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Neonatal capsaicin-treatment in mice: effects on pancreatic peptidergic nerves and 2-deoxy-d -glucose-induced insulin and glucagon secretion
Journal of the Autonomic Nervous System, 39 (1992) 51-60 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00
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JANS 01275
Neonatal capsaicin-treatment in mice: effects on pancreatic peptidergic nerves and 2-deoxy-D-glucose-induced insulin and glucagon secretion Sven Karlsson a, Frank Sundler Cand Bo Ahrdn a,b Departments of a Pharmacology, b Surgery and c Medical Cell Research, Lund University, Lund, Sweden (Received 4 December 1991) (Revision received 10 February 1992) (Accepted 3 March 1992)
Key words: Capsaicin-sensitive nerves; Pancreas; 2-Deoxy-D-glucose; Glucagon secretion; Insulin secretion; Glucose Abstract It is not known whether sensory nerves are involved in the insulin, glucagon or glucose responses to autonomic nerve activation induced by 2-deoxy-s-glucose (2-DG). We therefore treated mice neonatally with capsaicin which permanently destroys sensory afferent nerve fibers. Immunohistochemistry of the pancreas at 13-14 weeks of~age revealed a substantial reduction of calcitonin gene-related peptide (CGRP)-immunoreactive nerves and a partial reduction of substance P-immunoreactive nerves. In contrast, no effect was observed on galanin-immunoreactive nerves. At age 10-12 weeks, the mice were injected intravenously with 2-DG (500 mg/kg). In controls, 2-DG stimulated insulin and glucagon secretion and induced hyperglycemia ( P < 0.01). Capsaicin treatment partially reduced the glucose and glucagon responses to 2-DG ( P < 0.01). In contrast, the insulin response to 2-DG was not affected by capsaicin. It is concluded that the mouse pancreas contains capsaicin-sensitive sensory CGRP- and substance P-immunoreactive nerve fibers, whereas the galanin-immunoreactive nerve tibet's are not sensitive to capsaicin. Furthermore, capsaicin-sensitive sensory nerve fibers are partially involved in 2-DG-induced glucagon secretion and hyperglycemia, whereas sensory nerves are not involved in 2-DG-induced insulin secretion.
Introduction
The islets of Langerhans are innervated by cholinergic, adrenergic and peptidergic nerves and the secretion of insulin and glucagon may be under the influence of these nerves [2,20,31,34]. As a model to study this neural regulation of islet hormone secretion, the glucose analogue 2-deoxy-
Correspondence: S. Karlsson, Department of Pharmacology, S61vegatan 10, S-223 62 Lund, Sweden.
D-glucose (2-DG) is used [10-12,19,25]. 2-DG acts by competing with glucose for intra-cellular phosphorylation [6], which, in Mvo, activates the autonomic nervous system as a consequence of central neuroglycopenia [11,21,33]. This autonomic response also involves activation of pancreatic nerves [10]. In the mouse, 2-DG stimulates insulin and glucagon secretion by a mechanism that is largely cholinergic in its efferent pathway [16,17]. 2-DG also induces hyperglycemia and recently it was shown that this effect; as well as 2-DG-induced gastric acid secretion, is impaired
52 in rats treated neonatally with the neurotoxin capsaicin [9,35]. These results suggested that afferent sensory nerves are involved in these two responses to 2-DG, since capsaicin is known to destroy sensory afferent nerve fibers [4,15, for reviews]. However, whether capsaicin-sensitive sensory nerve fibers are involved in the effects of 2-DG also on glucagon and insulin secretion is not known. Therefore, in the present study, we investigated the effects of neonatal capsaicintreatment on the insulin, glucagon and glucose responses to 2-DG in mice. Furthermore, we investigated the effects of neonatal capsaicin treatment on calcitonin gene-related peptide (CGRP)-, substance P- and galanin-immunoreactive nerve fibers in the mouse pancreas.
Materials and Methods
Animals Female mice of the NMRI strain (Alab, Stockholm, Sweden) were used. The animals had free access to a standard pellet diet (Astra-Ewos, S6dert~ilje, Sweden) and tap water. Capsaicin (8methyl-N-vanillyl 6-nonenamide) (50 mg/kg) (Sigma Chemical Co., St. Louis, MO) was given subcutaneously to neonatal mice at the ages of two and five days, as previously described [26]. The volume load was 20 /a.1/animal. Capsaicin was dissolved in a vehicle containing 10% Tween 80, 10% ethanol in 0.9% saline. Control animals were given vehicle alone.
istry, using the indirect immunofluorescence procedure of Coons et al. [5]. In brief, sections were incubated with rabbit anti-rat a-CGRP antiserum (Code no 8427, Milab AB, Malm6, Sweden, working dilution 1:1280), rabbit anti-porcine galanin antiserum (code no RAGS 5777, dilution 1:640, a kind gift from Dr. B.E. Dunning, Sandoz Res Institute, East Hanover, N J) or rabbit anti-bovine substance P antiserum (code no. SP-7, dilution 1:320, kind gift from Dr. P.C. Emson, MCR, Cambridge, UK). The CGRP-antiserum does not cross-react with insulin, somatostatin or substance P [24,32]. The anti-galanin antiserum exhibits no cross-reactivity with insulin, glucagon, somatostatin, pancreatic polypeptide, or luteinizing hormone-releasing hormone, which is the peptide with the greatest homology with galanin currently known [7]. The anti-substance P antiserum does not cross-react with CGRP or with neurokinin A, although it cross-reacts with certain other tachykinins [32]. Fluorescein isothiocyanate (FITC)-labelled porcine antiserum against rabbit immunoglobulin G (IgG) was obtained from Dakopatts, Copenhagen, Denmark (working dilution 1:80). Control sections were incubated with each of the various antisera (in working dilution) preabsorbed with either synthetic rat a-CGRP, synthetic porcine galanin or synthetic porcine substance P at 10 tzg/ml of each (all three peptides were from Peninsula Labs, Belmont, MA). These control sections showed no immunoreactivity.
Experiments Immunocytochemistry Tissue specimens were taken from the pancreas at age 13-14 weeks and were fixed overnight in Stefanini's fixative (2% formaldehyde and 15% saturated solution of picric acid in 0.1 M phosphate-buffered saline, PBS). The tissue specimens were taken from the same animals that were previously subjected to autonomic nerve stimulation induced by 2-deoxy-D-glucose. After thorough rinsing in sucrose-enriched (10%) buffer, the specimens were frozen on dry ice. Sections were cut at 10 /~m in a cryostat and collected on chromalum-coated slides. The sections were then processed for immunocytochem-
Experiments were performed at 10-12 weeks of age. 2-DG (500 mg/kg) (Sigma Chemical Co., St Louis, MO) was injected intravenously to unanaesthetised mice. The volume load was 10 /a,1/g body weight. After either 2 or 10 min, blood (250 /.d) was collected from the retro-orbital venous plexus. Controls were injected with saline. The blood samples were centrifuged and plasma was removed and stored at -20°C.
Determination of insulin, glucagon and glucose Plasma levels of immunoreactive insulin were determined by radioimmunoassay, by the use of ~25I-labelled porcine insulin (Novo Res,
53
Fig. 1. Sections through mouse pancreas immunostained for calcitonin gene-related peptide (CGRP) (a and b), substance P (c) and galanin (d). (a) CGRP-immunoreactive nerve fibers (arrow) around blood vessel (v). (b) CGRP-immunoreactive nerve fibers (arrow) in the periphery of an islet (i). (e) Substance P-immunoreactive nerve fiber (arrow) in the exocrine parenchyma. (d) Galanin-immunoreactive nerve fibers (arrow) around blood vessel (v) ( × 200).
54 Bagsvaerd, Denmark), a guinea pig antibody raised against porcine insulin (Milab AB, Maim6, Sweden) and as a standard, porcine insulin (Novo Res Inst, Bagsvaerd, Denmark). The separation of bound and free radioactivity was performed
with the dextran-coated charcoal technique [13]. Plasma glucagon levels were determined by radioimmunoassay using samples of unextracted plasma as previously described [1]. Guinea pig anti-porcine glucagon antiserum, specific for pan-
Fig. 2. Section through mouse pancreas after neonatal capsaicin treatment showing nerve cell bodies within intrapancreatic ganglia immunostained for substance P (a) ( x 250) or galanin (b) ( × 200).
55 creatic glucagon g l u c a g o n a n d as a (Milab AB, Maim6, glucose levels w e r e o x i d a s e m e t h o d [3].
and t25I-labelled porcine standard, porcine glucagon S w e d e n ) , w e r e used. P l a s m a d e t e r m i n e d with t h e glucose
100
I 2 rain I I'---VEH--I--" CA P "- I
E c::
Statistics M e a n s + S.E.M. a r e shown. S t u d e n t ' s t-test for u n p a i r e d d a t a was u s e d for statistical evaluation o f t h e in vivo e x p e r i m e n t s .
1 10 rain I I~VEH'-'-F- CAP "-I ~--- n.s.---~,
50
In c m
Results
3000
Pancreatic CGRP-, substance P- and galanin-immunoreactivity In t h e p a n c r e a s o f c o n t r o l mice, C G R P - i m m u n o r e a c t i v e n e r v e fibers w e r e n u m e r o u s a r o u n d b l o o d vessels. In a d d i t i o n , a m o d e r a t e n u m b e r o f s c a t t e r e d fibers w e r e s e e n in t h e e x o c r i n e p a r e n c h y m a a n d in t h e islets. I n t r a - i s l e t C G R P i m m u n o r e a c t i v e fibers p r e d o m i n a t e d in t h e p e r i p h e r a l p a r t s o f t h e islets a n d only o c c a s i o n a l l y w e r e C G R P - i m m u n o r e a c t i v e fibers s e e n in t h e c e n t r a l part. S u b s t a n c e P- a n d g a l a n i n - i m m u n o r e a c t i v e fibers w e r e fairly n u m e r o u s a r o u n d b l o o d vessels a n d a few single fibers w e r e also s e e n in t h e e x o c r i n e p a r e n c h y m a a n d within, o r close to, islets (Fig. 1). C a p s a i c i n c a u s e d a s u b s t a n t i a l r e d u c t i o n in p a n c r e a t i c C G R P - i m m u n o r e a c t i v e nerves ( T a b l e
TABLE I Effects of neonatal capsaicin treatment on CGRP-, substance Pand galanin-immunoreactive nerve fibers in the mouse pancreas Type of nerve fiber
Capsaicin treated
Controls
CGRP Substance P Galanin
(+) + +
+++ ++ +
Mice were pretreated neonatally with capsaicin (50 mg/kg × 2) or vehicle alone (controls). At age 13-14 weeks pancreata were examined for evaluation of the occurrence of CGRP-, substance P- and galanin-immunoreactive nerve fibers. +++ = numerous nerve fibers; ++= moderate numbers of nerve fibers; + = few nerve fibers and (+) = occasional nerve fibers. Ten sections of each pancreas from 4 capsaicin-treated animals and from 4 vehicle-treated animals were examined.
~--- n.s.---~
{~P<0.01 1
2000 C 0
1000
--] Controts [ ~ 2-DG Fig. 3. Plasma insulin or glucagon levels in mice at 2 or 10 min after an intravenous injection of either 2-deoxy-D-glucose (2-DG) (500 mg/kg) or saline (controls). Mice were treated neonatally with either capsaicin (CAP) or with vehicle alone (VEH). Means+ S.E.M. are shown. There were 7-13 animals in each group, n.s. indicates lack of statistical significance (P > 0.05) in plasma insulin or glucagon levels after 2-DG-injection in capsaicin-treated vs. vehicle-treated controls. P < 0.01 indicates probability level of random difference in plasma glucagon levels at 10 min after 2-DG-injection in capsaicintreated animals vs. vehicle-treated controls. I). A l s o a r e d u c t i o n by c a p s a i c i n o f s u b s t a n c e P - i m m u n o r e a c t i v e nerves was o b s e r v e d , t h o u g h a few fibers r e m a i n e d . G a l a n i n - i m m u n o r e a c t i v e nerves r e m a i n e d virtually u n a f f e c t e d a f t e r c a p saicin t r e a t m e n t ( T a b l e I). B o t h s u b s t a n c e P- a n d g a l a n i n - i m m u n o r e a c t i v e nerve cell b o d i e s w e r e o b s e r v e d in i n t r a p a n c r e a t i c g a n g l i a in c o n t r o l s as well as in c a p s a i c i n - t r e a t e d a n i m a l s (Fig. 2). In contrast, no i n t r a p a n c r e a t i c ganglia c o n t a i n i n g C G R P - i m m u n o r e a c t i v e n e r v e cell b o d i e s w e r e observed. Plasma insulin 2 - D G i n c r e a s e d t h e p l a s m a insulin levels at 2 a n d 10 min a f t e r injection ( P < 0.01). This in-
56 TABLE II
Effects of capsaicin treatment on plasma glucose let,els after 2-deoxy-D-glucose (2-DG)-induced neuroglycopenia Time after 2-DG
Plasma glucose ( m m o l / l )
(min)
Capsaicin-treated
Vehicle-treated
2 10
12.9_+0.5 ** 15.5+0.4 *
15.5+_0.5 17.3+_0.5
Plasma glucose levels at 2 or 10 min after an intravenous injection of 2-DG (500 m g / k g ) in mice treated neonatally with capsaicin or vehicle. * P < 0.05 and ** P < 0.01 indicate probability level of random difference in plasma glucose levels in capsaicin-treated vs. vehicle-treated control animals. There were 7-13 animals in each group.
crease was not significantly affected by capsaicin treatment. Basal plasma insulin levels were not affected by capsaicin treatment (Fig. 3).
Plasma glucagon 2-DG increased the plasma glucagon levels at 2 and 10 min ( P < 0.001). Capsaicin treatment markedly diminished the 2-DG-induced increase in plasma glucagon levels observed at 10 min ( P < 0.01), but did not affect plasma glucagon levels at 2 min after 2-DG (Fig. 3).
Plasma glucose Plasma glucose levels were increased by 2-DG at both 2 and 10 min after injection ( P < 0.001). The hyperglycemic response was significanly lower in capsaicin-treated animals compared to controls ( P < 0.05). Capsaicin treatment did not affect the basal plasma glucose levels (Table II).
Discussion
The present study demonstrates that the mouse pancreas contains capsaicin-sensitive sensory nerve fibers, since most of the CGRP- and part of the substance P-immunoreactive nerve fibers disappeared after neonatal capsaicin treatment. These findings are in accordance with previous studies in the rat [27,28,30]. The reduction in the number of substance P-immunoreactive nerves induced by capsaicin pretreatment was less pro-
nounced than the reduction of CGRP-immunoreactive nerves. This also indicates the presence of substance P nerve fibers that are not sensitive to capsaicin treatment. The nature of this capsaicin-insensitive subpopulation of pancreatic substance P-immunoreactive nerves remains to be established. It has previously been demonstrated that most of the pancreatic galanin-immunoreactive nerve fibers are adrenergic [18]. We show here that the galanin-immunoreactive nerves remained virtually unaffected by capsaicin treatment. This confirms a previous study in the rat [30] and shows that the galanin-immunoreactive pancreatic nerve fibers are not capsaicin-sensitive. In the present study we also found that after capsaicin treatment, nerve cell bodies immunoreactive for substance P and galanin could be observed in local intrapancreatic ganglia, implying the existence of substance P- and galanin-immunoreactive nerve fibers of intrinsic origin in the mouse pancreas. The hyperglycemic response to autonomic nerve activation by 2-DG was partially impaired in capsaicin-treated mice, which is in accordance with a previous study in the rat [35]. This finding is also in line with a prevous study by Zhou and co-workers in which a delayed recovery of plasma glucose levels after insulin-induced hypoglycemia was demonstrated in capsaicin-treated rats [36]. In the present study we demonstrate also that the glucagon response to 2-DG is markedly inhibited in capsaicin-treated mice. This latter finding suggests that the impaired glucose response might be due in part to the reduced glucagon response. However, capsaicin treatment reduced the hyperglycemic response to 2-DG at both 2 and 10 min after 2-DG, whereas the glucagon response was inhibited by capsaicin treatment only at 10 min. This shows that capsaicin-sensitive nerves regulate plasma glucose levels after 2-DG by other mechanisms as well as through glucagon. Such a mechanism could involve, for example, hepatic glucose output via sympathetic nerves, which are known to be involved in 2-DG-induced hyperglycemia [29]. The increased plasma glucagon levels at 2 min after 2-DG seem to be regulated in a different way than those at 10 min. Hence, only at 10 min,
57 the plasma glucagon levels were partially reduced by capsaicin-treatement, whereas at 2 min, capsaicin had no effect. This shows that the glucagon response to 2-DG is mediated by at least two mechanisms, one of which is sensitive to capsaicin and thus most likely mediated by sensory afferent nerves. The general view of the mechanism whereby 2-DG induces hyperglycemia and stimulates islet hormone secretion is that the glucose analogue induces central neuroglycopenia which, in turn, activates the efferent autonomic output from the central nervous system [21]. The impaired glucagon and glucose responses to 2-DG after capsaicin as well as the results of a previous study [35] suggest that capsaicin-sensitive afferent nerve fibers are involved in the autonomic nerve activation by 2-DG. It has previously been shown that neonatal capsaicin treatment in the rat abolishes the increase in gastric acid secretion in response to 2-DG [9]. Although it has been speculated that sensory neurons in the hypothalmus might explain this phenomenon, there is no known (to us) experimental evidence for the presence of such fibers in the hypothalamus. It is therefore plausible that peripheral capsaicin-sensitive afferent nerve fibers are involved in the autonomic neural response to 2-DG. It has previously been reported that peripheral glucose-sensitive receptors exist in the liver [8,23]. Thus, 2-DG has previously been demonstrated to increase the vagal afferent discharge from the perfused guinea pig liver [23]. The possibility therefore exists that part of the hyperglycemic reponse to 2-DG is due to an effect on peripheral glucoceptors in the liver, which, via capsaicin-sensitive nerve fibers, transmit afferent signals to the central nervous system. However, since a large part of the glucose response to 2-DG was not affected by capsaicin treatment, the major mechanism behind 2-DG-stimulated hyperglycemia is most likely to be induction of central neuroglycopenia and an ensuing autonomic efferent activation without the involvement of capsaicin-sensitive sensory nerves. Regarding the glucagon response to 2-DG, a similar dual mechanism of 2-DG with a partial effect on peripheral glucoreceptors and activation
via afferent capsaicin-sensitive nerve fibers might be suggested. Indeed, it has previously been shown that systemic injection of 2-DG increases vagal afferent activity from the rabbit pancreas [22]. The involvement of pancreatic sensory neural mechanisms in the glucagon response to 2-DG could be supported by the immunohistochemical findings of the present study showing a reduction of CGRP- and substance P-immunoreactive pancreatic nerve fibers following capsaicin treatment. Thus, it might be speculated that neuropeptides from capsaicin-sensitive nerve fibers within the pancreas exert local effector functions as described for such nerves within other tissues [14]. Neuropeptides released from such capsaicin-sensitive nerves could thereby modulate glucagon secretion directly by its effects on the islet ceils or indirectly by affecting pancreatic neural activity or pancreatic blood flow. An indirect effect on neural activity could theoretically reside in activation of afferent neural signals to the central nervous system with a resulting activation of efferent cholinergic and adrenergic nerves. Such a hypothesis might be supported by previous studies demonstrating that the glucagon response to 2-DG is abolished by either ganglionic, nicotinic or muscarinic antagonists and is partially inhibited by a-adrenergic antagonists [16]. Whereas capsaicin pretreatment inhibited the glucagon response to 2-DG, the treatment had no effect on the insulin response. Hence, in contrast to the glucagon response, the insulin secretory reponse to 2-DG in mice does not involve capsaicin-sensitive sensory nerves. Thus, differences in the regulation of 2-DG-induced insulin and glucagon secretion exist. In conclusion, the present study has shown that 2-DG-induced glucagon secretion and 2DG-induced hyperglycemia are partially dependent on intact capsaicin-sensitive sensory nerve fibers. In contrast, 2-DG-induced insulin secretion is not dependent on such nerves. Furthermore, the study also shows that the mouse pancreas contains CGRP- and substance P-immunoreactive capsaicin-sensitive sensory nerve fibers and that the pancreatic galanin-immunoreactive nerve fibers are not capsaicin-sensitive.
58
Acknowledgements The technical assistance of Lena Kvist, Lilian Bengtsson and Doris Persson is gratefully acknowledged. The study was financially supported by The Swedish Medical Research Council (grants No 14X-6834, 17P-8453 and 4499), Nordisk Insulinfond, Swedish Diabetes Association, Diabetesf6reningen i Malm6, Swedish Hoechst Diabetes Fund, The Swedish Medical Society, The Swedish Society for Medical Research, Craafordska and Albert P[ihlssons Foundations, and by the Faculty of Medicine, Lund University.
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References 1 Ahr6n, B. and Lundquist, I., Glucagon immunoreactivity in plasma from normal and dystrophic mice, Diabetologia, 22 (1982) 258-263. 2 Ahr6n, B., Taborsky, G.J., Jr. and Porte, D., Jr., Neuropeptidergic versus cholinergic and adrenergic regulation of islet hormone secretion, Diabetologia, 29 (1986) 827836. 3 Bruss, M.L. and Black, A.L., Enzymatic microdetermination of glycogen, Analyt. Biochem., 84 (1978) 309-312. 4 Buck, S.H. and Burks, T.F., The neuropharmacology of capsaicin: review of some recent observations, Pharmacol. Rev., 38 (1986) 179-226. 5 Coons, A.H., Leduc, E.H. and Connolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit, J. Exp. Med., 102 (1955) 49-59. 6 Cramer, F.B. and Woodward, G.E., 2-Deoxy-o-glucose as an antagonist of glucose in yeast fermentation, J. Franklin. Inst., 253 (1952) 354-360. 7 Dunning B.E. and Taborsky G.J. Jr., Galanin release during pancreatic nerve stimulation is sufficient to influence islet function, Am. J. Physiol., 256 (1989) E191-E198. 8 Donovan, C.M., Halter J.B. and Bergman, R.N., Importance of hepatic glucoreceptors in sympathoadrenal response to hypoglycemia, Diabetes, 40 (1991) 155-158. 9 Evangelista, S., Santicioli, P., Maggi, C.A. and Meli, A., Increase in gastric secretion induced by 2-deoxy-D-glucose is impaired in capsaicin pretreated rats, Br. J. Pharmacol., 98 (1989) 35-37. 10 Havel, P.J., Veith, R.C., Dunning, B.E. and Taborsky, G.J., Jr., Pancreatic noradrenergic nerves are activated by neuroglycopenia but not by hypotension or hypoxia in the dog. Evidence for stress-specific regionally selective activation of the sympathetic nervous system, J. Clin. Invest., 82 (1988) 1538-1545. 11 Havel, P.J. and Taborsky, G.J., Jr., The contribution of the
17
18
19
20
21
22 23
24
25
26
27
autonomic nervous system to changes of glucagon and insulin secretion during hypoglycemic stress, Endocrine Rev., 10 (1989) 332-350. Hedo, J.A., Vitlanueva, M.L. and Marco, J., Stimulation of pancreatic polypeptide and glucagon secretion by 2-deoxyD-glucose in man: evidence for cholinergic mediation, J. Clin. Endocrinol. Metab., 47 (1978) 366-371. Herbert, V., Lau, K.S., Gottlieb, C.W. and Bleicher, S.J., Coated charcoal immunoassay of insulin. J. Clin. Endocrinol. Metab., 25 (1965) 1875-1884. Holzer P., Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides, Neuroscience, 24 (1988) 739-768. Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 143-201. Karlsson, S. and Ahr4n, B., Inhibition of 2-dexoxyglucose-induced glucagon secretion by muscarinic and t~adrenoceptor blockade in the mouse, Diabet. Res. Clin. Pract., 3 (1987) 239-242. Karlsson, S., Bood, M. and Ahr4n, B., The mechanism of 2-deoxy-glucose-induced insulin secretion in the mouse, J. Autonom. Pharmacol., 7 (1987) 135-144. Lindskog, S., Ahr6n, B., Dunning, B.E. and Sundler, F., Galanin-immunoreactive nerves in the mouse and rat pancreas, Celt Tiss. Res., 264 (1991) 363-368. Matsunaga, H., Iguchi, A., Yatomi, A., Uemura, K., Miura, H., Gotoh, M., Mano, T. and Sakamoto, N., The relative importance of nervous system and hormones to the 2-deoxy-D-glucose-induced hyperglycemia in fed rats, Endocrinology, 124 (1989) 1259-1264. Miller, R.E., Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the islets of Langerhans, Endocrine Rev., 2 (1981) 471-494. Muller, E.E., Cocchi, D. and Forni, A., A central site for the hyperglycemic action of 2-deoxy-D-glucose in mouse and rat, Life. Sci., 10 (1971) 1057-1067. Niijima, A., Visceral afferents and metabolic function, Diabetologia, 20 (1981) 325-330. Niijima, A., Glucose-sensitive afferent nerve fibers in the hepatic branch of the vagus nerve in the guinea-pig, J. Physiol., 332 (1982) 315-323. Pettersson, M., Ahr4n, B., B6ttcher, G. and Sundler, F., Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse, Endocrinology, 119 (1986)865-869. Raghu, P.K., Taborsky, G.J., Jr., Paquette, T.L., Halter, J.B. and Palmer, J.P., Evidence for noncholinergic ganglionic neural stimulation of B cell secretion, Am. J. Physiol., 247 (1984) E265-E270. Scadding, J.W., The permanent anatomical effects of neonatal capsaicin on somatosensory nerves, J. Anat., 131 (1980) 473-484. Sharkey, K.A., Williams, R.G. and Dockray G.J., Sensory substance P innervation of the stomach and pancreas, Gastroenterology, 87 (1984) 914-921.
59 28 Sternini, C. and Brecha, N., Immunocytochemical identification of islet cells and nerve fibers containing calcitonin gene-related peptide-like immunoreactivity in the rat pancreas, Gastroenterology, 90 (1986) 1155-1163. 29 Storlien, L.H., Grunstein, H.S. and Smythe, G.A., Guanethidine blocks the 2-deoxy-D-glucose-induced hypothalamic noradrenergic drive to hypoglycemia, Brain Res., 335 (1985) 144-147. 30 Su, N.C., Bishop, A.E., Power, R.F., Hamada, Y. and Polak, J.M., Dual intrinsic and extrinsic origins of CGRPand NPY-immunoreactive nerves of rat gut and pancreas, J. Neurosci., 7 (1987) 2674-2687. 31 Sundler, F. and B6ttcher, G., Islet innervation, with special reference to neuropeptides. In E. Samols, (Ed.), The endocrine pancreas, Raven Press, New York, 1991, pp. 29-52. 32 Sundler, F, Brodin, E., Ekblad, E., H~kansson, R. and Uddman R., Sensory nerve fibers: distribution of sub-
33
34 35
36
stance P, neurokinin A and calcitonin gene-related peptide. In R. H~ikansson and F. Sundler (Eds.), Tachykinin antagonists, Elsevier, Amsterdam, 1985, pp. 3-14. Taborsky, G.J., Jr., Halter, J.B. and Porte, D., Jr., Morphine suppresses plasma catecholamine responses to laparatomy but not to 2-deoxy-glucose, Am. J. Physiol., 242 (1982) E317-322. Woods, S. and Porte, D., Jr., Neural control of the endocrine pancreas, Physiol. Rev., 54 (1974) 596-619. Zhou, X.F. and Livett, B.G., Effect of capsaicin-sensitive sensory nerves on plasma glucose and catecholamine levels during 2-deoxy-glucose-induced stress in conscious rats, Br. J. Pharmacol., 100 (1990) 523-529. Zhou, X.F., Jhamadans, K.H. and Livett, B.G., Capsaicinsensitive nerves are required for glucostasis but not for catecholamine output during hypoglycemia in rats, Am. J. Physiol. 258 (1990) E212-E219.
51
JANS 01275
Neonatal capsaicin-treatment in mice: effects on pancreatic peptidergic nerves and 2-deoxy-D-glucose-induced insulin and glucagon secretion Sven Karlsson a, Frank Sundler Cand Bo Ahrdn a,b Departments of a Pharmacology, b Surgery and c Medical Cell Research, Lund University, Lund, Sweden (Received 4 December 1991) (Revision received 10 February 1992) (Accepted 3 March 1992)
Key words: Capsaicin-sensitive nerves; Pancreas; 2-Deoxy-D-glucose; Glucagon secretion; Insulin secretion; Glucose Abstract It is not known whether sensory nerves are involved in the insulin, glucagon or glucose responses to autonomic nerve activation induced by 2-deoxy-s-glucose (2-DG). We therefore treated mice neonatally with capsaicin which permanently destroys sensory afferent nerve fibers. Immunohistochemistry of the pancreas at 13-14 weeks of~age revealed a substantial reduction of calcitonin gene-related peptide (CGRP)-immunoreactive nerves and a partial reduction of substance P-immunoreactive nerves. In contrast, no effect was observed on galanin-immunoreactive nerves. At age 10-12 weeks, the mice were injected intravenously with 2-DG (500 mg/kg). In controls, 2-DG stimulated insulin and glucagon secretion and induced hyperglycemia ( P < 0.01). Capsaicin treatment partially reduced the glucose and glucagon responses to 2-DG ( P < 0.01). In contrast, the insulin response to 2-DG was not affected by capsaicin. It is concluded that the mouse pancreas contains capsaicin-sensitive sensory CGRP- and substance P-immunoreactive nerve fibers, whereas the galanin-immunoreactive nerve tibet's are not sensitive to capsaicin. Furthermore, capsaicin-sensitive sensory nerve fibers are partially involved in 2-DG-induced glucagon secretion and hyperglycemia, whereas sensory nerves are not involved in 2-DG-induced insulin secretion.
Introduction
The islets of Langerhans are innervated by cholinergic, adrenergic and peptidergic nerves and the secretion of insulin and glucagon may be under the influence of these nerves [2,20,31,34]. As a model to study this neural regulation of islet hormone secretion, the glucose analogue 2-deoxy-
Correspondence: S. Karlsson, Department of Pharmacology, S61vegatan 10, S-223 62 Lund, Sweden.
D-glucose (2-DG) is used [10-12,19,25]. 2-DG acts by competing with glucose for intra-cellular phosphorylation [6], which, in Mvo, activates the autonomic nervous system as a consequence of central neuroglycopenia [11,21,33]. This autonomic response also involves activation of pancreatic nerves [10]. In the mouse, 2-DG stimulates insulin and glucagon secretion by a mechanism that is largely cholinergic in its efferent pathway [16,17]. 2-DG also induces hyperglycemia and recently it was shown that this effect; as well as 2-DG-induced gastric acid secretion, is impaired
52 in rats treated neonatally with the neurotoxin capsaicin [9,35]. These results suggested that afferent sensory nerves are involved in these two responses to 2-DG, since capsaicin is known to destroy sensory afferent nerve fibers [4,15, for reviews]. However, whether capsaicin-sensitive sensory nerve fibers are involved in the effects of 2-DG also on glucagon and insulin secretion is not known. Therefore, in the present study, we investigated the effects of neonatal capsaicintreatment on the insulin, glucagon and glucose responses to 2-DG in mice. Furthermore, we investigated the effects of neonatal capsaicin treatment on calcitonin gene-related peptide (CGRP)-, substance P- and galanin-immunoreactive nerve fibers in the mouse pancreas.
Materials and Methods
Animals Female mice of the NMRI strain (Alab, Stockholm, Sweden) were used. The animals had free access to a standard pellet diet (Astra-Ewos, S6dert~ilje, Sweden) and tap water. Capsaicin (8methyl-N-vanillyl 6-nonenamide) (50 mg/kg) (Sigma Chemical Co., St. Louis, MO) was given subcutaneously to neonatal mice at the ages of two and five days, as previously described [26]. The volume load was 20 /a.1/animal. Capsaicin was dissolved in a vehicle containing 10% Tween 80, 10% ethanol in 0.9% saline. Control animals were given vehicle alone.
istry, using the indirect immunofluorescence procedure of Coons et al. [5]. In brief, sections were incubated with rabbit anti-rat a-CGRP antiserum (Code no 8427, Milab AB, Malm6, Sweden, working dilution 1:1280), rabbit anti-porcine galanin antiserum (code no RAGS 5777, dilution 1:640, a kind gift from Dr. B.E. Dunning, Sandoz Res Institute, East Hanover, N J) or rabbit anti-bovine substance P antiserum (code no. SP-7, dilution 1:320, kind gift from Dr. P.C. Emson, MCR, Cambridge, UK). The CGRP-antiserum does not cross-react with insulin, somatostatin or substance P [24,32]. The anti-galanin antiserum exhibits no cross-reactivity with insulin, glucagon, somatostatin, pancreatic polypeptide, or luteinizing hormone-releasing hormone, which is the peptide with the greatest homology with galanin currently known [7]. The anti-substance P antiserum does not cross-react with CGRP or with neurokinin A, although it cross-reacts with certain other tachykinins [32]. Fluorescein isothiocyanate (FITC)-labelled porcine antiserum against rabbit immunoglobulin G (IgG) was obtained from Dakopatts, Copenhagen, Denmark (working dilution 1:80). Control sections were incubated with each of the various antisera (in working dilution) preabsorbed with either synthetic rat a-CGRP, synthetic porcine galanin or synthetic porcine substance P at 10 tzg/ml of each (all three peptides were from Peninsula Labs, Belmont, MA). These control sections showed no immunoreactivity.
Experiments Immunocytochemistry Tissue specimens were taken from the pancreas at age 13-14 weeks and were fixed overnight in Stefanini's fixative (2% formaldehyde and 15% saturated solution of picric acid in 0.1 M phosphate-buffered saline, PBS). The tissue specimens were taken from the same animals that were previously subjected to autonomic nerve stimulation induced by 2-deoxy-D-glucose. After thorough rinsing in sucrose-enriched (10%) buffer, the specimens were frozen on dry ice. Sections were cut at 10 /~m in a cryostat and collected on chromalum-coated slides. The sections were then processed for immunocytochem-
Experiments were performed at 10-12 weeks of age. 2-DG (500 mg/kg) (Sigma Chemical Co., St Louis, MO) was injected intravenously to unanaesthetised mice. The volume load was 10 /a,1/g body weight. After either 2 or 10 min, blood (250 /.d) was collected from the retro-orbital venous plexus. Controls were injected with saline. The blood samples were centrifuged and plasma was removed and stored at -20°C.
Determination of insulin, glucagon and glucose Plasma levels of immunoreactive insulin were determined by radioimmunoassay, by the use of ~25I-labelled porcine insulin (Novo Res,
53
Fig. 1. Sections through mouse pancreas immunostained for calcitonin gene-related peptide (CGRP) (a and b), substance P (c) and galanin (d). (a) CGRP-immunoreactive nerve fibers (arrow) around blood vessel (v). (b) CGRP-immunoreactive nerve fibers (arrow) in the periphery of an islet (i). (e) Substance P-immunoreactive nerve fiber (arrow) in the exocrine parenchyma. (d) Galanin-immunoreactive nerve fibers (arrow) around blood vessel (v) ( × 200).
54 Bagsvaerd, Denmark), a guinea pig antibody raised against porcine insulin (Milab AB, Maim6, Sweden) and as a standard, porcine insulin (Novo Res Inst, Bagsvaerd, Denmark). The separation of bound and free radioactivity was performed
with the dextran-coated charcoal technique [13]. Plasma glucagon levels were determined by radioimmunoassay using samples of unextracted plasma as previously described [1]. Guinea pig anti-porcine glucagon antiserum, specific for pan-
Fig. 2. Section through mouse pancreas after neonatal capsaicin treatment showing nerve cell bodies within intrapancreatic ganglia immunostained for substance P (a) ( x 250) or galanin (b) ( × 200).
55 creatic glucagon g l u c a g o n a n d as a (Milab AB, Maim6, glucose levels w e r e o x i d a s e m e t h o d [3].
and t25I-labelled porcine standard, porcine glucagon S w e d e n ) , w e r e used. P l a s m a d e t e r m i n e d with t h e glucose
100
I 2 rain I I'---VEH--I--" CA P "- I
E c::
Statistics M e a n s + S.E.M. a r e shown. S t u d e n t ' s t-test for u n p a i r e d d a t a was u s e d for statistical evaluation o f t h e in vivo e x p e r i m e n t s .
1 10 rain I I~VEH'-'-F- CAP "-I ~--- n.s.---~,
50
In c m
Results
3000
Pancreatic CGRP-, substance P- and galanin-immunoreactivity In t h e p a n c r e a s o f c o n t r o l mice, C G R P - i m m u n o r e a c t i v e n e r v e fibers w e r e n u m e r o u s a r o u n d b l o o d vessels. In a d d i t i o n , a m o d e r a t e n u m b e r o f s c a t t e r e d fibers w e r e s e e n in t h e e x o c r i n e p a r e n c h y m a a n d in t h e islets. I n t r a - i s l e t C G R P i m m u n o r e a c t i v e fibers p r e d o m i n a t e d in t h e p e r i p h e r a l p a r t s o f t h e islets a n d only o c c a s i o n a l l y w e r e C G R P - i m m u n o r e a c t i v e fibers s e e n in t h e c e n t r a l part. S u b s t a n c e P- a n d g a l a n i n - i m m u n o r e a c t i v e fibers w e r e fairly n u m e r o u s a r o u n d b l o o d vessels a n d a few single fibers w e r e also s e e n in t h e e x o c r i n e p a r e n c h y m a a n d within, o r close to, islets (Fig. 1). C a p s a i c i n c a u s e d a s u b s t a n t i a l r e d u c t i o n in p a n c r e a t i c C G R P - i m m u n o r e a c t i v e nerves ( T a b l e
TABLE I Effects of neonatal capsaicin treatment on CGRP-, substance Pand galanin-immunoreactive nerve fibers in the mouse pancreas Type of nerve fiber
Capsaicin treated
Controls
CGRP Substance P Galanin
(+) + +
+++ ++ +
Mice were pretreated neonatally with capsaicin (50 mg/kg × 2) or vehicle alone (controls). At age 13-14 weeks pancreata were examined for evaluation of the occurrence of CGRP-, substance P- and galanin-immunoreactive nerve fibers. +++ = numerous nerve fibers; ++= moderate numbers of nerve fibers; + = few nerve fibers and (+) = occasional nerve fibers. Ten sections of each pancreas from 4 capsaicin-treated animals and from 4 vehicle-treated animals were examined.
~--- n.s.---~
{~P<0.01 1
2000 C 0
1000
--] Controts [ ~ 2-DG Fig. 3. Plasma insulin or glucagon levels in mice at 2 or 10 min after an intravenous injection of either 2-deoxy-D-glucose (2-DG) (500 mg/kg) or saline (controls). Mice were treated neonatally with either capsaicin (CAP) or with vehicle alone (VEH). Means+ S.E.M. are shown. There were 7-13 animals in each group, n.s. indicates lack of statistical significance (P > 0.05) in plasma insulin or glucagon levels after 2-DG-injection in capsaicin-treated vs. vehicle-treated controls. P < 0.01 indicates probability level of random difference in plasma glucagon levels at 10 min after 2-DG-injection in capsaicintreated animals vs. vehicle-treated controls. I). A l s o a r e d u c t i o n by c a p s a i c i n o f s u b s t a n c e P - i m m u n o r e a c t i v e nerves was o b s e r v e d , t h o u g h a few fibers r e m a i n e d . G a l a n i n - i m m u n o r e a c t i v e nerves r e m a i n e d virtually u n a f f e c t e d a f t e r c a p saicin t r e a t m e n t ( T a b l e I). B o t h s u b s t a n c e P- a n d g a l a n i n - i m m u n o r e a c t i v e nerve cell b o d i e s w e r e o b s e r v e d in i n t r a p a n c r e a t i c g a n g l i a in c o n t r o l s as well as in c a p s a i c i n - t r e a t e d a n i m a l s (Fig. 2). In contrast, no i n t r a p a n c r e a t i c ganglia c o n t a i n i n g C G R P - i m m u n o r e a c t i v e n e r v e cell b o d i e s w e r e observed. Plasma insulin 2 - D G i n c r e a s e d t h e p l a s m a insulin levels at 2 a n d 10 min a f t e r injection ( P < 0.01). This in-
56 TABLE II
Effects of capsaicin treatment on plasma glucose let,els after 2-deoxy-D-glucose (2-DG)-induced neuroglycopenia Time after 2-DG
Plasma glucose ( m m o l / l )
(min)
Capsaicin-treated
Vehicle-treated
2 10
12.9_+0.5 ** 15.5+0.4 *
15.5+_0.5 17.3+_0.5
Plasma glucose levels at 2 or 10 min after an intravenous injection of 2-DG (500 m g / k g ) in mice treated neonatally with capsaicin or vehicle. * P < 0.05 and ** P < 0.01 indicate probability level of random difference in plasma glucose levels in capsaicin-treated vs. vehicle-treated control animals. There were 7-13 animals in each group.
crease was not significantly affected by capsaicin treatment. Basal plasma insulin levels were not affected by capsaicin treatment (Fig. 3).
Plasma glucagon 2-DG increased the plasma glucagon levels at 2 and 10 min ( P < 0.001). Capsaicin treatment markedly diminished the 2-DG-induced increase in plasma glucagon levels observed at 10 min ( P < 0.01), but did not affect plasma glucagon levels at 2 min after 2-DG (Fig. 3).
Plasma glucose Plasma glucose levels were increased by 2-DG at both 2 and 10 min after injection ( P < 0.001). The hyperglycemic response was significanly lower in capsaicin-treated animals compared to controls ( P < 0.05). Capsaicin treatment did not affect the basal plasma glucose levels (Table II).
Discussion
The present study demonstrates that the mouse pancreas contains capsaicin-sensitive sensory nerve fibers, since most of the CGRP- and part of the substance P-immunoreactive nerve fibers disappeared after neonatal capsaicin treatment. These findings are in accordance with previous studies in the rat [27,28,30]. The reduction in the number of substance P-immunoreactive nerves induced by capsaicin pretreatment was less pro-
nounced than the reduction of CGRP-immunoreactive nerves. This also indicates the presence of substance P nerve fibers that are not sensitive to capsaicin treatment. The nature of this capsaicin-insensitive subpopulation of pancreatic substance P-immunoreactive nerves remains to be established. It has previously been demonstrated that most of the pancreatic galanin-immunoreactive nerve fibers are adrenergic [18]. We show here that the galanin-immunoreactive nerves remained virtually unaffected by capsaicin treatment. This confirms a previous study in the rat [30] and shows that the galanin-immunoreactive pancreatic nerve fibers are not capsaicin-sensitive. In the present study we also found that after capsaicin treatment, nerve cell bodies immunoreactive for substance P and galanin could be observed in local intrapancreatic ganglia, implying the existence of substance P- and galanin-immunoreactive nerve fibers of intrinsic origin in the mouse pancreas. The hyperglycemic response to autonomic nerve activation by 2-DG was partially impaired in capsaicin-treated mice, which is in accordance with a previous study in the rat [35]. This finding is also in line with a prevous study by Zhou and co-workers in which a delayed recovery of plasma glucose levels after insulin-induced hypoglycemia was demonstrated in capsaicin-treated rats [36]. In the present study we demonstrate also that the glucagon response to 2-DG is markedly inhibited in capsaicin-treated mice. This latter finding suggests that the impaired glucose response might be due in part to the reduced glucagon response. However, capsaicin treatment reduced the hyperglycemic response to 2-DG at both 2 and 10 min after 2-DG, whereas the glucagon response was inhibited by capsaicin treatment only at 10 min. This shows that capsaicin-sensitive nerves regulate plasma glucose levels after 2-DG by other mechanisms as well as through glucagon. Such a mechanism could involve, for example, hepatic glucose output via sympathetic nerves, which are known to be involved in 2-DG-induced hyperglycemia [29]. The increased plasma glucagon levels at 2 min after 2-DG seem to be regulated in a different way than those at 10 min. Hence, only at 10 min,
57 the plasma glucagon levels were partially reduced by capsaicin-treatement, whereas at 2 min, capsaicin had no effect. This shows that the glucagon response to 2-DG is mediated by at least two mechanisms, one of which is sensitive to capsaicin and thus most likely mediated by sensory afferent nerves. The general view of the mechanism whereby 2-DG induces hyperglycemia and stimulates islet hormone secretion is that the glucose analogue induces central neuroglycopenia which, in turn, activates the efferent autonomic output from the central nervous system [21]. The impaired glucagon and glucose responses to 2-DG after capsaicin as well as the results of a previous study [35] suggest that capsaicin-sensitive afferent nerve fibers are involved in the autonomic nerve activation by 2-DG. It has previously been shown that neonatal capsaicin treatment in the rat abolishes the increase in gastric acid secretion in response to 2-DG [9]. Although it has been speculated that sensory neurons in the hypothalmus might explain this phenomenon, there is no known (to us) experimental evidence for the presence of such fibers in the hypothalamus. It is therefore plausible that peripheral capsaicin-sensitive afferent nerve fibers are involved in the autonomic neural response to 2-DG. It has previously been reported that peripheral glucose-sensitive receptors exist in the liver [8,23]. Thus, 2-DG has previously been demonstrated to increase the vagal afferent discharge from the perfused guinea pig liver [23]. The possibility therefore exists that part of the hyperglycemic reponse to 2-DG is due to an effect on peripheral glucoceptors in the liver, which, via capsaicin-sensitive nerve fibers, transmit afferent signals to the central nervous system. However, since a large part of the glucose response to 2-DG was not affected by capsaicin treatment, the major mechanism behind 2-DG-stimulated hyperglycemia is most likely to be induction of central neuroglycopenia and an ensuing autonomic efferent activation without the involvement of capsaicin-sensitive sensory nerves. Regarding the glucagon response to 2-DG, a similar dual mechanism of 2-DG with a partial effect on peripheral glucoreceptors and activation
via afferent capsaicin-sensitive nerve fibers might be suggested. Indeed, it has previously been shown that systemic injection of 2-DG increases vagal afferent activity from the rabbit pancreas [22]. The involvement of pancreatic sensory neural mechanisms in the glucagon response to 2-DG could be supported by the immunohistochemical findings of the present study showing a reduction of CGRP- and substance P-immunoreactive pancreatic nerve fibers following capsaicin treatment. Thus, it might be speculated that neuropeptides from capsaicin-sensitive nerve fibers within the pancreas exert local effector functions as described for such nerves within other tissues [14]. Neuropeptides released from such capsaicin-sensitive nerves could thereby modulate glucagon secretion directly by its effects on the islet ceils or indirectly by affecting pancreatic neural activity or pancreatic blood flow. An indirect effect on neural activity could theoretically reside in activation of afferent neural signals to the central nervous system with a resulting activation of efferent cholinergic and adrenergic nerves. Such a hypothesis might be supported by previous studies demonstrating that the glucagon response to 2-DG is abolished by either ganglionic, nicotinic or muscarinic antagonists and is partially inhibited by a-adrenergic antagonists [16]. Whereas capsaicin pretreatment inhibited the glucagon response to 2-DG, the treatment had no effect on the insulin response. Hence, in contrast to the glucagon response, the insulin secretory reponse to 2-DG in mice does not involve capsaicin-sensitive sensory nerves. Thus, differences in the regulation of 2-DG-induced insulin and glucagon secretion exist. In conclusion, the present study has shown that 2-DG-induced glucagon secretion and 2DG-induced hyperglycemia are partially dependent on intact capsaicin-sensitive sensory nerve fibers. In contrast, 2-DG-induced insulin secretion is not dependent on such nerves. Furthermore, the study also shows that the mouse pancreas contains CGRP- and substance P-immunoreactive capsaicin-sensitive sensory nerve fibers and that the pancreatic galanin-immunoreactive nerve fibers are not capsaicin-sensitive.
58
Acknowledgements The technical assistance of Lena Kvist, Lilian Bengtsson and Doris Persson is gratefully acknowledged. The study was financially supported by The Swedish Medical Research Council (grants No 14X-6834, 17P-8453 and 4499), Nordisk Insulinfond, Swedish Diabetes Association, Diabetesf6reningen i Malm6, Swedish Hoechst Diabetes Fund, The Swedish Medical Society, The Swedish Society for Medical Research, Craafordska and Albert P[ihlssons Foundations, and by the Faculty of Medicine, Lund University.
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References 1 Ahr6n, B. and Lundquist, I., Glucagon immunoreactivity in plasma from normal and dystrophic mice, Diabetologia, 22 (1982) 258-263. 2 Ahr6n, B., Taborsky, G.J., Jr. and Porte, D., Jr., Neuropeptidergic versus cholinergic and adrenergic regulation of islet hormone secretion, Diabetologia, 29 (1986) 827836. 3 Bruss, M.L. and Black, A.L., Enzymatic microdetermination of glycogen, Analyt. Biochem., 84 (1978) 309-312. 4 Buck, S.H. and Burks, T.F., The neuropharmacology of capsaicin: review of some recent observations, Pharmacol. Rev., 38 (1986) 179-226. 5 Coons, A.H., Leduc, E.H. and Connolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit, J. Exp. Med., 102 (1955) 49-59. 6 Cramer, F.B. and Woodward, G.E., 2-Deoxy-o-glucose as an antagonist of glucose in yeast fermentation, J. Franklin. Inst., 253 (1952) 354-360. 7 Dunning B.E. and Taborsky G.J. Jr., Galanin release during pancreatic nerve stimulation is sufficient to influence islet function, Am. J. Physiol., 256 (1989) E191-E198. 8 Donovan, C.M., Halter J.B. and Bergman, R.N., Importance of hepatic glucoreceptors in sympathoadrenal response to hypoglycemia, Diabetes, 40 (1991) 155-158. 9 Evangelista, S., Santicioli, P., Maggi, C.A. and Meli, A., Increase in gastric secretion induced by 2-deoxy-D-glucose is impaired in capsaicin pretreated rats, Br. J. Pharmacol., 98 (1989) 35-37. 10 Havel, P.J., Veith, R.C., Dunning, B.E. and Taborsky, G.J., Jr., Pancreatic noradrenergic nerves are activated by neuroglycopenia but not by hypotension or hypoxia in the dog. Evidence for stress-specific regionally selective activation of the sympathetic nervous system, J. Clin. Invest., 82 (1988) 1538-1545. 11 Havel, P.J. and Taborsky, G.J., Jr., The contribution of the
17
18
19
20
21
22 23
24
25
26
27
autonomic nervous system to changes of glucagon and insulin secretion during hypoglycemic stress, Endocrine Rev., 10 (1989) 332-350. Hedo, J.A., Vitlanueva, M.L. and Marco, J., Stimulation of pancreatic polypeptide and glucagon secretion by 2-deoxyD-glucose in man: evidence for cholinergic mediation, J. Clin. Endocrinol. Metab., 47 (1978) 366-371. Herbert, V., Lau, K.S., Gottlieb, C.W. and Bleicher, S.J., Coated charcoal immunoassay of insulin. J. Clin. Endocrinol. Metab., 25 (1965) 1875-1884. Holzer P., Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides, Neuroscience, 24 (1988) 739-768. Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 143-201. Karlsson, S. and Ahr4n, B., Inhibition of 2-dexoxyglucose-induced glucagon secretion by muscarinic and t~adrenoceptor blockade in the mouse, Diabet. Res. Clin. Pract., 3 (1987) 239-242. Karlsson, S., Bood, M. and Ahr4n, B., The mechanism of 2-deoxy-glucose-induced insulin secretion in the mouse, J. Autonom. Pharmacol., 7 (1987) 135-144. Lindskog, S., Ahr6n, B., Dunning, B.E. and Sundler, F., Galanin-immunoreactive nerves in the mouse and rat pancreas, Celt Tiss. Res., 264 (1991) 363-368. Matsunaga, H., Iguchi, A., Yatomi, A., Uemura, K., Miura, H., Gotoh, M., Mano, T. and Sakamoto, N., The relative importance of nervous system and hormones to the 2-deoxy-D-glucose-induced hyperglycemia in fed rats, Endocrinology, 124 (1989) 1259-1264. Miller, R.E., Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the islets of Langerhans, Endocrine Rev., 2 (1981) 471-494. Muller, E.E., Cocchi, D. and Forni, A., A central site for the hyperglycemic action of 2-deoxy-D-glucose in mouse and rat, Life. Sci., 10 (1971) 1057-1067. Niijima, A., Visceral afferents and metabolic function, Diabetologia, 20 (1981) 325-330. Niijima, A., Glucose-sensitive afferent nerve fibers in the hepatic branch of the vagus nerve in the guinea-pig, J. Physiol., 332 (1982) 315-323. Pettersson, M., Ahr4n, B., B6ttcher, G. and Sundler, F., Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse, Endocrinology, 119 (1986)865-869. Raghu, P.K., Taborsky, G.J., Jr., Paquette, T.L., Halter, J.B. and Palmer, J.P., Evidence for noncholinergic ganglionic neural stimulation of B cell secretion, Am. J. Physiol., 247 (1984) E265-E270. Scadding, J.W., The permanent anatomical effects of neonatal capsaicin on somatosensory nerves, J. Anat., 131 (1980) 473-484. Sharkey, K.A., Williams, R.G. and Dockray G.J., Sensory substance P innervation of the stomach and pancreas, Gastroenterology, 87 (1984) 914-921.
59 28 Sternini, C. and Brecha, N., Immunocytochemical identification of islet cells and nerve fibers containing calcitonin gene-related peptide-like immunoreactivity in the rat pancreas, Gastroenterology, 90 (1986) 1155-1163. 29 Storlien, L.H., Grunstein, H.S. and Smythe, G.A., Guanethidine blocks the 2-deoxy-D-glucose-induced hypothalamic noradrenergic drive to hypoglycemia, Brain Res., 335 (1985) 144-147. 30 Su, N.C., Bishop, A.E., Power, R.F., Hamada, Y. and Polak, J.M., Dual intrinsic and extrinsic origins of CGRPand NPY-immunoreactive nerves of rat gut and pancreas, J. Neurosci., 7 (1987) 2674-2687. 31 Sundler, F. and B6ttcher, G., Islet innervation, with special reference to neuropeptides. In E. Samols, (Ed.), The endocrine pancreas, Raven Press, New York, 1991, pp. 29-52. 32 Sundler, F, Brodin, E., Ekblad, E., H~kansson, R. and Uddman R., Sensory nerve fibers: distribution of sub-
33
34 35
36
stance P, neurokinin A and calcitonin gene-related peptide. In R. H~ikansson and F. Sundler (Eds.), Tachykinin antagonists, Elsevier, Amsterdam, 1985, pp. 3-14. Taborsky, G.J., Jr., Halter, J.B. and Porte, D., Jr., Morphine suppresses plasma catecholamine responses to laparatomy but not to 2-deoxy-glucose, Am. J. Physiol., 242 (1982) E317-322. Woods, S. and Porte, D., Jr., Neural control of the endocrine pancreas, Physiol. Rev., 54 (1974) 596-619. Zhou, X.F. and Livett, B.G., Effect of capsaicin-sensitive sensory nerves on plasma glucose and catecholamine levels during 2-deoxy-glucose-induced stress in conscious rats, Br. J. Pharmacol., 100 (1990) 523-529. Zhou, X.F., Jhamadans, K.H. and Livett, B.G., Capsaicinsensitive nerves are required for glucostasis but not for catecholamine output during hypoglycemia in rats, Am. J. Physiol. 258 (1990) E212-E219.