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Modulation of insulin secretion by hepatic vagotomy in cirrhotic rats
Physiology&Behavior,Vol. 53, pp. 521-525, 1993
0031-9384/93 $6.00 + .00 Copyright© 1993PergamonPressLtd.
Printed in the USA.
Modulation of Insulin Secretion by Hepatic Vagotomy in Cirrhotic Rats HIROSHI OKAZAKI, K A T S U A K I TANAKA, HAJIME NAGASE AND SHUJI INOUE 1
The Third Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236, Japan Received 17 August 1992 OKAZAKI, H., K. TANAKA, H. NAGASE AND S. INOUE. Modulation tfinsulin secretion by hepatic vagotomy in cirrhotic' rats. PHYSIOL BEHAV 53(3) 521-525, 1993.--There is evidence that glucose sensors and arginine sensors are present in the hepato-portal system and exert a reflex regulation of the pancreatic neuroendocrine system in normal rats. To investigate the function of these sensor systems in liver cirrhosis, we examined the effect of hepatic vagotomy on plasma glucose and insulin concentrations after intraperitoneal (IP) injection of glucose or L-argininein carbon tetrachloride (CCl4)-inducedcirrhotic rats. After IP glucose injection, hepatic vagotomy decreased plasma insulin with elevation of plasma glucose in control rats; however, in cirrhotic rats, this procedure did not affect either plasma glucose or insulin concentrations. After IP arginine injection, hepatic vagotomy increased plasma insulin and reduced plasma glucose in control rats, although in cirrhotic rats, this procedure did not affect plasma glucose but did increase plasma insulin concentrations. These results suggest that function of the glucose sensor in CCI4-inducedcirrhotic rats is disturbed, although that of the arginine sensor is retained. Glucose sensor
Arginine sensor
Hepaticvagotomy
THERE have been a number of reports that vagal neural efferents from the brain stem to the pancreatic islets alter the secretion of insulin and glucagon (12,24); however, afferents that might affect this system have received little attention. The hepatic glucose sensor system was reported to enhance pancreatic insulin secretion through the afferent vagus (9,14). Lee and Miller (9) found that intraperitoneal (IP) glucose administered to normal rats increased plasma glucose concentrations and decreased plasma insulin concentrations in hepatic-vagotomized animals more than in sham-vagotomized animals. We found that IP Larginine enhanced plasma insulin and glucagon concentrations more in hepatic-vagotomized rats than in sham-vagotomized rats (20). We also reported the existence of arginine sensors, which cause a reflex inhibition of pancreatic vagus nerve activity, and a reflex activation of pancreatic sympathetic nerve activity when arginine is injected into the portal vein (22). Arginine sensors, thus, appear to inhibit arginine-induced insulin and glucagon secretion via afferent vagal nerves, whereas glucose sensors enhance glucose-induced insulin secretion. The function of these sensor systems under pathophysiological conditions has been little studied, although Nagase et al. (13) reported that streptozotoccin-induced mildly diabetic rats with a reserve of insulin release have hyperfunction of the arginine sensors and normofunction of the glucose sensors. It is recognized that hepatic cirrhosis is often accompanied by impaired glucose tolerance with higher plasma levels of insulin and glucagon (8,23). Thus, the manner in which these hepatic
Insulin
Cirrhosis
glucose and arginine sensor systems work in a cirrhotic liver condition is a matter of interest. The present study was carried out to examine how these receptor systems function in CC14induced cirrhotic rats. METHOD
Animals Twelve-week-old male Sprague-Dawley rats, weighing 220240 g, were housed in an environment with a constant temperature (23 + 2°C) and a 12-h light-dark cycle. The rats were allowed free access to laboratory chow and water, except on the night before the experiment.
Induction of Liver Cirrhosis and Surgery Rats received a subcutaneous injection of mixed CC14 and olive oil (3 ml/kg b. wt., two times/week, 12-14 weeks). Control rats received a subcutaneous injection of olive oil alone under the same conditions. Three days before the experiment, hepatic vagotomy was performed under hexobarbital anesthesia (50 mg/kg b. wt.) as previously described (20). The hepatic branch of the vagus nerve clearly branches off the main vagal trunk a few mm proximal to the cardia. It was exposed and completely sectioned at the proximal end using a dissecting microscope. Sham vagotomy was conducted by the same surgical procedure, except for the
Requests for reprints should be addressed to Shuji Inoue, MD, The Third Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan.
521
522
OKAZAKI E'I .'\I.
FIG. 1, Representative light micrograph of liver after treatment with C C I 4 ()'(20, Stained Azan).
sectioning of the vagus nerve. After the hepatic and sham vagotomies, a polyethylene-10 catheter (Clay Adams Co., Parsippany, N J) was installed in a jugular vein, and the open end was exposed to the posterior of the neck through the subcutaneous tissue (18). After the cannulation, rats were returned to their cages and allowed free access to food and water. At the end of the experiments, accomplishment of hepatic vagotomy was checked in all rats using a binocular microscope. Liver cirrhosis was verified by histologic examinations.
Experimental Procedures Four groups of seven animals each were prepared: 1. 2. 3. 4.
a a a a
group group group group
of hepatic-vagotomized, cirrhotic rats, of sham-vagotomized, cirrhotic rats, of hepatic-vagotomized, normal rats, and of sham-vagotomized, normal rats (control).
Three days after the surgery, three experiments were performed under unanesthetized and unrestrained conditions after 16 h of overnight food deprivation.
Experiment I: The effect of hepatic vagotomy on plasma glucose and insulin concentrations after intraperitoneal glucose injection. Warmed (37°C) glucose solution (0.5 g/kg b. wt., 20%,
Immediately after each blood sampling, the same amount of heparinized blood was replaced to minimize the influence of blood volume depletion (2). The plasma was separated immediately and stored at - 8 0 ° C until the assay. After the experiment, the pancreas was removed from each rat and weighed. Pancreatic insulin content was measured in a specimen after acid-alcohol extraction (1).
,4 ssa)' Glucose was measured by a Beckman glucose autoanalyzer (Palo Alto, CA), employing the glucose oxidase method. Immunoreactive insulin was measured by the modified double antibody method of Hales and Randle, using rat insulin as a standard (insulin assay kit, Amersham, Tokyo, Japan) (7).
Statistical Analysis Data were statistically analyzed by two-way (treatments × rain) analysis of variance. When the F values proved significant, Student's t-test was employed to locate significances between groups. The level of significance was P < 0.05. RESULTS
w/v) was injected intraperitoneally. Five-tenths of I ml of blood was withdrawn from the jugular vein catheter before and 5, 10, 15, 30, and 60 min after the injection to measure plasma glucose and insulin, and was poured into chilled tubes, each containing 10 mM EDTA.
Histologic examination of the livers of CCl4-induced rats showed a thin fibrous band connected this organ with the portal triad-central vein or the portal triad-portal triad, or both, which is characteristic of cirrhosis (Fig. 1).
Experiment 2: The effect of hepatic vagotomy on plasma glucose and insulin concentrations after intraperitoneal L-arginine injection. Warmed L-arginine solution (1.0 g/kg b. vet., 25%, w/
Experiment I: Intraperitoneal Glucose Stimulation Test
v; pH 7.4) was injected intraperitoneally. Five-tenths of 1 ml of blood was withdrawn from the jugular vein catheter before and 5, 10, 15, 30, and 60 rain after the injection to measure plasma glucose and insulin, and poured into chilled tubes, each containing 10 mM EDTA.
There were no significant differences in body weight changes among the four experimental groups 3 days after the operations. The responses of plasma glucose and insulin to glucose stimulation are shown in Fig. 2. Plasma glucose and insulin concentrations after glucose stimulation were significantly higher in all samples in cirrhotic rats than those in normal rats (Figs, 2A, B).
HEPATIC SENSORS IN CIRRHOSIS In normal rats, plasma glucose concentrations were significantly higher at 5, 10, and 15 min, and plasma insulin concentrations were significantly lower at 5, 10, 15, 30, and 60 min in the hepatic-vagotomized rats than in the sham-vagotomized rats. There were no significant differences in the basal glucose and insulin concentrations between the two groups. In cirrhotic rats, there were no significant differences in either basal or glucosestimulated glucose and insulin concentrations between hepaticvagotomized and sham-vagotomized, cirrhotic rats. Integrated glucose and insulin concentrations during IP glucose stimulation are shown in Table 1. Both plasma glucose and insulin after the stimulation were significantly higher in cirrhotic rats than in normal rats. Hepatic vagotomy significantly decreased integrated insulin concentration with elevation of integrated glucose concentration in normal rats, although in cirrhotic rats it showed a similar tendency without significant difference.
523
A (mo/tq ] T**
250-
DISCUSSION
In this study, we confirmed our previous findings in normal rats that glucose stimulation in hepatic-vagotomized animals is followed by a decrease in circulating insulin level than in shamvagotomized rats, and that arginine stimulation in the former animals is followed by an increase in circulating insulin level than in the latter (13,20). The hepatic branch of the vagus nerve contains glucose-sensitive afferent fibers that sense portal glucose concentrations after glucose stimulation and send signals to the brain stem center (14), which then stimulates the activities of the vagal efferent fibers, resulting in a stimulation of insulin secretion (15,17). In contrast, arginine-sensitive afferent fibers in the hepatic vagus nerve sense portal arginine concentrations following arginine stimulation and send signals to the brain stem center, which then inhibits the activities of the vagal efferent fibers, resulting in an inhibition of pancreatic hormone secretion (20,22). Hepatic vagotomy interrupts this mechanism, and suppresses glucose-induced insulin secretion or enhances arglnineinduced insulin secretion from the pancreatic islets (20). These explanations are consistent with the electrophysiologlcalevidence that glucose- and arginine-sensors are present in the liver and that these sensors are responsible for a reflex modulation of pancreatic vagus nerve activity (14,22).
** **~"X-~,~.~.,x~
sharn-vagotomized control rats /x hel~tic-vagotomized control rats •sham-vagotomized LC rats • hepatic-vagotomized LC rats
200t~ 150-
O
~ 100O 50-
Experiment 2: Intraperitoneal Arginine Stimulation Test The response of plasma glucose and insulin to arglnine stimulation is shown in Fig. 3. Concentrations of plasma glucose and insulin following arginine stimulation showed no significant differences between cirrhotic and normal rats. In normal rats, plasma glucose concentrations were significantly lower at 5 and 10 min, and plasma insulin concentrations were significantly higher at 5, 10, and 15 rain in hepatic-vagotomized rats than in sham-vagotomized rats. In cirrhotic rats, plasma insulin in hepatic-vagotomized, cirrhotic rats resulted in a significant increase at 10, 15, 30, and 60 rain compared to the values in shamvagotomized, cirrhotic rats. There were no significant differences in either basal glucose concentrations or arginine-stimulated plasma glucose concentrations in these two groups. Integrated glucose and insulin concentrations after arginine stimulation showed no significant differences between cirrhotic and normal rats. In normal rats, hepatic vagotomy increased integrated insulin concentrations although significantly decreasing integrated glucose concentrations, whereas in cirrhotic rats, it increased integrated insulin concentrations, but did not affect integrated glucose concentrations (Table 1). There were no significant differences in pancreatic insulin content among the four experimental groups.
0
6 5
1'01'5 3'0
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10-
ix
sham-vagotomized control rats hepatic-vagotomized control rats sham-vagotomized LC rats
• • hepatic-vagotomized LC rats 8"
** **
1i_
=-Iz4-6"~ * * ~ , Zm 2"
*
0
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*
5 1'0 |'5
3'0
TIME
6'0 minute
FIG. 2. Plasma glucose (A) and insulin (B) after IP glucose injection. *p < 0.05, **p < 0.01 compared to values at corresponding times in shamvagotomized control rats.
We further demonstrated in this study that I. the sectioning of the hepatic branch of the vagus nerve did not affect either plasma insulin or glucose concentration after glucose stimulation, and 2. this procedure enhanced plasma insulin concentration but not plasma glucose concentration after arginine stimulation in cirrhotic rats. The results indicated that function of the glucose-sensor is impaired, although function of the arglnine-sensor is retained. There are two possible explanations for the disturbance of function of the glucose sensor in these rats: 1. densitization of the sensor due to hyperglycemia or 2. blunted sensitivity of the sensor as a result of insulin resistance produced by hyperinsulinemia. Several abnormalities of the glucose metabolism have been described in liver cirrhosis, including impaired glucose tolerance
524
OkALZhl
and hyperinsulincmia (8.16). Creutzfeldt ct al. (5) previously reported that the overall incidence of diabetes mellitus in patients with cirrhosis varied between 7’R and 14%. Impaired glucose tolerance is also reportedly present in a large majoritv of cirrhotic patients-from 68% up to a high 92% (4.10). The cirrhotic rats in this study also showed higher glucose levels than normal rats. Thus, the hyperglycemia seen in cirrhotic rats appears to desensitize the glucose sensors that usually respond to glucose stimulation in the liver, in turn. disturbing the function of these sensors. The cause of hyperinsulinemia in cirrhosis may be increased insulin secretion or decreased insulin degradation, or a combination of the two (3.6). The presence of hepatic insulin resistance and impaired hepatic clearance of insulin would account for the hyperinsulinemia in cirrhotic rats treated with thioacetamide ( 19). Meyer-Alber et al. (I I) recently reported that the principal cause of hyperinsulinemia in rats with experimental cirrhosis appeared to be insulin resistance caused by a disturbance of insulin-stimulated glycogen formation in muscle tissues. Insulin resistance would, thus, blunt the hepatic sensor that responds to glucose, resulting in a disturbance of its function. The latter explanation. however. is unlikely because arginine-sensor mediated insulin secretion was retained in these rats. What role might these sensor systems play in the normal or cirrhotic state? Glucose is the most potent stimulant of insulin secretion among the major components of ingested carbohydrate foodstuffs. After the ingestion. glucose itself directly stimulates insulin release. resulting in a prevention of hyperglycemia. An excessive supply of glucose, however, could cause hyperglycemia. In normal rats. liver glucose-sensors sense an increase in portal glucose and send a signal to stimulate insulin release. thus. maintaining blood glucose homeostasis. However. in cirrhotic rats, elevated plasma glucose disturbs the function of the glucose sensor. and this may worsen hyperglycemia following carbohydrate intake. Arginine is also a potent stimulant and ingested as a component of protein foodstuffs. Following its ingestion. arginine
I I
\I
A (mg/dll
sham-vogotomlzed
250-
A hepatlc-vagotomued 0 sham-vagotomlzed A hepatlc-vagotomlzed
control
rats
control
rats
LC rats LC rats
200-
t8
150-
3 (3
loo-
>>
50-
O-
B .J sham-vagotomlzed
(r&ml1 lo-
A hepatac-vagotomized
control
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control
rats
0 sham-vagotomized
LC rats
A hepatx-vagotomued
LC
30
60 minute
rats
&
z
6-
$ rn z
42-
O-
TABLE
0
I
INTEGRATED GLUCOSE AND INSULIN RELEASE GLUCOSE OR ARGlNlNE STIMULATION IP Glucose
5
10 15
TIME
DURING
IP Arginine
FIG. 3. Plasma glucose (A) and insulin (B) after IP arginine injection. *p < 0.05, **p < 0.01 compared to values at corresponding times in sham-vagotomized control rats. tp < 0.05. $p < 0.01 differences between sham-vagotomized LC rats and hepatic-vagotomized LC rats.
Integrated glucose release (mg/dl min)* Control rats Sham vagotomy Hepatic vagotomy Cirrhotic rats Sham vagotomy Hepatic vagotomy integrated
7368.8 f 149.4 8049.6 + 249.7t
1659.2 t 541.6 6228.3 + 361.4t
9248.9 + 557.9$ 8856.3 k 457.3
6988.0 + 306. I 7136.0 + 181.5
insulin release (ng/ml
Control rats Sham vagotomy Hepatic vagotomy Cirrhotic rats Sham vagotomy Hepatic vagotomy Values are means + SE. *$ Compared to control rats,
min)*
146.3 ?I 95.7 *
4.5 10.5g
116.9 + 218.8 +
18.1 20.7t
255.8 f 183.0 +
23.4$ l8.6$
119.3 + 208.5 +
13.8 20.15
*p < 0.05, Sp < 0.0 I.
t$ Compared to sham-vagotomized rats. tp < 0.05. #P < 0.0 1.
directly stimulates insulin release from the pancreas; however, if present in an excessive quantity, arginine could cause exaggerated insulin release, In normal animals, liver arginine-sensors sense an increase in portal arginine and send a signal to prevent this exaggerated insulin secretion, thus maintaining blood glucose homeostasis. Similar to normal rats, function of the arginine sensor is preserved, resulting in the suppression of an overabundant release of insulin following protein intake in cirrhotic rats. Hepatic vagotomy did not affect plasma glucose concentration after arginine stimulation in cirrhotic rats, in contrast to the situation in normal rats. The insulin resistance present may explain this difference (I I). In summary, postprandial hyperglycemia may impair the glucose sensor system more than the arginine sensor system in cirrhotic rats. ACKNOWLEDGEMEN
This work was supported partly by the Nissin Seifun Foundation.
H E P A T I C SENSORS IN C I R R H O S I S
525
REFERENCES 1. Becker, K. L.; Sha, N. Pancreatic insulin content of nonpregnant, pregnant and postpartum rats and the developing rat fetus. Diabetes 18:268-272; 1969. 2. Berthoud, H. R.; Laughton, W. B.; Powley, T. L. A method for large volume blood sampling and transfusion in rats. Am. J. Physiol. 250: E331-E337; 1986. 3. Cavallo-Perin, P.; Bruno, A.; Nuccio, P.; Goria, M.; Pagano, G.; Lenti, G. Feedback inhibition of insulin secretion is altered in cirrhosis. J. Clin. Endocrinol. Metab. 63:1023-1027; 1986. 4. Collins, J. R.; Crofford, O. B. Glucose intolerance and insulin resistance in patients with liver disease. Arch. Int. Med. 124:142-148; 1969. 5. Creutzfeldt, W.; Frerichs, H.; Sickinger, K. Liver disease and diabetes mellitus. In: Popper H.; Schaffner F., eds. Progress in liver diseases. vol Ill. New York: Grune and Stratton; 1970:371-407. 6. Feingold, K. R.; Siperstein, M. D. Abnormalities of glucose metabolism in liver disease: In: Zakim, D.; Boyer, T. D., eds. Hepatology. Philadelphia: Saunders Co.; 1982:499-508. 7. Hales, C. N.; Randle, P. Immunoassay of insulin with antibody precipitate. Biochem. J. 90:620-624; 1963. 8. Johnston, D. G.; Arberti, K. G. M. M.; Faber, O. K.; Binder, C.; Wright, L. Hyperinsulinism of hepatic cirrhosis: Diminished degradation or hypersecretion? Lancet l : l 0-13; 1977. 9. Lee, K. C.; Miller, R. E. The hepatic vagus nerve and the neural regulation of insulin secretion. Endocrinology 117:307-314; 1985. l 0. Megyesi, C.; Samols, E.; Marks, V. Glucose tolerance and diabetes in chronic liver disease: Lancet 2:1051-1056; 1967. I1. Meyer-Alber, A.; Hartmann, H.; Stumpel, F.; Creutzfeldt, W. Mechanism of insulin resistance in CC14-induced cirrhosis of rats. Gastroenterology 102:223-229; 1992. 12. Miller, R. E. Pancreatic neuroendocrinology: Peripheral neural mechanisms in the regulation of the islets of Langerhans. Endocr. Rev. 2:471-494; 1981. 13. Nagase, H.; Inoue, S.; Tanaka, K.; Saitoh, T.; Takamura, Y. Hyperresponse of insulin release to arginine in streptozotocin-induced di-
abetic rats with hepatic vagotomy. Endocrinol. Jpn. 37:545-553; 1990. 14. Niijima, A. Glucose-sensitive afferent nerve fibers in the liver and regulation of blood glucose. Brain Res. Bull. (Suppl. 4):175-179; 1980. 15. Niijima, A. Nervous regulatory mechanism of blood glucose levels. In: Katsuki, Y.; Sato, M.; Takagi, S. F.; Oomura, Y., eds. Food intake and chemical senses. Tokyo: Japan Scientific Societies Press; 1977:413-416. 16. Riggio, 04 Merli, M.; Cangiano, C.; Capocaccia, R.; Cascino, A.; Lala, A.; Leonotti, F.; Mauceri, M.; Pepe, M.; Rossi Fanelli, F.; Savioli, M.; Tamburrano, G.; Capocaccia, L. Glucose intolerance in liver cirrhosis. Metabolism 31:627-634; 1982. 17. Sakaguchi, T.; Yamaguchi, K. Effect of electrical stimulation of the hepatic vagus nerve on the plasma insulin concentration in the rat. Brain Res. 164:314-316; 1979. 18. Shah, J. H.; Wongsurawat, N.; Aran, P. P.; Motto, G. S.; Bowser, E. N. A method for studying acute insulin secretion and glucose tolerance in unanesthetized rats. Diabetes 26:1-6; 1977. 19. Shankar, T. P.; Drake, S.; Solomon, S. S. Insulin resistance and delayed clearance of peptide hormones in cirrhotic rat liver. Am. J. Physiol. 252:E772-E777; 1987. 20. Tanaka, K.; Inoue, S.; Fujii, T.; Takamura, Y. Enhancement of insulin and glucagon secretion by arginine after hepatic vagotomy. Neurosci. Lett. 72:67-73; 1986. 21. Tanaka, K.; lnoue, S.; Nagase, H.; Takamura, Y. Modulation of arginine-induced insulin and glucagon secretion by the hepatic vagus nerves in the rats: Effects of celiac vagotomy and administration of atropine. Endocrinology 127:2017-2023; 1990. 22. Tanaka, K.; Inoue, S.; Takamura, Y.; Jiang, Z.-Y.; Niijima, A. Arginine sensors in the hepatoportal system and their reflex effect on pancreatic efferents in the rats. Neurosci. Lett. 72:67-73; 1986. 23. Taylor, R.; Heine, R. J.; Collins, J.; James, O. F. W.; Alberti, K. G. M. M. Insulin action in cirrhosis. Metabolism 5:64-71; 1985. 24. Woods, S. C.; Porte, D., Jr. Neural control of the endocrine pancreas. Physiol. Rev. 54:596-619; 1974.
0031-9384/93 $6.00 + .00 Copyright© 1993PergamonPressLtd.
Printed in the USA.
Modulation of Insulin Secretion by Hepatic Vagotomy in Cirrhotic Rats HIROSHI OKAZAKI, K A T S U A K I TANAKA, HAJIME NAGASE AND SHUJI INOUE 1
The Third Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236, Japan Received 17 August 1992 OKAZAKI, H., K. TANAKA, H. NAGASE AND S. INOUE. Modulation tfinsulin secretion by hepatic vagotomy in cirrhotic' rats. PHYSIOL BEHAV 53(3) 521-525, 1993.--There is evidence that glucose sensors and arginine sensors are present in the hepato-portal system and exert a reflex regulation of the pancreatic neuroendocrine system in normal rats. To investigate the function of these sensor systems in liver cirrhosis, we examined the effect of hepatic vagotomy on plasma glucose and insulin concentrations after intraperitoneal (IP) injection of glucose or L-argininein carbon tetrachloride (CCl4)-inducedcirrhotic rats. After IP glucose injection, hepatic vagotomy decreased plasma insulin with elevation of plasma glucose in control rats; however, in cirrhotic rats, this procedure did not affect either plasma glucose or insulin concentrations. After IP arginine injection, hepatic vagotomy increased plasma insulin and reduced plasma glucose in control rats, although in cirrhotic rats, this procedure did not affect plasma glucose but did increase plasma insulin concentrations. These results suggest that function of the glucose sensor in CCI4-inducedcirrhotic rats is disturbed, although that of the arginine sensor is retained. Glucose sensor
Arginine sensor
Hepaticvagotomy
THERE have been a number of reports that vagal neural efferents from the brain stem to the pancreatic islets alter the secretion of insulin and glucagon (12,24); however, afferents that might affect this system have received little attention. The hepatic glucose sensor system was reported to enhance pancreatic insulin secretion through the afferent vagus (9,14). Lee and Miller (9) found that intraperitoneal (IP) glucose administered to normal rats increased plasma glucose concentrations and decreased plasma insulin concentrations in hepatic-vagotomized animals more than in sham-vagotomized animals. We found that IP Larginine enhanced plasma insulin and glucagon concentrations more in hepatic-vagotomized rats than in sham-vagotomized rats (20). We also reported the existence of arginine sensors, which cause a reflex inhibition of pancreatic vagus nerve activity, and a reflex activation of pancreatic sympathetic nerve activity when arginine is injected into the portal vein (22). Arginine sensors, thus, appear to inhibit arginine-induced insulin and glucagon secretion via afferent vagal nerves, whereas glucose sensors enhance glucose-induced insulin secretion. The function of these sensor systems under pathophysiological conditions has been little studied, although Nagase et al. (13) reported that streptozotoccin-induced mildly diabetic rats with a reserve of insulin release have hyperfunction of the arginine sensors and normofunction of the glucose sensors. It is recognized that hepatic cirrhosis is often accompanied by impaired glucose tolerance with higher plasma levels of insulin and glucagon (8,23). Thus, the manner in which these hepatic
Insulin
Cirrhosis
glucose and arginine sensor systems work in a cirrhotic liver condition is a matter of interest. The present study was carried out to examine how these receptor systems function in CC14induced cirrhotic rats. METHOD
Animals Twelve-week-old male Sprague-Dawley rats, weighing 220240 g, were housed in an environment with a constant temperature (23 + 2°C) and a 12-h light-dark cycle. The rats were allowed free access to laboratory chow and water, except on the night before the experiment.
Induction of Liver Cirrhosis and Surgery Rats received a subcutaneous injection of mixed CC14 and olive oil (3 ml/kg b. wt., two times/week, 12-14 weeks). Control rats received a subcutaneous injection of olive oil alone under the same conditions. Three days before the experiment, hepatic vagotomy was performed under hexobarbital anesthesia (50 mg/kg b. wt.) as previously described (20). The hepatic branch of the vagus nerve clearly branches off the main vagal trunk a few mm proximal to the cardia. It was exposed and completely sectioned at the proximal end using a dissecting microscope. Sham vagotomy was conducted by the same surgical procedure, except for the
Requests for reprints should be addressed to Shuji Inoue, MD, The Third Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan.
521
522
OKAZAKI E'I .'\I.
FIG. 1, Representative light micrograph of liver after treatment with C C I 4 ()'(20, Stained Azan).
sectioning of the vagus nerve. After the hepatic and sham vagotomies, a polyethylene-10 catheter (Clay Adams Co., Parsippany, N J) was installed in a jugular vein, and the open end was exposed to the posterior of the neck through the subcutaneous tissue (18). After the cannulation, rats were returned to their cages and allowed free access to food and water. At the end of the experiments, accomplishment of hepatic vagotomy was checked in all rats using a binocular microscope. Liver cirrhosis was verified by histologic examinations.
Experimental Procedures Four groups of seven animals each were prepared: 1. 2. 3. 4.
a a a a
group group group group
of hepatic-vagotomized, cirrhotic rats, of sham-vagotomized, cirrhotic rats, of hepatic-vagotomized, normal rats, and of sham-vagotomized, normal rats (control).
Three days after the surgery, three experiments were performed under unanesthetized and unrestrained conditions after 16 h of overnight food deprivation.
Experiment I: The effect of hepatic vagotomy on plasma glucose and insulin concentrations after intraperitoneal glucose injection. Warmed (37°C) glucose solution (0.5 g/kg b. wt., 20%,
Immediately after each blood sampling, the same amount of heparinized blood was replaced to minimize the influence of blood volume depletion (2). The plasma was separated immediately and stored at - 8 0 ° C until the assay. After the experiment, the pancreas was removed from each rat and weighed. Pancreatic insulin content was measured in a specimen after acid-alcohol extraction (1).
,4 ssa)' Glucose was measured by a Beckman glucose autoanalyzer (Palo Alto, CA), employing the glucose oxidase method. Immunoreactive insulin was measured by the modified double antibody method of Hales and Randle, using rat insulin as a standard (insulin assay kit, Amersham, Tokyo, Japan) (7).
Statistical Analysis Data were statistically analyzed by two-way (treatments × rain) analysis of variance. When the F values proved significant, Student's t-test was employed to locate significances between groups. The level of significance was P < 0.05. RESULTS
w/v) was injected intraperitoneally. Five-tenths of I ml of blood was withdrawn from the jugular vein catheter before and 5, 10, 15, 30, and 60 min after the injection to measure plasma glucose and insulin, and was poured into chilled tubes, each containing 10 mM EDTA.
Histologic examination of the livers of CCl4-induced rats showed a thin fibrous band connected this organ with the portal triad-central vein or the portal triad-portal triad, or both, which is characteristic of cirrhosis (Fig. 1).
Experiment 2: The effect of hepatic vagotomy on plasma glucose and insulin concentrations after intraperitoneal L-arginine injection. Warmed L-arginine solution (1.0 g/kg b. vet., 25%, w/
Experiment I: Intraperitoneal Glucose Stimulation Test
v; pH 7.4) was injected intraperitoneally. Five-tenths of 1 ml of blood was withdrawn from the jugular vein catheter before and 5, 10, 15, 30, and 60 rain after the injection to measure plasma glucose and insulin, and poured into chilled tubes, each containing 10 mM EDTA.
There were no significant differences in body weight changes among the four experimental groups 3 days after the operations. The responses of plasma glucose and insulin to glucose stimulation are shown in Fig. 2. Plasma glucose and insulin concentrations after glucose stimulation were significantly higher in all samples in cirrhotic rats than those in normal rats (Figs, 2A, B).
HEPATIC SENSORS IN CIRRHOSIS In normal rats, plasma glucose concentrations were significantly higher at 5, 10, and 15 min, and plasma insulin concentrations were significantly lower at 5, 10, 15, 30, and 60 min in the hepatic-vagotomized rats than in the sham-vagotomized rats. There were no significant differences in the basal glucose and insulin concentrations between the two groups. In cirrhotic rats, there were no significant differences in either basal or glucosestimulated glucose and insulin concentrations between hepaticvagotomized and sham-vagotomized, cirrhotic rats. Integrated glucose and insulin concentrations during IP glucose stimulation are shown in Table 1. Both plasma glucose and insulin after the stimulation were significantly higher in cirrhotic rats than in normal rats. Hepatic vagotomy significantly decreased integrated insulin concentration with elevation of integrated glucose concentration in normal rats, although in cirrhotic rats it showed a similar tendency without significant difference.
523
A (mo/tq ] T**
250-
DISCUSSION
In this study, we confirmed our previous findings in normal rats that glucose stimulation in hepatic-vagotomized animals is followed by a decrease in circulating insulin level than in shamvagotomized rats, and that arginine stimulation in the former animals is followed by an increase in circulating insulin level than in the latter (13,20). The hepatic branch of the vagus nerve contains glucose-sensitive afferent fibers that sense portal glucose concentrations after glucose stimulation and send signals to the brain stem center (14), which then stimulates the activities of the vagal efferent fibers, resulting in a stimulation of insulin secretion (15,17). In contrast, arginine-sensitive afferent fibers in the hepatic vagus nerve sense portal arginine concentrations following arginine stimulation and send signals to the brain stem center, which then inhibits the activities of the vagal efferent fibers, resulting in an inhibition of pancreatic hormone secretion (20,22). Hepatic vagotomy interrupts this mechanism, and suppresses glucose-induced insulin secretion or enhances arglnineinduced insulin secretion from the pancreatic islets (20). These explanations are consistent with the electrophysiologlcalevidence that glucose- and arginine-sensors are present in the liver and that these sensors are responsible for a reflex modulation of pancreatic vagus nerve activity (14,22).
** **~"X-~,~.~.,x~
sharn-vagotomized control rats /x hel~tic-vagotomized control rats •sham-vagotomized LC rats • hepatic-vagotomized LC rats
200t~ 150-
O
~ 100O 50-
Experiment 2: Intraperitoneal Arginine Stimulation Test The response of plasma glucose and insulin to arglnine stimulation is shown in Fig. 3. Concentrations of plasma glucose and insulin following arginine stimulation showed no significant differences between cirrhotic and normal rats. In normal rats, plasma glucose concentrations were significantly lower at 5 and 10 min, and plasma insulin concentrations were significantly higher at 5, 10, and 15 rain in hepatic-vagotomized rats than in sham-vagotomized rats. In cirrhotic rats, plasma insulin in hepatic-vagotomized, cirrhotic rats resulted in a significant increase at 10, 15, 30, and 60 rain compared to the values in shamvagotomized, cirrhotic rats. There were no significant differences in either basal glucose concentrations or arginine-stimulated plasma glucose concentrations in these two groups. Integrated glucose and insulin concentrations after arginine stimulation showed no significant differences between cirrhotic and normal rats. In normal rats, hepatic vagotomy increased integrated insulin concentrations although significantly decreasing integrated glucose concentrations, whereas in cirrhotic rats, it increased integrated insulin concentrations, but did not affect integrated glucose concentrations (Table 1). There were no significant differences in pancreatic insulin content among the four experimental groups.
0
6 5
1'01'5 3'0
6'0minute B
[ng/mlJ
0
10-
ix
sham-vagotomized control rats hepatic-vagotomized control rats sham-vagotomized LC rats
• • hepatic-vagotomized LC rats 8"
** **
1i_
=-Iz4-6"~ * * ~ , Zm 2"
*
0
()
*
5 1'0 |'5
3'0
TIME
6'0 minute
FIG. 2. Plasma glucose (A) and insulin (B) after IP glucose injection. *p < 0.05, **p < 0.01 compared to values at corresponding times in shamvagotomized control rats.
We further demonstrated in this study that I. the sectioning of the hepatic branch of the vagus nerve did not affect either plasma insulin or glucose concentration after glucose stimulation, and 2. this procedure enhanced plasma insulin concentration but not plasma glucose concentration after arginine stimulation in cirrhotic rats. The results indicated that function of the glucose-sensor is impaired, although function of the arglnine-sensor is retained. There are two possible explanations for the disturbance of function of the glucose sensor in these rats: 1. densitization of the sensor due to hyperglycemia or 2. blunted sensitivity of the sensor as a result of insulin resistance produced by hyperinsulinemia. Several abnormalities of the glucose metabolism have been described in liver cirrhosis, including impaired glucose tolerance
524
OkALZhl
and hyperinsulincmia (8.16). Creutzfeldt ct al. (5) previously reported that the overall incidence of diabetes mellitus in patients with cirrhosis varied between 7’R and 14%. Impaired glucose tolerance is also reportedly present in a large majoritv of cirrhotic patients-from 68% up to a high 92% (4.10). The cirrhotic rats in this study also showed higher glucose levels than normal rats. Thus, the hyperglycemia seen in cirrhotic rats appears to desensitize the glucose sensors that usually respond to glucose stimulation in the liver, in turn. disturbing the function of these sensors. The cause of hyperinsulinemia in cirrhosis may be increased insulin secretion or decreased insulin degradation, or a combination of the two (3.6). The presence of hepatic insulin resistance and impaired hepatic clearance of insulin would account for the hyperinsulinemia in cirrhotic rats treated with thioacetamide ( 19). Meyer-Alber et al. (I I) recently reported that the principal cause of hyperinsulinemia in rats with experimental cirrhosis appeared to be insulin resistance caused by a disturbance of insulin-stimulated glycogen formation in muscle tissues. Insulin resistance would, thus, blunt the hepatic sensor that responds to glucose, resulting in a disturbance of its function. The latter explanation. however. is unlikely because arginine-sensor mediated insulin secretion was retained in these rats. What role might these sensor systems play in the normal or cirrhotic state? Glucose is the most potent stimulant of insulin secretion among the major components of ingested carbohydrate foodstuffs. After the ingestion. glucose itself directly stimulates insulin release. resulting in a prevention of hyperglycemia. An excessive supply of glucose, however, could cause hyperglycemia. In normal rats. liver glucose-sensors sense an increase in portal glucose and send a signal to stimulate insulin release. thus. maintaining blood glucose homeostasis. However. in cirrhotic rats, elevated plasma glucose disturbs the function of the glucose sensor. and this may worsen hyperglycemia following carbohydrate intake. Arginine is also a potent stimulant and ingested as a component of protein foodstuffs. Following its ingestion. arginine
I I
\I
A (mg/dll
sham-vogotomlzed
250-
A hepatlc-vagotomued 0 sham-vagotomlzed A hepatlc-vagotomlzed
control
rats
control
rats
LC rats LC rats
200-
t8
150-
3 (3
loo-
>>
50-
O-
B .J sham-vagotomlzed
(r&ml1 lo-
A hepatac-vagotomized
control
rats
control
rats
0 sham-vagotomized
LC rats
A hepatx-vagotomued
LC
30
60 minute
rats
&
z
6-
$ rn z
42-
O-
TABLE
0
I
INTEGRATED GLUCOSE AND INSULIN RELEASE GLUCOSE OR ARGlNlNE STIMULATION IP Glucose
5
10 15
TIME
DURING
IP Arginine
FIG. 3. Plasma glucose (A) and insulin (B) after IP arginine injection. *p < 0.05, **p < 0.01 compared to values at corresponding times in sham-vagotomized control rats. tp < 0.05. $p < 0.01 differences between sham-vagotomized LC rats and hepatic-vagotomized LC rats.
Integrated glucose release (mg/dl min)* Control rats Sham vagotomy Hepatic vagotomy Cirrhotic rats Sham vagotomy Hepatic vagotomy integrated
7368.8 f 149.4 8049.6 + 249.7t
1659.2 t 541.6 6228.3 + 361.4t
9248.9 + 557.9$ 8856.3 k 457.3
6988.0 + 306. I 7136.0 + 181.5
insulin release (ng/ml
Control rats Sham vagotomy Hepatic vagotomy Cirrhotic rats Sham vagotomy Hepatic vagotomy Values are means + SE. *$ Compared to control rats,
min)*
146.3 ?I 95.7 *
4.5 10.5g
116.9 + 218.8 +
18.1 20.7t
255.8 f 183.0 +
23.4$ l8.6$
119.3 + 208.5 +
13.8 20.15
*p < 0.05, Sp < 0.0 I.
t$ Compared to sham-vagotomized rats. tp < 0.05. #P < 0.0 1.
directly stimulates insulin release from the pancreas; however, if present in an excessive quantity, arginine could cause exaggerated insulin release, In normal animals, liver arginine-sensors sense an increase in portal arginine and send a signal to prevent this exaggerated insulin secretion, thus maintaining blood glucose homeostasis. Similar to normal rats, function of the arginine sensor is preserved, resulting in the suppression of an overabundant release of insulin following protein intake in cirrhotic rats. Hepatic vagotomy did not affect plasma glucose concentration after arginine stimulation in cirrhotic rats, in contrast to the situation in normal rats. The insulin resistance present may explain this difference (I I). In summary, postprandial hyperglycemia may impair the glucose sensor system more than the arginine sensor system in cirrhotic rats. ACKNOWLEDGEMEN
This work was supported partly by the Nissin Seifun Foundation.
H E P A T I C SENSORS IN C I R R H O S I S
525
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