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Division of Diabetes, Department of Medicine, University of Helsinki, Helsinki 00290, Finland
Address all correspondence and requests for reprints to: Hannele Yki-Järvinen, M.D., Department of Medicine, University of Helsinki, P.O. Box 340, 00029 HUCH, Helsinki, Finland. E-mail: ykijarvi{at}helsinki.fi
Abstract
It is unclear whether insulin sensitivity independent of body weight regulates control of heart rate variation (HRV) by the autonomic nervous system.
Insulin action on whole-body glucose uptake (M-value) and heart rate variability were measured in 21 normal men. The subjects were divided into 2 groups [normally insulin sensitive (IS, 8.0 ± 0.4 mg/kg·min) and less insulin sensitive (IR, 5.1 ± 0.3 mg/kg·min)] based on their median M-value (6.2 mg/kg·min). Spectral power analysis of heart rate variability was performed in the basal state and every 30 min during the insulin infusion.
The IS and IR groups were comparable, with respect to age (27 ± 2 vs. 26 ± 2 yr), body mass index (22 ± 1 vs. 23 ± 1 kg/m2), body fat (13 ± 1 vs. 13 ± 1%), systolic (121 ± 16 vs. 117 ± 14 mm Hg) and diastolic (74 ± 11 vs. 73 ± 11 mm Hg) blood pressures, and fasting plasma glucose (5.4 ± 0.1 vs. 5.5 ± 0.1 mmol/L) concentrations. Fasting plasma insulin was significantly higher in the IR (30 ± 4 pmol/L) than in the IS (17 ± 3 pmol/L, P < 0.05) group. In the IS group, insulin significantly increased the normalized low-frequency (LFn) component, a measure of predominantly sympathetic nervous system activity, from 36 ± 5 to 48 ± 4 normalized units (nu; 0 vs. 30–120 min, P < 0.001); whereas the normalized high-frequency (HFn) component, a measure of vagal control of HRV, decreased from 66 ± 9 to 48 ± 5 nu (P < 0.001). No changes were observed in either the normalized LF component [35 ± 5 vs. 36 ± 2 nu, not significant (NS)] or the normalized HF component (52 ± 6 vs. 51 ± 4 nu, NS) in the IR group. The ratio LF/HF, a measure of sympathovagal balance, increased significantly in the IS group (0.92 ± 0.04 vs. 1.01 ± 0.04, P < 0.01) but remained unchanged in the IR group (0.91 ± 0.04 vs. 0.92 ± 0.03, NS). Heart rate and systolic and diastolic blood pressures remained unchanged during the insulin infusion in both groups.
We conclude that insulin acutely shifts sympathovagal control of HRV toward sympathetic dominance in insulin-sensitive, but not in resistant, subjects. These data suggest that sympathetic overactivity is not a consequence of hyperinsulinemia.
ONE OF THE many actions of insulin is its action on the autonomic nervous system. These include stimulation of muscle sympathetic nerve activity (1), an effect which is observed already with physiological insulin doses and within a physiological time frame. In insulin-resistant states, such as in essential hypertension and obesity, basal sympathetic activity is increased (2); and in some studies, higher basal muscle sympathetic activity, as measured with microneurographic recordings, has correlated with the degree of insulin resistance (3, 4). This could mean that insulin stimulation of sympathetic nervous system activity is preserved in insulin resistant-subjects, i.e. that basal hyperinsulinemia in insulin-resistant individuals leads to sympathetic overactivation. When spectral power analysis of heart rate variation (HRV) has been used to study insulin action on autonomic nervous function (the ability of insulin to stimulate the sympathetic nervous system, as judged from an increase in the LF/HF ratio), a measure of so-called sympathovagal balance has also been blunted in obesity (5, 6), and basal sympathetic activity increased (5, 7). In insulin-resistant hypertensive and type 2 diabetic patients, blunted responses of the cardiac autonomic nervous system to insulin have also been recently described (7). It is, however, unclear whether the blunted response to insulin was a consequence of increased basal activity or to impaired insulin action per se. In the present study, we examine whether insulin regulation of components of HRV is influenced by insulin sensitivity that is independent of obesity.
Subjects and Methods
Subjects
Twenty-one male subjects were studied. All were healthy, and
none were taking any medications. In each subject, the effect of
insulin on whole-body glucose metabolism and on HRV were measured as
described below. For data analysis, the subjects were ranked according
to whole-body insulin sensitivity of glucose metabolism and were
divided into 2 groups based on the median M-value (6.2
mg/kg·min): a normally insulin-sensitive (IS); and a less
insulin-sensitive (IR) group. To verify that these terms accurately
characterized insulin sensitivity, we calculated the predicted insulin
sensitivity in these men, based on their age and body mass index (BMI),
using an equation generated from studies performed in 166 apparently
healthy Finnish men, 37 ± 1 yr old (range, 18–60 yr), with a BMI
of 25 ± 1 kg/m2 (range, 19–34
kg/m2), who have previously participated as
volunteers in studies assessing insulin sensitivity, with the same
method in our laboratory. As shown in Fig. 1
, the range (mean ± 95%
confidence intervals) of insulin sensitivity in the less
insulin-sensitive men fell clearly below the predicted range, whereas
the observed insulin sensitivity in the normally insulin-sensitive men
was close to that predicted to represent normal. Clinical
characteristics of these groups are given in Table 1. The nature and potential risks of the
study were explained to all subjects before obtaining their written
informed consent. The experimental protocol was approved by the Ethics
Committee of the Helsinki University Central Hospital.
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The euglycemic hyperinsulinemic clamp technique was used to assess tissue sensitivity to insulin (8). The clamp study was begun at 0800 h, after a 10- to12-h fast. Two 18-gauge catheters (Venflon, Viggo-Spectramed, Helsingborg, Sweden) were inserted, one in the left antecubital vein for infusions of insulin and glucose and another in the same arm retrogradely in a heated (60 C) dorsal hand vein for withdrawal of arterialized venous blood (8, 9). Insulin (Actrapid Human; Novo Nordisk, Copenhagen, Denmark) was infused in a primed continuous manner at a rate of 1 mU/kg·min for 120 min. Normoglycemia was maintained by adjusting the rate of a 20% glucose infusion, based on plasma glucose measurements that were performed every 5 min. Blood samples were taken for measurement of glycosylated hemoglobin and fasting serum free insulin concentrations before start of the insulin infusion, and serum free insulin concentrations were determined every 30 min during the insulin infusion. The rate of glucose infusion required to maintain normoglycemia, corrected for changes in the glucose pool size (M-value; Ref. 8), was used as a measure of whole-body insulin sensitivity.
Insulin action on HRV
Heart rate variation was measured using a 5-min controlled breathing test (10) at baseline and every 30 min during the insulin infusion. A quiet sound signal was given to pace the inspiration and expiration for 2 sec each. The controlled breathing pattern was maintained for 5 min, during which the electrocardiogram was recorded and R-R intervals were measured (R = R peak in QRS complex). Frequency domain analysis on heart rate variability was performed using spectral power analysis of R-R interval variability using CAFTS system (Medikro Oy, Kuopio, Finland). After detrending the R-R interval signal, a least-mean-square autoregressive model with a model order of 14 was used to obtain the power spectral estimate of R-R interval variability. Total power (TP) was determined in the frequency range from 0–0.5 times the heart rate, in Hz. LF power, which is thought to reflect both parasympathetic and sympathetic activity (11), was determined in the frequency range 0.04–0.15 Hz. HF power was determined in the frequency range 0.15–0.40 Hz. This component is thought to be determined by vagal activity (11). The signal powers were calculated as integrals under the respective part of the power spectral density function and were expressed in absolute units (ms2) and as a ratio (LF/HF) as a measure of sympathovagal balance (12). LF and HF were also expressed as normalized units (nu) [low- and HF components (LFn and HFn)], which are as follows: LFn = LF/(TP-VLF)·100, HFn = HF/(TP-VLF)·100. Normalization tends to minimize the effect of changes in total power on the LF and HF components (13).
Insulin action on peripheral blood flow
Because peripheral vasodilatation will induce changes in heart rate, we followed forearm blood flow during the insulin infusion. Forearm blood flow was measured in the right forearm using a mercury- in-rubber strain gauge venous occlusion plethysmography (Hokanson Plethysmograph model EC 4, Hokanson, Bellevue, WA), a rapid cuff inflator (E20, Hokanson), and a analog-to-digital converter (Maclab/4e, AD Instruments Pty Ltd., Castle Hill, Australia) connected to a personal computer. The gauge was attached to the widest, most muscular part of the forearm. Circulation to the hand was interrupted by inflating a pediatric blood pressure cuff around the wrist, 80 mm Hg above systolic blood pressure. Venous return was occluded by inflating a sphygmomanometer cuff around the upper arm to 50 mm Hg using a rapid cuff inflator. Forearm blood flow was recorded at 10- to 15-sec intervals during a 3-min period. Recordings were made under basal conditions and every 30 min during the insulin infusion. The mean of the final five measurements of each recording period was used for analysis (14).
Other measurements
Body composition (fat free mass and percentage of body fat) were determined using bioelectrical impedance plethysmography (BioElectrical Impedance Analyzer System model no. BIa-101A; RJL Systems, Detroit, MI; Refs. 15 and 16). Blood pressure was measured using a mercury sphygmomanometer. Waist circumference was measured at the level midway between spina iliaca superior and the lower rib margin, and hip circumference was measured at the level of greater trochanters. Plasma glucose concentrations were measured, in duplicate, with the glucose oxidase method (17), using a Glucose analyzer II (Beckman Coulter, Inc. Instruments, Fullerton, CA). Serum free insulin concentrations were determined by double-antibody RIA (Ref. 18 ; Pharmacia Insulin RIA kit, Pharmacia, Uppsala, Sweden) after precipitation with polyethylene glycol (19). Glycosylated hemoglobin was measured by high-performance liquid chromatography using a fully automated Glycosylated Hemoglobin Analyzer System (Bio-Rad Laboratories, Inc., Richmond, CA; Ref. 20). Serum total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol concentrations were measured with enzymatic colorimetric tests (Roche Molecular Biochemicals, Germany) using a 917 automatic analyzer (Hitachi Scientific Instruments, Inc., Osaka, Japan). Low-density lipoprotein (LDL) concentration was calculated using the formula of Friedewald.
Statistical analyses
The unpaired t test was used to compare mean values between the IS and IR groups. Fisher’s exact test was used to compare family history of diabetes and coronary heart disease between the groups. Because of their nonnormal distribution, power spectral parameters and fasting plasma insulin were transformed to their natural logarithms before statistical analysis and back-transformed for presentation in Figures and Tables. Comparison of parameters of insulin action on HRV was performed using repeated-measures ANOVA as described by Ludbrook et al. (21). Vertical pair-wise contrasts were performed using the unpaired t test followed by Bonferroni adjustment (21). Correlation analyses were performed using Spearman’s nonparametric correlation coefficient. The calculations were made using the Systat statistical package, version 7.0, (Systat, Evanston, IL) and Prism version 2.01 (GraphPad Software, Inc., San Diego, CA). A P-value less than 0.05 was considered statistically significant. Data are expressed as mean ± SEM.
Results
Clinical characteristics of the study subjects
The study groups were comparable, with respect to age, body
weight, BMI, waist/hip ratio, and several other characteristics (Table 1
). The mean M-values averaged 8.0 ± 0.4 mg/kg·min and 5.1
± 0.3 mg/kg·min in the IS and the IR groups. Figure 2 shows M-values, heart rate, mean
arterial pressure, and forearm blood flow at baseline and during the
insulin infusion in the IR and IS groups. All except M-value variables
remained unchanged during the insulin infusion, compared with baseline,
in both groups.
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At baseline, the fasting plasma insulin concentration was significantly higher in the IR (30 ± 3 pmol/L) than the IS (17 ± 2 pmol/L, P < 0.05) group. During the insulin infusion, the steady-state plasma free insulin concentration was also slightly higher in the IR (392 ± 15 pmol/L) than in the IS (354 ± 8 pmol/L, P < 0.05) group. Steady-state plasma glucose concentrations were comparable in the IR (5.1 ± 0.1 mmol/L) and IS (5.0 ± 0.1 mmol/L) groups.
Effect of insulin on HRV
At baseline, all heart rate spectral parameters were similar in
both groups (Table 2). During the insulin
infusion, the IS group showed a significant shift toward sympathetic
dominance of autonomic heart rate regulation (Table 2). Within the
first 30 min, insulin significantly increased LFn from 36 ± 5 to
50 ± 6 nu in the IS group (Fig. 3).
At the same time, the HFn decreased from 66 ± 9 to 48 ± 7
nu (Fig. 3), and LF/HF increased significantly. In the IR group, all
spectral power components remained unchanged during the insulin
infusion (Fig. 3). The changes in LFn, HFn, and LF/HF by insulin were
also significantly different between the groups (Fig. 3). Insulin
actions on whole-body glucose uptake (M-value) and components of HRV,
measured as changes in LFn, LF/HF, and HFn, were significantly
correlated (Fig. 4). The M-value was
positively correlated with the LFn component and the LF/HF ratio,
whereas M-value was inversely correlated with the HFn component (Fig. 4).
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We determined whether regulation of HRV by insulin is influenced by insulin sensitivity, defined as the ability of insulin to stimulate whole-body glucose metabolism. Under normoglycemic conditions, insulin caused a significant shift toward sympathetic activation, vagal deactivation, and thus, sympathetic predominance in insulin sensitive subjects. These changes were not a reflex response to peripheral vasodilatation, because peripheral blood flow, blood pressure, and heart rate all remained unchanged. In the less insulin-sensitive subjects, insulin failed to change any of the components of HRV. Because the sensitive and less-sensitive (or resistant) subjects were similar with respect to BMI, waist-to-hip ratio, and age, these data demonstrate that an individual’s inherent insulin sensitivity modulates the response of HRV to insulin.
The subjects were classified as insulin resistant and insulin sensitive based on the rate of whole-body glucose uptake. The insulin-resistant subjects had a mean rate of glucose uptake of 5.1 ± 0.3 mg/kg·min, which is higher than that characterizing older and more obese type 2 diabetic subjects (22) but lower than the average rate characterizing normal Finnish men of the same age and BMI (23). Thus, the term insulin resistant merely means less sensitive than the average, and it remains unclear whether a low value for this or associated parameters (including the inability of insulin to regulate HRV) has adverse effects on the subject’s subsequent health.
The present data in the insulin-sensitive subjects, showing an increase in the LF component and sympathovagal balance (as measured by the LF/HF ratio) is consistent with all (5, 7) except one (24) study using similar methodology in normal subjects. Insulin has also been previously shown to increase sympathetic activity by other methods in different anatomical locations. These include the ability of low physiological insulin concentrations to increase muscle sympathetic nerve activity (1, 25, 26, 27). At least, high insulin concentrations also have increased arterial plasma norepinephrine levels in most (1, 3, 7, 25, 28, 29), although not all (26, 30, 31), studies. Finally, insulin increases the norepinephrine spillover rate across forearm tissues (29, 32, 33).
Several studies have examined the ability of insulin to regulate autonomic nervous system activity in obesity. Using microneurography in skeletal muscle to record sympathetic nervous activity, obese subjects have had higher basal nerve activities (4, 34, 35) and a blunted subsequent stimulatory response to insulin (3). In keeping with data showing the LF component to correlate with muscle sympathetic nervous activity during sympathetic activation (36), the LFn component has also been higher in obese, than in nonobese, subjects under resting fasting conditions (5, 7) and failed to respond to insulin (5, 7). It is not, however, possible to conclude from these studies whether the failure of insulin to increase sympathetic activity was caused by a higher basal sympathetic activity or by insulin resistance. If anything, the paradoxical combination of elevated basal activity under fasting hyperinsulinemic conditions in obese subjects suggests preserved insulin action and contradicts the lack of an effect of higher insulin concentrations on sympathetic nervous activity. The present study may help to resolve this dilemma by demonstrating that regulation of the autonomic nervous system, as measured by spectral power analysis of HRV, is resistant to insulin, even in nonobese subjects. In our subjects, basal measures of autonomic control of HRV were similar between the sensitive and less-sensitive groups, despite fasting hyperinsulinemia in the latter group. This similarity of basal values is compatible with the idea that factors other than insulin are responsible for the increase in sympathetic nervous system activity in obesity. Hypothetically, such factors could include defective nitric oxide synthesis or action, which characterizes obese subjects (37). Nitric oxide inhibits central neural vasoconstrictor outflow, both in animals (38, 39, 40) and humans (41, 42). Alternatively, we have recently shown that, in young obese men, insulin fails to normally diminish arterial stiffness (43). The increased stiffness could diminish baroreceptor sensitivity and contribute to sympathetic hyperactivity in obesity (44).
Regarding the mechanism of insulin-induced activation of the sympathetic nervous system, it is known that the effect is mediated by insulin rather than stimulation glucose metabolism. Inhibition of glucose uptake and oxidation by lipid infusion (31) or fructose (26) does not interfere with insulin stimulation of sympathetic nervous system activity. The anatomical location of insulin’s effects is unclear. Possibly, the effect is centrally mediated (45, 46, 47), because intra-arterial infusions of insulin do not change the norepinephrine spillover rate, in contrast to iv infusions (29).
Reduced HRV predicts increased mortality in patients after myocardial infarction (48). Uncomplicated coronary artery disease is also associated with impaired circadian rhythm of cardiac neural regulation (49). In patients with type 2 (50) and type 1 diabetes (51) and neuropathy, power spectral components are reduced and resistant to respond to various stimuli such as breathing or changes in blood pressure. These patients are also at increased risk of sudden death (52). Thus, low and inflexible HRV seems to be associated with a poor cardiovascular prognosis and cardiovascular risk factors such as diabetes. Considering that insulin resistance is a strong predictor of type 2 diabetes (53) and that cardiovascular disease is its main complication, the present finding of unresponsiveness of HRV to insulin in insulin-resistant subjects could be considered yet another mechanism linking insulin resistance and cardiovascular dysfunction. This hypothesis assumes that failure of insulin to regulate HRV could be harmful, like autonomic dysfunction, defined as unresponsiveness of components of HRV to various other stimuli such as changes in posture, blood pressure, or breathing (54). On the other hand, sympathetic activation might trigger malignant arrhythmia, whereas vagal activity may exert a protective effect (55). Thus, although all known normal actions of insulin, including suppression of hepatic VLDL production (56), antiaggregatory effects on platelets (57), peripheral vasodilatation (37), and a decrease in arterial stiffness (58), are antiatherogenic, it is currently uncertain whether this also applies to insulin stimulation of the autonomic nervous system. Modulation of autonomic nervous control of HRV by chronic hyperinsulinemia may also differ from that caused by acute hyperinsulinemia. Interestingly, interventions known to enhance insulin sensitivity of glucose uptake, such as weight loss (59), physical training (60), and angiotensin-converting enzyme inhibitors (61), also increase basal HRV (62, 63, 64, 65) and decrease sympathetic activity. These data suggest that the unresponsiveness of HRV to insulin might be reversible.
Footnotes
1 Supported by grants from the Medical Society of Finland (to R.B.),
The Finnish Foundation for Cardiovascular Research (to A.S.-L.), the
Juselius Foundation (to H.Y. and R.B.), the Scandinavia-Japan Sasakawa
Foundation (to T.G.), the Academy of Finland (to H.Y.), and the
Helsinki University Central Hospital (to H.Y.). 
Received July 20, 2000.
Revised November 6, 2000.
Accepted November 9, 2000.
References
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