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Endocrinology Vol. 147, No. 6 2893-2901
Copyright © 2006 by The Endocrine Society

Direct Stimulation of Ghrelin Secretion by Sympathetic Nerves

Thomas O. Mundinger, David E. Cummings and Gerald J. Taborsky, Jr

Department of Medicine, Division of Metabolism, Endocrinology, and Nutrition, Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108; and University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: Dr. Thomas O. Mundinger, Division of Endocrinology and Metabolism (151), Veterans Affairs Puget Sound Health Care System, 1660 South Columbian Way, Seattle, Washington 98108. E-mail: mundin{at}u.washington.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hormone ghrelin is secreted mainly from the gut, rises in peripheral plasma before meals, and is implicated in stimulating hunger, initiating meals, and developing obesity. We hypothesize that activation of the sympathetic nervous system contributes to preprandial ghrelin surges. The present studies in isoflurane-anesthetized Wistar rats were designed to determine whether sympathetic nerves and neurohormones are capable of stimulating ghrelin secretion. We activated gut sympathetic nerves by two methods: electrical sympathetic nerve stimulation (SNS) and chemical sympathetic nerve activation with iv tyramine (TYR) administration. Portal venous blood was sampled before and during a 10-min sympathetic stimulation. Successful activation of gut sympathetic nerves was verified by increments in portal venous norepinephrine. SNS increased portal ghrelin by 206 ± 50%. In contrast, simply isolating gut sympathetic nerves without applying current had a minimal effect on ghrelin levels. TYR also increased portal ghrelin [change ({Delta}), +52 ± 11%], whereas saline infusion had little effect. We next determined whether the neural stimulation of ghrelin secretion was mediated indirectly via the suppression of insulin secretion during SNS and TYR. Streptozotocin-induced diabetes prevented a fall in insulin during TYR, yet the portal ghrelin response ({Delta} = +47 ± 18%) was similar to that in nondiabetic rats. Lastly, to test for humoral stimulation of ghrelin, we infused the sympathetic neurohormone, epinephrine, to achieve levels found during severe stress. Epinephrine failed to stimulate ghrelin secretion ({Delta} = +4 ± 35%). We conclude that the neural, but not the neurohumoral, branch of the sympathetic nervous system can directly stimulate ghrelin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN IS AN orexigenic peptide hormone that potently stimulates food intake in humans (1, 2) and rodents (3, 4). It is implicated as a participant in mealtime hunger and meal initiation (5). Prominent among the evidence supporting this assertion are marked premeal surges in circulating ghrelin levels in man (6), rats (7), and sheep (8), which are temporally linked to the perception of hunger in humans (9). In rats, exogenous ghrelin administration rapidly stimulates food intake, decreasing the latency to feed and increasing the number of meals initiated, without affecting meal size (10). Moreover, the specific food-related behaviors that are stimulated by ghrelin, including foraging, sniffing, hoarding, and performing work for food, are all examples of appetitive, rather than consummatory, feeding behavior. In other words, the effects of ghrelin appear to motivate animals to seek out and acquire food.

Endogenous ghrelin is synthesized primarily by the gastrointestinal tract, with minor expression in several other tissues (11, 12). The effects of gastrectomy or extensive small bowel resection in mice (13) and humans (11, 14) indicate that approximately 75–80% of circulating ghrelin is derived from the stomach, and the remainder is secreted predominantly from the proximal small intestine. Therefore, most ghrelin enters the circulation via the portal vein, which drains this portion of the gut. However, no direct stimulators of ghrelin secretion have been identified.

Two lines of evidence suggest that the rise in peripheral ghrelin levels before scheduled meals results from active stimulation of secretion, rather than from passive return to an elevated baseline. First, the sharp profile of preprandial ghrelin surges in man (6), sheep (8, 15), and rats (7) suggests an acute onset of stimulation, rather than a passive drift upward after release from previous suppression. Consistent with this assertion, people undergoing an acute 24-h fast, with no food-related ghrelin suppression, display spikes of circulating ghrelin levels immediately preceding their accustomed meal times (16). Second, the ability of different feeding regimens to alter the number and magnitude of preprandial ghrelin surges suggests that ghrelin secretion is actively regulated, probably by a conditioned reflex. For instance, when rats are habituated to a single daily meal period, circulating ghrelin surges are initiated 1 h before that meal (17). When sheep are placed on controlled feeding regimens with varying numbers of scheduled meals per day, yet consume the same total daily calories, the number of ghrelin surges matches the number of habitual feeding sessions (15). Furthermore, the magnitude of each premeal ghrelin surge in sheep increases when the number of meals per day is reduced (15); presumably, greater ghrelin surges drive greater hunger. These findings suggest that peripheral ghrelin secretion is actively regulated by cephalic mechanisms, as has been shown for feeding behavior and many metabolic responses that occur in anticipation of scheduled meals (18). Because most of these conditioned responses (e.g. salivation, gastric motility and acid secretion, and insulin secretion) are mediated by the autonomic nervous system (18, 19), preprandial ghrelin surges may also be subject to autonomic control, particularly in the setting of regularly scheduled meals.

We hypothesize that activation of the sympathetic nervous system, which is known to regulate the secretion of other gut hormones (20, 21, 22), is one mediator of increased ghrelin secretion before scheduled meals. The following evidence is consistent with sympathetic stimulation of ghrelin secretion. 1) In pathological states, circulating levels of catecholamines, the classic products of sympathetic output, correlate positively with ghrelin levels. For example, both norepinephrine and ghrelin levels are elevated in chronic obstructive pulmonary disease (23). Conversely, patients with poorly controlled phenylketonuria, in whom circulating levels of norepinephrine and epinephrine (EPI) are low due to deficient catecholamine synthesis, have reduced fasting ghrelin levels (24). 2) Cold exposure increases both sympathetic output (25, 26) and circulating ghrelin levels (27). 3) The human ghrelin gene promoter is activated by cAMP (28), the second messenger that mediates adrenergic signaling. 4) There is precedent for regional, peritoneal activation of sympathetic nerves to mobilize fuels during fasting, a setting in which ghrelin levels increase in rodents (3). For example, sympathetic activity to some white adipose tissue is increased during fasting in rats (29, 30, 31) and humans (32), helping to mobilize free fatty acids (33, 34). Likewise, there is sympathetic tone to the canine liver during fasting that is sufficient to stimulate net hepatic glucose production (35, 36) to maintain plasma glucose levels. A fall in plasma glucose or the resultant central glucopenia also increases sympathetic drive to the canine liver (37), which markedly stimulates hepatic glucose production (35). Therefore, it seems reasonable to speculate that sympathetic activation might participate in the feeding response to energy deficit by stimulating ghrelin secretion.

A cornerstone of our hypothesis, that there is an increase in sympathetic neural activity to the gut before scheduled meals, may seem at odds with previous reports. For instance, early work by Young and Landsberg (38, 39) demonstrated a decrease in sympathetic activity in the heart, liver, and pancreas of fasted rats, leading to the general impression that all sympathetic neural outflow from the brain is decreased during a fast. However, subsequent work in rats (40), steers (41), and humans (32, 42) consistently failed to demonstrate the expected decrease in systemic plasma norepinephrine levels after a prolonged fast. Finally, sympathetic activity to white adipose tissue was shown to actually increase during fasting in rats (29, 30, 31) and humans (32). Together, these studies demonstrate that one cannot predict the sympathetic activity to a specific organ using either the systemic levels of norepinephrine or the sympathetic activity to other organs. Therefore, because there is little information on stomach-specific sympathetic activity, the question of whether the sympathetic nerves to the stomach are activated before meals remains open. This particular question, however, is beyond the scope of the current studies.

As a first step toward testing our hypothesis that the sympathetic nervous system contributes to preprandial ghrelin surges, we determined whether this branch of the autonomic nervous system is indeed capable of stimulating ghrelin secretion. We assessed portal venous ghrelin responses to neural and humoral sympathetic stimulations. Postganglionic sympathetic neural stimulation of the gut was produced with both electrical and chemical stimuli. Because these neural stimuli also suppressed pancreatic insulin release, which, in theory, could stimulate ghrelin secretion indirectly (43), we performed additional nerve stimulation studies in rats rendered insulin deficient with the pancreatic ß-cell toxin, streptozotocin (STZ). Finally, to determine whether the sympathetic nervous system is capable of stimulating ghrelin humorally, we administered the sympathetic neurohormone, EPI, at a dose replicating the pathophysiological EPI levels typical of severe stress. Our results show that activation of gut sympathetic nerves is one possible mediator of the preprandial rise in ghrelin levels seen in humans and other animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and pretreatments
All experiments were performed on male Wistar rats (280–350 g; Simonsen Laboratories, Gilroy, CA). Animals were housed in groups under a 12-h light, 12-h dark cycle and had ad libitum access to pelleted chow until the evening before the experiments. Water was available at all times.

STZ (Sigma-Chemical Corp., St. Louis, MO) was administered to one group of rats to destroy pancreatic ß-cells and thereby prevent a fall of insulin secretion during sympathetic activation. STZ was dissolved in citric acid buffer (pH 4.5) immediately before sc administration to rats (40 mg/kg, once daily for 2 d). Twenty-four hours after the second injection, hyperglycemia was confirmed (blood glucose, >450 mg/dl). Rats were then briefly anesthetized with isoflurane and implanted with a sc insulin pellet (0.67 U/d; LinShin Canada, Inc., Toronto, Canada). Seven or 9 d later, rats underwent acute, terminal studies.

All rats included in these studies were certified as healthy by the Veterinary Medical Officer and exhibited normal grooming and feeding behavior on the day of the study. Research involving animals was conducted in an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited facility, and all protocols were approved by the institutional animal care and use committee of the Seattle Veterans Affairs Puget Sound Health Care System.

Surgery and experimental design
All studies were performed on overnight-fasted rats to determine the ghrelin response to sympathetic neural or humoral stimulation. Studies were performed as acute experiments under anesthesia, and the rats were killed after the experiment. Each rat was studied under one protocol, so that no rat received multiple infusions or stimulations. Animals were anesthetized with isoflurane (4% induction, 2% maintenance in 1 liter/min oxygen) and placed on a heating pad with a rectal thermometer (Harvard Apparatus, Holliston, MA) to maintain body temperature at 37 C throughout surgery and experimentation. A midline laparotomy was performed to implant sampling catheters, infusion catheters, and, in some studies, stimulus electrodes. Saline at 37 C was dripped on peritoneal tissue throughout surgery to minimize dehydration. Portal venous sampling catheters (polyethylene; BD Biosciences, Sparks, MD) were implanted to measure ghrelin, norepinephrine, EPI, insulin, and glucagon increments in response to sympathetic stimulation. Portal catheters were filled with heparinized saline, introduced into a jejunal vein adjacent to the cecum, advanced cranially through the portal vein, and secured with the tip 1 cm caudal to the liver. This location of the tip maximized sampling of gastric and proximal duodenal effluent. For electrical nerve stimulation, tyramine (TYR) in STZ rats, and iv EPI studies only, an arterial sampling catheter was introduced into the descending aorta caudal to the kidneys to measure systemic catecholamine increments. Peripheral infusion catheters (polyethylene; BD Biosciences) for donor blood and drug infusion were introduced into the inferior vena cava. For electrical sympathetic nerve stimulation and isolation studies, the lateral nerve trunk projecting from the celiac ganglion and running between the lienal and hepatic arteries was isolated. A bipolar electrode (Harvard Apparatus) was placed around the nerve and connected to an S-44 stimulator (Grass Instruments, Quincy, MA) and oscilloscope. Peritoneal tissue was again bathed in warm saline, the laparotomy was covered with plastic wrap, and a 30-min stabilization period preceded baseline samples.

Fifteen minutes before baseline blood sampling, an iv infusion of heparinized blood from fasted donor rats was begun. This infusion, designed to avoid hypovolemia, was continued throughout the study, so that the total volume of donor blood infused fully matched that which was withdrawn for portal and arterial samples. Experiments consisted of a 5-min baseline period, followed by a 10-min sympathetic stimulation period, followed by a 20- or 25-min recovery period. Five or seven portal samples were drawn per experiment: two basal, two or three stimulation, and one or two recovery. In the minority of studies where arterial samples were also drawn, only one sample was taken during the baseline, stimulation, and recovery periods. At the end of these terminal studies, correct catheter tip and electrode placements were confirmed, and some rats had the splenic lobe of the pancreas excised, snap-frozen on dry ice, and stored at –70 C to determine tissue catecholamine content. The ratio of EPI/norepinephrine content of the pancreas was taken as representative of gut tissue and was used to estimate the amount of releasable EPI in gut sympathetic nerves. Rats were killed immediately after tissue harvest.

Electrical sympathetic neural stimulation parameters were 8 Hz for 10 min; pulse duration and intensity were 1 msec and 10 mA, respectively. These stimulation parameters are considered in the high physiological range, because electrical stimulation of canine hepatic sympathetic nerves using these stimulus parameters increased the spillover of norepinephrine from the liver (35) only twice that seen during reflexive activation of hepatic sympathetic nerves during hypoxic stress (37). The current applied to postganglionic sympathetic nerves was verified by oscilloscope deflection. TYR (Sigma-Aldrich Corp.) was infused into the inferior cava at 320 µg/kg·min, and EPI was infused at 400 ng/kg·min. All iv drug infusions were given for 10 min at a rate of 0.12 ml/min. Infusion pumps for donor blood and drug administration (Harvard Apparatus) were calibrated before each study. TYR and EPI infusates were prepared immediately before administration. These amines were dissolved in saline plus ascorbic acid, and care was taken to protect infusates from light to avoid degradation.

Blood for ghrelin and catecholamine determinations was drawn on a mixture (20 µl/ml blood) of EGTA (0.09 mg/ml) and glutathione (0.06 mg/ml). Blood for insulin and glucagon measurements was drawn on benzamidine HCl (1 M; 50 µl/ml blood). Samples were immediately placed on ice and centrifuged (1730 x g, 20 min, 3 C), and plasma was frozen (–80 C) until assay.

Plasma and tissue analyses
Plasma ghrelin concentrations were measured in duplicate by RIA using a primary antibody against rat ghrelin and 131I-labeled ghrelin as the tracer (kit RK-031-31, Phoenix Pharmaceuticals, Belmont, CA). This assay detects both bioactive acylated ghrelin and inactive des-acyl ghrelin; however, acute and chronic changes in total ghrelin correlate with changes in the active form, and the ratio of the acylated/des-acyl forms remains constant under a wide variety of conditions that affect ghrelin levels (44, 45, 46). The intra- and interassay coefficients of variance (CVs) in this laboratory are 6% and 10%, respectively.

Plasma catecholamine concentrations were measured in duplicate using a sensitive and specific radioenzymatic assay (47). The intra- and interassay CVs in this laboratory are 6% and 12%, respectively. Frozen pancreatic tissue (0.4–0.6 g) was homogenized and boiled in 1 N acetic acid (4 ml/g tissue, 10 min). The homogenate was centrifuged twice (19,230 x g, 20 min), and the supernatant was dried, then reconstituted in 1 ml Tris buffer containing EGTA and glutathione. Extracts were stored at –30 C until assay.

Plasma insulin concentrations were measured in duplicate using a modification of the double-antibody RIA technique of Morgan and Lazarow (48). Plasma glucagon was measured in duplicate by RIA (Linco Research, Inc., St. Charles, MO).

Data presentation and statistics
There was little variation in baseline catecholamine values among individuals (CV, 28%; n = 29 rats) and among experimental groups (CV, 17%; range, 992 ± 145 to 1,517 ± 159 pg/ml; n = 6 groups). Because of this small degree of variability, catecholamine responses were easily compared between groups using raw data; therefore, EPI and norepinephrine data are presented in the figures as absolute values. In contrast, baseline ghrelin values varied greatly among individuals (CV, 56%) and groups (CV, 35%; range, 3663 ± 374 to 10,075 ± 2330 pg/ml). To accommodate for this variability and allow adequate comparisons between groups, ghrelin data are presented in the figures as the percent change (%{Delta}) from baseline.

EPI extraction across the gut was calculated as follows: extraction = [([EPI] artery – [EPI] portal vein)/([EPI] artery)] x 100%. Gut-specific release of norepinephrine was calculated only during SNS and TYR/STZ studies, because these were the only neural stimulation studies with arterial sampling catheters. Gut release of NE = [NE] portal vein – [[NE] artery x (1 – extraction)].

All data are expressed as the mean ± SEM. Responses between groups were compared using ANOVA with repeated measures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The data presented in the following section describe, in single values, the average of two basal samples, the average of two or three stimulation samples, and either a single or the average of two recovery samples. The accompanying figures describe data in more detail by including group averages at each individual time point.

Responses to sympathetic nerve stimulation (SNS)
To determine whether SNS of the gut increases ghrelin secretion, we measured the plasma concentration of ghrelin in the portal vein before and during electrical SNS of postganglionic axons emanating from the celiac ganglia. Portal venous ghrelin levels increased during SNS from a baseline of 6582 ± 691 to 20,146 ± 3706 pg/ml during SNS (%{Delta} = +206 ± 50%; n = 5; Fig. 1AGo). Ghrelin levels tended to fall during the recovery period, but remained elevated over baseline (%{Delta} = +119 ± 59%). In contrast, when sympathetic nerves were isolated, but not electrically stimulated (ISO), portal ghrelin levels increased only slightly from a baseline of 10,075 ± 2,330 pg/ml (%{Delta} = +27 ± 7%; n = 5; Fig. 1AGo) and continued to drift upward during the recovery period (%{Delta} = +52 ± 14%). The portal ghrelin response during SNS was markedly greater than that during ISO (P < 0.005), demonstrating sympathetic stimulation of gastrointestinal ghrelin secretion.


Figure 1
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  		FIG. 1. A, Increase in plasma ghrelin concentration in the portal vein (PV) during electrical SNS (bullet). Isolating the sympathetic nerves without applying current (Isolation; {circ}) had little effect on portal ghrelin. Data in all figures are expressed as the mean ± SEM. B, There was an increase in plasma norepinephrine concentration in the portal vein during electrical SNS (bullet). Isolating the sympathetic nerves without applying current (Isolation; {circ}) had no effect on portal norepinephrine.

 
To verify successful stimulation of sympathetic nerves during SNS, we measured concentrations of the classical sympathetic neurotransmitter, norepinephrine, in portal venous plasma. Portal norepinephrine concentrations rose from a baseline of 1020 ± 147 to 5257 ± 643 pg/ml during SNS ({Delta} = +4237 ± 593 pg/ml; Fig. 1BGo). Norepinephrine levels returned almost completely to baseline levels during recovery ({Delta} = +250 ± 118 pg/ml). In contrast, during ISO, portal norepinephrine did not change from a baseline of 992 ± 145 pg/ml ({Delta} = –39 ± 55 pg/ml; Fig. 1BGo), and norepinephrine levels remained unchanged during the recovery period ({Delta} = +80 ± 127 pg/ml). The portal norepinephrine response during SNS was markedly greater than that during ISO (P < 0.001), verifying successful stimulation of sympathetic nerves.

To define the magnitude of gut-specific sympathetic neural activation during SNS, we calculated gut-specific release of norepinephrine. Arterial norepinephrine increased from a baseline of 854 ± 95 to 1774 ± 175 pg/ml during SNS ({Delta} = +920 ± 119 pg/ml). Using this arterial norepinephrine increment, the 71% extraction of catecholamines across the gut determined during the EPI infusion experiments (see below) and the portal venous norepinephrine increment described above, the gut contribution to the portal venous norepinephrine increment during SNS was calculated to be +3970 ± 564 pg/ml (see Materials and Methods). Thus, SNS successfully stimulated gut-specific sympathetic nerves.

The concentration of EPI in the portal vein increased from a baseline of 390 ± 64 to 1354 ± 166 pg/ml during SNS ({Delta} = +964 ± 127 pg/ml). In contrast, portal EPI rose only slightly during ISO, from 322 ± 53 to 431 ± 38 pg/ml ({Delta} = +109 ± 20 pg/ml). Thus, the portal EPI response during SNS was substantially greater than that during ISO (P < 0.001). The EPI/norepinephrine increment ratio seen in portal venous plasma during SNS was 23%, which was much larger than that during TYR (see below).

To determine the magnitude of the expected inhibition of insulin secretion during SNS, we measured portal venous insulin concentrations. During SNS, portal insulin decreased from 30 ± 17 to 10 ± 4 pmol/liter ({Delta} = –21 ± 13 pmol/liter). As expected, portal insulin did not change during ISO ({Delta} = –4 ± 3 pmol/liter). The fall in insulin during SNS tended to be greater than that during ISO (P = 0.11).

Responses to iv TYR infusion
To verify, by a second independent method, that activation of sympathetic nerves stimulates gut ghrelin secretion, we infused the sympathomimetic drug, TYR iv. Portal ghrelin concentrations rose from a baseline of 9,321 ± 1662 to 13,340 ± 1435 pg/ml during TYR (%{Delta} = +52 ± 11%; n = 5; Fig. 2AGo). Ghrelin levels remained elevated during the recovery period (%{Delta} = +56 ± 13%). In contrast, when saline was infused iv (NaCl), portal ghrelin increased only slightly, from a baseline of 5,666 ± 1222 to 6603 ± 1,333 pg/ml (%{Delta} = +18 ± 7%; n = 5; Fig. 2AGo). Ghrelin levels continued to rise during the recovery period (%{Delta} = +58 ± 27%). The portal ghrelin response during the TYR stimulation period was significantly greater than that during NaCl (P < 0.05), verifying sympathetic neural stimulation of gut ghrelin secretion.


Figure 2
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  		FIG. 2. A, Increase in plasma ghrelin concentration in the portal vein during iv administration of TYR (bullet). Intravenous administration of saline (NaCl; {circ}) had little effect on portal ghrelin. B, There was an increase in plasma norepinephrine concentration in the portal vein during iv administration of TYR (bullet). Intravenous administration of saline (NaCl; {circ}) had no effect on portal norepinephrine.

 
Portal norepinephrine increased from a baseline of 1517 ± 159 to 6815 ± 412 pg/ml during TYR ({Delta} = +5298 ± 292 pg/ml; Fig. 2BGo) and returned to baseline during recovery ({Delta} = +22 ± 106 pg/ml). As expected, portal norepinephrine did not change from a baseline of 1204 ± 124 pg/ml during NaCl ({Delta} = +47 ± 33 pg/ml) and remained low during the recovery period ({Delta} = +223 ± 89 pg/ml). Thus, the portal norepinephrine response during TYR was markedly greater than that during NaCl (P < 0.001), verifying successful SNS.

Unlike SNS, TYR does not stimulate the adrenal medulla. Therefore, portal EPI increased only slightly during TYR, from 146 ± 23 to 310 ± 58 pg/ml ({Delta} = +164 ± 42 pg/ml). As expected, NaCl had no effect on portal EPI (basal, 99 ± 9 pg/ml; {Delta} = +4 ± 2 pg/ml). The slight, but significant, EPI response to TYR (P < 0.005 vs. NaCl) was only 3% of the corresponding norepinephrine response. Interestingly, this ratio was identical with the 3% ratio of EPI to norepinephrine content found in pancreatic tissue (EPI content, 5.18 ± 0.64 ng/g weight; norepinephrine content, 165.39 ± 20.37 ng/g weight; n = 12). These data suggest that the portal EPI response during TYR was exclusively due to neuronal release of EPI from noradrenergic nerves. In contrast, the portal EPI response during SNS was 23% of the norepinephrine response, verifying significant nonneural (i.e. adrenal) release of EPI during SNS. In support of this assertion, despite similar portal norepinephrine responses in SNS and TYR, the portal EPI response to SNS was significantly greater than that to TYR (P < 0.001).

As expected, TYR decreased portal insulin concentrations; however, this suppression did not occur either during TYR in STZ-induced diabetic rats or during NaCl. For instance, portal insulin levels fell in nondiabetic rats from 44 ± 10 to 19 ± 2 pmol/liter during TYR ({Delta} = –25 ± 9 pmol/liter), whereas portal insulin did not decrease from 59 ± 18 pmol/liter with TYR in STZ rats ({Delta} = +2 ± 5). Likewise, portal insulin did not decrease from 55 ± 8 pmol/liter during NaCl ({Delta} = –1 ± 5; P < 0.05 vs. TYR).

Although STZ prevented a fall in insulin during TYR, it did not impair the ghrelin response to TYR. In STZ rats, portal ghrelin increased from 3663 ± 374 to 5259 ± 541 pg/ml during TYR (%{Delta} = +47 ± 18%; n = 5; Fig. 3AGo) and remained moderately elevated during the recovery period ({Delta} = +28 ± 14%). Portal norepinephrine increased from 1378 ± 163 to 6619 ± 541 pg/ml during TYR in STZ rats ({Delta} = +5241 ± 462 pg/ml; n = 5; Fig. 3BGo) and returned nearly to baseline levels during recovery ({Delta} = +422 ± 145 pg/ml). Thus, the portal ghrelin and norepinephrine responses to TYR in STZ rats were similar to those in nondiabetic rats (both P = NS).


Figure 3
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  		FIG. 3. A, Similar increases in plasma ghrelin concentration in the portal vein during iv administration of tyramine in nondiabetic rats (bullet) and STZ-diabetic rats ({square}). B, There were similar increases in the plasma norepinephrine concentration in the portal vein during iv administration of TYR in nondiabetic rats (bullet) and STZ-diabetic rats ({square}).

 
Arterial norepinephrine increased from a baseline of 1148 ± 235 to 6156 ± 581 pg/ml during TYR in STZ rats ({Delta} = +5008 ± 385 pg/ml). Gut-specific release of norepinephrine during TYR in STZ rats was calculated to be +3789 ± 455 pg/ml. Thus, TYR in STZ rats successfully stimulated gut sympathetic nerves, and this gut norepinephrine release was not different from that during SNS (P = NS).

Responses to iv EPI infusion
To determine whether the adrenal EPI release during SNS could contribute to the ghrelin response, we measured the plasma concentration of ghrelin in the portal vein before and during iv EPI infusion. Portal ghrelin concentrations averaged 9770 ± 3125 pg/ml during the baseline period and remained unchanged during EPI infusion (%{Delta} = +4 ± 35%; n = 4; Fig. 4AGo; P = NS vs. NaCl). This suggests that sympathetic humoral stimulation did not alter ghrelin secretion. Similar to NaCl studies, ghrelin levels tended to rise moderately during the recovery period (%{Delta} = +24 ± 22%).


Figure 4
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  		FIG. 4. A, Lack of an increase in plasma ghrelin concentration in the portal vein during iv administration of either EPI (bullet) or saline (NaCl; {circ}). B, Differential increases in plasma EPI were achieved in the artery ({blacksquare}) vs. the portal vein (bullet) in response to iv EPI infusion. Intravenous saline infusion (NaCl) had no effect on portal venous EPI ({circ}).

 
To verify that EPI provided a marked humoral stimulus to the gut, we measured both the arterial increments in EPI and the portal increments in glucagon, a pancreatic hormone stimulated by circulating EPI (22). Arterial EPI concentrations increased from 1593 ± 323 to 7343 ± 1062 pg/ml during EPI ({Delta} = +5750 ± 758 pg/ml; Fig. 4BGo), confirming levels equal to or exceeding those found during severe stress (37, 49, 50). Arterial EPI then fell to near baseline during the recovery period ({Delta} = +613 ± 478). Furthermore, portal glucagon levels rose from a baseline of 303 ± 31 to 1379 ± 217 pg/ml during EPI infusion ({Delta} = +1076 ± 188 pg/ml) and fell to baseline (367 ± 78 pg/ml) during recovery. Therefore, the EPI levels achieved were capable of markedly stimulating gut hormone secretion.

Portal EPI increased from 421 ± 78 to 2224 ± 314 pg/ml during EPI infusion ({Delta} = +1803 ± 243 pg/ml; Fig. 4BGo), a response significantly greater than that during SNS (P < 0.01). Portal EPI then returned close to baseline levels during recovery ({Delta} = +316 ± 154 pg/ml). From the individual portal and arterial levels before and during EPI infusion, we calculated gastrointestinal EPI extraction to be 72 ± 2% at baseline and 71 ± 5% during EPI infusion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies in humans (6, 16), sheep (8, 15), and rodents (17) have shown that the increase in ghrelin levels before scheduled meals results from active stimulation of ghrelin secretion; however, no specific mediators of this preprandial ghrelin stimulation have been described. The precedent for sympathetic mediation of fuel mobilization during fasting (29, 31, 32, 35, 36) coupled with the positive correlation between ghrelin and norepinephrine levels in disease (23, 24) led us to hypothesize that activation of sympathetic nerves to the stomach and proximal intestine before meals stimulates ghrelin secretion. In support of this hypothesis, the current studies demonstrate that two independent methods of activating sympathetic nerves of the gastrointestinal tract increase ghrelin secretion. Therefore, sympathetic neural activation is a viable candidate for mediation of preprandial ghrelin surges, particularly in the context of regularly scheduled meals.

To test for sympathetic neural regulation of ghrelin secretion, we electrically stimulated postganglionic sympathetic axons projecting from the celiac ganglion of the rat. We chose this particular prevertebral sympathetic ganglion because the majority of sympathetic nerves innervating the stomach and duodenum, the major sources of circulating ghrelin (5), originate from cell bodies in the celiac ganglia (51). The substantial norepinephrine response during SNS verified successful stimulation of gastrointestinal sympathetic nerves, and the concomitant portal ghrelin response demonstrated marked stimulation of ghrelin secretion from the gut. Although the celiac ganglia project nerves to the stomach, proximal and distal intestine, spleen, pancreas, and liver, and all but the last of these organs drains into the portal vein, the portal ghrelin increment during SNS is most likely of stomach and duodenal origins, given the dominant ghrelin content and secretory ability of these two tissues (5). The portal venous norepinephrine increase, however, cannot be attributed mainly to stomach and duodenal effluent, because all SNS-stimulated tissues listed above are capable of significant norepinephrine release.

Although there was a simultaneous increase in both ghrelin and norepinephrine during SNS, there was a disparity in ghrelin and norepinephrine levels after discontinuing the 10-min stimulus. Ghrelin levels tended to decrease in the poststimulation period, but remained elevated over baseline. This incomplete recovery is probably due to the long plasma half-life of this peptide (7) and to an unknown factor causing a slow, progressive rise over time, a trend also seen in the isolation group. Conversely, norepinephrine levels returned close to baseline after discontinuation of SNS. This complete recovery of norepinephrine is due to its extremely short plasma half-life (52) and to the absence of a progressive rise over time. Regardless of the disparity in the recovery period, the sharp increases in ghrelin and norepinephrine during the short SNS period, compared with those in ISO animals, clearly demonstrates robustly stimulated ghrelin secretion during acute neural stimulation.

In addition to the portal norepinephrine response to SNS, there was an increase in EPI concentration in the portal vein. We conclude that most of this EPI was released from the adrenal medulla, with a minority released from sympathetic nerves. For instance, EPI is predominantly produced in and originally secreted from the adrenal medulla; however, this catecholamine can reside in and be released from peripheral sympathetic nerves (53, 54, 55). We presume that during the surgery, EPI was released from the adrenal medulla and subsequently taken up by and stored in sympathetic nerve terminals throughout the body. During SNS, this EPI was released along with norepinephrine from gut sympathetic nerves. Because EPI taken up by sympathetic nerves resides exclusively in secretory granules in the nerve terminal (56, 57), the amount of EPI available for rerelease from sympathetic nerves is limited to the tissue EPI content. In the current studies, the tissue EPI content was 3% of the norepinephrine content. Therefore, during sympathetic stimulation, any plasma EPI increment greater than 3% of the concomitant norepinephrine increment suggests nonneural (i.e. adrenal medullary) EPI release. This is the case with the SNS studies, in which the plasma EPI/norepinephrine increment ratio was 23%. Adrenal activation was unexpected, because neuronal cell bodies that project axons to the adrenal gland are located in the intermediolateral gray matter of the spinal cord (58), not in the celiac ganglia. Presumably, some spinal nerves projecting to the adrenals pass through, but do not synapse within, the celiac ganglia (59).

Because EPI is theoretically capable of stimulating the same {alpha}- and ß-adrenergic receptors as neurally released norepinephrine, we evaluated the possibility that humoral EPI contributed to the ghrelin response to SNS. We infused this neurohormone iv at a dose that produced markedly elevated systemic levels of EPI, similar to those seen during severe stress (37, 49, 50), and portal levels higher than those seen during SNS. This EPI dose provided a significant gastrointestinal stimulus, as demonstrated by the substantial glucagon response it elicited. However, there was no ghrelin response to this EPI infusion. Therefore, it is unlikely that the circulating EPI released during SNS contributed to its ghrelin response. Rather, the high synaptic levels of norepinephrine produced locally during neural activation seem to be necessary to produce sympathetic stimulation of ghrelin secretion. This demonstrates that the autonomic nervous system can indeed stimulate ghrelin secretion when sympathetic nerves to the stomach and proximal duodenum are activated. To gain additional support for neural stimulation of ghrelin secretion, we chose a second method to stimulate sympathetic nerves, one that avoids adrenal activation.

To limit our stimulus to the neural, rather than the humoral, branch of the sympathetic nervous system, we infused the sympathomimetic amine, TYR, iv. Doses of TYR infusions that produce marked increases in plasma norepinephrine in the rat have little effect on plasma EPI (60, 61). The sympathetic neural specificity of TYR results from its mechanism of action (62, 63). This sympathomimetic amine is taken up into noradrenergic nerve terminals by the norepinephrine transporter, which is located primarily on the presynaptic membrane (64). Once TYR is in the axoplasm of sympathetic nerves, it inhibits the uptake of norepinephrine into secretory vesicles causing norepinephrine concentrations in the axoplasm to increase dramatically. Norepinephrine then moves down its concentration gradient through the bidirectional norepinephrine transporter and into the synaptic cleft, thereby producing adrenergic effects. Nonadrenergic neurons do not express the norepinephrine transporter (64); therefore, TYR-induced activation should be specific for catecholamine release from sympathetic nerves. There was a large portal venous increase in norepinephrine during TYR infusion, verifying successful sympathetic neural activation. As expected, the EPI increment during TYR infusion was limited to 3% of the norepinephrine increment, suggesting that EPI was released exclusively from sympathetic nerves, rather than from the adrenal medulla. The significant portal ghrelin response to the neural stimulation of TYR infusion confirmed our conclusion that activation of sympathetic nerves to the gut stimulates ghrelin secretion.

A second TYR study was required before we could claim that this sympathetic neural stimulation of ghrelin resulted from direct stimulation of ghrelin cells by sympathetic neurotransmitters. Specifically, both SNS and TYR infusion suppressed insulin secretion, a known effect of activating pancreatic sympathetic nerves (21, 65). Because insulin can suppress ghrelin secretion (5), and particularly because chronic insulin deficiency increases ghrelin levels (43), we sought to rule out the possibility that the increased ghrelin secretion during SNS and TYR infusion was mediated indirectly via acute suppression of insulin secretion. Therefore, we repeated the TYR study in rats that were pretreated with the pancreatic ß-cell toxin, STZ, to stimulate ghrelin cells neurally in the absence of a fall in endogenous insulin. STZ pretreatment did indeed prevent a fall in insulin during TYR infusion, yet the ghrelin response was unaffected. Consistent with this finding, we performed trial SNS experiments in adrenalectomized rats with and without STZ (data not shown). Again, STZ pretreatment prevented a fall in insulin during SNS, but the ghrelin response was not diminished. We conclude that local neurotransmitter release during SNS and TYR infusion directly stimulates ghrelin secretion, independently of the acute decreases in insulin levels.

Interestingly, the magnitude of the ghrelin response to SNS was greater than that to TYR infusion despite similar gut-specific norepinephrine release during these two stimuli. One possible explanation for this difference is that SNS releases, in addition to norepinephrine, peptide neurotransmitters that may not be released by TYR. Sympathetic nerves apparently do not have bidirectional transporters for neuropeptides on the presynaptic membrane (66). Thus, neuropeptides are released from sympathetic nerves exclusively by regulated exocytosis, not by indirect sympathomimetics such as TYR. SNS, in contrast, is known to corelease neuropeptides and norepinephrine by the process of exocytosis (66, 67, 68, 69, 70). If sympathetic neuropeptides have the ability to stimulate ghrelin secretion, either directly or by potentiating norepinephrine action (71), this additional nonadrenergic stimulation may account for the greater ghrelin release during SNS.

In summary, we conclude that sympathetic neural activation can robustly stimulate gastrointestinal ghrelin secretion. Furthermore, this neural stimulation of ghrelin secretion results from direct effects of sympathetic nerves on ghrelin-producing cells, not from indirect inhibition of insulin secretion. Lastly, sympathetic stimulation of ghrelin secretion is restricted to its neural, as opposed to humoral, branch. Additional studies are required to determine the contribution that sympathetic neural activation makes to preprandial ghrelin surges.


   Acknowledgments
 
We thank Scott Frayo for ghrelin measurements, Jira Wade and Breanne Barrow for catecholamine and insulin measurements, and Aryana Zavosh for glucagon measurements.


   Footnotes
 
This work was supported by the Medical Research Service of the Department of Veterans Affairs and National Institutes of Health Grants DK-12829, DK-17047, DK-50154, DK-61516, and DK-68384.

T.O.M., D.E.C., and G.J.T. have nothing to declare.

First Published Online March 9, 2006

Abbreviations: CV, Coefficient of variance; {Delta}, change; EPI, epinephrine; ISO, isolated, but not electrically stimulated; NS, not significant; SNS, sympathetic nerve stimulation; STZ, streptozotocin; TYR, tyramine.

Received September 15, 2005.

Accepted for publication March 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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