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Effect of glibenclamide on pancreatic hormone release from isolated perifused islets of normal and cysteamine-treated rats
Effect of Glibenclamide on Pancreatic Hormone Release From Isolated Islets of Normal and Cysteamine-Treated Rats Yasuhiro Sako, Taro Wasada,
Fumio Umeda.
Perifused
and Hiroshi lbayashi
The effect of glibenclamide. a sulfonylurea agent, on islet hormone secretion, particularly on glucagon was studied using isolated perifused pancreatic islets of normal and cysteamine-treated rats. In the normal rat islets. glibenclamide enhanced both insulin and somatostatin release in normoglycemic 60 mg/dL) and glucopenic (0 mg/dL) states, as well as under the condition of arginine stimulation. In contrast, glibenclamide stimulated glucagon release only transiently, then suppressed it in a sustaining manner in each state. In the cysteamine-treated islets, as expected, somatostatin concentrations in the perifusate remained unchanged during the infusion of arginine and/or glibenclamide. Under this condition, glibenclamide enhanced insulin release to the same extent as seen in normal islets, and again markedly inhibited glucagon release. These observations indicate that in isolated perifused rat pancreatic islets, glibenclamide suppresses glucagon secretion independently of D cell stimulation. It is concluded that glibenclamide may exert its inhibitory effect directly on A cell rather than through paracrine action of concomitant somatostatin release, and that the suppression of glucagon secretion by glibenclamide may. in part, contribute to the antidiabetogenic effect of this compound. @ 1986 by Grune & Stratton, Inc.
ULFONYLUREAS are important adjuncts in the current treatment of diabetes mellitus. A hypoglycemic effect of these compounds has been primarily attributed to stimulation of insulin secretion. Certain extrapancreatic effects were also suggested.‘-’ Regarding the effects on other islet hormones, sulfonylureas stimulate somatostatin release,4l5 but the effect on glucagon has been contradictory: stimulation,5-9 suppression,‘&16 and no effect.‘7-‘9 In experiments using the isolated perfused rat pancreas, Laube et al’* reported that tolbutamide has an inhibitory effect on glucagon secretion, independent of the functional state of the B cell. Samols et al? Grodsky et aL2’ and EffendiE et al” demonstrated a transient stimulation of glucagon release followed by marked inhibition in the glucopenic state. EffendiC et al’o*‘5also suggested that sulfonylureas-induced glucagon inhibition is subject to the paracrine action of concomitant somatostatin release that is stimulated by these compounds. In the present investigation, we studied the effect of glibenclamide, one of the most widely used sulfonylureas, on insulin, glucagon, and somatostatin release from perifused isolated rat pancreatic islets. To determine whether glibenclamide-induced glucagon release is subject to the paracrine action of somatostatin, we also used islets from cysteaminetreated rats, in which somatostatin was depleted, but with no alteration in insulin and glucagon release.2’
S
MATERIALS
Isolation and PrecuIture
AND METHODS
of Islets
Adult male Wistar rats, weighing 250 to 350 g and fed ad libitum with Oriental chow (Oriental Yeast Co, Tokyo) were used in this study. Intact islets were isolated aseptically from the pancreas using the collagenase technique22 and were collected with the use of a discontinuous Ficoll-Conray gradient.23 To stabilize the function, the From the Third Department of Internal Medicine, Faculty of Medicine, Kyushu University. Fukuoka. Japan. Address reprint requests to Yasuhiro Sake. MD, the Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 8I2. Japan. o 1986 by Grune & Stratton, Inc. 0026-0495/86/3510-0011$03.00~0 944
islets were precultured in a CO2 incubator (95% Or, 5% CO*) at 37 “C for 72 hours in Dulbecco’s modified Eagle medium (Gibco, Long Island, NY), which contains penicillin (100 U/mL), streptomycin sulfate (100 gg/mL), and 100 mg/dL glucose with supplementation of 10% fetal calf serum. Preparation
of Cysteamine-Treated
Rats
According to the method of Sorenson et ai:’ cysteamine @mercaptoethylamine, Sigma Co, St. Louis) was prepared as an aqueous 6% solution and neutralized with NaOH immediately before administration subcutaneously at a dose of 300 mg/kg body wt to the rat. Cysteamine-treated rats were killed 24 hours later and the islets were isolated. Perifusion
System
The basic components of our perifusion system are similar to ones originally described by Lacy et al” and Gingerich et a1.25A known number of the cultured islets (averaging 200 islets) were placed in the perifusion chamber, which consisted of a plastic Millipore (5 rm) filter unit (Swinnex-13, Millipore, Bedford, Mass). The islets were perifused at a flow rate of 0.7 mL/min with a Krebs-Ringer Hepes buffer solution, pH 7.4 containing 0.2% bovine serum albumin, 20 mmoI/L Hepes (N-2-hydroxy ethyl piperazine-N’-2-ethane sulfonic acid, Sigma Co, St Louis), and 0 or 50 mg/dL glucose equilibrated with 100% 0, at 37 OC.After a 40-minute equilibration period, glibenclamide (final concentration: 0.1, 1, 10 Mg/mL) and/ or 20 mmol/L arginine was added to the basal perifusion buffer for 20 minutes. 0.7 mL aliquots of the perifusate were collected in chilled tubes containing 1 mg EDTA and 400 KIU Trasylol (Bayer, Leverkussen, Germany) and stored at - 20 “C until assay for insulin, glucagon, and somatostatin. Radioimmunoassay
Insulin (IRI) was measured by the double-antibody method using rat insulin (EIIi-Lilly Research Laboratory, Indianapolis) as a standard. Glucagon (IRG) was determined with a glucagon RIA kit using antiserum OAL-123’” (Daiichi Radioisotope Laboratory Ltd, Tokyo). Somatostatin (SLI) was measured by a modification of reported methods,27-29 using synthetic somatostatin-14 (Peptide Institute Co, Osaka, Japan) as a standard, ‘2*I-Tyr”-somatostatin14 (Amersham International plc, Buckinghamshire, UK) as a tracer, and antisomatostatin serum 8OC (kindly provided by Dr Roger H. Unger, Dallas), which was directed against the central portion of somatostatin. Metabolism, Vol35. No 10 (October). 1986: pp 944-949
GLIBENCLAMIDE
1
945
Glucose SOW/d1 1 Gltbsnclamlde
Statistical Analysis
1 ra/ml
I
MrSE.n=S
All data are shown as mean * SEM. Student’s t-test was used for paired or nonpaired
/ O.--q -01
200.
comparison.
0 /rg/ml ug/ml
RESULTS 150.
Dose-Response Eflects of Glibenclamide
Insulin release was stimulated significantly at all doses given (0.1, 1, 10 pg/mL) (Fig 1). There was no apparent acute insulin response with the smallest dose of glibenclamide (0.1 bg/mL). The peak was higher and occurred earlier with increasing doses. Total amounts of insulin released during the first six minutes reached a plateau at 1 Fg/mL of glibenclamide (Table I). Somatostatin release was stimulated significantly, and the response pattern was almost the same as that of insulin. In contrast, the glucagon release was initially stimulated and subsequently suppressed below control levels at all doses given; 0.1 pg/mL glibenclamide was as potent as 10 Gg/mL in suppressing glucagon. Therefore, we used 1 pg/mL of glibenclamide in further studies. Eflect of Clibenclamide on Pancreatic Hormone Release Under Glucopenia
Fig 1. Effect of glibenclamide at different concentrations on the release of (A) insulin, (Bl somatostatin. and (Cl glucagon. from the isolated perifused pancreatic islets of normal rats with 60 mg/dL glucose. Values are given as mean f SEM.
Table 1. Effect of Glibenclamide
at Different
Concentrations
To observe the effect of glibenclamide on glucagon release (Fig 2), we elevated the basal level of glucagon by lowering the glucose concentration (0 mg/dL of glucose). The basal glucagon levels under 0 mg/dL of glucose (90.3 + 2.6 pg/ mL) were significantly higher, as compared with those under 50 mg/dL of glucose (44.1 2 5.9 pg/mL, P < 0.05). With the introduction of glibenclamide, the elevated glucagon levels were clearly suppressed after a transient stimulation. The suppression of glucagon release was reversed after termination of the glibenclamide infusion. In case of 0 mg/dL of glucose, both insulin and somatostatin release were stimulated significantly and peaked at three minutes after infusion of glibenclamide.
on the Release of Insulin, Glucagon. and Somatostatin
From the Isolated
Perifused Rat Islets Concentrationof Glibenclamidein PerifusicnMedium Release insulin (rlJ/min) lo-16 min
A(0 ca/mL)
6 (0.1 aglmLJ
c f 1.o /IglmL)
D (10.0 pg/mL)
35.2
+ 2.5
50.4
zk 5.7’
97.1
f 9.1*t
99.3
k 6.2’t
17-30
min
32.4
+- 4.2
53.2
+ 8.0*
71.6
k 8.1’
65.8
* 7.1’
1O-30
min
33.3
+ 2.2
52.3
k 7.5’
80.1
+ 8.2*t
77.0
+ 4.0*t
Somatostatin (pg/min) lo-16 min
5.9 * 1.0
12.6 f 0.4*
16.2 i
min
7.0 * 0.5
12.5 + 0.6’
13.9 t 1.9’
14.6 c 1.9’
1O-30 min
6.7 + 0.5
12.5 i 0.4*
14.7 t 1.8’
16.2 k 1.6*t
44.2
48.9
17-30
Glucagon (pg/min) lo-13 min
1.8*t
19.3 2 1.3.t
34.7
* 2.0
49.1
14-30
min
33.0
t 3.7
19.7 + 2.6’
19.1 i 4.5’
18.2 t 1.2+
lo-30
min
33.3
k 3.6
26.3
23.9
24.0
* 9.0s 2 3.8
Values are given as mean + SEM of five parallel experiments in each group. Release of each hormone is expressed as secretion rate per min. lP < 0.05
“A.
tP < 0.05
Y B.
+ 4.0’ -t 3.9’
* 5.6’ + 2.0’
SAKO ET AL
946
Glucose
(
uU/rnl zoo-
Ghbenclam~de
I
1 ughl
M+SE
A
i
0-e
: contron *
: D\ao6
“=5 150.
:
B
sL’
w/ml
;
GlucoseOmg/dl
I
Omg/dl
IRI
[
Gl~benclamide
1 us/ml
IRI
M * SE
flu/ml
200
1
1
f
A
D--O: Cmtrol * : PC006
~
,“5”,,”
: *** ‘\I:
30.
Somatostatln
* I
** 77
20. A
lfifl
loo
__&.-
.
6--a 6-
r
d
‘A__&
* : 0’
10
*****
* *
20
I
-a_&
*
‘G
1
a,
a_*_
-_&__
*
*I* 30
40
20
10
Fig 2. Effect of glibenclamide on the release of (A) insulin, (B) somatostatin. and iC) glucagon from the isolated perifused pancreatic islets of normal rats with 0 mg/dL glucose. Values are given as me8n ‘_ SEM. lP < 0.05 (significant difference Y control values).
Efect of Glibenclamide on Pancreatic Hormone Release From the Isolated Islets of Cysteamine-Treated Rats
To determine if the glibenclamide-induced glucagon suppression is subject to the paracrine action of concomitant somatostatin secretion (Fig 3) we evaluated this effect in a somatostatin-depleted state. Somatostatin release from cysteamine-treated rat islets remained unaffected with glibenclamide infusion. In the cysteamine-treated islets, the basal glucagon release did not differ significantly from that of normal islets. The glucagon release was again stimulated initially and then suppressed markedly during the infusion of glibenclamide. Total glucagon release during the first peak (10 to 13 minutes) was significantly greater than that in the normal rat islets (362.5 i 49.6 v 258.6 +_44.2 pg/3 min, P < 0.05). Insulin release was comparable to that in the case of normal rat islets.
Trne
of perrfusnn
30
40
(mln)
Fig 3. Effect of gfibenclamide on the release of (A) insulin, (6) somatostatin. and (C) glucagon from the isolated perifused pancreatic islets of cysteamine-treated rats with 0 mg/dL glucose. Values ar8 given es mean + SEM. lP < 0.05 (significant difference Y control values).
from normal rats (Fig 4). The pattern of release was biphasic for all three hormones. Addition of glibenclamide (1 pg/mL) significantly enhanced the arginine-induced insulin and somatostatin release, but in contrast, it inhibited the release of glucagon. In islets isolated from cysteamine-treated rats (Fig 5), 20 mmol/L arginine significantly induced a biphasic insulin and glucagon release, and this was not significant as compared with that in the case of normal rat islets. The somatostatin release was not significantly affected by the administration of arginine. Addition of glibenclamide (1 wg/mL) significantly enhanced the arginine-induced insulin release but markedly suppressed the arginine-induced glucagon release from the isolated islets of cysteamine-treated rats. DISCUSSION
Ejject of Glibenclamide on Arginine-Induced Pancreatic Hormone Release From ?he Mets ofNormal and Cysteamine-Treated Rats
Arginine (20 mmol/L) significantly stimulated insulin, glucagon, and somatostatin release from the islets isolated
In the present investigation we studied the effect of glibenclamide, a representative sulfonylurea, on pancreatic hormone release, using isolated perifused pancreatic islets system. To our knowledge, there is no documentation using the perifusion system for this purpose.
GLIBENCLAMIDE
Effect of glibenclamide on arginine-induced release of Fig 4. (A) insulin, (B) somatostatin. and (C) glucagon from the isolated perifused pancreatic islets of normal rats. Values are given as mean ‘t- SEM.
Effect of glibenclamide on arginine-induced release of Fig 5. (A) insulin, (6) somatostatin, and IC) glucagon from the isolated perifused pancreatic islets of cysteamine-treated rats. Values are given as mean k SEM.
It is well-established that sulfonylureas stimulate not only B cell, but also D cell secretion,4” and this was reconfirmed in the present study. Our study demonstrated that in the presence of 50 mg/dL glucose, 1 pg/mL of glibenclamide induces a maximal stimulation of insulin and a near maximal stimulation of somatostatin secretion from isolated perifused rat islets. Similar results were reported by ostenson et a13’ using isolated perfused rat pancreas. We also demonstrated that glibenclamide potentiates an arginine-induced secretion of insulin and somatostatin in isolated perifused normal rat islets. Effendii: et al” reported that glibenclamide significantly enhances arginine-stimulated somatostatin release from the isolated perfused rat pancreas, whereas the insulin release is less affected. On the contrary, regarding the effect of sulfonylureas on glucagon release, both in vivo and in vitro results are inconsistent. In our experiment, glibenclamide markedly inhibited glucagon release under normoglycemic, glucopenit, and arginine-stimulated states. Similarly, Laube et al” found that tolbutamide has an inhibitory effect on glucagon secretion under the glucose-free and normoglycemic states. Samols et aI5,6and Grodsky et al*’ demonstrated a transient stimulation of basal glucagon release followed by marked inhibition in the glucopenic state, which is in good agreement with our results. They suggested that this inhibitory effect
may be mediated by paracrine somatostatin and/or insulin effects on A cell. However, with respect to insulin as a paracrine factor, experiments using exogenously added insulini and with the isolated perfused pancreas from streptozotocin-treated rats’2*“*3’ indicated that sulfonylureas inhibit glucagon secretion independently of B cell function. Therefore, we directed attention to the suggestion by Effendii: et al’ov’5that sulfonylurea-induced glucagon inhibition may be subjected to paracrine action of concomitant somatostatin release, which is stimulated by this compound. Szabo and Reichlin’* reported that cysteamine (@-mercaptoethylamine) markedly depletes somatostatin in the gastric and duodenal mucosa, hypothalamus, pancreas, and plasma. Recently, Sorenson et al *’ demonstrated that cysteamine depletes pancreatic somatostatin contents to 36% of the control level, but does not alter pancreatic insulin or glucagon contents and release. These studies indicate the possibility that the cysteamine-treated rat may serve as a model for further investigations into the regulation of islet hormone release in the absence of stimulated somatostatin release. In the present study, somatostatin release from cysteamine-treated rat islets was not significantly affected by arginine and/or glibenclamide, as compared with the control value. Under this experimental condition, glibenclamide
SAKO ET AL
948
enhanced insulin and inhibited glucagon. These data indicate that glibenclamide inhibits glucagon secretion independently of D cell stimulation. Therefore, providing that sulfonylurea affects glucagon secretion independently of B cell stimulation as reported,‘2~2”*3’ our results raise the possibility that the inhibitory effect of sulfonylurea upon A cell may be direct action by this compound rather than a secondary effect mediated by paracrine action of concomitant somatostatin and jnsulin release. In this study, we also demonstrated that glibenclamide stimulated basal glucagon secretion only transiently and that total glucagon release during the first peak was significantly greater in the cysteamine-treated rat islets in comparison with the normal rat islets. This finding may imply that
somatostatin has a tonic inhibitory effect on glucagon secretion. Concerning plasma concentration of glibenclamide in diabetic patients during its routine clinical administration, Balant et al33 reported that the maximum glibenclamide serum level was always attained after two to six hours, and was 240 to 460 ng/mL after the administration of 5 mg glibenclamide, values of which are similar to those reported by Sartor et a134and Matsuda et a1.35The dose we used (1 pg/mL) of glibenclamide is therefore supraclinical (two to three times); nevertheless, the suppression of glucagon secretion by glibenclamide may in part contribute to the antidiabetogenic effect of this compound.
REFERENCES
1. Feinglos MN, Lebovitz HE: Sulfonylureas increase the number of insulin receptors. Nature 276184-185, 1978 2. Blumenthal SA: Potentiation of the hepatic action of insulin by chlorpropamide. Diabetes 26:485-489, 1977 3. Pernet A, Trimble ER, Kuntschen F, et al: Sulfonylureas in insulin-dependent (Type 1) diabetes: Evidence for an extrapancreatic effect in vivo. J Clin Endocrinol Metab 61:247-251, 1985 4. Ipp E, Dobbs RE, Arimura A, et al: Release of immunoreactive somatostatin from the pancreas in response to glucose, amino acids, pancreozymin-cholecystokinin, and tolbutamide. J Clin Invest 60:76@-765,1977 5. Samols E, Weir GC, Ramseur R, et al: Modulation of pancreatic somatostatin by adrenergic and cholinergic agonism and by hyper and hypoglycemic sulfonamides. Metabolism 27:12191221,1978 (suppl 1) 6. Samols E, Harrison J: Intraislet negative insulin-glucagon feedback. Metabolism 251443-1447, 1976 (suppl 1) 7. Loubatitres AL, Loubatieres-Mariani MM, Alric R, et al: Tolbutamide and gluoagon secretion. Diabetologia 10:271-276, 1971 8. Hermansen K: Tolbutamide, glucose, calcium, and somatostatin secretion. Acta Endocrinol99:86-93, 1982 9. Loubatitres AL, Loubatitres-Mariani MM, Chapal J, et al: Action du glibenclamide sur les secretions de glucagon et d’insuline Btudiees sur le pancreas isole et perfus6 du rat. C R Sot Biol (Paris) 169:1568-1571,1975 10. EffendiC S, Enzmann F, NylCn A, et al: Effect of glucose/ sulfonylurea interaction on release of insulin, glucagon, and somatostatin from isolated perfused rat pancreas. Proc Nat1 Acad Sci USA 76:5901-5904,1979 11. Pfeifer MA, Beard JC, Halter JB, et al: Suppression of glucagon secretion during a tolbutamide infusion in normal and noninsulin-dependent diabetic subjects. J Clin Endocrinol Metab 56:586-591, 1983 12. Laube H, Fussglnger R, Goberna R, et al: Effects of tolbutamide on insulin and glucagon secretion of the isolated perfused rat pancreas. Horm Metab Res 3:238-242, 197 1 13. Ohneda A, Sato M, Matsuda K, et al: Suppression of pancreatic glucagon secretion by tolbutamide in dogs. Horm Metab Res 6:478483, 1974 14. Ohneda A, Ishii S, Horigome K, et al: Glucagon response to arginine after treatment of diabetes mellitus. Diabetes 24:81 l-819, 1975 15. Effendi6 S, Enzmann F, Nyltn A, et al: Sulphonylurea (Glibenclamide) enhances somatostatin and inhibits glucagon release induced by arginine. Acta Physiol Stand 108:231-233, 1980
16. Samols E, Tyler JM, Mialhe P: Suppression of pancreatic glucagon release by the hypoglycemic sulfonylureas. Lancet 1:17& 176,1969 17. Chesney TM, Schofield JG: Studies on the secretion of pancreatic glucagon. Diabetes 18:627-632, 1969 18. Unger RH, Aquillar-Parada E, Miiller WA, et al: Studies of pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest 49:837-848, 1970 19. Pek S, Fajans S, Floyd JC, et al: Failure of sulfonylureas to suppress plasma glucagon in man. Diabetes 21:216-223, 1972 20. Grodsky GM, Epstein GH, Fan&a R, et al: Pancreatic action of the sulfonylureas. Fed Proc 36:27 14-27 19, 1977 21. Sorenson RL, Grouse LH, Elde RP: Cysteamine blocks somatostatin secretion without altering the course of insulin or glucagon release: A new model for the study of islet function. Diabetes 32:377-379, 1983 22. Lacy PE, Kostianovsky M: Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 11:35-39, 1967 23. Okeda T, Ono J, Takaki R, et al: Simple method for the collection of pancreatic islets by the use of Ficoll-Conray gradient. Endocrinol Jpn 26:495-499, 1979 24. Lacy PE, Finke EH, Conant S, et al: Long-term perifusion of isolated rat islets in vitro. Diabetes 25:484-493, 1976 25. Gingerich RL, Aronoff SL, Kipnis DM, et al: Insulin and glucagon secretion from rat islets maintained in a tissue cultureperifusion system. Diabetes 28:276-281, 1979 26. Imagawa K, Nishino T, Shin S, et al: Production of antiglucagon sera with a C terminal fragment of pancreatic glucagon. Endocrinol Jpn 26:123-131, 1979 27. Harris V, Conlon JM, Srikant CB, et al: Measurements of somatostatin-like immunoreactivity in plasma. Clin Chim Acta 87:275-283,1978 28. Arimura A, Sato H, Coy DH, et al: Radioimmunoassay for GH-release inhibiting hormone. Proc Sot Exp Biol Med 148:78& 789,197s 29. Kronheim S, Berelowitz M, Pimstone BL: A radioimmunoassay for growth hormone release-inhibiting hormone: Method and quantitative tissue distribution. Clin Endocrinol (0x0 5619630, 1976 30. Gstenson CG, Grill V, Nylen A, et al: Modulation by IBMX, fasting and experimental diabetes of glibenclamide-induced islet hormone release from the perfused rat pancreas. Diabete Metab 9:58-65, 1983 31. Kadowaki S, Taminato T, Chiba T, et al: Effect of tolbutamide on insulin, glucagon, and somatostatin release from the
GLIBENCLAMIDE
diabetic rat pancreas with special reference to glucose concentration. Endocrinology 122:2 187-2 192,1983 32. Szabo S, Reichlin S: Somatostatin in rat tissues is depleted by cysteamine administration. Endocrinology 109:2255-2257, 1981 33. Balant L, Zahnd GR, Weber F, et al: Behavior of glibenclamide on repeated administration to diabetic patients. Eur J Clin Pharmacol I 1:19-25, 1977
949
34. Sartor G, Melander A, Schersten B, et al: Serum glibenclamide in diabetic patients, and influence of food on the kinetics and effects of glibenclamide. Diabetologia 18:17-22, 1980 35. Matsuda A, Kuzuya T, Sugita Y, et al: Plasma levels of glibenclamide in diabetic patients during its routine clinical administration determined by a specific radioimmunoassay. Horm Metab Res 15:425-428, 1983
Fumio Umeda.
Perifused
and Hiroshi lbayashi
The effect of glibenclamide. a sulfonylurea agent, on islet hormone secretion, particularly on glucagon was studied using isolated perifused pancreatic islets of normal and cysteamine-treated rats. In the normal rat islets. glibenclamide enhanced both insulin and somatostatin release in normoglycemic 60 mg/dL) and glucopenic (0 mg/dL) states, as well as under the condition of arginine stimulation. In contrast, glibenclamide stimulated glucagon release only transiently, then suppressed it in a sustaining manner in each state. In the cysteamine-treated islets, as expected, somatostatin concentrations in the perifusate remained unchanged during the infusion of arginine and/or glibenclamide. Under this condition, glibenclamide enhanced insulin release to the same extent as seen in normal islets, and again markedly inhibited glucagon release. These observations indicate that in isolated perifused rat pancreatic islets, glibenclamide suppresses glucagon secretion independently of D cell stimulation. It is concluded that glibenclamide may exert its inhibitory effect directly on A cell rather than through paracrine action of concomitant somatostatin release, and that the suppression of glucagon secretion by glibenclamide may. in part, contribute to the antidiabetogenic effect of this compound. @ 1986 by Grune & Stratton, Inc.
ULFONYLUREAS are important adjuncts in the current treatment of diabetes mellitus. A hypoglycemic effect of these compounds has been primarily attributed to stimulation of insulin secretion. Certain extrapancreatic effects were also suggested.‘-’ Regarding the effects on other islet hormones, sulfonylureas stimulate somatostatin release,4l5 but the effect on glucagon has been contradictory: stimulation,5-9 suppression,‘&16 and no effect.‘7-‘9 In experiments using the isolated perfused rat pancreas, Laube et al’* reported that tolbutamide has an inhibitory effect on glucagon secretion, independent of the functional state of the B cell. Samols et al? Grodsky et aL2’ and EffendiE et al” demonstrated a transient stimulation of glucagon release followed by marked inhibition in the glucopenic state. EffendiC et al’o*‘5also suggested that sulfonylureas-induced glucagon inhibition is subject to the paracrine action of concomitant somatostatin release that is stimulated by these compounds. In the present investigation, we studied the effect of glibenclamide, one of the most widely used sulfonylureas, on insulin, glucagon, and somatostatin release from perifused isolated rat pancreatic islets. To determine whether glibenclamide-induced glucagon release is subject to the paracrine action of somatostatin, we also used islets from cysteaminetreated rats, in which somatostatin was depleted, but with no alteration in insulin and glucagon release.2’
S
MATERIALS
Isolation and PrecuIture
AND METHODS
of Islets
Adult male Wistar rats, weighing 250 to 350 g and fed ad libitum with Oriental chow (Oriental Yeast Co, Tokyo) were used in this study. Intact islets were isolated aseptically from the pancreas using the collagenase technique22 and were collected with the use of a discontinuous Ficoll-Conray gradient.23 To stabilize the function, the From the Third Department of Internal Medicine, Faculty of Medicine, Kyushu University. Fukuoka. Japan. Address reprint requests to Yasuhiro Sake. MD, the Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 8I2. Japan. o 1986 by Grune & Stratton, Inc. 0026-0495/86/3510-0011$03.00~0 944
islets were precultured in a CO2 incubator (95% Or, 5% CO*) at 37 “C for 72 hours in Dulbecco’s modified Eagle medium (Gibco, Long Island, NY), which contains penicillin (100 U/mL), streptomycin sulfate (100 gg/mL), and 100 mg/dL glucose with supplementation of 10% fetal calf serum. Preparation
of Cysteamine-Treated
Rats
According to the method of Sorenson et ai:’ cysteamine @mercaptoethylamine, Sigma Co, St. Louis) was prepared as an aqueous 6% solution and neutralized with NaOH immediately before administration subcutaneously at a dose of 300 mg/kg body wt to the rat. Cysteamine-treated rats were killed 24 hours later and the islets were isolated. Perifusion
System
The basic components of our perifusion system are similar to ones originally described by Lacy et al” and Gingerich et a1.25A known number of the cultured islets (averaging 200 islets) were placed in the perifusion chamber, which consisted of a plastic Millipore (5 rm) filter unit (Swinnex-13, Millipore, Bedford, Mass). The islets were perifused at a flow rate of 0.7 mL/min with a Krebs-Ringer Hepes buffer solution, pH 7.4 containing 0.2% bovine serum albumin, 20 mmoI/L Hepes (N-2-hydroxy ethyl piperazine-N’-2-ethane sulfonic acid, Sigma Co, St Louis), and 0 or 50 mg/dL glucose equilibrated with 100% 0, at 37 OC.After a 40-minute equilibration period, glibenclamide (final concentration: 0.1, 1, 10 Mg/mL) and/ or 20 mmol/L arginine was added to the basal perifusion buffer for 20 minutes. 0.7 mL aliquots of the perifusate were collected in chilled tubes containing 1 mg EDTA and 400 KIU Trasylol (Bayer, Leverkussen, Germany) and stored at - 20 “C until assay for insulin, glucagon, and somatostatin. Radioimmunoassay
Insulin (IRI) was measured by the double-antibody method using rat insulin (EIIi-Lilly Research Laboratory, Indianapolis) as a standard. Glucagon (IRG) was determined with a glucagon RIA kit using antiserum OAL-123’” (Daiichi Radioisotope Laboratory Ltd, Tokyo). Somatostatin (SLI) was measured by a modification of reported methods,27-29 using synthetic somatostatin-14 (Peptide Institute Co, Osaka, Japan) as a standard, ‘2*I-Tyr”-somatostatin14 (Amersham International plc, Buckinghamshire, UK) as a tracer, and antisomatostatin serum 8OC (kindly provided by Dr Roger H. Unger, Dallas), which was directed against the central portion of somatostatin. Metabolism, Vol35. No 10 (October). 1986: pp 944-949
GLIBENCLAMIDE
1
945
Glucose SOW/d1 1 Gltbsnclamlde
Statistical Analysis
1 ra/ml
I
MrSE.n=S
All data are shown as mean * SEM. Student’s t-test was used for paired or nonpaired
/ O.--q -01
200.
comparison.
0 /rg/ml ug/ml
RESULTS 150.
Dose-Response Eflects of Glibenclamide
Insulin release was stimulated significantly at all doses given (0.1, 1, 10 pg/mL) (Fig 1). There was no apparent acute insulin response with the smallest dose of glibenclamide (0.1 bg/mL). The peak was higher and occurred earlier with increasing doses. Total amounts of insulin released during the first six minutes reached a plateau at 1 Fg/mL of glibenclamide (Table I). Somatostatin release was stimulated significantly, and the response pattern was almost the same as that of insulin. In contrast, the glucagon release was initially stimulated and subsequently suppressed below control levels at all doses given; 0.1 pg/mL glibenclamide was as potent as 10 Gg/mL in suppressing glucagon. Therefore, we used 1 pg/mL of glibenclamide in further studies. Eflect of Clibenclamide on Pancreatic Hormone Release Under Glucopenia
Fig 1. Effect of glibenclamide at different concentrations on the release of (A) insulin, (Bl somatostatin. and (Cl glucagon. from the isolated perifused pancreatic islets of normal rats with 60 mg/dL glucose. Values are given as mean f SEM.
Table 1. Effect of Glibenclamide
at Different
Concentrations
To observe the effect of glibenclamide on glucagon release (Fig 2), we elevated the basal level of glucagon by lowering the glucose concentration (0 mg/dL of glucose). The basal glucagon levels under 0 mg/dL of glucose (90.3 + 2.6 pg/ mL) were significantly higher, as compared with those under 50 mg/dL of glucose (44.1 2 5.9 pg/mL, P < 0.05). With the introduction of glibenclamide, the elevated glucagon levels were clearly suppressed after a transient stimulation. The suppression of glucagon release was reversed after termination of the glibenclamide infusion. In case of 0 mg/dL of glucose, both insulin and somatostatin release were stimulated significantly and peaked at three minutes after infusion of glibenclamide.
on the Release of Insulin, Glucagon. and Somatostatin
From the Isolated
Perifused Rat Islets Concentrationof Glibenclamidein PerifusicnMedium Release insulin (rlJ/min) lo-16 min
A(0 ca/mL)
6 (0.1 aglmLJ
c f 1.o /IglmL)
D (10.0 pg/mL)
35.2
+ 2.5
50.4
zk 5.7’
97.1
f 9.1*t
99.3
k 6.2’t
17-30
min
32.4
+- 4.2
53.2
+ 8.0*
71.6
k 8.1’
65.8
* 7.1’
1O-30
min
33.3
+ 2.2
52.3
k 7.5’
80.1
+ 8.2*t
77.0
+ 4.0*t
Somatostatin (pg/min) lo-16 min
5.9 * 1.0
12.6 f 0.4*
16.2 i
min
7.0 * 0.5
12.5 + 0.6’
13.9 t 1.9’
14.6 c 1.9’
1O-30 min
6.7 + 0.5
12.5 i 0.4*
14.7 t 1.8’
16.2 k 1.6*t
44.2
48.9
17-30
Glucagon (pg/min) lo-13 min
1.8*t
19.3 2 1.3.t
34.7
* 2.0
49.1
14-30
min
33.0
t 3.7
19.7 + 2.6’
19.1 i 4.5’
18.2 t 1.2+
lo-30
min
33.3
k 3.6
26.3
23.9
24.0
* 9.0s 2 3.8
Values are given as mean + SEM of five parallel experiments in each group. Release of each hormone is expressed as secretion rate per min. lP < 0.05
“A.
tP < 0.05
Y B.
+ 4.0’ -t 3.9’
* 5.6’ + 2.0’
SAKO ET AL
946
Glucose
(
uU/rnl zoo-
Ghbenclam~de
I
1 ughl
M+SE
A
i
0-e
: contron *
: D\ao6
“=5 150.
:
B
sL’
w/ml
;
GlucoseOmg/dl
I
Omg/dl
IRI
[
Gl~benclamide
1 us/ml
IRI
M * SE
flu/ml
200
1
1
f
A
D--O: Cmtrol * : PC006
~
,“5”,,”
: *** ‘\I:
30.
Somatostatln
* I
** 77
20. A
lfifl
loo
__&.-
.
6--a 6-
r
d
‘A__&
* : 0’
10
*****
* *
20
I
-a_&
*
‘G
1
a,
a_*_
-_&__
*
*I* 30
40
20
10
Fig 2. Effect of glibenclamide on the release of (A) insulin, (B) somatostatin. and iC) glucagon from the isolated perifused pancreatic islets of normal rats with 0 mg/dL glucose. Values are given as me8n ‘_ SEM. lP < 0.05 (significant difference Y control values).
Efect of Glibenclamide on Pancreatic Hormone Release From the Isolated Islets of Cysteamine-Treated Rats
To determine if the glibenclamide-induced glucagon suppression is subject to the paracrine action of concomitant somatostatin secretion (Fig 3) we evaluated this effect in a somatostatin-depleted state. Somatostatin release from cysteamine-treated rat islets remained unaffected with glibenclamide infusion. In the cysteamine-treated islets, the basal glucagon release did not differ significantly from that of normal islets. The glucagon release was again stimulated initially and then suppressed markedly during the infusion of glibenclamide. Total glucagon release during the first peak (10 to 13 minutes) was significantly greater than that in the normal rat islets (362.5 i 49.6 v 258.6 +_44.2 pg/3 min, P < 0.05). Insulin release was comparable to that in the case of normal rat islets.
Trne
of perrfusnn
30
40
(mln)
Fig 3. Effect of gfibenclamide on the release of (A) insulin, (6) somatostatin. and (C) glucagon from the isolated perifused pancreatic islets of cysteamine-treated rats with 0 mg/dL glucose. Values ar8 given es mean + SEM. lP < 0.05 (significant difference Y control values).
from normal rats (Fig 4). The pattern of release was biphasic for all three hormones. Addition of glibenclamide (1 pg/mL) significantly enhanced the arginine-induced insulin and somatostatin release, but in contrast, it inhibited the release of glucagon. In islets isolated from cysteamine-treated rats (Fig 5), 20 mmol/L arginine significantly induced a biphasic insulin and glucagon release, and this was not significant as compared with that in the case of normal rat islets. The somatostatin release was not significantly affected by the administration of arginine. Addition of glibenclamide (1 wg/mL) significantly enhanced the arginine-induced insulin release but markedly suppressed the arginine-induced glucagon release from the isolated islets of cysteamine-treated rats. DISCUSSION
Ejject of Glibenclamide on Arginine-Induced Pancreatic Hormone Release From ?he Mets ofNormal and Cysteamine-Treated Rats
Arginine (20 mmol/L) significantly stimulated insulin, glucagon, and somatostatin release from the islets isolated
In the present investigation we studied the effect of glibenclamide, a representative sulfonylurea, on pancreatic hormone release, using isolated perifused pancreatic islets system. To our knowledge, there is no documentation using the perifusion system for this purpose.
GLIBENCLAMIDE
Effect of glibenclamide on arginine-induced release of Fig 4. (A) insulin, (B) somatostatin. and (C) glucagon from the isolated perifused pancreatic islets of normal rats. Values are given as mean ‘t- SEM.
Effect of glibenclamide on arginine-induced release of Fig 5. (A) insulin, (6) somatostatin, and IC) glucagon from the isolated perifused pancreatic islets of cysteamine-treated rats. Values are given as mean k SEM.
It is well-established that sulfonylureas stimulate not only B cell, but also D cell secretion,4” and this was reconfirmed in the present study. Our study demonstrated that in the presence of 50 mg/dL glucose, 1 pg/mL of glibenclamide induces a maximal stimulation of insulin and a near maximal stimulation of somatostatin secretion from isolated perifused rat islets. Similar results were reported by ostenson et a13’ using isolated perfused rat pancreas. We also demonstrated that glibenclamide potentiates an arginine-induced secretion of insulin and somatostatin in isolated perifused normal rat islets. Effendii: et al” reported that glibenclamide significantly enhances arginine-stimulated somatostatin release from the isolated perfused rat pancreas, whereas the insulin release is less affected. On the contrary, regarding the effect of sulfonylureas on glucagon release, both in vivo and in vitro results are inconsistent. In our experiment, glibenclamide markedly inhibited glucagon release under normoglycemic, glucopenit, and arginine-stimulated states. Similarly, Laube et al” found that tolbutamide has an inhibitory effect on glucagon secretion under the glucose-free and normoglycemic states. Samols et aI5,6and Grodsky et al*’ demonstrated a transient stimulation of basal glucagon release followed by marked inhibition in the glucopenic state, which is in good agreement with our results. They suggested that this inhibitory effect
may be mediated by paracrine somatostatin and/or insulin effects on A cell. However, with respect to insulin as a paracrine factor, experiments using exogenously added insulini and with the isolated perfused pancreas from streptozotocin-treated rats’2*“*3’ indicated that sulfonylureas inhibit glucagon secretion independently of B cell function. Therefore, we directed attention to the suggestion by Effendii: et al’ov’5that sulfonylurea-induced glucagon inhibition may be subjected to paracrine action of concomitant somatostatin release, which is stimulated by this compound. Szabo and Reichlin’* reported that cysteamine (@-mercaptoethylamine) markedly depletes somatostatin in the gastric and duodenal mucosa, hypothalamus, pancreas, and plasma. Recently, Sorenson et al *’ demonstrated that cysteamine depletes pancreatic somatostatin contents to 36% of the control level, but does not alter pancreatic insulin or glucagon contents and release. These studies indicate the possibility that the cysteamine-treated rat may serve as a model for further investigations into the regulation of islet hormone release in the absence of stimulated somatostatin release. In the present study, somatostatin release from cysteamine-treated rat islets was not significantly affected by arginine and/or glibenclamide, as compared with the control value. Under this experimental condition, glibenclamide
SAKO ET AL
948
enhanced insulin and inhibited glucagon. These data indicate that glibenclamide inhibits glucagon secretion independently of D cell stimulation. Therefore, providing that sulfonylurea affects glucagon secretion independently of B cell stimulation as reported,‘2~2”*3’ our results raise the possibility that the inhibitory effect of sulfonylurea upon A cell may be direct action by this compound rather than a secondary effect mediated by paracrine action of concomitant somatostatin and jnsulin release. In this study, we also demonstrated that glibenclamide stimulated basal glucagon secretion only transiently and that total glucagon release during the first peak was significantly greater in the cysteamine-treated rat islets in comparison with the normal rat islets. This finding may imply that
somatostatin has a tonic inhibitory effect on glucagon secretion. Concerning plasma concentration of glibenclamide in diabetic patients during its routine clinical administration, Balant et al33 reported that the maximum glibenclamide serum level was always attained after two to six hours, and was 240 to 460 ng/mL after the administration of 5 mg glibenclamide, values of which are similar to those reported by Sartor et a134and Matsuda et a1.35The dose we used (1 pg/mL) of glibenclamide is therefore supraclinical (two to three times); nevertheless, the suppression of glucagon secretion by glibenclamide may in part contribute to the antidiabetogenic effect of this compound.
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GLIBENCLAMIDE
diabetic rat pancreas with special reference to glucose concentration. Endocrinology 122:2 187-2 192,1983 32. Szabo S, Reichlin S: Somatostatin in rat tissues is depleted by cysteamine administration. Endocrinology 109:2255-2257, 1981 33. Balant L, Zahnd GR, Weber F, et al: Behavior of glibenclamide on repeated administration to diabetic patients. Eur J Clin Pharmacol I 1:19-25, 1977
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