Acta Med Scand 202: 87-88, 1977
The Effect of Glucagon on Plasma Cyclic AMP and Glucose Concentrations in Patients with Alcoholic Cirrhosis R. C. Strange, 0. D. Mjm, Thale Henden and P. Jynge From the Department of Clinical Chemistry, University of Edinburgh, Edinburgh, United Kingdom, and the Department of Physiology, Institute of Medical Biology, University of Trams@, Tromss, Norway
ABSTRACT. Glucagon was infused intravenously into four patients with alcoholic cirrhosis and five healthy subjects and serial measurements were made of plasma cyclic AMP and glucose concentrations. The results in the cirrhotic patients did not differ significantly from those in healthy subjects.
Attempts to utilize diagnostically the association between liver disease and abnormal glucose metabolism (6) have proved disappointing. For example, Van Itallie and Bentley (5) were unable to differentiate patients with liver disease from healthy controls on the basis of differences in glucagoninduced hyperglycaemia. Hepatic glucose production is regulated by the plasma concentrations of glucagon and insulin. This control is exerted by hormone-induced changes in the intracellular concentration of the nucleotide cyclic adenosine 3'3'-monophosphate (cyclic AMP) (3. 4). Although several tissues, including liver, can release the nucleotide into plasma in the basal state and after hormonal stimulation (3, 4), it appears that the increased cyclic AMP concentrations found in plasma after i.v. administration of glucagon derive entirely from the liver (7). The possibility that differences in plasma cyclic AMP concentrations after i.v. injections of glucagon can be used to differentiate patients with various types of liver disease from normal subjects has been examined with some success by Davies et al. ( I ) . We now describe the results of the test in four patients with well compensated, alcoholic cirrhosis and in five matched healthy subjects.
STUDY POPULATION AND METHODS Four non-hospitalized male subjects (age range 44-59 years, weight range 71-110 kg) with well compensated alcoholic cirrhosis were studied. In all cases the diagnosis was based on the pathology of liver biopsy specimens. The control group (age range 49-63 years, weight range 66-87 kg) comprised one female and four male in-patients recuperating after minor orthopaedic surgery performed at least 10 days previously. In all subjects studied, the plasma concentrations of total bilirubin, total protein, albumin and thyroxine and the plasma activities of alanine aminotransferase and alkaline phosphatase were within reference ranges. Informed consent for the procedures was obtained from all subjects. Patients were studied after an overnight fast. After inserting a catheter into each antecubital vein, two blood samples were taken with a 15 min interval from one vein and glucagon (100 ng/kg b.wt./min) in NaCl (0.9% w/v) was then infused for 30 min into the other vein. Blood samples were taken at 5 min intervals over 30 min and then at 10 min intervals over a further 30 min. Blood was collected into heparinized tubes standing in ice, immediately centrifuged and the plasma concentrations of cyclic AMP and glucose were measured. Blood for cyclic AMP assay was collected in 10 ml tubes containing theophylline (50 mM). Cyclic AMP was measured with a protein-binding method (The Radiochemical Centre, Amersham, Bucks, UK) and glucose with a glucose oxidase method. Statistical analysis was performed using the Wilcoxon test (2).
RESULTS The mean plasma concentrations of glucose and cyclic AMP for both groups of subjects are shown in Fig. I. Although plasma glucose concentrations appeared to be higher in the cirrhotic patients, at no
R . C . Strange et al.
88
AMP concentrations after administration of glucagon between healthy subjects and patients with well compensated alcoholic cirrhosis. In contrast to our findings, however, Davies et al. noted significantly higher fasting plasma cyclic AMP concentrations in patients with cirrhosis than in control subjects. The reason for this difference is not clear, although their control group was considerably younger (mean age 27.4 years) than ours. The failure to discriminate between healthy controls and patients with cirrhosis may partly be explained by the suggestion that diffusible cyclic AMP represents only a small percentage of the total hepatic content of the nucleotide even after enhancement of adenyl cyclase activity by glucagon (3). Measurement of cyclic AMP clearance from plasma does not appear to be a useful diagnostic aid either, since although the liver can remove cyclic AMP from plasma (8), the clearance of cyclic AMP after cessation of the glucagon infusion was not significantly different in patients with cirrhosis compared with the normal subjects.
GLucagon 100 nglkglmin
200
I
,.
T
2
E
. 0
c
P
Y
u
100
3
m
9 E 0 VI
n
.8 2
I
I
0
I
I I
I I
I
I
200
E
u
n
f
u
; 100 >. 9
E
m P
L -1 5
I
I
0
30
60
min
Fig. 1. Effect of i.v. infusions of glucagon on the plasma
concentrations of cyclic AMP and gluco5e in patients with cirrhosis ( 0 )and in healthy subjects (0).Each point is the mean concentration k I S.E.M.
time during the study were they significantly different from those of the control group. Fasting plasma cyclic AMP concentrations were similar in both groups and although slightly higher, they were not significantly different from values previously reported in similar healthy subjects (9). During the course of the glucagon infusion there was a considerable variation in cyclic AMP concentrations between individuals. Thirty minutes after the start of the glucagon infusion the range of plasma cyclic AMP concentrations in the cirrhotic patients was 57-123 pmol/ml and in the healthy controls 34-342 pmol/ml. Throughout the study there was n o significant difference in cyclic AMP concentrations between the two groups.
DISCUSSION In agreement with the results of Davies et al. ( I ) we found no significant difference in plasma cyclic
REFERENCES I . Davies, T. F., Prudhoe, K. & Douglas, A. P.: Plasma cyclic adenosine-3',5'-monophosphate response to glucagon in patients with liver disease. Br Med J 1: 93 I, 1976. 2. Documenta Geigy: Scientific tables (ed. Diem and Lentner), p. 128.Geigy, Bade 1970. 3. Exton, J. H.,Lewis, S. B., Ho,R. J. & Park, C. R.: The role of cyclic AMP in the control of hepatic glucose production by glucagon and insulin. Adv Cyclic Nucleotide Res 1: 91, 1972. 4. Exton, J. H., Robison, G. A,, Sutherland. E. W. & Park. C. R.: Studies on the role of adenosine 3'3'-
monophosphate in the hepatic actions of glucagon and catecholamines. J Biol Chem 246:6166, 1971. 5. Van Itallie, T. B. & Bentley, W. B. A.: Glucagoninduced hyperglycaemiaas an index of liver function. J Clin Invest 34: 1730, 1955. 6. Megyesi, C., Samols, E. & Marks, V.: Glucose tolerance and diabetes in chronic liver disease. Lancet 2: 1051, 1967.
7. Strange, R. C. & Mjm, 0.D.: The sources of plasma cyclic AMP: studies in the rat using isoprenaline, nicotinic acid and glucagon. Eur J Clin Invest 5: 147, 1975. 8. Strange, R. C. & Percy-Robb, 1. W.: Hepatic clearance
of adenosine 3'5'-cyclic monophosphate from plasma in the rat. Biochem J 146: 509, 1975. 9. Strange, R. C., Rowe, M. J., Mjm, 0. D. & Oliver, M. F.: The effect of antilipolytic agents on cyclic AMP, free fatty acid and total catecholamine concentrations in plasma. Acta Med Scand 199:421, 1976.
The Effect of Glucagon on Plasma Cyclic AMP and Glucose Concentrations in Patients with Alcoholic Cirrhosis R. C. Strange, 0. D. Mjm, Thale Henden and P. Jynge From the Department of Clinical Chemistry, University of Edinburgh, Edinburgh, United Kingdom, and the Department of Physiology, Institute of Medical Biology, University of Trams@, Tromss, Norway
ABSTRACT. Glucagon was infused intravenously into four patients with alcoholic cirrhosis and five healthy subjects and serial measurements were made of plasma cyclic AMP and glucose concentrations. The results in the cirrhotic patients did not differ significantly from those in healthy subjects.
Attempts to utilize diagnostically the association between liver disease and abnormal glucose metabolism (6) have proved disappointing. For example, Van Itallie and Bentley (5) were unable to differentiate patients with liver disease from healthy controls on the basis of differences in glucagoninduced hyperglycaemia. Hepatic glucose production is regulated by the plasma concentrations of glucagon and insulin. This control is exerted by hormone-induced changes in the intracellular concentration of the nucleotide cyclic adenosine 3'3'-monophosphate (cyclic AMP) (3. 4). Although several tissues, including liver, can release the nucleotide into plasma in the basal state and after hormonal stimulation (3, 4), it appears that the increased cyclic AMP concentrations found in plasma after i.v. administration of glucagon derive entirely from the liver (7). The possibility that differences in plasma cyclic AMP concentrations after i.v. injections of glucagon can be used to differentiate patients with various types of liver disease from normal subjects has been examined with some success by Davies et al. ( I ) . We now describe the results of the test in four patients with well compensated, alcoholic cirrhosis and in five matched healthy subjects.
STUDY POPULATION AND METHODS Four non-hospitalized male subjects (age range 44-59 years, weight range 71-110 kg) with well compensated alcoholic cirrhosis were studied. In all cases the diagnosis was based on the pathology of liver biopsy specimens. The control group (age range 49-63 years, weight range 66-87 kg) comprised one female and four male in-patients recuperating after minor orthopaedic surgery performed at least 10 days previously. In all subjects studied, the plasma concentrations of total bilirubin, total protein, albumin and thyroxine and the plasma activities of alanine aminotransferase and alkaline phosphatase were within reference ranges. Informed consent for the procedures was obtained from all subjects. Patients were studied after an overnight fast. After inserting a catheter into each antecubital vein, two blood samples were taken with a 15 min interval from one vein and glucagon (100 ng/kg b.wt./min) in NaCl (0.9% w/v) was then infused for 30 min into the other vein. Blood samples were taken at 5 min intervals over 30 min and then at 10 min intervals over a further 30 min. Blood was collected into heparinized tubes standing in ice, immediately centrifuged and the plasma concentrations of cyclic AMP and glucose were measured. Blood for cyclic AMP assay was collected in 10 ml tubes containing theophylline (50 mM). Cyclic AMP was measured with a protein-binding method (The Radiochemical Centre, Amersham, Bucks, UK) and glucose with a glucose oxidase method. Statistical analysis was performed using the Wilcoxon test (2).
RESULTS The mean plasma concentrations of glucose and cyclic AMP for both groups of subjects are shown in Fig. I. Although plasma glucose concentrations appeared to be higher in the cirrhotic patients, at no
R . C . Strange et al.
88
AMP concentrations after administration of glucagon between healthy subjects and patients with well compensated alcoholic cirrhosis. In contrast to our findings, however, Davies et al. noted significantly higher fasting plasma cyclic AMP concentrations in patients with cirrhosis than in control subjects. The reason for this difference is not clear, although their control group was considerably younger (mean age 27.4 years) than ours. The failure to discriminate between healthy controls and patients with cirrhosis may partly be explained by the suggestion that diffusible cyclic AMP represents only a small percentage of the total hepatic content of the nucleotide even after enhancement of adenyl cyclase activity by glucagon (3). Measurement of cyclic AMP clearance from plasma does not appear to be a useful diagnostic aid either, since although the liver can remove cyclic AMP from plasma (8), the clearance of cyclic AMP after cessation of the glucagon infusion was not significantly different in patients with cirrhosis compared with the normal subjects.
GLucagon 100 nglkglmin
200
I
,.
T
2
E
. 0
c
P
Y
u
100
3
m
9 E 0 VI
n
.8 2
I
I
0
I
I I
I I
I
I
200
E
u
n
f
u
; 100 >. 9
E
m P
L -1 5
I
I
0
30
60
min
Fig. 1. Effect of i.v. infusions of glucagon on the plasma
concentrations of cyclic AMP and gluco5e in patients with cirrhosis ( 0 )and in healthy subjects (0).Each point is the mean concentration k I S.E.M.
time during the study were they significantly different from those of the control group. Fasting plasma cyclic AMP concentrations were similar in both groups and although slightly higher, they were not significantly different from values previously reported in similar healthy subjects (9). During the course of the glucagon infusion there was a considerable variation in cyclic AMP concentrations between individuals. Thirty minutes after the start of the glucagon infusion the range of plasma cyclic AMP concentrations in the cirrhotic patients was 57-123 pmol/ml and in the healthy controls 34-342 pmol/ml. Throughout the study there was n o significant difference in cyclic AMP concentrations between the two groups.
DISCUSSION In agreement with the results of Davies et al. ( I ) we found no significant difference in plasma cyclic
REFERENCES I . Davies, T. F., Prudhoe, K. & Douglas, A. P.: Plasma cyclic adenosine-3',5'-monophosphate response to glucagon in patients with liver disease. Br Med J 1: 93 I, 1976. 2. Documenta Geigy: Scientific tables (ed. Diem and Lentner), p. 128.Geigy, Bade 1970. 3. Exton, J. H.,Lewis, S. B., Ho,R. J. & Park, C. R.: The role of cyclic AMP in the control of hepatic glucose production by glucagon and insulin. Adv Cyclic Nucleotide Res 1: 91, 1972. 4. Exton, J. H., Robison, G. A,, Sutherland. E. W. & Park. C. R.: Studies on the role of adenosine 3'3'-
monophosphate in the hepatic actions of glucagon and catecholamines. J Biol Chem 246:6166, 1971. 5. Van Itallie, T. B. & Bentley, W. B. A.: Glucagoninduced hyperglycaemiaas an index of liver function. J Clin Invest 34: 1730, 1955. 6. Megyesi, C., Samols, E. & Marks, V.: Glucose tolerance and diabetes in chronic liver disease. Lancet 2: 1051, 1967.
7. Strange, R. C. & Mjm, 0.D.: The sources of plasma cyclic AMP: studies in the rat using isoprenaline, nicotinic acid and glucagon. Eur J Clin Invest 5: 147, 1975. 8. Strange, R. C. & Percy-Robb, 1. W.: Hepatic clearance
of adenosine 3'5'-cyclic monophosphate from plasma in the rat. Biochem J 146: 509, 1975. 9. Strange, R. C., Rowe, M. J., Mjm, 0. D. & Oliver, M. F.: The effect of antilipolytic agents on cyclic AMP, free fatty acid and total catecholamine concentrations in plasma. Acta Med Scand 199:421, 1976.
