity.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Extracellular metabolism of cyclic AMP.

Intravenously administered cyclic [8-3H]AMP to rats was quickly eliminated from the circulation. After 2 min 93% of the administered radioactivity disappeared from the plasues was recovered mainly in the form of nucleotides, ATP, ADP, AMP and IMP. In vitro contact of cyclic AMP with perfused liver, isolated liver cells and adipose tissue resulted in a rapid breakdown of the nucleotide, presumably on the outer surface of the cells. The degradation products have been identified mainly as adenosine and inosine. Incubation of adipose tissue and isolated liver cells with [3H] AMP also resulted in the breakdown of the nucleotide in themedium. The rate of AMP degradation by these tissues was faster than that for cyclic AMP degradation. The data suggest that cyclic AMP is readily metabolized on the outer surface of cells to products which may be converted within the cells to nucleotides. These findings seem of importance for the quantitative assessments of cellular cyclic AMP outflow during hormonal stimulation.     

Adenylate cyclase and phosphodiesterase activities in rat hepatocytes cultured in the presence and absence of dexamethasone

Summary The effects of glucagon and dexamethasone on the activities of the enzymes involved in cyclic adenosine 3′∶5′-monophosphate (cyclic AMP) metabolism in primary monolayer cell cultures of adult rat hepatocytes were examined. Short-term experiments indicated that the magnitude of the cultured cells' response to glucagon, as measured by production of cyclic AMP, was essentially the same as that for freshly isolated hepatocytes. However, the time course of this response was markedly different. Although the activity of adenylate cyclase is maintained throughout the culture period at a level similar to that of the freshly isolated hepatocytes, the activity of both low and highK _m forms of phosphodiesterase decreases rapidly with length of time in vitro. This is reflected by an increase in cyclic AMP produced in response to glucagon and theophylline by cells of different ages. Dexamethasone caused an increased loss of phosphodiesterase activity, as well as increased cyclic AMP accumulation in the presence or absence of theophylline. Various agents failed to restore the lost phosphodiesterase activity. These results may indicate that phosphodiesterase activity is more sensitive to the inevitable inadequacies of the in vitro environment of cultured hepatocytes than adenylate cyclase. It was also found that a modification of the method of Seglen (1) for the preparation of isolated hepatocytes yielded cells that had less phosphodiesterase activity than those prepared by the method of Berry and Friend (2). This work was supported by grants from the Medical Research Council of New Zealand and the Medical Research Distribution Committe.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Production of cyclic AMP from extracellular ATP by intact LM cells

Intact LM cells, a line of cultured mouse fibroblasts, exhibited and adenylate cyclase (APT pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence exogenous [α-³²]ATP which was 20–30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E₁ stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an endenylate cyclase activity whic is accessible to extracellular ATP.     

Nalpha-trinitrophenyl glucagon: an inhibitor of glucagon-stimulated cyclic AMP production and its effects on glycogenolysis.

Nalpha-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AM-independent process.     

The mechanism by which 2-deoxyglucose inhibits glucose-induced activation of Pilobolus longipes spores

Sporangiospores of Pilobolus longipes were activated by either glucose, 6-deoxyglucose, or derivatives of cyclic AMP. Cyclic AMP content increased after the addition of either glucose or 6-deoxyglucose and the increase preceded spore activation, indicating that glucose triggers germination via cyclic AMP. Activation, whether induced by glucose, 6-deoxyglucose, or cyclic nucleotides was inhibited by 2-deoxyglucose. However, cyclic AMP levels also increased after the addition of 2-deoxyglucose. Radioactive 2-deoxyglucose was recovered from spores mainly as 2-deoxyglucose 6-phosphate, suggesting that phosphorylation of 2-deoxyglucose may inhibit spore activation by trapping ATP. Support for the hypothesis came from ATP assays which showed that 2-deoxyglucose reduced intracellular ATP to undetectable levels. Moreover, when ATP levels were restored with exogenous fructose, 2-deoxyglucose was no longer inhibitory but was then an effective germination trigger.     

Adenosine Receptor Activation and the Regulation of Tyrosine Hydroxylase Activity in PC 12 and PC 18 Cells

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Change of Cyclic AMP Level in Synaptosomes from Cerebral Cortex; Increase by Adenosine Derivatives

The endogenous level of cyclic AMP in incubated synaptosomes from cerebral cortex of guinea pigs was investigated after the addition of various agents to the incubation medium. It appeared that the synaptosomal suspension already contained exogenous adenosine. Preincubation with theophylline or with adenosine deaminase (ADase) decreased both the exogenous level of adenosine and the intrasynaptosomal level of cyclic AMP. The level of cyclic AMP was reincreased by the addition of adenosine agonists, especially 2-chloroadenosine. This increase was antagonized by deoxyadenosine and was not inhibited by dipyridamole. These results suggest that the adenosine derivatives in the synaptic cleft regulate the level of cyclic AMP in nerve terminals through adenosine receptor on the presynaptic membrane. ADP, ATP, dopamine, and histamine also stimulate the formation of cyclic AMP in the ADase-treated synaptosomes.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.     

Extracellular metabolism of cyclic AMP

Intravenously administered cyclic [8-³H]AMP to rats was quickly eliminated from the circulation. After 2 min 93% of the administered radioactivity disappeared from the plasma, and most of it was recovered in the kidney, liver and muscles. The label in the tissues was recovered mainly in the form of nucleotides, ATP, ADP, AMP and IMP.In vitro contact of cyclic AMP with perfused liver, isolated liver cells and adipose tissue resulted in a rapid breakdown of the nucleotide, presumably on the outer surface of the cells. The degradation products have been identified mainly as adenosine and inosine.Incubation of adipose tissue and isolated liver cells with [³H]AMP also resulted in the breakdown of the nucleotide in the medium. The rate of AMP degradation by these tissues was faster than that for cyclic AMP degradation.The data suggest that cyclic AMP is readily metabolized on the outer surface of cells to products which may be converted within the cells to nucleotides. These findings seem of importance for the quantitative assessments of cellular cyclic AMP outflow during hormonal stimulation.     

Extracts of human articular cartilage contain an inhibitor of tissue metalloproteinases.

Vasopressin and angiotensin are able to lower the glucagon-induced increase of cyclic AMP levels in isolated hepatocytes. Results presented are in favour of an enhanced phosphodiesterase activity to account for this cyclic AMP lowering effect. In particular, vasopressin prevents exogenous cyclic AMP from activating glycogen phosphorylase: in the presence of phosphodiesterase inhibitors, the hormone becomes unable to decrease glucagon-induced cyclic AMP levels. This anti-glucagon effect of vasopressin and angiotensin might be physiologically more important than their glycogenolytic effect; indeed, the latter is very transient in nature and, in addition, requires higher hormone concentrations [Bréant, Keppens & De Wulf (1981) Biochem. J. 200, 509-514] than those needed for the anti-glucagon effect, as reported here.     

Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion.

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.     

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.     

Nα-Trinitrophenyl glucagon An inhibitor of glucagon-stimulated cyclic AMP production and its effects on glycogenolysis

N^α-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis.in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AMP-independent process.