Hannele Yki-Jrvinen

Satu Vehkavaara, Sari Mkimattila, Anna Schlenzka, Juha Vakkilainen, Jukka Westerbacka and
Insulin Therapy Improves Endothelial Function in Type 2 Diabetes
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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is published by the American Heart Association, 7272 Arteriosclerosis, Thrombosis, and Vascular Biology
doi: 10.1161/01.ATV.20.2.545
2000;20:545-550 Arterioscler Thromb Vasc Biol.
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Insulin Therapy Improves Endothelial Function in
Type 2 Diabetes
Satu Vehkavaara, Sari Makimattila, Anna Schlenzka, Juha Vakkilainen,
Jukka Westerbacka, Hannele Yki-Jarvinen
AbstractA total of 75 in vivo endothelial function tests (intrabrachial artery infusions of endothelium-dependent
[acetylcholine] and -independent [sodium nitroprusside] vasoactive agents) were performed in 18 type 2 diabetic
patients (aged 582 years, body mass index 28.50.6 kg/m
2
, and fasting plasma glucose 22911 mg/dL) and 27
matched normal subjects. These tests were performed before and 6 months after combination therapy with insulin and
metformin and before and 6 months after metformin therapy only. Before insulin therapy, blood flow responses to
acetylcholine (15 g/min) were significantly blunted in type 2 diabetic patients (7.50.7 mL dL
1
min
1
) compared
with normal subjects (11.60.9 mL dL
1
min
1
, P0.01). During insulin therapy, the acetylcholine response
increased by 44% to 10.81.6 mL dL
1
min
1
(P0.05). Insulin therapy also significantly increased the blood flow
responses to both low and high doses of sodium nitroprusside. We conclude that insulin therapy improves
endothelium-dependent and -independent vasodilatation. These data support the idea that insulin therapy has beneficial
rather than harmful effects on vascular function. (Arterioscler Thromb Vasc Biol. 2000;20:545-550.)
Key Words: endothelium

atherosclerosis

vasodilatation

insulin therapy

type 2 diabetes
T
he UK Prospective Diabetes Study (UKPDS) showed
that intensive blood glucose control with insulin or
sulfonylureas, both of which significantly increase circulating
free insulin concentrations, retards the development of mi-
crovascular complications.
1
The incidence of myocardial
infarction decreased by 16%, which was almost statistically
significant (P0.052). Neither insulin nor sulfonylureas had
adverse effects on cardiovascular outcome.
1
In contrast to
these drugs, a substudy of the UKPDS suggested metformin
to be cardioprotective in overweight patients.
2
The patients
treated with metformin were not better controlled but gained
less weight and had lower serum free insulin concentrations
than did those treated with other regimens.
2
Endothelium-dependent vasodilatation is diminished in
atherosclerotic coronary arteries
3,4
and characterizes patients
at risk of developing atherosclerosis, such as those with
hypercholesterolemia, type 2 diabetes, and hypertension.
57
In patients with type 2 diabetes, the impaired vasodilator
response to endothelium-dependent vasodilators, such as
acetylcholine (ACh), has been a rather consistent finding.
812
In addition, responses to endothelium-independent vasodila-
tor agents, such as sodium nitroprusside (SNP), have been
found to be impaired in many
8,11
although not all
9,13
studies.
These data suggest that the function of endothelial or vascular
smooth muscle cells or both may be abnormal in type 2
diabetic patients. It is also possible that inactivation of
exogenous and endogenous nitric oxide is increased.
14
It is
currently unknown, however, whether these defects are in-
herent features of type 2 diabetes or secondary to metabolic
alterations such as chronic hyperglycemia,
15,16
increases in
free fatty acid (FFA) concentrations,
1721
or other lipid
abnormalities.
18,22,23
Regarding insulin, acute studies have
shown that although physiological changes in circulating
insulin concentrations do not alter blood flow, they potentiate
ACh-induced vasodilatation.
24
The only treatment study hith-
erto performed in type 2 diabetes examined effects of
antioxidants on endothelial function.
10
The present study was undertaken to determine whether
insulin therapy changes in vivo endothelial function in
patients with type 2 diabetes. Because metformin diminishes
weight gain during insulin therapy and may improve cardio-
vascular outcome,
25
we chose to study the effects of 6 months
of combination therapy with insulin and metformin on endo-
thelial function. These data were compared with a group of
normal subjects and with a group of type 2 diabetic patients
chronically treated with metformin.
Methods
Study Design
In vivo endothelial function was measured in 18 type 2 diabetic
patients previously treated with metformin (1000 mg BID, n14;
500 mg BID, n4) before and 6 months after combination therapy
with bedtime human isophane insulin and metformin (effect of
insulin therapy). A control group of 27 normal subjects was studied
to determine whether endothelial function was abnormal in the type
2 diabetic patients.
Received August 5, 1999; revision accepted August 5, 1999.
From the Department of Medicine, Division of Endocrinology and Diabetology, Helsinki, Finland.
Correspondence to Hannele Yki-Jarvinen, MD, University of Helsinki, Department of Medicine, PO Box 340, 00029 HUCH, Helsinki, Finland. E-mail
ykijarvi@helsinki.fi
2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
545
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Inclusion and Exclusion Criteria
The type 2 diabetic patients were recruited from diabetes outpatient
clinics in the Helsinki area on the basis of the following criteria: (1)
aged 40 through 70 years, (2) treatment with metformin alone, (3)
glycosylated hemoglobin (HbA
1C
) 8.5% (reference range 4.0% to
6.0%), (4) current body mass index 35 kg/m
2
, (5) duration of
diabetes 3 years, and (6) no history of ketoacidosis. Exclusion
criteria included the following: (1) clinically significant cardiovas-
cular, hepatic, neurological, endocrinologic, or other major systemic
disease, (2) retinopathy requiring laser treatment, (3) an elevated
serum creatinine concentration, (4) history of drug or alcohol abuse,
and (5) mental illness rendering the subjects unable to understand the
nature, scope, and possible consequences of the study. Informed
written consent was obtained after the purpose, nature, and potential
risks were explained to the subjects. The experimental protocol was
designed and performed according to the principles of Helsinki
Declaration and was approved by the Ethical Committee of the
Helsinki University Central Hospital.
Insulin Therapy
Patients considered eligible to participate in the study met with the
doctor and the diabetes nurse 4 weeks before the start of insulin
treatment. At this visit, the patients underwent a complete history and
physical examination. The patients were instructed to measure their
fasting blood glucose concentrations and to record any episode of
symptomatic hypoglycemia daily. The patients then visited the
laboratory for measurement of fasting blood glucose, HbA
1c
, serum
concentrations of creatinine, liver enzymes, and the urinary albumin
excretion rate. An ECG was also recorded. The results of the
laboratory tests were checked, and if acceptable, an endothelial
function test was performed before the start of insulin treatment with
bedtime human isophane. Treatment with metformin was continued
without changing the metformin dose. The patients were taught
self-adjustment of the insulin dose on the basis of fasting plasma
glucose measurements. The patients were asked to increase the dose
by 2 and 4 IU/d if the fasting plasma glucose exceeded 144 and 180
mg/dL on 3 consecutive measurements. The goal was to lower
fasting plasma glucose to 108 mg/dL and HbA
1c
to 7.5%.
25
The
patients visited the hospital outpatient clinic monthly for 3 months
after start of insulin therapy and then at 3-month intervals. The
second endothelial function test was performed after the outpatient
visit at 6 months.
To determine the degree of variation in endothelial function
attributable to the method and continued treatment of type 2 diabetic
patients with metformin, 6 type 2 diabetic patients who had been
treated with metformin for 1.20.4 years (6 men, aged 532 years,
body mass index 281 kg/m
2
, duration of diabetes 51 years, and
HbA
1c
9.10.4%) were studied twice with a 6-month interval.
Measurements
In Vivo Endothelial Function Test
In vivo endothelial function was determined by measuring forearm
blood flow responses to intra-arterial infusions of endothelium-
dependent (ACh) and -independent (SNP) vasodilators. The study
was begun after a 10- to 12-hour fast at 7:30 AM. Venous blood
samples were withdrawn for measurement of plasma glucose and
serum free insulin, HbA
1C
, FFA, triglyceride, HDL, and total
cholesterol concentrations. A 27-gauge unmounted steel cannula
(Coopers Needle Works) connected to an epidural catheter (Portex)
was inserted into the left brachial artery. Drugs were infused at a
constant rate of 1 mL/min with infusion pumps (Braun AG). Subjects
rested in a supine position in a quiet environment for 30 minutes after
needle placement before blood flow measurements. Normal saline
was first infused for 18 minutes. Drugs were then infused in the
following sequence: 3 (low dose) and 10 (high dose) g/min SNP
(Roche) and 7.5 (low dose) and 15 (high dose) g/min ACh (Iolab
Corp). Each dose was infused for 6 minutes, and the infusion of each
drug was separated by infusion of normal saline for 18 minutes,
during which blood flow returned to basal values. Forearm blood
flow was recorded for 10 seconds at 15-second intervals during the
last 3 minutes of each drug and saline infusion period with
mercury-in-rubber strain-gauge venous occlusion plethysmography
(EC 4 Strain Gauge Plethysmograph, Hokanson), which was con-
nected to a rapid cuff inflator (E 20, Hokanson), an analog-to-digital
converter (McLab/4e, AD Instruments Pty Ltd), and a personal
computer, as previously described.
26
Blood flow measurements were
performed simultaneously in the infused (experimental) and control
arm. Means of the final 5 measurements of each recording period
were used for analysis. Metformin was discontinued for 2 days
before the endothelial function studies to avoid any acute effects on
vascular function.
Other Measurements
Plasma glucose concentrations were measured in duplicate by the
glucose oxidase method
27
with the use of a Beckman Glucose
Analyzer II (Beckman Instruments). HbA
1c
was measured by high-
performance liquid chromatography with the use of a fully auto-
mated Glycosylated Hemoglobin Analyzer System (Bio-Rad). Se-
rum free insulin concentrations were determined by double-antibody
radioimmunoassay (Pharmacia Insulin RIA kit) after precipitation
with polyethylene glycol.
28
Urine albumin was measured by an
immunoturbidimetric (Hitachi Ltd) method with the use of an
antiserum against human albumin (Orion Diagnostica). Microalbu-
minuria was defined as an albumin excretion rate of 20 to 200
g/min. FFA, serum total cholesterol, and triglycerides
29
along with
HDL cholesterol
30
were measured as previously described. Whole-
body fat and fat-free mass were measured by a single frequency
bioelectric impedance device (model BIA-101A, Bio-Electrical Im-
pedance Analyzer System, RJL Systems).
31
Statistical Analysis
Data between the type 2 diabetic patients and control subjects were
compared by the Student unpaired t test. Changes induced by insulin
therapy in endothelial function were calculated by ANOVA for
repeated measures because treatment groups were composed of the
same subjects, as described by Ludbrook.
32
Horizontal contrasts
were thereafter calculated by the paired t test with the Bonferroni
correction. Correlation analyses were performed by the Spearman
nonparametric correlation coefficient. All calculations were made
with the use of the Systat statistical package. All probability values
are 2-tailed. A value of P0.05 was considered statistically signif-
icant. Data are expressed as meanSEM.
Results
Glycemic Control, Body Composition, Blood
Pressure, and Lipids
During 6 months of insulin therapy, HbA
1c
decreased from
9.00.3% to 7.60.1% (P0.001). The bedtime insulin dose
averaged 324 IU (range 12 to 60 IU). Fasting serum free
insulin concentrations increased from 111 to 141 mU/L
(P0.05). Body weight remained unchanged during insulin
therapy (mean change 1.00.6 kg, PNS). The percentage
of body fat decreased from 29.51.6% to 28.41.7%
(P0.05), and fat-free mass increased from 58.02.0% to
59.62.4% (P0.05). Serum FFAs decreased significantly
during 6 months of insulin therapy from 74646 to
57430 mol/L (P0.001), and serum triglycerides de-
creased by 24% (P0.01). Other lipid concentrations and
blood pressure remained unchanged (Table 1).
Basal blood flow in the experimental arm was comparable
before (2.20.2 mL dL
1
min
1
) and after (2.10.2 mL
dL
1
min
1
) insulin therapy and was not different from that
in the normal subjects (2.20.2 mL dL
1
min
1
). Blood
flows in the control arm were similar to those in the
experimental arm basally in patients with type 2 diabetes
(2.10.2 and 2.00.1 mL dL
1
min
1
) and normal subjects
(2.00.2 mL dL
1
min
1
, PNS) throughout the study.
546 Arterioscler Thromb Vasc Biol. February 2000
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Endothelial Function
Before insulin therapy, blood flow during infusion of the low
(6.70.6 versus 9.30.8 mL dL
1
min
1
, P0.05) and
high (7.50.7 versus 11.60.9 mL dL
1
min
1
, P0.01)
doses of ACh was significantly blunted in the type 2 diabetic
patients compared with the normal subjects. During insulin
therapy, the blood flow response to the low dose of ACh did
not increase significantly, whereas that to the high dose of
ACh increased by 44% (7.50.7 versus 10.81.6 mL dL
1

min
1
, before versus after; P0.05). The responses to ACh
were not different from those of the normal subjects (Figure).
Forearm blood flow responses to both the low (7.80.4
versus 9.10.4 mL dL
1
min
1
, P0.05) and high
(11.00.8 versus 13.00.7 mL dL
1
min
1
, P0.05) doses
of SNP also increased significantly during insulin therapy.
Blood flows during SNP infusion were not significantly
different from those in normal subjects (Figure).
To determine the possible causes of enhanced endothelial
functions during insulin therapy, simple correlation coeffi-
cients (Spearman) were calculated between changes in met-
abolic parameters and those of endothelium-dependent and
-independent vasodilatation. No significant correlations were
observed between metabolic parameters, which changed sig-
nificantly during insulin therapy (HbA
1C
, fasting plasma
glucose, serum FFAs, serum triglycerides, and serum free
insulin concentrations), and the absolute or relative change in
blood flow during SNP and ACh infusions (Table 2).
In the type 2 diabetic patients, who were studied twice during
metformin therapy, basal flows were comparable (2.20.2 and
2.50.3 mL dL
1
min
1
, PNS). Blood flow responses to the
low (8.41.2 and 7.70.3 mL dL
1
min
1
, PNS) and high
(10.82.0 and 10.52.7 mL dL
1
min
1
, PNS) doses of
SNP also remained unchanged, as did the responses to the low
(7.21.0 and 7.31.1 mL dL
1
min
1
, PNS) and high
(8.91.4 and 9.11.0 mL dL
1
min
1
, PNS) doses of ACh.
The coefficients of variation for the 2 repeated measurements in
these patients were 101%, 143%, 134%, 102%, and
154% for infusion of saline, low-dose SNP, high-dose SNP,
low-dose ACh, and high-dose ACh.
Discussion
The present study is, to our knowledge, the first to examine
the effect of insulin therapy on endothelial function in any
type of diabetes. Before insulin therapy, endothelial function
was impaired.
811
Combination therapy with bedtime insulin
and metformin resulted in correction of several metabolic
abnormalities, including decreases in glucose, triglyceride,
Forearm blood ow responses to intra-arterial SNP and ACh
infusions in type 2 diabetic patients before (F) and after ()
insulin therapy and in normal subjects (E). *P0.05 for type 2
diabetic patients before vs after insulin therapy. P0.05 and
P0.01 for type 2 diabetic patients vs normal subjects.
TABLE 1. Physical and Biochemical Characteristics Before and After 6 Months of Insulin
Therapy in Patients With Type 2 Diabetes and in Normal Subjects
Diabetic Patients
Normal Subjects
(n27)
Before Treatment
(n18)
After Treatment
(n18)
Sex, female/male 4/14 4/14 7/20
Age, y 582 592 561
Body weight, kg 82.42.5 83.42.8 85.22.1
Body mass index, kg/m
2
28.50.6 28.90.7 27.60.4
Systolic blood pressure, mm Hg 1446 1446 1316
Diastolic blood pressure, mm Hg 792 792 793
Fasting plasma glucose, mg/dL 22911* 1325 1032
Glycosylated hemoglobin, % 9.00.3* 7.60.1 5.70.1
Insulin, mU/L 111* 141 61
Insulin dose, IU/kg

0.370.04

Total cholesterol, mmol/L 5.690.27 5.450.26 5.730.19
LDL cholesterol, mmol/L 3.630.18 3.640.22 3.750.15
HDL cholesterol, mmol/L 1.080.04 1.140.03 1.380.06
Total triglycerides, mmol/L 2.080.25 1.600.17 1.380.15
UAER, g/min (range) 3120 (0377)

61 (011)
Data are meanSEM. UAER indicates urinary albumin excretion rate.
*P0.01, P0.01, and P0.05 for type 2 diabetic patients before insulin therapy vs normal subjects; P0.01
and P0.05 for type 2 diabetic patients before vs after insulin therapy.
Vehkavaara et al Insulin Therapy and Endothelial Function 547
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and FFA concentrations, and in significant improvement of
both endothelium-dependent and -independent blood flows in
forearm resistance vessels. These changes were due to acute
or chronic effects of insulin therapy rather than to metformin,
in view of the fact that endothelial function remained un-
changed in a control group studied twice during metformin
therapy.
We chose to use combination therapy, with bedtime human
isophane and metformin as the insulin treatment regimen.
This regimen improves glycemic control without inducing
weight gain.
25
Lack of weight gain may be beneficial during
insulin therapy, because weight gain is associated with
increases in blood pressure and LDL cholesterol.
29
In the
UKPDS, patients treated with metformin gained less weight
than did those treated with other agents,
2
and the UKPDS also
suggested that metformin might be cardioprotective in over-
weight patients. So far, however, it is unknown whether the
decrease in cardiovascular events in the metformin-treated
patients was due to differences in weight gain between the
various treatment regimens. In the present study, endothelial
function remained unchanged when measured twice in pa-
tients on chronic metformin therapy. Because the patients had
already been treated for over a year with metformin before the
first endothelial function test, these data do not exclude
potential beneficial effects of metformin on endothelial
function.
In all previous studies, except that of Avogaro et al,
13
addressing endothelial function in type 2 diabetic patients by
using either the invasive technique used in the present
study
8,1012
or noninvasive measurement of the brachial artery
diameter,
9
endothelium-dependent vasodilatation has been
impaired. This defect was also identified in the present study.
The reason for normal endothelial function in the study of
Avogaro et al is unclear but could be due to the small number
of subjects studied (6 normal subjects and 10 patients with
type 2 diabetes). Regarding endothelium-independent but
guanylate cyclasedependent vasodilatation, endothelial
function has been found to be either impaired
8,11,12
or
normal.
9,13
Multiple causes could contribute to endothelial dysfunction
in patients with type 2 diabetes compared with nondiabetic
subjects matched for age, body weight, LDL cholesterol
levels, and blood pressure, as in the present study (Table 1).
Such factors could include those known to be associated with
increased cardiovascular risk, such as chronic hyperglyce-
mia,
23
hyperinsulinemia independent of insulin resistance,
33
insulin resistance, and its consequences (hypertriglyceride-
mia, increased concentrations of small dense LDL particles,
low HDL cholesterol, and increases in FFA concentrations).
Data on possible causes of endothelial dysfunction are sparse
in previous cross-sectional studies addressing endothelial
function in type 2 diabetic patients. We recently performed a
comprehensive search for such factors and found LDL size to
be weakly correlated with endothelium-dependent vasodila-
tation.
34
A similar relation was also described by Watts et al
11
within a group of type 2 diabetic patients. In other stud-
ies,
8,12,13
no significant correlations between cardiovascular
risk factors and endothelium-dependent vasodilatation have
been identified.
Insulin therapy induces several changes that potentially
could enhance endothelial function. Such changes include
decreases in serum triglyceride,
18,35,36
FFA,
17,18,21
and glu-
cose
37
concentrations, and all these parameters have been
associated with endothelial function.
21,35
Acute increases in
insulin concentrations also enhance ACh-induced vasodilata-
tion.
24
In the present study, changes in none of these param-
eters during insulin therapy were significantly associated with
enhanced endothelial function. Whether this reflects lack of a
dominant effect of a single metabolic parameter or an effect
of some factor not quantified in the present study, such as
growth and hemostatic factors and LDL size, remains
unclear.
Endothelium-independent vasodilatation, ie, vascular
smooth muscle celldependent vasodilatation, also increased
significantly during insulin therapy. Multiple factors, which
could be either direct or indirect consequences of insulin
therapy, could underlie the enhanced vascular smooth mus-
cledependent vasodilatation. Regarding direct effects of
insulin, insulin attenuates vascular contraction by inhibiting
voltage-dependent calcium channels
38
via a mechanism that
appears coupled to the ability of insulin to increase glucose
transport in vascular smooth muscle cells.
39
The ability of
insulin to attenuate angiotensin IImediated calcium tran-
sients is blunted in cultured unpassaged vascular smooth
muscle cells in insulin-resistant spontaneously hypertensive
rats.
40
Insulin therapy increases in vivo insulin sensitivity of
glucose uptake in type 2 diabetic patients.
17,4143
If this would
also happen in vascular smooth muscle, it might provide one
mechanism to explain enhanced vascular smooth muscle
celldependent vasodilatation after insulin therapy. Other
possible mechanisms include increased bioavailability of
exogenous (and endogenous) nitrates due to decreases in
oxidative stress,
44,45
advanced glycosylation end products,
46
small dense LDL particles,
22,36
and the susceptibility of
circulating LDL to oxidation.
47
Serum FFAs have also been
suggested to enhance endothelium-dependent,
21
and possibly
TABLE 2. Interrelations Between Absolute and Relative Changes in Forearm Blood Flows
During Intrabrachial Infusions of Endothelium-Dependent (ACh) and Endothelium-Independent
(SNP) Vasodilators With Changes in Metabolic Parameters
SNP (10 g/min) %SNP (10 g/min) ACh (15 g/min) %ACh (15 g/min)
Serum triglycerides 0.07 0.07 0.01 0.08
Serum FFAs 0.28 0.22 0.17 0.25
fP glucose 0.10 0.11 0.25 0.14
HbA
1C
0.03 0.05 0.16 0.18
fP free insulin 0.09 0.08 0.13 0.08
f P indicates fasting plasma levels.
548 Arterioscler Thromb Vasc Biol. February 2000
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endothelium-independent,
35
vasodilatation. FFAs are of inter-
est because of the exquisite sensitivity of lipolysis to
insulin.
17,48
To conclude, endothelium-dependent and -independent
vasodilatation improves during insulin therapy. Both im-
provements can be considered potentially antiatherogenic and
support the view that insulin therapy, either via direct or
indirect mechanisms, has beneficial rather than harmful
effects on vascular function.
Acknowledgments
This study was supported by grants from the Academy of Finland
(Drs Yki-Jarvinen and Vehkavaara), Novo-Nordisk (Dr Yki-
Jarvinen), and Sigfrid Juselius (Dr Yki-Jarvinen) Foundations. We
thank Kati Tuomola and Sari Haapanen for excellent technical
assistance and the volunteers for their help.
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