Inhibition by insulin of glucagon-dependent phospholipid methyltransferase phosphorylation in rat hepatocytes

Addition of insulin to isolated rat hepatocytes prelabeled with [32P]phosphate inhibited glucagon-dependent phospholipid methyltransferase phosphorylation and activation. Insulin alone had no effect on either the phosphorylation of the enzyme or on its activity. The effect of insulin on glucagon-dependent phospholipid methyltransferase phosphorylation was dose-dependent and occurred at physiological doses of the hormone (10(-11)-10(-10) M). Analysis of 32P-labeled peptides after digestion with trypsin revealed only one site of phosphorylation regulated by glucagon (10(-8) M) in isolated rat hepatocytes. This site, as analyzed by HPLC and thin-layer chromatography, coincided with that phosphorylated by the cAMP-dependent protein kinase using purified rat liver phospholipid methyltransferase.     

Phospholipid methyltransferase phosphorylation by intact hepatocytes: effect of glucagon

We have obtained a rabbit antiserum that specifically immunoprecipitates the 50K and 25K proteins of rat liver phospholipid methyltransferase. Exposure of intact rat hepatocytes preincubated with [32P]phosphate to glucagon induces a time-dependent phosphorylation of the 50K protein of phospholipid methyltransferase. The incorporation of 32P into the 50K protein was only on phosphoserine. These data support the concept that the activation of rat liver phospholipid methyltransferase by glucagon is mediated by phosphorylation of the enzyme.     

Beta-adrenergic control of phosphatidylcholine synthesis by transmethylation in hepatocytes from juvenile, adult and adrenalectomized rats.

Changes in isoprenaline-sensitive phospholipid methyltransferase were studied in hepatocytes isolated from juvenile, mature and adrenalectomized rats. Isoprenaline produced greater stimulation of cyclic AMP accumulation in juvenile and mature adrenalectomized rats than in mature animals. Similarly, isoprenaline stimulated phospholipid methyltransferase in juvenile and mature adrenalectomized rats but had no effect in mature animals. Isoprenaline-mediated activation of phospholipid methyltransferase in adrenalectomized rats was time- and dose-dependent. In hepatocytes isolated from adrenalectomized rats incubated with [Me-3H]methionine or [3H]-ethanolamine the addition of isoprenaline increased the amount of radioactivity incorporated into phosphatidylcholine. The activation by isoprenaline of phospholipid methyltransferase was abolished by the beta-blocker propranolol and by insulin. These results indicate that rat liver the occupation of functional beta-receptors causes a stimulation of phospholipid methylation. It is suggested that, as reported previously, cyclic AMP activates phospholipid methyltransferase.     

Stimulation by epidermal growth factor of phospholipid methyltransferase in isolated rat hepatocytes

Epidermal growth factor produces a time- and dose-dependent activation of phospholipid methyltransferase activity in hepatocytes isolated from juvenile and mature hepatectomized rats. This treatment however has no effect with hepatocytes isolated from mature or laparotomized rats. Dansylcadaverine (50μM), an inhibitor of receptor-mediated internalization of epidermal growth factor, has no effect on basal phospholipid methyltransferase but inhibits the stimulation of this enzyme by epidermal growth factor.

These results indicate a possible role of phospholipid methylation during liver proliferation.     

Adrenocorticotropic stimulation and insulin inhibition of adipocyte phospholipid methylation

Treatment of isolated rat adipocytes with adrenocorticotropin (ACTH) caused a 1.5-fold increase in phospholipid methyltransferase activity within 5 min. This effect of ACTH was concentration-dependent with maximal activation at 2 milliunits/ml ACTH, and was reproduced by dibutyryl cyclic AMP. ACTH (2 milliunits/ml) caused an increase in the Vmax value of phospholipid methyltransferase without changing the Km for S-adenosyl-L-methionine. Insulin caused a concentration-dependent inhibition of both control and ACTH-stimulated phospholipid methyltransferase. Half-maximal inhibition by insulin was demonstrated with 5 microunits/ml insulin in control cells and with 25 microunits/ml insulin in ACTH-stimulated cells. The rapid and sensitive activation of adipocyte phospholipid methyltransferase by ACTH and inhibition by insulin are consistent with a role for this pathway in the hormonal response of the adipocyte.     

Vasopressin-stimulated phosphorylation of rat liver phospholipid methyltransferase in isolated hepatocytes

Addition of vasopressin (1 microM) to isolated rat hepatocytes prelabeled with [32P]phosphate was accompanied by a 250% increase in the phosphorylation of phospholipid methyltransferase. Vasopressin-stimulated phospholipid methyltransferase phosphorylation was time- and dose-dependent. 32P-labeled phospholipid methyltransferase was recovered by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. After electrophoresis, phospholipid methyltransferase was electroeluted from the polyacrylamide gel and subjected to tryptic digestion or HCl hydrolysis. Analysis of 32P-labeled peptides reveals only one site of phosphorylation and the analysis of [32P]phosphoamino acids indicates that phosphoserine is the only labeled amino acid.     

The polar head group of a novel insulin-sensitive glycophospholipid mimics insulin action on phospholipid methyltransferase

A phospholipid has been purified from rat liver membranes which copurified with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this phospholipid was generated by treatment with a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus and purified through a C18 extraction column. Like insulin, the addition of this polar head group to isolated rat adipocytes inhibited the stimulatory effect of isoproterenol on phospholipid methyltransferase. The polar head group was also active on a subcellular fraction. The addition of the polar head group to microsomes isolated from isoproterenol-treated adipocytes produced a time-dependent inactivation of phospholipid methyltransferase, approaching basal activity. It is proposed that the effects of insulin on phospholipid methyltransferase may be mediated by this polar head group.     

Inactivation of phosphofructokinase by glucagon in rat hepatocytes.

Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.     

Glucagon binding by isolated chick hepatocytes

Studies have been made on the binding of 125I-glucagon by isolated chick hepatocytes. It was shown that pH and temperature dependence of the binding does not differ from that in rat hepatocytes. Optimum binding was observed at pH 7.6, the rate of binding being higher at 37 degrees C as compared to that at 20 degrees C, although the binding capacity increased with the decrease in the temperature. Unlabeled glucagon was able to compete with 125I-glucagon at the binding sites. Scatchard plot was found to be curvilinear revealing two classes of the binding sites with Kd values 10(-9) and 10(-7) M at temperatures 20 and 37 degrees C correspondingly. Earlier studies revealed in rats the binding sites of a sole class with Kd value 10(-9) M. Preincubation of cells with native glucagon results in changes of labeled glucagon binding, the effect being proportional to the concentration of native glucagon. Preincubation effect was observed at 37 degrees C, being absent at 20 degrees C; the effect was due to the decrease in the number of both high and low affinity binding sites. The presence of down-regulation of glucagon receptors in chick hepatocytes is suggested.     

Modulation of glucagon effects by changes in extracellular pH and calcium

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 microM Gpp[NH]p, 30 microM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component Ns and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase alpha activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.     

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.     

Phorbol ester effects on hormonal responses in freshly isolated short-term incubated and cultured hepatocytes

Beta-adrenergic, alpha-1-adrenergic and glucagon stimulation of glucose release were compared between hepatocytes which were freshly isolated, incubated for 3 h in suspension or cultivated for 4 or 24 h in plastic culture flasks in the presence and absence of the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast to the absence of an isoproterenol effect in freshly isolated hepatocytes, and increased sensitivity of glucose liberation towards isoproterenol could be observed 4 h after the start of culture, whereas the beta-receptor number was not found to be increased before 24h. TPA has no effect on isoproterenol-stimulated glucose release at all investigated conditions. The alpha-1-adrenergic responses tested by using the alpha-1-adrenergic agonist phenylephrine is blocked completely in freshly isolated hepatocytes preincubated with 10⁻⁶ M TPA. However, after 3 h incubation of hepatocytes in suspension or in primary culture, TPA had no effect on phenylephrine-stimulated glucose release. The effect of 10⁻⁹ M glucagon on glucose release from freshly isolated hepatocytes was not influenced by TPA, whereas after 90 and 180 min incubation a significant decrease could be observed. On the other hand, TPA inhibited stimulation of adenylate cyclase activity by glucagon concentrations of 10⁻⁵ M in freshly isolated hepatocytes, but not effect was found in hepatocytes incubated for 3 h in suspension or maintained for 24 h in primary culture. The different TPA effects may be an expression of changes of the accessibility of protein kinase C to TPA caused by translocation and/or intracellular activation of this enzyme at the tested experimental conditions.     

Correlation between cAMP, activation of cAMP-dependent protein kinase(s), and rate of glycogenolysis in isolated rat hepatocytes.

We have studied the correlation between cAMP-dependent protein kinase activation and rates of glycogenolysis in hepatocytes isolated from fed rats. With doses of 20 μM glucagon, the protein kinase was activated to a -cAMP/+cAMP ratio of 0.8 within 10 min and remained activated for up to 2 hours. A dose-response relationship between protein kinase activation and rates of glycogenolysis can be demonstrated to 0–20 μM glucagon. Glycogenolysis was stimulated greater than 2-fold after 2 hours of incubation with the higher doses of glucagon. Protein kinase activity ratios correlated well with the rates of glycogenolysis as the ratios varied from control levels of about 0.25 to the stimulated values of 0.5–0.6. However, as the ratios increased from 0.6 to 0.8, with higher doses of glucagon, there were no corresponding increases in the rates of glycogenolysis. These data may indicate (1) that activation of all of the protein kinase present in the liver cells is not necessary for maximal stimulation of glycogenolysis, or (2) that a specific protein kinase is involved in the intracellular control of glycogen breakdown in isolated rat hepatocytes.     

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.     

Effects of insulin-glucagon interactions on glycogenolysis and protein kinase activity in rat hepatocytes

The effects of insulin and glucagon on cAMP accumulation, protein kinase activation, and glycogenolysis were investigated in isolated rat hepatocytes. Glucagon (0.01 nM to 10 micro M) increased the activation state of protein kinase and the rate of glucose accumulation. Addition of 1.0 nM insulin to cells preincubated with 0.1 nM glucagon attenuated the rate of glucose accumulation, but did not alter the protein kinase activity ratio. Addition of 0.1 nM glucagon to cells preincubated with 1.0 nM insulin caused a rapid activation of protein kinase; however, glycogenolysis was not immediately affected. These effects were enhanced with pharmacological concentrations of glucagon and insulin. These data indicate that the degree of protein kinase activation does not always correlate temporally or quantitatively with rates of glycogenolysis in liver cells exposed to insulin and glucagon.     

Decrease by glucagon in hepatic succinyl-CoA

Succinyl-CoA has been determined in freeze-clamped rat liver tissue to amount to 9.5 ± 0.7 nmoles/g fresh wt. (n=11). Intravenous injection of glucagon lowers this level within 10 min to 4.5 nmoles/g fresh wt. (n=11; P < 0.0005). Isolated hepatocytes also show a significant decrease in their succinyl-CoA content when incubated with glucagon or dibutyryl cyclic AMP. This effect appears to be fully expressed already 1 min after exposure of isolated hepatocytes to glucagon.     

The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism.

Treatment of intact hepatocytes with glucagon, TH-glucagon [( 1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/loss of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in a washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein Gs, and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.     

The effect of glucagon on ureagenesis from ammonia by isolated rat hepatocytes

Glucagon, at a maximally effective concentration of 1 μM, stimulated by 35% the rate at which rat hepatocytes synthesized urea from 10 mM NH₄Cl in the presence of 10 mM ornithine. The rate at which citrulline accumulated in the incubations was relatively unchanged by the presence of glucagon.Mitochondria isolated from glucagon treated hepatocytes were observed to synthesize citrulline from 10 mM NH₄Cl and 10 mM ornithine more rapidly than did mitochondria isolated from untreated hepatocytes.The role of the intracellular malate concentration in the regulation of the rate of urea synthesis, and the changes observed in the cellular content of malate in response to glucagon are discussed.     

Flux through glycine cleavage system in isolated hepatocytes: effects of glucagon, cAMP, and calcium

The hepatic glycine cleavage system (GCS) is the principal route for the metabolism of glycine in mammals. Flux through the GCS in isolated rat hepatocytes was stimulated by about 100% by glucagon (10(-7) M), forskolin (10(-4) M), and dibutyryl cAMP (10(-4) M). The stimulation of flux through the GCS by these agents was accompanied by marked elevation of cellular cAMP levels. A significant correlation was observed between increased cellular cAMP levels induced by glucagon and stimulation of flux through the GCS by glucagon. Exclusion of calcium from the incubation medium reduced the basal flux by 38%, but did not affect the degree of stimulation of flux through the GCS by glucagon. A single intraperitoneal injection of glucagon to rats prior to isolation of hepatocytes resulted in a 76% stimulation of flux through the GCS. These hepatocytes with stimulated flux through the GCS showed reduced sensitivity for further stimulation by glucagon. Half-maximal stimulation of flux through the GCS occurred at 3.8 +/- 1.1 and 8.5 +/- 1.4 nM glucagon in hepatocytes isolated from control and glucagon-injected rats, respectively. We conclude that cAMP is involved in the regulation of flux through the GCS by glucagon.     

Glucagon regulation of amino acid transport in hepatocytes: Effect of cell enucleation

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na⁺-dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon-stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.