0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society
Vol. 85, No. 5 Printed in U.S.A.
Plasma Leptin Concentrations and Cardiac Autonomic Nervous System in Healthy Subjects with Different Body Weights GIUSEPPE PAOLISSO, DANIELA MANZELLA, NICOLA MONTANO, ANTONIO GAMBARDELLA, AND MICHELE VARRICCHIO Department of Geriatric Medicine and Metabolic Diseases II, University of Naples, I-80138 Naples; and Centro Ricerche Cardiovascolari, Medicina Interna II, Ospedale L. Sacco, University of Milan (N.M.), 20100 Milan, Italy ABSTRACT Previous studies have shown that leptin stimulates sympathetic nervous system; heart rate variability (HRV) is a widely used technique for assessing the sympathovagal balance at the cardiac level. The aim of our study was to investigate a possible relationship between plasma leptin levels and the autonomic regulation using spectral analysis of HRV. In 120 healthy nonobese subjects the plasma leptin concentration was determined, and HRV was recorded at baseline and during tilt. All subjects were categorized in quartiles of plasma leptin concentration. Analysis of data showed a significant increase in body mass index, body fat, fasting plasma insulin, triglyceride concentration, and homeostatic model assessment values throughout the different quartiles of plasma leptin concentration. Concerning cardiovascular parameters, heart rate, arterial blood pressures, and RR intervals were not significantly different among the quartiles. Total power and high frequency (HF) in normalized
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EPTIN IS THE peptide hormone product of the obese (ob) gene expressed in adipose tissue; it acts centrally, decreasing appetite and increasing energy expenditure (1). More recently, a possible impact of leptin on the autonomic nervous system (ANS) has been hypothesized. In fact, leptin has been shown to stimulate sympathetic nervous system (SNS) activity in thermogenic (2) and nonthermogenic organs (3) in animal models. In the same experimental model, chronic leptin infusion concentration has been also demonstrated to stimulate heart rate and increase arterial blood pressure (4). Whether such an effect of leptin is a physiological or pharmacological one still needs to be demonstrated. The latter question has important clinical implication, as SNS activation might promote atherosclerosis (5, 6) and trigger acute cardiovascular events (7, 8). Despite some criticism (9), spectral analysis of heart rate variability (HRV) is an well accepted noninvasive tool for assessing the state of the cardiac sympathovagal balance (10, 11). In fact, several studies have indicated that the powers of the low frequency (LF) and the high frequency (HF) component, occurring in synchrony with vasomotor waves and respiratory acts, respectively, appear to reflect in their reciprocal relationship
Received October 14, 1999. Revision received December 14, 1999. Accepted December 15, 1999. Address all correspondence and requests for reprints to: Giuseppe Paolisso, M.D., Department of Geriatric Medicine and Metabolic Diseases, Servizio di Astanteria Medica, Piazza Miraglia 2, I-80138 Naples, Italy. E-mail:
[email protected].
units were significantly decreased, whereas low frequency (LF) normalized units was progressively increased from the first to the fourth quartile. Thus, the LF/HF ratio rose gradually and significantly from the lowest to the highest quartile. Such results were independent of the body fat estimate (P ⬍ 0.03 for the trend). The change in the LF/HF ratio was significantly enhanced during tilt (P ⬍ 0.001 vs. rest values for all quartiles); the effect was stronger in subjects in the fourth quartile of plasma leptin concentration (P ⬍ 0.005 for the trend). The latter parameter was also independent of body fat content and distribution (P ⬍ 0.01). Our study shows that increasing fasting plasma leptin concentrations are associated with a shift of the sympathovagal balance toward a progressive increase in sympathetic activation and an increased response to orthostatic stimulus in nonobese subjects with different body fat contents. (J Clin Endocrinol Metab 85: 1810 –1814, 2000)
the state of sympathovagal balance in numerous physiological and pathophysiological conditions (12–15). Interestingly, correlation between body fat content and LF/HF ratio after glucose ingestion has been correlated in nonobese subjects with different degrees of body fat content (16). To the best of our knowledge, the possible relationship between different plasma leptin concentrations and HRV parameters has not yet been evaluated. Accordingly, we planned to investigate the possible association of physiologically occurring fasting leptin concentrations and cardiac sympathovagal balance in healthy nonobese subjects in baseline conditions and during the tilt test. Materials and Methods Subjects One hundred and twenty healthy young males volunteered for the study. All subjects were nonsmokers, normotensive, and receiving no medication and had no evidence of metabolic or cardiovascular diseases. Subjects with a change in body weight of more than 2 kg during the preceding year were excluded from the study. All volunteers had normal glucose tolerance to a 75-g oral glucose load (17). Insulin resistance was derived by homeostatic model assessment (HOMA) according to Matthews et al. (18). All tests were performed in the morning and after an overnight fast (at least 12 h). After clear explanation of potential risks of the study, each volunteer gave informed consent to participate in the study, which was approved by the ethical committee of our institution.
Anthropometric determinations Weight and height were measured using a standard technique. Body mass index (BMI) was calculated as body weight (kilograms)/height
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CARDIAC AUTONOMIC NERVOUS SYSTEM AND LEPTIN (meters)2. Body fat (BF) was measured using a four terminal bioimpedance analyzer (IA 101/SC, RJL Spectrum Bioelectrical Impedance, Akern, Italy; RJL System, Florence, Italy).
Study protocol All subjects were studied at 0800 h in a quiet comfortable room at a temperature ranging between 22–24 C. A venous blood sample for plasma metabolite determinations was immediately drawn. Then each subject rested in the supine position for at least 30 min before starting baseline Holter (Remco Italy Cardioline, Milan, Italy) recording, which lasted 60 min. Later the table was rotated to an upright position (head-up tilt test), which was maintained for 10 min. Transit from 0° at 90° took about 15 s. If hypotension developed during postural tilt, testing was stopped, and the subject was excluded from study (n ⫽ 4). Blood pressure and heart rate at baseline and during the studies were determined by Finapres (Omheda, Englewood, CO). Respiratory frequency was also calculated over a period of 2 min before the test. Subjects with a respiratory rate less than 10 breaths/min (i.e. ⬍0.15 Hz) were excluded from the study. Ambulatory electrocardiograph monitoring was performed with 2-channel frequency modulatory tape recorders (AD 35, recorder model LP103, Remco Italy Cardioline, Milan, Italy). After accurate skin preparation, the electrodes were placed on the chest to obtain the bipolar chest leads CM1 (modified V1) on the first channel and CM4 (modified V4) on the second channel. Two independent and blind experienced investigators analyzed the ambulatory electrocardiograph recording tapes by Holter AD35 TOP (Remco Italy Cardioline). Ectopic beats were corrected for linear interpolation with the adjacent complexes. Electrocardiograph tracings with more than 1% premature beats were eliminated from the analysis. Power spectral analysis was calculated from a consecutive series of 512 intervals. An autoregressive algorithm computed the power spectral densities. Autoregressive spectral analysis was undertaken after estimation of model coefficients by the LevinsonDurbin algorithm (19). The model order selection was performed according to the Akaike (19) information criterion. Spectral components were identified and estimated using the spectral decomposition algorithm proposed by Johnsen and Andersen (20) and were then assigned, on the basic of their central frequency, to 1 of the 3 bands: very low frequency (VLF) band (from 0 – 0.03 Hz), LF band (from 0.04 – 0.15 Hz), and HF band (from 0.16 – 0.45 Hz). As the physiological explanation of the VLF component is much less defined and the existence of a specific physiological process attributable to that heart period change has been strongly questioned (11), only LF and HF components were considered. LF and HF components are reported in absolute as well as normalized units (nu), which represent the relative value of each power component in proportion to the total power minus the VLF component (11). Normalized units tend to minimize the effect of the changes in total power on the values of LF and HF components (11).
Analytical techniques Plasma glucose was determined by the glucose oxidase method [Autoanalyzer, Beckman Coulter, Inc., Fullerton, CA; coefficient of variation (CV), 2.1 ⫾ 0.2%]. Plasma total cholesterol (CV, 3.3 ⫾ 0.3%), high density lipoprotein cholesterol (CV, 3.5 ⫾ 0.4%), and triglyceride (CV, 3.7 ⫾ 0.6%) concentrations were determined by routine methods (Ortho-Clinical Diagnostic, Milan, Italy). Blood samples for plasma hormone measurements were collected in heparinized tubes. After centrifugation, plasma insulin (Sorin Biomedical, Milan, Italy; CV, 3.2 ⫾ 0.2%) and leptin (Linco Research, Inc., St. Louis, MO; CV, 4.1 ⫾ 0.7%) concentrations were determined by RIA. To rule out the possible interference of day by day plasma leptin variation, we measured plasma leptin concentration at 24-h interval in 45 subjects. Plasma leptin concentrations had a very small interday variation (mean variation, 4.5 ⫾ 0.8%). Furthermore, to avoid circadian variation in plasma leptin, blood was always sampled between 0800 – 0900 h. Plasma catecholamine concentrations were determined by high performance liquid chromatography (epinephrine CV, 4.8 ⫾ 1.1%; norepinephrine CV, 5.1 ⫾ 1.2%).
Statistical analysis All results are the mean ⫾ sd. Because the distribution of fasting plasma triglycerides, insulin, and leptin concentrations and of the fre-
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quency domain measures of HRV are extremely skewed, each value was also log-transformed to improve normality for statistical testing and back-transformed for presentation in tables and figures. The ⌬ LF/HF ratio was calculated as difference between baseline values and those reported at the end of tilt. The nQuery test was used to predict the adequacy of sample size in each quartile. This test demonstrated that 22 subjects in each quartile were sufficient to obtain a significant difference in HRV parameters. ANOVA was used to assess differences among the leptin groups organized in quartiles. When P ⬍ 0.05 was found, Scheffe’s test was also performed. ANOVA for repeated measures was used for calculating the P value for the trend for each variable among the different groups. Pearson’s simple correlation allowed studying the association between two variables. Analysis of covariance allowed the study of differences in baseline LF/HF ratio among the different quartiles independently of body fat. Partial correlation allowed studying the relationship between two variables independently of covariates. Multivariate linear regression analysis allowed investigating the independent association among age, body fat, HOMAIR, fasting plasma triglyceride and leptin concentrations, and baseline LF/HF ratio. All data were analyzed on an IBM PC computer by SOLO (BMDP, Cork, Ireland) software.
Results Baseline data
The clinical characteristics of the study groups are reported in Table 1. All subjects were adult, nonobese, and normotensive. The fasting plasma leptin concentration was significantly correlated with BMI, body fat, HOMA, triglycerides, and the main index of cardiac ANS (Table 2). Metabolic and cardiovascular data stratified in quartiles are reported in Table 3. Analysis of data showed a significant increase in BMI, body fat, fasting plasma insulin and triglyceride concentrations, and HOMAIR values throughout the different quartiles. In contrast, fasting plasma glucose, catecholamine, and low and high density lipoprotein cholesterol levels were similar in all quartiles studied. As far as the cardiovascular parameters are concerned, heart rate, systolic and diastolic blood pressures, and RR intervals were not TABLE 1. Clinical characteristics of study groups (n ⫽ 120) Mean ⫾
Age BMI Body fat HOMA Fasting plasma glucose (mmol/L) Fasting plasma insulin (pmol/L) Fasting plasma leptin (ng/mL) Fasting plasma LDL cholesterol (mmol/L) Fasting plasma HDL cholesterol (mmol/L) Fasting plasma triglycerides (mmol/L) Fasting plasma norepinephrine (nmol/L) Fasting plasma epinephrine (pmol/L) HR (beats/min) SBP (mm Hg) DBP (mm Hg) Total power (ms2) RR interval (ms) LF (ms2) LF (nU) HF (ms2) HF (nU) LF/HF
SD
42.5 ⫾ 1.9 24 ⫾ 2 23 ⫾ 3 2.25 ⫾ 0.6 5.2 ⫾ 0.3 76.6 ⫾ 6.4 8.1 ⫾ 5.3 3.8 ⫾ 0.4 1.3 ⫾ 0.3 1.6 ⫾ 0.3 1.99 ⫾ 0.34 331 ⫾ 67 71 ⫾ 4 128 ⫾ 8 73 ⫾ 3 12021 ⫾ 1357 810 ⫾ 16 546 ⫾ 258 68.4 ⫾ 6 272 ⫾ 151 37.8 ⫾ 5.7 2.06 ⫾ 0.5
All results are the mean ⫾ SD. BMI, Body mass index; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; LF, low frequency; HF, high frequency.
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TABLE 2. Correlation between fasting plasma leptin and all variables studied (n ⫽ 120)
Age (yr) BMI (kg/m2) Body fat (kg) HOMA Glucose (mmol/L) Insulin (pmol/L) LDL (mmol/L) HDL (mmol/L) Triglycerides (mmol/L) Norepinephrine (nmol/L) Epinephrine (pmol/L) HR (beats/min) SBP (mm Hg) DBP (mm Hg) TP (ms2) RR interval (ms) LF (nU) HF (nU) LF/HF
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r
P
0.09 0.56 0.78 0.25 0.11 0.21 0.10 0.12 0.19 0.06 0.08 0.10 0.05 0.07 0.18 0.15 0.20 0.18 0.24
0.21 0.001 0.001 0.001 0.18 0.03 0.14 0.19 0.05 0.24 0.22 0.14 0.27 0.25 0.05 0.08 0.04 0.05 0.005
BMI, Body mass index; HOMA, homeostasis model assessment; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; TP, total power; LF, low frequency; HF, high frequency. All plasma metabolite and hormone samples were drawn in fasting conditions.
significantly different among the quartiles. Total power and HF (in nu) showed a significant declining trend, whereas LF was progressively increased from the first to the fourth quartile. Thus, the LF/HF ratio rose progressively and significantly from the lowest to the highest quartile. Interestingly, this finding was independent of body fat (P ⬍ 0.03 for the trend). In the whole group of subjects (n ⫽ 120), the fasting plasma leptin concentration and baseline LF/HF ratio were significantly correlated (r ⫽ 0.43; P ⬍ 0.001); this relation was independent of body fat and waist/hip ratio (r ⫽ 0.24; P ⬍ 0.009). The independent role of fasting plasma leptin concentration on the baseline LF/HF ratio was also investigated by multivariate linear regression analysis in the whole group of subjects (n ⫽ 120; Table 4). This analysis demonstrated that age, body fat, HOMAIR values, and fasting plasma triglycerides and leptin concentrations were all significantly and independently associated with the baseline LF/HF ratio. Furthermore, stepwise multivariate analysis indicated that the whole model explained 63% of the variability in the baseline LF/HF ratio, and fasting plasma leptin explained 28% of the variability in the dependent variable. Effects of tilt on heart rate variability
Changes in HRV parameters in different quartiles of plasma leptin concentration are reported in Table 5. Briefly, changes in total power, RR intervals, and HF were significantly reduced during the tilt stimulus (P ⬍ 0.01 vs. rest values for all parameters and quartiles); the effect was stronger in subjects in the fourth quartile than those in the first quartile. Changes in LF and LF/HF ratio appeared significantly increased during tilt (P ⬍ 0.001 vs. rest values for all parameters and quartiles), with subjects in the fourth quartile having the strongest response. After adjusting for body fat content and distribution, all trends were still significant (Ta-
ble 5). Plasma catecholamine concentrations showed a similar response in all groups (Table 5). In the entire group of subjects, the ⌬ LF/HF ratio correlated with the fasting plasma leptin concentration (r ⫽ 0.55; P ⬍ 0.001) and body fat (r ⫽ 0.61; P ⬍ 0.001). Nevertheless, the correlation between the ⌬ LF/HF ratio and the fasting plasma leptin concentration (r ⫽ 0.31; P ⬍ 0.001) was independent of body fat content, body fat distribution, and changes in plasma catecholamine concentration. Discussion
Our study demonstrates that the plasma leptin concentration is associated with an increase in the LF/HF ratio, an index of cardiac sympathovagal balance, independently of anthropometric characteristics and of insulin resistance in healthy subjects. Furthermore, we demonstrate an association between the extent of the stimulation of the LF/HF ratio by tilt test parallels and the rise in plasma leptin concentration, a finding independent of body fat distribution and changes in plasma catecholamine concentrations. Previous studies have shown that plasma leptin may affect ANS activity. In particular, leptin has been shown to enhance norepinephrine turnover in interscapular brown adipose tissue, suggesting increased sympathetic outflow to this thermogenic organ in animal models (2). This effect had a slow onset, suggesting an influence of leptin on the central nervous system (4). The effect of leptin on sympathetic nerve activity was dose dependent, with a threshold dose of 100 g/kg (plasma concentration, ⬃5 ng/mL). Leptin-induced sympathetic activation was still apparent after transaction of the sympathetic nerve distal to the recording site, implying that the increase in activity was from efferent, not afferent, nerves. This was confirmed by the disappearance of sympathetic activity after ganglion blockade with iv chlorisondamine (30 mg/kg). In contrast, leptin did not cause SNS activation in obese Zucker rats, which are known to possess a mutation in the leptin receptor gene. Such a phenomenon implies that functional receptors (and possibly secondary signaling mechanisms) are necessary to elicit a nervous system response. Evidence has been also accumulated that leptin increased sympathetic nerve activity in nonthermogenic organs, such as kidney, hindlimb, and adrenal gland (21). In humans, the relationship between plasma leptin concentration and ANS has been shown in Pima Indians, in whom the plasma leptin concentration correlated with basal muscle sympathetic nerve activity (22). The results concerning the effect of plasma leptin on the ANS at the cardiovascular level are controversial. In rats, iv leptin infusion did not affect heart rate or arterial blood pressure (23, 24). Nevertheless, in this study the rats were anesthetized, and thus the effect of leptin on the cardiovascular ANS might be offset. On the other hand, others have shown that a chronic rise in circulating leptin cause sustained increases in arterial blood pressure and heart rate, consistent with a possible role of leptin in obesity hypertension (4). Why the acute rise in the plasma leptin concentration increased heart rate was not explained; nevertheless, a possible impact of plasma leptin on cardiac sympatovagal balance was hypothesized (25). A possible interference between plasma lep-
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TABLE 3. Baseline values before and after distribution in quartiles of fasting plasma leptin concentration
Age BMI Body fat HOMA Glucose (mmol/L) Insulin (pmol/L) Leptin (ng/ml) LDL (mmol/L) HDL (mmol/L) Triglycerides (mmol/L) Norepinephrine (nmol/L) Epinephrine (pmol/L) HR (beats/min) SBP (mm Hg) DBP (mm Hg) TP (ms2) RR interval (ms) LF (nU) HF (nU) LF/HF
Quartiles of plasma leptin conc.
All (n ⫽ 120)
1 (n ⫽ 33)
2 (n ⫽ 26)
3 (n ⫽ 32)
4 (n ⫽ 29)
P for trend
42.5 ⫾ 1.9 24 ⫾ 2 23 ⫾ 3 2.53 ⫾ 0.6 5.2 ⫾ 0.3 76.6 ⫾ 6.4 8.1 ⫾ 5.3 3.8 ⫾ 0.4 1.3 ⫾ 0.3 1.6 ⫾ 0.3 1.9 ⫾ 0.3 331 ⫾ 67 71 ⫾ 4 128 ⫾ 8 73 ⫾ 3 12021 ⫾ 1357 810 ⫾ 16 68.4 ⫾ 6 37.8 ⫾ 5.7 2.06 ⫾ 0.5
42.3 ⫾ 1.6 22.1 ⫾ 0.3 21 ⫾ 0.4 2.22 ⫾ 0.1 5.0 ⫾ 0.3 70.6 ⫾ 0.8 4.5 ⫾ 1.7 3.6 ⫾ 0.1 1.3 ⫾ 0.1 1.5 ⫾ 0.1 1.9 ⫾ 0.3 321 ⫾ 47 70 ⫾ 2 125 ⫾ 5 70.3 ⫾ 0.8 12912 ⫾ 450 805 ⫾ 10 63.3 ⫾ 1.2 43.1 ⫾ 0.2 1.67 ⫾ 0.5
41.4 ⫾ 0.5 23.8 ⫾ 0.5 24 ⫾ 0.3 2.43 ⫾ 0.2 5.1 ⫾ 0.4 75.3 ⫾ 0.7 5.1 ⫾ 1.5 3.7 ⫾ 0.2 1.3 ⫾ 0.2 1.5 ⫾ 0.1 2.0 ⫾ 0.2 348 ⫾ 39 72 ⫾ 1 130 ⫾ 3 71.3 ⫾ 0.4 12215 ⫾ 386 810 ⫾ 10 66.6 ⫾ 2 40.2 ⫾ 0.5 1.95 ⫾ 0.6
42.3 ⫾ 1.8 25.1 ⫾ 0.2 25 ⫾ 0.3 2.60 ⫾ 0.2 5.2 ⫾ 0.3 78.9 ⫾ 0.5 7.8 ⫾ 2.7 3.8 ⫾ 0.5 1.2 ⫾ 0.1 1.6 ⫾ 0.2 1.7 ⫾ 0.4 324 ⫾ 59 73 ⫾ 2 133 ⫾ 3 73.4 ⫾ 0.6 11843 ⫾ 321 795 ⫾ 20 69.9 ⫾ 3.2 37.4 ⫾ 1.1 2.22 ⫾ 0.8
41.3 ⫾ 1.3 26.2 ⫾ 0.3 26 ⫾ 0.3 2.78 ⫾ 0.1 5.3 ⫾ 0.2 82.6 ⫾ 0.6 11.8 ⫾ 2.2 4.1 ⫾ 0.4 1.1 ⫾ 0.2 1.6 ⫾ 0.2 1.8 ⫾ 0.1 346 ⫾ 50 70 ⫾ 3 131 ⫾ 2 75.3 ⫾ 0.9 10821 ⫾ 189 799 ⫾ 9 73.2 ⫾ 1.1 34.5 ⫾ 0.7 2.59 ⫾ 0.5
0.78 0.001 0.001 0.005 0.33 0.007 0.001 0.31 0.09 0.05 0.38 0.51 0.77 0.65 0.58 0.03 0.81 0.05 0.05 0.001
All results are the mean ⫾ SD. BMI, Body mass index; HOMA, homeostasis model assessment; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; TP, total power; LF, low frequency; HF, high frequency. All plasma metabolite and hormone samples were drawn in fasting conditions. TABLE 4. Multivariate analysis using baseline LF/HF as the dependent variable (n ⫽ 120) Variable
t
P
Age Body fat HOMA Fasting plasma triglycerides Leptin
⫺3.38 3.45 3.81 2.11 4.48
0.03 0.02 0.05 0.05 0.005
The r2 of the model was 0.63.
tin and the cardiovascular apparatus is also strengthened by the evidence that hyperleptinemia might be a component of a metabolic syndrome and also a cardiovascular risk factor (26). In fact, fasting plasma leptin was significantly correlated with arterial blood pressure, BMI, fasting plasma triglycerides, and serum uric acid (26). Our data for HRV are in agreement with the data demonstrating an excitatory effect of plasma leptin on cardiac ANS activity. Interestingly, our data demonstrated that the association between the variation in plasma leptin concentration and the cardiac ANS activity was independent of body fat. Despite the fact that the plasma leptin concentration is strictly correlated with body fat content, the relationship between plasma leptin concentration and cardiac ANS activity independently of body fat might be supported by the following experimental evidence: 1): direct activation of SNS activity (4, 27); 2) increase in norepinephrine turnover, as demonstrated in interscapular brown tissue (2, 23); and 3) decline of cholinergic activity at the cardiac level (25). All of those possibilities are also strengthened by evidence that leptin receptor messenger ribonucleic acid is expressed in the heart (28). Our data are in agreement with the prospective data showing a strong predictive role of obesity in sudden death (29). In fact, one can hypothesize that increased body fat content might be associated with a rise in plasma leptin concentration, which, in turn, could make the cardiac ANS
more sensitive and thus contribute to sudden death. Of course, only longitudinal future studies specifically designed to address this point can provide prove our hypothesis. Nevertheless, one cannot rule out that an increased body fat content might affect cardiac ANS through overactivity of the SNS (30, 31). Indeed, our study did not assess either muscle sympathetic nerve activity or plasma catecholamine turnover, so the latter hypothesis cannot be completely excluded. Notwithstanding, we measured the plasma catecholamine concentration at baseline and after orthostatic stimulus, and no significant difference among the study groups was found on either occasion. A potential limitation of our study was that only association among different variables were determined, and thus no cause-effect relationship would be drawn. Notwithstanding, human leptin is still not commercially available, and thus the effect of acute change in plasma leptin concentration, independently of other metabolic variables, cannot be investigated. Our data were only related to Caucasian men. Indeed, the limitation in race was only due to geographic reasons, as Caucasians represent more than 98% of the populatin of Italy. As far as gender is concerned, we only focused on men because of the sexual dimorphism (32–34) in plasma leptin concentrations. Indeed, it has been hypothesized that changes in the quality and quantity of sex hormones may significantly affect the plasma leptin concentration (33, 34). Thus, a study in women should also take into account menstrual cycle variations and include pre- and postmenopausal women; these variables make it difficult to compare women and men, and thus women should be investigated in a more appropriate experimental design. An unexpected finding of our study was that the association between the plasma leptin concentration and the cardiac ANS was independent of insulin resistance. The relationship between insulin resistance and cardiac sympathovagal balance is still debated. Briefly, it has been shown that hyper-
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TABLE 5. Changes in HRV parameters and fasting plasma catecholamine concentrations in different quartiles of plasma leptin concentration Quartiles of plasma leptin conc.
Total power (ms2) RR interval (ms) LF (nU) HF (nU) LF/HF Norepinephrine (nmol/L) Epinephrine (pmol/L)
1 (n ⫽ 33)
2 (n ⫽ 26)
3 (n ⫽ 32)
4 (n ⫽ 29)
P for trend
⫺1455 ⫾ 50 ⫺140 ⫾ 22 13.6 ⫾ 0.6 ⫺30.3 ⫾ 1.5 10.5 ⫾ 1.2 3.2 ⫾ 0.2 665 ⫾ 50
⫺1828 ⫾ 84 ⫺187 ⫾ 15 14.1 ⫾ 0.8 ⫺27.8 ⫾ 1.4 12.2 ⫾ 1.2 3.5 ⫾ 0.1 672 ⫾ 20
⫺1986 ⫾ 75 ⫺193 ⫾ 21 15.6 ⫾ 2.4 ⫺27.2 ⫾ 1.1 13.3 ⫾ 2.1 3.4 ⫾ 0.3 691 ⫾ 30
⫺2284 ⫾ 68 ⫺212 ⫾ 17 17.3 ⫾ 2.5 ⫺24.7 ⫾ 1.8 17.1 ⫾ 1.3 3.3 ⫾ 0.5 665 ⫾ 40
0.003 0.05 0.05 0.01 0.005 0.32 0.59
All results are the mean ⫾ SD. LF, Low frequency; HF, high frequency. P values for trends are adjusted for body fat content and distribution.
insulinemia/insulin resistance might attenuate the variability of the sinoatrial node activity in response to both sympathetic and parasympathetic influences in obese subjects (35). Our results do not confirm this finding, as we found that subjects high HOMAIR values and high plasma leptin concentrations had the strongest LF/HF response to tilt. The apparent discrepancy between our study and others may be due to the fact the subjects have different degrees of body fat, but they were not obese. Thus, one can hypothesize the occurrence of a threshold for plasma insulin concentration before the variability in sinoatrial node activity would be reduced. Nevertheless, only a doseeffect curve between plasma insulin concentration and variability in sinoatrial node activity would resolve such question. In conclusion, our study demonstrates that increasing fasting plasma leptin concentrations are associated with a shift of the sympathovagal balance toward a progressive increase in sympathetic activation and an increased response to orthostatic stimulus in nonobese subjects with different body fat contents. Nevertheless, only future studies made by acute changes in the human plasma leptin concentration will be able to demonstrate a cause-effect relationship. References 1. Pelleymounter MA, Cullen MJ, Baker MB, et al. 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 269:540 –543. 2. Collins S, Kuhn CM, Petro AE, Swick AG, Chrynk BA, Surwit RS. 1996 Role of leptin in fat regulation. Nature. 380:677. 3. Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI. 1997 Receptormediated regional sympathetic nerve activation by leptin. J Clin Invest. 100:270 –278. 4. Shek EW, Brands MW, Hall JE. 1998 Chronic leptin infusion increases arterial pressure. Hypertension. 31:409 – 414. 5. Sloan JA, Hooper M, Izzo JLJ. 1989 Effects of circulating norepinephrine on platelets, leukocyte and RBC counts by ␣1-adrenergic stimulation. Am J Cardiol. 63:1140 –1142. 6. Clayton S, Cross M. 1963 The aggregation of blood platelets by catecholamine and by thrombin. J Physiol. 169:82. 7. Kleiger RE, Miller JP, Bigger JT, Moss AJ. 1987 Decreases heart rate variability and its association with increase mortality after acute myocardial infarction. Am J Cardiol. 59:256 –262. 8. Martin GJ, Magid NM, Myers G, et al. 1987 Heart rate variability and sudden death secondary to coronary artery disease during ambulatory electrocardiographic monitoring. Am J Cardiol. 60:86 – 89. 9. Eckberg DL. 1997 Sympathovagal balance: a critical appraisal. Circulation. 96:3224 –3232. 10. Malliani A, Pagani M, Montano N, Mela GS. 1998 Sympathovagal balance: a reappraisal. Circulation. 98:2640 –2643. 11. Task Force of the European Society of Cardiology and North American Society of Pacing and Electrophysiology. 1996 Heart rate variability. Standards of measurement, physiological interpretation and clinical use. Circulation. 93:1043–1065. 12. Kamath MV, Fallen EL. 1993 Power spectral analysis of heart rate variability: a non-invasive signature of cardiac autonomic function. Crit Rev Biomend Engin. 21:245–311.
13. Montano N, Gnecchi Ruscone T, Porta A, Lombardi F, Pagani M, Malliani A. 1994 Power spectrum analysis of heart rate variability to assess the changes in sympathovagal balance during graded orthostatic tilt. Circulation. 90:1826 –1831. 14. Malliani A, Pagani M, Lombardi F, Cerruti S. 1991 Cardiovascular neural regulation explored in the frequency domain. Circulation. 84:482– 492. 15. Pagani M, Montano N, Porta A. 1997 Relationship between spectral components of cardiovascular variabilities and direct measures of muscle sympathetic nerve activity in humans. Circulation. 95:1441–1448. 16. Paolisso G, Manzella D, Ferrara N, et al. 1997 Glucose ingestion affects cardiac ANS in healthy subjects with different amounts of body fat. Am J Physiol 273:E471–E478. 17. WHO Study Group. 1985 Diabetes mellitus: report of a WHO Study Group. WHO Tech Rep Ser. 727:9 –17. 18. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. 1985 Homeostasis model assessment: insulin resistance and -cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia. 28:412– 419. 19. Kay SM. 1977 Modern spectral estimation–theory and application. Englewood Cliffs: Prentice Hall; 221–237. 20. Jonsen SJ, Andersen N. 1978 On power estimation in maximum entropy spectral analysis. Geophysics. 43:681– 690. 21. Haynes WG, Sivitz WI, Morgan DA, Walsh SA, Mark AL. 1997 Sympathetic and cardiorenal action of leptin. Hypertension. 30:619 – 623. 22. Snitker S, Pratley RE, Nicolson M, Tataranni PA, Ravussin E. 1997 Relationship between muscle sympathetic nerve activity and plasma leptin concentration. Obes Res. 5:338 –340. 23. Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI. 1997 Leptin increase sympathetic nerve activity to brown adipose tissue and kidney. FASEB J. 11(Suppl 3):A4. 24. Reams G, Villarreal D, Taraben A, Freman RH, Knoblich P. 1997 Renal effects of leptin in normotensive and spontaneously hypertensive rats. FASEB J 11:A258. 25. Van Vliet BN, Hall JE, Mizelle HL, Montani JP, Smith Jr MJ. 1995 Reduced parasympathetic control of heart rate in obese dogs. Am J Physiol. 269:H629 –H637. 26. Leyva F, Godsland IF, Ghatei M. 1998 Hyperleptinemia as a component of a metabolic syndrome of cardiovascular risk. Arterioscler Thromb Vasc Biol. 18:928 –933. 27. Hopkins PN, Hunt SC, Wu LL, Williams GH, Williams RR. 1996 Hypertension, dyslipidemia, and insulin resistance: link in a chain or spokes on a wheel? Curr Opin Lipidol. 7:241–253. 28. Tartaglia LA, Dembski M, Weng X. 1995 Identification and expression cloning of a leptin receptor, OB-R. Cell. 83:1263–1271. 29. Messerli FA, Numer BD, Ventura HO, Snyder DW. 1987 Overweight and sudden death. Arch Intern Med. 174:1725–1728. 30. Berne C, Fagius J, Niklasson F. 1989 Sympathetic response to oral carbohydrate administration. Evidence from microelectrode nerve recordings. J Clin Invest. 84:1403–1409. 31. Peterson HR, Rothshild M, Weinberg CR, Fell RD, McLeish KR, and Pfeifer MA. 1988 Body fat and the activity of the autonomic nervous system. N Engl J Med. 318:1077–1083. 32. Haffern SM, Gingerich RL, Miettinen H, Stern MP. 1996 Leptin concentrations in relation to overall adiposity and regional body fat distribution in Mexican Americans. Int J Obes. 20:904 –908. 33. Saad MF, Damiani S, Gingerich RL. 1997 Sexual dimorphism in plasma leptin concentration. J Clin Endocrinol Metab. 82:579 –584. 34. Paolisso G, Rizzo MR, Mone CM. 1998 Plasma sex hormones are significantly associated with plasma leptin concentration in healthy subjects. Clin Endocrinol (Oxf). 48:291–297. 35. Muscelli E, Edmin M, Natali L. 1998 Autonomic and hemodynamic response to insulin in lean and obese humans. J Clin Endocrinol Metab. 83:2084 –2089.
