DOI: 10.1111/j.1464-5491.2004.01241.

Vascular dysfunction and autonomic neuropathy in

Vascular
Original
Oxford, UK
Diabetic
DME
Blackwell
0742-3071
21 article
dysfunction
Publishing,and
Article
Medicine Ltd.autonomic neuropathy in Type 2 diabetes C. Meyer et al.
2004

Type 2 diabetes
C. Meyer, F. Milat, B. P. McGrath, J. Cameron, D. Kotsopoulos and H. J. Teede

Abstract
Monash University Department of Medicine, Aims To test the hypothesis that arterial dysfunction in Type 2 diabetes is
Dandenong Hospital, Melbourne, Victoria, Australia related to autonomic neuropathy.
Accepted 8 October 2003 Methods Arterial function and autonomic neuropathy were assessed over two
consecutive days in 45 Type 2 diabetic and control subjects. Systemic arterial
compliance (SAC), arterial stiffness (pulse-wave velocity, PWV) and carotid
intima thickness (IMT) were assessed; these markers reflect early vascular disease
and predict clinical vascular events. Autonomic neuropathy was assessed using
heart rate variability with continuous ECG recording during various breathing
and postural manoeuvres and an overall autonomic score was generated. Fast-
ing metabolic parameters including glucose, insulin, HbA1c and lipid profile
were measured.
Results Autonomic neuropathy tests were all repeatable in diabetic subjects.
Compared with controls, diabetic subjects had arterial dysfunction with in-
creased PWV (P = 0.009), IMT (P < 0.001) and reduced SAC (P = 0.053). After
adjustment for age, central PWV correlated with fasting insulin (r 2 = 0.45,
P < 0.05) and autonomic score (r 2 = 0.44, P < 0.05), peripheral PWV correlated
with autonomic score (r 2 = 0.51, P < 0.005) and IMT correlated with fasting
insulin (r 2 = 0.5, P < 0.005). The presence of autonomic neuropathy correlated
with fasting insulin (P = 0.015), but not age, duration diabetes, lipids or blood
pressure.
Conclusion Using repeatable measures of autonomic neuropathy and vascular
function in Type 2 diabetic subjects, we have demonstrated associations
between autonomic neuropathy, vascular dysfunction and hyperinsulinaemia.
This may help to explain the excess cardiovascular mortality seen in diabetic
subjects with autonomic neuropathy.
Diabet. Med. 21, 746751 (2004)
Keywords arterial stiffness, arterial compliance, autonomic function,
hyperinsulinemia, Type 2 diabetes mellitus

cardiovascular risk. Other factors common in diabetes, which

Introduction
Patients with Type 2 diabetes mellitus have increased cardio-
vascular morbidity and mortality [1]. Clustering of conven-
tional cardiovascular risk factors such as hypertension, obesity
and dyslipidaemia explains some, but not all, of this increased
Correspondence to: Dr Helena Teede, Department of Vascular Science and
Medicine, Dandenong Hospital, David St, Dandenong, Victoria, 3175,
Australia. E-mail: h.teede@southernhealth.org.au
may also contribute to premature cardiovascular disease,
include autonomic neuropathy [2 4], vascular dysfunction [5]
and hyperinsulinaemia [6,7].
Autonomic neuropathy is a frequent complication in diabe-
tes with a reported prevalence of 46% in diagnosed Type 2
diabetes for less than 5 years and up to 70% with > 20 years of
diabetes [2]. It is a predictor of increased cardiovascular mor-
tality in diabetic subjects [2 4,8]. Although the pathophysio-
logical mechanism of this increased mortality is unclear, it has

746 2004 Diabetes UK. Diabetic Medicine, 21, 746751


been associated with fatal arrhythmias [9] and subclinical
coronary atherosclerosis [10].
Arterial dysfunction occurs early in atherosclerotic vascular
disease. It has been demonstrated in subjects with Type 2 dia-
betes and insulin resistance, as shown by increased arterial
stiffness [11] and endothelial dysfunction [12]. Recent pro-
spective studies have shown that pulse-wave velocity (PWV)
predicts cardiovascular mortality in those with chronic renal
failure [13] and in essential hypertension [14,15]. Despite the
influence of insulin and the autonomic nervous system on
vascular function, their relationships have not been studied
in Type 2 diabetes.
In this cross-sectional study, non-invasive measures of
arterial and autonomic function were examined in a group of
patients with Type 2 diabetes to test the hypothesis that auto-
nomic neuropathy is a determinant of arterial dysfunction in
diabetes. We also examined the role of hyperinsulinaemia to
determine whether insulin and autonomic neuropathy have
independent effects on arterial function.
Patients and methods
Study design
In this cross-sectional study, 45 participants with Type 2 dia-
betes mellitus, aged 4570 years, were recruited from community
advertisements. Exclusion criteria included: those on insulin,
smokers (last 10 years), prior stroke, ischaemic heart disease,
peripheral vascular disease and major co-existent illness. A
control group of 45 healthy subjects matched for age, sex and
body mass index (BMI) was recruited from the same suburban
population. Controls were normotensive, non-smokers and
had no history of cardiovascular disease. They were not on
any anti-hypertensive or lipid-lowering therapy which could
potentially alter arterial function. The Dandenong Hospital
Research Advisory and Ethics Committee approved the study
and all participants gave written informed consent.
Participants were assessed by an endocrinologist (CM) with
a baseline medical examination and history. In the diabetic
subjects, autonomic and vascular function tests were assessed
over two consecutive days to determine the repeatability. The
results presented are an average of the values obtained.
Autonomic function tests
In the diabetic group, autonomic function tests were performed
in the morning after a 12-h fast by the same two operators (FM
and CM) and were all analysed by one clinician (CM). The use
of alcohol was prohibited 24 h before the study and all medica-
tions were withheld for 12 h prior to the study. Tests were
preceded by a 5-min resting period, with patients in the supine
position.
The diagnosis of autonomic neuropathy was based on an
autonomic score generated from the results of heart-rate re-
sponse to single deep breathing, six consecutive breaths, Valsalva
manoeuvre, heart-rate change from lying to standing and postural
blood pressure change [2].
2004 Diabetes UK. Diabetic Medicine, 21, 746751
Original article 747
The heart-rate response to single deep breathing was taken as
the maximum minus the minimum rate during the breathing
cycle [2]. During six consecutive breaths (at a rate of six breaths/
minute), the result was taken as the mean difference in the
maximum and minimum heart rate during the last three breath-
ing cycles [16]. Heart-rate responses to the Valsalva manoeuvre
were measured from the difference between the shortest R-R
interval reflecting the tachycardia during the strain and the
longest R-R interval during the manoeuvre reflecting overshoot
bradycardia during release [16]. The heart-rate response during
the change from lying to standing was the difference between
the shortest R-R interval at or around the 15th beat and the
longest R-R interval at or around the 30th beat after standing
[16]. The postural fall in blood pressure was taken as the differ-
ence between the systolic blood pressure lying and standing
[16]. The heart-rate response to deep breathing, the Valsalva
manoeuvre and standing were used to reflect cardiac parasym-
pathetic integrity, while blood pressure response to standing
was used to reflect sympathetic function.
The results of the tests for autonomic function were analysed
for repeatability as recommended by Bland and Altman [17].
Repeatability coefficients were calculated using the formula
RC = 2 ( Di2 / n)
where Di is the absolute difference between the measurements
taken at visit 1 and visit 2 and n is the number of measurements.
Tests of autonomic function were all repeatable over the
2 days of testing. The correlation coefficient for the single
breath test between the two visits was 0.76; the coefficient of
variation was 10.1% and the repeatability coefficient was 3.54.
The corresponding values for the six breaths test were: 0.80,
8.6% and 3.38, for postural change; 0.78, 12.8% and 5.34,
and for Valsalva 0.81, 6.4% and 5.32. Bland Altman plots
showed no significant trends in the differences between meas-
urements for increasing values for any of the indices.
Arterial parameters
All arterial parameters were measured by an experienced
research assistant (DK). Studies were performed after a 12-h
fast in participants from the diabetic group and after at least an
8-h fast in the controls. During this period, caffeine-containing
drinks were avoided. All studies were performed in a darkened,
quiet, air-conditioned clinical laboratory following 10 min rest
in the supine position.
Total systemic arterial compliance
Non-invasive determination of blood flow and pressure wave-
forms were applied to determine systemic arterial compliance
(SAC) as previously described [18]. Aortic volumetric blood
flow was measured from a hand-held 3.5-MHz continuous
wave Doppler flow velocimeter (Multidoplex MD1, Huntleigh
Technology, Cardiff, UK) at the suprasternal notch. Simultane-
ous driving pressure was ascertained by applanation tonometry
with a pressure transducer (Millar Mikro-tip, Millar Instru-
ments, Houston, TX, USA) over the carotid artery, with
pressures calibrated against Dinamap brachial artery pressures
 748 Vascular dysfunction and autonomic neuropathy in Type 2 diabetes C. Meyer et al.
(CRITIKON 1846 SX). Compliance over the total systemic
arterial tree was calculated by the following formula according
to the method of Liu et al. [19].
Ad
(1) SAC =
R(Ps Pd )
R = MAP/Qmean = MAP/ [ r 2 Fmean]
r = 0.25 BSA + 0.52
Ad = area under the BP diastolic decay curve from end-systole
to end-diastole; Ps = end-systolic BP; Pd = end-diastolic BP
(carotid); MAP is mean arterial pressure; R = total peripheral
resistance; Qmean is mean flow; Fmean is mean velocity; r is aortic
radius and BSA is body surface area [20].
Pulse-wave velocity
Pulse-wave velocity was determined from recorded pressure
waveforms (applanation tonometry) over both the aorto-
femoral (central) and the femoro-dorsalis pedis (peripheral)
arterial segments [18]. Central pulse-wave velocity was meas-
ured by simultaneous applanation at the carotid and femoral
arteries with transit distance defined as the distance from
the manubrium to the femoral applanation point minus that
from the manubrium to the carotid applanation point [21].
Peripheral pulse-wave velocity was derived over the femoral-
dorsalis pedis arterial segment by simultaneous applanation
tonometry at these points. Transit distance was the distance
between applanation points. Pulse transit time was defined
as the time between the foot of simultaneously recorded
pressure waves, occurring at the end of diastole, and the begin-
ning of systole, averaged over 10 cardiac cycles. Velocity was
derived from computer-generated pulse transit times and
measured distances between the two applanation sites, as pre-
viously described [18]. Pulse-wave velocity was calculated
based on the formula:
Pulse wave velocity = D/t (m /s) where D = distance, t = time
interval.
Intima-media thickness (IMT)
This parameter was derived from non-invasive ultrasound of
the common carotid arteries, using a high-resolution ultra-
sound machine (Diasonics DRF-400, USA) with 7.5-MHz
mechanical sector transducer (7.5-SPC). The IMT was defined
as the distance between the blood-intima and media-adventitia
boundaries on B-mode imaging [ 22]. The far wall of the right
common carotid artery, 1 cm proximal to the origin of the bulb,
was selected, as it has been shown to be the most reproducible
[23]. Three B-mode images were recorded from different angles
then digitized and saved on computer via a customized com-
puter program (A House of Windows, C. Smith, Auckland,
New Zealand) as previously described [24]. Brachial blood
pressure recordings were recorded every 5-min throughout the
imaging period using a Dinamap device (CRITIKON, 1846 SX).
Image analysis was performed using a standardized measure-
ment protocol, using 30 data points per subject, by the same
sonographer. Measurements were automatically transferred and
saved in a database (Quest for Windows, version 2.1). The results
are reported as mean common carotid IMT.
Laboratory methods
Fasting blood samples were taken from all participants in both
groups on the morning of the first study. Total cholesterol and
triglycerides were measured using enzymatic reagents (DADE
Diagnostics, Brisbane, Australia), HDL cholesterol was meas-
ured by homogeneous assay techniques (HDLC-Plus, DADE
Diagnostics) adapted to a DADE Dimension RXL chemistry
analyser (DADE Diagnostics). LDL cholesterol was calcul-
ated using the Friedewald equation [LDL-C = (TC-HDL-
C)-(triglycerides/2.2)], adapted to SI units. The insulin assay
is the AxSYM assay based on the Microparticle Enzyme
Immunoassay (MEIA) technology. The sensitivity of the assay
is 1.0 U insulin /ml, the cross reactivity with Proinsulin is
0.016% and there is no cross reactivity with C-peptide. Plasma
glucose was determined with the glucose oxidase method.
Statistical analysis
Statistical calculations were performed using the SPSS Inc.
statistical package, version 11 (SPSS Inc., Chicago, IL, USA).
Results are expressed as mean SE. Repeatability of measure-
ments of autonomic function in the diabetic group were exam-
ined by constructing Bland Altman plots and determining
repeatability coefficients for each variable [17]. The differences
between groups were assessed by Students t-test. Stepwise re-
gression analyses were performed to examine the determinants
of vascular dysfunction and autonomic neuropathy. Signific-
ance was accepted at P < 0.05.
Results
In total, there were 45 Type 2 diabetic and 45 control subjects
matched for age, sex and body mass index. In the diabetic
group, the average duration of diabetes was 10.6 years, 34/45
were taking one or more anti-hypertensives (nine on a beta
blocker, 22 on an ACE inhibitor and/or angiotensin II antago-
nist, 11 on a calcium channel antagonist) and 15/45 were tak-
ing lipid lowering medication. None of the control subjects
were on anti-hypertensive or lipid-lowering medication.
Table 1 gives a comparison of subject characteristics and
mean values for clinical chemistry and blood pressure meas-
urements in all participants. Subjects with diabetes had elev-
ated fasting glucose, total cholesterol, LDL and triglycerides
and reduced HDL compared with controls. The diabetic group
had higher systolic and diastolic pressures based on the average
of three readings taken whilst the subject was resting supine
prior to vascular function testing (Table 1).
Autonomic function tests
Autonomic function was tested in the 45 subjects with Type 2
diabetes. The incidence of autonomic neuropathy in the study
2004 Diabetes UK. Diabetic Medicine, 21, 746751
 Original article 749

Table 1 Subject characteristics, blood pressure and lipid parameters Table 3 Age-adjusted inter-relationship of parameters

Diabetics Controls P value AN score Plasma insulin HbA1c Duration of DM2

n 45 45 PWV central 0.44* 0.45* NS NS

Men 22 22 PWV peripheral 0.51** NS NS NS
Women 23 23 Carotid IMT NS 0.50** NS NS
Age (years) 60.9 1.2 59.5 1.1 NS SAC NS NS NS NS
BMI (kg/m2) 28.9 0.7 27.2 0.84 NS
Fasting plasma glucose (mmol/ l) 7.98 0.3 5.03 0.2 < 0.001 Partial correlation coefficient values *P < 0.05, **P < 0.005.
Total cholesterol (mmol/ l) 6.04 0.2 4.78 0.1 < 0.001
Triglycerides (mmol/ l) 1.88 0.1 1.28 0.1 0.002
diabetic and control groups. Diabetes was associated with
HDL (mmol/ l) 1.19 0.05 1.59 0.06 0.005
LDL (mmol/ l) 3.88 0.2 2.84 0.1 0.005
significant arterial dysfunction with elevated central and
Systolic BP (mmHg) 135.3 2.2 125.1 1.8 0.001 peripheral pulse wave velocity (arterial stiffness) and reduced
Diastolic BP (mmHg) 88.5 1.4 75.2 1.1 0.005 SAC (arterial elasticity) compared with controls. Because of
the difference in resting blood pressure in the two groups,
pulse-wave velocity and SAC measurements were adjusted for
Table 2 Arterial function parameters adjusted for MAP mean arterial pressure. The differences between the groups
persisted for central and peripheral pulse-wave velocity, but
Diabetics Controls P value
not for SAC ( Table 2).
PWV central (m/s) 10.09 0.26 9.06 0.29 0.009 Diabetic subjects also had increased carotid IMT compared
PWV peripheral (m/s) 11.24 0.27 10.23 0.26 0.008 with controls, consistent with earlier atherosclerotic arterial
SAC (units/mmHg) 0.45 0.03 0.52 0.02 0.053 disease in diabetes.
Carotid IMT (mm) 0.88 0.02 0.68 0.02 < 0.001

Autonomic neuropathy, plasma insulin and arterial structure

population was 33/45 (73%) as defined by an overall auto-
nomic score of > 3 (maximum score 6) [2]. The average overall
score in the study population was 4.1. The heart rate variabil-
ity to Valsalva manoeuvre, deep breathing and standing which
largely reflect cardiac parasympathetic integrity, were ab-
normal in 33/45 subjects, while the blood-pressure response to
standing, a reflection of sympathetic function, was abnormal
in only 1/45 subjects. This patient had an overall autonomic
score of 5, reflecting more severe autonomic neuropathy.
Of the diabetic subjects, there were no significant differeces
between those with and without autonomic neuropathy in the
number taking each medication ( Blockers P = 0.72, ACEI /
AGII antagonists P = 0.57, calcium channel blockers P = 0.57).
and function in diabetic subjects
Autonomic neuropathy (total score), was correlated with
fasting insulin levels (P = 0.015), but not with age, duration
of diabetes, lipids, blood pressure or HbA1c.
On stepwise linear regression modelling age, autonomic
score and insulin were all independently associated with central
pulse wave velocity ( Table 4) (sequential r = 0.64, r 2 = 0.41;
P = 0.025). Similar modelling with peripheral pulse-wave veloc-
ity identified that the autonomic score was the only predictive
variable (r = 0.53, r 2 = 0.23; P < 0.001), while independent
predictive variables for IMT were age and insulin (sequential
r = 0.57, r 2 = 0.33; P = 0.001). None of the arterial indices
correlated with duration of diabetes or HbA1c levels ( Table 3).

Arterial structure and function Discussion

Table 2 compares mean ( SEM) for SAC, central pulse-wave In the current study, we have demonstrated arterial dys-
velocity, peripheral pulse-wave velocity and carotid IMT in function in Type 2 diabetic subjects compared with controls

Table 4 Summary of stepwise linear regression for central PWV (dependent variable) with the following variables: age, autonomic score and insulin

Std. error Change statistics

Adjusted of the
Model R R square R square estimate R square change F change df1 df2 Sig. F change

1 0.41* 0.17 0.15 1.59 0.17 8.16 1 41 0.007

2 0.57 0.33 0.29 1.45 0.16 9.68 1 40 0.003
3 0.64 0.41 0.36 1.37 0.08 5.40 1 39 0.025

*Predictors: (Constant), AGE.

Predictors: (Constant), AGE, ANSCORE.
Predictors: (Constant), AGE, ANSCORE, INSULIN.

2004 Diabetes UK. Diabetic Medicine, 21, 746751

 750 Vascular dysfunction and autonomic neuropathy in Type 2 diabetes C. Meyer et al.
matched for age, sex and body mass index. Both central and
peripheral pulse-wave velocity were significantly and inde-
pendently correlated with the presence of autonomic neuro-
pathy in those with diabetes, in addition we found a significant,
independent association between central pulse-wave velocity
and hyperinsulinaemia.
Carotid IMT is an established marker for early atheroscle-
rotic disease [22]. IMT was significantly increased in the
diabetic population compared with controls and was inde-
pendently associated with age and fasting hyperinsulinaemia.
This is consistent with other studies which have demonstrated
that IMT is positively correlated with fasting C-peptide and
insulin levels [25] and with insulin resistance [26,27]. These
findings support the hypothesis that hyperinsulinaemia per se
may contribute to accelerated atherosclerosis in diabetes in-
dependently of other cardiovascular risk factors.
Central pulse-wave velocity, reflecting central elastic
arterial stiffness, is predictive of cardiovascular mortality in
chronic renal failure [13] and essential hypertension [14,15].
In a cohort of patients with chronic renal failure followed
for a mean of 60 months, an increase in central pulse-wave
velocity of 1 m/s was associated with a 39% increase in
mortality [28]. Arterial stiffness has been correlated with
metabolic parameters of glycaemic control and hyper-
insulinaemia in both diabetic [29,30] and non-diabetic
subjects [31,32].
In this study, we have shown a greater central pulse-wave
velocity and after correction for blood pressure and a trend
towards a lower SAC in diabetic compared with control sub-
jects. Calculation of central pulse-wave velocity as described
(with subtraction of the manubrium-carotid distance [21])
effectively precludes the most elastic proximal aortic segment
from the measured central pulse-wave velocity. Differential
effects between the most proximal and more distal thoraco-
abdominal aorta related to increasing smooth muscle content
likely explain the discrepancies seen in the degree of difference
between SAC and pulse-wave velocity in our groups.
Peripheral pulse-wave velocity reflects arterial stiffness in
smaller, more muscular arteries. Patients with diabetes were
found to have an increase in peripheral pulse-wave velocity
compared with normal subjects matched for age, sex and body
mass index. This difference persisted after adjustment for
mean blood pressure. As with the increase in central pulse
wave velocity, this may be representative of an increased
burden of atherosclerotic disease in the diabetic subjects.
However, peripheral pulse-wave velocity did not correlate with
other traditional cardiovascular risk factors such as age, lipids
or hyperinsulinaemia, and a more likely possibility is that
peripheral pulse-wave velocity is influenced by autonomic
dysfunction in diabetes, as supported by the relationship with
the autonomic score.
In the current study, we demonstrated a novel relationship
between markers of arterial dysfunction, hyperinsulinaemia
and autonomic neuropathy in a Type 2 diabetic population.
Previous studies have identified an association between
arterial stiffness and autonomic neuropathy [33], whilst
others have demonstrated a link between autonomic neuropathy
and hyperinsulinaemia [34,35]. This, however, is the first
study which has demonstrated an association between all of
these three factors in Type 2 diabetes.
Our results suggest that autonomic neuropathy is an import-
ant determinant of arterial dysfunction in Type 2 diabetes.
Moreover hyperinsulinaemia and autonomic function have
independent, direct effects which appear to be occurring in
parallel, on the vasculature.
The role of hyperinsulinaemia as an independent predictor
of increased arterial stiffness and atherosclerosis has been
established in both diabetic and non-diabetic populations
[25,31]. In addition, autonomic dysfunction has also been
independently associated with aortic stiffness in a Type 1 dia-
betic population [33]. Whilst hyperinsulinaemia is thought to
influence vascular stiffness and atherosclerosis through direct
trophic effects [36], stimulation of vascular smooth muscle cell
growth [37] and by altering lipid metabolism within the vessel
wall [36], the mechanism by which autonomic dysfunction
may lead to increased arterial stiffness is less clear.
The autonomic nervous system is known to have an impor-
tant role in the generation of the circadian blood pressure and
heart-rate pattern [38,39]. In conditions that affect autonomic
nerve function, such as pure autonomic failure [39] or diabetic
neuropathy [40], the circadian variation of blood pressure has
been shown to be blunted or reversed, leading to an overall
increase in the 24-h blood pressure load. As blood pressure
load increases, the distending pressure within elastic central
arteries rises leading to a greater portion of the load bearing
function shifting from the elastic fibres of the aortic wall to less
distensible collagen fibres [41]. In healthy arteries, the elastic
vessels distend with increased intraluminal pressure serving a
buffering function (compliance). As these fibres are lost, so
too is this compliance function, resulting in increased vessel
stiffness. Elevated heart rate has also been associated with
increased arterial stiffness in elderly normotensive and hyper-
tensive subjects [32,42]. Potentially, a loss of nocturnal dip in
heart rate as occurs in autonomic neuropathy, might lead to
accelerated fatigue of elastic stretch fibres and thus increased
arterial stiffness, by either increasing the number of stretch
cycles or by not allowing sufficient relaxation time for large
arteries between ventricular contractions [32].
Our novel finding of the association between autonomic
neuropathy, hyperinsulinaemia and vascular dysfunction
may help to explain the excess cardiovascular mortality seen
in those Type 2 diabetic subjects with autonomic neuropathy.
We hypothesize that both hyperinsulinaemia and autonomic
neuropathy have pathogenic roles in the development of
cardiovascular disease. The results of our study indicate that
they may be occurring as parallel, independent processes,
however, further studies are required to characterize these
relationships, in particular the state of sympathetic activation
in Type 2 diabetes, which may be an important prognostic
indicator.
2004 Diabetes UK. Diabetic Medicine, 21, 746751

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