of Pancreatic Hormones Isolated Chicken D.R.
39, 521-526 (1979)
on the Cyclic AMP Content Hepatocytes
of Biochemistry. Royal (Dick) School of Veterinary Studies. Unirsersity of Edinburgh. Edinburgh EH9 IQH. and TDepartment of Medical Biochemistry, Welsh National School of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom
Accepted June 12, 1979 The hormonal control of cyclic AMP content was investigated in hepatocytes isolated by collagenase digestion from chicken liver. Glucagon (2.9 x lo-’ M) increased cyclic AMP content within 1 min. and the concentration continued to increase for at least 30 min. More than 90% of the total cyclic AMP measured remained within the cells during this time. Significant stimulation of glycogenolysis was obtained with a glucagon concentration (2.9 x 10-l’ M) which had no detectable effect on cyclic AMP content. Half-maximal increases in cyclic AMP content and glucose production were produced by glucagon concentrations of approximately 10mRand 10-l” M. respectively. Insulin (0.17- 17 x lo+ M) and avian pancreatic polypeptide (0.24-240 x lo-” M) had no effect on cyclic AMP content either in the presence or absence of glucagon. Adrenaline increased cyclic AMP content, the minimum effective concentration being 5 x lo-” M. The cyclic AMP response to adrenaline reached a peak within 2.5 min. and the maximum increase was much less than that produced by glucagon.
Both glucagon and adrenaline stimulate glycogenolysis and gluconeogenesis in isolated chicken hepatocytes. The cells are more sensitive to glucagon than to adrenaline while dibutyryl cyclic AMP mimics the effect of both hormones (Anderson and Langslow, 1975; Dickson and Langslow, 1977; Dickson, 1978; Dickson et al., 1978). Neither insulin nor avian pancreatic polypeptide (APP) influences glucose production by chicken hepatocytes in bitt-o although APP injection into chickens depletes liver glycogen (Hazelwood et al., 1973; Anderson and Langslow, 1975). Most chicken tissues are insensitive to insulin and chickens effectively resist insulininduced hypoglycaemia (Langslow and Hales, 1971). The aim of the experiments in the present paper was to relate the rate and amount of cyclic AMP production to the biological actions of glucagon, adrenaline, insulin, and APP on chicken hepatocytes. MATERIALS
system (G. Cramb, I. E. O’Neill, and D. R. Langslow, unpublished method). Cells were prepared, and incubated with different hormone additions, as described previously (Dickson and Langslow, 1975; Dickson et al., 1978.) Chicken insulin and APP were kind gifts from Professor J. R. Kimmel, Department of Biochemistry, University of Kansas Medical Center, and porcine glucagon was a gift from Dr. M. Root, Eli Lilly & Co., Indianapolis, Ind.). Between 5 and 15 x lo6 hepatocytes were present in 1.5 ml incubation medium. (The wet weight of 10” cells was 1.27 mg). The incubations were terminated by the addition of 1 ml of 10% TCA to cells plus medium (for total cyclic AMP) or medium alone following removal of cells by centrifugation (for extracellular cyclic AMP). The precipitate was removed by centrifugation and the supernatant was extracted four times with 5 ml of ether. A portion of the resulting solution was freeze dried and redissolved in 100 mM potassium phosphate. pH 7. for the assay of cyclic AMP. Cyclic AMP was measured by the radioimmunoassay method of Siddle et al. (1973). The assay detected cyclic AMP concentrations down to 0.1 pmol per 40+1 sample. In each experiment, the responsiveness of the cells to glucagon was tested by measuring glucose production from the activation of glycogenolysis in cells during a 30-min incubation with hormones (Dickson et al.. 1978).
Isolated chicken hepatocytes were prepared from the liver of fed chickens initially by using a nonrecirculating perfusion and, recently. using a recirculating
The cyclic AMP content of chicken hepatocytes was increased by glucagon at 521 001~6480/79/12052 l-06$01.00/0 Copyrighr ,c 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
0 0.00029 0.0029 0.029 0.29 1.45 2.9 29 290
cyclic AMP (pmoI/lO” cells at 5 min)
Glucagon concentration WW
Cyclic AMP (pmoVi0” cells at 10 min)
Glucose (&IO” cells/ 30 mitt)
4.8 4.2 4.3 4.0 4.9
2 0.5 -+ 0.5 t 0.2 t 0.2 2 0.5 16.2 + 0.7* 72.1 t 5.9* 115.1 2 3.3*
2.8 k 0.3 2.1 + 0.1 2.9 k 0.3 3.0 -c 0.2 7.9 k 0.4* 12.7 k 0.3* 30.5 _’ 2.4* -
1.15 1.00 0.98 1.58 1.66
* 0.21 2 0.08 k 0.20 2 0.17* 2 0.14* 2.38 -r- 0.23* 2.26 2 0.19* 2.62 -t 0.08*
” Hepatocytes from a fed chicken were incubated as described under Materials and Methods, with glucagon at the concentrations shown. Cyclic AMP was measured after 5 or 10 min, and glucose output after 30 min. Results are means 2 SEM of four observations. * Significantly different from 0 glucagon (P c 0.05).
concentrations of 2.9 x lo-lo to 2.9 x lo-’ M. After 5-min incubation, a significant rise in cyclic AMP content was always observed with 1.45 x lop9 M glucagon while 2.9 x lo-lo M sometimes produced an increase (Tables 1 and 2). A stimulation of glucose production by glucagon was consistently found at glucagon concentrations below those at which a significant increase in cyclic AMP content could be detected above basal cyclic AMP content (Table 1). In chicken hepatocytes the production of cyclic AMP stimulated by 2.9 x lo-’ M glucagon continued to increase for at least
30 min (Fig. l), unlike rat hepatocytes where cyclic AMP production peaks after 5 to 10 min (Christofferson and Berg, 1974; Pilkis et al., 1975). The total cyclic AMP response to this concentration of glucagon varied between different batches of cells but the time course of response was always the same. Such variability is commonly encountered in isolated cell preparations, but whether it reflects differences between donor animals, or a differential effect of enzyme digestion, is unknown. Glucagon rapidly stimulated cyclic AMP production and significant effects were
COURSE TO VARIOUS
Cyclic AMP (pmoY 10’ cells) Glucagon (t&f)
0 0.0029 0.029 0.29 2.9
5.9 L 0.2 -
1 min 6.4 4.3 5.4 7.7 8.7
_f 0.2 T 1.1 k 0.5 2 0.3 k 0.7
4 min 4.4 5.5 5.4 8.4 26.6
2 2 5 ? i
0.9 0.1 0.4 1.6 4.4
Glucose (ugil0” cells) 10 min 3.6 4.3 5.3 10.4 29.7
k 2 t k zt
0.4 0.3 0.3 0.5 1.2
30 min 5.25 6.27 7.67 8.84 9.39
k 0.07 f 0.15 c 0.06 2 0.23 I!z 0.10
U Hepatocytes from a fed chicken were incubated as described under Materials and Methods, with glucagon at the concentrations shown. Cyclic AMP was measured after various times and glucose output after 30 min. Results are means t SEM of four observations.
CYCLIC AMP IN CHICKEN
but the effect was not as great as that produced by a maximally effective concentration of glucagon (Dickson et al., 1978). Since the cyclic AMP content of cells plus medium was normally measured, accumulation of cyclic AMP by transfer from the hepatocytes to the medium could ac3 0 li_i__2;i count for the continuing increase in cyclic 0 10 30 AMP over 30 min following exposure to Time (mn) glucagon. The proportion of cyclic AMP in FIG. 1. Time course of cyclic AMP response of the cells and medium at 2.9 x 1O-s M glucachicken hepatocytes to glucagon. Hepatocytes were incubated with 290 nM glucagon, and cyclic AMP was gon over 20 min was measured. A relatively of the cyclic AMP apmeasured after the times shown. Results are means small proportion peared in the medium and most (>90%) ? SEM of four observations. cyclic AMP was intracellular at all time points (results not shown). found after 1 min with 0.29 and 2.9 nM Insulin is antagonistic to the effects of glucagon (Table 2). Little effect of glucagon glucagon on hepatocytes from mammalian on the cyclic AMP content of the cells was liver (Pilkis et nl., 1975). However, in observed below 0.29 nM at 1, 4, or 10 min chicken hepatocytes, insulin (0.17- 17 nM) although 0.0029 nM gave a significant failed to reduce the cyclic AMP increase stimulation of glucose production within 30 induced by glucagon (0.29-2.9 nM) (results min. not shown). Avian pancreatic polypeptide Adrenaline stimulated cyclic AMP ac- (APP) was also tested for its effect on cumulation in the cells but the time course chicken hepatocytes. APP alone (2.4 PM) and maximum response were different from did not affect basal cyclic AMP content those induced by glucagon. Maximal over a period of 30 min, nor did APP stimulation was achieved with 5.5 x 10e6 M (0.24-240 nM) either potentiate or inhibit adrenaline and the minimum concentration the cyclic AMP responses to glucagon to elevate cyclic AMP was 5.5 x lo-* M (0.29-2.9 n&Z) or adrenaline (0.5-5 pM) (Table 3). At all concentrations the (results not shown). Neither insulin nor maximum cyclic AMP content was found APP affected glucose production in chicken after 2.5 min and further incubation only hepatocytes (results not shown). maintained the cyclic AMP content at the DISCUSSION higher adrenaline concentrations (Table 3). The change in cyclic AMP content of Significant stimulation of glycogenolysis chicken hepatocytes following the addition was produced by 5.5 x 10m6 M adrenaline, . THE
0 2.5 5 10
2.9 t 0.2
” Results are
2.1 c 0.3 cyclic
2.9 -+ 0.3 2.6 2 0.2 1.8 ? 0.1
4.1 2 0.2 3.6 + 0.2 3.6 t_ 0.1
5.1 k 0.1 5.2 ? 0.3 5.9 * 0.3
8.2 2 0.2 7.1 + 0.2 8.3 + 0.3
AMP content (pmoV10” cells), means + SEM of four observations.
of glucagon was rapid (within 1 min, Table 2), substantial, and prolonged (at least 30 min, Fig. 1). By contrast, adrenaline caused a much smaller change in cyclic AMP content which was maximal at the first time measured (2.5 min, Table 3). These responses in cyclic AMP production contrast with those observed in hepatocytes from mammalian systems, especially rats, where both the adrenaline and glucagon effects peak rapidly (Christofferson and Berg, 1974; Pilkis ef al., 1975). A prolonged cyclic AMP response to hormones has been observed with adrenaline action on other avian cells, including pigeon erythrocytes, although the physiological significance of this response remains unknown (Campbell and Siddle, 1976). While the sensitivity of pigeon erythrocytes to adrenaline was similar to chicken hepatocytes, the time course was quite different. Chicken adipocytes also show a prolonged response of cyclic AMP production to 2.9 x lop9 M glucagon (Langslow et al., 1979) similar to that observed in hepatocytes although Boyd et al. (1975) reported a peak cyclic AMP content after only 7 min with 3 x lo-’ M glucagon. A different time course of cyclic AMP production between rat and chicken hepatocytes could be brought about in several ways. There may be different adenylate cyclase to phosphodiesterase ratios, or activation of rat hepatocyte phosphodiesterase by hormones or cyclic AMP (Allan and Sneyd, 1975), or differences in sensitivity to feedback inhibition of cyclic AMP production. Since the cyclic AMP content of cells plus medium was measured in most experiments, it was possible that the continuing production of cyclic AMP up to 30 min (Fig. 1) was due to the transfer of cyclic AMP from the cells to the medium. Measurement of the cyclic AMP content of the medium or medium plus cells demonstrated clearly that cyclic AMP was continuing to accumulate within the cells throughout the
entire incubation period and that leakage of cyclic AMP from the cells was minimal. Either continuing stimulation of the adenylate cyclase was occurring without any feedback inhibition by cyclic AMP, or the large concentration of glucagon employed overrode potential feedback controls. About 10% of the cyclic AMP had leaked from the cells after 30-min incubation. Leakage of cyclic AMP at this rate would explain the changes in blood and total liver and muscle cyclic AMP contents observed by Frohlich and Marquardt (1972) following the injection of 1 mg of glucagonikg body wt into chickens. The concentration of cyclic AMP in liver was 6 nmol/g wet wt after glucagon injection, similar to the 3-10 nmol/g wet wt observed in the liver cells 20 min after exposure to glucagon. The response to adrenaline in \vivo was less than that to glucagon, as found in vitro, although the time courses of the initiated responses to adrenaline and glucagon were similar in viw . Insulin did not influence basal or glucagon-stimulated cyclic AMP production, unlike the situation in rat hepatocytes where 10 nM insulin prevented increases in cyclic AMP due to 1.5 nM glucagon (Pilkis et al., 1975). This accords with the general low or zero response of many chicken tissues to insulin (Langslow and Hales, 1971). Frohlich and Marquardt (1972) found that insulin slowly elevated the cyclic AMP content of chicken blood. That effect might be due to the stimulation of endogenous glucagon production caused by insulin (Hazelwood and Langslow, 1978). APP is secreted following food ingestion and might be expected to be antagonistic to glucagon. However, it did not influence basal cyclic AMP content and did not change the stimulation of cyclic AMP production caused by glucagon or adrenaline. Unlike insulin, the injection of APP into chicken lowers the plasma glucagon concentration (Hazelwood and Langslow, 1978) and, as it
CYCLIC AMP IN CHICKEN
is secreted following refeeding (Langslow et al., 1973), is more likely to be associated with a fall than a rise in cyclic AMP. Glycogenolysis in hepatocytes was stimulated by 2.9 x lo-” M glucagon while the increase in cyclic AMP was minimal at 2.9 x lo-lo M and only became significant at 2.9 x lop9 M. The concentrations of glucagon which would give half-maximal stimulation of glucose output and cyclic AMP content were estimated to be of the order of lo-lo and lops M, respectively (Table 1). The lack of correlation between biological responsiveness and cyclic AMP content has been noted in other systems such as chick adipocytes (Boyd et al., 1975; Langslow et al., 1979) and rat hepatocytes (Birnbaum and Fain, 1977; Assimacopoulos-Jeannet et al., 1977). In chicken hepatocytes, it seems that quite a small change in cyclic AMP content may be capable of fully activating a cyclic AMP mediated process. Alternatively, another message may be generated which activates glycogenolysis and might also cause an increase in cyclic AMP. The sensitivity of chicken hepatocytes to cyclic AMP stimulation by glucagon is similar to rat hepatocytes (Christofferson and Berg, 1974; Westwood et al., 1979). Pilkis et al. (1975) quote a lower figure for half-maximal stimulation by glucagon but it is distorted by the presence of theophylline which will increase the apparent sensitivity. If the action of glucagon on glycogenolysis in chicken hepatocytes is mediated by cyclic AMP then only a very small (or transient) increase in cyclic AMP is required to stimulate phosphorylase. ACKNOWLEDGMENTS D R. L. thanks the Medical Research Council for financial support and the authors are grateful to Caroline Anderson, Alan Dickson. and Iris O’Neill for their help in the preparation of hepatocytes.
REFERENCES Allan. E. H., and Sneyd. J. G. T. (1975). An effect of glucagon on 3’, S-cyclic AMP phosphodiesterase
hepatocytes. Biochem. 62, 594-601. Anderson, C. E.. and Langslow, D. R. (1975). Glucose production and its hormonal control in isolated chicken hepatocytes. Biochrm. SW. Trans. 3, 1037- 1039. Assimacopoulos-Jeannet, F. D., Blackmore, P. F., and Exton, J. H. (1977). Studies on a-adrenergic activation of hepatic glucose output: Studies on role of calcium in a-adrenergic activation of phosphorylase. J. Biol. Chem. 252, 2662-2669. Bimbaum, M. J., and Fain, J. N. (1977). Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines. J. Biol. Chem. 252, 528-535. Boyd, T. A., Wieser, P. B.. and Fain, J. N. (1975). Lipolysis and cyclic AMP accumulation in isolated fat cells from chicks. Cm. Camp. Endocrinol.
26, 243 -247.
Campbell, A. K., and Siddle K. (1976). The effect of intracellular calcium ions on adrenalinestimulated adenosine 3’:s’~cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187. Bioc,hc,m. J. 158, 21 I-221. Christofferson, T., and Berg, T. (1974). Glucagon control of cyclic AMP accumulation in isolated intact rat liver parenchymal cells in vitro. Biochem.
Dickson, A. J. (1977). “The Preparation and Properties of Isolated Chicken Hepatocytes.” Ph.D. Thesis, University of Edinburgh. Dickson, A. J., Anderson, C. E., and Langslow. D. R. (1978). The use of viable hepatocytes to study the hormonal control of glycogenolysis in the chicken. Mol. Cell. Biochem. 19, 81-92. Dickson, A. J., and Langslow. D. R. (1975). The preparation and assessment of the physiological competence of isolated chicken hepatocytes. Biochem. Sac. Trans. 3, 1034-1037. Dickson, A. J., and Langslow, D. R. (1977). Gluconeogenesis in isolated chicken hepatocytes. Biochrm.
Frohlich. A. A., and Marquardt, R. R. (1972). Cyclic AMP levels in various tissues of the domestic fowl (Callus domesficas) as affected by glucagon, epinephrine and insulin. Biochem. Biophys. Acta 286, 396-405. Hazelwood, R. L., and Langslow, D. R. (1978). Intrapancreatic regulation of hormone secretion in the domestic fowl, Gal/as domesticas. J. Endoc~+~ol. 76, 449-459. Hazelwood, R. L., Turner, S. D., Kimmel, J. R., and Pollock, H. G. (1973). Spectrum effects of a new polypeptide (third hormone ?) isolated from chicken pancreas. Gen Camp. Endocrinol. 21. 485 -497.
Langslow, D. R., and Hales, C. N. (1971). The role of the endocrine pancreas and catecholamines in the control of carbohydrate and lipid metabolism. In “Physiology and Biochemistry of the Domestic Fowl” (D. J. Bell, and B. M. Freeman, eds.), pp. 52 I-547. Academic Press, London. Langslow, D. R., Cramb, G., and Siddle, K. (1979). Possible mechanisms for the increased sensitivity to glucagon and catecholamines of chicken adipose tissue during hatching. Gen. Camp. Endocrinol.
Langslow, D. R., Kimmel, J. R., and Pollock, H. G. (1973). Studies of the distribution of a new avian pancreatic polypeptide and insulin among birds, reptiles, amphibians and mammals. Endocrinology
AND SIDDLE 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. &I[. Chem. 2.50, 6328 -6336. 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.
Westwood, S. A., Luzio, J. P., Flockhart, D. A., and Siddle. K. (1979). Investigation of the subcellular distribution of cyclic AMP phosphodiesterase in rat hepatocytes, using a rapid immunological procedure for the isolation of plasma membrane. Biochim.