1 Clinical Diabetes and Nutrition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona
2 Ophthalmology Department, Phoenix Indian Medical Center, Phoenix, Arizona
3 Department of Nutrition, University of California-Davis, Davis, California
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ABSTRACT |
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The Pima Indians of Arizona have one of the highest reported prevalence rates of obesity and type 2 diabetes in the world (1). Compared with Caucasians, Pima Indians have, on average, lower insulin sensitivity and higher plasma insulin concentrations, both fasting and in response to intravenous and oral glucose loads (2). Prospectively, fasting hyperinsulinemia predicts the development of diabetes in both Pima Indians (35) and other populations (6,7). The hyperinsulinemia in Pima Indians appears to be an early characteristic because Pima Indian children have 5070% higher fasting insulin concentrations than Caucasian children (8).
The precise mechanisms contributing to hyperinsulinemia in Pima Indians remain unknown. In part, the higher fasting insulin concentrations may be a secondary adaptation of the ß-cell to compensate for the high degree of insulin resistance. However, the latter does not appear to entirely account for the hyperinsulinemia (2).
Hyperinsulinemia is also an early feature of various animal models of both genetic and hypothalamic obesity and type 2 diabetes, such as the ob/ob mouse (9), the fa/fa rat (10), and rodents with lesions of the ventromedial hypothalamus (11,12). In these animals, the hyperinsulinemia is thought to be due in large part to an exaggerated parasympathetic drive to the pancreatic ß-cells because acute administration of parasympathicolytic drugs such as atropine (a blocker of muscarinic receptors) (911) or truncal vagotomy (12,13) can almost completely normalize insulin levels.
Compared with Caucasians, Pima Indians have been reported to have higher plasma concentrations of pancreatic polypeptide (PP) both in the fasting state and especially in response to a meal (14,15). PP is a peptide hormone produced by the pancreatic F-cells, the secretion of which is largely under vagal control (16). In response to a meal, plasma PP concentrations increase, in parallel to an increase in plasma insulin concentrations (15,17). Vagotomy (17) or administration of parasympatholytic drugs such as atropine completely abolish the postprandial increase in PP in humans (18,19). Conversely, electrical stimulation of the vagus nerve or administration of parasympathicomimetic drugs such as pyridostigmin (a cholinesterase inhibitor) increase PP secretion (19,20). Based on these findings, it is generally agreed that the plasma PP concentration represents a valid surrogate marker of the parasympathetic drive to the pancreas (16).
Based on these findings, we hypothesized that an increased parasympathetic drive to the pancreas might contribute to hyperinsulinemia in Pima Indians. To test this hypothesis, we examined the effect of graded doses of the parasympathetic nervous system (PNS) blocker atropine on postprandial PP and insulin secretory responses in 17 Caucasian and 17 Pima Indian men with normal glucose tolerance.
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RESEARCH DESIGN AND METHODS |
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Methods and experimental protocol.
Subjects were admitted for 810 days to the National Institutes of Health Clinical Research Unit in Phoenix, AZ, were fed a weight-maintaining diet (50, 30, and 20% of daily calories provided as carbohydrate, fat, and protein, respectively), and abstained from strenuous exercise.
Body composition was estimated by total body dual-energy X-ray absorptiometry (DPX-L; Lunar Radiation, Madison, WI) with calculations of percentage of body fat, fat mass, and fat-free mass (FFM) as previously described (22).
At least 3 days after admission and after a 12-h overnight fast, subjects underwent a 2-h 75-g oral glucose tolerance test. Plasma glucose concentrations were determined by the glucose oxidase method (Beckman Instruments, Fullerton, CA) and plasma insulin concentrations by an automated immunoassay (Access; Beckman Instruments).
On day 3, before administration of atropine, subjects underwent a series of four standard tests for the assessment of cardiovascular autonomic nervous system activity (23). During these tests, subjects were in a sitting position and blood pressure and electrocardiogram were monitored continuously. In the first two tests, which assessed PNS activity, the change in heart rate was assessed in response to deep breathing (the difference between minimum and maximum heart rate during six deep breathing cycles per min) and a Valsalva maneuver (the ratio of the longest R-R interval after the maneuver to the shortest R-R interval during the maneuver). In the third and fourth tests, which assessed sympathetic nervous system activity, the change in blood pressure was measured in response to standing up (the difference between the lowest systolic pressure in the first 2 min after standing up and the average systolic pressure while lying down) and in response to a hand-grip test (using a dynamometer, 30% of maximum voluntary contraction over 5 min).
On 4 separate days after a 12-h overnight fast, subjects were fed a standardized liquid meal (Ensure Plus, 20% of daily caloric requirements [64% carbohydrate, 22% fat, and 14% protein, identical on all 4 days]) (Fig. 1). The room was maintained at mesopic illumination levels, and the volunteer was instructed to abstain from near-centered visual tasks. During each test, two intravenous catheters were placed in an antecubital vein on the left and right arm, one for the saline/atropine infusion and one for blood sampling. After a 30-min baseline period, a primed-continuous infusion of the study medication was started and maintained for 150 min. To avoid the risk of untoward effects of high-dose atropine in hypersensitive individuals, infusions were not randomized. The infusions contained saline on the first occasion, always followed by three increasing doses of atropine. Atropine was administered in doses of 2.5, 5, or 10 µg/kg FFM for priming (injected over 2 min), followed by a continuous infusion at doses of 2.5, 5, or 10 µg · kg FFM-1 · h-1, respectively. At 0 min, subjects ingested the standardized liquid meal within 5 min. Since atropine was shown to affect gastrointestinal motility, we estimated the rate of gastric emptying by use of the acetaminophen absorption test (24,25). For this purpose, the liquid meal contained 20 mg/kg of acetaminophen, the appearance of which in the plasma was determined at subsequent time points (-60, 30, 60, and 120 min). Blood pressure, heart rate, and electrocardiogram were monitored continuously throughout the experiment and documented at the same time points as the blood samples, and they were collected to measure PP, insulin, and glucose (-60, -45, -30, -20, 0, 5, 10, 15, 20, 30, 45, 60, and 120 min). Plasma PP concentrations were determined by a radioimmunoassay (Linco, St. Charles, MO). The within and between errors in this assay were 7 and 10%, respectively. Immediately before, immediately after, and 30 min after injection of the priming dose (2 min) of saline or atropine, ocular responses were examined by a pupillometer (Iscan, Burlington, MA), which allowed us to measure the pupil area and the near point of accommodation.
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Racial differences in autonomic nervous system tests were assessed by unpaired t test. ANOVA for repeated measures was used to assess the effect of race and intervention (to exclude possible carryover effects of consecutive atropine studies) on fasting plasma insulin, glucose, and PP concentrations at baseline (-60 to -30 min of the study) and during infusion of atropine (-30 to 0 min).
Areas under the curve (AUC) for early (AUC030 min) and total (AUC0120 min) plasma insulin, glucose, PP, and acetaminophen were calculated using the trapezoid method. To account for differences in peripheral glucose uptake and any residual effect of gastric emptying rate not captured by the acetaminophen test and racial differences in percentage of body fat, all time points for insulin and glucose of all four atropine studies were used to adjust plasma insulin for plasma glucose. AUCs for early (AUC030 min) and total (AUC0120 min) adjusted plasma insulin secretory responses were then calculated. ANOVA for repeated measures was used to assess the effects of race and intervention and the interaction between race x intervention on secretory responses for plasma insulin, glucose, PP, and acetaminophen. The race effect within each atropine study was assessed post hoc by unpaired t test. The effect of atropine on blood pressure, heart rate, pupillometry, and near point of accommodation was similarly assessed by ANOVA.
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RESULTS |
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Fasting plasma insulin and PP concentrations decreased (both P < 0.0001 for intervention effect), whereas fasting plasma glucose concentration did not change with increasing dose of atropine (P > 0.5). Fasting plasma insulin, PP, and glucose concentrations during the atropine infusions but before consumption of the meal did not differ between Pima Indians and Caucasians (all P > 0.05, for race effect), and there were no differences in response to atropine between Pima Indians and Caucasians (P = 0.9 for insulin, P = 0.06 for PP, and P = 0.5 for glucose and for race x intervention effect).
Postprandial changes of plasma insulin, glucose, PP, and acetaminophen.
The time course of the change of plasma PP, insulin, and glucose in response to the meal at all doses of atropine for both Pima Indians and Caucasians is shown in Fig. 2.
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Early postprandial adjusted insulin and PP secretory responses (AUC030 min) were 2.5-fold and 2-fold higher in Pima Indians compared with Caucasians, respectively (Fig. 4). Secretion of adjusted insulin and PP was dose-dependently inhibited by atropine (Fig. 4). Increasing doses of atropine attenuated the ethnic difference in PP but not in adjusted insulin responses (Fig. 4).
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Systolic and diastolic blood pressure AUCs (AUC0120 min) in response to meal ingestion did not change with increasing doses of atropine and did not differ between Pima Indians and Caucasians, and there was no difference in response to atropine between Pima Indians and Caucasians (P = 0.9.). Heart rate AUC in response to the meal ingestion (AUC 0120 min) increased with increasing dose of atropine (P = 0.001) and was higher in Pima Indians than in Caucasians (P = 0.02), and there was a greater response to atropine in Caucasians than in Pima Indians (P = 0.04). There was no effect of race or intervention on the pupil size or near point of accommodation (all P > 0.1).
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DISCUSSION |
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What is the significance of hyperinsulinemia in Pima Indians? We have shown that the variability in fasting plasma insulin concentrations is only partially (55%) explained by the difference in insulin sensitivity (2) and that "relative" hyperinsulinemia in Pima Indians with normal glucose tolerance is an independent predictor of type 2 diabetes, probably due to a deleterious effect on insulin secretion (5). Hyperinsulinemia is also an independent risk factor for cardiovascular disease (26) and may increase the risk of Alzheimers disease (27).
Consistent with previous reports (14), we found that Pima Indians had larger postprandial plasma PP responses than Caucasians. As expected, increasing doses of atropine led to a dose-dependent reduction of the postprandial increase of PP. Finally, the highest dose of atropine completely abolished the postprandial increase of PP, an effect that was observed in previous studies (1719,2830) of other populations. Unlike previous reports, we did not observe higher fasting plasma PP levels in Pima Indians than in Caucasians. This may be due to the relatively small sample size in the present study. Interestingly, muscarinic blockade tended to produce a more marked dose-dependent decrease in fasting plasma PP levels in Pima Indians than in Caucasians.
Hyperinsulinemia in Pima Indians is most pronounced during the first 30 min of a meal (2). In the present study, we showed higher early postprandial insulin secretion and a trend for higher total postprandial insulin secretion in Pima Indians compared with Caucasians. Both early postprandial insulin and total postprandial insulin secretion were inhibited by increasing doses of atropine. The significant contribution of the PNS to insulin secretion in response to meal ingestion has been demonstrated in both animals (28,30) and humans (29,31). Despite the ethnic difference, insulin secretion, and the decrease in plasma insulin response with increasing doses of atropine, there was no ethnic difference in the ability of cholinergic blockade to reduce postprandial insulin secretion. Thus, while the lowest dose of atropine normalized the PP response in Pima Indians to the magnitude measured during saline administration in Caucasians, this same dose did not appreciably reduce the difference in insulin secretion between Pima Indians and Caucasians. Furthermore, with PP completely suppressed at the highest dose of atropine, insulin secretion was still present and was still significantly higher in the Pima Indians than in Caucasians. Taken together, these data suggest that PNS contributes to an increase of plasma insulin secretion in response to a meal; however, the enhanced insulin secretion in adult Pima Indians is not due to an exaggerated PNS stimulation of the ß-cells.
There are some limitations to our study. First, we did not achieve a complete blockade of PNS activation by atropine, which antagonizes only cholinergic muscarinic receptors but not the release or actions of other PNS-signaling neuropeptides (26). However, it was shown that nicotinic blockade provides only a further 9% decrease in insulin secretion compared with muscarinic blockade (28). A second limitation is that we did not have a more accurate measure of insulin sensitivity (i.e., glucose uptake measured by euglycemic clamp) available in our study than glucose and percentage of body fat to adjust plasma insulin and thereby account for ethnic differences in insulin sensitivity. It could be argued that Pima Indians did not decrease their postprandial plasma insulin more than Caucasians because they are more insulin resistant. Finally, we do not have estimates of insulin clearance in this study. However, in previous studies insulin clearance was not found to be different in Pima Indians compared with Caucasians (2) and the effect of atropine on C-peptide paralleled the effects on insulin, suggesting that insulin secretion and insulin clearance are not differentially affected by PNS blockade (29).
There are data to suggest that PNS input contributes directly to enhanced insulin secretion of obesity in young animals (obese Zucker rats), but that in older animals with established obesity the primary contributor to hyperinsulinemia is increased ß-cell mass (10). This suggests the possibility of a hyperstimulation of the pancreas by the PNS chronically leading to an increase in ß-cell mass that may no longer be acutely affected by pharmacological blockade once established. To our knowledge, no studies have addressed this possibility.
If an increased PNS drive to the pancreas does not explain the hyperinsulinemia of Pima Indians then the question arises of what other hormones might contribute to this ethnic difference. In this respect, it should be pointed out that incretins (glucagon-like peptide 1 and gastric inhibitory peptide) and catecholamines play an important role in the modulation of postprandial insulin secretion (2831). Other possible etiologic factors contributing to hyperinsulinemia in Pima Indians may include downregulation of insulin receptors in pancreatic ß-cells leading to impaired glucose sensing, plasma free fatty acid concentrations, ß-cell insulin receptor expression, metabolic clearance rate of insulin, and abnormalities in central (hypothalamic) regulatory pathways (5). However, the contribution of these mechanisms was not addressed this study.
Despite an ethnic difference in the PNS input to the pancreatic F-cells, there was no ethnic difference in measures of PNS activity to other organs (e.g., stomach, eye, or cardiovascular system). This suggests that PNS activity is not globally upregulated in Pima Indians compared with Caucasians. It is well established that PNS outflow is selective to different tissues and that the input to PNS-innervated organs is discretely activated under differing physiological conditions (32). Therefore, it is not too surprising that activation of the parasympathetic input to pancreatic F-cells can be exaggerated in response to meal ingestion in Pima Indians compared with Caucasians but that PNS activity to other organs or under other conditions is not exaggerated. Furthermore, the results of this study indicating increased cholinergic stimulation to PP-secreting F-cells but not to B-cells in Pima Indians provides evidence that there are likely additional differences in the PNS input to different cell types within an individual organ. Whether this cell-specific difference within the islet is anatomical (i.e., more parasympathetic nerve fibers adjacent to F-cells than ß-cells), or functional, with enhanced F-cell sensitivity to acetylcholine, is not known.
In conclusion, results from this study confirm that compared with Caucasians, Pima Indians have an exaggerated PNS drive to the pancreas and more specifically to the pancreatic F-cells. Thus, the hyperinsulinemia of this population does not appear to be due to increased vagal input to pancreatic ß-cells.
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ACKNOWLEDGMENTS |
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Address correspondence to P. Antonio Tataranni, MD, Clinical Diabetes and Nutrition Section, National Institutes of Health, 4212 N. 16th St., Rm. 5-41, Phoenix, AZ 85016. E-mail: antoniot{at}mail.nih.gov
Received for publication August 13, 2003 and accepted in revised form November 17, 2003
AUC, area under the curve; FFM, fat-free mass; PNS, parasympathetic nervous system; PP, pancreatic polypeptide
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REFERENCES |
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