Pancreatic endocrine function in cirrhotic rats

Pancreatic Takahiko

Nakamura,

Makoto

Endocrine

Function in Cirrhotic

Otsuki, Satoshi Tani, Yoshinori

Okabayashi,

Masatoshi

Rats

Fujii, Toru Oka, Takashi

Fujisawa,

and Shigeaki Baba Pancreatic endocrine function in liver cirrhosis was examined in rats both in vivo and in vitro. Experimental liver cirrhosis was induced by subcutaneous injections of 60% carbon tetrachloride in a dose of 2 mL/kg body weight twice a week for 16 weeks. Control rats received a similar dose of olive oil during the same period. In cirrhotic rats, immunoreactive insulin contents in the pancreas were significantly lower, whereas immunoreactive glucagon contents were about threefold higher than those of control rats. In the first part of this study, insulin and glucagon concentrations in both jugular and portal venous blood at basal conditions and after oral glucose loading were simultaneously determined in vivo. Peripheral insulin levels, both before and after glucose loading, were higher, whereas portal insulin concentrations were lower in cirrhotic rats than in the control rats. In contrast, glucagon levels in both the peripheral and portal veins were significantly higher in cirrhotic rats than in control rats. In the second part, isolated perfused pencreata were prepared from cirrhotic and control rats to further characterize the endocrine function of cirrhotic rat pancreas. Insulin secretion in response to 16.7 mmol/L glucose and 1 DO pmol/L cholecystokinin-octapeptide both were 40% lower in cirrhotic rats than in controls. In contrast, there was no significant difference in arginine-stimulated insulin release between the two groups. However, glucagon secretion in response to 20 mmol/L arginine was 40% higher in cirrhotic rats. If sensitivity is defined as the hormone release proportional to the pancreatic contents, then A and B cells in the cirrhotic rats had normal sensitivity to both glucose and cholecystokinin-octapeptide. In contrast, the responsiveness of B cells to arginine was greatly increased in cirrhotic rats, although that of A cells remained normal. These results suggest that the abnormal pancreatic endocrine secretory function in liver cirrhosis might be due mainly to alterations in the pancreatic hormone contents rather than an altered hormone release mechanism. o 1988 by Grune & Stratton, inc.

T

HE LIVER is the major site of degradation

of insulin and of its action. A consistent finding in patients with liver cirrhosis is diminished tolerance to oral or intravenous (IV) glucose associated with an absolute or relative increase in peripheral insulin concentrations.‘-5 That the hyperinsulinemia does not merely result from hyperglycemia is suggested by the presence of fasting hyperinsulinemia when blood glucose is normal.6 Until recently, the mechanism of this hyperinsulinemia has been obscure. Diminished insulin degradation by the diseased liver, with insulin resistance as an independent or secondary phenomenon, is the most likely explanation.‘-’ Approximately 60% of the insulin secreted by the pancreas is removed in the first transhepatic passage.‘oS” Diminished degradation by the diseased liver could therefore be a major factor in the hyperinsulinemia of liver disease. The liver also contributes to glucagon removal,‘2 although to a considerably lesser degree. Hepatic removal of insulin and of glucagon is important for the control of the circulating concentrations of these hormones. Impaired liver function could influence plasma glucagon levels as well as insulin concentrations independently of their secretions. It is not suprising, therefore, that changes observed peripherally do not reflect identical changes in pancreatic secretion. In the present study, insulin and glucagon concentrations in both portal and peripheral blood, before and after glucose loading, were determined in order to establish whether portal measurements could give more accurate information con-

cerning the pancreatic secretion of these hormones in rats rendered cirrhotic by carbon tetrachloride treatment. In addition, since liver cirrhosis is associated with hyperammonemia,” and since ammonia has been shown to influence insulin and glucagon secretion,‘c’6 we further examined the ability to glucose, cholecystokinin-octapeptide (CCK-I), and arginine to stimulate insulin and glucagon release in the isolated perfused pancreas prepared from cirrhotic rats to determine whether pancreatic endocrine function is irreversibly altered in the cirrhotic rats. MATERIALS

AND METHODS

Animals

Male Wistar rats initially weighing approximately 230 g were divided into two groups and injected subcutaneously with either 50% carbon tetrachloride (Ccl,: olive oil, 1:l; cirrhotic group) or olive oil alone (control group) in doses of 2 mL/kg body weight twice a week for 16 weeks.All injections were administered at 10 AM and the last injection was always administered 48 hours before the start of the experiment. The animals in both groups were kept at 23°C on a 12-hour light-dark cycle with free access to water and a standard rat chow (Oriental Yeast Co, Tokyo). Carbon tetrachloride produced the histological appearance of advanced micronodular cirrhosis (Fig 1, A) without apparent changes in the pancreas (Fig 1, B). On this evidence, we hereafter refer to the carbon-tetrachloride-treated rat as cirrhotic. Glucose Loading

From the Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku. Kobe, Japan. Address reprint requests to Makoto Otsuki, MD, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho. Chuo-ku, Kobe 650 Japan. o 1988 by Grune & Stratton, Inc. 0026-0495/88/3709-0015$03.00/0

892

Control and cirrhotic rats were subjected to an oral glucose load of 3 g/kg body weight 48 hours after the last injection of either olive oil or carbon tetrachloride solution. After a 1Chour fast, a glucose solution (50 g/100 mL) was administered via an orogastric tube under pentobarbital (50 mg/kg body weight) anesthesia, and blood samples for glucose, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG) determination were collected from the jugular and portal veins in microtest tubes containing 1.2 mg EDTA and Metabolism, Vol37,

No 9 (September), 1988: pp 892-899

893

LIVER CIRRHOSIS AND PANCREATIC FUNCTION

Fig 1. Light micrographs of the liver (A x 40) and the pancreas of 2 mL/kg body weight twice a week for 16 weeks.

500 kallidinogenase inactivator units (KIU) aprotinin (Bayer Yakuhin, Osaka, Japan) per 1.OmL of blood at 0,30, and 60 minutes. Pancreatic Endocrine Secretory

Studies

The isolated perfused rat pancreas was prepared as reported previously.” The perfusate used was a Krebs-Ringer bicarbonate (KRB) solution containing 4.6% Dextran T-70 (Pharmacia Fine Chemicals, Uppsala, Sweden), 0.25% bovine plasma albumin (fraction V; Armour Pharmaceutical Co, Phoenix), and 2.8 mmol/L glucose. It was gassed with 95% Or 5% CO2 and adjusted to pH 7.4. The inlets of the vascular perfusion were the superior mesenteric and celiac arteries, and the outlet was the portal vein. The flow rate through the pancreas was kept constant at 2.0 mL/min. After a single passage through the pancreas, the complete portal venous effluent was collected in chilled tubes at one-minute intervals for the measurement of IRI and IRG concentrations. After surgery, there was an equilibration period of 40 minutes before the start of an experiment. After a ten-minute basal period in the presence of 2.8 mmol/L glucose, the glucose concentration in the perfusate was changed to 16.7 mmol/L. Ten minutes later, CCK-8 (Squibb Institute for Medical Research, Princeton, NJ) was added for 20 minutes. CCK-8 was then removed, and the infusion was continued for an additional 20 minutes. In another experiment, but in a similar manner, 20 mmol/L arginine was added for ten minutes in the presence of 5.6 mmol/L glucose, and insulin and glucagon concentrations in the portal effluent were determined.

(6 x 100) in a rat given subcutaneous

injections of 50% Ccl, in a dose

Hormone and Enzyme Content After 16 weeks from the start of CCll injection, the rats were killed by decapitation; the whole pancreas was removed rapidly, freed from fat and lymph nodes, and weighed. A splenic portion of the pancreas (about 200 mg wet weight) was homogenized at 4°C in acid ethanol with a motor-driven glass-Teflon homogenizer at 2,000 rpm (eight passes). Insulin and glucagon were then extracted from this homogenate by a modification of the method of Davoren.‘* A portion of the pancreas (about 500 mg wet weight) was also homogenized in 0.15 mol/L NaCl solution in a similar manner. The aqueous phase was used to determine protein and DNA concentrations, and amylase, lipase, and trypsin activity. Assay Serum glucose concentrations were measured by the glucose oxidase method with the Glucose-E Reagent kit (International Reagents Co, Kobe, Japan). SGOT and SGPT levels in the serum were determined by an enzymatic method using VX-1000 (Nihon Denshi Co Ltd. Tokyo). Blood ammonia concentrations were measured by the Amitest-N (Daiichi Kagaku Co, Kyoto, Japan). IRI was measured by polyethylene-glycol radioimmunoassay using crystalline rat insulin as a reference standard.” IRG was measured using radioimmunoassay kit (Glucagon kit Daiichi; Daiichi Radioisotope Laboratory Ltd, Tokyo). Protein was determined by the method of Lowry et al*’ with bovine plasma albumin as a standard. DNA was

894

NAKAMURA

ET AL

Table 1. Body Weight, Pancreatic Wet Weight. Pancreatic Protein, DNA, Enzyme, Insulin, and Glucagon Content, and Laboratory Data in Cirrhotic and Control Rats LiverCkrhcsisfn - 7)

Controlfn - 5)

P vallle

Body weight fg) Initial

222.1

i 3.6

222.5

f 6.5

Final

367.8

f 10.5

470.0

f

NS

14.1

,001

Pancreatic wet weight (a per rat)

1.58 f 0.06

(mg/ 100 g body weight)

453.8

1.48 f 0.07

NS

f 31.5

315.0

f

17.2

247.1

f 12.8

239.4

f

12.7

NS

5.42

f 0.26

5.97

f 0.52

NS

,001

Content in the pancreas Protein (mg) DNA (ma) Amylass (x 10’ SU)

154.8

i

12.3

157.8

f 8.2

Lipase (IU1

228.2

c 24.1

302.8

+ 16.3

Tvpsin (me)

20.4

f

Insulin (pg)

74.2

f 3.9

Glucagon (Rg)

1.8

12.4 f 121.6

7.4 f 0.5

NS .05

1.3

.OOl

+ 16.2

.Ol

2.1 + 0.6

,001

Laboratory data SGOT (KU/L)

640.4

f 54.0

131.4

SGPT (KU/L)

122.2

+ 12.3

23.4

Albumin (g/ 100 mL)

3.5 f 0.1

NH, (tie/ 100 mL)

223.4

f 21.8

f

12.8

.OOl

+ 3.4

.OOl

4.5 + 0.1

.OOl

100.8

f

12.7

.Ol

Values are the mean + SE of the number of rats indicated in parentheses.

measured fluorometrically by the reaction between 35diaminobenzoic acid and deoxyribose sugar using calf thymus DNA as a standard.*’ Amylase activity was determined by a chromogenic method with blue-dyed starch polymer (Amylase Test A, Shionogi Pharmaceutical Co Ltd, Osaka, Japan)** and expressed as Somogyi unit (Xl). Lipase activity was determined according to Whitakar using a-naphtyl palmitate as a substrate (DIACOLOR Lipase, Ono Pharmaceutical Ltd, Osaka, Japan) and expressed in International units.” Trypsinogen was determined as trypsin after activation with enterokinase (Sigma Chemical Co, St Louis) by the method of Erlanger et alz4 using a-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA: Sigma Chemical Co, St Louis) as a substrate.

Data Analysis Comparison of the difference between the mean values of the two groups of experiments was made by ANOVA. A difference with P < nglml

mg/lOOml

I

0

.05 was considered statistically significant. Results are expressed as mean * SE. RESULTS

Although the initial body weight was similar in both groups, the final body weights of cirrhotic rats were significantly lower than those of control rats. In spite of the reduced body-weight gain, the pancreatic wet weights, and pancreatic protein and DNA contents of the rats receiving Ccl, injections were not significantly different from those of the control group. Consequently, the injections of Ccl, led to an increase in the pancreatic wet weight per body weight (Table 1). Pancreatic content of amylase in cirrhotic rats was not significantly different from that in control rats. whereas

f@ml

*

,

30

60

BO

30 Time

60 (min)

c

0

30

60

Fig 2. Glucose (AL IRI (Bj, and IRG (Cj concentrations in jugular venous blood after an oral glucose load of 3.0 g/kg body weight in control (O----O. n = 5) and cirrhotic rats (M, n = 6). Each value represents the mean f BE. *A significant difference from the corresponding value in control rats. Plasma glucose concentrations of 150 and 300 mgl 100 mL are equivalent to 8.3 and 18.7 mmol/L, respectively.

895

LIVER CIRRHOSIS AND PANCREATIC FUNCTION

mg/lOOml

Fig 3. Glucose (A), IRI (B). and IRG (C) concentrations in portal venous blood after an oral glucose load of 3.0 g/kg body weight in control (O----O, n = 5) and cirrhotic rats (O--O, n = 5). Each value represents the mean * SE. *A significant difference from the corresponding value in control rats. Plasma glucose concentrations of 150 and 300 mgl 100 mL are equivalent to 8.3 and 16.7 mmol/L.

12

300

9

200

6

100

n

“J A0

pg/ml

nglml

400

30

60

respectively.

pancreatic content of trypsin was significantly increased compared with that in control rats. In cirrhotic rats, pancreatic IRI contents were significantly lower, whereas pancreatic IRG contents were about three-fold higher than those of control rats (Table 1). Serum levels of SGOT and SGPT in cirrhotic rats were about five-fold higher than those in the control rats. Blood ammonia level had already increased 12 weeks after the start of CC& injection and was about twice as high as in the control rats at the time of the experiment (Table 1). Jugular and portal venous blood glucose concentrations, both before and after glucose loading, in cirrhotic rats were not significantly different from those in the control (Fig 2, A and Fig 3, A). The cirrhotic rats, however, had significantly higher peripheral IRI levels, both before and after glucose loading, and significantly lower portal IRI levels (Fig 2, B and Fig 3, B). On the other hand, cirrhotic rats had significantly higher IRG levels in both their jugular and portal veins (Fig 2, C and Fig 3, C). In the second set of experiments, isolated perfused pancreata were prepared from rats that received subcutaneous injections of CC& for 16 weeks to examine the pancreatic endocrine function directly. Figure 4 shows the time course of IRI secretion in response to 16.7 mmol/L glucose and 100 pmol/L CCK-8 from cirrhotic and control rats. Sharp rises in IRI release were observed after either increasing the glucose concentration or adding CCK-8. The peak and the cumulative IRI output over ten minutes (from 11 to 20 minutes) in response to 16.7 mmol/L glucose were lower by approximately 20% and 40%, respectively, in cirrhotic rats (peak, 7.1 + 0.7 ng/mL; cumulative output, 83.0 + 3.7 ng/ 10 min) compared with the control group (peak, 9.2 t_ 0.9 ng/mL, P < .Ol; cumulative output, 146.4 * 8.3 ng/lO min, P
600-

I

B 0

30 Time

60

I

b6

io

do

(min)

whereas that from the rats injected with Ccl, was only 321.1 + 19.9 ng/20 min (PC ,001). However, when IRI release was related to the pancreatic content, secretory ability of IRI in cirrhotic rat pancreas was similar to that in control rat pancreas. Figure 5 shows the time course of IRI and IRG release in response to 20 mmol/L arginine in the presence of 5.6 mmol/L glucose in cirrhotic and control rats. A sharp rise in IRI release was observed in the first one-minute sample after the addition of arginine. In contrast to IRI release in response to 8.3 mmol/L glucose and 100 pmol/L CCK-8 (Fig 4) there was no significant difference in argininestimulated IRI release between cirrhotic and control rats CCK-g(lOOpM)

15= E z

lo-

E 5-

1

0

10

20

30

40

50

Time (min) Fig 4. Time course of IRI release in response to 16.7 mmol/L glucose and 100 pmol/L CCK-B stimulation from isolated perfused pancreata prepared from control (O----O) and cirrhotic rats (O---O) that received subcutaneous injections of Ccl, for 16 weeks. Each value represents the mean * SE of four seperate experiments. *A significant difference from the corresponding value in control rats.

NAKAMURA

896

Arginine

(mM)

Glucose

(mM)

5.6

-

ET AL

-

h E ‘M 6

50

E

A

0 0 Time

(min)

(Fig 5, A). However, the peak and the ten-minute-cumulative IRG output (from six to 15 minutes) in response to arginine were higher by roughly 60% and 90%, respectively, in the cirrhotic rats (peak, 129 1.O + 218.4 pg/mL; cumulative output, 3.5 + 0.4 ng/ 10 min) compared with the control rats (peaks, 788.1 * 124.8 pg/mL, P -c.Ol; cumulative output, 1.8 + 0.4 ng/lO min, P -c.Ol). However, when related to total pancreatic hormone content, the pancreata from cirrhotic rats had normal secretory responsiveness of IRG to arginine and increased secretory responsiveness of IRI.

DISCUSSION

The present study has demonstrated that chronic treatment with Ccl, reduces the pancreatic content and secretion of insulin in rats, whereas it increases that of glucagon. While CC&-induced cirrhosis in rats is not completely analogous to cirrhosis in man, these results are in agreement with previous reports of altered secretory function of the endocrine pancreas in liver cirrhosis.‘-5,25-2*The present study extends these observations by demonstrating the persistent secretory alterations in isolated perfused pancreata from CC&-treated rats. Carbon tetrachloride is a frequently used hepatotoxin to induce liver cirrhosis in experimental animals, but it also affects other organs as we11.29.30 Although a possible direct effect of CC& on pancreatic A and B cells cannot be ruled out, histology of the pancreas by light microscopic examination appeared to be normal. Furthermore, despite the reduced body weight gain, the pancreatic wet weights and pancreatic protein contents of the cirrhotic rats were not significantly different from those of the control group.

5

10 Time

(min)

15

20

Fig 5. Time course of IRI (A) and IRG lB) release in response to 20 mmol/L srginine from isolated perfused pancreas prepared from control (O----O) and cirrhotic rats W--W that received subcutaneous injections of Ccl, for 16 weeks. Each value represents the mean f SE of four separate experiments. *A significant diiference from the corresponding value in control rats.

The following mechanisms could provide a plausible explanation for the increased glucagon and the decreased insulin contents in the pancreata of CC&-induced cirrhotic rats: (1) CC& may have a direct effect on the structure and function of islets. (2) Since pancreatic trypsin content was increased in cirrhotic rats, there is a possibility that trypsin released by acinar cells may damage the more vulnerable components of islet-cell plasma membranes. This mechanism is supported by the observation of selective impairment of islet secretory function after treatment with agents that inflict mild damage on membranes.3’ (3) Gastrointestinal (GI) hormones may affect pancreatic hormone contents. Previous study has demonstrated that repeated in vivo injections with caerulein (a synthetic analogue of CCK-8) and secretin increase pancreatic glucagon contents in the rat.32 Circulating plasma concentrations of CCK are supposed to be elevated in cirrhotic rats due to diminished hepatic elimination.33T34This hypothesis is supported by the following observations in the present study: In spite of the reduced body weight gain, the pancreatic wet weights of the cirrhotic rats were similar to those of control rats, resulting in significant increases in the pancreatic wet weight per body weight. This may suggest pancreatic hypertrophy and/or hyperplasia. In addition, pancreatic trypsin content of the cirrhotic rats was significantly greater than that of the control group. These alterations of pancreatic weight and enzyme contents in cirrhotic rats are completely analogous to those in rats treated with either repeated injections of CCK-835J6 or with chronic oral trypsin inhibitor to induce endogenous CCK release.37*3*(4) Hyperammonemia due to decreased conversion of enteric ammonia to urea, or due to the entry of ammonia into the systemic circulation via portal-systemic

LIVER CIRRHOSIS AND PANCREATIC

FUNCTION

shunting, may damage vulnerable components of B cells. Ammonia is shown to accumulate in islets under a hyperammonemic situation.*’ In addition, ammonia is known to decrease the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) in islet cells,28 to affect the efflux of K’ and Ca*+ from islets,*’ and to increase intracellular pH (PH~).‘~,~’We assume, therefore, that the observed changes in pancreatic hormone content in cirrhotic rats are induced by direct effects of possibly increased GI hormones or by damage to vulnerable membrane components either directly, by pancreatic digestive enzymes, or indirectly, by increased plasma ammonia levels. Damage to the B cells could impair insulin biosynthesis and decrease insulin production, resulting in inadequate stores of hormone. Conversely, the increased pancreatic glucagon content probably reflects increased synthesis, although the possibility of decreased intracellular degradation cannot be ruled out. The liver is a major site of insulin degradation, and it is now well established that 40% to 60% of the insulin presented to the liver is removed during a single transhepatic passage.“*” Peripheral insulin concentrations, therefore, reflect a balance between insulin secretion and degradation. Our in vivo findings in cirrhotic rats of decreased portal insulin concentration and increased peripheral insulin levels are consistent with previous reports attributing the phenomenon to reduced removal of insulin by the diseased liver.7-9 Cirrhotic rats in the present study began to show increased plasma ammonia levels as early as 12 weeks after the start of Ccl, injection. Their pancreata, therefore, had been exposed to high levels of ammonia for at least 4 weeks before the experiment. Ammonia is shown to inhibit glucose-induced insulin secretion both in vivo in rats4’ and human,41 and in vitro in isolated islets.42 The presence of high levels of ammonia in the cirrhotic rat might account for the low levels of insulin release in portal vein following glucose ingestion. However, since the comparisons of peripheral and portal insulin levels were performed in vivo under pentobarbital anesthesia, and since anesthesia is known to affect GI motility43 and insulin secretion,44 possible effects of anesthesia on plasma insulin levels could not be ruled out. While insulin hyposecretion as well as hypodegradation are demonstrated in the present study in anesthetized cirrhotic rats, most recent studies in patients with liver cirrhosis have found significantly elevated fasting C-peptide levels, suggesting that hypersecretion is responsible for the peripheral hyperinsulinemia (for review, see reference 45). The impairment of insulin secretion persisted in isolated perfused pancreata from Ccl,-treated rats. It is unlikely that the ammonia remained even after the 40-minute equilibration period that preceded perfusion of isolated pancreata. Nonetheless, these pancreata had a persistent decrease in insulin release to glucose and CCK-8 stimulation. This suggests that the changes in insulin secretion in the cirrhotic rat are due not to ammonia, but to persisting alterations in the endocrine pancreas. It is known that alterations in membrane fluidity affects the availability of certain receptors for binding and effector systems.& Either Ccl, or ammonia may affect islet membranes through such a mechanism. Alternatively, the impaired insulin response to glucose

897

in cirrhotic rats, both in vivo and in vitro perfused pancreas, could be caused by destruction of vulnerable membrane components either by pancreatic digestive enzymes released in the course of the disease or by somatostatin released under possibly high plasma CCK levels.‘* The failure of glucose and CCK-8 to increase insulin output in pancreata from Ccl,-treated rat contrasts with the notable stimulatory ability of arginine. This may reflect either a difference in insulin receptor for glucose and arginine or an effect of the high concentration of arginine used. If sensitivity is defined as the hormone release proportional to the pancreatic content, then these cirrhotic rats had normal sensitivity to both glucose and CCK-8, and increased sensitivity to arginine. The reduced insulin secretion from the cirrhotic rat pancreas may be due merely to reduced content in the pancreas rather than impaired insulin release mechanism. The excessive glucagon response to glucose in vivo and the persistent increase in response to arginine in isolated perfused pancreas suggest that the hyperglucagonemia does not result merely from release of stored hormone from damaged A cells. The liver contributes to glucagon removal,‘* although the kidneys seem to be of major importance.47 Hepatic dysfunction could also influence plasma glucagon levels independent of its secretion. The half-life of immunoreactive glucagon in perfused normal rat liver has been shown to be 9.6 + 2.1 minutes, whereas that in cirrhotic liver is 25.0 + 7.1 minutes.48 Therefore, decreased hormonal degradation may be responsible for peripheral hyperglucagonemia in liver cirrhosis. In the present study, however, chronic treatment with Ccl, caused a significant enhancement of stimulated as well as basal glucagon secretion in both in vivo and in vitro isolated perfused pancreas. Thus, the marked hyperglucagonemia may result not only from decreased hormonal degradation, but also from increased glucagon secretion. The mechanism for the marked hyperglucagonemia remains unclear. The observations of increased basal and poststimulation glucagon secretion in cirrhotic rats are consistent with previous reports of hyperglucagonemia both in patients with pancreatitis49,s0 and rats receiving ethionine.5’ It has been proposed that a lower-than-normal food intake or ammonia administration produces the elevation in circulating glucagon levels in humans and animals.‘4~‘5~49~5’ However, the mechanisms seem not to be responsible for the enhanced glucagon secretion by perfused pancreata in the present study. Rats were fed standard laboratory chow until before pancreatic isolation, and the isolated pancreata were perfused with fresh KRB solution for more than 40 minutes before the start of the experiment. There is a possibility that Ccl, acts on the liver and may suppress gluconeogenesis and decrease hepatic glycogen storage, causing a tendency towards hypoglycemia and hyperglucagonemia. Chronic Ccl4 treatment in vivo produced significant increases in both glucagon secretory response and islet glucagon content. Thus, the secretory responsiveness of glucagon was not significantly altered when related to the pancreatic content. Taken together, it is most likely that the increased glucagon secretion in liver cirrhosis is a reflection of the increased pancreatic content of the hormone rather than altered secretory mechanism.

NAKAMURA

This study has clearly demonstrated that, in liver cirrhosis, pancreatic content of insulin decreases, whereas that of glucagon increases. These changes in pancreatic hormone contents induce an alteration in the secretory function of endocrine pancreas. However the present results obtained in cirrhotic rats do not necessarily apply to the situation in humans, because liver cirrhosis induced in rats by Ccl4 is not completely comparable to the cirrhotic liver in man. REFERENCES

1. Samaan NA, Stone DB, Eckhardt RD: Serum glucose, insulin and growth hormone in chronic hepatic cirrhosis. Arch Intern Med 124:149-152, 1969 2. Berkowitz D: Glucose tolerance, free fatty acid, and serum insulin responses in patients with cirrhosis. Am J Dig Dis 14:691699,1969 3. Kasperska T, Lawecki J, Rogala H, et al: The behavior of insulinemia in patients with liver cirrhosis after intravenous administration of glucose, tolbutamide and glucagon. Diabetologia 7:391394,197l 4. Shurberg JL, Resnick RH, Ross E, et al: Serum lipid, insulin and glucagon after portacaval shunt in cirrhosis. Gastoenterology 72:301-304, 1977 5. Shankar TP, Solomon SS, Duckworth WC, et al: Studies of glucose intolerance in cirrhosis. J Lab Clin Med 102:459-469, 1983 6. Johnston DG, Alberti KGMM, Wright R, et al: C-peptide and insulin in liver disease. Diabetes 27:201-206, 1978 7. Johnston DC, Alberti KGMM, Faber OK, et al: Hyperinsulinemia of cirrhosis: Diminished degradation or hypersecretion? Lancet l:lO-13,1977 8. Iwasaki Y, Ohkubo A, Kajinuma H, et al: Degradation and secretion of insulin in hepatic cirrhosis. J Clin Endocrinol Metab 471774-779, 1978 9. Greco AV, Crucitti F, Chirlanda G, et al: Insulin and glucagon concentrations in portal and peripheral veins in patients with hepatic cirrhosis. Diabetologia 17:23-28, 1979 10. Kaden M, Harding P, Field JB: Effect of intraduodenal glucose administration on hepatic extraction of insulin in the anesthetized dog. J Clin Invest 54:2016-2028, 1973 11. Mondon CE, Olefsky JM, Dolkas CB, et al: Removal of insulin by perfused rat liver: Effect of concentration. Metabolism 24:153-160.1975 12. Jaspan JB, Huen AHJ, Morley CG, et al: The role of the liver in glucagon metabolism. J Clin Invest 60:421-428, 1977 13. Strombeck DR, Weiser MG, Kaneko JJ: Hyperammonemia and encephalopathy in the dog. J Am Vet Med Assoc 166:11051108,1975 14. Marchesini G, Zoli M, Dondi C, et al: Ammonia-induced changes in pancreatic hormones and plasma amino acids in patients with liver cirrhosis. Dig Dis Sci 27:406-412, 1982 15. Strombeck DR, Rogers QR, Stern JS: Effects of ammonia infusion on plasma glucagon, insulin and amino acids in intact pancreatectomized, and adrenalectomized dogs. Am J Vet Res 42:810-818, 1981 16. Smith JS, Pace CS: Modification of glucose-induced insulin release by alteration of pH. Diabetes 32:61-66, 1983 17. Otsuki M, Sakamoto C, Yuu H, et al: Discrepancies between the doses of cholecystokinin or caerulein-stimulating exocrine and endocrine response in perfused isolated rat pancreas. J Clin Invest 63:478-484, 1979 18. Davoren PR: The isolation of insulin from a single cat pancreas. Biochim Biophys Acta 63:150-153, 1962 19. Desbuquois B, Aurbach GD: Use of polyethyleneglycol to

ET AL

separate free and antibody-bound peptide hormones in radioimmunoassay. J Clin Endocrinol Metab 33:732-738, 1971 20. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with Folin phenol reagent. J Biol Chem 193:265-275, 1951 21. Thomas PS, Farquhar MN: Specific measurement of DNA in nuclei and nucleic acids using diaminobenzoic acid. Anal Biochem 89:35-44, 1978 22. Ceska M, Birath K, Brown B: A new and rapid method for the clinical determination of a-amylase activities in human serum and urine. Clin Chim Acta 26:437-444, 1969 23. Whitaker JF: A rapid and specific method for the determination of pancreatic lipase in serum and urine. Clin Chim Acta 44:133-138, 1973 24. Erlanger BF, Kokowaky N, Cohen W: The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271-278,196l 25. Marco J, Diego J, Villanueva ML, et al: Elevated plasma glucagon levels in cirrhosis of the liver. N Engl J Med 289:11071111,1973 26. Sherwin RS, Fisher M, Bessof J: Hyperglucagonemia in cirrhosis: Altered secretion and sensitivity to glucagon. Gastroenterology 74:1224-1228, 1978 27. Smith-Laing G, Orskov H, Gore MBR, et al: Hyperglucagonemia in cirrhosis, relationship to hepatccellular damage. Diabetologia 19:103-108, 1980 28. Kadabi UM, Eisenstein AB, Tucci J, et al: Hyperglucagonemia in hepatic cirrhosis: Its relation to hepatocellular dysfunction and normalization on recovery. Am J Gastroenterol 79:143-149, 1984 29. Cawthorne MA, Palmer ED, Bunyan J, et al: In vivo effects of carbon tetrachloride and chloroform on liver and kidney glucose6-phosphatase in mice. Biochem Pharmacol20:494-496, 197 1 30. Furukawa T, Yasumoto K, Inokuchi K: Pulmonary interstitial edema in experimental cirrhosis of the liver in rats. Eur Surg Res 16:366-371, 1984 31. Lambert AE, Henquin JC, Orci L, et al: Enzyme-induced modifications of beta cell function. I. Effect of pronase on insulin secretion. Eur J Clin Invest 4:459-468, 1974 32. Yamada T, Brunstedt J, Solomon T: Chronic effects of caerulein and secretion on the endocrine pancreas of the rats. Am J Physiol244:G541-G545, 1983 33. Kanayama S, Himeno S, Kurokawa M, et al: Marked prolongation in disappearance half-time of plasma cholecystokininoctapeptide in patients with hepatic cirrhosis. Am J Gastroenterol 80:557-560, 1985 34. Doyle JW, Wolfe MM, McGuigan JE: Hepatic clearance of gastrin and cholecystokinin peptides. Gastroenterology 87:60-68, 1984 35. Solomon TE, Petersen H, Elashoff J, et al: Interaction of caerulein and secretin on pancreatic size and composition in rat. Am J Physiol235:E714-E719, 1978 36. Folsch UR, Winckler K, Wormsley KG: Influence of repeated administration of cholecystokinin and secretin on the pancreas of the rat. Stand J Gastroenterol 13:663-67 1, 1978 37. Goke B, Pritz H, Koop I, et al: Endogenous CCK release and pancreatic growth in rats after feeding a proteinase inhibitor (camostate). Pancreas 1:509-5 15, 1986 38. Otsuki M, Ohki A, Okabayashi Y, et al: Effect of synthetic trypsin inhibitor camostate on pancreatic exocrine function in rats. Pancreas 2:164-169, 1987 39. Wanke E, Carbone E, Testa PL: K+ conductance modified by a titrable group accessible to protons from the intracellular side of the squid axon membrane. Biophys J 26:319-324, 1979 40. Schlienger JL, Imler M, Stahl J: Diabetogenic effect and

LIVER CIRRHOSIS AND PANCREATIC

FUNCTION

inhibition of insulin secretion induced in normal rats by ammonium infusions. Diabetologia 11:439-443, 1975 4 1. Schlienger JL, Imler M, Stahl J: Diminution de la tolerance glucosee et de l’insulino-secretion appres perfusion de sels d’ammonium chez I’homme normal et chez des malades attenits de steatose hepatique ou de cirrhose. Biol Gastroenterol7:101-110, 1974 42. Sener A, Hutton JC, Kawazu S, et al: The stimulus-secretion coupling of glucose-induced insulin release, metabolic and functional effects of NH,+ in rat islets. J Clin Invest 62:868-878,1978 43. Reynell PC, Spray GH: The effect of ether and pentobarbitone sodium on gastrointestinal function in the intact rat. Br J Pharmacol 12:104-106, 1957 44. Bailey CJ, Flatt PR: Insulin and glucagon during pentobarbitone anesthesia. Diabete Metab 6:91-95, 1980 45. Taylor R, Johnston DG, Alberti KGMM: Glucose homeostasis in cirrhotic liver disease. Clin Sci 70:317-320, 1986 46. Strittmatter WJ, Hirata F, Axelrod J: Phospholipid methyla-

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tion unmasks cryptic B-adrenergic receptors in rat reticulocytes. Science 204:1205-1207,1979 47. Lefebvre PJ, Luyckx AS: Effect of acute kidney exclusion of renal arteries on peripheral plasma glucagon levels and pancreatic glucagon production in the anesthetized dog. Metabolism 24:11691183,1975 48. Shankar TP, Drake S, Solomon SS: Insulin resistance and delayed clearance of peptide hormones in cirrhotic rat liver. Am J Physiol252:E772-E777, 1987 49. Donowitz M, Hender R, Spiro HM, et al: Glucagon secretion in acute and chronic pancreatitis. Ann Intern Med 83:778-781, 1975 50. Kalk WJ, Vink AI, Paul M, et al: Immunoreactive glucagon responses to intravenous tolbutamide in chronic pancreatitis. Diabetes 24:851-855, 1915 51. Axen KV, Pi-Sunyer FX: Altered insulin and glucagon secretion in perfused ethionine-treated rat pancreas. Am J Physiol 243:E505-E511,1982