Novel gastroinsular axis involving a gastric transmural glucose flux and vagal mediation

Atsushi Nakagawa1, Shigeru Azuma2, and Hajime Nakabayashi3

1 Division of Endocrinology, Department of Internal Medicine, Kanazawa Medical University, Uchinada 920-0293; 2 Department of Internal Medicine, Shinminato Municipal Hospital, Shinminato 934-0053; and Department of Internal Medicine (II), School of Medicine, Kanazawa 920-8641; and 3 Division of Life Science, Graduate School of Natural Science and Technology and Health Science Service Center, Kanazawa University, Kanazawa 920-1192, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether the appearance of nutrients into the gastric lumen per se provokes insulin secretion, glucose solution was instilled into the pylorus-cannulated stomach via an orogastric tube in anesthetized dogs. When 200 ml of 0, 5, 10, and 20% glucose solution were sequentially instilled, transgastric gradients (TGG) of plasma glucose concentration across the fundus [short gastric vein (SGV) - femoral artery, TGG(SGV)] and insulin levels in the superior pancreaticoduodenal vein (SPDV) increased stepwise. Upon instillation of 300 ml of 10% glucose, but not 1.8% saline, for 12 min followed by 48-min spontaneous drainage via the cannula (n = 5 each), TGG(SGV) and insulin levels in the SPDV increased concomitantly and significantly by 0.95 mM and 1,334 pM (mean), respectively, regardless of unaltered arterial glucose levels. The amount of secreted insulin (area under the curve) significantly correlated with the maximum TGG(SGV) (r = 0.693). In selectively gastric-vagotomized dogs (n = 5), insulin levels in the SPDV did not increase upon instillation despite a TGG(SGV) rise comparable to that in normal dogs. These results indicate that intragastric glucose appearance provokes vagus-mediated insulin secretion probably related to the transfundic glucose flux, suggesting the presence of a novel neurogenic gastroinsular axis.

intragastric glucose instillation; short gastric vein; insulin secretion; gastric vagotomy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN SECRETION in response to meal ingestion is required to be appropriate in the postprandial time course of digestive and absorptive process and adequate in amount according to quality and quantity of ingested nutrients. To achieve these requirements, a variety of humoral and neural signals evoked upon food passage along the gastrointestinal tract must operate in an integrated fashion. However, the manner in and the extent to which interactions of the humoral and neural signals play a role(s) in integration of functions of the gastrointestinal tract, the liver, and the endocrine pancreas are still not well understood. A link between sensing organs and pancreatic islets or between the intestinal tract and islets has been the focus of many investigations (5, 6, 41, 57). The cephalic phase of insulin secretion occurs at the sight, smell, and expectation of food by mediation of neural reflexes (41). Moreover, the intestinal phase of insulin secretion becomes predominant in absorptive process of nutrients as a result of the incretin effects mediated by gut peptide hormones and by altered neural activities in the peripheral autonomic system (PANS) and in the central nervous system (CNS) (5, 6, 57). Nevertheless, the role of the stomach in insulin secretion has been poorly elucidated, even though ingested nutrients inevitably pass through the stomach before being absorbed in the intestine.

The existence of the neural (vagal) sensing system for levels of metabolites (e.g., glucose), as well as osmolality and temperature, in the portal vein (35-37, 44) (for review see Refs. 38, 46), and the existence of the neural (vagal) monitoring system for glucose in the intestinal lumen (27) have been reported. We have been interested in the vagal sensing system for gastrointestinal hormones, such as glucagon-like peptide-1-(7-36) amide (34, 39, 40) and somatostatin-14 (28, 32, 33) in the portal vein, as the collective outflow from the splanchnic organs, and in the system for somatostatin-14 in the pancreatic vein, as the local outflow from the organ secreting somatostatin-14 itself (31).

The aim of the present study was to determine whether insulin secretion is induced by the glucose appearance confined to the gastric lumen, and if so, whether selective gastric vagotomy, i.e., a blockade of vagal glucose sensing in the stomach, abolishes the insulin secretion. The hypothesis was specifically tested in view of our interest in the vagal chemoreception for a substance whose concentration is changing in the locally draining vein of an organ. Thus glucose solution was instilled into the pylorus-cannulated stomach through an orogastric tube in normal and gastric-vagotomized dogs. Plasma glucose concentrations in the short gastric vein (SGV), the right gastroepiploic vein (GEV), and the femoral artery (FA) were then determined simultaneously to evaluate regional (i.e., fundic and antral) transgastric gradients of plasma glucose levels. Plasma insulin levels in the superior pancreaticoduodenal vein (SPDV) and FA were also measured to verify the occurrence of insulin secretion. The present results suggest the existence of a previously unknown gastroinsular axis mediated by the vagus, providing a unique insight into the role of the stomach in postprandial glucose homeostasis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparations. After an overnight fast (14-16 h), mongrel dogs weighing 18-25 kg were anesthetized by intravenous injection of pentobarbital sodium (30 mg/kg). The dog was secured on a table in a supine position, mounted on a respirator and ventilated with room air, and maintained on an automonitored heating pad to keep the rectal temperature in the range of 38.0 ± 0.1°C until the end of the experiment. A small piece of Silastic tubing was inserted into the femoral vein, through which physiological saline was infused for maintaining the circulating blood volume as we will describe below.

The following four tubes for blood collection were placed (Fig. 1). First, an 18-gauge polyethylene cannula was inserted into the FA. Second, after laparotomy, a Silastic tube (1.55 mm OD, 0.8 mm ID) was placed in one of the SGVs. The tubing was inserted into a small branch of the splenic vein just after draining of the spleen, advanced ~10 mm into the SGV accompanying the splenic branch, and secured to the branch. Special attention was paid to keep the bloodstream in the SGV flowing freely into the splenic vein through the peritubing space (Fig. 1, inset). A third Silastic tube (1.55 mm OD, 0.8 mm ID) was inserted into the right GEV at a position 15-20 mm apart from the pyloric ring and introduced ~10 mm in the direction of the gastric corpus. This procedure enabled the bloodstream to flow into the left GEV. Finally, a polyethylene tube (1.3 mm OD, 0.9 mm ID) was inserted into one of the small duodenal branches of the SPDV. The tube was positioned so that its tip remained just before the entrance to the main trunk of SPDV, by which means the bloodstream in the trunk was not perturbed. To keep these tubes patent, they were filled with heparinized saline (20 units/ml) except during blood collection periods.


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Fig. 1.   Diagrammatic localization of tubes for selective blood sampling in one of the short gastric veins (SGV), the right gastroepiploic vein (GEV), and the superior pancreaticoduodenal vein (SPDV) in a dog. Inset: detailed positioning of the tubing in the SGV.

To perform an intragastric instillation of glucose or saline solution confined to the gastric lumen, an orogastric tube (16 French) was inserted perorally into the stomach. The tip of the tube was positioned in the middle of the gastric corpus, and the tube was fixed to the teeth with a ligature. Thereafter, 50 ml of distilled water (38°C ) were infused into the stomach and drained to confirm that the gastric lumen was free from meal remnants and to keep the stomach empty until the start of the experiment. A small incision was made in the ventral wall of the duodenal bulb near the pyloric ring, and a gastric outflow fistula was created by placing a cannula (16-Fr) with double ligation, one around the pyloric ring and the other around the pyloroduodenal junction just aboral to the incision. The extension tube connected to the cannula was exteriorized through a small incision in the abdominal wall, and the outlet of the tube was secured at the pyloric level. These procedures completely prevented gastric contents from entering the duodenum.

In a separate series of experiments, dogs underwent selective gastric vagotomy. Upon performance of the selective, gastric vagotomy, the ventral gastric bundles (from the ventral vagal trunk), the dorsal gastric bundles, and the bundles along with the left gastric artery and vein (from the dorsal vagal trunk), were all severed as completely as possible. In addition, the serosal and subserosal tissues around the esophagogastric junction were electrically cauterized. Special attention was paid not to damage the vagal branches distributed to the liver and the pancreas. Completeness of the vagotomy was substantiated by autopsy after the experiment. The intrathoracic vagal trunks and their all supra- and subdiaphragmatic branches were exposed after median thoracotomy and removal of the diaphragm; then it was macroscopically confirmed that no branches to the stomach were left and that branches to the liver and pancreas remained intact. Dogs that underwent incomplete gastric vagotomy were excluded from the study.

The abdominal wall was closed, leaving the tubes outside the abdominal cavity, and then the dog was kept in a 15° head-up position until the end of the experiment. To maintain the circulating blood volume, each dog received immediate substitution of physiological saline in volume equal to drawn blood and continuous infusion of saline (35-40 ml/kg) through the femoral vein over the experimental period, by which hematocrit levels in each dog were kept constant. After completion of the experiment, each animal was killed with an overdose of potassium chloride under deep anesthesia. All of the procedures and protocols were approved by the Ethics Committee for Animal Experiments of Kanazawa University.

Materials and intragastric instillation. D-Glucose and sodium chloride were purchased (Wako Pure Chemicals Industries, Osaka, Japan), and each of them was dissolved in sterile distilled water for clinical use (Otsuka Pharmaceutical, Tokyo, Japan) to make solutions of various concentrations. Each solution, warmed at 38°C, was instilled by gravity into the stomach through an orogastric tube (usually within 2 min), where the pylorus cannula was clamped and the solution container was placed at a level 20 cm above the pylorus.

Protocols. A blood sample from the SGV, GEV, or SPDV for a given time point was drawn over 2 min (from -1 to +1 min of a time point) with a constant speed by hand, and a sample from the FA was taken quickly at a given time point. In the SGV and GEV, the tubes were left open for blood to flow out spontaneously for >= 30 s before each blood sampling was started. After a 45- to 60-min postsurgery equilibration period, one of the two sets of experiments was conducted on each dog. First, 200 ml of 0 (distilled water), 5, 10, and 20% glucose solution were sequentially introduced into the pylorus-cannulated stomach with an instillation duration of 30 min for each solution. Before the start of instillation of the subsequent solution, both the pylorus cannula and the orogastric tube were opened to drain the stomach of the antecedent solution as completely as possible (total drainage of an instilled solution was 96-100% in volume). Blood samples for determination of plasma glucose concentrations in the SGV and FA and of insulin in the SPDV and FA were taken simultaneously at -5 and 0 min in the basal state and at 25 and 30 min after the start of each instillation. The transgastric gradient (TGG) of plasma glucose concentrations across the fundus [SGV - FA; TGG(SGV)] was evidenced by calculation. Second, 300 ml of 10% glucose solution (pH 5.5-6.2) or 1.8% saline (as a control; pH 6.0-7.1) were introduced in normal dogs or in dogs with selective gastric vagotomy (the reason for our employing this concentration of glucose solution is described in RESULTS). The solution was retained in the gastric lumen for the initial 12 min of the instillation and thereafter was allowed to flow out spontaneously through the pylorus cannula with its outlet kept at the pylorus level. The total volume of an efflux of intragastric solution was measured at the end of the experiment on each dog. Blood samples were taken at -15, -10, -5, and 0 min as a baseline and at 5, 10, 15, 30, 45, and 60 min after start of the instillation. TGG of plasma glucose concentrations in the fundic and antral areas were obtained in the SGV and GEV [GEV - FA; TGG(GEV)], respectively.

Plasma analyses. Collected blood was transferred into an ice-cold tube containing 1.2 mg EDTA · 2Na and 1,000 kallikrein inhibitor units of aprotinin/ml of blood. Blood samples were centrifuged at 2°C, and the separated plasma samples were stored at -30°C until the assays were performed. Samples of the same set of experiments (i.e., sequential glucose instillation group and 10% glucose or 1.8% saline instillation groups) were measured in one assay. Plasma glucose concentration was measured using a glucose oxidase method, and plasma insulin was measured by previously described radioimmunoassay with a dog insulin standard (lot no. H7574, a gift of Dr. L. Heding, Novo Industri, Copenhagen, Denmark) (29, 30).

Data presentation and statistical analysis. Results are presented as means ± SE. Incremental area under the curve (AUC) of plasma glucose or insulin concentration from a basal value (the mean of values before instillation, i.e., -15, -10, -5, and 0 min) was calculated using a trapezoidal method. Repeated-measures analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, was employed for detection of statistically significant changes from a baseline within a group (Figs. 3-5 and 7). In addition, statistical differences in time course profiles between two groups were evaluated using two-way repeated-measures ANOVA. Correlation between two variables was examined using methods of Pearson's correlation and Spearman's correlation by ranks (Fig. 6). Values in saline-instilled normal dogs and in glucose-instilled gastric-vagotomized dogs were compared with those in glucose-instilled normal dogs by one-way ANOVA followed by Dunnett's multiple comparison test (Fig. 8). Values of P < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in TGG(SGV) and in insulin level in SPDV upon sequential intragastric instillation of glucose solutions graded by concentration. Sequential instillation of 0, 5, 10, and 20% glucose solutions into the stomach was performed in two dogs (Fig. 2). TGG(SGV) was negative at baseline and did not change meaningfully on instillation of distilled water (i.e., 0% glucose solution). As the glucose concentration of the intragastric solution increased, TGG(SGV) increased stepwise, turning eventually positive in both dogs. This suggests that transmural glucose flux occurred in the fundic stomach. With the TGG(SGV) increases, plasma insulin concentrations in the SPDV did increase simultaneously. Because glucose levels in the FA showed no discernible changes as long as <= 10% glucose solution was used for instillation (data not shown), it is unlikely that the insulin increases upon any such glucose instillation were induced by the arterial (systemic) glycemic changes. Upon 20% glucose instillation, glucose levels in the FA increased by 0.18 and 0.56 mM from baseline in dogs 1 and 2, respectively, reinforcing the existence of the transmural glucose flux influenced by the intraluminal glucose concentration.


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Fig. 2.   Transgastric gradients (TGG) of glucose concentrations in the fundus [SGV - femoral artery (FA); TGG(SGV); bars] and plasma insulin concentrations (--- and ---) in the SPDV upon instillation of 0 (distilled water), 5, 10, or 20% glucose solution (200 ml) into the pylorus-cannulated stomach in 2 dogs. Intragastric instillation of 30-min duration for each solution was sequentially performed, and blood samples were drawn over 2 min. A pair of data corresponding to each solution indicates 2 samples collected at a 5-min interval before termination of each instillation.

Changes in TGG of plasma glucose concentration and insulin level in SPDV upon intragastric instillation of 10% glucose or 1.8% saline solution in normal dogs. This set of experiments was done to determine whether glucose levels in the GEV (antral vein) as well as the SGV (fundic vein) increase upon intragastric glucose instillation and which one (or both) of the vein's glucose levels correlates with insulin secretion in the SPDV when arterial glycemic levels are unaltered. For those purposes, a 10% glucose solution was employed for the instillation, as discussed in MATERIALS AND METHODS, because the results of our preliminary experiments and those of the aforementioned experimental series have consistently shown that 10% glucose instillation induces an increase of plasma glucose levels in the SGV but not in the FA. It was also necessary to obtain more detailed evidence for the time course changes of glucose and insulin levels over 60 min. The 48-min spontaneous drainage of 10% glucose or 1.8% saline solution after the 12-min intragastric retention resulted in ~40 or 33% efflux of the instilled solution in volume, respectively (n = 5 each).

When a 10% glucose solution was instilled into the pylorus-cannulated stomach in normal dogs (Fig. 3), plasma glucose concentrations in the SGV started to increase at 5 min, showed a significant rise at 30 min, and reached a peak plateau level of 0.85 ± 0.24 and 0.85 ± 0.28 mM above baseline (means of increments) at 45 and 60 min, respectively (P < 0.01 vs. mean basal value at 30, 45, and 60 min). In contrast, plasma glucose levels in the GEV did not change significantly until the end of the experiment. Glucose levels in the FA, as expected, remained unchanged throughout the experiment. Upon intragastric 1.8% saline instillation (Fig. 3), glucose levels in the FA rather decreased slightly but significantly for the last 30 min, showing a change of -0.44 ± 0.16 mM from baseline at 60 min (P < 0.05 at 30 min, P < 0.01 at 45 and 60 min). Glucose concentrations in the SGV and GEV decreased similarly in the time course after saline instillation, reflecting the changes of arterial glucose levels. The time course profiles of glucose levels in the SGV and GEV induced by the saline were significantly different from those induced by the glucose (P < 0.01), but not in the FA (P = 0.08).


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Fig. 3.   Changes in plasma glucose levels in the SGV, right GEV, and FA upon intragastric instillation of 300 ml of 10% glucose (open circle ) or 1.8% saline () solution in dogs with the pylorus-cannulated stomach (n = 5 each). Results are expressed as means ± SE. Double circles indicate significant changes from the mean basal value. P or n.s. (not significant), statistical difference in time course profiles between glucose- and saline-instilled dogs.

Because an obvious difference between the changes of glucose levels in the SGV and those in the GEV was observed on 10% glucose instillation, as mentioned previously, the time course profiles of the TGG of glucose concentrations occurring in the veins were further evaluated (Fig. 4). In the basal state, there were no significant differences between levels of TGG(SGV) and TGG(GEV) in each of the groups instilled with glucose or saline (P = 0.10 and P = 0.28, respectively). This strongly suggests that our catheterization method for the veins was not accompanied by blood flow obstruction in the different areas of the stomach. Upon glucose instillation, TGG(SGV) increased gradually but obviously (P < 0.05 vs. mean basal value at 30 min, P < 0.01 at 45 and 60 min), whereas TGG(GEV) did not change significantly. Moreover, the increasing time course profile of TGG(SGV) on glucose instillation was clearly different compared with the rather decreasing profile on 1.8% saline instillation (P < 0.01). TGG(GEV) changes upon glucose instillation were not statistically different from those upon saline instillation in time course (P = 0.87). These results clearly show the regional difference in the transmural glucose flux.


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Fig. 4.   Changes in TGG in the fundic (SGV) and the antral (GEV) stomach upon intragastric instillation of 300 ml of 10% glucose (open circle ) or 1.8% saline () solution in dogs with the pylorus-cannulated stomach (n = 5 each). Results are expressed as means ± SE. Double circles indicate significant changes from the mean basal value. P or n.s., statistical difference in time course profiles between glucose- and saline-instilled dogs.

Plasma insulin levels in the SPDV upon 10% glucose instillation showed an increase at 30 min and reached a significant mean peak (212 ± 39% of basal) at 45 min, remaining at an above-basal level at 60 min (P < 0.05 vs. mean basal value at 45 and 60 min; Fig. 5). However, insulin levels in the FA remained unchanged throughout the experiment, not reflecting the insulin secretion observed in the SPDV. Because insulin levels in the SPDV and FA did not change upon saline instillation, the time course profile of insulin in the SPDV was significantly different from that upon glucose instillation (P < 0.05).


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Fig. 5.   Changes in plasma insulin concentrations in the SPDV and FA upon intragastric instillation of 300 ml of 10% glucose (open circle ) or 1.8% saline () solution in dogs with the pylorus-cannulated stomach (n = 5 each). Results are expressed as means ± SE. Double circles indicate significant changes from the mean basal value. P or n.s., statistical difference in time course profiles between glucose- and saline-instilled dogs.

We further examined the relationship between the magnitude of the glucose flux into the SGV and the amount of insulin secreted into the SPDV; the insulin secretion may be a concomitant of the SGV glucose level. As shown in Fig. 6, there was a significant correlation between the maximum level of TGG(SGV) [max-TGG(SGV)] and the incremental AUC of insulin (AUC-insulin) in the SPDV (Pearson's correlation coefficient of r = 0.693, P < 0.05; Spearman's correlation coefficient by ranks of rS = 0.768, P < 0.05). No significant correlation was observed between the incremental AUC of glucose in the FA and AUC-insulin in the SPDV (r = 0.334; rS = 0.285).


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Fig. 6.   Correlation between the maximum TGG(SGV) and the incremental area under the curve of plasma insulin concentrations (AUC-Insulin) in the SPDV observed upon intragastric instillation of 300 ml of 10% glucose (open circle ) and 1.8% saline () solutions in dogs with the pylorus-cannulated stomach. Pearson's coefficient of correlation is shown.

Changes of TGG of plasma glucose concentration and insulin level in SPDV upon intragastric instillation of 10% glucose solution in gastric-vagotomized dogs. To provide further insight into the relationship between the max-TGG(SGV) and AUC-insulin, the contributory role of gastric vagal afferent signals for inducing the insulin secretion was examined. To this end, the same experiment in dogs with selective gastric vagotomy was conducted. Upon intragastric instillation of 300 ml of 10% glucose solution in gastric-vagotomized dogs (n = 5), TGG(SGV) increased significantly by ~0.6 mM from the mean basal of -0.24 ± 0.07 mM (P < 0.05 at 15 min, P < 0.01 at 30, 45 and 60 min; Fig. 7). The time course profile of TGG(SGV) in this group was not significantly different from that in normal dogs (P = 0.45). Glucose levels in the FA again remained unchanged throughout the experiment, and the time course profile was essentially identical with that in normal dogs (P = 0.99). Despite these facts, plasma insulin concentrations in the SPDV did not change significantly from the mean basal of 1,413 ± 227 pM. The results on max-TGG(SGV) and AUC-insulin in normal and gastric-vagotomized dogs upon glucose and saline instillation are shown in Fig. 8. Upon glucose instillation in selectively gastric-vagotomized dogs, AUC-insulin was apparently small, showing a nearly negligible level when compared with that upon glucose instillation in normal dogs (P < 0.05), whereas max-TGG(SGV) was comparable to that in normal dogs. Upon saline instillation in normal dogs, both max-TGG(SGV) and AUC-insulin were significantly smaller than those on glucose instillation in normal dogs (P < 0.01 and P < 0.05, respectively). These results clearly show the importance of the gastric vagal innervation in inducing the insulin secretion.


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Fig. 7.   Changes in TGG(SGV), plasma insulin concentrations in the SPDV, and plasma glucose levels in the FA upon intragastric instillation of 300 ml of 10% glucose solution in normal dogs (open circle ) or in selectively gastric-vagotomized dogs (diamond ) (n = 5 each; data in normal dogs are quoted from those in Figs. 3, 4, and 5 for comparison). Results are expressed as means ± SE. Double circles and double diamonds indicate significant changes from the mean basal value. P or n.s., statistical difference in time course profiles between normal and gastric-vagotomized dogs.



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Fig. 8.   Maximum TGG(SGV) (top) and incremental AUC-insulin in the SPDV (bottom) observed upon intragastric instillation of 300 ml of 1.8% saline (filled bar) and 10% glucose (open bar) solution in normal dogs and of 10% glucose solution in selectively gastric-vagotomized dogs (hatched bar) (n = 5 each). Results are expressed as means ± SE. P or n.s., statistical difference between groups.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It has been reported that insulin release in response to meal ingestion is composed of two phases of insulin secretion according to its physiological mechanism: the cephalic phase, triggered by neural signals, and the intestinal phase, induced by humoral and neural information related to passage, digestion, and absorption of food (5, 6, 41, 57). However, little is known about insulin secretion elicited by glucose appearance confined to the gastric lumen. It has come to our knowledge that only one report pertinent to this issue has been listed, and its results are not available in detail (18). In that report, the authors mentioned briefly that intragastric glucose instillation (1.0 g/kg) increased insulin levels in the peripheral vein without changes of circulating glucose levels in 5 of 10 dogs with esophageal inflow and pyloric outflow fistulae. They assumed that contact of glucose with the gastric mucosa might have caused insulin secretion. In the present study, we instilled glucose solution (of different concentrations) into the gastric lumen in normal dogs and in gastric-vagotomized dogs, measuring the regional transgastric glucose gradients as well as insulin secretion. The results showed the following. 1) When intragastric instillation of 0, 5, 10, and 20% glucose solution was sequentially performed over 30 min for each solution in normal dogs, TGG(SGV) and insulin levels in the SPDV increased stepwise according to the graded increases in glucose concentration of intragastric solution, even when arterial glucose levels remained unaltered; that was typically observed on instillation of 5 and 10% glucose solution. 2) Upon 10% glucose instillation for 60 min in normal dogs, glucose levels in the SGV, but not in the GEV and in the FA, increased gradually, accompanying an increase of insulin levels in the SPDV. The amount of insulin secreted into the SPDV (AUC) showed a significant correlation with the max-TGG(SGV). 3) Insulin secretion upon intragastric 10% glucose instillation was no longer observed in selectively gastric-vagotomized dogs, despite the increase of TGG(SGV) being comparable to that in normal dogs. These results have revealed the existence of a novel neurogenic gastroinsular axis mediated by the vagal nerve.

It is noteworthy that, as glucose concentration of intragastric solution multiplied, plasma glucose levels in the SGV increased. Because it is known that the gastric epithelium has no active transport system for glucose and the gastric tissues have no ability to produce glucose, the present results strongly suggest that glucose traverses the gastric wall, in a way like "diffusion," down the concentration gradient from the lumen to the draining veins. Moreover, this glucose traverse seems to exist in a localized region of the stomach, because an increase of plasma glucose concentrations was observed only in the SGV (draining mainly the fundus), not in the GEV (draining the antrum). The present observations, however, do not seem to be compatible with the general concept currently believed of a diffusion barrier in the gastrointestinal tract: the epithelia covering the luminal surface throughout the gastrointestinal tract are impermeable to hydrophilic molecules such as glucose, except where the membrane-associated transport system exists. Thus we have attempted to examine the gastric tissue histologically but have been unsuccessful either in finding out new observations on the specific structure pertinent to the glucose flux in the control stomach, or in verifying the disrupted lining of the mucosa and vasculature in SGV regions of the postexperimental organs. However, as for the gastric mucosal barrier, functional differences to intragastric acidity between the fundic and antral mucosa have been reported, suggesting involvement of regional differences in the nature of apical cellular membrane and tight junction (8, 17). Recently, it has been reported that functions of tight junction involved in paracellular transport of water, ions, and substances like glucose might be regulated by occludin and claudins, which were discovered as two distinct types of tight-junction-specific integral membrane proteins (56). Glucose flux across the fundic wall, to our knowledge, has not been reported previously, but glucose absorption from the autotransplanted canine antral pouch, although influenced by acidity in the fundic pouch, was reported (9). This issue of transcellular and paracellular movement of glucose across the gastric mural tissues remains to be clarified in future studies.

The present observation that an increase of insulin concentrations in the SPDV upon intragastric glucose instillation was abolished by the selective gastric vagotomy strongly suggests two important aspects of the frame of the insulin secretion: 1) an appearance of glucose solution into the gastric lumen is sensed by the vagus; 2) the vagal information on the glucose appearance is conveyed to the beta -cells of the islets and causes insulin secretion in a reflex fashion. In this context, the following possibilities related to the frame must be considered as well. First, the vagal monitoring system for intravascular glucose levels outside the stomach, i.e., intraportal, could evoke such a vagal efferent signaling to the islets, because glucose entering from the gastric lumen to the SGV reaches the hepatoportal area where the glucose-sensitive vagal nerve resides (36-38). However, we paid meticulous attention not to damage the hepatic and pancreatic bundles of the vagus (the afferent and efferent components of the speculated reflex, respectively) when performing the gastric vagotomy. In addition, because the blood flow rate in portal circulation is much larger than that in the SGVs, intraportal glucose concentration may show hardly discernible changes, at least upon intragastric instillation of <= 10% glucose solution, resulting in no arterial glycemic excursions. Thus the glucose sensor in the hepatoportal area does not appear to be involved in the present observations.

Second, regarding the gastric vagal afferents, it has been reported that the vagal afferent fibers related to the gastric mechanoreceptor are electrophysiologically distinct from those related to the chemoreceptors (25). In the present study, intragastric instillation of 1.8% saline as a control, with the use of the same volume and temperature and almost the same osmolality and pH as those of 10% glucose solution, was ineffective in eliciting insulin secretion. This excluded the possibility of involvement of a gastric mechanoreceptor, as well as other receptors for temperature and acidity in the stomach, in inducing the insulin secretion. As to a coincidental observation on glucose levels upon intragastric saline instillation, it is difficult to explain at present that a slight but significant decline of systemic plasma glucose levels occurred in the late phase of the experiment. However, this small decline of arterial glucose levels upon saline instillation would at least not be the main evidence for explaining the differences of insulin levels between glucose-instilled and saline-instilled dogs. In gastric-vagotomized dogs, insulin levels upon glucose instillation were strikingly different from those in normal dogs, despite the fact that arterial glucose levels in both dog groups were actually identical. This stresses again the importance of the gastric vagus pertaining to the chemoreception in the increased insulin secretion.

Third, it should be kept in mind that changes of insulin concentrations in the SPDV observed in the present study may not mean simply changes of insulin output into the vein; hormone output consists of two parameters, hormone concentration and blood flow in the draining vein. We thus attempted to measure SPDV blood flow by using an ultrasonic flow probe over the time course of the experiments, but we often failed to obtain reliable and reproducible results of venous blood flow and insulin levels with confidence because of some difficulties in keeping the proper position of the probe to such a thin-walled and rather small vein, which was also easily compressible and flexible with the probe in anesthetized, head-up supine-positioned, and mechanically ventilated dogs with the stomach nearly full of solution. Accordingly, by the present method employing slow blood collection (for 2 min) from the SPDV without the probe, we rather succeeded in obtaining reliable samples for insulin determination, as shown by the stable and constant insulin levels throughout the experiments in saline-instilled normal and glucose-instilled gastric-vagotomized dogs. In this regard, blood flow of the pancreas as well as the duodenum measured by the radioactive microsphere technique (13) and pancreatic blood flow (microcirculation) by the thermoelectric method (20) are known to increase postprandially. It is thus quite conceivable that the increased insulin levels in the SPDV after glucose instillation correspond to increased insulin output rather than simply resulting from decreased blood flow.

Glucose-sensing mechanism in the stomach relevant to the present observation is intriguing. Because the presence of the gastric vagal innervation was crucial for eliciting insulin secretion in response to intragastric glucose instillation, the manner in which the gastric vagal afferent system monitors glucose appearance into the gastric lumen and/or glucose flux into the SGV deserves consideration. As for the vagus sensing glucose in the gastric luminal space, only one report, that by Ouazzani and Mei (42), is, to our knowledge, available. That report was published as one of the results in a research series on glucoreceptor in the gastrointestinal tract by Mei (27). In the report, when glucose solution was infused into the pylorus-ligated stomach and then aspirated through a large fenestra made in the gastric wall in cats, facilitation of single-unit firing in the neuronal body in the nodose ganglion was evoked within several seconds after the start of intragastric glucose infusion. As a result of the style of the experimental design, the gastric region responsible for the observation was not specified; however, the facilitated firing was observed, albeit only on two occasions when a cotton ball soaked with glucose was applied on the antral mucosa, which was performed as different experiments in the paper. Judging from the promptness of the vagal afferent response in the paper, such intraluminal glucose sensing may not be involved, at least not primarily, in eliciting the present insulin secretion, i.e., the secretion occurring slowly after intragastric instillation of 10% glucose. Moreover, the amount of secreted insulin correlated significantly with the magnitude of increase in plasma glucose concentrations in the SGV, namely the magnitude of TGG(SGV). This observation thus seems to support the hypothesis that the putative gastric glucose sensor is receptive to glucose in the SGV rather than in the gastric lumen; the glucose-sensing site is in or related to the wall of the SGV. However, because levels of TGG(SGV) depend on intraluminal glucose concentrations, the possibility that the insulin secretion occurred concurrently, not concomitantly, with the changes of TGG(SGV) cannot be excluded in the present experimental design. In this context, the sensor system for intraportal (intravascular) glucose levels (36, 37) has recently been reported to exist in the wall of the extrahepatic, not the intrahepatic, portal vein (16) as an example of a glucose-sensing site in the splanchnic area. In any event, the existence of a vagal chemoreception system for intragastric glucose appearance sounds quite plausible, favoring an idea that the system operates to monitor glucose levels changing in the SGV rather than a fixed concentration in the gastric lumen.

It is, then, tempting to speculate on the physiological role of this gastric neurochemoreception to glucose in the SGV and/or in the gastric lumen. The proximal stomach has a characteristic function, called receptive and adaptive relaxation, where the stomach retains a relatively large amount of food as a reservoir for a fairly long period of time without appreciable changes in intragastric pressure, contrasting with the vigorous contractions and emptying function of the distal stomach. In this situation, signals from the vagal mechanoreceptor for stretch of the proximal stomach (volume signal) and from the putative chemoreceptor for glucose concentration in the SGV and/or in the lumen (concentration signal) become available. If the signals are integrated by the CNS or by the PANS in and/or related to the stomach as "the little brain," then an energy content of nutrient (glucose) in the stomach should be known as if the stomach were equipped with a "fuel level gauge" in the SGV region. Such information on intragastric energy content, if it exists at all, would contribute to control of appetite, regulation of eating behavior, and maintenance of fuel homeostasis. It is also conceivable that the information may regulate gastric emptying if the information returns to the stomach via the CNS or the PANS in a reflex manner. In this context, it is known that the rate of gastric emptying of a liquid meal is constant with regard to energy content in chyme leaving the stomach, and it is an interesting but hitherto unsolved concept that the total energy content of an intragastric liquid meal behaves as a determinant of gastric emptying time (15, 19, 26). Such a vagal gastrogastric reflex, if it occurs, might offer another possible explanation for the concept that has been explained from the point of view of postpyloric feedback involving duodenointestinal humoral and neural factors (3, 24, 58).

As for a link between the stomach and the endocrine pancreas, there have been previous reports suggesting that such links are attributable mainly to humoral factors operating locally and/or systemically (45, 47). The concepts of links in those papers are distinct from the concept of a putative link, i.e., the insulin secretion triggered by the vagal chemoreception of glucose in the stomach. Regarding the neural gastropancreatic links, it has been reported that different types of afferent signals arising from regionally different parts of the stomach, different pathways, and distinct transmission mechanisms are involved in establishing the various links. For example, the well-known facilitation of pancreatic exocrine secretion in the gastric phase consists of oxyntopancreatic and antropancreatic components (2), the former known to be mediated by the cholinergic pathways and the latter not, although both components start with mechanoreception in the corresponding part of the stomach (51). Second, postprandial secretion of pancreatic polypeptide, whose secreting cells are located in both the exocrine and the endocrine pancreas (1, 22), is known to be under the strong influence of vagal regulation (12, 49, 50). It has also been reported that the secretion of pancreatic polypeptide induced by intragastric instillation of glucose solution, saline, or a mixed-liquid meal is partly due to gastric distension through the mechanoreceptor (52). The secretion of the hormone, upon intragastric balloon inflation, showed the more striking cholinergic nature of a vagovagal reflex on the fundic rather than the antral distension (48, 52). Those previously reported links between the stomach and the exocrine or endocrine pancreas had their afferent limbs depending mainly on the vagal mechanoreception in the fundus, suggesting preferential regional distribution of the vagal afferent system leading to the neural links. In contrast, our present results stress the unique character of the gastropancreatic link; the neural link initiated with the vagal chemoreception, probably of a transmural glucose flux in the fundic stomach, culminates in a purely endocrine effect (insulin secretion). As to the underlying mechanism of the putative link, pituitary adenylate cyclase-activating polypeptide (PACAP) was recently reported to be present in the visceral afferent nerves supplying the rat digestive tract such as the stomach and the pancreas (10) and, furthermore, in the efferent nervous system of the pancreas as a regulatory neurotransmitter in the exo- and endocrine pancreas (53). More recently, the contribution of PACAP and its receptor in insulin response to gastric glucose administration as a model of prandial insulin secretion has been reported in mice (11). Whether such actions of PACAP are involved in the present results remains a subject for further studies. In addition, it was revealed that glucose-dependent insulinotropic polypeptide (GIP), one of the physiological incretin hormones released from the small intestine, is also expressed in the human and mouse stomach (59). Because GIP secreted on oral glucose promotes rapid release of insulin (23), and GIP secretion is under poor regulation of vagal or muscarinic pathways (14), whether gastric GIP is secreted upon intragastric glucose instillation and the extent to which gastric GIP is related to the present link depending strongly on the vagal pathways should be fully appreciated.

The novel gastroinsular axis seems to have a physiologically important implication. One of the interesting characteristics of the axis is that a rise of plasma glucose concentrations in the SGV and the rise of TGG(SGV) occurred gradually and lasted for a long duration of 60 min (or probably more) after intragastric glucose instillation was started, and a rise of insulin concentrations in the SPDV occurred concomitantly, even when arterial glucose levels did not change. This profile of insulin secretion evoked by the axis contrasts with the prompt and short-period insulin secretion in the cephalic phase and with the sharply rising and prominent insulin secretion in the intestinal phase accompanied by apparent changes in arterial glucose and insulin levels. Thus it would be more prudent not to call the present insulin secretion "the gastric phase" until the secretion is studied in more detail. In this context, it is plausible that the unaltered peripheral arterial insulin levels in glucose-instilled normal dogs could reflect increased hepatic insulin extraction by the mechanism that the hepatic efferent neural (probably vagal) signals originating from the putative gastric vagal afferents may lead to augmented insulin extraction. In any event, insulin secreted thus by the axis may more conveniently and efficiently control hepatic glucose output in a late postprandial stage, where exogenous glucose appearing into the circulation from the intestine is tapering off with a concomitant rapid decrease of arterial insulin levels, and endogenous glucose appearing from the liver is eventually starting to increase. In such a situation, if the fundic vagus monitors calorie content of chyme still remaining in the stomach and signals to secrete the appropriate amount of insulin according to the intragastric calorie content, then the hepatic glucose output would be optimally regulated by insulin secreted into the portal vein. In fact, plasma insulin in a relatively low concentration is known to effectively inhibit hepatic glucose output rather than to stimulate glucose uptake by the peripheral tissues (4, 7, 43). The calculated changes of intraportal insulin concentrations in the current study, particularly on intragastric instillation of <= 10% glucose solutions, are well within the range of insulin concentrations that effectively inhibit hepatic glucose output (4, 43), accompanying actually no changes in arterial insulin levels for peripheral action.

Finally, the current findings on the gastroinsular axis will provide new insight into the role of the stomach in postprandial glucose homeostasis, particularly in postprandial glucose counterregulation (21, 54, 55). This extends our previous observations on integration of insulin release induced by the neural cross talk between (or among) the visceral organs through the vagal chemoreception of nutrients and gut hormones (28, 34).


    ACKNOWLEDGEMENTS

We appreciate Drs. Akira Niijima and Yoriaki Kurata for suggestions and critical discussions in preparation of the manuscript.


    FOOTNOTES

Address for reprint requests and other correspondence: H. Nakabayashi, Health Science Service Center, Kanazawa University, 1-1 Kakuma-machi, Kanazawa 920-1192, Japan (E-mail: nakabaya{at}kenroku.kanazawa-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 September 2000; accepted in final form 20 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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