Adrenoceptor antagonists, but not guanethidine, reduce glucopenia-induced glucagon secretion from perfused rat pancreas

ELSEVIER

Diabetes Research and Clinical

Practice 30 (1995) 173- 180

Adrenoceptor antagonists, but not guanethidine, reduce glucopenia-induced glucagon secretion from perfused rat pancreas Katsuhiko

Itoa,*, Hiroshi Hiroseaab, Koichi Kido”, Kazunori Hiroshi Maruyamaa, Takao Sarutaa

Koyama”,

“Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan bDepartment of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Received 18 August 1995; revised 9 November

1995; accepted 22 November

1995

Abstract This study was designed to investigate (1) whether norepinephrine is released in response to glucopenia in vitro, thereby stimulating glucagon secretion and, (2) the modulating effects of norepinephrine on insulin and glucagon secretion, using isolated perfused rat pancreas preparations. Simultaneous addition of the adrenergic receptor antagonists yohimbine, prazosin and propranolol, each at a concentration of lop5 mol/l, significantly potentiated glucose-stimulated insulin secretion (6.23 + 0.76 vs. 2.11 f 0.72 (control) nmol/min, P < O.Ol), and suppressed glucopenia-induced glucagon secretion (0.59 f 0.10 vs. 1.34 f 0.18 (control) ng/min, P < 0.05). Also, 10 - 5 mol/l yohimbine alone significantly potentiated glucose-stimulated insulin secretion (4.86 f 0.50 nmol/min, P < 0.05). The norepinephrine release inhibitor, guanethidine, significantly inhibited tyramine-induced secretion of both norepinephrine (7.86k 0.77 vs. 49.7 + 2.3 nmol/min, P< 0.01) and glucagon (0.31 f0.08 vs. 1.21 f0.15 ng/min, P < O.Ol), but exerted no effects on glucopenia-induced secretion of either norepinephrine or glucagon. We conclude that these results further support the concept that the neurotransmitter norepinephrine is released in response to glucopenia in vitro, and modulates insulin and glucagon secretion. Our data do not, however, provide evidence indicating that glucopenia-induced glucagon secretion is mainly mediated by activation of sympathetic nerve terminals around the cr-cells in the isolated perfused rat pancreas. Keywords:

Norepinephrine;

Insulin; Glucagon; Perfused rat pancreas

1. Introduction

* Corresponding

author.

Elsevier Science Ireland Ltd. SSDI 0021-9150(95)01189-S

Glucagon plays an important role in glucose homeostasis. Glucopenia-induced glucagon secretion is thought to be due mainly to the autonomic nervous system activation that accompanies hypoglycemia in vivo [l-9] (also reviewed in Refs.

174

K. Ito et al. / Diabetes Research and Clinical Practice 30 (1995) 173- 180

[ 10,111). It has also been demonstrated that direct sympathetic innervation of the pancreas [ 12,131 can be activated by glucopenic stress [14]. Recently, we have reported that aZA- and p-adrenergic agonism stimulate glucagon secretion from perfused pancreata of normal and streptozotocininduced diabetic rats [ 15,161. However, there are few reports indicating that the peripheral autonomic nervous system in the pancreas is activated independently of the central nervous system. Christensen et al. [17] showed that endogenous norepinephrine (NE) is released in response to glucopenia from the perfused canine pancreas. Hisatomi et al. [18] showed that the a-adrenoceptor antagonist phentolamine markedly suppressed glucagon release in response to glucopenia from the perfused rat pancreas. They concluded that glucagon secretion in response to glucopenia is controlled within the pancreas, through a direct glucopenic enhancement of NE release from sympathetic nerve endings, stimulating glucagon secretion from islet a-cells. Howmight influence insulin ever, phentolamine secretion via pre- and post- synaptic effects of alpha receptors [19]. Moreover, Plant et al. [20] reported that phentolamine inhibits ATP-sensitive K + -channels in pancreatic a-cells thereby stimulating insulin release. Thus, phentolamine acts not only as an cc-adrenergic antagonist but also as an insulin stimulator. There is a possibility that glucopenia-induced phentolamine suppresses glucagon secretion as a result of stimulating endogenous insulin secretion. The present study was designed to evaluate (1) whether NE is released in response to glucopenia in vitro, thereby stimulating glucagon secretion and, (2) the modulating effects of NE on insulin and glucagon secretion in the isolated perfused rat pancreas, under conditions free of central nervous system influence. 2. Materials

and methods

2.1. Animals Male Wistar rats weighing 300-400 g were used. Our institution’s guidelines for the care and use of laboratory animals were followed.

2.2. Experimental procedure Pancreata were isolated and perfused using a modification of the method of Grodsky and Fanska [21] after an 18-24-h fast, as described previously [22]. Rats were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg). The perfusate was Krebs-Ringer bicarbonate buffer supplemented with 4.5% (weight/volume) dextran T-70 (Pharmacia LKB Biotechnology AB, Uppsala, Sweden), 1% (weight/volume) bovine serum albumin (Miles, Kankakee, IL), 5 mmol/l sodium pyruvate, sodium fumarate and sodium glutamate (Sigma, St. Louis, MO), and the flow rate was set at a constant 3.0 ml/min. The partial pressure of oxygen was maintained between 450 and 550 mmHg by a bubble oxygenator using a 95% 0,/5% CO, gas mixture. The perfusate pH was maintained between 7.35 and 7.45. A preperfusion period, sufficient to stabilize insulin and glucagon levels, was used in all protocols. Over the course of a 65- or 75-min experimental period, the perfused pancreata were successively exposed to glucose concentrations of 5.6 mmol/l as the basal level, 16.7 mmol/l for high glucose stimulation (10 min) and 1.4 mmol/l for low glucose stimulation (15 min). Insulin and glucagon in the perfusate were measured during the entire experimental period. Concentrations of NE were also measured in controls and tyramine experiments. In the first experiment, the ol,-adrenoceptor antagonist yohimbine hydrochloride (Sigma), the or,blocker prazosin hydrochloride (Pfizer Pharm., Tokyo, Japan) and B-antagonist propranolol hydrochloride (ICI Pharma, Osaka, Japan) were added simultaneously at 5 min, to achieve perfusate concentrations of 10 pmol/l for all three drugs (Y.P.P.). In the second experiment, only yohimbine (10 pmol/l) was added at 5 min. In the third experiment, in order to identify whether NE is released from sympathetic nerve terminals in response to glucopenia through the activation of sympathetic nerves in the pancreas, we used guanethidine (1-[2-guanyldinoathyl] octahydroazocine; Sigma), which inhibits the release of NE from sympathetic nerve terminals. Tyramine hy-

K. Ito et al. /Diabetes

115

Research and Clinical Practice 30 (1995) 173-180

drochloride (4-hydroxyphenethylamine HCl; Sigma), which promotes NE release from sympathetic nerve terminals was added at 65 min, in order to determine the effects of guanethidine on tyramine-induced glucagon and NE release. 2.3. Hormone measurements

Immunoreactive insulin was measured with a commercially available kit (Eiken, Tokyo, Japan) based on a radioimmunoassay using rat insulin (Novo Research Institute, Bagsvaerd, Denmark) as the standard. Immunoreactive glucagon was measured by a previously described method using antiserum to synthetic glucagon 19-29 [15,22]. NE was measured by the high performance liquid chromatography (HPLC) method.

suppressed glucagon secretion in response to glucopenia as compared to controls (0.59 f 0.10 vs. 1.22 f 0.16 ng/min, P < 0.05). As shown in Fig. 2, 10 pmol/l yohimbine alone promoted both the first (2.83 + 0.34 nmol/min, P < 0.01) and second (4.86 + 0.50 nmol/min, P < 0.05) phases of insulin secretion which occurred in response to a high glucose concentration. After the addition of yohimbine, basal glucagon secretion decreased significantly at a glucose concentration of 5.6 mmol/l (0.82 + 0.06 vs. 1.28 + 0.10 ng/min, P < 0.05). Under the influ-

1.4

v

mM

2.4. Statistical analyses

All data are expressed as means + 1SEM. Statistical significance of differences was evaluated using one and two-way analysis of variance followed by Dunnett’s multiple comparison test, if the null hypothesis was rejected by the former, and by Student’s unpaired t-test for two groups. Differences were considered statistically significant when P < 0.05.

1600

% '-

1200

E

600

3. Results 3.1. Efsects of yohimbine, prazosin and propranolol (Y.P.P., each at a concentration of 10 ,umol/l) on insulin and glucagon secretion from the perfused rat pancreas

Fig. 1 shows insulin and glucagon secretion in response to changes in glucose concentration in controls and the Y.P.P.-perfused group. After the addition of Y.P.P., basal glucagon secretion at a glucose concentration of 5.6 mmol/l decreased significantly (0.52 f 0.08 vs. 1.18 + 0.20 ng/min, P < 0.05). Y.P.P. promoted the first (4.07 f 0.35 vs. 1.60 + 0.23 (control) nmol/min, P < 0.01) and second (6.23 + 0.76 vs. 2.11 f 0.72 nmol/min, P < 0.01) phases of insulin secretion which occurred in response to high glucose. Y.P.P. also

0

5

20

30

40

55

65

Time (min) Fig. 1. Effects of simultaneous addition of yohimbine, prazosin and propranolol CyPP, each at a concentration of 10 pmol/l) on insulin (IRI) and glucagon (IRG) secretion from the perfused rat pancreas. Shaded area depicts controls (n = 9) and solid lines with open circles depict results with Y.P.P. in the perfusate (n = 6). Values are means + SEM.

K. Ito et al. /Diabetes Research and Clinical Practice 30 (1995) 173-180

176

tyramine. In controls, glucopenia and 10 mg/l tyramine caused roughly the same glucagon secretion response. Although glucagon levels were slightly potentiated by guanethidine for the initial few minutes at a glucose concentration of 5.6 mmol/l, baseline levels were significantly decreased thereafter (0.68 f 0.06 vs. 1.06 + 0.17 ng/

tyramine

ID mg/L

m

2400

600 3

600

G g

400

1800

% g

200

1200

600

0 0

5

20

30

40

55

65

lime (min) Fig. 2. Effects of 10 pmol/l yohimbine on insulin (IRI) and glucagon (IRG) secretion from the perfused rat pancreas. Shaded area depicts controls (n = 9) and solid lines with open circles depict results obtained with yohimbine in the perfusate (n = 6). Values are means f SEM.

ence of yohimbine, insulin secretion during glucopenia was potentiated (though not significantly). Yohimbine alone also reduced glucagon secretion in response to glucopenia at some time points. However, there was no significant difference in the area under the concentration curve, with glucopenic stimulation, between the yohimbine-perfused group and the control. 3.2. E#ect of 10 ,umolll guanethidine on insulin and glucagon secretion from the perfused rat pancreas in response to changes in glucose concentration and 10 mg/l tyramine

Fig. 3 shows insulin and glucagon secretion in response to different glucose concentrations and

c

1000 -

;

aoo-

/

600-

;

0 5

20

30

40

55

65

75

Time (min) Fig. 3. Effects of 10 flmol/l guanethidine on insulin (IRI) and glucagon (IRG) secretion, in response to changes in the glucose concentration and tyramine, from the perfused rat pancreas. Shaded area depicts controls (n = 9) and solid lines with open circles depict data obtained with guanethidine in the perfusate (n = 6). Values are means f SEM.

K. Ito et al. /Diabetes

111

Research and Clinical Practice 30 (1995) 173-180

Table 1 Effects of 10 pmol/l guanethidine on glucagon (IRG) release in response to glucopenia (1.4 mmol/l) or tyramine (10 mg/l) from the perfused rat pancreas (baseline glucose consentration = 5.6 mM)

Glucopenia Control (n = Guanethidine Tyramine Control (n = Guanethidine *P
secretion rate IRG (ng/min)

Change IRG (ng/min)

Baseline IRG (ng/min)

Maximal

9) (n = 5)

1.18 * 0.12 0.70 f 0.09

2.44 k 0.30 2.55 f 0.50

1.24 & 0.23 1.51 +0.39

9) (n = 5)

0.70 f 0.08 0.53 f 0.04

1.91 f 0.16 0.83 k 0.10

1.21 kO.15 0.31 * 0.08*

vs. change in control.

min, P < 0.05). Guanethidine exerted no influence on high-glucose-inhibited and low-glucose-stimulated glucagon secretion, while it significantly suppressed the glucagon secretion in response to tyramine (Table 1). When the glucose concentration was raised from 1.4 to 5.6 mmol/l, insulin secretion was potentiated in the guanethidine-perfused group. 3.3. Eflect of guanethidine on endogenous norepinephrine release in response to glucopenia or tyramine from the perfused rat pancreas Glucopenia did significantly increase endogenous NE release from the perfused rat pancreas (Table 2). Tyramine evoked a marked increase in NE release and guanethidine significantly inhibited this NE release. However, guanethidine did not influence glucopenia-induced NE release.

4. Discussion Pharmacological blockade with hexamethonium, or a vagotomy with cervical spinal cord section, can suppress glucopenia-induced glucagon secretion in vivo [23]. Glucopenia by itself does not induce glucagon secretion from canine gastric a-cells [24] or purified rat a-cells [25]. Furthermore, only a small amount of glucagon is secreted from perifused rat islets in response to glucopenia [26]. The possibility that promoting factors are generated not only in vivo but also in the perfused pancreas, in response to glucopenia, which does not exist around gastric a-cells,

purified cc-cells or in the isolated islets, must be considered. In the present study, endogenously released NE was detectable even in the normoglycemic state, and a significant increase in NE release was seen in response to glucopenic stimulation. Tyramine actually induced NE release, from sympathetic nerve terminals, including those around g-cells, causing glucagon secretion [27]. However, .a large part of the tyramine-induced NE increase in the perfusate seems to come from sympathetic nerve terminals not associated with islets; apparently, numbers of sympathetic nerve terminals around acinar tissues or vessels far excells. Although ceed those around islet guanethidine caused NE leakage and the concentration of NE in the perfusate was higher than that in controls, in this study, the leaked NE did not induce glucagon secretion. Furthermore, the basal glucagon secretion significantly decreased after the addition of guanethidine. Thus, our data suggest that only a small part of the NE released from sympathetic nerve terminals, in response to tyramine, potentiated glucagon secretion, and a large part of the NE leakage caused by tyramine or guanethidine seems to have come from sympathetic nerve terminals unrelated to islet function. If there was a functional release of NE from the sympathetic nerve terminals in response to glucopenia, guanethidine should have reduced glucopenia-induced glucagon secretion. However, guanethidine did not reduce glucopenia-induced NE or glucagon release. These results contradict the hypothesis that the NE release which occurs in response to glucopenia in the perfused pancreas is from sympathetic nerve terminals around the CI-

178

K. Ito et al. 1 Diabetes Research and Clinical Practice 30 (1995) 173-180

Table 2 Effects of 10 pmol/l guanethidine on endogenous norepinephrine release in response to glucopenia (1.4 mmol/l) mg/l) from the perfused rat pancreas (NE, norepinephrine). Baseline glucose consentration = 5.6 mM

Glucopenia Control (n = Guanethidine Tyramine Control (n = Guanethidine *Pt0.05

or tyramine

Baseline NE (nmol/min)

Maximal

9) (n = 5)

0.53 * 0.12 1.60 + 0.35

1.06+0.18* 4.43 k 0.65*

0.59 rl: 0.18 2.42 k 0.71

9) (n = 5)

0.89 rf 0.24 3.61 f 0.35

50.4 k 2.36** 12.1 * 0.35**

49.1 k 2.3 7.86 f 0.77***

and **PiO.Ol

vs. basal secretion rate. ***P
cells. Although we used tyramine and guanethidine in this investigation, further study may be necessary to clarify these issues. Baetens et al. [28] reported that in pancreatic ganglia, small cells with catecholamine-like granules were often identified in clusters around fenestrated capillaries, and they speculated that these cells are a source of catecholamines, acting either as interneurons or neuroendocrine cells, as part of a local regulatory mechanism for islet hormone secretion. These cells might be a site which responds to glucopenia, independently of the central nervous system. Weigert et al. reported that glucopenia-induced glucagon secretion from the perfused rat pancreas is suppressed by the neurotoxin tetrodotoxin [29], suggesting that tetrodotoxin can suppress glucopenia-induced NE release from these cells. In the present study, we used yohimbine and prazosin as a-adrenergic antagonists instead of phentolamine, because phentolamine stimulates insulin release by itself. We have recently reported that exogenous insulin suppresses glucopenia-induced glucagon secretion in a dose-dependent manner from the perfused rat pancreas [30]. Plant et al. reported that yohimbine also inhibits ATPsensitive K + -channels, though the effect is much weaker than that of phentolamine [20]. However, the primary mechanism of the action of yohimbine is an a,-adrenoceptor blocking effect [31]. Thus, we speculate that glucose-stimulated insulin secretion was potentiated while basal glucagon secretion was suppressed by Y.P.P. or yohimbine alone because p- and ~-cells are continuously

secretion rate NE (nmol/min)

(10

Change NE (nmol/min)

vs. change in control.

influenced by endogenous NE. Yohimbine alone did not completely suppress (approximately 30%) the glucagon secretion induced by glucopenia, suggesting that the /I-adrenergic effect of endogenous NE also plays a role in stimulation of glucagon secretion. Our data do not agree with the results of Weigert et al. [29] in regards to the effect of adrenoceptor antagonists on basal glucagon secretion. The discrepancy may be due to differences in the perfusate, as we added pyruvate, fumarate and glutamate to the perfusate which Weigert et al. did not. To summarize, we have shown in this study that (1) adrenoceptor antagonists potentiate glucose-stimulated insulin secretion and suppress glucopenia-induced glucagon secretion, and that (2) glucopenia increases endogenous NE release from the perfused rat pancreas; the NE release inhibitor, guanethidine, inhibits tyramine-induced secretion of both endogenous NE and glucagon, while exerting no effects on glucopenia-induced secretion of either endogenous NE or glucagon. We conclude that these results further support the concept that the neurotransmitter NE plays an important role in modulating insulin and glucagon secretion. Our results do not, however, support the hypothesis that glucopenia-induced glucagon secretion is mainly mediated by activation of sympathetic nerve terminals around the a-cells in the isolated perfused rat pancreas. We would rather emphasize reduction of the inhibitory effect of insulin [32-351 on glucopenia-induced glucagon secretion in the in vitro pancreas.

K. Ito et al. I Diabetes Research and Clinical Practice 30 (1995) 173-180

Acknowledgements The authors thank Roger H. Unger, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, for critical review of the manuscript. We also thank Takashi Mamizuka, Shuji Oguchi and Eiko Takeshita (Clinical Laboratories, Keio University School of Medicine) for technical assistance with the radioimmunoassay and HPLC. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan, and by grants to KI and HH from Keio University School of Medicine, Tokyo, Japan. Part of this work was presented at the 54th Annual Meeting and Scientific Sessions of the American Diabetes Association in New Orleans, on June 14, 1994.

References [l] Unger, R.H., Eisentraut, A.M., McCall, MS. and Madison, L.L. (1962) Measurements of endogenous glucagon in plasma and the influence of blood glucose concentration upon the secretion. J. Clin. Invest. 41, 682-689. [2] Marliss, E.B., Girardier, L., Seydoux, J. et al. (1973) Glucagon release induced by pancreatic nerve stimulation in the dog. J. Clin. Invest. 52, 124661259. [3] Gerich, J.E., Langlois, M., Noacco, C., Karam, J. and Forsham, P. (1973) Lack of glucagon response to hypoglycemia in diabetes: evidence for an intrinsic alpha-cell defect. Science 182, 171-173. [4] Bloom, S.R., Edwards, A.V. and Vaughan, N.J.A. (1973) The role of sympathetic innervation in the control of plasma glucagon concentration in the calf. J. Physiol. 233, 457-466. [5] Gerich, J.E., Langlois, M., Noacco, C., Schneider, V. and Forsham, P.H. (1974) Adrenergic modulation of pancreatic glucagon secretion in man. J. Clin. Invest. 53, 14411446. [6] Palmer, J., Henry, D., Bensen, J., Johnson, D. and Ensinck, J. (1976) Glucagon response to hypoglycemia in sympathectomized man. J. Clin. Invest. 57, 5222525. [7] Benson, J., Johnson, D., Palmer, J., Werner, P. and Ensinck, J. (1977) Glucagon and catecholamine secretion during hypoglycemia in normal and diabetic man. J. Clin. Endocrinol. Metab. 44, 459-464. [8] Rizza, R., Cryer, P. and Gerich, J.E. (1979) Role of glucagon, catecholamines, and growth hormone in human glucose counterregulation: effects of somatostatin and combined OL-and P-adrenergic blockade on plasma glu-

119

case recovery and glucose flux rates following insulin-induced hypoglycemia. J. Clin. Invest. 64, 62-71. [91 Gerich, J.E., Davis, J., Lorenzi, M. et al. (1979) Hormonal mechanisms of recovery from insulin-induced hypoglycemia in man. Am. J. Physiol. 236, 380-385. 1101Cryer, P.E. and Gerich, J.E. (1990) Hypoglycemia in insulin-dependent diabetes mellitus: Insulin excess and defective glucose counterregulation. In: H. Rilkin. and D. Porte Jr. (Eds.), Diabetes mellitus: theory and practice. Fourth edition, Elsevier, New York, Amsterdam, London, pp. 5266546. 1111Cryer, P.E. (1992) Glucose homeostasis and hypoglycemia. In: J.D. Wilson and D.W. Foster (Eds.), Textbook of endocrinology. 8th Edition, W.B. Saunders Co., Philadelphia, London, Toronto, Montreal, Sydney, Tokyo, pp. 1223-1253. 1121Woods, S.C. and Porte, D. (1974) Neural control of the endocrine pancreas. Physiol. Rev. 54, 596-619. [I31 Woods, S.C. and Porte, D. (1978) The central nervous system, pancreatic hormones, feeding, and obesity. Adv. Metab. Dis. 9, 2822312. [I41 Havel, P.J., Veith, R.C., Dunning, B.E. and Taborsky, G.J. (1988) Pancreatic noradrenergic nerves are activated by neuroglucopenia but not by hypotension or hypoxia in the dog: evidence for stress-specific and regionally selective activation of the sympathetic nervous system. J. Clin. Invest. 82, 1538-1545. [I51 Hirose, H., Maruyama, H., Ito, K., Koyama, K., Kido, K. and Saruta, T. (1992) Alpha-2A adrenergic agonism stimulates islet glucagon release from perfused rat pancreas: possible involvement of alpha-2A adrenergic receptor subtype. Acta Endocrinol. 127, 279-283. 1161Hirose, H., Maruyama, H., Ito, K., Kido, K., Koyama, K. and Saruta, T. (1993) Effects of x2- and /3-adrenergic agonism on glucagon secretion from perfused pancreata of normal and streptozocin-induced diabetic rats. Metabolism 42, 1072-1076. [I71 Chiristensen, N.J. and Iversen, J. (1973) Release of large amount of noradrenaline from the isolated perfused canine pancreas during glucose deprivation. Diabetologia 9, 396-399. 1181Hisatomi, A., Maruyama, H., Orci, L., Vasko, M. and Unger, R.H. (1985) Adrenergically mediated intrapancreatic control of the glucagon response to glucopenia in the isolated rat pancreas. J. Clin. Invest. 75, 420-426. [I91 Samols, E. and Weir, G.C. (1979) Adrenergic modulation of pancreatic A, B, and D cells: a-Adrenergic suppression and /3-adrenergic stimulation of somatostatin secretion, I-adrenergic stimulation of glucagon secretion in the perfused dog pancreas. J. Clin. Invest. 63, 230-238. WY Plant, T.D. and Henquin, J.C. (1990) Phentolamine and yohimbine inhibit ATP-sensitive K+ channels in mouse pancreatic /?-cells. Br. J. Pharmacol. 101, 115-120. WI Grodsky, G.M. and Fanska, R.E. (1975) The in vitro perfused pancreas. Methods Enzymol. 39, 364-373. [-=I Koyama, K., Hirose, H., Maruyama, H., Ito, K., Kido, K. and Saruta, T. (1992) Arachidonic acid metabolites

180

[23]

[24]

[25]

[26]

[27]

[28]

[29]

K. Ito et al. 1 Diabetes Research and Clinical Practice 30 (1995) 173-180 and cc2-adrenoceptor-mediated glucagon secretion in rats. Diabetes Res. Clin. Pratt. 16, 229-232. Havel, P.J., Veith, R.C., Dunning, B.E. and Taborsky, G.J. (1991) Role for autonomic nervous system to increase pancreatic glucagon secretion during marked insulin-induced hypoglycemia in dogs. Diabetes 40, 1107-l 114. Matsuyama, T., Tanaka, R., Shima, K., Nonaka, K. and Tarui, S. (1978) Lack of gastrointestinal glucagon response to hypoglycemia in depancreatized dogs. Diabetologia 15, 471-474. Pipeleers, D.G., Schuit, F.C., Schravendijk, C.F.H.V. and Winkel, M.V.D. (1985) Interplay of nutrients and hormones in the regulation of glucagon release. Endocrinology 117, 8177823. Oliver, J.R., Williams, V.L. and Wright, P.H. (1976) Studies on glucagon secretion using isolated islets of Langerhans of the rat. Diabetologia 12, 301-306. Tominaga, M., Maruyama, H., Vasko, M., Baetens, D., Orci, L. and Unger, R.H. (1987) Morphologic and functional changes in sympathetic nerve relationships with pancreatic a-cells after destruction of B-cells in rats. Diabetes 36, 365-373. Baetens, D., Vasko, M., Unger, R.H. and Orci, L. (1985) Ultrastructural detection of granulated cells in the autonomic ganglia of the rat pancreas. Diabetologia 28, 841846. Weigert, N., Dollinger, M., Schmid, R. and Schusdziarra, V. (1992) Contribution of neural intrapancreatic non-

[30]

[31]

[32]

[33]

[34]

[35]

cholinergic non-adrenergic mechanisms to glucoseinduced insulin release in the isolated rat pancreas. Diabetologia 35, 113331139. Ito, K., Maruyama, H., Hirose, H. et al. (1995) Exogenous insulin dose-dependently suppresses glucopeniainduced glucagon secretion from perfused rat pancreas. Metabolism 44, 358-362. Ohneda, K., Ohneda, A. and Koizumi, F. (1993) Mechanism of insulin secretion by midaglizole. Diabetes Res. Clin. Pratt. 19, 1277132. Ito, K., Hirose, H., Maruyama, H., Fukamachi, S., Tashiro, Y. and Saruta, T. (1995) Neurotransmitters partially restore glucose sensitivity of insulin and glucagon secretion from perfused streptozotocin-induced-diabetic rat pancreas. Diabetologia, 38, 1276-1284. Maruyama, H., Hisatomi, A., Orci, L., Grodsky, G.M. and Unger, R.H. (1984) Insulin within islet is a physiologic glucagon release inhibitor. J. Clin. Invest. 74, 22962299. Samols, E., Stagner, J.I., Ewart, R.B.L. and Marks, V. (1988) The order of islet microvascular cellular perfusion is B --f A --t D in the perfused rat pancreas. J. Clin. Invest. 82, 350-353. Tominaga, M., Maruyama, H., Bolli, G., Helderman, J.H. and Unger, R.H. (1986) Simulation of the glucopenia-induced decline in insulin partially restores the glucagon response to glucopenia in isolated perfused pancreata of streptozotocin-diabetic rats. Endocrinology 118, 8866887.