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Glucose tolerance and gastric emptying in thyrotoxic rats
Glucose Tolerance and Gastric Emptying in Thyrotoxic Tadasu
Ikeda, Katsumi
Fujiyama,
Tazue Hoshino,
Tatsuo Takeuchi,
Masato
Tominaga,
Rats
and Hiroto Mashiba
To clarify the contribution of gastrointestinal function to impaired oral glucose tolerance in hyperthyroidism, gastric emptying rate and portal and peripheral blood glucose responses to intragastric or intraduodenal glucose administration were investigated in experimental thyrotoxic rats. Glucose absorption from perfused intestine of thyrotoxic rats was also examined. Thyrotoxicosis was induced by subcutaneous (SC) thyroxine injection (SO pg/kg/dj for seven days. In intragastric glucose tolerance test, although insulin and glucagon responses were not significantly altered, increments in portal and peripheral blood glucose were significantly higher in thyrotoxic rats than in controls at 30 minutes. This phenomenon was almost normalized by the preadministration of phentolamine (2 mg/kg SC). In intraduodenal glucose tolerance test, blood glucose, insulin, and glucagon responses were similar in thyrotoxic and control rats. Gastric emptying rate in thyrotoxic rats was significantly higher than that in controls at 30 minutes, and that was also normalized by phentolamine administration. Absorption of glucose from perfused intestine was similar in thyrotoxic and control rats. These results suggest that an altered glucose tolerance to intragastric glucose load in thyrotoxic rats may primarily be due to rapid gastric emptying induced by increased a-adrenoceptor responses, and that glucose absorption from small intestine was not increased in short-term thyrotoxic @ 1989 by W.8. Saunders Company.
rats.
I
T IS WELL KNOWN that hyperthyroid patients frequently exhibit impaired oral glucose tolerance.‘A3 The impaired glucose tolerance in hyperthyroidism might be produced by enhanced gastric emptying and increased intestinal absorption4 altered pancreatic secretion of insulinz~5~6 and glucagon,’ augmented hepatic glucose production,* and insulin resistance.‘,” Holdsworth and Besser4 have reported that rapid gastric emptying was associated with elevated peak blood glucose in patients with hyperthyroidism. However, the influence of gastric and intestinal function on glucose tolerance in hyperthyroidism has not been fully elucidated. To elucidate the contribution of gastrointestinal function to the abnormalities of glucose tolerance in hyperthyroidism, we investigated gastric emptying rate and portal and peripheral blood glucose responses to intragastric or intraduodenal glucose infusion in experimental thyrotoxic rats. Glucose absorption from perfused intestine of thyrotoxic rats was also investigated. MATERIALS AND METHODS
Materials Dextran T-70 was purchased from Green Cross Co, Tokyo. Bovine serum albumin (BSA, fraction V) and l-thyroxine (T,) were
obtained from Sigma Chemical Co, St Louis. Phentolamine (Regitin), a-adrenergic antagonist, was obtained from Ciba Geigy, Takarazuka, Japan. Animals and Induction of Thyrotoxicosis Male Wistar albino rats weighing approximately 100 g were used in the present study. The rats were divided into two groups: control and experimental. Thyroxine was dissolved in a small volume of O.OlN NaOH and brought to a concentration of 100 pg/mL with saline. The experimental rats were administered T, (50 ag/kg body
From the First Department of Internal Medicine, Tottori University School of Medicine, Yonago, Japan. Address reprint requests to Tadasu Ikeda, MD, First Department of Internal Medicine, Tottori University School of Medicine, Nishi-machi 36-1, Yonago 683, Japan. o 1989 by W.B. Saunders Company. 0026-0495/89/3809-0010$03.00/0 874
weight/d SC) for seven days. The control rats were injected vehicle alone. The rats were housed in an air conditioned room at 22 + 2°C with a lighting schedule of 12-hour light and 12-hour darkness, and were allowed free access to laboratory chow and tap water. The last injection was performed two hours before the experiments. After overnight fast, the experimental and control rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg) and the blood was drawn from the femoral vein to measure T,, T,, insulin, gastrin, glucagon, and secretin concentration. Then the rats were used in the following experiments. Intragastric or Intraduodenal Glucose Tolerance Test The abdomen was opened and polyethylene cannula was inserted into the stomach or duodenum to infuse glucose solution. Glucose (1.75 g/kg in 20% solution) was then administered into the stomach or duodenum. The blood was drawn from portal and tail vein at 30, 60, 120, and I80 minutes. Some of the thyrotoxic rats were administered SC phentolamine (2 mg/kg) 45 minutes before glucose administration, and an intragastric glucose tolerance test was performed. Perfusion of Rat Intestine A modification” of the method of Levin et al’* was used for isolation and perfusion of the rat intestine. The pylorus was ligated and transsected. The spleen was resected and the pancreas was removed from the duodenum with minimal trauma. The superior mesenteric artery and portal vein were cannulated. The entire preparation, consisting of duodenum, jejunum, ileum, and colon, was removed and placed in the perfusion chamber. Then a polyethylene tube was inserted into the duodenum to instillate glucose solution, and the cecum was incised to allow the removal of intestinal content. The isolated intestine was perfused without recirculation with a synthetic medium consisting of a Krebs-Ringer bicarbonate medium containing 0.5% BSA, 4.6% Dextran T-70, and 5.5 mmol/L glucose. During perfusion the perfusion medium and the chamber were warmed and kept at 37OC, and the medium was bubbled with a mixture of 95% 0, and 5% CO,. The pH level was maintained constant at 7.4. The flow rate was maintained at 6.0 mL/min. After basal perfusion for 15 minutes, glucose was intraluminally instillated (100 mg/min in 10% solution) for three minutes. Then the intestine was perfused for 30 minutes. The effluent was collected every minute for 30 minutes, and stored at -20°C until the time of glucose measurement. Intestinal glucose absorption was calculated by the following formula: (glucose concentration of effluent after luminal glucose instillation - glucoseconcenMetabolism, Vol38. No 9 Eeptember), 1989: pp 874-877
575
GLUCOSE TOLERANCE IN THYROTOXIC RATS
tration of effluent before luminal glucose instillation) x perfusion
mgldl
mgldl
volume. Gastric Emptying Rate
Nonabsorbable indocyanine green solution was administered into the stomach, and residual indocyanine green in the stomach was measured at 30, 60, 120, and 180 minutes. Disappearance rate of indocyanine green from the stomach was regarded as gastric emptying rate. Measurements
..
c ._
Glucose concentration was measured by glucose oxidase method.
012 >
T, and T, were assayed by respective commercial RIA kit. Insulin,‘3 glucagon,14gastrin,” and secretin16were measured by respective RIA.
~illoo
Analysis of Data
;ik m
:
m
0
0
60
120
160 mi”
Two-tailed Student’s unpaired t-test was used for statistical analysis. The data were expressed as the mean * SD. RESULTS
Body Weight and Plasma Concentrations of Various Hormones The body weight of thyrotoxic rats (114 + 10 g) was significantly (P < .OOl) lower than that (131 f 9 g) of
control rats. T, (9.2 f 2.1 pg/dL), and T, (359 + 105 ng/dL) levels in thyrotoxic rats were significantly (P < .OOl) higher than those (1.5 + 0.3 rg/dL and 104 f 18 ng/dL) in control rats, respectively. Peripheral and portal glucagon (79 + 16 pg/mL and 98 + 20 pg/mL) and secretin (250 * 30 pg/mL and 310 + 49 pg/mL) levels in thyrotoxic rats were similar to those (83 + 18 pg/mL and 101 + 19 pg/mL, and 240 f 25 pg/mL and 295 f 45 pg/mL) in control rats, respectively. Peripheral and portal gastrin level (249 f 59 pg/mL and 362 f 71 pg/mL) in thyrotoxic rats was significantly (P < .OOl) higher than that (178 * 35 pg/mL and 288 t 45 pg/mL) in control rats, respectively. Peripheral and portal insulin level (16 f 4 rU/mL and 3 1 f 7 pU/mL) in thyrotoxic rats was also significantly (P < .05) higher than that (8 + 2 MU/mL and 18 + 5 $J/mL) in controls, respectively.
Fig 1. Mood sugar (left panel) and increment in blood sugar (right panel) after intragastric glucose infusion tept. ABS, increment in blood sugar after intragartric glucose. (0). Control (n = 7). (0). Thyrotoxic fn = 71. (A), Phentolamine-treated thyrotoxic (n = 7). lP i .02. l*P < .Ol, l**P < Ml, significantly different from control rats. ‘P < .02, signiffcantly~different from thyrotoxic rats.
increments in peripheral and portal insulin after intragastric glucose were similar in three groups, and decrements in peripheral and portal glucagon after intragastric glucose were also similar in three groups. lntraduodenal Glucose Tolerance Test
As shown in Fig 3, fasting blood glucose in thyrotoxic rats was significantly higher than that in controls; however, blood glucose response to intraduodenal glucose in thyrotoxic rats was similar to that in controls. Similarly, increments in blood glucose after intraduodenal glucose in thyrotoxic rats were not significantly different from that in controls. As shown in Fig 4, response of insulin and glucagon after intraduodenal glucose in thyrotoxic rats was not significantly different from that in controls. 80
( tail I
vein)
T
Intragastric Glucose Tolerance Test
As shown in Fig 1, peripheral blood glucose levels in thyrotoxic rats were significantly higher than those in control rats at 0, 30, and 60 minutes, and portal blood glucose levels in thyrotoxic rats were also significantly higher than those in control rats at 0 and 30 minutes. Peripheral and portal blood glucose levels in phentolamine-treated thyrotoxic rats were significantly higher than those in controls at 0 minutes. Peripheral and portal blood glucose responses after intragastric glucose infusion were similar in phentolamine-treated thyrotoxic and control rats, and those in phentolaminetreated thyrotoxic rats were significantly lower than those in thyrotoxic rats at 30 minutes. Increments in peripheral and portal blood glucose after intragastric glucose in thyrotoxic rats were significantly higher than those in controls at 30 minutes. Those in phentolamine-treated thyrotoxic rats were significantly lower than those in thyrotoxic rats at 30 min. As shown in Fig 2,
-1001 . 0
1
60
120
180
Fig 2. Insulin and glucagon response after intragastric glucose infusion test; (left panel), peripheral blood; fright1panel), portal blood. AIRI, increment in plasma insulin. AIRG.I increment in plasma glucagon. (0). Control fn = 7). (0). Thyrotoxic In = 7). (A), Phentolamine-treated thyrotoxic fn = 7).
876
IKEDA ET AL
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0. 300'
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120
As shown in Fig 5, gastric emptying rate (65% k 7%) in thyrotoxic rats was significantly higher than that (48% + 7%) in control rats at 30 minutes. That (52% + 7%) in phentolamine-treated thyrotoxic rats was significantly lower than that in thyrotoxic rats at 30 minutes. Glucose Absorption From Perfused Rat Intestine As shown in Fig 6, basal effluent glucose concentration was similar in both groups. Effluent glucose concentration was increased by intraduodenal glucose instillation, and the increase in glucose concentration during perfusion was similar in thyrotoxic and control rats. Glucose absorption (39 k 11 mg/30 min) in thyrotoxic rats was slightly but not significantly lower than that (48 + 6 mg/30 min) in control rats. DISCUSSION
In the present thyrotoxic rats, blood glucose, insulin, and glucagon responses to intraduodenal glucose administration (tail
f@::
vein)
120
ti
f::
9
60
min
min
Gastric Emptying Rate
t
T( portal
vein)
60
120
180
120
160
Fig 3. Blood sugar (left panel) and increment in blood sugar fright panel) after intraduodenal glucose infusion teat. ( 0). Control In = 7). 101, Thyrotoxic fn = 7). lP -z .02, significantly different from control.
100 t
.
0
Fig 6. Gastric emptying rate. (0). Control (n = 71. (0). Thyrotoxic (n = 7). (A), Phentolamine-treated thyrotoxic (n = 7). lP < .06, significantly different from control rats. ‘P < .05. significantly different from thyrotoxic rats.
were not significantly different from those in controls, and glucose absorption from perfused small intestine of thyrotoxic rats was similar to that of controls, suggesting that glucose absorption from small intestine may not be increased in short-term thyrotoxic rats. Overnight fast is a considerable stress in the rat and causes loss of about 20% of the mucosa of the small intestine. It is possible that greater loss of mucosa could have occurred in the thyrotoxic than in the control animals. It is possible that more glucose was metabolized by the intestine in the thyrotoxic than the control. Thus, glucose absorption in thyrotoxic rats might have been underestimated in the present study. Peripheral and portal blood glucose responses after intragastric glucose infusion were significantly higher in thyrotoxic rats at 30 minutes, and gastric emptying rate in thyrotoxic rats was significantly higher than that in controls at 30 minutes. Insulin and glucagon responses after intragastric glucose were similar in thyrotoxic and control rats. These results suggest that higher blood glucose response after intragastric glucose in thyrotoxic rats may be due to rapid gastric emptying. The present
~glucose
1OOmglmin
*
.3 0
60
120
160 min
0
180 ml"
Fig 4. Insulin and glucagon response after intraduodenal glucose infusion test; (left panel), peripheral blood: fright panel). portal blood. AIRI, increment in plasma insulin. AIRG, increment in plasma glucagon. (0). Control fn = 7). (0). Thyrotoxic (n = 7).
.
0
,
30
15 min
Fig 6. Glucose absorption from perfused intestine. (0). Control (n = 7). (0). Thyrotoxic In = 7).
877
GLUCOSE TOLERANCE IN THYROTOXIC RATS
results agreed with the report of Holdsworth and Besser4 that rapid gastric emptying was associated with elevated blood glucose in hyperthyroid patients. The blood glucose levels are higher at 0, 30, and 60 minutes in the peripheral blood with hyperthyroidism, whereas in the portal blood the levels are higher at 0 and 30 minutes only. These indicate that glucose utilization may be slightly impaired in the periphery in thyrotoxic rats. Gastrin, glucagon, and secretin have been known to influence gastric emptying rate.‘7-20 Plasma glucagon and secretin concentrations in thyrotoxic rats were not significantly different from those in controls. However, plasma gastrin level in thyrotoxic rats was significantly higher than that in controls. This result was consistent with the report of Seino et al” that plasma gastrin levels are elevated in patients with hyperthyroidism. Although the mechanism by which hypergastrinemia occurs in thyrotoxic rats is unclear, gastrin may not contribute to rapid gastric emptying in the present study because gastrin is known to slow gastric emptying.‘“l’9 Basal plasma insulin levels in thyrotoxic rats were significantly higher than that in controls. However, there have been no reports that small amounts of insulin directly influence gastric emptying. Other intestinal hormones may be responsible for rapid gastric emptying in thyrotoxic rats. Further studies are necessary to elucidate
whether some intestinal hormones enhance gastric emptying in thyrotoxic rats. It has been well known that increased adrenoceptor response was observed in certain tissues in experimental thyrotoxic animals,22~23 and it has been reported that cu-adrenoceptors may be involved in the control of gastric emptying in the rat. 24 in the present study, phentolamine, cY-adrenoceptor antagonist administration did not alter insulin and glucagon response to glucose but normalized rapid gastric emptying and blood glucose responses after intragastric glucose infusion in thyrotoxic rats. Although we do not know whether rapid gastric emptying and impaired oral glucose tolerance in hyperthyroid patients are normalized by cu-adrenergic antagonist administration, rapid gastric emptying via ru-adrenergic stimulation may have an important role in glucose intolerance in short-term thyrotoxic rats. Increased adrenergic tone responsiveness in thyrotoxicosis are generally /3-adrenergic in nature.22*23 Further studies are necessary to elucidate more precisely the adrenergic modulation in hyperthyroidism. In summary, we conclude that an altered glu?ose tolerance to intragastric glucose in thyrotoxic rats may be due to rapid gastric emptying induced by increased a-adtienoceptor responses, and that glucose absorption from small intestine may not be increased in short-term thyrotoxic rats.
REFERENCES
1. Andreani D, Menziger G, Fallucca F, et al: Insulin levels in thyrotoxicosis and primary myxedema: Response to intravenous glucose and glucagon. Diabetologia 6:1-7, 1977 2. Anderson 00, Friis T, Ottessen B: Glucose intolerance and insulin secretion in hyperthyroidism. Acta Endocrinol 84:576-587, 1977 3. Moxon HR, Kreines KW, Goldsmith RE, et al: Long term observations of glucose tolerance in thyrotoxic patients. Arch Intern Med 135:1477-1480, 1975 4. Holdsworth CD, Besser GM: Influence of gastric emptyingrate and of insulin response on oral glucose tolerance in thyroid disease. Lancet 2:700-702, 1968 5. Malaisse W, Malaisse-Lagae F, McGraw E: Effects of thyroid function upon insulin secretion. Diabetes 16:643-646, 1967 6. Doar J, Stamp T, Wynn V, et al: Effects of oral and intravenous glucose loading in thyrotoxicosis: Studies of plasma glucose, free fatty acid, plasma insulin, and blood pyruvate levels. Diabetes 18:633-639, 1969 7. Kabadi U, Eisenstein A: Glucose intolerance in hyperthyroidism: Role of glucagon. J Clin Endocrinol Metab 50:392-396, 1980 8. Wennlund A, Felig P, Hagenfeldt L, et al: Hepatic glucose production and splanchnic glucose exchange in hyperthyroidism. J Clin Endocrinol Metab 62: 174- 179, 1986 9. Dimitriades G, Baker B, March H, et al: Effect of thyroid hormone excess on action, secretion and metabolism of insulin in humans. Am J Physiol248:E593-E601, 1985 10. Shen DC, Davidson MB: Graves disease causes insulin antagonism. J Clin Endocrinol Metab 60:1038-1041, 1985 11. Ikeda T, Mokuda 0, Kuno S, et al: Enhanced intestinal insulinotropic effect in streptozotocin-diabetic rats. Am J Physiol 248:E304-E308,1985 12. Levin SR, Pehlevanian MZ, Lavee AE, et al: Secretion of an
insulinotropic factor from isolated, perfused rat intestine. Am J Physiol236:E710-E720, 1979 13. Yalow RS, Berson SA: Immunoassay of endognous insulin in man. J Clin Invest 39:1157-l 175, 1960 14. Nishino T, Kodaira T, Shin S, et al: Glucagon mdioimmunoassay with use of antiserum to glucagon C-terminal fragment. Clin Chem27:1690-1697,198l 15. Yalow RS, Berson SA: Radioimmunoassay of gastrin. Gastroenterology 58:1-14, 1970 16. Yanaihara N, Sato H, Kubota M, et al: Radtoimmunoassay for secretin using N’-tyrosylsecretin and [Tyr’]]Tsecretin. Endocrinol Jpn 23:87-90, 1976 17. Hunt JN, Ramsbottom N: Effect of gastrin II on gastric emptying and secretion during a test meal. Br Med J 4:386-390, 1967 18. Dozois RR, Kelly KA: Effect of a gastrin pentapeptide on canine gastric emptying of liquids. Am J Physiol 22 1:113- 117, 1971 19. Johansson H, Segerstriim A: Glucagon and gastrointestinal motility in relation to thyroid-parathyroid function. Upsala J Med Sci77:183-188, 1972 20. Chey WY, Hitanant S, Hendricks J, et al: EtTect of secretin and cholecystokinin on gastric emptying and gastric secretion in man. Gastroenterology 58:820-827, 1970 21. Seino Y, Matsukura S, Miyamoto Y, et al: Hypergastrinemia in hyperthyroidism. J Clin Endocrinol Metab 43:8524855, 1976 22. Giudicelli Y: Thyroid hormone modulation of the number of @-adrenergic receptors in rat fat-cell membranes. Biochem J 176: 1007-l 1lo,1978 23. Ho K, Lloyd BL, Taylor RR: Cardiac @adrenoceptors in the thyroxine-treated dog. Clin Exp Pharmacol Physiol81183- 187, 198 1 24. Cooper SM, McRitchie B: Role of dopamine and a-adrenoceptors in the control of gastric emptying in the rat: Possible involvement in the mechanism of action of metoclopramide. J Auton Pharmacol 5:325-331, 1981
Ikeda, Katsumi
Fujiyama,
Tazue Hoshino,
Tatsuo Takeuchi,
Masato
Tominaga,
Rats
and Hiroto Mashiba
To clarify the contribution of gastrointestinal function to impaired oral glucose tolerance in hyperthyroidism, gastric emptying rate and portal and peripheral blood glucose responses to intragastric or intraduodenal glucose administration were investigated in experimental thyrotoxic rats. Glucose absorption from perfused intestine of thyrotoxic rats was also examined. Thyrotoxicosis was induced by subcutaneous (SC) thyroxine injection (SO pg/kg/dj for seven days. In intragastric glucose tolerance test, although insulin and glucagon responses were not significantly altered, increments in portal and peripheral blood glucose were significantly higher in thyrotoxic rats than in controls at 30 minutes. This phenomenon was almost normalized by the preadministration of phentolamine (2 mg/kg SC). In intraduodenal glucose tolerance test, blood glucose, insulin, and glucagon responses were similar in thyrotoxic and control rats. Gastric emptying rate in thyrotoxic rats was significantly higher than that in controls at 30 minutes, and that was also normalized by phentolamine administration. Absorption of glucose from perfused intestine was similar in thyrotoxic and control rats. These results suggest that an altered glucose tolerance to intragastric glucose load in thyrotoxic rats may primarily be due to rapid gastric emptying induced by increased a-adrenoceptor responses, and that glucose absorption from small intestine was not increased in short-term thyrotoxic @ 1989 by W.8. Saunders Company.
rats.
I
T IS WELL KNOWN that hyperthyroid patients frequently exhibit impaired oral glucose tolerance.‘A3 The impaired glucose tolerance in hyperthyroidism might be produced by enhanced gastric emptying and increased intestinal absorption4 altered pancreatic secretion of insulinz~5~6 and glucagon,’ augmented hepatic glucose production,* and insulin resistance.‘,” Holdsworth and Besser4 have reported that rapid gastric emptying was associated with elevated peak blood glucose in patients with hyperthyroidism. However, the influence of gastric and intestinal function on glucose tolerance in hyperthyroidism has not been fully elucidated. To elucidate the contribution of gastrointestinal function to the abnormalities of glucose tolerance in hyperthyroidism, we investigated gastric emptying rate and portal and peripheral blood glucose responses to intragastric or intraduodenal glucose infusion in experimental thyrotoxic rats. Glucose absorption from perfused intestine of thyrotoxic rats was also investigated. MATERIALS AND METHODS
Materials Dextran T-70 was purchased from Green Cross Co, Tokyo. Bovine serum albumin (BSA, fraction V) and l-thyroxine (T,) were
obtained from Sigma Chemical Co, St Louis. Phentolamine (Regitin), a-adrenergic antagonist, was obtained from Ciba Geigy, Takarazuka, Japan. Animals and Induction of Thyrotoxicosis Male Wistar albino rats weighing approximately 100 g were used in the present study. The rats were divided into two groups: control and experimental. Thyroxine was dissolved in a small volume of O.OlN NaOH and brought to a concentration of 100 pg/mL with saline. The experimental rats were administered T, (50 ag/kg body
From the First Department of Internal Medicine, Tottori University School of Medicine, Yonago, Japan. Address reprint requests to Tadasu Ikeda, MD, First Department of Internal Medicine, Tottori University School of Medicine, Nishi-machi 36-1, Yonago 683, Japan. o 1989 by W.B. Saunders Company. 0026-0495/89/3809-0010$03.00/0 874
weight/d SC) for seven days. The control rats were injected vehicle alone. The rats were housed in an air conditioned room at 22 + 2°C with a lighting schedule of 12-hour light and 12-hour darkness, and were allowed free access to laboratory chow and tap water. The last injection was performed two hours before the experiments. After overnight fast, the experimental and control rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg) and the blood was drawn from the femoral vein to measure T,, T,, insulin, gastrin, glucagon, and secretin concentration. Then the rats were used in the following experiments. Intragastric or Intraduodenal Glucose Tolerance Test The abdomen was opened and polyethylene cannula was inserted into the stomach or duodenum to infuse glucose solution. Glucose (1.75 g/kg in 20% solution) was then administered into the stomach or duodenum. The blood was drawn from portal and tail vein at 30, 60, 120, and I80 minutes. Some of the thyrotoxic rats were administered SC phentolamine (2 mg/kg) 45 minutes before glucose administration, and an intragastric glucose tolerance test was performed. Perfusion of Rat Intestine A modification” of the method of Levin et al’* was used for isolation and perfusion of the rat intestine. The pylorus was ligated and transsected. The spleen was resected and the pancreas was removed from the duodenum with minimal trauma. The superior mesenteric artery and portal vein were cannulated. The entire preparation, consisting of duodenum, jejunum, ileum, and colon, was removed and placed in the perfusion chamber. Then a polyethylene tube was inserted into the duodenum to instillate glucose solution, and the cecum was incised to allow the removal of intestinal content. The isolated intestine was perfused without recirculation with a synthetic medium consisting of a Krebs-Ringer bicarbonate medium containing 0.5% BSA, 4.6% Dextran T-70, and 5.5 mmol/L glucose. During perfusion the perfusion medium and the chamber were warmed and kept at 37OC, and the medium was bubbled with a mixture of 95% 0, and 5% CO,. The pH level was maintained constant at 7.4. The flow rate was maintained at 6.0 mL/min. After basal perfusion for 15 minutes, glucose was intraluminally instillated (100 mg/min in 10% solution) for three minutes. Then the intestine was perfused for 30 minutes. The effluent was collected every minute for 30 minutes, and stored at -20°C until the time of glucose measurement. Intestinal glucose absorption was calculated by the following formula: (glucose concentration of effluent after luminal glucose instillation - glucoseconcenMetabolism, Vol38. No 9 Eeptember), 1989: pp 874-877
575
GLUCOSE TOLERANCE IN THYROTOXIC RATS
tration of effluent before luminal glucose instillation) x perfusion
mgldl
mgldl
volume. Gastric Emptying Rate
Nonabsorbable indocyanine green solution was administered into the stomach, and residual indocyanine green in the stomach was measured at 30, 60, 120, and 180 minutes. Disappearance rate of indocyanine green from the stomach was regarded as gastric emptying rate. Measurements
..
c ._
Glucose concentration was measured by glucose oxidase method.
012 >
T, and T, were assayed by respective commercial RIA kit. Insulin,‘3 glucagon,14gastrin,” and secretin16were measured by respective RIA.
~illoo
Analysis of Data
;ik m
:
m
0
0
60
120
160 mi”
Two-tailed Student’s unpaired t-test was used for statistical analysis. The data were expressed as the mean * SD. RESULTS
Body Weight and Plasma Concentrations of Various Hormones The body weight of thyrotoxic rats (114 + 10 g) was significantly (P < .OOl) lower than that (131 f 9 g) of
control rats. T, (9.2 f 2.1 pg/dL), and T, (359 + 105 ng/dL) levels in thyrotoxic rats were significantly (P < .OOl) higher than those (1.5 + 0.3 rg/dL and 104 f 18 ng/dL) in control rats, respectively. Peripheral and portal glucagon (79 + 16 pg/mL and 98 + 20 pg/mL) and secretin (250 * 30 pg/mL and 310 + 49 pg/mL) levels in thyrotoxic rats were similar to those (83 + 18 pg/mL and 101 + 19 pg/mL, and 240 f 25 pg/mL and 295 f 45 pg/mL) in control rats, respectively. Peripheral and portal gastrin level (249 f 59 pg/mL and 362 f 71 pg/mL) in thyrotoxic rats was significantly (P < .OOl) higher than that (178 * 35 pg/mL and 288 t 45 pg/mL) in control rats, respectively. Peripheral and portal insulin level (16 f 4 rU/mL and 3 1 f 7 pU/mL) in thyrotoxic rats was also significantly (P < .05) higher than that (8 + 2 MU/mL and 18 + 5 $J/mL) in controls, respectively.
Fig 1. Mood sugar (left panel) and increment in blood sugar (right panel) after intragastric glucose infusion tept. ABS, increment in blood sugar after intragartric glucose. (0). Control (n = 7). (0). Thyrotoxic fn = 71. (A), Phentolamine-treated thyrotoxic (n = 7). lP i .02. l*P < .Ol, l**P < Ml, significantly different from control rats. ‘P < .02, signiffcantly~different from thyrotoxic rats.
increments in peripheral and portal insulin after intragastric glucose were similar in three groups, and decrements in peripheral and portal glucagon after intragastric glucose were also similar in three groups. lntraduodenal Glucose Tolerance Test
As shown in Fig 3, fasting blood glucose in thyrotoxic rats was significantly higher than that in controls; however, blood glucose response to intraduodenal glucose in thyrotoxic rats was similar to that in controls. Similarly, increments in blood glucose after intraduodenal glucose in thyrotoxic rats were not significantly different from that in controls. As shown in Fig 4, response of insulin and glucagon after intraduodenal glucose in thyrotoxic rats was not significantly different from that in controls. 80
( tail I
vein)
T
Intragastric Glucose Tolerance Test
As shown in Fig 1, peripheral blood glucose levels in thyrotoxic rats were significantly higher than those in control rats at 0, 30, and 60 minutes, and portal blood glucose levels in thyrotoxic rats were also significantly higher than those in control rats at 0 and 30 minutes. Peripheral and portal blood glucose levels in phentolamine-treated thyrotoxic rats were significantly higher than those in controls at 0 minutes. Peripheral and portal blood glucose responses after intragastric glucose infusion were similar in phentolamine-treated thyrotoxic and control rats, and those in phentolaminetreated thyrotoxic rats were significantly lower than those in thyrotoxic rats at 30 minutes. Increments in peripheral and portal blood glucose after intragastric glucose in thyrotoxic rats were significantly higher than those in controls at 30 minutes. Those in phentolamine-treated thyrotoxic rats were significantly lower than those in thyrotoxic rats at 30 min. As shown in Fig 2,
-1001 . 0
1
60
120
180
Fig 2. Insulin and glucagon response after intragastric glucose infusion test; (left panel), peripheral blood; fright1panel), portal blood. AIRI, increment in plasma insulin. AIRG.I increment in plasma glucagon. (0). Control fn = 7). (0). Thyrotoxic In = 7). (A), Phentolamine-treated thyrotoxic fn = 7).
876
IKEDA ET AL
mgldl -
100,
300'
%
.' : ZOO* z L ",lOQ. In m
0. 300'
E .; ZOO. >
jj 100. Lo m
o-
P h
50b
.
0
Ok 120
60
0
160 ml"
60
120
As shown in Fig 5, gastric emptying rate (65% k 7%) in thyrotoxic rats was significantly higher than that (48% + 7%) in control rats at 30 minutes. That (52% + 7%) in phentolamine-treated thyrotoxic rats was significantly lower than that in thyrotoxic rats at 30 minutes. Glucose Absorption From Perfused Rat Intestine As shown in Fig 6, basal effluent glucose concentration was similar in both groups. Effluent glucose concentration was increased by intraduodenal glucose instillation, and the increase in glucose concentration during perfusion was similar in thyrotoxic and control rats. Glucose absorption (39 k 11 mg/30 min) in thyrotoxic rats was slightly but not significantly lower than that (48 + 6 mg/30 min) in control rats. DISCUSSION
In the present thyrotoxic rats, blood glucose, insulin, and glucagon responses to intraduodenal glucose administration (tail
f@::
vein)
120
ti
f::
9
60
min
min
Gastric Emptying Rate
t
T( portal
vein)
60
120
180
120
160
Fig 3. Blood sugar (left panel) and increment in blood sugar fright panel) after intraduodenal glucose infusion teat. ( 0). Control In = 7). 101, Thyrotoxic fn = 7). lP -z .02, significantly different from control.
100 t
.
0
Fig 6. Gastric emptying rate. (0). Control (n = 71. (0). Thyrotoxic (n = 7). (A), Phentolamine-treated thyrotoxic (n = 7). lP < .06, significantly different from control rats. ‘P < .05. significantly different from thyrotoxic rats.
were not significantly different from those in controls, and glucose absorption from perfused small intestine of thyrotoxic rats was similar to that of controls, suggesting that glucose absorption from small intestine may not be increased in short-term thyrotoxic rats. Overnight fast is a considerable stress in the rat and causes loss of about 20% of the mucosa of the small intestine. It is possible that greater loss of mucosa could have occurred in the thyrotoxic than in the control animals. It is possible that more glucose was metabolized by the intestine in the thyrotoxic than the control. Thus, glucose absorption in thyrotoxic rats might have been underestimated in the present study. Peripheral and portal blood glucose responses after intragastric glucose infusion were significantly higher in thyrotoxic rats at 30 minutes, and gastric emptying rate in thyrotoxic rats was significantly higher than that in controls at 30 minutes. Insulin and glucagon responses after intragastric glucose were similar in thyrotoxic and control rats. These results suggest that higher blood glucose response after intragastric glucose in thyrotoxic rats may be due to rapid gastric emptying. The present
~glucose
1OOmglmin
*
.3 0
60
120
160 min
0
180 ml"
Fig 4. Insulin and glucagon response after intraduodenal glucose infusion test; (left panel), peripheral blood: fright panel). portal blood. AIRI, increment in plasma insulin. AIRG, increment in plasma glucagon. (0). Control fn = 7). (0). Thyrotoxic (n = 7).
.
0
,
30
15 min
Fig 6. Glucose absorption from perfused intestine. (0). Control (n = 7). (0). Thyrotoxic In = 7).
877
GLUCOSE TOLERANCE IN THYROTOXIC RATS
results agreed with the report of Holdsworth and Besser4 that rapid gastric emptying was associated with elevated blood glucose in hyperthyroid patients. The blood glucose levels are higher at 0, 30, and 60 minutes in the peripheral blood with hyperthyroidism, whereas in the portal blood the levels are higher at 0 and 30 minutes only. These indicate that glucose utilization may be slightly impaired in the periphery in thyrotoxic rats. Gastrin, glucagon, and secretin have been known to influence gastric emptying rate.‘7-20 Plasma glucagon and secretin concentrations in thyrotoxic rats were not significantly different from those in controls. However, plasma gastrin level in thyrotoxic rats was significantly higher than that in controls. This result was consistent with the report of Seino et al” that plasma gastrin levels are elevated in patients with hyperthyroidism. Although the mechanism by which hypergastrinemia occurs in thyrotoxic rats is unclear, gastrin may not contribute to rapid gastric emptying in the present study because gastrin is known to slow gastric emptying.‘“l’9 Basal plasma insulin levels in thyrotoxic rats were significantly higher than that in controls. However, there have been no reports that small amounts of insulin directly influence gastric emptying. Other intestinal hormones may be responsible for rapid gastric emptying in thyrotoxic rats. Further studies are necessary to elucidate
whether some intestinal hormones enhance gastric emptying in thyrotoxic rats. It has been well known that increased adrenoceptor response was observed in certain tissues in experimental thyrotoxic animals,22~23 and it has been reported that cu-adrenoceptors may be involved in the control of gastric emptying in the rat. 24 in the present study, phentolamine, cY-adrenoceptor antagonist administration did not alter insulin and glucagon response to glucose but normalized rapid gastric emptying and blood glucose responses after intragastric glucose infusion in thyrotoxic rats. Although we do not know whether rapid gastric emptying and impaired oral glucose tolerance in hyperthyroid patients are normalized by cu-adrenergic antagonist administration, rapid gastric emptying via ru-adrenergic stimulation may have an important role in glucose intolerance in short-term thyrotoxic rats. Increased adrenergic tone responsiveness in thyrotoxicosis are generally /3-adrenergic in nature.22*23 Further studies are necessary to elucidate more precisely the adrenergic modulation in hyperthyroidism. In summary, we conclude that an altered glu?ose tolerance to intragastric glucose in thyrotoxic rats may be due to rapid gastric emptying induced by increased a-adtienoceptor responses, and that glucose absorption from small intestine may not be increased in short-term thyrotoxic rats.
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