Meal-induced insulin secretion in dogs is mediated by both branches of the autonomic nervous system

Lambertus Benthem, Thomas O. Mundinger, and Gerald J. Taborsky Jr.

Division of Metabolism, Endocrinology, and Nutrition, Seattle Veterans Affairs Puget Sound Health Care System, and the University of Washington, Seattle, Washington 98108


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the relationship between autonomic activity to the pancreas and insulin secretion in chronically catheterized dogs when food was shown, during eating, and during the early absorptive period. Pancreatic polypeptide (PP) output, pancreatic norepinephrine spillover (PNESO), and arterial epinephrine (Epi) were measured as indexes for parasympathetic and sympathetic nervous activity to the pancreas and for adrenal medullary activity, respectively. The relation between autonomic activity and insulin secretion was confirmed by autonomic blockade. Showing food to dogs initiated a transient increase in insulin secretion without changing PP output or PNESO. Epi did increase, suggesting beta 2-adrenergic mediation, which was confirmed by beta -adrenoceptor blockade. Eating initiated a second transient insulin response, which was only totally abolished by combined muscarinic and beta -adrenoceptor blockade. During absorption, insulin increased to a plateau. PP output showed the same pattern, suggesting parasympathetic mediation. PNESO decreased by 50%, suggesting withdrawal of inhibitory sympathetic neural tone. We conclude that 1) the insulin response to showing food is mediated by the beta 2-adrenergic effect of Epi, 2) the insulin response to eating is mediated both by parasympathetic muscarinic stimulation and by the beta 2-adrenergic effect of Epi, and 3) the insulin response during early absorption is mediated by parasympathetic activation, with possible contribution of withdrawal of sympathetic neural tone.

cephalic phase of insulin release; feeding; pancreatic polypeptide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

FOOD INTAKE is generally considered as the primary physiological stimulus for insulin secretion. In this view, nutrients, such as glucose, are absorbed into the systemic circulation and play the major role in stimulating the pancreatic beta -cell to secrete insulin. However, careful analysis of the time course of insulin secretion during carbohydrate ingestion has shown that insulin secretion can start even before glucose is actually absorbed (11, 34). This so-called early insulin response (EIR) is initiated through stimulation of sensory receptors in the oropharyngeal region as well as through visual and olfactory stimulation (21). This afferent neural input is conveyed to the central nervous system, which mediates autonomic outflow to the pancreatic islet beta -cell, resulting in a rapid, transient increase in insulin release.

The EIR is traditionally attributed to parasympathetic stimulation of pancreatic beta -cells, because, at least in rats, muscarinic blockade diminishes the EIR (4, 19, 31, 32, 33). However, pancreatic endocrine function is under control of both the parasympathetic and the sympathoadrenal branch of the autonomic nervous system. Sympathetic stimulation of beta 2-adrenoceptors on pancreatic islets, most profoundly by circulating epinephrine (Epi), can also increase insulin secretion (25, 41), even though alpha 2-adrenoceptor stimulation, most profoundly by neurally released norepinephrine (NE), leads to inhibition of insulin secretion (31). Sympathetic activation has been reported during feeding (7, 30). Therefore, it is possible that sympathoadrenal stimulation of beta 2-adrenoceptors could also play a part in meal-related stimulation of insulin secretion.

It was the aim of this study to determine the autonomic contribution to meal-related insulin secretion in dogs. To this end, two sets of experiments were performed. In the first set, pancreatic norepinephrine spillover (PNESO), pancreatic polypeptide (PP) output, circulating catecholamines (Epi and NE), and pancreatic insulin output were determined in conscious dogs bearing chronically implanted arterial and pancreatic venous catheters. This was done during the following three phases of the meal: 1) when food was shown to the dogs, without allowing them to eat; 2) during actual eating; and 3) during the early absorptive phase.

PNESO was used as an index for sympathetic neural activity to the pancreas. The measurement of PNESO during hypoglycemia in anesthetized dogs (14, 15), and recently in conscious dogs as well (9), demonstrates the activation of the sympathetic nerves innervating the pancreas during this type of stress. Pancreatic ACh spillover would provide an index for parasympathetic neural activity to the pancreas. However, the extremely rapid degradation of ACh by cholinesterases in plasma and in tissue precludes this option. Measurement of PP output provides an alternative. PP is secreted by pancreatic F cells and is predominantly under vagal control, because muscarinic blockade with atropine as well as vagotomy prevents almost all PP responses (26, 27, 37). PP output has been used as an index for parasympathetic activity to the pancreas and has been correlated with the EIR (26, 37). Finally, circulating Epi serves as an index for adrenal medullary activity. Comparison of the time courses of PNESO, PP output, and Epi with that of insulin secretion during the three phases of feeding should allow one to decide if pancreatic sympathetic nerves, pancreatic parasympathetic nerves, or the adrenal medullary neurohormone Epi could mediate the observed insulin responses.

Proving the suspected mediator, however, would require blockade of the appropriate autonomic receptors. This was performed in a second set of experiments in conscious dogs bearing only arterial catheters. In these experiments, arterial concentrations of PP and insulin were determined during the three phases of a meal in dogs that were treated with the muscarinic blocker methylatropine and/or the beta -adrenoceptor blocker timolol.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All surgical and experimental protocols were approved by The Seattle Veterans Affairs Puget Sound Health Care System Animal Use Committee.

Animals and Surgical Preparation

After an overnight fast (~18 h), adult male dogs (28-34 kg) were anesthetized with the short-acting barbiturate thiamylal sodium (thiopental; Parke-Davis, Morris Plains, NJ). Anesthesia was subsequently maintained with isoflurane (2.0%) administered from a calibrated vaporizer (Draeger) by mechanical ventilation with pure oxygen.

All animals (experiments 1 and 2) had indwelling catheters (Tygon microbore S-54-HL, ID 0.5 mm, OD 1.5 mm) inserted in the right omocervical vein and artery. The tips of the catheters were advanced in the pulmonary artery for drug infusion and in the descending aorta for blood sampling and blood pressure measurement (23). The catheters were tunneled subcutaneously and externalized in the midscapular region. There they were secured to arterial valve connectors (Harvard Apparatus) and sutured to the skin.

Dogs used for the experiments involving direct measurement of pancreatic hormone output and neurotransmitter spillover (experiment 1) received a midline laporotomy and had an additional indwelling catheter (Tygon microbore S-54-HL, ID 2.0 mm, OD 3.0 mm) inserted in the main duodenal input to the superior pancreaticoduodenal vein (SPDV). The tip of this catheter was placed 1 mm proximal to the junction of the main duodenal input with the SPDV, allowing unrestrained blood flow from the duodenal lobe of the pancreas into the SPDV. Additionally, an ultrasonic blood flow probe (Transonic Systems) was positioned around the SPDV 1 cm proximal to the junction of the SPDV with the portal vein. Both the catheter and the wire of the blood flow probe were tunneled subcutaneously and externalized in the midscapular region.

The catheters were kept patent by daily flushing with heparinized (50 U/ml) saline, whereafter they were filled with heparin (1,000 U/ml). The arterial catheters remained patent for months; the pancreatic venous catheters remained patent for up to 1 mo.

The combination of simultaneous sampling of arterial and pancreatic venous blood with blood flow measurement in the pancreatic vein allowed the calculation of hormone output and neurotransmitter spillover from the right lobe (duodenal lobe and uncinate process) of the canine pancreas (35-50% of the total canine pancreas).

Experimental Protocols

To avoid novelty stress, the dogs were habituated to the laboratory surroundings and personnel and were trained to stand in a Pavlovian sling before the start of the actual experiments. To minimize external influences, the dogs were habituated to the moderate sound level of a radio. To avoid excitatory visual stimulation during muscarinic blockade, the experimental room was dimly lit.

Experiment 1. The first set of experiments was designed to test whether the adrenal medulla, the pancreatic parasympathetic, or the pancreatic sympathetic nerves were activated during the three phases of a meal. After an overnight fast, the dogs (n = 7) entered the experimental room and were placed in a Pavlovian sling. The arterial catheter was, via a blood pressure transducer (Statham P23Db), connected to a blood pressure monitor (Propac 106 EL; Protocol Systems) for measurement of mean arterial pressure (MAP). The socket of the blood flow probe was connected to the blood flowmeter (T101 ultrasonic bloodflow meter; Transonic Systems) to measure pancreatic venous blood flow. After a cooldown period of ~60 min, during which MAP had stabilized, baseline arterial and pancreatic venous blood samples were taken [time (t) = -10 and -1 min]. At t = 0, food [2 cans of Western Family Chicken Flavored Dog Food (protein >8%, fat >3%, fiber >1%, water <78%, ash <5%) +50 ml of D50 glucose)] was shown to the animals, without allowing them to eat. Blood samples were then taken at t = 2 and 4 min. At t = 5 min, the animals were allowed to eat the meal. In general, the dogs finished the meal within 3 min. When the meal was not finished at t = 11 min, the remaining food was taken away to maintain clear separation between the eating phase and the early absorptive phase. Additional blood samples were taken during eating (t = 7.5 and 10 min) and early absorption (t = 12.5, 15, 20, 25, and 35 min).

Experiment 2. The second set of experiments was designed to determine which autonomic input mediated the insulin responses to the sight and smell of the food, to eating the food, and early during absorption of the food. To this end, each dog participating in this study underwent a series of four experiments: one with muscarinic blockade, one with beta -adrenoceptor blockade, one with combined muscarinic and beta -adrenoceptor blockade, and one, as a control, without blockade. Each dog (n = 8) underwent these four experiments in a random order. Muscarinic blockade was induced using methylatropine (0.5 mg/kg + 0.5 mg · kg-1 · h-1; Sigma; see Ref. 23); beta -adrenoceptor blockade was induced using timolol (0.2 mg/kg + 0.2 mg · kg-1 · h-1; Sigma; see Ref. 23). Drugs were dissolved in saline and were administered through the cannula in the pulmonary artery. Infusion of saline alone served as the control. The experimental protocol was similar to the one used for experiment 1. A baseline sample was taken at t = -15 min, whereafter blockade was induced and maintained throughout the experiment. Baseline blood samples were then taken at t = -7.5 and -1 min. At t = 0, food was shown to the animals, and blood samples were taken at t = 2 and 4 min. At t = 5 min, the animals were allowed to eat the meal (see above for the procedure). Additional blood samples were taken during eating (t = 7.5 and 10 min) and during early absorption (t = 12.5, 15, 20, 25, and 35 min).

Assays

Blood samples were immediately placed on ice in tubes containing EDTA for determination of immunoreactive insulin, glucose, and PP and in tubes containing glutathione and EGTA for catecholamine determination. Samples were centrifuged (20 min at 2,600 g and 4°C), whereafter the plasma was decanted and frozen at -70°C until assay.

Plasma insulin and PP concentrations were determined using RIAs described previously (6, 36). Plasma NE and Epi concentrations were determined in duplicate with a highly sensitive and specific radioenzymatic assay (10). The intra- and interassay coefficients of variation for the plasma catecholamine assay in this laboratory are 6 and 12%, respectively. Plasma glucose concentration was determined using a glucose oxidase technique.

Data Analysis

Pancreatic insulin and PP output were calculated using the formula
output = ([SPDV] − [art]) × blood flow<SUB>SPDV</SUB> × (1 − hematocrit)
where [SPDV] is SPDV plasma concentration, [art] is arterial plasma concentration, and blood flowSPDV is SPDV blood flow.

Duodenal glucose absorption was calculated as
absorption = ([glucose]<SUB>SPDV</SUB> − [glucose]<SUB>art</SUB>) 

× blood flow<SUB>SPDV</SUB> × (1 − hematocrit)
where [glucose]SPDV and [glucose]art are the SPDV and arterial plasma glucose concentrations, respectively. PNESO was calculated by the formula
PNESO = {[NE]<SUB>SPDV</SUB> − (arterial contribution)} 

× blood flow<SUB>SPDV</SUB> × (1 − hematocrit)
where [NE]SPDV is the plasma concentration of NE in the SPDV. The arterial contribution was calculated as [NE]art × ([Epi]SPDV/[Epi]art), as described by Dunning and colleagues (9), with [Epi]SPDV/[Epi]art being the actual fractional passage of Epi through the pancreas. The use of this term for the fractional passage of NE through the pancreas assumes that pancreatic extractions of Epi and NE are equivalent (1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Baseline values for arterial hormone and glucose concentrations in Experiment 2 involving muscarinic blockade (methylatropine), beta -adrenoceptor blockade (timolol), or combined muscarinic and beta -adrenoceptor blockade (methylatropine + timolol)

Data are expressed as means ± SE for experiment 1 and as mean change ± SE from baseline [delta (Delta )] obtained at t = -1 min for experiment 2. Wilcoxon's matched-pairs signed-rank test was used to test for significant changes from baseline at t = -1 min (experiment 1). ANOVA and the Mann-Whitney U-test were used to test for significant differences between responses in the control experiment and those during autonomic blockade (experiment 2). The level of significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1

Figure 1 shows that insulin output increased when food was shown to the animals, even though they were not yet allowed to eat. This increase was transient; insulin output had already returned to the baseline by t = 4 min. When the animals were allowed to eat (t = 5 min), insulin output increased again (+24.0 mU/min at t = 7.5 min). After this second transient increase, insulin output declined below the baseline (t = 12.5-15 min). Twenty minutes after the onset of the meal (t = 25 min), a third more modest but more sustained increase in insulin output was recognized (P < 0.05 at t = 2, 7.5, 12.5, 15, and 20-35 min).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Activity of pancreas beta -cells related to glucose absorption from the duodenum and arterial glucose concentration when food is shown [time (t) = 2 and 4 min], during eating (t = 5 and 7.5 min), and during the early absorptive phase (t = 12.5-35 min). Data are expressed as means ± SE.

The arterial insulin concentration increased when food was shown and increased further immediately after the onset of the meal before returning toward the baseline. Twenty minutes after the onset of the meal, arterial insulin increased again and remained elevated throughout the experiment (P < 0.05 at t = 2-12.5 and 20-35 min).

Duodenal glucose absorption was negligible before feeding and was not affected by the sight and smell of the food. Eating had no immediate effect on duodenal glucose absorption. However, duodenal glucose absorption increased significantly at t = 10 min, i.e., 5 min after the start of the meal, and was elevated throughout the remainder of the experiment. It is noteworthy that the increase in duodenal glucose absorption occurred after the second transient increase in insulin output, in fact when insulin output was declining.

The arterial glucose concentration increased progressively above the baseline level when the food was shown to the animals and later when the animals ate the food. Then it declined before another sustained increase occurred. The first increase of plasma glucose occurred before the increase of duodenal glucose absorption; the second increase of plasma glucose occurred during the increase of duodenal glucose absorption.

PP output was not significantly affected by the sight and smell of food (Fig. 2). However, eating the meal did initiate a large increase in PP output that reached a maximal value of 492 ± 128 ng/min at t = 7.5 min before it declined to 135 ± 31 ng/min at t = 15, still significantly above baseline. Thereafter, it increased modestly again and remained elevated for the duration of the experiment. After the start of the meal, the time course of PP output was similar to that of insulin output. Arterial PP showed a significant and sustained increase from t = 7.5 min throughout the remainder of the experiment.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2.   Activity of pancreatic autonomic nerves and pancreatic blood flow when food is shown (t = 2 and 4 min), during eating (t = 5 and 7.5 min), and during the early absorptive phase (t = 12.5-35 min). PP, pancreatic polypeptide; SPDV flow, blood flow in superior pancreaticoduodenal vein; PNESO, pancreatic norepinephrine spillover. Data are expressed as means ± SE.

PNESO was not significantly affected by the presentation of the food or immediately by eating the meal. However, at t = 10 min, PNESO had decreased below baseline and remained suppressed throughout the experiment.

Showing food did not significantly affect SPDV blood flow. However, during eating, SPDV blood flow increased significantly. Thereafter, it gradually declined to reach a value of 83 ± 13 ml/min at t = 25 min, still significantly above baseline (P < 0.05 at t = 7.5-35 min).

The arterial Epi concentration (Fig. 3) increased sharply when food was shown. Thereafter, it gradually declined to return to baseline at t = 12.5 min, where it remained for the rest of the experiment (P < 0.05 at t = 2-7.5 min). Arterial NE also increased when food was shown, but unlike Epi it increased further when the animals were allowed to eat (P < 0.05 at t = 2-10 min). NE reached its maximal value at t = 7.5 min (vs. t = 2 min for Epi). Thereafter, it declined to reach the baseline at t = 12.5 min. The time course of MAP was similar to that of NE; MAP increased when food was shown to the animals, increased further when the food was actually eaten, and reached its maximum at t = 7.5 min (P < 0.05 at t = 2, 7.5, and 10 min).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Arterial catecholamines and blood pressure when food is shown (t = 2 and 4 min), during eating (t = 5 and 7.5 min), and during the early absorptive phase (t = 12.5-35 min). Epi, epinephrine; NE, norepinephrine; MAP, mean arterial pressure. Data are expressed as means ± SE.

Experiment 2

The arterial insulin response to showing food to the animals was not reduced by muscarinic blockade (Fig. 4). In contrast, the arterial insulin response to the sight and smell of food was significantly reduced by beta -adrenoceptor blockade and by combined beta -adrenoceptor and muscarinic blockade (Figs. 5 and 6, respectively; P < 0.05 at t = 2 and 4 min).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of muscarinic blockade on the arterial PP, insulin, and glucose concentration when food is shown (t = 2 and 4 min), during eating (t = 5 and 7.5 min), and during the early absorptive phase (t = 12.5-35 min). Data are expressed as mean ± SE change (Delta ) from the baseline as obtained at t = -1 min, i.e., just before food was shown.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of beta -adrenoceptor blockade on the arterial PP, insulin, and glucose concentration when food is shown (t = 2 and 4 min), during eating (t = 5 and 7.5 min), and during the early absorptive phase (t = 12.5-35 min). Data are expressed as mean ± SE change from the baseline as obtained at t = -1 min, i.e., just before food was shown.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of combined muscarinic and beta -adrenoceptor blockade on the arterial PP, insulin, and glucose concentration when food is shown (t = 2 and 4 min), during eating (t = 5 and 7.5 min), and during the early absorptive phase (t = 12.5-35 min). Data are expressed as mean ± SE change from the baseline as obtained at t = -1 min, i.e., just before food was shown.

The immediate arterial insulin response to eating was significantly reduced, but not totally abolished, after either muscarinic blockade or beta -adrenoceptor blockade (P < 0.05 at t = 5 min). Combined muscarinic and beta -adrenoceptor blockade, however, totally prevented the eating-induced increase of arterial insulin (P < 0.05 at t = 5 and 7.5 min).

During the early absorptive phase (t = 10-35 min), the insulin response was not affected by beta -adrenoceptor blockade (Fig. 5). Muscarinic blockade and combined muscarinic and beta -adrenoceptor blockade prevented insulin from increasing during the early absorptive phase (P < 0.05 at t = 25 and 35 min).

No significant differences in the arterial PP responses were observed between the experiment with beta -adrenoceptor blockade and the control experiment (Fig. 5). Muscarinic blockade with or without beta -adrenoceptor blockade, on the contrary, totally prevented the responses in arterial PP to eating and during the early absorptive phase (P < 0.05 a t = 7.5-35 min).

In the experiment with muscarinic blockade (Fig. 4), the arterial blood glucose response during the early absorption phase was significantly decreased compared with that in the control experiment (P < 0.05 at t = 20-35 min). In the experiment with beta -adrenoceptor blockade (Fig. 5) and in the experiment with combined beta -adrenoceptor and muscarinic blockade (Fig. 6), the blood glucose response during eating and during the early absorptive phase was significantly increased compared with the glucose response in the control experiment (P < 0.05 at t = 7.5-15 and 25-35 min for the experiment with beta -adrenoceptor blockade and P < 0.05 at t = 10-20 min for the experiment with combined blockade).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have examined the meal-related insulin responses in dogs and have assessed which branch of the autonomic nervous system mediates these responses. We separated the insulin responses to a meal into the following three phases: 1) the response to the sight and smell of food [when the food was shown to the animals without allowing them to eat (t = 2 and 4 min)], 2) the immediate response to eating the meal (t = 7.5 and 10 min), and 3) the response to the early absorptive phase after eating (t = 12.5-35 min). We sought to characterize the autonomic contribution to each of these insulin responses. We found that showing food to the animals induced a rapid and transient increase in insulin secretion. Surprisingly, the data suggest that this response is not mediated by either glucose or direct parasympathetic stimulation but rather by circulating Epi. When the animals ate the meal, a second rapid and transient insulin response occurred. The data suggest that this response is mediated by combined parasympathetic, muscarinic, and sympathetic beta 2-adrenergic stimulation. During the early phase of absorption, a third, and now sustained insulin response occurred. Surprisingly, the data suggest that this response is mediated by parasympathetic stimulation rather than by glucose. Thus, in dogs, all three early insulin responses to a meal appear to be autonomically mediated.

Showing food to the dogs, without allowing them to eat, induced a rapid and transient insulin response. Such an early insulin response to the sight and smell of food has been observed in a variety of species, including humans (24, 28, 29, 42). It has been suggested to be mediated by a conditioned increase in parasympathetic activity (24, 29, 42). However, in the experiments presented here, PP output did not increase when food was shown, suggesting that vagal activity to the pancreas had not increased at that moment. This conclusion was confirmed by the failure of methylatropine to block or even reduce the arterial insulin response to showing food. Thus, in dogs, the insulin response to the sight and smell of food cannot be attributed to increased parasympathetic cholinergic stimulation.

Although the plasma glucose concentration rose slightly when food was shown, it is unlikely that this increase triggered the insulin response. First, at that time, there was no increase in glucose absorption from the duodenum (see Fig. 1). Second, the arterial insulin response to showing food was still present after muscarinic blockade, even though plasma glucose did not increase.

Another possible mediator of the insulin response to the sight and smell of food could be withdrawal of inhibitory sympathetic neural tone. Such tone is apparently present in rats, since alpha -adrenergic blockade increases plasma insulin concentrations (3, 31). However, in the present experiments, PNESO, an index for sympathetic neural activity to the pancreas (9, 14, 15), did not decrease significantly when food was shown, suggesting that any inhibitory sympathetic neural tone was still present. Alternatively, because our measurement of NE spillover includes both pancreas and attached duodenum, one could argue that NE spillover from the proximal duodenum might have increased, masking a potential decrease of NE spillover from the pure pancreatic tissue. However, duodenal dilution of pancreatic blood is estimated at only 24% (9), making the above explanation unlikely.

Ruling out parasympathetic stimulation, withdrawal of sympathetic tone and stimulation by plasma glucose as the mediators of the insulin response to showing food leaves sympathoadrenal beta 2-adrenoceptor (18) stimulation as the likely mediator. Arterial NE is increased in response to showing food, despite unchanged PNESO, indicating that the sympathetic nerves to other organs beside the pancreas, for example the heart, are activated. This observation confirms the poor relation between circulating NE and sympathetic drive to individual organs, in particular the pancreas (8). Arterial Epi is also increased in response to showing food, indicating that the adrenal branch of the sympathetic system was activated as well. Feeding-associated activity of the sympathoadrenal system has been reported for dogs (7) and for rats (30) and has been related to the increased energy expenditure noticed during the cephalic phase of feeding (7). A direct link to the stimulation of feeding-associated insulin secretion, however, has to the best of our knowledge not been made before. Still, we would argue that the increase in insulin secretion is due to beta 2-adrenoceptor-mediated stimulation, with the arterial insulin response to the sight and smell of food being blocked by the beta -adrenoceptor antagonist timolol, despite the higher arterial glucose concentration present during beta -blockade.

The beta 2-adrenoceptor-mediated stimulation of insulin secretion is most likely to be mediated via Epi rather than via NE, because beta 2-adrenoceptors have a much higher affinity for Epi than for NE (17). This means that Epi at low concentrations, such as present in the current experiments, has profound beta 2-adrenoceptor-stimulating properties and can stimulate insulin secretion (2). Epi is released primarily in situations having excitatory emotional components (12), a situation clearly reflected in the behavior of the animals when food was presented without allowing them to eat. beta -Adrenoceptor-mediated stimulation of insulin secretion is suggested to prevent total alpha -adrenoceptor-mediated suppression of insulin release during sympathetic (neural) activity, thus securing a minimal release of insulin (35).

Eating induces a rapid and transient insulin response similar to that produced by just showing the food. This early insulin response to eating has been observed in a variety of species, including humans (4, 7, 11, 16, 30-35, 36, 39). Like the insulin response to showing food, the early response to eating has been attributed to a neural reflex, because it is also observed in response to sham feeding conditions (4, 5, 38) or taste stimulation (20, 21). This reflex has been associated with hypothalamically mediated parasympathetic stimulation of the pancreas (22). In rats, the insulin response to eating can be prevented by vagotomy (19) or atropinization (34), confirming the parasympathetic mediation in that species.

In dogs, the mechanism underlying the immediate increase in insulin during eating appears to be more complex. As in other species, there is activation of the parasympathetic nervous system, as indicated by a rapid increase of the arterial PP concentration. This parasympathetic activation does contribute to the meal-induced increase of the arterial insulin concentration, since methylatropine abolishes the PP response and reduces the insulin response to eating. However, some of the eating-induced insulin response remains during muscarinic blockade (see Fig. 4). This remaining insulin response is not due to a decrease in pancreatic sympathetic nervous activity, since PNESO dropped only after the insulin response to eating had already peaked. Rather, it is due to beta -adrenergic stimulation of the beta -cell, because the addition of beta -adrenoceptor blockade to muscarinic blockade abolished the insulin response to eating. This total suppression of the eating-induced insulin response occurs despite the higher glucose levels present during combined blockade. This observation excludes the small increase in plasma glucose, which is observed immediately at the start of eating, as a mediator of the immediate insulin response. However, the present experiments do not rule out the alternative possibility that the effect of the small rise of plasma glucose to contribute to eating-induced insulin secretion is inhibited by the unopposed alpha -adrenergic effect of catecholamines during beta -adrenoceptor blockade.

During eating in the control experiment, a small increase in glucose can be recognized, although glucose absorption has not yet increased. This small increase in glucose in the control experiment is probably mediated by direct or indirect (via glucagon) sympathoadrenal stimulation of hepatic glucose production (13). Epi, a potent stimulus for glucagon, is still increased during eating. The elevated glucose concentrations during eating after combined blockade were most likely allowed by the decrease in baseline insulin, resulting from the combined withdrawal of beta -adrenoceptor and muscarinic stimulation of the beta -cell, leaving unopposed alpha -adrenoceptor-mediated inhibition of insulin secretion.

After the immediate response to eating, insulin output fell rapidly below the baseline and then rose slowly. This slow rise occurred during increasing glucose absorption and is traditionally thought to be due to the rising arterial glucose level. However, the importance of the small increase in circulating glucose for stimulation of insulin secretion can be argued. Based on a vast number of in vitro studies, it is beyond dispute that glucose directly stimulates insulin secretion. However, it is also true that most of the in vitro studies challenged the beta -cells with glucose concentrations that by far exceed the small increase of the arterial glucose concentration observed in the present study.

Alternatively, stimulation of insulin secretion during the early absorption phase could be due to parasympathetic stimulation of the beta -cell, either directly or indirectly via interaction with gastrointestinal hormones, because PP remains elevated during this early absorptive phase. The blockade of the PP response and the lowering of the insulin level by methylatropine during this phase are consistent with this hypothesis. However, muscarinic blockade also lowers the glucose level, probably by impairing glucose absorption. Therefore, it is unclear from these data if it is the decrease in parasympathetic stimulation or the decreased glucose level that causes the decreased insulin response during early absorption in the experiments with muscarinic blockade. However, combined muscarinic and beta -adrenoceptor blockade totally abolishes the insulin response during absorption, despite higher glucose levels (see Fig. 6). Therefore, it is our current opinion that the insulin response during absorption is mediated by increased parasympathetic activity rather than by the small rise in glucose level. One cannot exclude, however, that small increases in glucose may still play a role in potentiating the beta -cell response to parasympathetic stimulation. Another possible contributor to this insulin response could be the withdrawal of inhibitory pancreatic sympathetic neural tone, since PNESO appears to be decreased throughout this time period (see Fig. 2).

Based upon these results, we conclude that, in the dog, 1) the insulin response to showing food is mediated by the beta 2-adrenergic effects of Epi; 2) the immediate insulin response to eating is mediated both by parasympathetic cholinergic stimulation and by the beta 2-adrenergic effects of Epi; and 3) the insulin response during early absorption is mediated via parasympathetic cholinergic stimulation, with a possible contribution of withdrawal of sympathetic neural tone.


    ACKNOWLEDGEMENTS

We thank Richard Chang, Ruth Hollingworth, Rix Kuester, Hong Nguyen, and Jira Wade for expert technical assistance.


    FOOTNOTES

This research was supported by the Medical Research Service of the Department of Veterans Affairs and by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-12829, DK-12047, and DK-50154. L. Benthem was supported by an Albert Renold Fellowship awarded by the European Association for the Study of Diabetes and by a grant from the Diabetes Foundation Netherlands.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Benthem, Dept. of Human Biology, University of Maastricht, PO Box 616, NL-6200 MD Maastricht, The Netherlands (E-mail: B.Benthem{at}HB.UNIMAAS.NL).

Received 10 May 1999; accepted in final form 4 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahren, B, Dunning BE, Havel PJ, Veith RC, and Taborsky Jr GJ. Extraction of epinephrine and norepinephrine by the dog pancreas in vivo. Metabolism 37: 68-73, 1988[ISI][Medline].

2.   Ahren, B, Veith RC, and Taborsky Jr GJ The effects of epinephrine on islet hormone secretion in the dog. Int. J. Pancreat. 3: 375-388, 1988[ISI].

3.   Benthem, L, van der Leest J, Steffens AB, and Zijlstra WG. Metabolic and hormonal responses to adrenoceptor antagonists in exercising rats. Metabolism 44: 245-253, 1995[ISI][Medline].

4.   Berthoud, HR, and Jeanrenaud B. Sham feeding-induced cephalic phase insulin release in the rat. Am J Physiol Endocrinol Metab 242: E280-E285, 1982[Abstract/Free Full Text].

5.   Bruce, DG, Storlien LH, Furler SM, and Chrisholm DJ. Cephalic phase metabolic responses in normal weight adults. Metabolism 36: 721-725, 1987[ISI][Medline].

6.   Chance, RE, Moon NE, and Johnson MG. Human pancreatic polypeptide (HPP) and bovine pancreatic polypeptide (BPP). In: Methods of Hormone Radioimmunoassay. New York: Academic, 1979, p. 657-672.

7.   Diamond, P, and LeBlanc J. Hormonal control of postprandial thermogenesis in dogs. Am J Physiol Endocrinol Metab 253: E521-E529, 1987[Abstract/Free Full Text].

8.   Dunning, BE, Havel PJ, Veith RC, and Taborsky Jr GJ. Pancreatic and extrapancreatic galanin release during sympathetic neural activation. Am J Physiol Endocrinol Metab 258: E436-E444, 1990[Abstract/Free Full Text].

9.   Dunning, BE, Scott MF, Neal DW, and Cherrington AD. Direct quantification of norepinephrine spillover and hormone output from the pancreas of the conscious dog. Am J Physiol Endocrinol Metab 272: E746-E755, 1997[Abstract/Free Full Text].

10.   Evans, MI, Halter JB, and Porte Jr D. Comparison of double- and single-isotope enzymatic derivative methods for measuring catecholamines in human plasma. Clin Chem 24: 567-570, 1978[Abstract/Free Full Text].

11.   Fischer, U, Hommel H, Fiedler H, and Bibergeil H. Reflex mechanism on insulin secretion. Endocrinol Exp 8: 137-146, 1974[ISI][Medline].

12.   Frankenhaeuser, M. Experimental approaches to the study of catecholamines and emotion. In: Emotions---Their Parameters and Measurement, edited by Levi L.. New York: Reaven, 1976, p. 209-234.

13.   Havel, PJ, Scott M, Allen E, and Neal D. Rapid increase of hepatic glucose output in response to meal anticipation and to feeding in dogs; mediation via increased glucagon secretion. Diabetes 44, Suppl 1: 90A, 1995.

14.   Havel, PJ, and Taborsky Jr GJ. The contribution of the autonomic nervous system to changes of glucagon and insulin secretion during hypoglycemic stress. Endoc Rev 10: 332-350, 1989[ISI][Medline].

15.   Havel, PJ, Veith RC, Dunning BE, and Taborsky Jr GJ. Pancreatic noradrenergic nerves are activated by neuroglucopenia but not hypotension or hypoxia in the dog. J Clin Invest 82: 1538-1545, 1988[ISI][Medline].

16.   Jong, A de, Strubbe JH, and Steffens AB. Hypothalamic influence on insulin and glucagon release in the rat. Am J Physiol Endocrinol Metab 235: E380-E388, 1977.

17.   Lands, AM, Arnold A, Mcauliff JP, Luduena FP, and Brown G. Differentiation of receptor systems activated by sympathomimetic amines. Nature Lond 214: 597-598, 1967[ISI][Medline].

18.   Loubatières, A, Mariani M-M, Sorel G, and Savi L. The action of beta -adrenergic blocking and stimulating agents on insulin secretion. Characterization of the type of beta -receptor. Diabetologia 7: 127-132, 1971[ISI][Medline].

19.   Louis-Sylvestre, J. Preabsorptive insulin release and hypoglycemia in rats. Am J Physiol 230: 56-60, 1976[ISI][Medline].

20.   Niijima, A, Togiyama T, and Adachi A. Cephalic phase insulin release induced by taste stimulus of monosodium glutamate (unami tatste). Physiol Behav 48: 905-908, 1990[ISI][Medline].

21.   Powers, MA, Schiffman SS, Lawson DC, Pappas TN, and Taylor IL. The effect of taste on gastric and pancreatic responses in dogs. Physiol Behav 47: 1295-1297, 1990[ISI][Medline].

22.   Powley, TL. The ventromedial hypothalamic syndrome, satiety, and the cephalic phase hypothesis. Physiol Rev 84: 89-126, 1977.

23.   Roossien, A, Brunsting JR, Nijmeijer A, Zaagsma J, and Zijlstra WG. Effects of vasoactive intestinal polypeptide on heart rate in relation to vagal cardioacceleration in conscious dogs. Cardiovasc Res 33: 392-399, 1997[ISI][Medline].

24.   Roozendaal, B, Oldenburger WP, Strubbe JH, Koolhaas JM, and Bohus B. The central amygdala is involved in the conditioned but not in the meal-induced cephalic insulin response in the rat. Neurosci Lett 116: 210-215, 1990[ISI][Medline].

25.   Samols, E, and Weir GC. Adrenergic modulation of pancreatic A, B, and D cells. J Clin Invest 63: 230-238, 1979[ISI][Medline].

26.   Schwartz, TW. Pancreatic polypeptide: a hormone under vagal control. Gastroenterology 85: 1411-1425, 1983[ISI][Medline].

27.   Schwartz, TW, Holst JJ, Fahrenkrug J, Lindkær Jensen S, Nielsen OV, Rehfeld JF, Schaffalitzky OB, and Stadil F. Vagal, cholinergic regulation of pancreatic polypeptide secretion. J Clin Invest 61: 781-789, 1978[ISI][Medline].

28.   Simon, C, Schlienger JL, Sapin R, and Imler M. Cephalic phase insulin secretion in relation to food presentation in normal and overweight subjects. Physiol Behav 36: 465-469, 1986[ISI][Medline].

29.   Sjøstrøm, L, Garellick G, Krotkiewsky M, and Luyckx L. Peripheral insulin in response to the sight and smell of food. Metabolism 29: 901-909, 1980[ISI][Medline].

30.   Steffens, AB, Van Der Gugten J, Godeke J, Luiten PGM, and Strubbe JH. Meal-induced increases in parasympathetic and sympathetic activity elicit simultaneous rises in plasma insulin and free fatty acids. Physiol Behav 37: 119-122, 1986[ISI][Medline].

31.   Strubbe, JH. Central nervous system and insulin secretion. Neth J Med 34: 154-167, 1989[ISI][Medline].

32.   Strubbe, JH. Parasympathetic involvement in rapid meal-associated conditioned insulin secretion in the rat. Am J Physiol Endocrinol Metab 263: ER615-R618, 1992.

33.   Strubbe, JH, Prins AJA, Bruggink J, and Steffens AB. Daily variation of food induced changes in blood glucose and insulin in the rat and the control by the suprachiasmatic nuscleus and the vagus nerve. J Auton Nerv Syst 20: 113-119, 1987[ISI][Medline].

34.   Strubbe, JH, and Steffens AB. Rapid insulin release after ingestion of a meal in the unanesthetized rats. Am J Physiol 229: 1019-1022, 1975[ISI][Medline].

35.   Strubbe, JH, and Steffens AB. Neural control of insulin secretion. Horm Metab Res 25: 507-512, 1993[ISI][Medline].

36.   Taborsky Jr, GJ. Evidence of a paracrine role for pancreatic somatostatin in vivo. Am J Physiol Endocrinol Metab 245: E598-E603, 1983[Abstract/Free Full Text].

37.   Taylor, IL, Impicciatore M, Carter DC, and Walsh JH. Effect of atropine and vagotomy on pancreatic polypeptide response to a meal in dog. Am J Physiol Endocrinol Metab 235: E443-E447, 1978[ISI][Medline].

38.   Teff, KL, Levin BE, and Engelman K. Oral sensory stimulation in man: effects on insulin, C-peptide, and catecholamines. Am J Physiol Regulatory Integrative Comp Physiol 265: R1223-R1230, 1993[Abstract/Free Full Text].

39.   Teff, KL, Mattes RD, and Engelman K. Cephalic phase insulin release in normal weight males: verification and reliability. Am J Physiol Endocrinol Metab 261: E430-E436, 1991[Abstract/Free Full Text].

40.   Teff, KL, Mattes RD, Engelman K, and Mattern J. Cephalic-phase insulin in obese and normal-weight men: relation to postprandial insulin. Metabolism 42: 1600-1608, 1993[ISI][Medline].

41.   Woods, SC, and Porte Jr D. Neural control of the endocrine pancreas. Physiol Rev 54: 596-619, 1974[Free Full Text].

42.   Woods, SC, Vasselli JR, Kaestner R, Szakmary GA, Milburn P, and Vitiello MV. Conditioned insulin secretion and meal feeding in rats. J Clin Endocrinol Metab 55: 1114-1117, 1982[Abstract].


Am J Physiol Endocrinol Metab 278(4):E603-E610
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society