Hormonal effects on cyclic nucleotides and carbohydrate and lipid metabolism in isolated chicken hepatocytes

Hormonal effects on cyclic nucleotides and carbohydrate and lipid metabolism in isolated chicken hepatocytes

GENERAL AND COMPARATIVE ENDOCRINOLOGY 46, 310-321 (1982) Hormonal Effects on Cyclic Nucleotides and Carbohydrate Lipid Metabolism in Isolated Ch...

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310-321 (1982)

Hormonal Effects on Cyclic Nucleotides and Carbohydrate Lipid Metabolism in Isolated Chicken Hepatocytes GORDON Department

CRAMB,~ of Biochemistry,



University Edinburgh EH8

LANGSLOW,’ of Edinburgh 9XD. United

AND Medical Kingdom

JOHN School,






Accepted June 4, 1981 The basal CAMP content of isolated chicken hepatocytes (0.5- 1.5 pmol 106 cells) was increased transitorily by both glucagon (up to 60-fold) and adrenaline (up to 2t?-fold). These hormones stimulated cellular glycogenolysis by up to 200% and gluconeogenesis from lactate by up to 30%. and inhibited lipogenesis from acetate by up to 90%. Half-maximal metabolic effects were induced by hormone concentrations that were less than 10% of the concentrations producing a half-maximal rise in CAMP. Physiological concentrations of insulin and avian pancreatic polypeptide (APP) failed to alter basal or hormone-elevated CAMP concentrations, and were also without effect on glycogenolysis, gluconeogenesis. and lipogenesis. Insulin failed to stimulate glycogen synthesis. None of these hormones affected the cGMP content of the hepatocytes (0.03 pmoVIOG cells), or altered hepatocyte phosphodiesterase activity. Since high-affinity insulin receptors are present on these chicken hepatocytes we conclude that the insulin resistance of chickens in vivo is due to lack of a hormone effector system in the liver; the lack of metabolic effects resulting from APP is consistent with its lack of receptors on the hepatocytes.

While circulating levels of glucagon and insulin are of crucial importance in the maintenance of carbohydrate and lipid homeostasis in mammals, their roles are much less clear in birds (Hazelwood, 1976). Although insulin can decrease the high avian blood glucose concentration (lo- 15 mM in the chicken), its effects are less intense than in mammals. Glucagon is the most important pancreatic hormone, as total pancreatectomy of chickens induces severe hypoglycaemia rather than hyperglycaemia (Mikami and Ono, 1962; Sitbon, 1967). The possible role of the most recently discovered pancreatic hormone, avian pancreatic polypeptide (APP), has yet to be elucidated: injection of high concentrations decreases liver glycogen stores and decreases blood glycerol and free fatty acids (Hazelwood and Langslow, 1978), but ’ Present address: Department of Physiology, University of St. Andrews, St. Andrews, Fife KY16 9TS, U.K. * Present address: Nature Conservancy Council, Godwin House, George Street, Huntingdon PE18 6BU,


00166480/82/030310-12$01.00/O Copyright AU rights

@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

no direct effect on chicken hepatocytes has been demonstrated (McCumbee and Hazelwood, 1977). The liver is the main site of fatty acid synthesis in birds (Goodridge and Ball, 1967; O’Hea and Leveille, 1969a). Since the activity of the pentose phosphate pathway is low in the chicken (O’Hea and Leveille. 1969b), the NADPH necessary for lipogenesis is supplied from enhanced malic enzyme activity (Goodridge, 1969). Lipid metabolism in general plays a more significant role in birds than in mammals, perhaps due to weight conservation in flight, especially in migrating birds (Blem, 1976); it also acts as an energy source for embryonic development within the egg. There is little decrease in blood glucose when chickens are fasted for 7 days (Hazelwood and Lorenz, 1959). Glycogen stores which are low in birds (Langslow and Hales, 1971) are quickly depleted, but partial replenishment occurs after a few days. A rise in blood nonprotein nitrogen (Hazelwood and Lorenz, 1959) suggests that gluconeogenesis is responsible for the




maintenance of plasma glucose. In the present study isolated chicken hepatocytes have been used in order to study the role of the liver in regulating blood metabolite concentrations. MATERIALS


Male Thornber chickens, 3 to 5 weeks old, were obtained from the Poultry Research Centre. Roslin, Edinburgh, and housed under a constant 12-hr-dark. 12-hr-light cycle. Birds were either allowed free access to food and water or deprived of food for 24 hr prior to use. Collagenase and other enzymes used were obtained from the Boehringer Corporation, London. Biochemicals were obtained from Sigma (London). Cyclic AMP and cyclic GMP antibodies were a kind gift from Dr. Ken Siddle. Department of Medical Biochemistry. Cambridge University. Anion-exchange resin AG 1 x 2. 200-400 mesh, chloride form. was obtained from Bio-Rad (Richmond, Calif.). [8-“H]cAMP, [8“H]cGMP, and [14C]acetate were obtained from the Radiochemical Centre (Amersham). Monocomponent porcine insulin was obtained from Wellcome Laboratories (Beckenham, Kent) and chicken insulin was a gift from Dr. J. R. Kimmel, Department of Biochemistry. University of Kansas. Other hormones and reagents were obtained as described previously (Cramb rf al., 1982). Ml?thods

Cell isolution and incubation. Chicken hepatocytes were isolated by the method outlined in the accompanying paper (Cramb et (II., 1982). When isolating the cells from livers of fed chickens, a Krebs-Ringer bicarbonate buffer (Krebs and Henseleit, 1932) containing a high potassium and low sodium content was used: the normal concentrations of these two cations were interchanged. The normal high-sodium buffer was then used after cell isolation for all experimental purposes. All buffers also contained 1% bovine serum albumin. The high-potassium medium inhibited the breakdown of intracellular glycogen in the hepatocytes, possibly by inhibiting phosphorylase activity (Hue rt cl/.. 1975). Preparations using the highpotassium medium did not alter any of the other parameters measured with the hepatocytes. After isolation concentrated cell suspensions ( 10 to 20 x IO6 cells/ml) were preincubated in an orbital shaking water bath (60 cyclesimin) for 15 min at 37” with constant gassing with 95% Oz/5% CO* before experiments in which CAMP was measured. This allowed elevated cyclic nucleotide levels to stabilize. For all other experiments, cells were used immediately after isolation. Incubations were initiated by adding 0.5 ml



of cell suspension to 1 ml of pregassed Krebs-Ringer bicarbonate buffer containing hormone or effector, in 7.5 x 2.5-cm plastic pots fitted with air-tight caps. Pots were then gassed for 10 to 20 set with 95% 0,/S% CO,, caps fitted, and then incubated in a reciprocal shaking water bath for the desired time. Cyclic tzucleotide determinations. Determination of CAMP and cGMP was carried out by radioimmunoassay as described by Siddle rf al. (1973, 1976). For CAMP determinations, cell suspensions were deproteinised by adding an equal volume of 6% PCA and neutralised with 3 M K,CO,. Standard CAMP solutions were also acid extracted before radioimmunoassay. Cross reactivity between CAMP antisera and other nucleotides was so low that further purification of CAMP from the extracts was unnecessary. For cGMP determination. the cell suspensions were extracted with an equal volume of 10% trichloroacetic acid and neutralised by four washes in water-saturated ether. Due to the cross-reactivity of endogenous CAMP with cGMP antisera, it was necessary to further purify the cGMP from extracts using anion-exchange chromatography (Siddle rt (11.. 1976) followed by concentration by freeze drying. Resuspension of lyophylised cGMP from the tubes was greatly enhanced by refreezing and thawing the sample (C. J. Davies, personal communication). Adrnylufe c~clos~ crssav. The method used was that given by Luzio ul (11. (1976). Cell suspensions were centrifuged (275Og. 1 min) and the cells homogenised in 10 mM Tris -HCl, pH 7.4, containing 1 mM dithiothreitol, 10 mM theophylline. and 0.2% bovine serum albumin. CAMP produced from cell extracts incubated for 10 min at 30” in the presence of 10 mM theophylline and 0.5 mM ATP was measured by radioimmunoassay as described above. Phosphodiest~r~fsr ~ssa>l. Phosphodiesterase activity in crude cell homogenates was measured by the method of Arch and Newsholme (1976). Cell suspensions were centrifuged as above and the pellet homogenised in 50 mM Tris- HC 1, pH 7.4. containing 10 mM MgC12, 1 mM dithiothreitol. and 0.2% bovine serum albumin. Triton X-100 was also present at 0.2% (w/v) in some homogenising buffers. Glucose release und glycoget~ detertninafiott. The cell suspension was centrifuged (275Og, 30 set) and a sample of supernatant (0.2-0.5 ml) removed for the determination of glucose by the glucose oxidase method, using 4-aminophenazone as the chromogenic oxygen acceptor in place of o-dianisidine (Hugget and Nixon, 1957; Trinder, 1969). Glycogen in the cell pellet was determined as glucose equivalents by the method of Walaas and Walaas (1950). Lipogetzesis. Cell suspension was incubated with 2.5 mM[‘4C]acetate for 1 hr at 37”. A sample of cell suspension (1 ml) was then removed and centrifuged (2750~. 30 set) to sediment the cells. Lipid was extracted by the method of Folch ef crl. (1957). Radioac-




tivity in the resulting triacylglycerol pellet was determined and lipogenic activity expressed as micromoles of acetate incorporated per [email protected] cells. Gluconeogenesis. Gluconeogenesis was studied by monitoring the rate of incorporation of lactate into glucose by hepatocytes isolated from 24 hr-starved birds. Glucose release was determined as above. The glycogen content was always below measurable amounts under the conditions used (less than 0.5 pg glucoseeq/106 celis).


Immediately after isolation, concentrated cell suspensions were found to have elevated intracellular CAMP concentrations, ranging from 1 to 4 pmol/lO’ cells. Incubation of the suspensions for 15 min at 37” with constant gassing with 95% Oz/5% CO, generally reduced the basal concentration to a new steady level of around 1 pmol/106 cells, which was maintained for at least an hour. Cell suspensions for all experiments in which CAMP was measured were therefore preincubated for 15 min to allow the CAMP concentration to stabilise. At least 95% of this CAMP was found to be intracellular. Addition of glucagon (7 nM) to cell suspensions induced a rapid accumulation of intracellular CAMP, reaching a peak at about 5 min (Fig. 1). Production of CAMP

FIG. 1. The effect of glucagon on hepatocyte CAMP-Hepatocytes (6 x 106 cells/ml) were incubated with 7 nM glucagon. Total CAMP (0) and extracellular CAMP (A) were measured; intracellular CAMP (0) was found by difference.



then stopped and the intracellular concentration rapidly declined, with up to 70% of the CAMP slowly escaping into the surrounding medium. Intracellular CAMP had returned to the basal level by 60 min. Degradation of extracellular CAMP continued throughout this period, presumably due to phosphodiesterase in the medium; this was shown by incubating exogenous CAMP in medium from which cells had been removed. In the presence of theophylline (1 or 5 mM), CAMP levels remained elevated for longer periods. The basal concentration of CAMP was not increased by the phosphodiesterase inhibitor, nor was the amount of glucagon-stimulated CAMP. Adrenaline (5 PM) also elevated CAMP in the suspension, with a similar time course (Fig. 2). Although the rate of initial production of CAMP was just as rapid, the maximum adrenaline-stimulated level was only one-third of that found with glucagon. Neither insulin (0.8-85 nM) nor APP (1.2- 120 nM) had any effect on basal CAMP levels in the hepatocytes. However, addition of high concentrations of insulin (85 nM) to cells in the presence of submaximal amounts of glucagon (3 nM) resulted in changes in the time course of CAMP production (Fig. 3). Initial rates were identical, but the CAMP degradation rate was decreased. This effect of insulin on

FIG. 2. The effect of adrenaline on hepatocyte CAMP-Hepatocytes (8 x [email protected] cells/ml) were incubated with 5 @4 r-adrenaline hydrochloride and CAMP was measured (0); a control incubation in the absence of hormone is also shown (0).






FIG. 3. The effect of insulin on glucagon-stimulated hepatocyte CAMP. Hepatocytes (12 x 10’ cells/ml) were incubated with 3 r&f glucagon in the presence (0) and absence (0) of 85 r&I insulin.

total CAMP degradation was not observed at concentrations of 17 nA4 or below, even when lower glucagon concentrations (1.5 nM) were employed. APP (120 nM) failed to show any effect on glucagon-stimulated cells. Cell suspensions were incubated in the presence of varying concentrations of glucagon or adrenaline for 2 min at 37” and the CAMP produced was determined. Cell sensitivities were highly dependent upon the batch of collagenase used during the isolation procedure. With one particular batch of collagenase half-maximal activation was found at approx 9 nJ4 glucagon (Fig. 4). In the presence of 5 mM theophylline, very low glucagon concentrations (0.2 nM) were found to elevate basal nucleotide levels by approximately 30% (from 1.96 + 0.21 to 2.51 L 0.35 pmol/106 cells (SD for triplicate determinations), Fig. 4). Adrenaline had a half-maximal effect at 1.2 @I, using cells prepared with the same batch of collagenase. Dose responses to glucagon or adrenaline were not affected by simultaneous addition or preincubation for 10 min with insulin (0.85-85 nM) or APP (I .2- 120 nM). Furthermore, the maximum response to glucagon alone was not increased by the simultaneous addition of adrenaline.

FIG. 4. Concentration dependence of glucagonstimulated CAMP formation. Hepatocytes (7 X 10” cells/ml) were incubated with glucagon and CAMP was measured after 2 min at 37” (0): low concentrations of glucagon were also investigated in a suspension containing 14 x 10” cells/ml and 5 mM theophylline (0).

A basal rate of CAMP production of 0.06 pmol/min/lO” cells in a crude cell homogenate was stimulated to 0.68 pmoYmin/lO” cells in the presence of glucagon (30 nM). Neither insulin (17 nM) nor APP (24 nM) had any direct effect on basal or glucagonstimulated adenylate cyclase activity. Heprrtocyte


Basal cGMP concentrations of 31.3 I 25.0 fmol/lO” cells (SD for four separate cell preparations) showed no significant changes on incubation of cells for 10 min with insulin (17 n/V), glucagon (30 nM), APP (24 nM). or adrenaline (5 FM). Phosphodiesterase


The phosphodiesterase activity in cell extracts was measured using 0.5, 5.0, and 250 FM CAMP as substrate. Cell suspensions were preincubated for 20 min with in-




sulin (0.85-17 nM), glucagon (3-30 nM), or APP (1.2- 24 nM) before centrifugation and disruption of the cell pellet by homogenization. Preincubation with hormones failed to alter the phosphodiesterase activity; basal activities were 0.76 + 0.05, 2.43 ? 0.19. and 6.75 ? 0.59 pmol CAMP/ minilO cells (SD, II = 4, for all observations) with 0.5, 5.0, and 250 pM CAMP, respectively. Inclusion of 0.2% (w/v) Triton X-100 increased the phosphodiesterase activity in the cell extracts by 50-70%, but no hormone-induced variation in activity was found.

Glycogen depletion and glucose release were stimulated by glucagon (Fig. 5). In the absence of hormone, cellular glycogen breakdown was not completely accounted for by the appearance of glucose in the medium, although equivalence was seen when glucagon was added. Glucagon dose-response curves for glucose release and glycogen depletion were superimposable (Fig. 6). The sensitivity of cells to glucagon varied with the batch of collagenase employed during the cell isolation: half-maximal stimulation of glucose


release occurred at 0.2,0.8, 1.1, and 2.8 nM glucagon (means for two cell preparations with each of four batches of collagenase). Incorporation of soya bean trypsin inhibitor (20 pg/ml) throughout the initial perfusion and preparative stages did not affect hormone sensitivity, nor cell yield or viability. and no effect was found when bacitracin (0.5 mgiml) was added to cell incubations. The values found were always an order of magnitude lower than the glucagon concentrations found to half-maximally stimulate CAMP production; only very small increases in CAMP (measured after 2 min) were detected at glucagon doses that stimulated glucose release maximally (measured over 30 min). Similar results were obtained with adrenaline (half-maximal stimulation at 95- 140 nM), again IO-fold lower than the concentration required for half-maximal stimulation of CAMP. Maximum effects of glucagon and adrenaline on glucose release were similar in magnitude, but were not additive to each other. Exogenous CAMP and dibutyryl CAMP also stimulated glucose release (Fig. 7). The latter was more effective, giving a maximum stimulation of more than 200% at

FIG. 5. Glycogenolysis in hepatocytes. Hepatocytes (6 x 106 cells/ml) were incubated at 37” in the presence 10, l ) or absence (0, El) of 2.9 t&Z glucagon. The total glucose content of the suspension following glycogen hydrolysis (a, 0) and the glucose content of the incubation medium (m, 0) were measured. The difference between these represents the glycogen content of the cells.





50 pM, with a half-maximum response at 1.6 pM. Half-maximum stimulation with CAMP was found at 50 PM, with a maximum response at 200 ,uM. Neither porcine nor chicken insulin (1.7-85 nM), nor APP (2.4- 120 nM), showed any significant effect on basal or glucagon-stimulated glucose release when incubated with hepatocytes from fed birds.

FIG. 6. Concentration dependence of glucagonstimulated glucose release and glycogen depletion. Hepatocytes (6 x lo6 cells/ml) were incubated with glucagon for 30 min at 37”. Cell glycogen (0) and medium glucose (0) were determined. Values obtained in the absence of hormone have been subtracted from the glucose measurements; in the case of glycogen, which is expressed in terms of glucose equivalents. values have been subtracted from the control value measured in the absence of hormone.


Incubation of cells resulted in a progressive decrease in glycogen content (Fig. 5). Addition of physiological glucose concentrations (15 mM) to cell incubations reduced glycogen loss after 1 hr by 25 to 30%. Further addition of either porcine or chicken insulin (0.17 pM to 17 nM) to the medium did not increase the glycogen content of the cells. Incubation of cells with APP resulted in a slight preservation of glycogen content, although the glucose released remained constant. Glucmeogenesis Gluconeogenic rates were investigated in hepatocytes isolated from birds fasted for 24

-. --CAMPcOnCent&nwo FIG. 7. Glucose release from hepatocytes in response to exogenous CAMP. Hepatocytes (8 x 10’ cells/ml) were incubated with CAMP (0) or dibutyryl CAMP (0) for 30 min at 37”. and medium glucose was measured.



I 109


I loo Clucagon o* adrenaline



I Id coneen1ration

t IO2

I lo3

I ld


FIG. 8. Stimulation of hepatocyte gluconeogenesis by glucagon and adrenaline. Hepatocytes (9 x lo6 cells/ml) were incubated for 20 min at 37” in the presence of 1.5 mM lactate and glucagon (0) or L-adrenaline hydrochloride (0). The glucose content of the incubation medium was measured; bars indicate SD of triplicate determinations.

hr by following the incorporation of lactate into glucose. Cell suspensions were incubated in the presence of 1.5 mM lactate for 20 min in the presence of varying concentrations of glucagon or adrenaline (Fig. 8). Both hormones induced maximum increases in gluconeogenic rate of 30% above basal. Half-maximal activation was found at 0.48 nM glucagon or 21 nM adrenaline. High concentrations of adrenaline, but not glucagon, resulted in a slight reduction in the stimulated gluconeogenic rate. Neither insulin (1.7- 17 nM) nor APP (2.4-24 nM) had any effect on basal or hormone-stimulated gluconeogenesis. Lipogenesis Hepatocytes isolated from fed birds were used in ail experiments. Cells were incubated for 1 hr in the presence of 2.5 mM [14C]acetate with varying concentrations of glucagon or adrenaline (Fig. 9). Basal rates of incorporation into triacylglycerol under these conditions were normally in the range of 20-25 nmol acetate incorporated/lOfi cells/hr. Both glucagon and adrenaline reduced acetate incorporation by up to 90%. Half-maximal effects were at 1 .O nM gluca-

gon (maximum inhibition at 3.5 nM) and at 70 nM adrenaline (maximum at 160 nM). DISCUSSION



and Metabolism

The principle difficulty encountered in assessing our data for hormone binding and associated metabolic effects, and in making comparisons with the work of others, is the variability of the collagenase preparations used in the hepatocyte isolation. This led to variable hormone sensitivity, presumably as a result of proteolytic activity. Results obtained with glucagon, adrenaline, and insulin. using cells made with a single batch of collagenase, are collected in Table 1. APP showed no high-affinity binding and no metabolic responses. The basal CAMP level of the hepatocytes was influenced by the collagenase batch and method of cell preparation. A preliminary investigation using a different cell isolation method has been reported (Langslow and Siddle, 1979). In general, values of 1-4 pmoY106 cells found on isolation dropped to 0.5- 1.5 pmol/lO’ cells after a brief incubation, but with certain collagenase batches






FIG. 9. Inhibition of triacylglycerol synthesis by glucagon and adrenaline. Hepatocytes t 11 X 10” cells/ml) were incubated in the presence of 2.5 mk’ [“Clacetate (60 @/mmol) and glucagon (0) or L-adrenaline hydrochloride (0). After 60 min at 37” the incorporation of acetate into triacylglycerol was measured.

levels it remained at two or three times this value. This could be due to adenylate cyclase activation by a proteolytic enzyme in the collagenase preparation (Hanoune et 01.. 1977), especially since serine proteases have been shown to stimulate various adenylate cyclase preparations (Richert and Ryan, 1977; Wallach rt nl., 1978). It is thus impossible to state the basal CAMP level of unstimulated hepatocytes with certainty. Adrenaline and glucagon maximally increased CAMP levels up to 20-fold and 60-fold, respectively. Peak levels were reached within 2 min (at low hormone concentrations) to 10 min and were followed by a rapid drop in level (which could be inhibited with theophylline) both inside and out-

side the cells. Theophylline did not increase the levels reached; it is noteworthy that the adenylate cyclase activation was transient, in spite of prolonged glucagon binding to receptors (Cramb et pi., 1982). With cells produced by the collagenase batch shown in Table 1, 2.9 nh4 glucagon measurably stimulated the CAMP level, with a half-maximum effect at 12.9 nM, comparable with the value of 9.4 nM found for its dissociation constant at high-affinity sites on the hepatocyte surface (Cramb et (11.. 1982). Values between 1 and 6 nM are reported for half-maximal stimulation of CAMP levels in rat hepatocytes (Rosselin et crl., 1974; Pilkis rt al., 1975; Sonne et al.. 1978), consistent with higher affinities






Glucagon (nM) High-affinity binding CAMP production Stimulation of glycogenolysis Stimulation of gluconeogenesis Inhibition of lipogenesis

9.4 12.9 1.1 0.48 1.0




Adrenaline (PM) nm 1.7 0.14 0.02 0.07

Note. All results were obtained using cells prepared with the same batch of collagenase. binding are from Cramb et LI/. (1982). Values are concentrations giving half-maximal binding bolic response. nm, Not measured: -. no effect.

Insulin (nM) 0.3 Data for hormone or effect on meta-




found for glucagon binding to the mammalian cells. Our data suggest that glucagon binding to every receptor molecule is capable of activating adenylate cyclase; by contrast, half-maximal stimulation by adrenaline occurs at 1.7 FM, but only a subset of cyclase molecules appears to be activated, since stimulated CAMP levels are much lower, and the maximal effects of the two hormones are not additive. Nevertheless, adrenaline was capable of stimulating glycogenolysis and gluconeogenesis and of inhibiting lipogenesis to the same maximal extent as glucagon. As found with the rat, only small increases in CAMP concentration are necessary for maximum metabolic effect (Butcher et crl., 1968; Exton et al., 1971). Immunoreactive glucagon in peripheral blood of starved chickens has been reported to be about 0.3-0.4 nM (Samols et al., 1969; Krug et crl., 1976); about half of this is derived from the pancreas, and half from the ileum and jejunum of the gut. Presumably the portal blood concentration is likely to be higher than this value. It has been suggested that gut glucagon-like immunoactivity is not active in carbohydrate or lipid metabolism in birds (Krug and Miahle, 1971; Langslow, 1973) so that it seems safe to conclude that only a small proportion of the high-affinity sites on the chicken hepatocyte are likely to be occupied under the highest physiological concentrations of the hormone. In an effort to assess the physiological response to such a concentration we found that the CAMP level of a sensitive cell preparation could be raised by 30% by as little as 0.2 nM glucagon as long as 5 mM theophylline was present; under these conditions about 1% of the receptors are expected to be occupied (130 sites per cell). In contrast to the effect of glucagon on CAMP, half-maximum activation of glycogenolysis and inhibition of lipogenesis in the hepatocytes occurred at a concentration as low as I nM; it is clear that maximal



effects were produced with only about 10% of receptor sites filled, and 10% of maximal CAMP production, when CAMP is about threefold higher than the basal level in the cells (Fig. 4). The 90% inhibition of lipogenesis by glucagon and adrenaline compares with the 90% reduction in intracellular citrate reported in the presence of these hormones (Watkins and Lane, 1976), suggesting that the elevated CAMP may exert some of its effects through the level of this metabolite. The effect of glucagon on gluconeogenesis occurred over a narrower concentration range; stimulation was maximal when only 5% of glucagon receptors were occupied and the peak level of CAMP was less than twice the basal level. This small effect on CAMP may suggest that the cyclic nucleotide is sequestered in one cellular compartment, or remains bound in order to exert its effect on gluconeogenesis. Our results are consistent with previous studies showing different glucagon concentration dependence for gluconeogenesis and glycogenolysis in chicken hepatocytes (Dickson and Langslow, 1978; Dickson et al., 1978), although the concentration dependence appears to be the same in rat hepatocytes (Lewis et uf., 1970; Garrison and Haynes, 1973). Essentially similar results were obtained with adrenaline: while 1.7 FM adrenaline had a half-maximal effect on CAMP production, 0.14 @4 adrenaline half-maximally stimulated glycogenolysis. and 0.02 pM had a half-maximal effect on gluconeogenesis. Dibutyryl CAMP and exogenous CAMP mimicked glucagon and adrenaline in stimulating glycogenolysis. In the case of CAMP, high concentrations were required (half-maximal stimulation with 50 PM). Half-maximal stimulation by glucagon occurs when the intracellular CAMP concentration is about 2 ~.LM, assuming that the CAMP formed is distributed evenly through a spherical cell of diameter 12 pm. The dibutyryl CAMP concentration producing a



half-maximal effect (1.6 PM) compares rather well with this. Intracellular CAMP levels appear to respond directly to hormonal activation of adenylate cyclase, as shown by in vitro assay: glucagon and adrenaline had no effect on phosphodiesterase activity. Furthermore, these hormones had no effect on cGMP. Iruulin

and APP



there; and that the marginal effect that is observed results from an extra-hepatic action. APP failed to affect cyclic nucleotide metabolism or carbohydrate metabolism in the hepatocytes. This is consistent with our failure to detect a high-affinity receptor: peripheral blood levels of l-3 nM suggest that the low-affinity receptors found are of no physiological significance. The physiological effects of APP found ipr vivo must result from its effect on other tissues.

Submaximal glucagon-stimulated CAMP levels of rat hepatocytes or perfused liver ACKNOWLEDGMENTS are decreased by insulin (Exton and Park, We are grateful to Iris O’Neill and Ishbel Jack for 1972; Pilkis et ul., 1975; Westwood and technical assistance. This work was supported by the Siddle, 1979). Preincubation of the chicken Medical Research Council. hepatocytes with 85 nM insulin for 10 min only slightly reduced the size of the REFERENCES glucagon-induced CAMP peak, and simultaneous addition of insulin (0.85585 nM) Arch, J. R. S., and Newsholme, E. A. (1976). Activities and some properties of adenylate cyclase failed to decrease it. The highest concenand phosphodiesterase in muscle, liver and nertrations used, however, did markedly supvous tissues from vertebrates and invertebrates in relation to the control of the concentration of press the CAMP degradation which noradenosine 3’:5’ cyclic monophosphate. Biochrm. mally followed its rapid rise. The reason for .I. 158, 603-622. this, and its possible physiological signifiBlem. C. R. (1976). Patterns of lipid storage and utilicance, are quite uncertain. Insulin had no zation in birds. Amer. Zoo/. 16, 671-684. effect on phosphodiesterase activity in Butcher, R. W., Robinson, G. A., Hardman, J. G.. and Sutherland, E. W. (1968). The role of cyclic crude cell homogenates (stimulation of a AMP in hormone actions. Advan. Enzyme Regd. particulate phosphodiesterase from rat 6. 357-389. hepatocytes has been reported by Loten et Cramb, G., Langslow, D. R., and Phillips, J. H. al., 1978), nor did it have an effect on the (1982). The binding of pancreatic hormones to cGMP level. isolated chicken hepatocytes. Gen. Camp. Endocrinol. 46, 297-309. Insulin similarly failed to affect the other metabolic parameters measured. This is Dickson, A. J., and Langslow, D. R. (1978). Hepatic gluconeogenesis in chickens. Mol. Cell. B&hem. particularly interesting in view of the exis22. 167-181. tence of insulin receptors on these cells Dickson. A. J., Anderson. C. A., and Langslow. D. R. with affinity for insulin (K,, 0.3 nM) in the ( 19781. The use of viable hepatocytes to study the hormonal control of glycogenolysis in the physiological concentration range of 0. l- 2 chicken. Mol. Cell. Biochem. 19, 81-92. nM (Cramb et al., 1982). Since insulin can decrease the blood glucose concentration of Exton. J. H.. and Park, C. R. (1972). Interaction of insulin and glucagon in the control of liver mebirds in vivo, its effect must be exerted on tabolism. III “Handbook of Physiology,” Sect. 7. another tissue, possibly muscle; it seems “Endocrinology” (R. 0. Greep and E. B. unlikely that its effector system in hepatoAstwood. eds.). Vol. 1, pp. 437-455. Waverly, Baltimore. cytes is lost during isolation. We therefore Exton. I. H., Robison. G. A., Sutherland, E. W.. and conclude that the well-known resistance of Park, C. R. (1971). Studies on the role of birds to high concentrations of intravenous adenosine 3’:5’-monophosphate in the hepatic acinsulin results from its lack of effectiveness tions of glucagon and catecholamines. J. Biol. in the liver, in spite of the receptors found Chem. 246, 6166-6177.

320 Folch,



J.. Lees, M., and Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipid from animals tissues. J. Biof. Chem. 266,497 - 509. Garrison, J. C., and Haynes, R. C.. Jr. (1973). Hormonal control of glycogenolysis and gluconeogenesis in isolated rat liver cells. J. Biol. Chem. 248, 5333-5343. Goodridge, A. G. (1969). The effects of dietary fat on fatty acid synthesis and malic enzyme activity in liver from growing chicks. Canud. J. Biochem. 47, 743- 746. Goodridge. A. G., and Ball. E. G. (1967). Lipogenesis in the pigeon-Zn viva studies. Amer. J. Physiol. 213, 245-249. Hanoune, J., Feldman, G., Stengel, D.. Lancombe, M.-L., and Coudrier. E. (1977). Proteolytic activation of rat liver adenylate cyclase by a contaminant of crude collagenase from clostridium histolyticum. J. Biol. Chem. 252, 2039-2045. Hazelwood, R. L. (1976). The pancreas. Ztr “Avian Physiology” (P. D. Sturkie, ed.). 3rd Ed., pp. 210-232. Springer-Verlag. New York. Hazelwood. R. L., and Langslow, D. R. (1978). Intrapancreatic regulation of hormone secretion in the domestic fowl, Gallus domesticus. /. Endocrinol. 76. 449-459. Hazelwood, R. L.. and Lorenz, F. W. (1959). Effects of fasting and insulin on carbohydrate metabolism of the domestic fowl. Amer. .I. Phvsiol. 197, 47-51. Hue. L., Bontemps, F., and Hers, H.-G. (1975). The effect of glucose and potassium ions on the interconversion of the two forms of glycogen phosphorylase and glycogen synthetase in isolated rat liver preparations. Biochem. J. 152, lO5- 114. Hugget. A. St. G., and Nixon, D. A. (1957). Enzymatic determination of blood glucose. BiocZtr/n. .Z. 66. 12P. Krebs, H. A., and Henseleit, J. (1932). Untersuchungen tiber die Harnstoffbildung im Tierkorper. Hoppe Seyler Z. Physiol. Chem. 210, 33-66. Krug, E.. and Miahle, P. (1971). Pancreatic and intestinal glucagon in the duck. Harm. Metnb. Rrs. 3. 24-21. Krug, E., Gross, R., and Miahle, P. (1976). The contribution of the pancreas and the intestine to the regulation of lipolysis in birds. Harm. Merab. Res. 8, 345-350. Langslow, D. R. (1973). The action of gut glucagonlike immunoreactivity and other intestinal hormones on lipolysis in chicken adipocytes. Harm. Mefob. Res. 5, 428-432. Langslow, D. R., and Hales, C. N. (1971). The role of the endocrine pancreas and catecholamines in the control of carbohydrate and lipid metabolism. It7 “Physiology and Biochemistry of the Domestic



Fowl” (D. J. Bell and B. M. Freeman, eds.), pp. 521-547. Academic Press, New York/London. Langslow, D. R., and Siddle, K. (1979). The action of pancreatic hormones on the cyclic AMP content of isolated chicken hepatocytes. Gen. Comp. Endocrirwl. 39, 521-526. Lewis, S. B., Exton, J. H., Ho, R. S., and Park, C. R. (1970). Dose responses of glucagon (2 x lo-*’ to 1 x lo-” M) in the perfused rat liver. Fed. Proc. 29. 379. (Abstract) Loten. E. G., Assimacopoulos-Jeannet, F. D., Exton, J. H., and Park, C. R. (1978). Stimulation of a low K, phosphodiesterase from liver by insulin and glucagon. J. Biol. Chem. 253, 746-757. Luzio, J. P., Newby, A. C., and Hales, C. N. (1976). A rapid procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane. B&hem. J. 154. 11-21. McCumbee. W. D.. and Hazelwood, R. L. (1977). Biological evaluation of the third pancreatic hormone (APP): Hepatocyte and adipocyte effects. Gen. Cot77p. Endocrirwl., 33, 518-525. Mikami. S. I., and Ono, K. (1962). Glucagon deliciency induced by extirpation of alpha islets of the fowl pancreas. Endocrinology. 71, 464-473. O’Hea. E. R., and Leveille. G. A. (1969a). Lipid biosynthesis and transport in the domestic chick (Gallus domesticus). Con7p. Biochem. Physiol. 30. 149- 159. O’Hea, E. R., and Leveille, G. A. t 1969b). Significance of adipose tissue and liver as sites of fatty acid synthesis in the pig and the efficiency of utilization of various substrates for lipogenesis. J. Nutr. 99. 338-344. Pilkis, S. J., Claus, T. H., Johnson, R. A., and Park, C. R. (1975). Hormonal control of cyclic 3’:5’AMP levels and gluconeogenesis in isolated hepatocytes from fed rats. J. Biol. Chem. 250, 6328-6336. Richert, N. D.. and Ryan, R. J. (1977). Proteolytic enzyme activation of rat ovarian adenylate cyclase. Proc. Nat. Acud. Sci. USA 74. 4857-4861. Rosselin, G.. Freychet, P., Fouchereau, M., Rancon, F., and Broer, Y. (1974). Interactions of insulin and glucagon with isolated rat liver cells. II. Dynamic changes in the cyclic AMP induced by hormones. Harm. Metob. Res. 5, 78-86. Samols. E., Tyler. J. M., Marks, V., and Miahle, P. ( 1969). The physiological role of glucagon in different species. It7 “Proceedings of the Third International Conference on Endocrinology, Mexico.” pp. 206-219. Excerpta Medica. Amsterdam. Siddle, K.. Davies, C. J., Shetty, K. J., and Elkeles. R. S. (1976). The effect of insulin on adenosine 3’:5’-monophosphate and guanosine 3’:5’monophosphate concentration in human plasma. Clin. Sci. Mol. Med. 50. 487-491.




Siddle, K., Kane-Maguire, B.. and Campbell, A. K. (1973). The effects of glucagon and insulin on adenosine 3’:5’-cyclic monophosphate concentrations in an organ culture of mature rat liver. Biochem.

J. 132, 765-773.

Sitbon, G. (1967). La pancreatectomie Totale chez I’oie. Dicrbetologio. 3, 427-434. Sonne, 0.. Berg, T., and Christoffersen, T. (1978). Binding of ‘“51-labelled glucagon and glucagonstimulated accumulation of adenosine 3’:5’monophosphate in isolated intact rat hepatocytes. J. Biol. Chem. 253, 3203-3210. Trinder, P. (1969). Determination of blood glucose using 4-aminophenazone as oxygen acceptor. J. Clirl.


22, 246-249.



Walaas, O., and Walaas, E. (1950). Effect of epinephrine on rat diaphragm. J. Biol. Chm. 187, 769776. Wallach. D., Anderson, W., and Pastan. I. (1978). Activation of adenylate cyclase in cultured fibroblasts by trypsin. J. Biol. Chm. 253. 24-26. Watkins, P. A.. and Lane, M. D. (1976). Regulation of fatty acid synthesis in chicken liver cells by CAMP-mediated control of intracellular citrate levels. Frd. PIYX. 35, 1428. (Abstract) Westwood, S. A., and Siddle, K. (1979). Calcium ions and adenosine 3’:5’-cyclic monophosphate in hepatocytes: Effects of ethanedioxybis (ethylamine) tetra-acetate (EGTA) and ionophore A23187. Biochem. Sac. Truns. 7. 1032- 1033.