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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0153
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3646-3652
Copyright © 2005 by The Endocrine Society

The Leu7Pro Polymorphism of PreproNPY Is Associated with Decreased Insulin Secretion, Delayed Ghrelin Suppression, and Increased Cardiovascular Responsiveness to Norepinephrine during Oral Glucose Tolerance Test

Ulriikka Jaakkola, Tom Kuusela, Tuomas Jartti, Ullamari Pesonen, Markku Koulu, Tero Vahlberg and Jaana Kallio

Departments of Pharmacology and Clinical Pharmacology (U.J., U.P., M.K., J.K.), Physics (T.K.), and Biostatistics (T.V.), University of Turku, FI-20520 Turku, Finland; and Department of Ophthalmology (U.J.), Turku City Hospital, FI-20700 Turku, Finland; and Department of Pediatrics (T.J.), Turku University Hospital, FI-20520 Turku, Finland

Address all correspondence and requests for reprints to: Jaana Kallio, M.D., Ph.D., Department of Pharmacology and Clinical Pharmacology, University of Turku, Itäinen Pitkäkatu 4, FI-20520 Turku, Finland. E-mail: jaana.kallio{at}utu.fi.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Neuropeptide Y (NPY) plays a role in angiogenesis, cardiovascular regulation, and hormone secretion. The leucine7 to proline7 (Leu7Pro) polymorphism of preproNPY is associated with vascular diseases and has an impact on hormone levels in healthy subjects.

Objective: The current study investigated the role of the Leu7Pro polymorphism in metabolic and cardiovascular autonomic regulation.

Design and Subjects: A 5-h oral glucose tolerance test was performed on 27 healthy volunteers representing two preproNPY genotypes (Leu7/Pro7 and Leu7/Leu7) matched for age, sex, body mass index and physical activity.

Main Outcome Measures: Simultaneously we performed cardiovascular autonomic function tests and plasma measurements of sympathetic transmitters, glucose, insulin, and ghrelin.

Results: The subjects with Leu7/Pro7 genotype had decreased plasma NPY, norepinephrine (NE), and insulin concentrations and insulin to glucose ratios. The suppression of ghrelin concentrations after glucose ingestion was delayed in these subjects. They also had increased heart rate variability indices and baroreflex sensitivity. However, they displayed significant negative association of NE concentration with variability of low-frequency R-R-intervals and with baroreflex sensitivity.

Conclusions: The Leu7Pro polymorphism of preproNPY is related to decreased level of basal sympathetic activity, decreased insulin secretion, and delayed ghrelin suppression during oral glucose tolerance test. The increased responsiveness of autonomic functions to NE associated with the polymorphism may be connected to increased cardiovascular vulnerability.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDE Y (NPY), a neurotransmitter of peripheral and central nervous systems, has various effects in different physiological functions. It is stored together with norepinephrine (NE) and released from sympathetic nerve endings (1). NPY is an angiogenic factor (2), which also has influence in vasoconstriction (3) and vasodilatation (4), function of kidneys (3), and glucose metabolism (5) as well as secretion and release of many hormones. In experimental animals the effects of NPY on insulin secretion are inhibitory when given peripherally and stimulatory when given centrally (6, 7). In isolated human pancreatic islets, NPY inhibits insulin secretion (8).

The Leucine 7 to Proline 7 (Leu7Pro) polymorphism of preproNPY is rather common in the Finnish and other Caucasian populations in which the carrier frequency of the polymorphism is 7–12%, whereas this polymorphism has not been found in Asian populations (9, 10, 11). The Leu7Pro polymorphism was first reported to associate with increased lipid concentrations in blood, especially in obese subjects (10, 12, 13) and rapid progression of atherosclerosis (12, 14). Later the Leu7Pro polymorphism has also been associated with increased risk for diabetic retinopathy in type 2 diabetes patients (9, 15) and diabetic nephropathy in type 1 diabetes patients (16). Recently, it has been associated also with myocardial infarctation and stroke in patients with hypertension (17). Therefore, it is important to investigate possible mechanisms linking the increased cardiovascular morbidity and the Leu7Pro polymorphism.

There is evidence that the Leu7Pro polymorphism affects metabolic, hormonal, and autonomic functions of young healthy subjects (18, 19, 20). The subjects with Leu7Pro polymorphism (Leu7/Pro7 genotype) have higher glucose and lower insulin concentrations after standard meals, compared with the subjects without this polymorphism (Leu7/Leu7 genotype) (18, 20). The diurnal levels of sympathetic transmitters NPY and NE are decreased during resting conditions in the subjects with Leu7/Pro7 genotype (20), but they have increased NPY release during exercise (18).

We have previously detected that the subjects with Leu7/Pro7 genotype have increased heart rate during daytime, despite decreased NE levels, suggesting that these subjects may have altered cardiovascular autonomic regulation (20). Therefore, the spectral analyses of R-R-interval (RRI; time between successive heartbeats) variability and baroreflex sensitivity are worth evaluating in these subjects. These measures give quantitative data about sympathetic and parasympathetic control of cardiovascular organ functions (21, 22). The spectrum displays relative densities of periodic components of signal variability on different frequency regions: high-frequency (HF) variability at respiratory frequency is related to parasympathetic modulation and low-frequency (LF) variability (10-sec waves) to both parasympathetic and sympathetic modulation. The arterial baroreflex is an important negative feedback system in readjusting acute deviations in blood pressure to its physiological set point (22).

The present study was undertaken to evaluate effects of the Leu7Pro polymorphism of preproNPY on insulin, glucose, and sympathetic transmitter concentrations in blood as well as cardiovascular autonomic regulation during a 5-h oral glucose tolerance test in carefully pair-matched healthy study subjects having either the Leu7/Pro7 or Leu7/Leu7 genotype. We also wanted to study ghrelin concentrations during oral glucose tolerance test because ghrelin is an anabolic hormone that acts partly via NPY system (23) and has also a regulatory interplay with insulin (24).


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

The Ethical Committee of the Hospital District of Southwest Finland approved the study. Written informed consent was received from each study subject for genotyping and the oral glucose tolerance test together with the measurement of autonomic functions. From a cohort of Finnish Caucasian healthy volunteers genotyped for the Leu7Pro polymorphism, 14 subjects with Leu7/Pro7 genotype and 14 control subjects with Leu7/Leu7 genotype [matched by sex, age, body mass index, and physical activity] were chosen for the study. Exclusion criteria were age under 18 or over 40 yr, smoking, drug or alcohol abuse, acute or chronic disease, medication other than oral contraceptives, abnormal laboratory test results, and abnormal clinical signs or symptoms. Before entering the study, all study subjects went through a careful interview and physical examination (cardiac and pulmonal auscultation, blood pressure measurement, abdominal palpation, weight and height measurements) performed by a physician. Also basic laboratory measurements were performed [blood hemoglobin concentration, leukocyte count, erythrocyte sedimentation rate, total cholesterol, low-density lipoprotein and high-density lipoprotein cholesterol concentrations, triglycerides, HbA1c, fasting plasma glucose, alanine transferase and creatinine concentrations] by standard methods in Turku University Hospital. The results of one study subject with Leu7/Pro7 genotype were excluded from the data analyses because she had a pathological 2-h plasma glucose concentration in the oral glucose tolerance test. Clinical characteristics of the study subjects according to preproNPY genotype are shown in Table 1Go.


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  		TABLE 1. Comparison of clinical characteristics of study subjects in screening tests in the two study groups of different preproNPY genotypes

 
Genotyping

For genotyping, a blood sample of 10 ml was taken from an antecubital vein. Blood leukocyte DNA was extracted by using a DNA isolation kit (Puregene, Gentra Systems, Minneapolis, MN), and the manufacturer’s instructions were followed. The genotype was determined as described earlier (9).

Study protocol

Oral glucose tolerance test. The study subjects were asked not to use any alcohol or caffeine-containing drinks or tobacco products over 24 h preceding the study. They were also asked to avoid strenuous physical exercise for 2 preceding days. The study subjects came to the laboratory at 0800 h having fasted for 10 h. An iv cannula was inserted into a vein of left arm for blood sampling. The study subjects were given 1 g/kg glucose as solution (0.375 g/ml); however, the maximum amount of glucose was 75 g per person. The subjects weighing more than 75 kg, and their matched pairs (eight subjects), were given glucose 0.9 g/kg. For measurements of plasma NPY, glucose, insulin, insulin to glucose ratio, ghrelin, NE, and its metabolite dihydroxyphenylglycol (DHPG), blood samples were taken at 13 time points until 5 h after (–30, –5, +15, +30, +45, +60, +75, +90, +120, +150, +180, +240 and +300 min), the zero point that was the time of glucose ingestion. The blood samples were kept on ice before the immediate centrifugation, after which the plasma samples were stored at –70 C until analyzed.

Cardiovascular autonomic function test. A continuous precordial three-channel electrocardiogram was obtained via cardioscope (Olli Monitor 432, Kone Instrument Division, Espoo, Finland). A continuous noninvasive blood pressure signal was recorded from the right middle finger that was held at heart level (Ohmeda 2300 BP monitor, Ohmeda Inc., Louisville, CO). To obtain a controlled 0.25 Hz breathing during the recordings, a paced sound signal was used and flow-volume spirometer signal (M909, Medikro Oy, Kuopio, Finland) was recorded to calculate pulmonary air volume and control the breathing frequency. The first recording lasted 5 min and was performed in standing position at –30 min after adjusting for 5 min to standing position. The following five recordings lasted 15 min each and were performed at supine position at –15, +30, +60, +120, and +240 min after 15 min lying down. After each data recording, blood pressure and heart rate were measured at a brachial artery level (sphygmomanometer/printer BP-203 M II, Instrumentarium, Helsinki, Finland). The blood samples were always taken before recordings.

Data analyses for cardiovascular autonomic function test. All data analyses were performed using special software (WinCPRS, Absolute Aliens Oy, Turku, Finland). The heart rate (R-R intervals) and systolic arterial blood pressure (SAP) variability indices were computed for user-defined stationary data sets of either 600 (in supine position) or 300 R-R-intervals (in standing position) using the fast Fourier transform method for spectral analysis. Before spectral computations the signals were linearly detrended, resampled with the sampling frequency of 5 Hz, and windowed using Hanning window. The total variabilities of R-R intervals and SAP were computed integrating the spectra from 0.003 to 0.40 Hz, the LF variabilities from 0.04 to 0.15 Hz, and the HF variabilities from 0.15 to 0.40 Hz. Sympathovagal balance was estimated by calculating the LF to HF ratio for R-R intervals and SAP. Baroreflex sensitivity was obtained using the sequence method in humans (22) for slopes of rising SAP and lengthening R-R intervals.

Analytical methods

Plasma NPY concentrations were determined by using a commercial RIA kit EURIA-NPY (Euro-Diagnostica Inc., Malmö, Sweden). Plasma insulin and acylated ghrelin (active form) concentrations were determined by using commercial RIA kits from Linco Research (St. Charles, MO). NE and DHPG concentrations in plasma were determined using a HPLC method with electrochemical detection (25).

Statistical analyses

Statistical analysis was performed with SAS System for Windows (version 8.2/2001; SAS Institute Inc., Cary, NC) using linear mixed models (MIXED-procedure) (26). Regression analyses for associations of NPY and NE concentrations with other measured parameters and the differences in the associations between the genotypes were analyzed taking into account repeated measurements and matched pairs by defining time as a repeated factor and pair as a random factor in models (26). Regression coefficients for genotype groups were calculated with regression models in which time was used as a repeated factor. Baseline characteristics and mean concentrations at certain time points were compared between the groups using linear mixed model in which pair was used as a random effect. A two-sided P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Decreased NPY, NE, DHPG, and insulin concentrations in the subjects with Leu7/Pro7 genotype

The subjects with Leu7/Pro7 genotype had significantly lower [on average 38.9 pg/ml (9.1 pmol/liter)] NPY concentrations, compared with the subjects with Leu7/Leu7 genotype (Fig. 1AGo and Table 2Go). The subjects with Leu7/Pro7 genotype also had significantly lower plasma NE (Fig. 1BGo) and DHPG (Fig. 1CGo) concentrations (Table 2Go). No differences in plasma glucose concentrations were detected (Fig. 2AGo and Table 2Go). However, plasma insulin concentrations (Fig. 2BGo) and the insulin to glucose ratios (Fig. 2CGo) were significantly lower in the subjects with Leu7/Pro7 genotype, compared with the subjects with Leu7/Leu7 genotype whereas plasma NPY concentrations did not associate with plasma glucose, insulin, or ghrelin concentrations (Table 3Go). Also, no association between insulin and ghrelin concentrations was detected (data not shown). The associations of plasma NE concentrations with ghrelin concentrations were significantly different between the genotypes; a strong positive association was found in the subjects with Leu7/Pro7 genotype but no association was found in the subjects with Leu7/Leu7 genotype (Table 3Go). The regression coefficients of plasma NPY and NE concentrations vs. other measured variables are shown in Table 3Go.



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  		FIG. 1. During oral glucose tolerance test, the subjects with Leu7/Pro7 genotype (open triangles) displayed significantly lower plasma NPY (A), NE (B), and DHPG (C) concentrations, compared with the subjects with Leu7/Leu7 genotype (solid triangles). The vertical line at 0 min represents the glucose ingestion. Error bars, SEM values. NPY, 1 pg/ml = 0.000234 nmol/liter; NE, 1 pg/ml = 0.005911 nmol/liter; DHPG, 1 pg/ml = 0.005875 nmol/liter.

 

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  		TABLE 2. Genotype effect and genotype-by-time interaction effect on each measurement during oral glucose tolerance test (MIXED-procedure) in supine position

 


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  		FIG. 2. During oral glucose tolerance test, no significant differences were detected in overall plasma glucose (A) concentrations between the NPY genotypes. The subjects with Leu7/Pro7 genotype (open triangles) displayed significantly lower insulin concentrations (B) and the insulin to glucose ratios (C), compared with the subjects with Leu7/Leu7 genotype (solid triangles). Suppression of plasma ghrelin concentration (D) after glucose ingestion was significantly delayed in the subjects with Leu7/Pro7 genotype. The vertical line at 0 min represents the glucose ingestion. Errors bars, SEM values. Glucose, 1 mg/dl = 0.05551 mmol/liter; insulin, 1 µU/ml = 6 pmol/liter; ghrelin, 1 pg/ml = 3.371 pmol/liter.

 

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  		TABLE 3. Linear regression coefficients between plasma NPY and NE concentrations vs. other measurements within genotype groups and comparisons of the regression coefficients (P) between Leu7/Leu7 and Leu7/Pro7 genotypes in supine position

 
Delayed ghrelin suppression response in the subjects with Leu7/Pro7 genotype

The mean plasma ghrelin concentrations were similar in the two genotypes (Fig. 2DGo). However, the nadir ghrelin concentrations after glucose load were significantly later in the subjects with Leu7/Pro7 genotype, compared with the subjects with Leu7/Leu7 genotype (P = 0.037, Fig. 2DGo). The time between the maximum glucose concentration and the nadir ghrelin concentration was significantly longer in the subjects with Leu7/Pro7 genotype, compared with the subjects with Leu7/Leu7 genotype (P = 0.011, Fig. 2DGo), and the time between the maximum insulin concentrations and the nadir ghrelin concentrations was almost significantly longer in the subjects with Leu7/Pro7 genotype (P = 0.054). The decrease in ghrelin concentration from basal to nadir values was not significantly different between the subjects with Leu7/Pro7 and Leu7/Leu7 genotype [137.3 ± 43.6 pg/ml (462.8 ± 147.0 pmol/liter) and 250.6 ± 42.0 pg/ml (844.8 ± 141.6 pmol/liter), respectively, P = 0.073].

Increased heart rate variability and baroreflex sensitivity in the subjects with Leu7/Pro7 genotype

The total variability and the LF and HF variabilities of R-R intervals were significantly increased in supine position in the subjects with Leu7/Pro7 genotype, but no differences in the LF to HF ratio of R-R intervals were found between the genotypes (Table 2Go). Heart rate was similar between the genotypes (Table 2Go). Baroreflex sensitivity was significantly higher (on average 8.29 msec/mm Hg) in the subjects with Leu7/Pro7 genotype than in the subjects with Leu7/Leu7 genotype (Table 2Go) in supine position. No differences in heart rate variability indices between the genotypes were found in standing position (data not shown). Also, no significant differences were found in LF variability of SAP (finger blood pressure) in supine (Table 2Go) or standing positions (data not shown). Tidal volume and minute ventilation indices did not have significant changes during the oral glucose tolerance test (data not shown).

A significant genotype-by-time effect of systolic brachial blood pressure was detected (Table 2Go); the subjects with Leu7/Pro7 genotype had significantly lower blood pressure at 120 min after glucose load than the subjects with Leu7/Leu7 genotype (Fig. 3Go).



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  		FIG. 3. A significant genotype-by-time effect was seen in brachial systolic blood pressure at 120 min (*) during oral glucose tolerance test, the subjects with Leu7/Pro7 genotype (open triangles) having lower values than the subjects with Leu7/Leu7 genotype (closed triangles). The vertical line at 0 min represents the glucose ingestion. Errors bars, SEM values.

 
Negative association of NE concentrations with LF variability of heart rate and with baroreflex sensitivity in the subjects with Leu7/Pro7 genotype

In the subjects with Leu7/Pro7 genotype, there was a significant negative association of NE concentrations with LF variability of R-R intervals but not in the subjects with Leu7/Leu7 genotype; the difference in these associations between the genotypes was statistically significant (Table 3Go). Furthermore, there was a significant difference in the associations of NE concentrations with baroreflex sensitivity between the genotypes: in the subjects with Leu7/Pro7 genotype, the association was negative, whereas no significant association was detected in the subjects with Leu7/Leu7 genotype (Table 3Go).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study we detected decreased plasma levels of sympathetic transmitters NPY and NE in the subjects with Leu7/Pro7 genotype, compared with the subjects with Leu7/Leu7 genotype. Also, the concentrations of DHPG, a metabolite of NE, were decreased, which further indicates lower neuronal sympathetic activity in the subjects with Leu7/Pro7 genotype. This is in agreement with an earlier diurnal study, which showed that subjects with this genotype have low NPY and NE concentrations in rest (20). This may reflect decreased basal sympathetic tone in these subjects, which is further supported by present results of autonomic function tests.

We observed lower insulin concentrations and lower insulin to glucose ratios during oral glucose tolerance test in the subjects with Leu7Pro polymorphism than in the subjects without this polymorphism. This is in agreement with earlier studies that have shown that the Leu7Pro polymorphism is associated with decreased insulin, increased glucose concentrations, and decreased insulin to glucose ratio after meals (18, 20). In a study on a large group of middle-aged subjects, iv or oral glucose tolerance tests did not reveal any differences in insulin secretion between the preproNPY genotypes when area under the curve values were used for comparisons (27). This may be because of a heterogeneous study population with no pair matching and because the study subjects were given the same amount of glucose per os instead of weight-adjusted dosing as in the present study. The primary mechanism leading to decreased insulin levels in the subjects having the Leu7Pro polymorphism in preproNPY is not known. No change in insulin sensitivity has been observed due to the Leu7Pro polymorphism (27). NPY has been shown to have inhibitory effects on glucose-stimulated insulin secretion in rat, human, and mouse islet cells (6, 8, 28), and NPY has been shown to cause ß-cell hyperplasia and dysfunction in mice (28). We have earlier detected that human endothelial cells with the Leu7/Pro7 genotype display increased immunoreactivity of mature NPY (19), which may indicate that NPY concentrations are increased also in pancreas and possibly have a deteriorating effect on insulin secretion. Central mechanisms regulating sympathovagal balance may also be involved in decreased insulin secretion because there is evidence that the magnitude of vagal component in heart rate variability power spectral analysis and insulin levels have inverse relationship in healthy subjects (29).

We detected that the subjects with Leu7/Pro7 genotype displayed significantly delayed ghrelin suppression response after glucose load, compared with the subjects with Leu7/Leu7 genotype. Ghrelin is a gut-origin peptide that stimulates eating and GH release (30, 31). Several studies indicate that eating and glucose intake are followed by suppression of ghrelin concentrations in blood (32, 33, 34). After an oral glucose tolerance test, nadir ghrelin concentrations have been reported between 30 and 60 min after oral glucose intake. Also iv glucose infusions have been shown to suppress ghrelin levels, yet the minimum ghrelin concentrations are reached faster (32, 35). Many studies have shown that insulin is a key regulator of postprandial ghrelin suppression response in nondiabetic and diabetic subjects (24, 36, 37). Therefore, the slightly delayed ghrelin response in the subjects with Leu7/Pro7 genotype during oral glucose tolerance test could be due to the slightly lower insulin response in these subjects. Leptin inhibits both ghrelin and insulin secretion and could be a common regulator of these two hormones. However, our earlier study on matched healthy subjects could not indicate differences in diurnal leptin levels between subjects of the two preproNPY genotypes (20). It is also possible that changed GH secretion observed in the subjects with Leu7Pro polymorphism (19) also have a role in the changed ghrelin response because high GH and delayed ghrelin depression response during oral glucose tolerance test has been linked together in an earlier study with anorectic patients (33). The association of plasma ghrelin concentrations with plasma NE concentrations has not been observed earlier, but a significant positive association of these two was observed in the current study in the subjects with Leu7/Pro7 genotype. This finding may indicate coregulation of noradrenergic and ghrelin pathways in these subjects.

The results show increased variability of both LF and HF bands of R-R intervals as well as increased baroreflex sensitivity in the subjects with Leu7/Pro7 genotype, which can be interpreted as increased cardiovascular modulation (22). However, because ß-blockade has a similar effect, it can also be interpreted as sympathetic withdrawal (38), which is in agreement with the finding of decreased NPY, NE, and DHPG levels and decreased systolic blood pressure in the subjects with Leu7/Pro7 genotype. Earlier animal data indicate that NPY mediates central inhibition of sympathetic outflow (39), which supports an idea of central sympathetic withdrawal in human subjects having a polymorphic NPY gene and altered secretion of NPY.

Clinically increased cholinergic and decreased adrenergic cardiovascular reactivity (measured by R-R interval variability indices) can be considered as beneficial effects. However, the results of this study show that the cardiovascular autonomic regulation is much more sensitive to changes in plasma NE concentrations in the subjects with Leu7Pro polymorphism than in the subjects without this polymorphism. The strong negative association of NE concentrations with LF variability of R-R intervals and with baroreflex sensitivity [which is mostly a vagal test (22)] in the subjects with Leu7/Pro7 genotype suggests that the subjects with this genotype are likely to be more prone to undesired effects of NE when it is released in sympathetic stress. Higher heart rate during daytime, but not during night, and increased positive association of NE with both systolic and diastolic blood pressure in our earlier study (20) also suggested that the subjects with Leu7Pro polymorphism are more sensitive to the changes of plasma NE than the subjects without this polymorphism. It has been shown that low heart rate variability is a risk factor for cardiac mortality (40). Earlier studies have also shown that type 2 diabetic patients with Leu7Pro polymorphism in preproNPY have decreased heart rate variability and increased risk for atherosclerosis (14). Furthermore, hypertensive patients having the Leu7Pro polymorphism have increased risk for stroke and infarctation (17). These data suggest that although the subjects with Leu7/Pro7 genotype display increased heart rate variability when young, the high sensitiveness to NE could cause an increased risk for cardiovascular morbidity if they develop hypertension or type 2 diabetes later in life (14, 17). Therefore, in future studies it would be important to also investigate the role of the Leu7Pro polymorphism in cardiovascular mortality in nondiabetic elderly subjects and type 2 diabetes patients.

It is concluded that the genetic Leu7Pro polymorphism of preproNPY is associated with low basal sympathetic activity, low insulin secretion, and delayed ghrelin response but increased responsiveness of cardiovascular autonomic system to NE during oral glucose tolerance test. The increased responsiveness of autonomic functions to NE associated with the polymorphism may be connected to increased cardiovascular vulnerability of subjects having this polymorphism.


   Acknowledgments
 
We thank Ms. Raija Kaartosalmi and Ms. Elina Kahra for skillful technical assistance. Dr. Eriika Savontaus is acknowledged for valuable scientific comments.


   Footnotes
 
This work was supported by the National Technology Agency of Finland, Academy of Finland, EVO grants of Turku City Hospital, and the Finnish Diabetes Research Society.

First Published Online March 29, 2005

Abbreviations: DHPG, Dihydroxyphenylglycol; HF, high-frequency; Leu7Pro, leucine7 to proline7; LF, low-frequency; NE, norepinephrine; NPY, neuropeptide Y; RRI, R-R-interval, time between successive heartbeats; SAP, systolic arterial blood pressure.

Received January 25, 2005.

Accepted March 21, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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S. T. Ruohonen, U. Pesonen, N. Moritz, K. Kaipio, M. Roytta, M. Koulu, and E. Savontaus
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