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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5606-5613
Copyright © 2004 by The Endocrine Society

Prolonged Mild Hyperglycemia Induces Vagally Mediated Compensatory Increase in C-Peptide Secretion in Humans

Karen L. Teff and Raymond R. Townsend

Monell Chemical Senses Center (K.L.T.), and University of Pennsylvania School of Medicine, University of Pennsylvania (K.L.T., R.R.T.), Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Dr. Karen L. Teff, Monell Chemical Senses Center, 3500 Market Street, Philadelphia, Pennsylvania 19104. E-mail: kteff{at}pobox.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Experimentally induced prolonged hyperglycemia increases insulin release in humans, and in animals has been demonstrated to increase vagal efferent activity. The objective of the present experiment was to determine whether in humans, the compensatory increase in insulin release in response to short-term mild hyperglycemia is mediated by an induction of vagal efferent activity. Lean male subjects (n = 11; body mass index, 23.6 ± 0.8 kg/m2) underwent a frequently sampled iv glucose tolerance test (FSIGT) to determine B cell function and insulin sensitivity. Subjects were then tested under four conditions over 4 months. Subjects were infused for 48 h with either glucose (15% dextrose at 200 mg/m2·min) or saline (50 ml/h). Three hours after termination of the infusion, an FSIGT was administered in the presence of either saline or atropine (0.4 mg/m2 bolus: 0.4 mg/m2·h). Glucose (117 ± 14 vs. 98 ± 5 mg/dl) and insulin (49.5 ± 10 vs. 23 ± 5 µU/ml) levels were significantly elevated during the 48-h glucose infusion compared with those during saline treatment. Forty-eight-hour glucose infusions increased insulin and C-peptide levels during the FSIGT. When the FSIGT was conducted in the presence of atropine after glucose infusion, C-peptide levels were significantly attenuated during the period of endogenous insulin secretion (0–20 min; 31.8 ± 13 vs. 39.2 ± 11.9, atropine vs. no atropine) and exogenous insulin administration [20–40 min; 18.8 ± 10.8 vs. 31.6 ± 12.9., atropine vs. no atropine; F(3,9) = 4.99; P < 0.026]. A significant negative correlation was found between the repression of C-peptide by muscarinic blockade and the magnitude of the C-peptide response to the glucose infusion (r = 0.60; P < 0.045). Insulin AUC was not significantly altered by the presence of muscarinic blockade. In summary, we found that prolonged mild hyperglycemia results in a compensatory increase in C-peptide secretion, which is partially mediated by an induction in vagal efferent activity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
ELEVATED BASAL AND stimulated plasma insulin levels in the presence of either normal or mild hyperglycemia are physiological hallmarks of the insulin resistance associated with obesity. The progression of the insulin-resistant state to overt type II diabetes is thought to be an interaction between impaired B cell function and the degree of insulin resistance. Thus, the ability of an obese individual to maintain adequate B cell function in the face of chronic stimulation is a critical determinant to the development of type II diabetes. Understanding the compensatory mechanisms contributing to sustained B cell function may provide insight into individual predisposition to type II diabetes.

Experimentally induced hyperglycemia has been used to examine the consequences of chronic stimulation of the B cell on B cell function and insulin sensitivity. Although prolonged hyperglycemia has been shown to impair insulin secretion in some in vitro experiments (1) and various animal models of compromised B cell function (2, 3, 4, 5), other animal (6, 7) and human studies (8, 9, 10) have reported increased insulin secretion and insulin sensitivity. Discrepancies between data have led to the suggestion that an extra pancreatic factor, possibly neural, may be contributing to insulin secretion after prolonged hyperglycemia (7). In fact, animal studies suggest that sustained increases in plasma glucose can alter tonic activity of the vagus nerve, part of the parasympathetic nervous system (11, 12). Because vagal efferent activation elicits the release of acetylcholine at the level of the B cell and has stimulatory effects on insulin secretion, changes in neural activity may influence insulin release. Typically, vagal activity contributes to insulin release during food ingestion, but not under acute noningestive conditions such as iv glucose administration (13). In contrast, prolonged and substantial elevations of plasma glucose (≥350 mg/dl) in animals appear to alter the normal stimulatory pathways that are involved in vagal activation, such that a vagally mediated component of insulin release in response to iv glucose administration is induced (12, 14).

In humans, only a few studies have examined the effect of prolonged hyperglycemia on insulin secretion and sensitivity (9, 15, 16), and none has investigated the potential contribution of neural activation to these parameters. However, if physiological elevations in plasma glucose could be demonstrated to alter vagal efferent activity in humans, this would have important implications for the regulation of B cell function. Specifically, it would suggest that the parasympathetic nervous system is involved in maintaining B cell function during periods of chronic stimulation. Furthermore, it would provide evidence that changes in peripheral metabolism are recognized by the brain and result in compensatory efferent responses to maintain glucose homeostasis. The relationship between hyperglycemia and vagal efferent activity would be comparable to the prototypic feedback loop represented by leptin, which circulates peripherally, acts on specific areas within the central nervous system, and ultimately influences sympathetic nervous system activity (17, 18, 19). However, to date, there are no human data specifically demonstrating that elevations in blood glucose and/or insulin can alter neural efferent pathways that subsequently influence peripheral metabolism.

The objective of the present experiment was to determine whether in humans, prolonged hyperglycemia within the range of mildly impaired glucose tolerance could elicit a vagal contribution to insulin secretion and sensitivity. To address this objective, we conducted a within-subject experiment in which each individual first underwent a frequently sampled iv glucose tolerance test (FSIGT) to establish their insulin sensitivity under basal conditions. Subjects then underwent four experimental conditions, involving either 48-h glucose infusions or 48-h saline infusions. Subsequently, the FSIGT was conducted in the presence of saline or atropine. We found that elevations of plasma glucose within the range of impaired glucose tolerance induced a vagally mediated increase in C-peptide secretion.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

After a telephone interview to assess eligibility, subjects underwent screening for body mass index (BMI) and percent body fat and were then scheduled for a physical examination, including an electrocardiogram and a medical history to ensure they had no chronic illness, abnormal heart rhythms, hypertension, or family history of diabetes or obesity. A blood sample was taken after an overnight fast to evaluate clinical blood chemistries. Subjects whose fasting blood glucose was more than 90 mg/dl or whose blood pressure was more than 140/90 mm Hg were excluded from the study. Eleven normal weight male subjects, 21–34 yr of age (mean, 26.0 ± 4.9 yr), with BMIs ranging from 22.2–24.5 kg/m2 (mean, 23.6 ± 0.8) and percent lean body mass ranging from 13–18% (mean, 16.1 ± 1.8), completed the experiment and comprised the dataset shown. An additional three subjects did not complete the study and were not included in the analysis. These studies were approved by the committee on studies involving human beings at University of Pennsylvania.

Experimental design

Each subject underwent five experimental trials. The first trial consisted of a FSIGT conducted after an overnight fast. The purpose of this trial was to establish baseline values for all measured variables derived from the FSIGT under experimental conditions comparable to those in previous studies (10, 20, 21). The other four experimental trials were conducted over a 4-month period, administered once per month in a random order. The four trials consisted of 1) a 48-h saline infusion, followed by an FSIGT administered during saline infusion (48-h saline:FSIGT/saline; i.e. saline-infused, saline-treated); 2) a 48-h saline infusion, followed by an FSIGT administered during atropine infusion (48-h saline:FSIGT/atropine; i.e. saline-infused, atropine-treated); 3) a 48-h glucose infusion, followed by a FSIGT administered during saline infusion (48-h glucose:FSIGT/saline; i.e. glucose-infused, saline-treated); and 4) a 48-h glucose infusion, followed by an FSIGT administered during atropine infusion (48-h glucose:FSIGT/atropine; i.e. glucose-infused, atropine-treated). Trials 1 and 2 were included to control for the effect of the saline infusion and to verify that under nonhyperglycemic conditions, muscarinic blockade with atropine has no effect on insulin secretion in response to iv glucose. Trial 3 permitted evaluation of the effect of the glucose infusion on insulin and C-peptide release, whereas trial 4 allowed determination of the neural contribution to insulin secretion after glucose infusion by examining the effects of atropine on parameters determined by the FSIGT. The FSIGTs were initiated 3 h after termination of the 48-h infusions.

Before each experimental condition, subjects arrived at the General Clinical Research Center of the Hospital of University of Pennsylvania at 1700 h. Subjects were given dinner (1800 h) and a snack (2000 h), after which they remained fasting until the following morning. The caloric content of the dinner was determined based on the Harris-Benedict equation, which takes into account body weight and height. Basal energy expenditure is calculated and multiplied by an activity factor of 1.3 to determine the total number of calories required per day. Fifty percent of the total number of calories per day was assumed to be required for dinner. The dinner meal was composed of 50% carbohydrate, 20% protein, and 30% fat.

48-h infusion protocols

At 0600 h on the morning after admission, an iv infusion of either saline (0.9% saline at 50 ml/h) or glucose (15% dextrose solution at a rate of 200 mg/m2·min; for a 70-kg man, the flow rate would be 144 ml/h) was initiated and sustained for a 48-h period. Glucose infusions were supplemented with 8 mEq/liter potassium phosphate to prevent hyperglycemia-induced hypokalemia. Saline was infused at a lower rate to avoid effects of saline on insulin sensitivity (Townsend, R. R., unpublished observations). Blood samples were taken every 2 h. During the infusion periods, subjects were permitted to eat three meals and a snack per day ad libitum. Subjects were permitted to walk around the General Clinical Research Center, but were not allowed to leave the unit. At the end of the 48-h period (0600 h on the second day), the infusion was terminated. Three hours after the termination of the infusion, at 0900 h, an FSIGT was conducted during either saline or atropine infusion.

FSIGT

The FSIGT was conducted as previously described (22, 23), except a third catheter was inserted for the infusion of either saline or atropine. The catheters were kept patent by a slow infusion of saline. A three-way stopcock allowed for blood sampling and flushing. Subjects sat quietly for 30 min to acclimatize to the insertion of the catheters before initiation of the FSIGT. Briefly, four baseline samples were collected at 5-min intervals (–15, –10, –5, and –1 min). At time zero, a bolus of 50% glucose (0.3 g/kg) was injected over a 1-min period. At 20 min, insulin was injected as an iv bolus (0.03 U/kg). Blood samples were collected at 2, 3, 4, 5, 6, 8, 10, 14, 19, 22, 25, 30, 40, 50, 70, 100, 140, and 180 min after the injection of glucose. Each blood collection involved 1 ml withdrawn and discarded as waste. A 5-ml sample was then collected into a Vacutainer (BD Vacutainer, Franklin Lakes, NJ) with EDTA. Trasylol and leupeptin, proteinase inhibitors, were added immediately, and the sample was placed on ice. After each sample was obtained, the catheter was flushed with saline to keep the line patent. Samples were centrifuged, and the serum was aliquoted and frozen at –80 C within 1 h of collection.

Atropine was infused iv as a 0.4 mg/m2 bolus, followed by 0.4 mg/m2·h starting 30 min before initiation of the FSIGT and for the duration of the blood sampling (180 min). We found that this dose inhibits the effects of parasympathetic activity, as indicated by a total suppression of plasma pancreatic polypeptide levels after food ingestion (24).

Biochemical analysis

Plasma immunoreactive insulin and C-peptide were measured in duplicate by a double-antibody RIAs. All antibodies were purchased from Linco Research, Inc. (St. Charles, MO). The insulin antibody used is the human-specific antibody from Linco Research, Inc., with no cross-reactivity (<0.2%) to human proinsulin or the primary circulating split form. Analyses of insulin and C-peptide were performed by the Diabetes Research Center of University of Pennsylvania. The interassay coefficient of variation for insulin was 8%, and the intraassay variation was 6%. Plasma glucose was analyzed by a YSI automated glucose/lactate monitor (YSI, Inc., Yellow Springs, OH) at the Monell Chemical Senses Center.

Statistical analysis

The area under the curve (AUC) was calculated as an estimate of the combined consequence of hormonal secretion and degradation. AUC was determined by first calculating the integrated hormone area over baseline. This was done by subtracting the mean baseline value (first four blood samples) from plasma insulin and C-peptide concentrations at each time point. First phase insulin release was determined as insulin released between 2 and 10 min after glucose administration. Later phase insulin release was estimated by insulin released between 10 and 19 min after glucose administration. These data were plotted, and the area under the curve was calculated by a point to point method using computer software (Origin Lab Corp., Northampton, MA). C-peptide AUCs were calculated for the same time periods. Multiple ANOVAs were performed to determine statistically significant differences between treatments. Post hoc tests were conducted using Tukey’s t test. Data shown are the mean ± SD for a total of 11 subjects. Data from one subject were statistically omitted from the AUC analysis because their C-peptide levels dropped below baseline, resulting in negative areas.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effects of 48-h glucose and saline infusions on circulating fuels and hormone levels during infusion periods

The prolonged glucose infusion resulted in a 20% increase in plasma glucose over the 48-h period compared with the saline infusion. Mean glucose and insulin levels over the 48-h period are shown in Table 1Go. The increases in glucose were modest despite the presence of continued glucose infusion, because of the doubling of plasma insulin levels. Plasma insulin levels were increased 2-fold during the glucose infusion compared with those during the saline infusion. Three hours after termination of the infusion and before initiation of the FSIGT in the presence of either atropine or saline, plasma glucose levels were slightly elevated above baseline, but were within the normal range. During the same period, plasma insulin levels remained slightly elevated and within the normal range (Table 1Go).


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  		TABLE 1. Mean plasma insulin and glucose levels during 48-h infusions and 3-h after termination of the infusion before FSIGT

 
Effect of prolonged mild hyperglycemia on C-peptide levels during the FSIGT in the presence and absence of muscarinic blockade

After the 48-h saline infusions, plasma C-peptide levels during the FSIGT were not significantly different from those on the control day at any time period measured regardless of whether the FSIGT was conducted in the presence or absence of the muscarinic antagonist, atropine (Table 2Go). These results indicate that there is not a neurally mediated component of insulin release in response to acute iv glucose when the B cell is not chronically activated (Figs. 1Go and 2Go).


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  		TABLE 2. C-Peptide, insulin, and glucose area under the curves during five experimental conditions

 


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  		FIG. 1. Mean plasma C-peptide levels (mean ± SD; n = 11) during the FSIGT conducted in the presence or absence of the muscarinic antagonist, atropine after either saline (50 ml/hour) or 50% dextrose (200 mg/m2·min) infusions for a 48-h period. Treatment effect: F(3,9) = 4.99; P < 0.026. There were four experimental trials: 48-h Sal:FSIGT/Sal, 48-h saline infusion, followed 3 h later by FSIGT conducted in the presence of saline (Sal); 48-h Sal: FSIGT/Atr, 48-h saline infusion, followed 3 h later by FSIGT conducted in the presence of atropine (Atr); 48-h Glu:FSIGT/Sal, 48-h glucose infusion, followed 3 h later by FSIGT conducted in the presence of saline (Sal); and 48-h Glu:FSIGT/Atr, 48-h glucose infusion, followed 3 h later by FSIGT conducted in the presence of atropine (Atr).

 


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  		FIG. 2. A, C-Peptide AUC (nanograms per milliliter per 40 min) during the first 40 min of the FSIGT conducted in the presence or absence of the muscarinic antagonist, atropine (Atr), after either saline (50 ml/h) or 50% dextrose (200 mg/m2·min) infusion for a 48-h period. Treatment effect: F(4,40) = 5.2; P < 0.002. *, Tukey’s post hoc test determined that 48 h-G:F/Sal is significantly different from the control day (P < 0.007), 48 h-S:F/Sal (P < 0.03), 48 h-S:F/Atr (P < 0.002), and 48 h-G:F/Atr (P < 0.05). There were five experimental trials: control, FSIGT screening; 48 h-S: F/Sal, 48-h saline infusion, followed 3 h later by FSIGT conducted in the presence of saline (Sal); 48 h-S:F/Atr, 48-h saline infusion, followed 3 h later by FSIGT conducted in the presence of atropine (Atr); 48 h-G:F/Sal, 48-h glucose infusion, followed 3 h later by FSIGT conducted in the presence of saline (Sal); 48 h-G:F/Atr, 48-h glucose infusion, followed 3 h later by FSIGT conducted in the presence of atropine (Atr). B, C-Peptide AUC (nanograms per milliliter per 21 min) during the period of exogenous insulin administration (19–40 min) of the FSIGT conducted in the presence or absence of the muscarinic antagonist, atropine (Atr), after either saline (50 ml/h) or 50% dextrose (200 mg/m2·min) infusions for a 48-h period. Treatment effect: F(4,40) = 4.1; P < 0.007. *, Tukey’s post hoc test determines that 48 h-G:F/Sal is significantly different from 48 h-S:F/Atr (P < 0.005) and 48 h-G:F/Atr (P < 0.025). No significant differences were found between the other experimental conditions.

 
After the 48-h glucose infusion, plasma C-peptide levels during the FSIGT (saline-treated) were significantly elevated compared with those in the controls, the saline-infused, saline-treated group, as well as the saline-infused, atropine-treated group (Fig. 1Go). To control for elevated basal levels of C-peptide after the glucose infusion, AUCs were compared.

The C-peptide AUC during the FSIGT after glucose infusion was significant elevated during the periods of endogenous insulin secretion (2–19 min; 39.2 ± 11.9 vs. 24.3 ± 8.7 ng/ml·19 min; P < 0.01; Table 2Go) and exogenous insulin administration (19–40 min; 31.8 ± 13.8 vs. 20.5 ± 6.8 ng/ml·21 min; P < 0.03) compared with the same time periods during 48-h saline infusion (Fig. 2Go).

When the FSIGT was conducted in the presence of atropine after the 48-h glucose infusion, a significant attenuation [treatment effect, F(3,9) = 4.99; P < 0.026] in plasma C-peptide levels was observed compared with the glucose-infused, saline-treated levels (Fig. 1Go). Atropine administration inhibited C-peptide secretion to the extent that the C-peptide AUC for the total 40 min period was significantly decreased compared with the glucose-infused, saline-treated levels (P < 0.03) and was not significantly different from the control or saline-infused level (Fig. 2AGo). Similarly, during the period of exogenous insulin administration (Fig. 2BGo), atropine significantly inhibited the increase in C-peptide AUC observed after the glucose infusion (P < 0.05). During the period of endogenous insulin release, atropine had no significant effect on C-peptide AUC. In the 2–10 min and 10–19 min periods of endogenous insulin secretion, C-peptide AUCs in the glucose-infused, atropine-treated condition were not significantly different from either the control values or the saline-infused, saline-treated levels, nor were they significantly different compared with the glucose-infused, saline-treated values (Table 2Go).

Effects of prolonged mild hyperglycemia on insulin and glucose levels during the FSIGT in the presence and absence of muscarinic blockade

After the 48-h saline infusions, plasma insulin levels during the FSIGT were not significantly different from those on the control day regardless of whether the FSIGT was conducted in the presence or absence of the muscarinic antagonist, atropine (Table 2Go). During the FSIGT after the 48-h glucose infusion, the acute insulin response was significantly increased (Table 2Go), indicating an increase in B cell function (AUC, 2–10 min: glucose, 630.6 ± 452.4 µU/ml·10 min; saline, 305.7 ± 159.3 µU/ml·10 min; P < 0.001). The insulin AUC during the total period of endogenous insulin release (2–19 min) was also significantly elevated compared with that after the saline-infused, saline-treated condition (glucose, 972.6 ± 667 µU/ml·19 min; saline, 437.8 ± 212.7 µU/ml·19 min; P < 0.001) and that on the control day. However, no significant effect of the 48-h glucose infusion was observed on insulin AUC during the total 40-min period (Fig. 3AGo). After exogenous insulin administration (19–40 min) in the glucose-infused, saline-treated condition, the insulin AUC was not significantly different from controls and, in fact, exhibited a trend to a decrease (glucose, 1294.4 ± 670.2 µU/ml·21 min; saline, 1642.2 ± 994.6 µU/ml·21 min; Fig. 3BGo). Atropine administration during the FSIGT after the 48-h glucose infusion had no significant effect on insulin levels at any of the time periods compared with when the FSIGT was conducted in the absence of muscarinic blockade (Fig. 3Go).



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  		FIG. 3. A, Insulin AUC (nanograms per milliliter per 40 min) during the first 40 min of the FSIGT conducted in the presence or absence of the muscarinic antagonist, atropine (Atr), after either saline (50 ml/h) or 50% dextrose (200 mg/m2·min) infusion for a 48-h period. No significant differences were found between treatments. There were five experimental trials: control, FSIGT screening; 48 h-S:F/Sal, 48-h saline infusion, followed 3 h later by FSIGT conducted in the presence of saline (Sal); 48 h-S:F/Atr, 48-h saline infusion, followed 3 h later by FSIGT conducted in the presence of atropine (Atr); 48 h-G:F/Sal, 48-h glucose infusion, followed 3 h later by FSIGT conducted in the presence of saline (Sal); and 48 h-G:F/Atr, 48-h glucose infusion, followed 3 h later by FSIGT conducted in the presence of atropine (Atr). B, Insulin AUC (nanograms per milliliter per 21 min) during the period of exogenous insulin administration (19–40 min) of the FSIGT conducted in the presence or absence of the muscarinic antagonist, atropine (Atr), after either saline (50 ml/h) or 50% dextrose (200 mg/m2·min) infusion for a 48-h period. No significant differences were found between treatments.

 
No significant differences were found in plasma glucose levels or glucose area under the curves in any of the experimental conditions during the period of endogenous insulin secretion (Table 2Go). Similarly, during the period of exogenous insulin administration (19–40 min), glucose AUCs were not significantly altered (control day, 1020.2 ± 421.7; 48-h saline, FSIGT/saline, 1135.6 ± 396.1; 48-h saline, FSIGT/atropine, 914.8 ± 459.4; 48-h glucose, FSIGT/saline, 342.6 ± 269.7; 48-h glucose, FSIGT/atropine, 513.1 ± 628.7). However, a significant number of subjects (seven of 11) became hypoglycemic under the glucose-infused, atropine-treated condition. In comparison, no hypoglycemia was observed during the control day and the saline-infused, saline-treated condition, whereas two subjects became hypoglycemic during the saline-infused, atropine-treated and glucose-infused, saline-treated conditions. Bedside glucose monitoring permitted rapid identification of hypoglycemia, at which point the FSIGT was terminated, and the subject was given glucose tablets. Due to the termination of the FSIGT, typically at 50 min, data and graphs illustrate full datasets (n = 11) up to 45 min postglucose administration. Because of the foreshortened sampling period in the glucose-infused conditions, the data could not be modeled, and insulin sensitivity (SI) and glucose effectiveness (SG) could not be generated.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present study investigated the effects of a 48-h glucose infusion on the vagal efferent contribution to C-peptide secretion in normal weight men. Only a handful of studies have explored the effects of prolonged mild hyperglycemia in humans (8, 9, 16), and to date, none has examined the role of the parasympathetic nervous system as a potential mechanism in compensatory B cell responses to a chronic glucose challenge. Our data suggest that the previously reported increase in C-peptide secretion that occurs after experimentally induced prolonged mild hyperglycemia is partially mediated by vagal efferent activity. Activation of vagal efferent activity by prolonged mild hyperglycemia may contribute to adaptive B cell responses and could potentially provide a mechanism for individuals to compensate for insulin resistance.

Evidence for a vagally mediated increase in C-peptide secretion after prolonged stimulation of the B cell is provided by the significant attenuation of circulating C-peptide levels by muscarinic blockade [Fig. 1Go; F(3,9) = 4.99; P < 0.026]. Neural mediation of C-peptide secretion was only observed after the 48-h glucose infusion because atropine had no effect on C-peptide under other conditions (48-h saline infusions or control day), verifying previous reports that there is no neurally mediated component to insulin release after acute iv glucose. Muscarinic blockade attenuated the increase in C-peptide after prolonged glucose infusion, but did not completely inhibit the response, suggesting that induction of vagal efferent activity is only one of multiple compensatory mechanisms contributing to C-peptide secretion when the B cell is chronically challenged.

The majority of subjects exhibited a decrease in C-peptide AUC during atropine administration (Table 3Go). However, a wide range of individual responses to muscarinic blockade was observed, reflecting the known high variability in measures of autonomic activity (25, 26). The magnitude of inhibition of C-peptide ranged from less than 1 to 105%, with a mean of 16%, and was proportional to the amount of C-peptide released in response to the 48-h glucose infusion. In fact, a significant correlation was found between C-peptide release after the glucose infusion and the magnitude of inhibition by atropine (r = –0.88; P < 0.001; Fig. 4Go). Thus, the greater the C-peptide response to the glucose infusion, the greater the vagal contribution to C-peptide secretion. These data suggest that the ability of an individual to increase C-peptide secretion in response to sustained stimulation of the B cell is related to the individual’s ability to mount a vagally mediated response.


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  		TABLE 3. Individual C-peptide AUC (nanograms per milliliter per 40 min) response to glucose infusion (n = 10)

 


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  		FIG. 4. Correlation between the C-peptide AUC during the FSIGT after the 48-h glucose infusion (x-axis) and the percent inhibition of C-peptide by the muscarinic antagonist, atropine (y-axis), expressed as the difference in C-peptide AUC during the FSIGT conducted in the presence of saline minus C-peptide AUC during the FSIGT conducted in the presence of atropine, both after the 48-h glucose infusion (r = –0.88; P < 0.001; n = 10). One subject was removed from the data due to being a statistical outlier.

 
Individual vagal responsivity to prolonged stimulation of the B cell may account for why some obese individuals maintain insulin resistance for extended periods of time while others become diabetic. Evidence for increased parasympathetic activity in human obesity is limited (24, 27), although most animal models of obesity exhibit increased vagal efferent activity at the level of the pancreas (28, 29, 30, 31, 32, 33, 34). Increased levels of pancreatic polypeptide, a hormone released from the F cells of the pancreas and whose release is thought to be dependent on vagal activation, have been reported in obese Pima Indians (35). Recently, it was shown that the vagal contribution to the elevated levels of pancreatic polypeptide was not proportional to the vagal contribution to insulin release (36). However, it should be noted that comparing the vagal contribution to insulin release in a population that has been chronically obese since childhood with the vagal contribution to C-peptide secretion during a relatively short manipulation, such as that in the present study, is very different, particularly in light of the plasticity of the central and peripheral nervous systems. The induced increase in vagally mediated C-peptide secretion observed in our study may be an acute response, which may actually down-regulate with chronic B cell stimulation, such as occurs during a hypercaloric or chronically obese state.

As reported by others (8, 9, 16), we found that the 48-h glucose infusion significantly increased insulin release during both the acute phase (2–10 min) and the later phase of endogenous insulin secretion (10–20 min) compared with the 48-h saline infusion condition. However, when comparing insulin AUCs over the entire 40-min period, no significant effect of the glucose infusion was observed. This was because during the period of exogenous insulin administration (20–40 min), the 48-h glucose infusion resulted in a nonsignificant decrease (21%) in insulin AUC relative to the saline infusion condition (Fig. 3Go). In contrast, C-peptide levels remained relatively constant during periods of both endogenous insulin secretion and exogenous insulin administration (61% and 55%, respectively, increased compared with controls). Thus, circulating peripheral insulin levels decreased despite a sustained increase in insulin secretion.

In contrast to the attenuation in C-peptide levels observed with muscarinic blockade after the 48-h glucose infusion, atropine had no significant effect on insulin AUC during the period of endogenous insulin secretion or exogenous insulin administration. Atropine did appear to prevent the decline in insulin AUC (1917.0 ± 787.0 µU/ml·21 min) during the period of exogenous insulin administration that was observed after the glucose-infused, saline-treated condition (glucose, 1294.4 ± 670.2 µU/ml·21 min; saline, 1664.2 ± 794.6). We postulate that the discrepant effects of muscarinic blockade on insulin and C-peptide point toward a vagal mediation of hepatic insulin degradation. Vagal mediation of hepatic insulin degradation has previously been demonstrated in dogs by Chap et al. (37), but has not been examined in humans. We propose that the prolonged glucose infusion resulted in a vagally mediated increase in insulin secretion and hepatic insulin degradation. Administration of the muscarinic antagonist partially inhibited insulin release as well as the rate of insulin degradation. This resulted in sustained levels of circulating insulin that not only masked the effect of atropine on insulin release, but resulted in the hypoglycemia observed in the subjects. Additional studies will have to be conducted to directly address this hypothesis.

One of the questions raised, and not addressed by the present study, concerns the circulating signal responsible for the activation of vagal efferent activity. Glucose (38), insulin (39), leptin (40), and free fatty acids (41) are recognized by the central nervous system, and evidence exists supporting each as a potential signal to alter central nervous system neuronal activity and neuropeptide levels (42). In the present study both glucose and insulin were elevated and therefore were potential direct mediators of the effects observed. Circulating free fatty acids have also been shown to alter central nervous system neuronal firing (41) and decrease vagal firing (43). However, in the present study free fatty acids were suppressed by the 48-h glucose infusion (data not shown) and therefore are unlikely candidates for the circulating signal responsible for the change in vagal efferent activity.

In summary, we found that prolonged mild hyperglycemia results in a compensatory increase in C-peptide secretion, which is partially mediated by an induction in vagal efferent activity. The induced changes in vagal efferent activity as a result of chronically elevated glucose and insulin levels comprise a feedback loop, analogous to the relationship existing between leptin and the sympathetic nervous system. Few studies, particularly those conducted in humans, have examined the relationship between changes in peripheral metabolism and the parasympathetic nervous system. Additional investigation is required into this potentially important regulatory component of glucose homeostasis.


   Acknowledgments
 
We acknowledge the help of the nurses of the General Clinical Research Center of University of Pennsylvania, and the expert technical assistance of Dr. Heather Collins at the Diabetes Research Center of University of Pennsylvania. In addition, we thank Drs. Raymond Boston and Maja Petrova for their help in modeling the data.


   Footnotes
 
This work was supported by Grants DK-58003-05 (to K.L.T.), DK-19525, and M01-RR-00042.

Abbreviations: AUC, Area under the curve; BMI, body mass index; FSIGT, frequently sampled iv tolerance test.

Received December 5, 2003.

Accepted July 20, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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