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Effects of nitric oxide synthase blockade on dorsal vagal stimulation-induced pancreatic insulin secretion
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Research Report
Effects of nitric oxide synthase blockade on dorsal vagal stimulation-induced pancreatic insulin secretion Bashair M. Mussa, Daniela M. Sartor, Christian Rantzau, Anthony J.M. Verberne⁎ University of Melbourne, Department of Medicine, Austin Health, Heidelberg 3084, Victoria, Australia
A R T I C LE I N FO
AB S T R A C T
Article history:
We and others have previously shown that the dorsal motor nucleus of the vagus (DMV) is
Accepted 9 April 2011
involved in regulation of pancreatic exocrine secretion. Many pancreatic preganglionic
Available online 16 April 2011
neurons within the DMV are inhibited by pancreatic secretagogues suggesting that an inhibitory pathway may participate in the control of pancreatic exocrine secretion.
Keywords:
Accordingly, the present study examined whether chemical stimulation of the DMV
Dorsal motor nucleus of the vagus
activates the endocrine pancreas and whether an inhibitory pathway is involved in this
Insulin secretion
response. All experiments were conducted in overnight fasted isoflurane/urethane-
Nitric oxide
anesthetized Sprague Dawley rats. Activation of the DMV by bilateral microinjection of bicuculline methiodide (BIM, GABAA receptor antagonist, 100 pmol/25 nl; 4 mM) resulted in a significant and rapid increase in glucose-induced insulin secretion (9.2± 0.1 ng/ml peak response) compared to control microinjection (4.0± 0.6 ng/ml). Activation of glucose-induced insulin secretion by chemical stimulation of the DMV was inhibited (2.1 ± 1.1 ng/ml and 1.6 ± 0.1 ng/ml 5 min later) in the presence of the muscarinic receptor antagonist atropine methonitrate (100 μg/kg/min, i.v.). On the other hand, the nitric oxide (NO) synthesis inhibitor L-nitroarginine methyl ester (30 mg/kg, i.v.) significantly increased the excitatory effect of DMV stimulation on glucose-induced insulin secretion to 15.3± 3.0 ng/ml and 16.1± 3.1 ng/ml 5 min later. These findings suggest that NO may play an inhibitory role in the central regulation of insulin secretion. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
The role of the dorsal motor nucleus of the vagus (DMV) in the regulation of pancreatic secretion (PS) has been confirmed by several studies (Buijs et al., 2001; Ionescu et al., 1983; Love et al., 2006; Mussa and Verberne, 2008; Viard et al., 2007). The
DMV is the site of origin of vagal efferent neurons that innervate both the endocrine and exocrine pancreas (Berthoud and Powley, 1991; Jansen et al., 1997; Rinaman and Miselis, 1987). The compact formation of the nucleus ambiguus also contains vagal motor neurons but these mainly innervate the upper GI tract including the striated muscles of the soft palate, pharynx,
⁎ Corresponding author at: University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Department of Medicine, Austin Health, Heidelberg, Victoria 3084, Australia. Fax: +61 3 9459 3510. E-mail address: [email protected] (A.J.M. Verberne). Abbreviations: AMN, atropine methonitrate; BIM, bicuculline methiodide; CCK-8S, cholecystokinin sulphated octapeptide; DMV, dorsal motor nucleus of the vagus; L-NAME, L-nitroarginine methyl ester; NO, nitric oxide; NANC, nonadrenergic-noncholinergic; PBG, phenylbiguanide; PS, pancreatic secretion; RVLM, rostral ventrolateral medulla 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.04.015
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esophagus and larynx while cardiovagal motor neurons are found in the loose formation of the nucleus ambiguus (Loewy and Spyer, 1990). We have previously shown that chemical activation of the DMV induced by blockade of local GABAA receptors produced profound excitatory effects on pancreatic exocrine secretion. These effects were sensitive to the muscarinic acetylcholine receptor antagonist atropine methonitrate, emphasizing that the DMV influences pancreatic exocrine secretion via a cholinergic pathway (Mussa and Verberne, 2008). In view of these findings, we wished to determine whether chemical stimulation of the DMV also modulates pancreatic endocrine secretion via a cholinergic pathway. It is well-documented that DMV vagal efferents synapse onto cholinergic and non-cholinergic postganglionic neurons in the pancreas. Stimulation of these efferents directly influences both endocrine and exocrine secretions (Bergman and Miller, 1973; Berthoud and Powley, 1991; Roze, 1991). A recent investigation of DMV-pancreatic preganglionic neurons has shown that the discharge rate of these neurons was differentially affected by the pancreatic secretagogues, cholecystokinin sulfated octapeptide (CCK-8S) and the 5-HT3 receptor agonist phenylbiguanide (PBG) (Mussa et al., 2010). Although some of the DMV-pancreatic preganglionic neurons were activated by CCK-8S and PBG, the majority were inhibited. Despite their differential responsiveness to CCK or PBG these neurons could not be distinguished on the basis of other physiological properties e.g. axonal conduction velocity. These findings suggest that the link between the DMV and PS is more complex than previously believed and raises the possibility that an inhibitory pathway is implicated in regulation of PS (Mussa et al., 2010). An inhibitory vagal pathway arising from the DMV is not a novel concept since it has been previously proposed that parallel excitatory and inhibitory vagal efferent pathways control gastric function (Browning and Travagli, 2010; Hornby, 2001; Owyang and Logsdon, 2004; Travagli et al., 2003; Travagli et al., 2006). A potential inhibitory neurotransmitter in this pathway is nitric oxide (NO). Although NO has been implicated in neurally mediated PS, there is a lack of consensus in regard to the exact role of this agent (Holst et al., 1994; Vaquero et al., 1998). In addition, neural regulation of pancreatic exocrine secretion has been investigated in greater detail than neural control of pancreatic endocrine secretion. Therefore, in the present study, we have examined the effects of chemical stimulation of the DMV on glucose-induced insulin secretion and the role of NO in these responses.
2.
Results
2.1. Effects of chemical stimulation of the DMV on glucose-induced secretion At baseline, glucose levels were 7.2 ± 0.4 mM and insulin levels were 2.1 ± 0.2 ng/ml and in all experiments similar basal glucose and insulin levels were observed. Intravenous administration of glucose produced a substantial, immediate and short-lasting elevation in insulin levels (from 2.1 ± 0.2 to 9.8 ±
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0.7 ng/ml, n = 6, P < 0.0001). The results of the first group of experiments showed that chemical stimulation of the DMV by bilateral microinjection of BIM produced significant and rapid increases in glucose-induced insulin secretion compared to control microinjection (Fig. 1). Insulin levels were measured twice at 5 min intervals after bilateral microinjection of BIM and after control (vehicle) microinjection. After the first 5 min period insulin levels were 8.9 ± 1.2 ng/ml and 5.5 ± 0.7 ng/ml after BIM and control microinjections, respectively. After the second 5 min period insulin levels were 9.2 ± 1.0 ng/ml after BIM and 4.0 ± 0.6 ng/ml after control microinjections, respectively (n = 6, *P < 0.05, and ***P < 0.001) (Fig. 1). Furthermore, there were no further changes in insulin levels when the BIM or control microinjections were repeated 40 min later. As shown in Fig. 1B, basal levels of glucose increased rapidly after intravenous administration of glucose (from 7.2 ± 0.4 mM to 30.2 ± 0.1 mM, n = 6; P < 0.0001) but this increase lasted only for 5 min. No changes in glucose levels were observed after microinjection of BIM into the DMV and glucose levels continued to fall until the end of the experiment. Histological analysis of the microinjection sites showed that control microinjection of BIM outside the DMV or microinjection of ACSF inside the DMV did not affect insulin levels emphasizing that insulin secretion occurred only after microinjection of BIM into the DMV (Figs. 1 and 4B). In addition, the increase in insulin secretion occurring in response to chemical activation of the DMV was inhibited after infusion of AMN compared to infusion of saline (Fig. 2A). In the saline treated group, insulin levels after bilateral microinjection of BIM were 8.9 ± 1.2 ng/ml and 9.2 ± 0.9 ng/ml 5 min later (n = 6) whereas those after AMN were 2.1 ± 1.1 ng/ ml and 1.6 ± 0.1 ng/ml, respectively (n = 3 and P < 0.05). As shown in Fig. 2B, neither infusion of AMN or saline affected the blood glucose levels and, as noted previously, glucose levels were only increased after intravenous administration of glucose and returned approximately to baseline at the end of the experiment.
2.2. Chemical stimulation of the DMV and glucose-induced insulin secretion: effects of NO inhibition In the second group of experiments, the hypothesis that nitrergic nerves are involved in regulation of glucose-induced insulin secretion was tested. Fig. 3 shows that increased insulin secretion in response to chemical activation of the DMV was significantly enhanced (from 8.9± 1.2 ng/ml to 15.3 ± 3.0 ng/ml and from 9.2± 0.1 ng/ml to 16.1± 3.1 ng/ml 5 min later, n = 6, *P < 0.05) in the presence of the nitric oxide synthesis inhibitor L-NAME. The enhanced secretion appeared to be due to NO synthase inhibition induced by L-NAME since infusion of saline did not alter the secretory response to chemical stimulation of the DMV (Fig. 3A). Fig. 3B shows that the glucose levels declined more rapidly (n = 6, **P < 0.01, and ***P < 0.001) after infusion of L-NAME compared to that observed after infusion of normal saline. Fig. 4 summarizes the microinjection sites of BIM within the DMV during infusion of saline (Fig. 4A), AMN (Fig. 4C) and L-NAME (Fig. 4D) and microinjection sites of ACSF within the DMV and BIM microinjections sites outside the DMV (Fig. 4B).
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A 12
Glucose bolus injection
BIM Microinjection
Insulin (ng/ml)
*
BIM Microinjection
***
8
BIM microinjection 4 Control microinjection
Glucose level (mmol/l)
B
0 40
20 BIM microinjection Control microinjection
0 Basal
5
10
15
20
60
65
120
Post-bolus injection of glucose (min) Fig. 1 – Effects of chemical stimulation of the DMV on glucose-induced insulin secretion. A. Bicuculline methiodide (BIM) microinjection into the DMV produced a significant increase in the levels of glucose-stimulated insulin secretion (filled squares, n = 6, *P < 0.05, and ***P < 0.001). Control microinjection did not change the level of glucose-induced insulin secretion (open squares, n = 7). B. BIM microinjection into the DMV (filled squares, n = 6) or control microinjection (open squares, n = 7) did not affect the blood glucose levels. Data are presented as mean ± S.E.M. Note the breaks in the time-scale on the x-axis and that some error bars are obscured by the symbol. Vertical bars denote times of glucose and drug administration.
3.
Discussion
Several lines of evidence implicate the DMV as an important controller of PS (Ionescu et al., 1983; Kerr and Preshaw, 1969; Mussa and Verberne, 2008). Our previous investigations have confirmed that chemical activation of this nucleus directly stimulates pancreatic exocrine secretion via a cholinergic pathway (Mussa and Verberne, 2008). The results of the current investigation have clearly shown, for the first time, that insulin secretion was stimulated in response to chemical activation of the DMV. Blockade of GABAA receptors within the caudal to intermediate DMV using BIM caused an immediate increase in glucose-induced insulin secretion and this effect lasted for at least 10 min. Despite the increase in insulin secretion glucose levels did not decline more rapidly. Perhaps this is because there is a simultaneous increase in glucagon levels which has been reported previously (Berthoud and Powley, 1987). Microinjection of ACSF into the DMV did not alter insulin levels. Additional control experiments which showed that blockade of GABAA receptors outside the DMV did not stimulate insulin secretion, support the view that the DMV is the critical region for this response. Our data also shows that a second microinjection of BIM into the DMV did not alter
insulin levels. This may suggest that the majority of the GABAA receptors were blocked by the first microinjection of BIM and the maximal excitatory response had already been achieved. Alternatively, perhaps the second microinjection was ineffective because it was not preceded by a priming bolus of glucose. It is known that infusion of glucose stimulates the secretion of preformed insulin but newly synthesized insulin may be insensitive to chemical activation of the DMV (Curry et al., 1968; Rorsman et al., 2000). GABAA receptors are found in high concentration in the DMV and blockade of these receptors has profound effects on gastrointestinal function and pancreatic exocrine secretion (Ashworth-Preece et al., 1997; Feng et al., 1990; Mussa and Verberne, 2008; Washabau et al., 1995). The results of the present study indicate that insulin secretion is also modulated by GABAergic neurons that probably terminate within the DMV. In agreement with previous reports, the present study has demonstrated that the blockade of muscarinic acetylcholine receptors using AMN abolished the excitatory effects of chemical stimulation of the DMV on insulin secretion (Ionescu et al., 1983; Mussa and Verberne, 2008). This finding supports the hypothesis that a cholinergic pathway plays a major role in mediating the effect of DMV neurons on pancreatic function.
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Insulin (ng/ml)
A 12
Glucose bolus injection
BIM Microinjection
BIM Microinjection
8
4
*
BIM+Saline
* BIM+AMN
0
Glucose level (mmol/l)
B 40
BIM+Saline 20
BIM+AMN
0 Basal
5
10
15
20
60
65
120
Post-bolus injection of glucose (min) Fig. 2 – Effects of atropine methonitrate (AMN) on excitatory effects of chemical activation of the DMV on glucose-stimulated insulin secretion. A. Systemic administration of AMN inhibited the excitatory effects of DMV-chemical stimulation on glucose-induced insulin secretion, significantly (filled squares, n = 3, *P < 0.05). B. Blood glucose levels in the presence (filled squares, n = 3) and absence (open squares, n = 6) of AMN. Note the breaks in the time-scale on the x-axis and that some error bars are obscured by the symbol. Vertical bars denote times of glucose and drug administration.
Earlier studies suggested that the nucleus ambiguus is also involved in regulation of insulin secretion (Bereiter et al., 1981). However, since this study used electrical stimulation to activate the nucleus ambiguus it is possible that passing fibers from the DMV were activated simultaneously. In addition, the increase in plasma insulin produced by electrical stimulation of the nucleus ambiguus was enhanced by blockade of αadrenoceptors suggesting that the electrical stimulation also activated the sympathetic outflow. Nevertheless, these authors went on to show that chemical stimulation of the ambiguus region using bicuculline also produced a vagally-mediated increase in insulin secretion that was unmasked only after αadrenoceptor blockade (Bereiter et al., 1982). Perhaps stimulation of the nucleus ambiguus region also activated nearby presympathetic neurons in the rostral ventrolateral medulla (RVLM). The RVLM probably controls the sympathetic vasomotor outflow to the pancreas (Verberne and McInerney, 2006). Electrical stimulation the DMV provoked insulin secretion (Ionescu et al., 1983). However, as indicated above, electrical stimulation activates passing fibers as well as neurons. To obviate this problem many investigators have used glutamate receptor agonists to stimulate neuronal cell bodies selectively. Unfortunately, this approach also has several disadvantages. Firstly, most glutamate receptor agonists have a short duration of action and so are not very useful when the parameter under
investigation is the level of a circulating hormone. Secondly, glutamate receptor activation is sometimes ineffective as a method of neuronal activation because of simultaneous activation of local GABAergic neurons. On the other hand, bicuculline has the advantage that blockade of somatodendritic GABA receptors produces selective neuronal excitation and is long-lasting. One of the main findings of this study is that the increase in insulin secretion produced by chemical stimulation of the DMV was enhanced after blockade of peripheral NO synthase using L-NAME. It is noteworthy that after a second microinjection of BIM into the DMV insulin secretion remained significantly elevated suggesting that newly synthesized insulin is sensitive to DMV-chemical stimulation in the presence of LNAME. This suggests that NO exerts an inhibitory influence on the two phases of insulin secretion. Interestingly, a significant reduction in blood glucose was also observed in the presence of L-NAME. This suggests that the augmented secretion of insulin that occurred after NO synthase inhibition also resulted in enhanced glucose uptake by the liver, muscle and fat. An inhibitory role for NO in insulin secretion has been described previously but most of these studies were conducted in vitro and used high concentrations of NO inhibitors that are unlikely to be attained in vivo (Panagiotidis et al., 1995; Salehi et al., 1996). In addition, it has been suggested that NO is involved in
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Insulin (ng/ml)
A
30 Glucose bolus
BIM Microinjection
injection
20
*
BIM Microinjection
* *
10 BIM+L-NAME BIM+Saline
Glucose level (mmol/l)
B
0 40
20
***
BIM+L-NAME
*** **
**
**
60
65
120
BIM+Saline
0 Basal
5
10
15
20
Post-bolus injection of glucose (min) Fig. 3 – Effects of NO inhibition on excitatory effects of DMV-chemical stimulation on glucose-induced insulin secretion. A. Systemic administration of L-NAME increased the excitatory effects of chemical stimulation on glucose-induced insulin secretion, significantly (filled squares, n = 6 and *P < 0.05). B. Blood glucose levels in the presence (filled squares, n = 6, **P < 0.01, and ***P < 0.001) and absence (open squares, n = 6) of L-NAME. Note the breaks in the time-scale on the x-axis and that some error bars are obscured by the symbol. Vertical bars denote times of glucose and drug administration.
modulation of insulin secretion via stimulation of a negative feedback mechanism since NO donors were found to produce inhibitory effects on insulin secretion in mice, rats, pigs and humans (Antoine et al., 1993; Corbett et al., 1993; Cunningham et al., 1994; Mosen et al., 2006; Sjoholm, 1996). Although it is evident that NO and NOS are present in the DMV, central effects of NO can be excluded in this study since L-NAME does not cross the blood brain barrier (Kaufmann et al., 2004; Travagli and Gillis, 1994). The present data showed that blockade of peripheral NO potentiated the excitatory effects of chemical stimulation of the DMV on insulin secretion. Given that both nitrergic and cholinergic nerves innervate the pancreas and the pancreatic vagal postganglionic neurons containing ACh and NO, it is reasonable to postulate that the latter normally inhibits the release of ACh and subsequently insulin release (Wang et al., 1999). An alternative explanation is that pancreatic nitrergic nerves are tonically involved in inhibition of insulin secretion. Recently, we found that DMV-pancreatic preganglionic neurons responded differentially to pancreatic secretagogues (Mussa et al., 2010). It is possible that the DMV also contains neurons that have both excitatory and inhibitory effects on insulin secretion. In the light of these findings, we propose that the β-
cells of the endocrine pancreas receive both excitatory and inhibitory inputs from the DMV and these modulate insulin secretion. In conclusion, chemical activation of the DMV led to an increase in insulin secretion via a cholinergic mechanism. This response was augmented by inhibition of peripheral NO synthase suggesting an involvement of nitrergic pathways and NO release in regulation of pancreatic insulin secretion.
4.
Experimental procedures
4.1.
General anesthetic and surgical procedures
All experiments were approved by the Ethical Review Committee of Austin Health (Heidelberg, Victoria, Australia) and complied with the principles outlined in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Male Sprague Dawley (SD) rats weighing between 250 and 450 g obtained from the Animal Resource Centre (ARC, Perth, Western Australia) were used in all experiments. SD
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B
A AP
AP
NTS
NTS
NTS
NTS CC XII
CC XII
DMV
DMV
C
D AP
AP
NTS
NTS CC
NTS
NTS CC XII
XII DMV
DMV
Fig. 4 – Coronal sections of the rat dorsal medulla showing a summary of the bicuculline methiodide (BIM) microinjection sites within the DMV during infusion of saline (A), atropine methonitrate (AMN) (C) and L-nitroarginine methyl ester (L-NAME) (D). Section B shows a summary of control microinjections of BIM made outside the DMV and ACSF microinjections made inside the DMV. Abbreviations: AP; area postrema; CC, central canal; DMV, dorsal motor nucleus of the vagus; NTS, nucleus of solitary tract; and XII, hypoglossal nucleus.
rats were housed in a temperature-controlled environment with a 12 hour light–dark cycle with overnight food deprivation and continuous access to water. SD rats were initially anesthetized in a chamber saturated with isoflurane vapor (Isoflurane, Delvet, Seven Hills, NSW, Australia). After checking that the depth of anesthesia was appropriate (no response to firm paw pinch), the rat was quickly tracheostomized to allow artificial ventilation with 100% O2 (1 ml/100 g body weight, 50–60 breaths/min) containing 1.5–1.7% isoflurane. Throughout the experiment the body temperature was maintained at 36–38 °C with a servo-controlled heating pad (Yellow Springs Instruments, Yellow Springs, OH, USA). The adequacy of the anesthesia was assessed by noting the absence of a withdrawal reflex to firm paw pinch as well as the absence of an eye blink response to gentle probing of the cornea. These tests were made every 15 min and the anesthetic vapor concentration was increased if necessary. After the completion of all surgery, urethane (1.2–1.5 g/kg, i.v.; Sigma Chemical Co., St Louis, MO, USA) was administered over 20–30 min and the inspired isoflurane concentration was gradually reduced to zero. Throughout the experiment additional urethane was added as required to maintain an adequate level of anesthesia.
4.2.
Chemical stimulation of the DMV
Polyethylene catheters (ID 0.58 x OD 0.96 mm, Critchley Electrical Products, Castle Hill, NSW, Australia) were inserted into the left carotid artery and the left jugular vein to monitor the arterial blood pressure and to allow intravenous admin-
istration of drugs, respectively. Anesthetized rats were placed into a stereotaxic frame and the right cervical vagus was isolated through a lateral cervical incision. The vagus was isolated and placed carefully onto a pair of silver wire hook electrodes and insulated from the surrounding tissue using a two component silicone sealant (Kwik-Cast, World precision Instruments, Sarasota, FL, USA). A midline incision was made over the scalp and the connective tissue lying over the longus capitus muscles and interparietal bone was removed. The longus capitus muscles were divided longitudinally and the atlantooccipital membrane overlying the dorsal medulla oblongata was exposed. The membrane was carefully dissected away and a small section of the occipital bone was removed to allow easier access to the dorsal medulla. A recording electrode (2 mm OD, Harvard Apparatus Ltd., Kent, England), filled with 0.5 M sodium acetate/2% pontamine sky blue (Koch-Light Labs., Coinbrook, Berks. UK) was positioned over the DMV using the following stereotaxic coordinates: 400 μm lateral and 700 μm rostral to the calamus scriptorius which was used as reference point to localize the DMV. The recording microelectrode, with impedances ranging between 5 and 10 MΩ, was advanced into the dorsal medulla while electrical stimulation (0.5 Hz, 0.5 ms pulse width, 500 μA) was applied to the ipsilateral vagus nerve. DMV action potentials were amplified (×1000) and filtered (400–4000 Hz) using an extracellular single unit amplifier and window discriminator (Fintronics, Orange, CT, USA) and monitored using an audio amplifier and oscilloscope (Tektronix Inc., Beaverton, Oregon, USA). The antidromic nature of constant latency spikes
68
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elicited by stimulation of the vagus nerve was verified using the collision test (Lipski, 1981). After the location of the DMV was identified for subsequent microinjection of BIM, the recording electrode was exchanged for a microinjector filled with BIM (100 pmol) containing 10% rhodamine beads (Fluospheres, Invitrogen, Molecular Probes, Eugene, OR, USA), which were used to mark the microinjection sites. The microinjector, which consisted of a glass micropipette cemented to the tip of a 1 μl glass microsyringe (Scientific Glass Engineering, Ringwood, Victoria, Australia), was advanced into the medulla and positioned at the coordinates identified in the last recording procedure. BIM (GABAA receptor antagonist, 100 pmol/25 nl; 4 mM) was microinjected bilaterally into the DMV.
4.3. Effect of chemical stimulation of the DMV on glucose-induced insulin secretion To examine the effects of chemical stimulation of the DMV on glucose-induced insulin secretion, blood glucose and plasma insulin levels were measured prior to, and after bilateral microinjection of BIM. After the completion of all anesthetic and surgical procedures, a stabilization period of 30 min was allowed and basal glucose and insulin levels were measured. Two basal samples were collected 5 min apart and then insulin secretion was induced by a bolus injection of glucose (1 g/kg, i.v., Mallinckrodt, Paris, KY, USA). Following the induction of insulin secretion, insulin and glucose levels were measured twice 5 min apart. This was followed by chemical stimulation of the DMV by bilateral microinjection of BIM (100 pmol/25 nl) into the DMV and thereafter insulin and glucose levels were measured every 5 min. Forty minutes after the last set of blood glucose and insulin measurements were made, bilateral microinjection of BIM into the DMV and subsequent measurement of glucose and insulin levels were repeated. Final measurements of insulin and glucose levels were made 60 min after the last microinjection of BIM into the DMV.
4.4. Effect of peripheral muscarinic receptor blockade on glucose-induced insulin secretion produced by chemical stimulation of the DMV To establish the specificity of the results, two control experiments were performed using the above-mentioned protocol: (i) BIM was microinjected outside the DMV and (ii) ACSF was microinjected into the DMV. In addition, the involvement of a cholinergic pathway in the effects of DMV activation on insulin secretion was investigated by administration of AMN (100 μg/ kg/min, i.v. for 20 min). The DMV was stimulated using BIM after 20 min of intravenous infusion of AMN and glucose and insulin levels were measured prior to, and after intravenous infusion of AMN.
5 min apart. Then insulin secretion was induced by a bolus injection of glucose (1 g/kg, i.v.) and another set of insulin and glucose measurements were performed. This was followed by 20-min of intravenous infusion of L-NAME (a NO synthase inhibitor, 30 mg/kg, i.v.) at a rate of 50 μl/min. Measurement of insulin and glucose was performed prior to, and after microinjection of BIM in the presence of L-NAME. The specificity of the results was confirmed by control experiments in which normal saline was infused intravenously instead of L-NAME.
4.6.
Measurement of blood glucose and insulin levels
Blood glucose was measured in blood taken from the arterial cannula. A drop of arterial blood was placed onto the test strip of a glucometer (Optium Xceed, MediSense, Abbott Laboratories, Bedford, MA, U.S.A.). This device is calibrated against a Yellow Springs International Glucose Analyser (YSI Inc., Yellow Springs OH, USA). Blood samples (500 μl) for insulin assay were collected in plastic tubes, immediately centrifuged and frozen for subsequent measurement of insulin using rat insulin radioimmunoassay (RIA; LINCO Research, Inc. St Charles, MO, USA).
4.7.
Histological identification of the microinjection sites
At the end of all experiments, rats were anesthetized deeply with isoflurane and perfused transcardially with 160 ml of normal saline (0.9% NaCl w/v) followed by 100 ml of 10% formalin (formaldehyde; Riedel-de-Haen, Seelze, Germany, diluted 1:4 with normal saline). Brains were removed and postfixed in 10% formalin for at least 24 h and then were snapfrozen by dipping into liquid nitrogen. Frozen coronal sections (40 μm thickness) of the brainstem were cut using a cryostat (Cryocut 1800, Reichert-Jung, Germany). Coronal brain sections that contained microinjection sites were mounted onto glass slides coated with gelatin and coverslipped with Vectashield (Vector laboratories, Burlingame, CA, USA). Fluorescence microscopy using an excitation wavelength of 550 nm was used to visualize the microinjection sites. The sections were then stained with cresyl violet and examined using light microscopy. The exact locations of the microinjection sites were identified using standard maps of the rat brain that were reproduced from the atlas of Paxinos and Watson (Paxinos and Watson, 1986).
4.8.
Data and statistical analysis
All data are expressed as S.E.M and differences between individual means were assessed and compared using one way analysis of variance (ANOVA) followed by the Tukey Multiple Comparisons tests. Probability values of P < 0.05 were considered statistically significant.
4.5. Effect of NO synthase inhibition on glucose-induced insulin secretion produced by chemical stimulation of the DMV
4.9.
This group of experiments was performed to determine the role of NO, if any, in mediating the insulin secretory responses to chemical activation of the DMV. After a 30 min stabilization period, basal levels of insulin and glucose were measured
Atropine methonitrate (AMN) and L-nitroarginine methyl ester (L-NAME) were obtained from Sigma Chemical Co. (St Louis, MO, USA) and were dissolved in normal saline (0.9% NaCl w/v; Pharmacia & Upjohn Ltd., Perth, Australia).
Materials
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Bicuculline methiodide (BIM; Sigma Chemical Co., St Louis, MO, USA) was dissolved in artificial cerebrospinal fluid (ACSF) with the following composition (mM): NaCl, 128; KCl, 2.6; CaCl2·2H2O, 1.3; MgCl2·6H2O, 0.9; NaHCO3, 20; and Na2HPO4, 1.3.
Acknowledgments This study was supported by a grant from the Austin Hospital Medical Research Foundation and a post-graduate scholarship from the University of Melbourne to B.M.M. The authors declare no conflict of interest.
REFERENCES
Antoine, M.H., Hermann, M., Herchuelz, A., Lebrun, P., 1993. Sodium nitroprusside inhibits glucose-induced insulin release by activating ATP-sensitive K+ channels. Biochim. Biophys. Acta 1175, 293–301. Ashworth-Preece, M., Krstew, E., Jarrott, B., Lawrence, A.J., 1997. Functional GABAA receptors on rat vagal afferent neurones. Br. J. Pharmacol. 120, 469–475. Bereiter, D.A., Berthoud, H.R., Brunsmann, M., Jeanrenaud, B., 1981. Nucleus ambiguus stimulation increases plasma insulin levels in the rat. Am. J. Physiol. 241, E22–E27. Bereiter, D.A., Berthoud, H.R., Becker, M.J., Jeanrenaud, B., 1982. Brain stem infusion of the gamma-aminobutyric acid antagonist bicuculline increases plasma insulin levels in the rat. Endocrinology 111, 324–328. Bergman, R.N., Miller, R.E., 1973. Direct enhancement of insulin secretion by vagal stimulation of the isolated pancreas. Am. J. Physiol. 225, 481–486. Berthoud, H.R., Powley, T.L., 1987. Characteristics of gastric and pancreatic responses to vagal stimulation with varied frequencies: evidence for different fiber calibers? J. Auton. Nerv. Syst. 19, 77–84. Berthoud, H.R., Powley, T.L., 1991. Morphology and distribution of efferent vagal innervation of rat pancreas as revealed with anterograde transport of Dil. Brain Res. 553, 336–341. Browning, K.N., Travagli, R.A., 2010. Plasticity of vagal brainstem circuits in the control of gastric function. Neurogastroenterol. Motil. 22, 1154–1163. Buijs, R.M., Chun, S.J., Niijima, A., Romijn, H.J., Nagai, K., 2001. Parasympathetic and sympathetic control of the pancreas: a role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake. J. Comp. Neurol. 431, 405–423. Corbett, J.A., Sweetland, M.A., Wang, J.L., Lancaster Jr., J.R., McDaniel, M.L., 1993. Nitric oxide mediates cytokine-induced inhibition of insulin secretion by human islets of Langerhans. Proc. Natl Acad. Sci. USA 90, 1731–1735. Cunningham, J.M., Mabley, J.G., Delaney, C.A., Green, I.C., 1994. The effect of nitric oxide donors on insulin secretion, cyclic GMP and cyclic AMP in rat islets of Langerhans and the insulin-secreting cell lines HIT-T15 and RINm5F. Mol. Cell. Endocrinol. 102, 23–29. Curry, D.L., Bennett, L.L., Grodsky, G.M., 1968. Dynamics of insulin secretion by the perfused rat pancreas. Endocrinology 83, 572–584. Feng, H.S., Lynn, R.B., Han, J., Brooks, F.P., 1990. Gastric effects of TRH analogue and bicuculline injected into dorsal motor vagal nucleus in cats. Am. J. Physiol. 259, G321–G326. Holst, J.J., Rasmussen, T.N., Schmidt, P., 1994. Role of nitric oxide in neurally induced pancreatic exocrine secretion in pigs. Am. J. Physiol. 266, G206–G213.
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Hornby, P.J., 2001. Receptors and transmission in the brain-gut axis. II. Excitatory amino acid receptors in the brain-gut axis. Am. J. Physiol. 280, G1055–G1060. Ionescu, E., Rohner-Jeanrenaud, F., Berthoud, H.R., Jeanrenaud, B., 1983. Increases in plasma insulin levels in response to electrical stimulation of the dorsal motor nucleus of the vagus nerve. Endocrinology 112, 904–910. Jansen, A.S., Hoffman, J.L., Loewy, A.D., 1997. CNS sites involved in sympathetic and parasympathetic control of the pancreas: a viral tracing study. Brain Res. 766, 29–38. Kaufmann, P.A., Rimoldi, O., Gnecchi-Ruscone, T., Bonser, R.S., Luscher, T.F., Camici, P.G., 2004. Systemic inhibition of nitric oxide synthase unmasks neural constraint of maximal myocardial blood flow in humans. Circulation 110, 1431–1436. Kerr, F.W., Preshaw, R.M., 1969. Secretomotor function of the dorsal motor nucleus of the vagus. J. Physiol. Lond. 205, 405–415. Lipski, J., 1981. Antidromic activation of neurones as an analytical tool in the study of the central nervous system. J. Neurosci. Meth. 4, 1–32. Loewy, A.D., Spyer, K.M., 1990. Vagal preganglionic neurons. Vol. In: Loewy, A.D., Spyer, K.M. (Eds.), Central Regulation of Autonomic Functions. Oxford University Press, New York, pp. 68–87. Love, J.A., Yi, E., Smith, T.G., 2006. Autonomic pathways regulating pancreatic exocrine secretion. Autonom. Neurosci. 133, 19–34. Mosen, H., Salehi, A., Henningsson, R., Lundquist, I., 2006. Nitric oxide inhibits, and carbon monoxide activates, islet acid alpha-glucoside hydrolase activities in parallel with glucose-stimulated insulin secretion. J. Endocrinol. 190, 681–693. Mussa, B.M., Verberne, A.J., 2008. Activation of the dorsal vagal nucleus increases pancreatic exocrine secretion in the rat. Neurosci. Lett. 433, 71–76. Mussa, B.M., Sartor, D.M., Verberne, A.J., 2010. Dorsal vagal preganglionic neurons: differential responses to CCK1 and 5-HT3 receptor stimulation. Autonom. Neurosci. 156, 36–43. Owyang, C., Logsdon, C.D., 2004. New insights into neurohormonal regulation of pancreatic secretion. Gastroenterology 127, 957–969. Panagiotidis, G., Akesson, B., Rydell, E.L., Lundquist, I., 1995. Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues. Br. J. Pharmacol. 114, 289–296. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney. Rinaman, L., Miselis, R.R., 1987. The organization of vagal innervation of rat pancreas using cholera toxin-horseradish peroxidase conjugate. J. Auton. Nerv. Syst. 21, 109–125. Rorsman, P., Eliasson, L., Renstrom, E., Gromada, J., Barg, S., Gopel, S., 2000. The cell physiology of biphasic insulin secretion. News Physiol. Sci. 15, 72–77. Roze, C., 1991. Central regulation of pancreatic secretion. In: Tache, Y., Wingate, D.L. (Eds.), Brain–Gut Interactions. CRC Press, Boca Raton, Florida, pp. 187–198. Salehi, A., Carlberg, M., Henningson, R., Lundquist, I., 1996. Islet constitutive nitric oxide synthase: biochemical determination and regulatory function. Am. J. Physiol. 270, C1634–C1641. Sjoholm, A., 1996. Nitric oxide donor SIN-1 inhibits insulin release. Am. J. Physiol. 271, C1098–C1102. Travagli, R.A., Gillis, R.A., 1994. Nitric oxide-mediated excitatory effect on neurons of dorsal motor nucleus of vagus. Am. J. Physiol. 266, G154–G160. Travagli, R.A., Hermann, G.E., Browning, K.N., Rogers, R.C., 2003. Musings on the wanderer: what's new in our understanding of vago-vagal reflexes? Am. J. Physiol. 284, G180–G187.
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Travagli, R.A., Hermann, G.E., Browning, K.N., Rogers, R.C., 2006. Brainstem circuits regulating gastric function. Annu. Rev. Physiol. 68, 279–305. Vaquero, E., Molero, X., Puig-Divi, V., Malagelada, J.R., 1998. Contrasting effects of circulating nitric oxide and nitrergic transmission on exocrine pancreatic secretion in rats. Gut 43, 684–691. Verberne, A.J., McInerney, K., 2006. Pancreatic vasoconstrictor responses are regulated by neurons in the rostral ventrolateral medulla. Brain Res. 1102, 127–134.
Viard, E., Zheng, Z.L., Wan, S., Travagli, R.A., 2007. Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion. Am. J. Physiol. 293, G493–G500. Wang, J., Zheng, H., Berthoud, H.-R., 1999. Functional vagal input to chemically identified neurons in pancreatic ganglia as revealed by Fos expression. Am. J. Physiol. 277, E958–E964. Washabau, R.J., Fudge, M., Price, W.J., Barone, F.C., 1995. GABA receptors in the dorsal motor nucleus of the vagus influence feline lower esophageal sphincter and gastric function. Brain Res. Bull. 38, 587–594.
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Research Report
Effects of nitric oxide synthase blockade on dorsal vagal stimulation-induced pancreatic insulin secretion Bashair M. Mussa, Daniela M. Sartor, Christian Rantzau, Anthony J.M. Verberne⁎ University of Melbourne, Department of Medicine, Austin Health, Heidelberg 3084, Victoria, Australia
A R T I C LE I N FO
AB S T R A C T
Article history:
We and others have previously shown that the dorsal motor nucleus of the vagus (DMV) is
Accepted 9 April 2011
involved in regulation of pancreatic exocrine secretion. Many pancreatic preganglionic
Available online 16 April 2011
neurons within the DMV are inhibited by pancreatic secretagogues suggesting that an inhibitory pathway may participate in the control of pancreatic exocrine secretion.
Keywords:
Accordingly, the present study examined whether chemical stimulation of the DMV
Dorsal motor nucleus of the vagus
activates the endocrine pancreas and whether an inhibitory pathway is involved in this
Insulin secretion
response. All experiments were conducted in overnight fasted isoflurane/urethane-
Nitric oxide
anesthetized Sprague Dawley rats. Activation of the DMV by bilateral microinjection of bicuculline methiodide (BIM, GABAA receptor antagonist, 100 pmol/25 nl; 4 mM) resulted in a significant and rapid increase in glucose-induced insulin secretion (9.2± 0.1 ng/ml peak response) compared to control microinjection (4.0± 0.6 ng/ml). Activation of glucose-induced insulin secretion by chemical stimulation of the DMV was inhibited (2.1 ± 1.1 ng/ml and 1.6 ± 0.1 ng/ml 5 min later) in the presence of the muscarinic receptor antagonist atropine methonitrate (100 μg/kg/min, i.v.). On the other hand, the nitric oxide (NO) synthesis inhibitor L-nitroarginine methyl ester (30 mg/kg, i.v.) significantly increased the excitatory effect of DMV stimulation on glucose-induced insulin secretion to 15.3± 3.0 ng/ml and 16.1± 3.1 ng/ml 5 min later. These findings suggest that NO may play an inhibitory role in the central regulation of insulin secretion. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
The role of the dorsal motor nucleus of the vagus (DMV) in the regulation of pancreatic secretion (PS) has been confirmed by several studies (Buijs et al., 2001; Ionescu et al., 1983; Love et al., 2006; Mussa and Verberne, 2008; Viard et al., 2007). The
DMV is the site of origin of vagal efferent neurons that innervate both the endocrine and exocrine pancreas (Berthoud and Powley, 1991; Jansen et al., 1997; Rinaman and Miselis, 1987). The compact formation of the nucleus ambiguus also contains vagal motor neurons but these mainly innervate the upper GI tract including the striated muscles of the soft palate, pharynx,
⁎ Corresponding author at: University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Department of Medicine, Austin Health, Heidelberg, Victoria 3084, Australia. Fax: +61 3 9459 3510. E-mail address: [email protected] (A.J.M. Verberne). Abbreviations: AMN, atropine methonitrate; BIM, bicuculline methiodide; CCK-8S, cholecystokinin sulphated octapeptide; DMV, dorsal motor nucleus of the vagus; L-NAME, L-nitroarginine methyl ester; NO, nitric oxide; NANC, nonadrenergic-noncholinergic; PBG, phenylbiguanide; PS, pancreatic secretion; RVLM, rostral ventrolateral medulla 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.04.015
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esophagus and larynx while cardiovagal motor neurons are found in the loose formation of the nucleus ambiguus (Loewy and Spyer, 1990). We have previously shown that chemical activation of the DMV induced by blockade of local GABAA receptors produced profound excitatory effects on pancreatic exocrine secretion. These effects were sensitive to the muscarinic acetylcholine receptor antagonist atropine methonitrate, emphasizing that the DMV influences pancreatic exocrine secretion via a cholinergic pathway (Mussa and Verberne, 2008). In view of these findings, we wished to determine whether chemical stimulation of the DMV also modulates pancreatic endocrine secretion via a cholinergic pathway. It is well-documented that DMV vagal efferents synapse onto cholinergic and non-cholinergic postganglionic neurons in the pancreas. Stimulation of these efferents directly influences both endocrine and exocrine secretions (Bergman and Miller, 1973; Berthoud and Powley, 1991; Roze, 1991). A recent investigation of DMV-pancreatic preganglionic neurons has shown that the discharge rate of these neurons was differentially affected by the pancreatic secretagogues, cholecystokinin sulfated octapeptide (CCK-8S) and the 5-HT3 receptor agonist phenylbiguanide (PBG) (Mussa et al., 2010). Although some of the DMV-pancreatic preganglionic neurons were activated by CCK-8S and PBG, the majority were inhibited. Despite their differential responsiveness to CCK or PBG these neurons could not be distinguished on the basis of other physiological properties e.g. axonal conduction velocity. These findings suggest that the link between the DMV and PS is more complex than previously believed and raises the possibility that an inhibitory pathway is implicated in regulation of PS (Mussa et al., 2010). An inhibitory vagal pathway arising from the DMV is not a novel concept since it has been previously proposed that parallel excitatory and inhibitory vagal efferent pathways control gastric function (Browning and Travagli, 2010; Hornby, 2001; Owyang and Logsdon, 2004; Travagli et al., 2003; Travagli et al., 2006). A potential inhibitory neurotransmitter in this pathway is nitric oxide (NO). Although NO has been implicated in neurally mediated PS, there is a lack of consensus in regard to the exact role of this agent (Holst et al., 1994; Vaquero et al., 1998). In addition, neural regulation of pancreatic exocrine secretion has been investigated in greater detail than neural control of pancreatic endocrine secretion. Therefore, in the present study, we have examined the effects of chemical stimulation of the DMV on glucose-induced insulin secretion and the role of NO in these responses.
2.
Results
2.1. Effects of chemical stimulation of the DMV on glucose-induced secretion At baseline, glucose levels were 7.2 ± 0.4 mM and insulin levels were 2.1 ± 0.2 ng/ml and in all experiments similar basal glucose and insulin levels were observed. Intravenous administration of glucose produced a substantial, immediate and short-lasting elevation in insulin levels (from 2.1 ± 0.2 to 9.8 ±
63
0.7 ng/ml, n = 6, P < 0.0001). The results of the first group of experiments showed that chemical stimulation of the DMV by bilateral microinjection of BIM produced significant and rapid increases in glucose-induced insulin secretion compared to control microinjection (Fig. 1). Insulin levels were measured twice at 5 min intervals after bilateral microinjection of BIM and after control (vehicle) microinjection. After the first 5 min period insulin levels were 8.9 ± 1.2 ng/ml and 5.5 ± 0.7 ng/ml after BIM and control microinjections, respectively. After the second 5 min period insulin levels were 9.2 ± 1.0 ng/ml after BIM and 4.0 ± 0.6 ng/ml after control microinjections, respectively (n = 6, *P < 0.05, and ***P < 0.001) (Fig. 1). Furthermore, there were no further changes in insulin levels when the BIM or control microinjections were repeated 40 min later. As shown in Fig. 1B, basal levels of glucose increased rapidly after intravenous administration of glucose (from 7.2 ± 0.4 mM to 30.2 ± 0.1 mM, n = 6; P < 0.0001) but this increase lasted only for 5 min. No changes in glucose levels were observed after microinjection of BIM into the DMV and glucose levels continued to fall until the end of the experiment. Histological analysis of the microinjection sites showed that control microinjection of BIM outside the DMV or microinjection of ACSF inside the DMV did not affect insulin levels emphasizing that insulin secretion occurred only after microinjection of BIM into the DMV (Figs. 1 and 4B). In addition, the increase in insulin secretion occurring in response to chemical activation of the DMV was inhibited after infusion of AMN compared to infusion of saline (Fig. 2A). In the saline treated group, insulin levels after bilateral microinjection of BIM were 8.9 ± 1.2 ng/ml and 9.2 ± 0.9 ng/ml 5 min later (n = 6) whereas those after AMN were 2.1 ± 1.1 ng/ ml and 1.6 ± 0.1 ng/ml, respectively (n = 3 and P < 0.05). As shown in Fig. 2B, neither infusion of AMN or saline affected the blood glucose levels and, as noted previously, glucose levels were only increased after intravenous administration of glucose and returned approximately to baseline at the end of the experiment.
2.2. Chemical stimulation of the DMV and glucose-induced insulin secretion: effects of NO inhibition In the second group of experiments, the hypothesis that nitrergic nerves are involved in regulation of glucose-induced insulin secretion was tested. Fig. 3 shows that increased insulin secretion in response to chemical activation of the DMV was significantly enhanced (from 8.9± 1.2 ng/ml to 15.3 ± 3.0 ng/ml and from 9.2± 0.1 ng/ml to 16.1± 3.1 ng/ml 5 min later, n = 6, *P < 0.05) in the presence of the nitric oxide synthesis inhibitor L-NAME. The enhanced secretion appeared to be due to NO synthase inhibition induced by L-NAME since infusion of saline did not alter the secretory response to chemical stimulation of the DMV (Fig. 3A). Fig. 3B shows that the glucose levels declined more rapidly (n = 6, **P < 0.01, and ***P < 0.001) after infusion of L-NAME compared to that observed after infusion of normal saline. Fig. 4 summarizes the microinjection sites of BIM within the DMV during infusion of saline (Fig. 4A), AMN (Fig. 4C) and L-NAME (Fig. 4D) and microinjection sites of ACSF within the DMV and BIM microinjections sites outside the DMV (Fig. 4B).
64
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A 12
Glucose bolus injection
BIM Microinjection
Insulin (ng/ml)
*
BIM Microinjection
***
8
BIM microinjection 4 Control microinjection
Glucose level (mmol/l)
B
0 40
20 BIM microinjection Control microinjection
0 Basal
5
10
15
20
60
65
120
Post-bolus injection of glucose (min) Fig. 1 – Effects of chemical stimulation of the DMV on glucose-induced insulin secretion. A. Bicuculline methiodide (BIM) microinjection into the DMV produced a significant increase in the levels of glucose-stimulated insulin secretion (filled squares, n = 6, *P < 0.05, and ***P < 0.001). Control microinjection did not change the level of glucose-induced insulin secretion (open squares, n = 7). B. BIM microinjection into the DMV (filled squares, n = 6) or control microinjection (open squares, n = 7) did not affect the blood glucose levels. Data are presented as mean ± S.E.M. Note the breaks in the time-scale on the x-axis and that some error bars are obscured by the symbol. Vertical bars denote times of glucose and drug administration.
3.
Discussion
Several lines of evidence implicate the DMV as an important controller of PS (Ionescu et al., 1983; Kerr and Preshaw, 1969; Mussa and Verberne, 2008). Our previous investigations have confirmed that chemical activation of this nucleus directly stimulates pancreatic exocrine secretion via a cholinergic pathway (Mussa and Verberne, 2008). The results of the current investigation have clearly shown, for the first time, that insulin secretion was stimulated in response to chemical activation of the DMV. Blockade of GABAA receptors within the caudal to intermediate DMV using BIM caused an immediate increase in glucose-induced insulin secretion and this effect lasted for at least 10 min. Despite the increase in insulin secretion glucose levels did not decline more rapidly. Perhaps this is because there is a simultaneous increase in glucagon levels which has been reported previously (Berthoud and Powley, 1987). Microinjection of ACSF into the DMV did not alter insulin levels. Additional control experiments which showed that blockade of GABAA receptors outside the DMV did not stimulate insulin secretion, support the view that the DMV is the critical region for this response. Our data also shows that a second microinjection of BIM into the DMV did not alter
insulin levels. This may suggest that the majority of the GABAA receptors were blocked by the first microinjection of BIM and the maximal excitatory response had already been achieved. Alternatively, perhaps the second microinjection was ineffective because it was not preceded by a priming bolus of glucose. It is known that infusion of glucose stimulates the secretion of preformed insulin but newly synthesized insulin may be insensitive to chemical activation of the DMV (Curry et al., 1968; Rorsman et al., 2000). GABAA receptors are found in high concentration in the DMV and blockade of these receptors has profound effects on gastrointestinal function and pancreatic exocrine secretion (Ashworth-Preece et al., 1997; Feng et al., 1990; Mussa and Verberne, 2008; Washabau et al., 1995). The results of the present study indicate that insulin secretion is also modulated by GABAergic neurons that probably terminate within the DMV. In agreement with previous reports, the present study has demonstrated that the blockade of muscarinic acetylcholine receptors using AMN abolished the excitatory effects of chemical stimulation of the DMV on insulin secretion (Ionescu et al., 1983; Mussa and Verberne, 2008). This finding supports the hypothesis that a cholinergic pathway plays a major role in mediating the effect of DMV neurons on pancreatic function.
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Insulin (ng/ml)
A 12
Glucose bolus injection
BIM Microinjection
BIM Microinjection
8
4
*
BIM+Saline
* BIM+AMN
0
Glucose level (mmol/l)
B 40
BIM+Saline 20
BIM+AMN
0 Basal
5
10
15
20
60
65
120
Post-bolus injection of glucose (min) Fig. 2 – Effects of atropine methonitrate (AMN) on excitatory effects of chemical activation of the DMV on glucose-stimulated insulin secretion. A. Systemic administration of AMN inhibited the excitatory effects of DMV-chemical stimulation on glucose-induced insulin secretion, significantly (filled squares, n = 3, *P < 0.05). B. Blood glucose levels in the presence (filled squares, n = 3) and absence (open squares, n = 6) of AMN. Note the breaks in the time-scale on the x-axis and that some error bars are obscured by the symbol. Vertical bars denote times of glucose and drug administration.
Earlier studies suggested that the nucleus ambiguus is also involved in regulation of insulin secretion (Bereiter et al., 1981). However, since this study used electrical stimulation to activate the nucleus ambiguus it is possible that passing fibers from the DMV were activated simultaneously. In addition, the increase in plasma insulin produced by electrical stimulation of the nucleus ambiguus was enhanced by blockade of αadrenoceptors suggesting that the electrical stimulation also activated the sympathetic outflow. Nevertheless, these authors went on to show that chemical stimulation of the ambiguus region using bicuculline also produced a vagally-mediated increase in insulin secretion that was unmasked only after αadrenoceptor blockade (Bereiter et al., 1982). Perhaps stimulation of the nucleus ambiguus region also activated nearby presympathetic neurons in the rostral ventrolateral medulla (RVLM). The RVLM probably controls the sympathetic vasomotor outflow to the pancreas (Verberne and McInerney, 2006). Electrical stimulation the DMV provoked insulin secretion (Ionescu et al., 1983). However, as indicated above, electrical stimulation activates passing fibers as well as neurons. To obviate this problem many investigators have used glutamate receptor agonists to stimulate neuronal cell bodies selectively. Unfortunately, this approach also has several disadvantages. Firstly, most glutamate receptor agonists have a short duration of action and so are not very useful when the parameter under
investigation is the level of a circulating hormone. Secondly, glutamate receptor activation is sometimes ineffective as a method of neuronal activation because of simultaneous activation of local GABAergic neurons. On the other hand, bicuculline has the advantage that blockade of somatodendritic GABA receptors produces selective neuronal excitation and is long-lasting. One of the main findings of this study is that the increase in insulin secretion produced by chemical stimulation of the DMV was enhanced after blockade of peripheral NO synthase using L-NAME. It is noteworthy that after a second microinjection of BIM into the DMV insulin secretion remained significantly elevated suggesting that newly synthesized insulin is sensitive to DMV-chemical stimulation in the presence of LNAME. This suggests that NO exerts an inhibitory influence on the two phases of insulin secretion. Interestingly, a significant reduction in blood glucose was also observed in the presence of L-NAME. This suggests that the augmented secretion of insulin that occurred after NO synthase inhibition also resulted in enhanced glucose uptake by the liver, muscle and fat. An inhibitory role for NO in insulin secretion has been described previously but most of these studies were conducted in vitro and used high concentrations of NO inhibitors that are unlikely to be attained in vivo (Panagiotidis et al., 1995; Salehi et al., 1996). In addition, it has been suggested that NO is involved in
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Insulin (ng/ml)
A
30 Glucose bolus
BIM Microinjection
injection
20
*
BIM Microinjection
* *
10 BIM+L-NAME BIM+Saline
Glucose level (mmol/l)
B
0 40
20
***
BIM+L-NAME
*** **
**
**
60
65
120
BIM+Saline
0 Basal
5
10
15
20
Post-bolus injection of glucose (min) Fig. 3 – Effects of NO inhibition on excitatory effects of DMV-chemical stimulation on glucose-induced insulin secretion. A. Systemic administration of L-NAME increased the excitatory effects of chemical stimulation on glucose-induced insulin secretion, significantly (filled squares, n = 6 and *P < 0.05). B. Blood glucose levels in the presence (filled squares, n = 6, **P < 0.01, and ***P < 0.001) and absence (open squares, n = 6) of L-NAME. Note the breaks in the time-scale on the x-axis and that some error bars are obscured by the symbol. Vertical bars denote times of glucose and drug administration.
modulation of insulin secretion via stimulation of a negative feedback mechanism since NO donors were found to produce inhibitory effects on insulin secretion in mice, rats, pigs and humans (Antoine et al., 1993; Corbett et al., 1993; Cunningham et al., 1994; Mosen et al., 2006; Sjoholm, 1996). Although it is evident that NO and NOS are present in the DMV, central effects of NO can be excluded in this study since L-NAME does not cross the blood brain barrier (Kaufmann et al., 2004; Travagli and Gillis, 1994). The present data showed that blockade of peripheral NO potentiated the excitatory effects of chemical stimulation of the DMV on insulin secretion. Given that both nitrergic and cholinergic nerves innervate the pancreas and the pancreatic vagal postganglionic neurons containing ACh and NO, it is reasonable to postulate that the latter normally inhibits the release of ACh and subsequently insulin release (Wang et al., 1999). An alternative explanation is that pancreatic nitrergic nerves are tonically involved in inhibition of insulin secretion. Recently, we found that DMV-pancreatic preganglionic neurons responded differentially to pancreatic secretagogues (Mussa et al., 2010). It is possible that the DMV also contains neurons that have both excitatory and inhibitory effects on insulin secretion. In the light of these findings, we propose that the β-
cells of the endocrine pancreas receive both excitatory and inhibitory inputs from the DMV and these modulate insulin secretion. In conclusion, chemical activation of the DMV led to an increase in insulin secretion via a cholinergic mechanism. This response was augmented by inhibition of peripheral NO synthase suggesting an involvement of nitrergic pathways and NO release in regulation of pancreatic insulin secretion.
4.
Experimental procedures
4.1.
General anesthetic and surgical procedures
All experiments were approved by the Ethical Review Committee of Austin Health (Heidelberg, Victoria, Australia) and complied with the principles outlined in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Male Sprague Dawley (SD) rats weighing between 250 and 450 g obtained from the Animal Resource Centre (ARC, Perth, Western Australia) were used in all experiments. SD
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B
A AP
AP
NTS
NTS
NTS
NTS CC XII
CC XII
DMV
DMV
C
D AP
AP
NTS
NTS CC
NTS
NTS CC XII
XII DMV
DMV
Fig. 4 – Coronal sections of the rat dorsal medulla showing a summary of the bicuculline methiodide (BIM) microinjection sites within the DMV during infusion of saline (A), atropine methonitrate (AMN) (C) and L-nitroarginine methyl ester (L-NAME) (D). Section B shows a summary of control microinjections of BIM made outside the DMV and ACSF microinjections made inside the DMV. Abbreviations: AP; area postrema; CC, central canal; DMV, dorsal motor nucleus of the vagus; NTS, nucleus of solitary tract; and XII, hypoglossal nucleus.
rats were housed in a temperature-controlled environment with a 12 hour light–dark cycle with overnight food deprivation and continuous access to water. SD rats were initially anesthetized in a chamber saturated with isoflurane vapor (Isoflurane, Delvet, Seven Hills, NSW, Australia). After checking that the depth of anesthesia was appropriate (no response to firm paw pinch), the rat was quickly tracheostomized to allow artificial ventilation with 100% O2 (1 ml/100 g body weight, 50–60 breaths/min) containing 1.5–1.7% isoflurane. Throughout the experiment the body temperature was maintained at 36–38 °C with a servo-controlled heating pad (Yellow Springs Instruments, Yellow Springs, OH, USA). The adequacy of the anesthesia was assessed by noting the absence of a withdrawal reflex to firm paw pinch as well as the absence of an eye blink response to gentle probing of the cornea. These tests were made every 15 min and the anesthetic vapor concentration was increased if necessary. After the completion of all surgery, urethane (1.2–1.5 g/kg, i.v.; Sigma Chemical Co., St Louis, MO, USA) was administered over 20–30 min and the inspired isoflurane concentration was gradually reduced to zero. Throughout the experiment additional urethane was added as required to maintain an adequate level of anesthesia.
4.2.
Chemical stimulation of the DMV
Polyethylene catheters (ID 0.58 x OD 0.96 mm, Critchley Electrical Products, Castle Hill, NSW, Australia) were inserted into the left carotid artery and the left jugular vein to monitor the arterial blood pressure and to allow intravenous admin-
istration of drugs, respectively. Anesthetized rats were placed into a stereotaxic frame and the right cervical vagus was isolated through a lateral cervical incision. The vagus was isolated and placed carefully onto a pair of silver wire hook electrodes and insulated from the surrounding tissue using a two component silicone sealant (Kwik-Cast, World precision Instruments, Sarasota, FL, USA). A midline incision was made over the scalp and the connective tissue lying over the longus capitus muscles and interparietal bone was removed. The longus capitus muscles were divided longitudinally and the atlantooccipital membrane overlying the dorsal medulla oblongata was exposed. The membrane was carefully dissected away and a small section of the occipital bone was removed to allow easier access to the dorsal medulla. A recording electrode (2 mm OD, Harvard Apparatus Ltd., Kent, England), filled with 0.5 M sodium acetate/2% pontamine sky blue (Koch-Light Labs., Coinbrook, Berks. UK) was positioned over the DMV using the following stereotaxic coordinates: 400 μm lateral and 700 μm rostral to the calamus scriptorius which was used as reference point to localize the DMV. The recording microelectrode, with impedances ranging between 5 and 10 MΩ, was advanced into the dorsal medulla while electrical stimulation (0.5 Hz, 0.5 ms pulse width, 500 μA) was applied to the ipsilateral vagus nerve. DMV action potentials were amplified (×1000) and filtered (400–4000 Hz) using an extracellular single unit amplifier and window discriminator (Fintronics, Orange, CT, USA) and monitored using an audio amplifier and oscilloscope (Tektronix Inc., Beaverton, Oregon, USA). The antidromic nature of constant latency spikes
68
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elicited by stimulation of the vagus nerve was verified using the collision test (Lipski, 1981). After the location of the DMV was identified for subsequent microinjection of BIM, the recording electrode was exchanged for a microinjector filled with BIM (100 pmol) containing 10% rhodamine beads (Fluospheres, Invitrogen, Molecular Probes, Eugene, OR, USA), which were used to mark the microinjection sites. The microinjector, which consisted of a glass micropipette cemented to the tip of a 1 μl glass microsyringe (Scientific Glass Engineering, Ringwood, Victoria, Australia), was advanced into the medulla and positioned at the coordinates identified in the last recording procedure. BIM (GABAA receptor antagonist, 100 pmol/25 nl; 4 mM) was microinjected bilaterally into the DMV.
4.3. Effect of chemical stimulation of the DMV on glucose-induced insulin secretion To examine the effects of chemical stimulation of the DMV on glucose-induced insulin secretion, blood glucose and plasma insulin levels were measured prior to, and after bilateral microinjection of BIM. After the completion of all anesthetic and surgical procedures, a stabilization period of 30 min was allowed and basal glucose and insulin levels were measured. Two basal samples were collected 5 min apart and then insulin secretion was induced by a bolus injection of glucose (1 g/kg, i.v., Mallinckrodt, Paris, KY, USA). Following the induction of insulin secretion, insulin and glucose levels were measured twice 5 min apart. This was followed by chemical stimulation of the DMV by bilateral microinjection of BIM (100 pmol/25 nl) into the DMV and thereafter insulin and glucose levels were measured every 5 min. Forty minutes after the last set of blood glucose and insulin measurements were made, bilateral microinjection of BIM into the DMV and subsequent measurement of glucose and insulin levels were repeated. Final measurements of insulin and glucose levels were made 60 min after the last microinjection of BIM into the DMV.
4.4. Effect of peripheral muscarinic receptor blockade on glucose-induced insulin secretion produced by chemical stimulation of the DMV To establish the specificity of the results, two control experiments were performed using the above-mentioned protocol: (i) BIM was microinjected outside the DMV and (ii) ACSF was microinjected into the DMV. In addition, the involvement of a cholinergic pathway in the effects of DMV activation on insulin secretion was investigated by administration of AMN (100 μg/ kg/min, i.v. for 20 min). The DMV was stimulated using BIM after 20 min of intravenous infusion of AMN and glucose and insulin levels were measured prior to, and after intravenous infusion of AMN.
5 min apart. Then insulin secretion was induced by a bolus injection of glucose (1 g/kg, i.v.) and another set of insulin and glucose measurements were performed. This was followed by 20-min of intravenous infusion of L-NAME (a NO synthase inhibitor, 30 mg/kg, i.v.) at a rate of 50 μl/min. Measurement of insulin and glucose was performed prior to, and after microinjection of BIM in the presence of L-NAME. The specificity of the results was confirmed by control experiments in which normal saline was infused intravenously instead of L-NAME.
4.6.
Measurement of blood glucose and insulin levels
Blood glucose was measured in blood taken from the arterial cannula. A drop of arterial blood was placed onto the test strip of a glucometer (Optium Xceed, MediSense, Abbott Laboratories, Bedford, MA, U.S.A.). This device is calibrated against a Yellow Springs International Glucose Analyser (YSI Inc., Yellow Springs OH, USA). Blood samples (500 μl) for insulin assay were collected in plastic tubes, immediately centrifuged and frozen for subsequent measurement of insulin using rat insulin radioimmunoassay (RIA; LINCO Research, Inc. St Charles, MO, USA).
4.7.
Histological identification of the microinjection sites
At the end of all experiments, rats were anesthetized deeply with isoflurane and perfused transcardially with 160 ml of normal saline (0.9% NaCl w/v) followed by 100 ml of 10% formalin (formaldehyde; Riedel-de-Haen, Seelze, Germany, diluted 1:4 with normal saline). Brains were removed and postfixed in 10% formalin for at least 24 h and then were snapfrozen by dipping into liquid nitrogen. Frozen coronal sections (40 μm thickness) of the brainstem were cut using a cryostat (Cryocut 1800, Reichert-Jung, Germany). Coronal brain sections that contained microinjection sites were mounted onto glass slides coated with gelatin and coverslipped with Vectashield (Vector laboratories, Burlingame, CA, USA). Fluorescence microscopy using an excitation wavelength of 550 nm was used to visualize the microinjection sites. The sections were then stained with cresyl violet and examined using light microscopy. The exact locations of the microinjection sites were identified using standard maps of the rat brain that were reproduced from the atlas of Paxinos and Watson (Paxinos and Watson, 1986).
4.8.
Data and statistical analysis
All data are expressed as S.E.M and differences between individual means were assessed and compared using one way analysis of variance (ANOVA) followed by the Tukey Multiple Comparisons tests. Probability values of P < 0.05 were considered statistically significant.
4.5. Effect of NO synthase inhibition on glucose-induced insulin secretion produced by chemical stimulation of the DMV
4.9.
This group of experiments was performed to determine the role of NO, if any, in mediating the insulin secretory responses to chemical activation of the DMV. After a 30 min stabilization period, basal levels of insulin and glucose were measured
Atropine methonitrate (AMN) and L-nitroarginine methyl ester (L-NAME) were obtained from Sigma Chemical Co. (St Louis, MO, USA) and were dissolved in normal saline (0.9% NaCl w/v; Pharmacia & Upjohn Ltd., Perth, Australia).
Materials
BR A IN RE S E A RCH 1 3 94 ( 20 1 1 ) 6 2 –7 0
Bicuculline methiodide (BIM; Sigma Chemical Co., St Louis, MO, USA) was dissolved in artificial cerebrospinal fluid (ACSF) with the following composition (mM): NaCl, 128; KCl, 2.6; CaCl2·2H2O, 1.3; MgCl2·6H2O, 0.9; NaHCO3, 20; and Na2HPO4, 1.3.
Acknowledgments This study was supported by a grant from the Austin Hospital Medical Research Foundation and a post-graduate scholarship from the University of Melbourne to B.M.M. The authors declare no conflict of interest.
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