Effect of thiazolidinediones and metformin on LDL oxidation
and aortic endothelium relaxation in diabetic GK rats
Kaoruko Tada
Iida1,
Yasushi
Kawakami2,
Masatsune
Suzuki1,
Hitoshi
Shimano1,
Hideo
Toyoshima1,
Hirohito
Sone1,
Kazunori
Shimada3,
Yoshitaka
Iwama3,
Yoshiro
Watanabe3,
Hiroshi
Mokuno3,
Katsuo
Kamata4, and
Nobuhiro
Yamada1
1 Division of Endocrinology and Metabolism,
Department of Internal Medicine, and 2 Department of
Clinical Pathology, Institute of Clinical Medicine, University of
Tsukuba, Ibaraki 305-8575; 3 Department of
Cardiology, Juntendo University, Tokyo 113-8421; and
4 Departments of Physiology and Morphology,
Institute of Medical Chemistry, Hoshi University, Tokyo 142-8501, Japan
 |
ABSTRACT |
In this study, using GK
diabetic rats, we compared the effects of three insulin sensitizers on
lipid oxidation and the aortic relaxation response. Eight-week-old rats
were treated for 4 wk with either troglitazone or pioglitazone, both of
which are thiazolidinediones, or with metformin. Despite the fact that
only troglitazone has a similarity in structure to
-tocopherol, a
potent antioxidant, the level of thiobarbituric acid-reactive substance
was lower, and the lag time of the conjugated dienes was longer, in the
blood samples from the rats in both troglitazone- and
pioglitazone-treated groups. In contrast, another insulin sensitizer,
metformin, failed to inhibit the oxidation of blood samples. The aortic
vasorelaxation response was increased in both troglitazone- and
metformin-treated groups compared with the untreated group.
These findings suggest that thiazolidinediones have a beneficial effect
on lipid oxidation irrespective of the drug's structural similarity to
-tocopherol. It is also suggested that the thiazolidinediones and
metformin improve vascular function in diabetes. These effects may play a role in the prevention of atherosclerosis in diabetic patients.
diabetes mellitus; low-density lipoprotein; endothelium; Goto-Kakizaki rats
 |
INTRODUCTION |
DIABETES MELLITUS IS A
STRONG RISK FACTOR for all manifestations of atherosclerotic
vascular diseases. Although the pathophysiological mechanism of the
development of atherosclerosis in diabetic patients has not yet been
fully elucidated, it is thought that hyperlipidemia, increased
oxidation of low-density lipoproteins (LDL), and impaired vascular
function promote atherogenesis in diabetic patients. In addition,
several cohort studies on diabetic and nondiabetic patients
have shown an association between insulin resistance and
atherosclerotic disorders such as cardiovascular disease (11, 19), which is one of the major causes of death in such patients. In particular, the insulin-resistant state is often accompanied by
other risk factors, including hyperinsulinemia, hypertension, glucose
intolerance, and hypertriglyceridemia, which increase the risk for
cardiovascular disease, presumably by promoting atherosclerosis (20, 30).
Thiazolidinediones are a new class of oral antidiabetic agents that
reduce the blood glucose level by improving insulin resistance in
diabetic patients. This class of agents works by binding to peroxisome
proliferator-activated receptor-
(PPAR
), thereby promoting the
synthesis of glucose transporters and affecting lipid metabolism
(13, 21). In contrast, metformin hydrochloride, a
biguanide, improves the insulin-resistant state by unknown mechanisms, reducing gluconeogenesis and enhancing peripheral glucose
uptake. As a result, metformin hydrochloride promotes reduction
of the plasma glucose level (3, 26). Despite their actions
through different mechanisms, these two insulin sensitizers have been reported to prevent the development of atherosclerosis (5, 14,
17, 28). However, the precise mechanisms by which these drugs
prevent the development of atherosclerotic disorders have not yet been
fully elucidated.
It has been suggested that oxidation of LDL is a key initial event in
the progression of atherosclerotic lesion formation, which is followed
by the uptake of oxidized LDL by macrophages via their scavenger
receptors (25). Moreover, it has been reported that an
increase in modified LDL impairs vascular functions such as
endothelium-dependent vasodilatation (12). One kind of
thiazolidinedione, troglitazone, has been reported to act as an
antioxidant and inhibit the oxidation of LDL (6, 16);
therefore, this drug has been considered to play a beneficial role in
preventing the development of atherosclerosis. In this study, to
elucidate the mechanisms through which insulin sensitizers prevent
atherosclerotic disorders in diabetic patients, we compared the effects
of three different insulin sensitizers, troglitazone, pioglitazone, and
metformin, on lipid metabolism and the oxidized state of blood lipids
in Goto-Kakizaki (GK) rats, a model of type 2 diabetes. We also
examined the relaxation response in strips of the aorta to investigate the effect of these drugs on vascular function.
 |
MATERIALS AND METHODS |
Materials and experimental protocols.
Troglitazone, pioglitazone, and metformin were obtained from Sankyo
Pharmaceuticals (Tokyo, Japan), Takeda Pharmaceuticals (Tokyo, Japan),
and Sigma (St. Louis, MO), respectively. Twenty-four male GK rats bred
in a closed colony were obtained from Charles River Laboratories
(Wilmington, MA). They were fed standard powdered rodent chow (Oriental
Yeast, Tokyo, Japan) and maintained in an environmentally controlled
room under a 12:12-h light-dark cycle. Before drug administration, a
blood sample was taken from the tail vein, and fasting plasma glucose
was measured in each sample. Age-matched, male, nondiabetic Wistar rats
(obtained from Charles River Laboratories) were used as the controls
for measurement of plasma glucose to confirm that the diabetic state
was completely established in the GK rats. The GK rats were divided
into four groups (n = 6 in each group). There were no
significant differences in diabetic parameters among the four groups:
the untreated control group, the Troglitazone group [which had the
powdered chow containing 0.2% (wt/wt) troglitazone], the Pioglitazone
group [which had the powdered chow containing 0.02% (wt/wt)
pioglitazone], and the Metformin group [which had the powdered chow
containing 0.5% (wt/wt) metformin]. Administration of the drug was
started at 8 wk of age, when the diabetic state was already
established, and was continued for 4 wk. The weight of the residual
chow was measured every day for 1 wk after drug administration
commenced, and the average food intake of each animal was estimated by
dividing the amount of consumed chow by the number of animals per cage (3 animals/cage). After drug administration, the animals were kept off
chow for
12 h and were then killed. Blood samples were collected from
the abdominal aorta with an anticoagulant agent to measure various
metabolic markers.
Assay methods.
The plasma concentrations of glucose, cholesterol, and triglycerides
were measured by the enzymatic colorimetric method using a commercially
available kit (Wako Chemicals, Tokyo, Japan) and the Hitachi Auto
Analyzer (type 7170; Hitachi Electronics, Hitachi, Japan). The free
fatty acid (FFA) level was measured by the enzymatic colorimetric
method using a commercial kit (NEFA-SSAK, Eiken Chemicals, Tokyo,
Japan). The plasma insulin level was measured with a radioimmunoassay kit (Linco Research, St. Charles, MO). The percentage of glycosylated hemoglobin A1C (Hb A1C) was calculated using a
commercial kit (Unimate HbA1c; Roche Diagnostics, Basel,
Switzerland) according to the manufacturer's protocol. All
parameters in the plasma samples were assayed in duplicate.
Isolation of LDL.
LDL was isolated by sequential density ultracentrifugation of the
collected plasma from blood samples containing EDTA (at a 2 mM final
concentration). Briefly, the plasma samples were overlaid with normal
saline (d = 1.006 g/ml) and then centrifuged in a Beckman TLA
100.4 rotor (Beckman Coulter, Fullerton, CA) at 100,000 rpm at 15°C
for >3 h to float the very low-density lipoproteins (VLDL). After the
VLDL layer was removed, the salt density of the sample was adjusted to
1.063 g/ml by adding NaBr. The samples were then recentrifuged at
100,000 rpm at 15°C for >3 h. The layer of isolated LDL (d = 1.006-1.063 g/ml) was collected and dialyzed three times against
phosphate-buffered saline containing 1 mM EDTA and sterilized using a
22-µm filter. The protein content was measured with a modified
procedure of the Lowry assay as an estimate of the level of total LDL
in each isolated sample.
Biochemical markers of lipid peroxidation.
The levels of thiobarbituric acid-reactive substance (TBARS) formation
in the plasma and in the LDL samples were assayed as described by Yagi
(29). The concentration of TBARS was calculated as the
malondialdehyde (MDA) equivalent (nmol/ml plasma or nmol/100 µg
protein) by use of the standard curve of MDA generated by the hydrolysis of 1,1,3,3-tetraethoxypropane. The lag time of the formation of conjugated dienes was determined as follows. The protein
concentration was adjusted to 20 µg/ml in each isolated LDL sample.
Aliquots of LDL sample were then mixed with CuSO4 (at a 5 µM final concentration) and incubated at 37°C. The absorbance level
at 234 nm was continuously measured at 5-min intervals until the
absorbance reached the plateau level. The lag time was defined as the
interval of time up to the time at which the absorbance started to increase.
Measurement of isometric force.
The section of the aorta from between the aortic arch and the diaphragm
was removed from the euthanized rats and placed in oxygenated, modified
Krebs-Henseleit solution (KHS; in mM: 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.8 CaCl2, 1.2 NaH2PO4, 1.2 MgSO4, 11 dextrose).
The aorta was cut into helical strips 3 mm wide and 20 mm long and then
placed in a well-oxygenated (95% O2-5% CO2) bath of 10-ml KHS with one end connected to a tissue holder and the
other to a force displacement transducer (Nihon-Koden, TB-611T). The
tissue was equilibrated for 60 min under a resting tension of 1.0 g. After equilibration, each strip was precontracted with norepinephrine (NE; 5 × 10
8-3 × 10
7 M) to develop a tension of ~95 mg/mg tissue. When
the NE-induced contraction reached the plateau level, acetylcholine
(ACh) or sodium nitroprusside (SNP) was added in a cumulative manner.
The relaxation response was expressed as a percentage of the
contraction induced by NA.
Statistical analysis.
The results are expressed as means ± SD. Group means were
compared using the one-way ANOVA. A P value of <0.05 was
considered to be statistically significant.
 |
RESULTS |
Diabetic statement.
Before drug administration was started, the body weight and fasting
plasma glucose (FPG) level of each animal were measured. The body
weight was significantly lower, and the FPG levels were significantly
higher, in the four groups of GK rats than in the age-matched, control
Wistar rats (Table 1) at 8 wk of age,
indicating that the diabetic state was already established in the GK
rats at that time. There were no significant differences in body weight and FPG level among these four groups of GK rats. Several blood parameters of the diabetic state after the 4-wk drug administration are
shown in Table 2. At 12 wk of age,
although the GK rats in all four groups exhibited hyperglycemia that
was more pronounced than at 8 wk of age, the FPG levels in the
Troglitazone, Pioglitazone, and Metformin groups were significantly
lower than in the untreated group. The Hb A1C levels in the
three drug-treated groups were also significantly lower than the levels
in the untreated group. In addition, all of the three antidiabetic
drugs that we used significantly reduced the fasting insulin level,
which was associated with the reduction in the FPG level, suggesting
that these drugs improved the susceptibility to insulin in GK rats.
We also measured the food consumption of the animals in each group.
Among the GK rats, the food consumption of the Metformin group (17.9 g · rat
1 · day
1)
was lower than that of the three other groups (21.9 g · rat
1 · day
1
in the untreated group, 23.4 g · rat
1 · day
1
in the Troglitazone group, and 24.5 g · rat
1 · day
1
in the Pioglitazone group).
Blood lipid statement.
To evaluate the effect of each antidiabetic drug on lipid metabolism,
we measured the fasting levels of plasma cholesterol (total or HDL),
triglyceride, and FFA in the GK rats 4 wk after treatment (Table
3). The two antidiabetic
thiazolidinedione agents troglitazone and pioglitazone caused a
significant reduction in the total cholesterol level. However, the
HDL-cholesterol levels of the untreated GK rats and the
thiazolidinedione-treated rats did not significantly differ, indicating
that the non-HDL cholesterol level in the GK rats was decreased by the
treatment with thiazolidinediones. In addition, in these two groups,
the fasting triglyceride and FFA levels dramatically fell to <60% of
the respective value in the untreated group (triglyceride:
P < 0.05, P < 0.05; FFA:
P < 0.01, P < 0.01). In contrast, the
values of the lipid parameters in the Metformin group did not
significantly differ from those in the untreated group.
Lipid oxidation.
To assess the effect of each drug on lipid oxidation, the TBARS level,
which represents the plasma lipid peroxide content, was measured in the
four groups of GK rats. As shown in Table 4, the two thiazolidinedione agents
significantly reduced the TBARS level in the plasma samples of the GK
rats after treatment for 4 wk. The TBARS levels in the LDL samples of
the rats in the thiazolidinedione groups were also reduced but not
significantly. We also measured the level of conjugated dienes in the
LDL samples and analyzed the lag time to evaluate the effect of each
drug on the susceptibility of the LDL samples to oxidation (Fig.
1). The lag times were significantly
longer in the Troglitazone and Pioglitazone groups than in the
untreated group. In contrast, there was no significant difference in
lag time between the Metformin group and the untreated group.
Therefore, the therapeutic use of thiazolidinediones appeared to reduce
the susceptibility of LDL to oxidation.

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Fig. 1.
A: time course of the conjugated diene
formation of LDL from blood samples obtained from Wistar rats
(n = 6, ), Goto-Kakizaki (GK) rats
(n = 6, ), or GK rats that had been
treated with troglitazone (n = 6, ),
pioglitazone (n = 6, ) or metformin
(n = 6, ) for 4 wk. Each data point
represents the mean of 6 samples in each group. B: results
are expressed as means ± SD of the lag time of 6 samples in each
group. *P < 0.01 vs. untreated control.
|
|
Relaxation response to ACh in aorta.
To assess the effect of thiazolidinediones and metformin on vascular
function, the vasorelaxation response to ACh was examined in the
following three groups: the Troglitazone group, the Metformin group,
and the untreated group. The maximum relaxation responses were
significantly increased in the aortic strips of the animals in the
drug-treated groups compared with those in the untreated group
(74.5 ± 3.6% in the Troglitazone group and 71.6 ± 3.2% in the Metformin group vs. 61.2 ± 7.8% in the untreated group,
P < 0.01). The concentration of ACh achieving 50%
relaxation (log EC50) was significantly lower in the
drug-treated groups than in the untreated group [Troglitazone:
7.12 ± 0.08 and Metformin:
7.09 ± 0.05 vs. control:
6.84 ± 0.08 (
log M), P < 0.01; Fig. 2]. These results indicated that the
treatments with troglitazone and metformin similarly improved the
vasorelaxation response in the GK rats.

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Fig. 2.
Concentration-response curves for acetylcholine-induced
relaxation of aortic strip obtained from untreated GK rats
(n = 6, ), or GK rats that had been
treated with troglitazone (n = 6, ) or
metformin (n = 6, ) for 4 wk.
Relaxation of aorta was shown as a %contraction induced by an
equieffective concentration of norepinephrine. Each data point
represents the mean ± SD of 6 samples in each group.
|
|
We also assessed the effect of each drug in vasorelaxation response
induced by nitric oxide donor. Although the relaxation responses of
aortic strips induced by SNP were significantly increased in both
drug-treated groups compared with the untreated group (91.3 ± 3.9% in the Troglitazone group, P < 0.05; 107.5 ± 0.8% in the Metformin group, P < 0.01, vs.
77.5 ± 14.5% in the untreated group), the relaxation responses
were more intensified in the Metformin group than in the Troglitazone
group (Metformin group vs. Troglitazone group, P < 0.05; Fig. 3). Taken together, these findings suggest that the treatment with metformin increases not only
the endothelium-dependent but also the endothelium-independent relaxation in the aorta.

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Fig. 3.
Concentration-response curves for sodium nitroprusside
(SNP)-induced relaxation of aortic strip obtained from untreated GK
rats (n = 6, ) or GK rats that had been
treated with troglitazone (n = 6, ) or
metformin (n = 6, ) for 4 wk.
Relaxation of aorta was shown as a %contraction induced by an
equieffective concentration of norepinephrine. Each data point
represents the mean ± SD of 6 samples in each group.
|
|
 |
DISCUSSION |
In this study, we attempted to compare the effects of three
insulin sensitizers on lipid metabolism in GK rats, an animal model of
spontaneous diabetes. GK rats exhibit moderate hyperglycemia that does
not progress to a ketotic state, and they have a normal or slightly
elevated plasma insulin level (18, 23). In addition, a
euglycemic-clamp study showed that these animals have insulin resistance (4). Therefore, this animal model has been
established as a model of type 2 diabetes possessing insulin
resistance. In this study, we demonstrated that troglitazone,
pioglitazone, and metformin improved the fasting plasma glucose and Hb
A1C levels, which were associated with the reduction in the
plasma insulin level in GK rats. These results indicate that these
drugs improve insulin resistance in GK rats to a similar degree.
The clinical use of thiazolidinediones, including troglitazone and
pioglitazone, has been known to reduce the plasma triglyceride level in
diabetic patients and also in various genetically obese and diabetic
rodent models (8, 9). Our data showed that troglitazone
and pioglitazone reduced the plasma levels not only of triglyceride but
also of cholesterol and FFA in GK rats. The mechanism through which
thiazolidinediones affect these plasma lipids has not yet been
elucidated; however, our data showed that not only troglitazone but
also pioglitazone regulated the levels of plasma lipids in vivo to a
similar extent, suggesting that PPAR
is involved in the reduction of
these plasma lipid levels. In contrast, the 4-wk metformin treatment
did not reduce the plasma levels of cholesterol and triglyceride in the
GK rats despite the improvement in glycemic control. In addition, in
several previous clinical studies (2, 27), no significant
improvement was observed in the blood lipid profiles in type 2 diabetes
treated with metformin, suggesting that metformin was not as effective as thiazolidinediones in reducing hyperlipidemia.
Not only an elevated LDL level but also the oxidative modifications of
LDL have recently been considered to be an important factor in
atherosclerotic diseases (25). Therefore, the effect of
several antidiabetic drugs on the susceptibility of LDL to oxidation
has been investigated, and troglitazone has been reported to inhibit
the oxidation of LDL in vitro and in vivo (6, 16). It has
been considered that the mechanism of the inhibitory effect of
troglitazone on oxidation was based on its molecular similarity to
-tocopherol (Fig. 4), which is one of
the potent antioxidant-scavenging ROS (10, 16).
However, in the present study, another thiazolidinedione, pioglitazone,
inhibited the oxidation of LDL to a degree similar to that of
troglitazone despite the fact that its structure differs from the
structure of
-tocopherol. In contrast, another insulin sensitizer,
metformin, failed to inhibit the oxidation of LDL. These results point
to a novel finding that one particular effect of thiazolidinediones may
be to improve the susceptibility of LDL to oxidation irrespective of
the drug's structural similarity to
-tocopherol.
An impairment of vascular function has been considered to be involved
in diabetic vascular complications. In some previous reports, it was
observed that the functions of the cells that construct the arterial
walls were impaired in diabetic animal models (7, 22). It
was also reported that oxidized LDL itself impaired vascular function,
such as endothelium-dependent vasodilatation (12, 24). In
the present study, we hypothesized that thiazolidinediones ameliorate
vascular function by decreasing the native and oxidized LDL in diabetic
animals. As was expected, the treatment with troglitazone significantly
improved ACh-induced vasodilatation in GK rats. However, it was
reported that troglitazone did not improve the impaired vasorelaxation
in obese but nondiabetic Wistar rats, although it improved
obesity-induced lipid-metabolic changes (15). In the
present study, we also showed a novel finding that the treatment with
metformin improved the endothelium-dependent vasorelaxation to a
similar degree compared with thiazolidinediones despite the fact that
metformin did not inhibit the oxidation of LDL. These results suggest
this possible explanation: in diabetic rats, not only oxidized LDL but
also high plasma glucose or glucose derivatives such as abnormal
glycosylated proteins lead directly to vascular dysfunction, and
treatment with troglitazone or metformin improves the diabetes-induced
vascular dysfunction by decreasing the plasma glucose level. It is also
possible that the improvement of insulin resistance with these drugs
directly ameliorates vascular function, because it has been reported
that insulin resistance itself causes impairment of endothelium
function (1). In addition, we found that metformin
increased endothelium-independent vasorelaxation to a similar or
greater extent compared with troglitazone. The effects of metformin
reported in the present study may play a role in preventive effects of
this drug against the diabetic atherosclerosis, demonstrated in some
previous clinical studies such as the UK Prospective Diabetes Study
(28).
In conclusion, the present study demonstrated a novel finding that the
thiazolidinediones troglitazone and pioglitazone reduce the plasma
lipid peroxide levels and the susceptibility of LDL to oxidation in GK
rats irrespective of the drug's structural similarity to
-tocopherol. In contrast, another insulin sensitizer, metformin, had
a much smaller effect on lipid metabolism, even though this drug
improved the impairment of glucose metabolism in GK rats to a similar
extent to the two thiazolidinediones. However, metformin improved
vascular function as well as troglitazone, as analyzed by measuring the
relaxation response in strips of the aorta. Recently, evidence has been
presented from epidemiological studies showing that thiazolidinediones
and metformin reduce the risk of atherosclerotic diseases such as
coronary artery disease in diabetic patients (5, 14, 17,
28). The protective effects of thiazolidinediones and metformin
may be due to their beneficial effects on the metabolism of lipids and
the oxidation of LDL, or on vascular functions in diabetic patients.
 |
ACKNOWLEDGEMENTS |
This study was supported by the Japan Foundation for Aging and Health.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: N. Yamada, Div. of Endocrinology and Metabolism, Dept. of Internal Medicine, Inst. of Clinical Medicine, Univ. of Tsukuba,
1-1-1 Tennodai, Tsukuba-shi, Ibaraki 305-8575,
Japan (E-mail: ymdnbhr{at}md.tsukuba.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 4, 2003;10.1152/ajpendo.00430.2002
Received 4 October 2002; accepted in final form 28 January 2003.
 |
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Am J Physiol Endocrinol Metab 284(6):E1125-E1130
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Copyright © 2003 by the American Physiological Society.