Download Increased high-frequency heart rate variability during insulin

Clinical Science (2004) 106, 583–588 (Printed in Great Britain)
Increased high-frequency heart rate variability
during insulin-induced hypoglycaemia
in healthy humans
Hartmut SCHÄCHINGER∗ , Johannes PORT†, Stuart BRODY‡, Lilly LINDER∗ ,
Frank H. WILHELM§, Peter R. HUBER, Daniel COX¶ and Ulrich KELLER∗∗
∗
Department of Internal Medicine, Clinical Research Center and Division of Psychosomatic Medicine, University Hospital,
4031 Basel, Switzerland, †Biomedical Institute, University of Stuttgart, D70174 Stuttgart, Germany, ‡Institute of Medical
Psychology and Behavioral Neurobiology, University of Tübingen, D72074 Tübingen, Germany, §Institute of Psychology,
University of Basel, 4031 Basel, Switzerland, Central Laboratories, University Hospital, 4031 Basel, Switzerland,
¶Department of Psychiatric Medicine, University of Virginia, Charlottesville, VA 22908, U.S.A., and ∗∗ Division of
Endocrinology, Diabetology and Clinical Nutrition, University Hospital, 4031 Basel, Switzerland
A
B
S
T
R
A
C
T
Despite causing sympathetic activation, prolonged hypoglycaemia produces little change in HR
(heart rate) in healthy young adults. One explanation could be concurrent parasympathetic activation, resulting in unchanged net effects of autonomic influences. In the present study, hypoglycaemic (2.7 mmol/l) and normoglycaemic (4.7 mmol/l) hyperinsulinaemic clamp studies were
performed after normoglycaemic baseline clamp periods with 15 healthy volunteers (seven male;
mean age, 27 years) on two occasions in a randomized single-blind cross-over design. Noninvasive indices of cardiac autonomic activity and hormones were measured at baseline and 1 h
after the beginning of hypoglycaemia or control normoglycaemia. Plasma insulin levels and mean
HR were similar during both conditions. During hypoglycaemia, there was a 485 % increase in
plasma adrenaline (epinephrine). A shortening of the pre-ejection period by 45 % suggested strong
sympathetic cardiac activation. High-frequency (0.15–0.45 Hz) HRV (HR variability) increased,
indicating a concomitant increase in parasympathetic tone. Thus, during hypoglycaemia-induced
sympathetic cardiac activation in healthy adults, parasympathetic mechanisms are involved in
stabilizing mean HR.
INTRODUCTION
The sympathetic counter-regulatory response to hypoglycaemia is characterized by systemic adrenaline (epinephrine) and noradrenaline release [1] and enhanced sympathetic neural activity [2,3]. It is assumed that such
sympathetic activation induces tachycardia [4]; however,
several early studies described healthy individuals displaying a different response pattern of stable HR (heart
rate), despite clear hypoglycaemia [5], or a return of
HR to baseline levels, despite continuing increases in
plasma adrenaline concentration [6]. Recent studies (i.e.
[7,8]) confirm these early reports in that minimal nonsignificant HR changes were observed during hypoglycaemia, despite the presence of the classical sympathetic counter-regulatory response that is known to involve
cardiac sympathetic activation [9]. Such findings could
be explained by increased vagal transmission to the heart
preventing increases in HR, but available reports are
controversial, describing hypoglycaemia-induced parasympathetic augmentation leading to atropine-sensitive
sinus bradycardia [10], and hypoglycaemia-induced
parasympathetic withdrawal leading to tachycardia in
sympathectomized humans [11]. As there is no direct
Key words: autonomic nervous system, glucose, heart rate variability, hypoglycaemia, impedance cardiography.
Abbreviations: HF, high frequency; HR, heart rate; HRV, HR variability; IBI, interbeat intervals; i.v., intravenous; LVET, left
ventricular ejection time; PEP, pre-ejection period; RMSSD, root mean square of successive differences.
Correspondence: Dr Hartmut Schächinger MD (e-mail [email protected]).
C
2004 The Biochemical Society
583
584
H. Schächinger and others
method to assess parasympathetic cardiac activity, studies
of HF (high-frequency) HRV (HR variability) can
provide further information on this issue; however, the
two available studies report conflicting results: a case
report [12] indicated increased HRV during hypoglycaemia, whereas a recent study [8] suggested no change in
HRV.
The aim of the present study was to clarify this issue
and examined parasympathetic HR control by spectral
analysis of HR time series, which were obtained in the
presence of cardiac sympathetic activation triggered by
experimental hypoglycaemia of extended (1 h) duration.
Time-domain-based indices of HRV, such as S.D. and
RMSSD (root mean square of successive differences) of
IBI (interbeat intervals) [13,14] were also used, because
they are simple and readily understood time domain
measures of parasympathetic cardiac control [15] that
are rather resistant to the effects of changing breathing
patterns [14].
It was hypothesized that parasympathetic cardiac
influences would increase during hypoglycaemia, as
reflected by increased HF HRV. Furthermore, it was
hypothesized that sympathetic cardiac influences (either
effected neurally or via systemic adrenaline) would increase during hypoglycaemia, as reflected by shortening
of the cardiac PEP (pre-ejection period). Plasma catecholamines and glucagon were examined to demonstrate
the initiation of normal counter-regulatory responses
to hypoglycaemia. Because hyperinsulinaemia activates
the sympathetic nervous system [16] and interacts
with the parasympathetic nervous system [17], similar
plasma insulin levels were implemented during target
hypoglycaemia and target normoglycaemia.
METHODS
Subjects
Fifteen healthy volunteers (seven male; mean age,
27 years) with normal findings on physical examination,
routine blood chemistry and haematology and standard
electrocardiography participated in the study. Exclusion
criteria were current smoking or evidence of any illicit
drug or sedative use. Subjects were asked to refrain from
alcohol, caffeine and food for 12 h before the laboratory
sessions. Subjects read and signed an Institutional
Review Board-approved informed consent form prior to
participation. The research was in accordance with the
Declaration of Helsinki and was approved by the Ethics
Committee of the Basel University Hospital.
Experimental procedures
Normo- and hypo-glycaemic clamp studies were
performed at the Basel University Hospital 4 weeks
apart, at 08:00 hours, according to a randomized singleblinded cross-over design. An i.v. (intravenous) catheter
C
2004 The Biochemical Society
was inserted into a dorsal vein of the left hand, which was
placed in a heated box (52 ◦ C) to arterialize the venous
sample. Another i.v. catheter was inserted into a cubital
vein of the same arm for glucose and insulin infusion.
A loading dose of human insulin (0.01 unit/kg of body
weight; Novo Nordisk, Mainz, Germany) was given,
followed by continuous insulin infusion (1 milliunit ·
min−1 · kg−1 of body weight). Glucose infusion (20 %
dextrose solution) was started at 2.5 mg · min−1 · kg−1 of
body weight and adjusted according to the plasma glucose
level, which was measured every 5–10 min by a glucose analyser (2300 STAT Plus; Yellow Springs Instruments,
Yellow Springs, OH, U.S.A.). During the 120 min baseline period of the clamp study, glucose was maintained at
fasting levels. During the 90 min target period, insulin
infusion was increased to 2 milliunits · min−1 · kg−1
of body weight. Glucose infusion rate was adjusted
on the first day to maintain a normoglycaemic level
(normoglycaemic target period) and, on the other
experimental day (counterbalanced design), it was
rapidly decreased to a glucose target of 2.7 mmol/l
(hypoglycaemic target period; the levels achieved are
presented in Table 1). Subjects were not informed of the
glucose target level (single-blinded design).
Plasma adrenaline and noradrenaline (determined
by enzyme immunoassay; IBL-Hamburg, Hamburg,
Germany), insulin (determined by immunometric assay;
DPC, Los Angeles, CA, U.S.A.) and glucagon concentrations (determined by RIA; Diagnostic Products,
Los Angeles, CA, U.S.A.) were assessed during baseline
(90 min) and after achieving glucose target levels
(75 min). Cardiovascular beat-to-beat data were assessed
at baseline (80 min) and 65 min after achieving target
levels. A standard three-lead ECG, intermittent blood
pressure (determined by Dinamap; Criticon, Tampa, FL,
U.S.A.), impedance cardiogram (determined by a modified Minnesota device; Diefenbach, Frankfurt, Germany)
and respiratory frequency (determined by a respiration
belt; Medizintechnik KSB, Basel, Switzerland) were
recorded. Analogue-to-digital conversion was performed
at 1000 Hz for off-line analysis of IBI and beat-to-beat
systolic time intervals from the impedance cardiogram
output (dz/dt signal; where z is the thoracic impedance). The B-point (upward deflection, representing the
beginning of ventricular ejection) and X-point (minimum, corresponding to the second heart sound and
closure of aortic valve) of the inverted dz/dt signal of each
single beat were identified by manual control assisted by
customized PC algorithms [18]. PEP was calculated from
the ECG Q-onset to B-point. LVET (left ventricular
ejection time) was calculated from B-point to X-point.
PEP and LVET were averaged per person and over a
5 min period.
A customized computer program written in MATLAB
[19] was used for computation of HF HRV (an index
of cardiac vagal control). The beat-to-beat values of
Cardiac autonomic control in hypoglycaemia
Table 1 Physiological variables during baseline and the target periods
Values are means (S.E.M.). BL1, baseline on the day of normoglycaemia, T-N, target period on the day of normoglycaemia, BL2, baseline on the day of
hypoglycaemia; T-H, target period on the day of hypoglycaemia. SBP, systolic blood pressure; DBP, diastolic blood pressure; RF, respiratory frequency.
F (degrees of freedom) and P values are shown.
Glucose (mmol/l)
Mean HR (beat/min)
Mean IBI (ms)
S.D. IBI (ms)
PEP (ms)
LVET (ms)
HF-HRV (ln ms2 )
RMSSD IBI (ms)
SBP (mmHg)
DBP (mmHg)
RF (Hz)
Adrenaline (pmol/ml)
Noradrenaline (nmol/ml)
Glucagon (pg/ml)
Insulin (pmol/l)
BL1
T-N
BL2
T-H
F (1,14)
P
4.7 (0.1)
69.4 (1.8)
875 (22)
57 (5.4)
104 (4)
271 (24)
6.2 (0.1)
39 (4.6)
120 (4)
66 (2)
0.27 (0.02)
0.44 (008)
2.9 (0.25)
73 (3)
363 (20)
4.8 (0.1)
71.7 (1.9)
847 (22)
57 (6.2)
92 (3)
279 (18)
6.2 (0.2)
40 (5.6)
121 (4)
66 (2)
0.28 (0.01)
0.45 (0.15)
2.68 (0.36)
60 (3)
809 (41)
4.7 (0.1)
71.4 (1.7)
849 (19)
52 (4.2)
101 (3)
273 (17)
6.1 (0.2)
36 (4.2)
125 (5)
69 (3)
0.28 (0.01)
0.49 (0.14)
2.5 (0.23)
65 (4)
335 (20)
2.7 (0.1)
71.6 (3.1)
863 (36)
70 (8.6)
54 (4)
281 (28)
6.6 (0.2)
47 (5.6)
124 (5)
58 (4)
0.31 (0.02)
2.84 (0.39)
4.69 (0.86)
110 (9)
782 (45)
160
0.8
2.3
8.2
78.2
0.0
6.35
4.6
0.7
19.7
2.7
52.7
6.6
58.5
0.0
0.0001
0.4
0.15
0.01
0.0001
0.99
0.02
0.05
0.42
0.0006
0.13
0.0001
0.02
0.0001
0.95
IBI were edited for outliers due to artefacts or ectopic
myocardial activity, linearly interpolated and converted
into instantaneous time series with a resolution of 4 Hz.
IBI series were linearly detrended, and the power spectral
densities derived for each experimental period using
the Welch algorithm [20], which assembles averages
of successive periodograms (a total of nine 240 point
segments, overlapping by 50 %, were analysed using
a Hanning window, zero-padded to length 256, and
subjected to fast Fourier transformation; estimates of
power were adjusted to account for attenuation produced
by the Hanning window). HF HRV was computed by
summing power spectral density values in the 0.15–
0.45 Hz frequency range, and resulting values were
normalized using the natural logarithm.
During the 5 min assessment of cardiovascular state,
subjects were seated in a semi-recumbent position,
instructed to close their eyes, relax and to neither
move nor speak. SAS software (release 8.0; SAS, Cary,
NC, U.S.A.) was used. Repeated measures ANOVA
tested hypoglycaemia effects (interaction term). Nonadjusted two-tailed P values are shown. Values are
means +
− S.E.M.
RESULTS
The details of key variables during baseline and the target
periods as shown in Table 1. Hypoglycaemia did not
alter HR or LVET, but HRV increased (spectral estimate
of HF HRV, as well as S.D. and RMSSD of IBI), PEP
shortened (Figure 1), diastolic blood pressure decreased
Figure 1 Signal averaged impedance cardiogram (− dz /dt
output) referenced to the R-wave maximum during baseline
and the target period (60 min)
Hypoglycaemia, but not normoglycaemia, produces shorter PEP and greater
− dz /dt output maximum. The signal-averaged ECG is provided. BL1, baseline
on the day of normoglycaemia; BL2, baseline on the day of hypoglycaemia;
T-N, target period on the day of normoglycaemia; T-H, target period on the day
of hypoglycaemia.
and adrenaline, noradrenaline and glucagon increased.
Mean respiratory frequency was not significantly greater
during hypoglycaemia. As expected, the doubling of the
insulin infusion rate from baseline to the target period
increased plasma insulin concentrations approx. 2-fold.
C
2004 The Biochemical Society
585
586
H. Schächinger and others
Plasma insulin levels were equal during hypoglycaemia
and the target period on the day of normoglycaemia.
DISCUSSION
In the present study, prolonged hyperinsulinaemic hypoglycaemic and normoglycaemic control clamp studies
were performed on two separate days according to a randomized single-blinded cross-over design. Cardiovascular variables and hormones were measured after 1 h. Increased plasma glucagon, adrenaline and noradrenaline
indicated effective initiation of the counter-regulatory
responses during hypoglycaemia. Shortened cardiac
PEP suggested sympathetic cardiac activation during
hypoglycaemia. If such a sympathetic cardiac response
was induced by adrenaline infusion, it would lead to
increased HR [18]. However, HR remained largely
unchanged, as in other hypoglycaemic clamp studies,
i.e. [7,8]. Such a response was to be expected if
efferent parasympathetic cardiac nerve activity prevented
tachycardia during hypoglycaemia. Indeed, increased HF
HRV suggests that such a vagal mechanism helped to
stabilize HR.
HR non-linearly modulates autonomic indexes [21],
potentially complicating the interpretation of HRV when
HR changes significantly. However, in the present study
of healthy volunteers, we demonstrate that HR did not
change during hypoglycaemia and, thus, this is not an
issue. Respiratory frequency is a potential confounder
of HRV [22], and respiratory frequency increased marginally during hypoglycaemia. If anything, this response
might result in diminished HRV [22,23]; however, we
found the opposite pattern, which argues against the increase in HRV during hypoglycaemia being attributable
to changes in respiratory frequency.
PEP derived from non-invasive impedance cardiography has been used as an index of cardiac sympathetic
nervous system activation [18,24] in previous autonomic
research. Although PEP is affected by pre- and afterload changes, it is unlikely that the change in diastolic
blood pressure found in the present study, as well as other
hypoglycaemia studies (i.e. [9]), sufficiently explains the
45 % shortening of PEP. A decrease in blood pressure
greater than those obtained in the present study was
related to a < 20 % shortening of PEP [18]. Furthermore,
systematic afterload changes induced by noradrenaline
or tyramine infusions were found to have little impact
on PEP when β-sympathetic influences were blocked
pharamacologically [25]. Thus β-sympathetic stimulation
seems to be far more important for the effects of PEP than
blood pressure changes. Radionuclide ventriculography
has revealed stable end-diastolic volumes during hypoglycaemia [9]. Thus preload changes do not play a role
in hypoglycaemia effects on the cardiovascular system.
The impact of glucagon on PEP seems to be of limited
C
2004 The Biochemical Society
importance [26]; however, its impact merits further
investigation.
Insulin itself impacts on neuroendocrine responses to
hypoglycaemia [27], can activate the sympathetic nervous
system [16] and decrease vagal influence on the heart [17].
Thus this hormone could potentially confound the results
of our present study. However, plasma levels of insulin
were the same during hypoglycaemia and during the
appropriate control condition. Thus our findings cannot
be explained by different insulin levels.
Normally functioning heart cells prefer fatty acids
as fuel. In normal heart cells, carbohydrate depletion
is insufficient to disturb heart cell metabolism severely
enough to deteriorate pacemaker function [28], but this
is not absolutely certain. However, current knowledge
suggests it is unlikely that, during mild-to-moderate
hypoglycaemia, HR is substantially influenced by factors
other than autonomic efferents or humoral influences.
Our present findings differ from the recent study by
Laitinen and co-workers [8], which suggested that cardiac
parasympathetic regulation of HR is not affected by
hypoglycaemia. Several methodological differences may
explain this difference. The study by Laitinen et al. [8]
used a paced breathing protocol at 0.2 Hz. In general,
this method has some advantages, because respiratory
frequency clearly has an impact on HRV [22,29] and,
thus, may confound findings. However, under conditions
of impaired cognitive function (such as during hypoglycaemia), it might be quite difficult to follow the
external pacing protocol and, therefore, this procedure
could very well be more stressful, resulting in ‘artificially’
decreased HRV [30]. More importantly, Laitinen et al. [8]
used a single day fixed sequence study design (normoglycaemia, followed by hypoglycaemia). Thus there was
no appropriate period controlling for influences of the
time of day and duration of hyperinsulinaemia. In our
experience, HRV tends to decrease during longer experimental protocols. In the case of the study by Laitinen
et al. [8], this burden would result in the underestimation of HRV during hypoglycaemia. This is not the
case in our present study that involved a 2 day singleblinded cross-over design in which the appropriate control period was matched for time of day, duration of
hyperinsulinaemia and other factors. Another potentially
relevant issue is that Laitinen et al. [8] targeted higher
plasma glucose values (3.0 compared with 2.7 mmol/l
in our present study; values are means +
− S.E.M.) and,
consequently, achieved lower plasma adrenaline concentrations (1.68 +
− 0.32 compared with 2.84 +
− 0.39 pmol/l).
Finally, plasma insulin levels during normoglycaemia
were higher compared with their hypoglycaemia period
(999 +
− 57 compared with 862 +
− 33 pmol/l; values are
means +
− S.E.M.), thus confounding autonomic nervous
system responses.
There are reports indicating that HR increases during
hypoglycaemia which seem to contradict our findings.
Cardiac autonomic control in hypoglycaemia
Bolus administration of insulin tended to be associated
with pronounced HR increases [9,31] and might have
been due to more severe peak hypoglycaemia. In the two
studies cited above, hypoglycaemia remained substantial
(blood glucose < 2.5 mmol/l) 30 min after bolus insulin
application and, similarly, adrenaline was significantly increased, as was cardiac sympathetic activation (increased
stroke volume and left ventricular ejection fraction).
Despite this cardiac sympathetic activation, HR had
already returned to baseline levels. Importantly, this was
not the case when the parasympathetic responses were
blocked with the muscarinergic inhibitor atropine. HR
did not return to baseline in subjects pretreated in this
way during normo- and hypo-glycaemia [31]. Instead,
HR remained elevated relative to normoglycaemia, suggesting that a functioning parasympathetic nervous system is required to blunt the initial hypoglycaemiainduced HR increase during prolonged hypoglycaemia.
Hypoglycaemia-induced parasympathetic activation
has been shown previously in the parotid glands [32],
gastrointestinal tract [33,34] and pancreas [35]; however,
the present report is the first controlled study demonstrating increased parasympathetic restraint at the cardiovascular level during hypoglycaemia.
Limitations
The present study does not elucidate whether the increased vagal cardiac drive results from a change in the
baroreflex or other influences. Baroreflex mechanisms
might be sensitive to increased stroke volume and increased pulse pressure even when mean blood pressure is
decreased.
The study design also does not allow the determination
of whether the sympathetic cardiac activation originates
from neural sympathetic discharge at the pacemaker cell
(clearly implying dual autonomic co-activation) or from
systemic adrenaline. Neural cardiac parasympathetic and
sympathetic co-activation has been reported much less
frequently than the dominance of one system. Previous
examples of mild co-activation include responses to an
aversive tone in the rat [36], in response to human
touch [37] and water ingestion [13]. Such results are
consistent with previous findings [38] and support the
use of autonomic models allowing for co-activation and
co-inhibition [24]. Whether the results of the present
study provide an example of much more substantial coactivation might be clarified by more invasive research
methods.
Clinical implications
There is experimental and epidemiological evidence
linking sympathetic cardiac activation during severe
hypoglycaemia to increased cardiac vulnerability of lifethreatening arrhythmias [39] and subsequent mortality
[40]. Compensatory or balancing parasympathetic effects
might be quite beneficial [41]. However, parasympathetic
compensation requires an intact parasympathetic neural
transmission system that is not present in DAN (diabetic
autonomic neuropathy). Thus it is possible that a proportion of the increased cardiovascular mortality in DAN
might be explained by insufficient parasympathetic tone,
leaving the heart subject to unmitigated sympathetic
activation during hypoglycaemia. This possibility merits
further investigation.
ACKNOWLEDGMENTS
This study was supported by the ‘Stiftung der Diabetes
Gesellschaft beider Basel’ and the University Hospital
Basel.
REFERENCES
1 Cryer, P. E. (1981) Glucose counterregulation in man.
Diabetes 30, 261–264
2 Fagius, J. and Berne, C. (1991) Rapid resetting of human
baroreflex working range: insights from sympathetic
recordings during acute hypoglycaemia. J. Physiol.
(Cambridge, U.K.) 442, 91–101
3 Paramore, D., Fanelli, C., Shah, S. and Cryer, P. (1999)
Hypoglycemia per se stimulates sympathetic neural as well
as adrenomedullary activity, but, unlike the
adrenomedullary response, the forearm sympathetic neural
response is not reduced after recent hypoglycemia.
Diabetes 48, 1429–1436
4 Fisher, B. M. and Frier, B. M. (1993) Haemodynamic
responses and functional changes in major organs.
Hypoglycaemia and Diabetes: Clinical and Physiological
Aspects (Fisher, B. M. and Frier, B. M., eds), pp. 144–155,
Edward Arnold, London
5 Ernestene, A. C. and Altschule, M. D. (1931) The effect of
insulin hypoglycemia on the circulation. J. Clin. Invest. 10
521–529
6 Christensen, N. J. (1974) Plasma norepinephrine and
epinephrine in untreated diabetics, during fasting and after
insulin administration. Diabetes 23, 1–8
7 Fruehwald-Schultes, B., Kern, W., Born, J., Fehm, H. L.
and Peters, A. (2000) Comparison of the inhibitory effect
of insulin and hypoglycemia on insulin secretion in
humans. Metab., Clin. Exp. 49, 950–953
8 Laitinen, T., Huopio, H., Vauhkonen, I. et al. (2003) Effects
of euglycaemic and hypoglycaemic hyperinsulinaemia on
sympathetic and parasympathetic regulation of
haemodynamics in healthy subjects. Clin. Sci. 105, 315–322
9 Fisher, B. M., Gillen, G., Dargie, H. J., Inglis, G. C. and
Frier, B. M. (1987) The effects of insulin-induced
hypoglycaemia on cardiovascular function in normal man:
studies using radionuclide ventriculography. Diabetologia
30, 841–845
10 Pollock, G., Brady, Jr, W. J., Hargarten, S., DeSilvey, D.
and Carner, C. T. (1996) Hypoglycemia manifested by
sinus bradycardia: a report of three cases. Acad. Emerg.
Med. 3, 700–707
11 Mathias, C. J., Frankel, H. L., Turner, R. C. and
Christensen, N. J. (1979) Physiological responses to insulin
hypoglycaemia in spinal man. Paraplegia 17, 319–326
12 Malpas, S. C. and Maling, T. J. (1989) Heart rate variability
during hypoglycaemia. Diabet. Med. 6, 822–823
13 Routledge, H. C., Chowdhary, S., Coote, J. H. and
Townend, J. N. (2002) Cardiac vagal response to water
ingestion in normal human subjects. Clin. Sci. 103, 157–162
14 Penttila, J., Helminen, A., Jartti, T. et al. (2000) Time
domain, geometrical, and frequency domain analysis of
cardiac vagal outflow: effects of various respiratory
patterns. Clin. Physiol. 21, 365–376
C
2004 The Biochemical Society
587
588
H. Schächinger and others
15 Hayano, J., Sakakibara, Y., Yamada, A. et al. (1991)
Accuracy of assessment of cardiac vagal tone by heart
rate variability in normal subjects. Am. J. Cardiol. 67,
199–204
16 Tack, C. J., Lenders, J. W., Willemsen, J. J. et al. (1998)
Insulin stimulates epinephrine release under euglycemic
conditions in humans. Metab., Clin. Exp. 47, 243–249
17 Bellavere, F., Cacciatori, V., Moghetti, P. et al. (1996) Acute
effect of insulin on autonomic regulation of the
cardiovascular system: a study by heart rate spectral
analysis. Diabet. Med. 13, 709–714
18 Schachinger, H., Weinbacher, M., Kiss, A., Ritz, R. and
Langewitz, W. (2001) Cardiovascular indices of peripheral
and central sympathetic activation. Psychosom. Med. 63,
788–796
19 Wilhelm, F. H., Grossman, P. and Roth, W. T. (1999)
Analysis of cardiovascular regulation. Biomed. Sci.
Instrum. 35, 135–140
20 Welch, P. D. (1967) The use of fast Fourier transform for
the estimation of power spectra: a method based on time
averaging over short modified periodograms. IEEE Trans.
Audio Electroacoust. 15, 70–73
21 Zaza, A. and Lombardi, F. (2001) Autonomic indexes based
on the analysis of heart rate variability: a view from the
sinus node. Cardiovasc. Res. 50, 434–442
22 Cooke, W. H., Cox, J. F., Diedrich, A. M. et al. (1998)
Controlled breathing protocols probe human autonomic
cardiovascular rhythms. Am. J. Physiol. 274, H709–H718
23 Pitzalis, M. V., Mastropasqua, F., Massari, F. et al. (1998)
Effect of respiratory rate on the relationships between
RR interval and systolic blood pressure fluctuations:
a frequency-dependent phenomenon. Cardiovasc. Res.
38, 332–339
24 Berntson, G. G., Cacioppo, J. T., Quigley, K. S. and Fabro,
V. T. (1994) Autonomic space and psychophysiological
response. Psychophysiology 31, 44–61
25 Schafers, R. F., Poller, U., Ponicke, K. et al. (1997) Influence
of adrenoceptor and muscarinic receptor blockade on the
cardiovascular effects of exogenous noradrenaline and of
endogenous noradrenaline released by infused tyramine.
Naunyn–Schmiedebergs Arch. Pharmakol. 355, 239–249
26 Ibler, M. and Dalsgaard, M. (1975) Influence of glucagon
on systolic time intervals during induction of anaesthesia
with barbiturate. Acta Anaesthesiol. Scand. 19, 206–209
27 Galassetti, P. and Davis, S. N. (2000) Effects of insulin per
se on neuroendocrine and metabolic counter-regulatory
responses to hypoglycaemia. Clin. Sci. 99, 351–362
28 Senges, J., Brachmann, J., Pelzer, D., Kramer, B. and
Kubler, W. (1980) Combined effects of glucose
and hypoxia on cardiac automaticity and conduction.
J. Mol. Cell. Cardiol. 12, 311–323
29 Badra, L. J., Cooke, W. H., Hoag, J. B. et al. (2001)
Respiratory modulation of human autonomic rhythms.
Am. J. Physiol. Heart Circ. Physiol. 280, H2674–H2688
30 Stark, R., Schienle, A., Walter, B. and Vaitl, D. (2000)
Effects of paced respiration on heart period and heart
period variability. Psychophysiology 37, 302–309
31 Fisher, B. M., Gillen, G., Hepburn, D. A., Dargie, H. J. and
Frier, B. M. (1990) Cardiac responses to acute insulininduced hypoglycemia in humans. Am. J. Physiol. 258,
H1775–H1779
32 Corrall, R. J., Frier, B. M., Davidson, N. M., Hopkins,
W. M. and French, E. B. (1983) Cholinergic manifestations
of the acute autonomic reaction to hypoglycaemia in man.
Clin. Sci. 64, 49–53
33 Schvarcz, E., Palmer, M., Aman, J. and Berne, C. (1995)
Atropine inhibits the increase in gastric emptying during
hypoglycemia in humans. Diabetes Care 18, 1463–1467
34 Hollander, F. (1968) The insulin test for the presence of
intact nerve fibers after vagal operations for peptic ulcer.
Gastroenterology (Suppl.) 54, 719–720
35 Cardin, S., Jackson, P. A., Edgerton, D. S., Neal, D. W.,
Coffey, C. S. and Cherrington, A. D. (2001) Effect of
vagal cooling on the counterregulatory response to
hypoglycemia induced by a low dose of insulin in the
conscious dog. Diabetes 50, 558–564
36 Quigley, K. and Berntson, G. (1990) Autonomic origins
of cardiac responses to nonsignal stimuli in the rat.
Behav. Neurosci. 104, 751–762
37 Wilhelm, F., Kochar, A., Roth, W. and Gross, J. (2001)
Social anxiety and response to touch: incongruence
between self-evaluative and physiological reactions.
Biol. Psychol. 58, 181–202
38 Eckberg, D. (1997) Sympathovagal balance: a critical
appraisal. Circulation 96, 3224–3232
39 Marques, J. L., George, E., Peacey, S. R. et al. (1997)
Altered ventricular repolarization during hypoglycaemia in
patients with diabetes. Diabet. Med. 14, 648–654
40 Sovik, O. and Thordarson, H. (1999) Dead-in-bed
syndrome in young diabetic patients. Diabetes Care 22,
B40–B42
41 Zamotrinsky, A., Kondratiev, B. and de Jong, J. (2001)
Vagal neurostimulation in patients with coronary artery
disease. Auton. Neurosci. 88, 109–116
Received 15 October 2003/6 January 2004; accepted 13 January 2004
Published as Immediate Publication 13 January 2004, DOI 10.1042/CS20030337
C
2004 The Biochemical Society