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Cardiovascular Research 2005 66(1):150-161; doi:10.1016/j.cardiores.2004.12.025
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Copyright © 2005, European Society of Cardiology

Pioglitazone, a PPAR{gamma} agonist, restores endothelial function in aorta of streptozotocin-induced diabetic rats

Jayesh B. Majithiya*, Arvind N. Paramar and R. Balaraman

Pharmacy Department, Faculty of Technology and Engineering, M. S. University of Baroda, Kalabhavan, Baroda-390001, Gujarat, India

* Corresponding author. Tel.: +91 265 2434187; fax: +91 265 2418927. Email address: jayeshbm{at}yahoo.com rbalaraman2000{at}yahoo.com

Received 19 July 2004; revised 23 December 2004; accepted 28 December 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: To study the effect of pioglitazone (a PPAR gamma agonist) treatment on blood pressure, endothelial function, and oxidative stress in streptozotocin (STZ)-induced diabetic rats.

Methods: Sprague–Dawley rats were randomized into control (n=32) and STZ-diabetic (n=32) groups. Rats were further randomized to receive pioglitazone (10 mg/kg) or placebo for 4 weeks, and the following protocols were carried out. Blood pressure, blood glucose level, and body weight were measured. Thoracic aorta was isolated and the dose–response curve of phenylephrine (PE) in the presence or absence of N{omega}-nitro-L-arginine-methyl ester (L-NAME) was recorded. The dose–response curve of acetylcholine (Ach) in the presence or absence of indomethacin, L-NAME, and methylene blue was recorded. Tone-related basal nitric oxide release experiments were carried out. Lipid peroxidation, superoxide dismutase, catalase, and reduced glutathione were estimated in liver, kidney, and aorta. Aortic nitrite levels were also measured. Further, in vitro effects of PE and Ach in the presence of pioglitazone (0.1 M–10 mM) were measured in aortic rings of nondiabetic and STZ-diabetic rats. Pioglitazone-induced relaxations were recorded in PE-contracted rings (with intact and denuded endothelium) in the presence of L-NAME and in KCl-contracted rings.

Results: Pioglitazone treatment reduced blood pressure without having any significant effect on blood glucose level and body weight of STZ-diabetic rats. Enhanced PE-induced contraction and impaired Ach-induced relaxations in STZ-diabetic rats were restored to normal by pioglitazone treatment. The presence of L-NAME but not indomethacin blocked Ach-induced relaxation in pioglitazone-treated STZ-diabetic rats. Basal nitric oxide release was significantly higher in pioglitazone-treated STZ-diabetic rats. Oxidative stress was significantly higher in STZ-diabetic rats and pioglitazone treatment significantly reduced it. High aortic nitrite levels of STZ-diabetic rats were significantly reduced by pioglitazone treatment. The presence of pioglitazone at higher concentrations (>10 µM), but not at lower concentrations, significantly changed the dose–response curve of PE or Ach. Pioglitazone relaxations at lower concentrations but not at higher concentrations were blocked by endothelium removal or by the presence of L-NAME.

Conclusion: Pioglitazone administration reduced oxidative stress, which prevented the breakdown of nitric oxide and increased nitric oxide levels, thereby restoring the endothelial function in aorta of STZ-diabetic rat. Hence, from the present study it can be concluded that pioglitazone administration in STZ-diabetic rats lowers blood pressure, protects against oxidative stress, and restores endothelial function.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiovascular disease is one of the leading causes of death in the western world and diabetes mellitus has been identified as a primary risk factor [1], due to which there is alteration in vascular responsiveness to several vasoconstrictors and vasodilators [2]. Most of the complications in diabetes are due to increased serum glucose and increased generation of oxygen-derived free radicals, which lead to endothelium dysfunction. It has been shown that vessels from diabetic animals exhibited abnormal endothelium dependent vascular relaxation to acetylcholine [3,4]. This endothelium-dependent vasodilation is reduced in diabetes largely due to excessive oxidative stress and decreased bioavailability of nitric oxide.

Pioglitazone, a PPAR (peroxisome proliferators activated receptors) gamma agonist, improves insulin-mediated glucose uptake into skeletal muscle without increasing endogenous insulin secretion [5], and has been demonstrated to be effective in the treatment of non-insulin dependent diabetes mellitus with insulin resistance. It belongs to the thiazolidinedione class of drug and is an insulin sensitizer widely used in treatment of non-insulin-dependent diabetes mellitus. Pioglitazone lowers blood pressure and restores blunted endothelium-dependent vasodilatation in fructose-fed rats [6], insulin-resistant rheus monkey [7], SHR [8] and sucrose-fed SHR [9]. PPAR gamma agonists troglitazone, rosiglitazone, and pioglitazone all improve endothelial cell function in humans when measured by brachial artery responses to acetylcholine or when analyzed by small-vessel compliance [10]. Recently, a protective effect of pioglitazone against oxidative stress in liver and kidney of diabetic rabbits [11] has been reported. So far the effect of pioglitazone on blood pressure and endothelial function on aorta of streptozotocin-induced diabetic rats has not been studied. Hence, the purpose of the present study was to instigate the effect of pioglitazone treatment on blood pressure, endothelial function, and oxidative stress in streptozotocin-induced diabetic rats.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Drugs
Pioglitazone hydrochloride was obtained as a gift sample from Alembic, Baroda. Streptozotocin, phenylephrine, acetylcholine, N{omega}-nitro-L-arginine-methyl ester (L-NAME), indomethacin, epinephrine, 1,1,3,3,-tetra ethoxy propane, superoxide dismutase, catalase and glutathione standard were obtained form SIGMA, St. Louis, MO, USA. All other chemicals and reagents used in the study were of analytical grade. Indomethacin was dissolved in 0.2 M Na2CO3. Stock solution of phenylephrine, acetylcholine, methylene blue, sodium nitroprusside, potassium chloride and L-NAME were prepared in double distilled water. For oral administration pioglitazone suspension was prepared in 0.5% sodium carboxy methyl cellulose and for in vitro studies, stock solution of pioglitazone was prepared in 0.1% DMSO [12]. The final concentration of DMSO in organ bath was less than 0.05% vol/vol. Stock solution of phenylephrine was stabilized with L-(+) ascorbic acid (1 µM), final concentration of ascorbic acid in organ bath was less than 0.1 pM.

2.2. Experimental protocol
All experiments and protocols described in present study were approved by the Institutional Animal Ethics Committee (IAEC) of M.S. University, Baroda and are in accordance with guidelines as per "Guide for the care and use of laboratory animals" published by NIH publication (No. 85-23 revised 1996) and with permission from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Male Sprague–Dawley rats (200 ± 15 g) were housed in-group of 3 animals and maintained under standardized condition (12-h light/dark cycle, 24 °C) and provided free access to palleted CHAKKAN diet (Nav Maharashtra Oil Mills Pvt., Pune) and purified drinking water ad libitium. Diabetes was induced by single intravenous injection of streptozotocin (55 mg/kg, STZ) dissolved in normal saline. The control animals were injected with equal volume of vehicle. After 3 days following streptozotocin administration, blood was collected from tail vein and serum samples were analyzed for blood glucose (Enzymatic kits, GOD/POD method, SPAN diagnostics Pvt., India). Animals showing fasting blood glucose higher than 250 mg/dl were considered as diabetic rats and used for the study. Four weeks after induction of diabetes, blood pressure was measured by tail cuff method and rats with systolic blood pressure higher than 135 mmHg were selected, randomized into groups and used for the study. Age matched nondiabetic rats with systolic blood pressure less than 120 mmHg were randomized in to nondiabetic groups. Diabetic and nondiabetic rats were divided in to following groups: nondiabetic control (n=16, ND-CON), STZ-diabetic control (n=16, STZ-CON), nondiabetic group treated with pioglitazone (10 mg/kg/day) for 4 weeks (n=16, ND-PIO) and STZ-diabetic rats treated with pioglitazone (10 mg/kg/day) for 4 weeks (n=16, STZ-PIO). Respective control groups were orally administered with vehicle (1 ml/kg/day of 0.5% sodium carboxy methyl cellulose solution) for 4 weeks.

2.3. Blood pressure
Blood pressure was measured non invasively at the start of study and at weekly intervals by tail cuff method using LE 5002 storage pressure meter (LETICA scientific instruments, SPAIN) in all the above mentioned groups. For the blood pressure measurements animals were trained for at least 1 week until blood pressure was steadily recorded with minimal stress and restraint. The mean of 7–8 measurements of trained animals was recorded.

2.4. Preparation of aortic rings
The thoracic aorta of rats was isolated immediately after decapitation and carefully cleaned of fat and connective tissues. The aorta was cut into rings of 3 mm width. Extreme care was taken not to stretch or damage the luminal surface of the aorta to ensure the integrity of endothelium. In some rings endothelium was denuded by gently rubbing the aortic rings with forceps. Aortic rings were suspended between two 'S' shaped platinum loops in jacketed organ bath containing 20 ml krebs bicarbonate solution (pH 7.4) maintained at 37 ± 0.5 °C and continuously aerated with 95% oxygen and 5% carbon dioxide. The composition of the Krebs solution (mM) was NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 22.0, and glucose 11.0. The rings were connected to isometric force displacement transducer connected to Gemini pen recorder (UGO-BASILE, Italy). The rings were maintained under tension of 2 g and equilibrated for 90 min before initiating experimental protocol. During this period, the krebs solution was changed at every 15 min interval. After the equilibration period rings were maximally contracted with phenylephrine (PE, 1 µM) to test their contractile capacity, three recordings were carried out to find out a constant and reproducible contraction. The presence of functional endothelium was assessed by the ability of acetylcholine (Ach, 0.1 µM) to induce more than 60% of relaxation of rings precontracted submaximally with PE. Aortic rings were considered denuded when there was less than 10% relaxation to Ach.

2.5. Effect of PE and Ach on aortic rings obtained from control and pioglitazone treated rats
After 4 weeks of treatment aortic rings of ND-CON group, STZ-CON group, STZ-PIO group and ND-PIO group were isolated and mounted in organ bath as described above. Concentration–response curves to increasing concentrations of PE (1 nM–10 µM) were performed in rings with intact endothelium. Concentration–response curve of PE with the presence and absence of 100 µM of L-NAME was also recorded. Indomethacin (10 µM) was added to prevent the involvement of prostaglandins. Endothelium mediated relaxation was measured as a concentration–response curve to Ach (1 nM–10 µM) in rings precontracted with PE (80–90% of maximum response). Endothelium independent aortic relaxation to sodium nitroprusside (SNP, 0.001 nM–10 µM) was also measured in rings with denuded endothelium. Concentration dependent relaxation to Ach was recorded in precontracted rings 30 min after incubation with and in continued presence of 10 µM indomethacin (a non-selective cyclo-oxygenase inhibitor), 10 µM of methylene blue (a cGMP blocker) and 100 µM of L-NAME (a non-selective nitric oxide synthase inhibitor)+10 µM indomethacin. To investigate the effect of pioglitazone treatment on tone related basal nitric oxide release, aortic rings were submaximally (about 30–35%) contracted with PE (3 µM) and then response to addition to L-NAME (1–100 µM) was recorded as described by Hayashi et al. [13].

2.6. Measurement of superoxide dismutase, catalase, reduced glutathione and lipid peroxidation
After 4 weeks of treatment animals were sacrificed, liver, kidney and aorta were isolated and weighed [14]. The tissues were finely sliced and homogenized in chilled tris buffer at a concentration of 10% (w/v). The homogenates were centrifuged at 10,000 x g at 0 °C for 20 min using Remi C-24 high speed cooling centrifuge. The clear supernatant was used for estimation of lipid peroxidation (MDA content), endogenous antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) and reduced glutathione (GSH)). Superoxide dismutase was determined by the method of Mishra and Fridovich [15]. Catalase was estimated by the method of Hugo Aebi as given by Hugo [16]. Reduced glutathione was determined by the method of Moron et al. [17]. Lipid peroxidation or malondialdehyde formation was estimated by the method of Slater and Sawyer [18].

2.7. Aortic nitrite levels
Nitric oxide (NO) easily breaks down with the presence of free radicals, hence aortic nitrite levels were measured as a level of NO inactivated due to superoxide radical (O2–). Nitrite was estimated colorimetrically with the Griess reagent [19] in aortic homogenate. Briefly equal volumes of aortic homogenate and Griess reagent (sulfanilamide 1% w/ v, naphthylethylenediamine dihydrochloride 0.1% w/v, and orthophosphoric acid 2.5% v/v) were mixed and incubated at room temperature for 10 min and the absorbance was determined at 540 nm wavelength. Nitrite was determined from the standard curve obtained using sodium nitrite as standard. The amount of nitrite formed was normalized to the protein content of the respective aorta.

2.8. Effect of the presence of pioglitazone on dose–response curves of PE, Ach and SNP in aortic rings of untreated animals
Aortic rings of untreated age matched nondiabetic (n=28) and STZ-diabetic (n=28) were mounted in organ bath as previously described. Concentration–response curve of PE and Ach were measured in rings (with intact endothelium), as described earlier, with the presence of DMSO (vehicle) or with pioglitazone (0.1 µM–10 mM). Relaxation to SNP (1 pM–100 µM) in endothelium denuded rings was also measured after pioglitazone incubation.

2.9. Effect of pioglitazone perse on PE and KCl contracted aortic rings of untreated animals
Concentration dependent relaxation of pioglitazone (10 nM–10 mM) in PE contracted rings with intact and denuded endothelium was recorded. Concentration dependent relaxation was recorded in precontracted (PE) rings (with intact endothelium) with the presence of 100 µM of L-NAME. Concentration dependent relaxation of pioglitazone (10 nM–10 mM) in endothelium denuded rings precontracted with 60 mM KCl was also recorded.

2.10. Statistical analysis
All the data are expressed as mean ± SEM. Data were analyzed by ANOVA for repeated measurements followed by Bonferroni multiple comparison tests. Differences were considered to be statistically significant when p<0.05. The agonist pD2 value (–log EC50) was calculated from concentration–response curve by non-linear regression analysis of the curve using computer based fitting program (Prism, Graphpad).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Blood glucose and body weight
All streptozotocin injected animals developed diabetes. The changes in blood glucose levels are shown in Table 1. Blood glucose levels remained unchanged in nondiabetic animals (ND-CON and ND-PIO groups). There was significant (p<0.05) increase in blood glucose levels of streptozotocin injected animals. Pioglitazone treatment did not have any significant effect on blood glucose level of STZ-diabetic rats. Body weight of nondiabetic rats and STZ-diabetic rats showed a moderate increase. Pioglitazone treatment had no significant effect on the bodyweight of STZ-diabetic rat (Table 1).


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  		Table 1 Blood glucose level and body weight of rats before treatment (initial) and after 4 weeks of treatment (final) with pioglitazone

 
3.2. Blood pressure
There was a significant (p<0.05) increase in systolic (142 ± 11.41, mmHg), diastolic (104.5 ± 10.165, mmHg) and mean blood pressure (117 ± 10.58, mmHg) in STZ-CON group as compared to ND-CON (Fig. 1). Pioglitazone (10 mg/kg/day) treatment for 4 weeks significantly reduced systolic (124 ± 9.23, mmHg), diastolic (94.5 ± 9.2, mmHg) and mean blood pressure (107 ± 9.21, mmHg) of STZ-PIO group as compared to STZ-CON group (Fig. 1). There was no significant change in systolic, diastolic or mean blood pressure of ND-PIO group treated with pioglitazone.


Figure 1
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  		Fig. 1 Effect of pioglitazone treatment for 4 weeks on systolic (a), diastolic (b) and mean (c) blood pressure of ND-CON (-{square}-), STZ-CON (-bullet-), ND-PIO (-{circ}-) and STZ-PIO (-lhblk-) groups. Values are expressed as mean ± SEM. * P<0.05, compared to STZ-CON group (n=5–8).

 
3.3. Contractile response to PE on aorta obtained from control and pioglitazone treated rats with the presence and absence of L-NAME
Cumulative addition of PE (1 nM–10 µM) resulted in concentration dependent contraction of aorta in all the groups (Fig. 2). There was a significant (p<0.05) increase in maximal response (Emax) of PE in aorta obtained from STZ-CON group as compared to ND-CON group (Fig. 2). pD2 value of PE in aorta obtained from STZ-CON group was significantly (p<0.05) higher as compared to ND-CON (Table 2). Contractile response of PE in aorta obtained from STZ-PIO group was attenuated as compared to STZ-CON group. Maximal response (Emax) and pD2 value of PE in aorta obtained from STZ-PIO group was significantly (P<0.05) reduced as compared to STZ-CON group (Table 2). Maximal response (Emax) of PE in aortic rings of ND-CON, ND-PIO and STZ-PIO groups were significantly (p<0.05) increased due to the presence of L-NAME. pD2 value of PE in aortic rings of ND-CON, ND-PIO and STZ-PIO groups were significantly (p<0.05) changed due to the presence of L-NAME (Table 2, Fig. 3). There was no significant change in maximal response (Emax) or pD2 value of PE in aortic rings of STZ-CON group due to the presence of L-NAME (Table 2, Fig. 3).


Figure 2
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  		Fig. 2 Concentration response curve of phenylephrine on aortic rings obtained from ND-CON (-{square}-), STZ-CON (-bullet-), ND-PIO (-{circ}-) and STZ-PIO (lhblk) group. Values are expressed as mean ± SEM. * P<0.05, compared to STZ-CON group. (n=5–8).

 

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  		Table 2 Maximal response (Emax) and pD2 (–Log EC50) values of phenylephrine in presence and absence of L-NAME

 

Figure 3
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  		Fig. 3 Concentration response curve of phenylephrine on aortic rings obtained from (a) ND-CON (-{blacktriangleup}-), ND-PIO (-bullet-) and (b) STZ-CON (-bullet-), STZ-PIO (lhblk) group in presence (light legends) and absence of 100 µM of L-NAME (dark legends). Values are expressed as mean ± SEM. * p<0.05, compared to presence of L-NAME.

 
3.4. Relaxation response to Ach and SNP on aorta obtained from control and pioglitazone treated rats
Addition of Ach to all aortic rings with intact endothelium resulted in concentration dependent relaxation of rings that were precontracted with PE. Ach induced relaxation in aorta obtained from STZ-CON group was significantly (p<0.05) lower as compared to ND-CON group (Fig. 4). pD2 value of Ach in STZ-CON group was significantly (p<0.05) lower as compared to ND-CON group (Table 3). Pioglitazone treatment significantly (p<0.05) increased Ach induced relaxation in aorta obtained from STZ-PIO group as compared to STZ-CON (Fig. 4). pD2 value of Ach in STZ-PIO group was significantly (p<0.05) increased as compared to STZ-CON group (Table 3). Addition of SNP completely relaxed aortic rings of all the groups. There was no significant change in SNP induced relaxation on endothelium-denuded rings in any of the groups (Fig. 5).


Figure 4
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  		Fig. 4 Concentration dependent relaxation of acetylcholine on aortic rings obtained from ND-CON (-{square}-), STZ-CON (-bullet-), ND-PIO (-{circ}-) and STZ-PIO (lhblk) groups on aortic rings (with intact endothelium) precontracted with PE. Tension is expressed as % relaxation on initial contraction with PE. Values are expressed as mean ± SEM. * P<0.05, compared to STZ-CON group. (n=5–8).

 

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  		Table 3 Maximal response (Emax) and pD2 (–Log EC50) values of acetylcholine

 

Figure 5
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  		Fig. 5 Concentration dependent relaxation of sodium nitroprusside on endothelium denuded aortic rings obtained from ND-CON (-{square}-), STZ-CON (-bullet-), ND-PIO (-{circ}-) and STZ-PIO (lhblk) group. Tension is expressed as percentage relaxation of initial response to PE. Values are expressed as mean ± SEM. (n=5–8).

 
3.5. Effects of indomethacin, L-NAME and methylene blue on Ach induced endothelium dependent relaxation on aorta obtained from control and pioglitazone treated rats
Ach completely relaxed precontracted aortic rings in ND-CON and ND-PIO groups and relaxation was completely blocked due to the presence of L-NAME or methylene blue (Fig. 6a, b). The presence of indomethacin decreased the relaxation of Ach in ND-CON and ND-PIO group. Ach induced relaxation which was impaired in STZ-CON group, was significantly increased in STZ-PIO group. Relaxation in STZ-CON and STZ-PIO was unaltered due to the presence of indomethacin, while it was completely blocked due to the presence of L-NAME or methylene blue (Fig. 6c, d).


Figure 6
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  		Fig. 6 Concentration dependent relaxation of acetylcholine alone (-bullet-) and in the presence of 10 µM of indomethacin (-{square}-), 100 µM of L-NAME+10 µM of indomethacin (-{circ}-) and 10 µM methylene blue(lhblk) in aortic rings (with intact endothelium) obtained from (a) ND-CON, (b) ND-PIO, (c) STZ-CON and (d) STZ-PIO group. Tension is expressed as percentage relaxation of initial response to PE. Values are expressed as mean ± SEM. * P<0.05, compared to Ach+methylene blue and Ach+indomethacin+L-NAME. (n=5–8).

 
3.6. Basal nitric oxide release
Addition of L-NAME to aortic preparation caused increase in contraction in all the groups. Contraction was significantly higher in STZ-PIO group aortic rings as compared to STZ-CON group (Fig. 7).


Figure 7
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  		Fig. 7 Percent contraction of endothelium intact aortic rings to different concentration of L-NAME. The aortic rings obtained from ND-CON (-{square}-), STZ-CON (-bullet-), ND-PIO (-{circ}-) and STZ-PIO (lhblk) group were moderately contracted with phenylephrine before obtaining cumulative responses to L-NAME. Values are expressed as mean ± SEM. * P<0.05, compared to STZ-CON.

 
3.7. Superoxide dismutase, catalase, reduced glutathione and lipid peroxidation
Oxidative stress was significantly (p<0.001) increased in liver, kidney and aorta of STZ-CON group as compared to ND-CON group (Table 4). SOD, CAT and GSH were significantly decreased while lipid peroxidation was significantly increased in STZ-CON group. Pioglitazone treatment significantly (p<0.01) increased levels of endogenous antioxidants (SOD, CAT and GSH) in liver, kidney and aorta as compared to STZ-CON (Table 4). Moreover lipid peroxidation was significantly (p<0.01) decreased in liver, kidney and aorta of STZ-PIO group as compared to STZ-CON (Table 4). There was no significant change in SOD, CAT, GSH and lipid peroxidation on ND-PIO group as compared to ND-CON.


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  		Table 4 Effect of pioglitazone treatment on superoxide dismutase, catalase, reduced glutathione and lipid peroxidation levels in liver, kidney and aorta

 
3.8. Aortic nitrite levels
Aortic nitrite levels of various groups are shown in Fig. 8. Aortic nitrite levels were significantly (p<0.05) higher in STZ-CON group as compared to ND-CON group. Pioglitazone treatment significantly (p<0.05) reduced aortic nitrite content of STZ-PIO group as compared to STZ-CON group. There was no significant change in aortic nitrite levels of ND-PIO group as compared to ND-CON group (Fig. 8).


Figure 8
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  		Fig. 8 Aortic nitrite levels determined from ND-CON, STZ-CON, ND-PIO and STZ-PIO groups. Values are expressed as mean ± SEM. * P<0.05, compared to STZ-CON, #P<0.05, compared to ND-CON.

 
3.9. Contractile effect of PE with the presence of pioglitazone on aorta obtained from untreated nondiabetic and STZ diabetic rats
The presence of pioglitazone (10 nM–10 mM) showed a similar pattern on contractile effect of PE in untreated ND and STZ-diabetic rats. Maximal response (Emax) and pD2 value of ND and STZ-diabetic rats are shown in Table 5. Maximal response (Emax) was significantly higher in case of STZ-diabetic rats as compared to ND rats (Table 5). Contractile response of PE in high concentration (greater than 10 µM) of pioglitazone was significantly (p<0.05) decreased as compared to vehicle (Fig. 9). Contractile response of PE was unaltered with the presence of low concentration (less than 10 µM) of pioglitazone, while the maximal response (Emax) to PE was decreased as the concentration of pioglitazone exposed to the aortic rings was increased (Table 5). The presence of pioglitazone at higher concentrations significantly (p<0.05) decreased the pD2 values of PE induced contractile response (Table 5). The presence of pioglitazone caused a concentration dependent rightward shift in PE response (Fig. 9).


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  		Table 5 Maximal response (Emax) and pD2 (–Log EC50) values of phenylephrine and acetylcholine on aortic rings of untreated age-matched non-diabetic and STZ-diabetic rats in presence of various concentration of pioglitazone

 

Figure 9
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  		Fig. 9 Concentration response curve of phenylephrine on aortic rings obtained from untreated age matched non-diabetic rats (a) and STZ diabetic rats (b) in presence of varying concentration of pioglitazone ((lhblk) vehicle-DMSO and 0.1 µM ({triangledown}), 1 µM (bullet), 10 µM ({blacktriangleup}), 0.1 mM ({diamond}), 1 mM ({circ}) and 10 mM ({square}) of pioglitazone). Values are expressed as mean ± SEM. * P<0.05, compared to vehicle. (n=5–8).

 
3.10. Relaxation response to Ach and SNP with the presence of pioglitazone on aorta obtained from untreated nondiabetic and STZ diabetic rats
Ach completely relaxed aorta obtained from untreated ND rats while relaxation in aorta obtained from STZ-diabetic rats was impaired with the presence of vehicle (Emax,% relaxation: 55.4 ± 1.45%). Relaxation response to Ach in low concentration of pioglitazone (less than 10 µM), was unaltered in ND or STZ-diabetic rats (Fig. 10). There was significant (P<0.05) increase in Ach induced relaxation in aortic rings in higher concentration of pioglitazone (greater than 10 µM) in aortic rings of ND and STZ-diabetic rats (Fig. 10). Percent relaxation produced by Ach was significantly (p<0.05) enhanced with the presence of pioglitazone (greater than 10 µM) as compared to vehicle in ND as well as STZ-diabetic rats (Table 5). The presence of higher concentration of pioglitazone caused significant (p<0.05) change in pD2 values of relaxation response to Ach (Table 5). Concentration–response curve of Ach in aorta of untreated ND rats was shifted towards left with the presence of pioglitazone (Fig. 10). SNP induced relaxation in case of ND rats (data not shown) and STZ-diabetic rats were similar and there was no significant effect of SNP induced relaxation due to the presence of pioglitazone (10 nM–10 mM) as compared to vehicle (Fig. 11).


Figure 10
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  		Fig. 10 Concentration dependent relaxation of acetylcholine on aortic rings (with intact endothelium) of untreated age matched non-diabetic rats (a) and STZ diabetic rats (b), precontracted with PE and in presence of varying concentration of pioglitazone ((lhblk) vehicle-DMSO and 0.1 µM ({triangledown}), 1 µM (-bullet-), 10 µM ({blacktriangleup}), 0.1 mM ({diamond}), 1 mM (-{circ}-) and 10 mM (-{square}-) of pioglitazone). Tension is expressed as percentage relaxation of initial response to PE. Values are expressed as mean ± SEM. * P<0.05, compared to vehicle. (n=5–8).

 

Figure 11
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  		Fig. 11 Concentration dependent relaxation of sodium nitroprusside on endothelium denuded aortic rings of untreated age matched STZ diabetic rats precontracted with PE and in presence of varying concentration of pioglitazone ((lhblk) vehicle-DMSO and 0.1 µM ({triangledown}), 1 µM (-bullet-), 10 µM ({blacktriangleup}), 0.1mM ({diamond}), 1 mM (-{circ}-) and 10 mM (-{square}-) of pioglitazone). Tension is expressed as percentage relaxation of initial response to PE. Values are expressed as mean ± SEM. (n=5–6).

 
3.11. Relaxation response to pioglitazone on precontracted aorta obtained from untreated nondiabetic and STZ diabetic rats
Addition of pioglitazone (10 nM–10 mM) in PE contracted rings (with intact endothelium) produced concentration dependent relaxation in ND and STZ-diabetic rats (Fig. 12). The relaxation was blocked due to the presence of L-NAME at lower concentration (less than 10 µM), but not at higher concentration. Pioglitazone did not produce relaxation at lower concentration (less than 10 µM) in endothelium denuded aortic rings of ND and STZ-diabetic rats, whereas at concentration higher than 10 µM pioglitazone produced relaxation (Fig. 12). Addition of pioglitazone at higher concentrations (greater than 10 µM) produced relaxation in endothelium denuded aortic rings that were contracted by 60 mM K+, but did not relax at lower concentration (Fig. 12).


Figure 12
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  		Fig. 12 Concentration dependent relaxation of pioglitazone on untreated age matched non diabetic rats (a) and STZ diabetic rats (b), precontracted with (-bullet-) PE with intact endothelium (+E), (-{circ}-) PE with denuded endothelium (-E), (-{square}-) PE with intact endothelium (+E) in presence of 100 µM L-NAME, (lhblk) K+60 mM with denuded endothelium (-E) and ({blacktriangleup}) vehicle. Values are expressed as mean ± SEM. (n=5–8).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Administration of STZ caused significant elevation in blood glucose level of diabetic rats and treatment with pioglitazone had no significant effect on blood glucose concentration in both nondiabetic and diabetic rats. The reason for this could be that the streptozotocin-induced diabetic animals were not insulin-resistant but insulin-deficient. Glitazone reduce plasma glucose levels by increasing peripheral insulin sensitivity (GLUT 4) and by additional effects on liver and skeletal muscle. Hence in STZ-diabetic animals (insulin-deficient), there was no significant effect of blood glucose levels. Similarly, pioglitazone did not affect the body weight, which moderately increased in nondiabetic as well as in diabetic rats. The blood pressure of 8-week STZ-diabetic rats was significantly higher as compared to nondiabetic control.

The results from the isolated aortic studies demonstrated that aortas from 8-week STZ-diabetic rats (STZ-CON) are more responsive to PE, while the relaxation response to Ach was significantly decreased than those from ND-CON. Similar results showing the increased vascular responsiveness to PE and decreased Ach induced relaxation in STZ-diabetic rats have been reported in previous studies [20–22]. Moreover the levels of endogenous antioxidants (SOD, CAT and GSH) were significantly reduced and lipid peroxidation significantly increased in STZ-CON group showing increased oxidative stress. Similar results showing increased oxidative stress (increased lipid peroxidation and reduced SOD, CAT and GSH) have been reported [23] in previous studies in STZ model.

Enhanced contractility could be due to deficient endothelial activity [24,25], enhancement of oxidative stress due to excessive production of oxygen-free radicals and decreased antioxidant defense systems [26,27]. It is a well-known fact that endothelium-dependent relaxation response to agonists such as Ach is impaired in diabetic rat aorta [3,4]. There are two possible mechanisms for reductions in Ach-induced relaxation, first nitric oxide (NO)-dependent vasodilatation, i.e. a decrease in NO release (or production) from the endothelium and a decreased reactivity of vascular smooth muscle to NO in diabetic animals. Another possible mechanism of reduced responses to Ach in diabetic animals is that oxidative degradation and inactivation of NO may be increased in vessels of such rats. Several studies have indicated the increased production of superoxide anions in vessels of diabetic animals. Further it is suggested that this active form of oxygen can inactivate NO to attenuate NO-dependent vasodilatory response [28] in the diabetic rabbit aorta [29]. It has also been reported that oxidative stress increases diacylglycerol-protein kinase activity in aorta of hyperglycemic rats [30] and leads to impaired endothelium-dependent relaxation in STZ-diabetic rat aorta. STZ-diabetic rats showed increased oxidative stress along with enhanced vascular contractility and decreased Ach induced relaxation. Therefore, the oxidative stress in diabetic animals might be responsible for increased contractility together with deficient endothelial function [25,31].

Administration of pioglitazone for 4 weeks restored the elevated blood pressure, reduced the enhanced contractility to PE and Ach induced relaxation was restored. In pioglitazone treated STZ-diabetic rats there was an increase in Ach induced relaxation which may be due to involvement of NO pathway since the relaxation was blocked with the presence of L-NAME and not with the presence of indomethacin. Moreover relaxation to Ach was also blocked with the presence of cGMP blocker methylene blue suggesting role of cGMP in elevated relaxation to Ach in STZ-diabetic aorta. Further tone related basal nitric oxide studies showed that pioglitazone treatment significantly increased the basal nitric oxide release in aortas of STZ-diabetic rats.

Various authors have shown that pioglitazone directly dilates blood vessels by blocking calcium channels [32,33]. In vitro studies on aorta of nondiabetic and STZ-diabetic rats were carried out to investigate whether the blood pressure lowering effect is due to direct effect of pioglitazone by blocking calcium channels. PE induced contraction and Ach induced relaxation studies with various concentrations of pioglitazone showed that the presence of low concentration of pioglitazone did not have any effect on the PE induced contraction or Ach induced relaxation. But the presence of higher concentration (greater than 10 µM) of pioglitazone caused significant changes in dose–response curves of PE and Ach in STZ-diabetic and nondiabetic aortas, showing direct effect of pioglitazone exists at concentration higher than 10 µM. Moreover as concentration increases from 10 µM to 100 mM direct vasodilator effect of pioglitazone increases. This was further evidenced as pioglitazone induced relaxation in rings with intact endothelium was blocked with the presence of L-NAME at lower concentrations but not at higher concentrations. Further relaxation due to pioglitazone in endothelium denuded rings depolarized by 60 mM K+ also supported this fact. Pharmacokinetic study in male rats have shown that maximum plasma concentration (Cmax) of pioglitazone after oral administration of 10 mg/kg is 35 µM [34]. As maximum plasma concentration after oral administration of 10 mg/kg of pioglitazone is 35 µM and in vitro data shows direct effect of pioglitazone at concentration greater than 10 µM, hence in the present study some direct effects of pioglitazone dose exist. But blood pressure lowering effect cannot be completely attributed to direct effect of pioglitazone on calcium channels as blood pressure was not lowered after the first week of treatment, significant blood pressure lowering effect was observed only after the third week of pioglitazone treatment. This may be due to the fact that though there may be direct effect of pioglitazone at week 1, but as STZ induced endothelial dysfunction is prominent and not restored, as a result blood pressure is not lowered after 1 week of pioglitazone treatment. Blood pressure lowering effect is observed only after 3 weeks when endothelial function is restored. Hence restored endothelial function together with direct effect of pioglitazone on calcium channels may be the reason for the decrease in blood pressure after pioglitazone treatment.

The reduction in oxidative stress may also be one of the reasons of the decrease in blood pressure coupled with restored endothelium function of STZ-diabetic rats treated with pioglitazone. Nitric oxide is rapidly inactivated by O2– and it has been reported that an enhanced formation of O2– radical may be involved in the accelerated breakdown of nitric oxide [35,36]. Moreover it has been shown that rapid destruction of nitric oxide occurs in streptozotocin induced diabetic rats [37]. The protective effect of pioglitazone against oxidative stress may prevent the breakdown of nitric oxide, which may improve vascular function. Similar observations were reported that pioglitazone reduces oxidative stress and increases NO bioavailability in coronary arterioles of mice [38]. Dobrian et al. [39] have reported that pioglitazone administration prevents hypertension and reduces oxidative stress in diet induced obesity. Similarly Kanie et al. [40] have reported bezafibrate, a PPAR alpha agonist improves endothelium-dependent relaxation by increasing expressions of the mRNAs for PPAR alpha and PPAR gamma. This may lead to a decrease in the expression of prepro ET-1, and the consequent decrease in plasma ET-1 may cause a decline in the expression of NAD(P)H oxidase, thereby resulting in a decrease in superoxide anion and a normalization of the endothelial dysfunction. It is also reported that PPAR gamma agonists reduce blood pressure in patients with type 2 diabetes and hypertension [41] and obese patients without diabetes [42,43].

Hence the restored endothelial function could be attributed to the protective effect of pioglitazone against oxidative stress and blood pressure lowering effect could be attributed to combined effect of restored endothelial function and direct effect of pioglitazone. Hence from the present study it can be concluded that pioglitazone administration in STZ-diabetic rats lowers blood pressure, protects against oxidative stress and restores endothelial function.


   Acknowledgement
 
Financial assistance provided by M.S. University of Baroda for the first and second author is highly acknowledged.


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Uemura S., Matsushita H., Li W., Glassford A.J., Asagami T., Lee K.H., et al. Diabetes mellitus enhances vascular matrix metalloproteinase activity: role of oxidative stress. Circ. Res. (2001) 88:1291–1298.[Abstract/Free Full Text]
  2. Senses V., Ozyazgan S., Ince E., Tuncdemir M., Kaya F., Ozturk M., et al. Effect of 5-aminoimidazole-4-carboxamide riboside (AICA-r) on isolated thoracic aorta responses in streptozotocin-diabetic rats. J. Basic Clin. Physiol. Pharmacol. (2001) 12:227–248.[Medline]
  3. Oyama Y., Kawasaki H., Hattori Y., Kanno M. Attenuation of endothelium-dependent relaxation in aorta from diabetic rats. Eur. J. Pharmacol. (1986) 131:75–78.[CrossRef][Web of Science][Medline]
  4. Kamata K., Miyata N., Kasuya Y. Impairment of endothelium-dependent relaxation and changes on levels of cyclic GMP in aorta from streptozotocin-induced diabetic rats. Br. J. Pharmacol. (1989) 97:614–618.[Web of Science][Medline]
  5. Ikeda H., Taketomi S., Sugiyamam Y., Shimura Y., Sohda T., Meguro K., et al. Effects of pioglitazone on glucose and lipid metabolism in normal and insulin resistant animals. Arzneim.-Forsch. (1990) 40:156–162.[Medline]
  6. Kotchen T.A., Reddy S., Zhang H.Y. Increasing insulin sensitivity lowers blood pressure in the fructose-fed rat. Am. J. Hypertens. (1997) 10:1020–1026.[CrossRef][Web of Science][Medline]
  7. Kemnitz J.W., Elson D.F., Roecker E.B., Baum S.T., Bergman R.N., Meglasson M.D. Pioglitazone increases insulin sensitivity, reduces blood glucose, insulin, and lipid levels, and lowers blood pressure in obese, insulin-resistant Rhesus monkeys. Diabetes (1994) 43:204–211.[Abstract]
  8. Grinsell J.W., Lardinois C.K., Swislocki A., Gonzalez R., Sare J.S., Michaels J.R., et al. Pioglitazone attenuates basal and postprandial insulin concentrations and blood pressure in the spontaneously hypertensive rat. Am. J. Hypertens. (2000) 13:370–375.[CrossRef][Web of Science][Medline]
  9. Uchida A., Nakata T., Hatta T., Kiyama M., Kawa T., Morimoto S., et al. Reduction of insulin resistance attenuates the development of hypertension in sucrose-fed SHR. Life Sci. (1997) 61(4):455–464.[CrossRef][Web of Science][Medline]
  10. Caballero A.E., Saquaf R., Lim S.C., Hamdy O., O'Connor C., Abuelenin K., et al. The effects of troglitazone on the endothelial function of the micro and macrocirculation in patients with early or late type 2 diabetes. Diabetes (2001) 50(Suppl. 2):A149. [Abstract].
  11. Gumieniczek A. Effect of the new thiazolidinedione-pioglitazone on the development of oxidative stress in liver and kidney of diabetic rabbits. Life Sci. (2003) 74:553–562.[CrossRef][Web of Science][Medline]
  12. Eto K., Ohya Y., Nakamura Y., Abe I., Fujishima M. Comparative actions of insulin sensitizers on ion channels in vascular smooth muscle. Eur. J. Pharmacol. (2001) 423:1–7.[CrossRef][Web of Science][Medline]
  13. Hayashi T., Fukuto J.M., Ignarro L.J., Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc. Natl. Acad. Sci. U. S. A. (1992) 89:11259–11263.[Abstract/Free Full Text]
  14. Bafna P.A., Balaraman R. Anti-ulcer and antioxidant activity of DHC-1, a herbal formulation. J. Ethnopharmacol. (2004) 90:123–127.[CrossRef][Web of Science][Medline]
  15. Mishra H.P., Fridovich I. The role of superoxide anion in the auto-oxidation of epinephrine and a simple assay for superoxide dismutase. J. Biochem. (1972) 247:3170–3175.
  16. Hugo E.B. Methods in Enzymology. Colowick S.P., Kaplan N.O., Packer L., eds. (1984) vol. 105. London: Academic Press. 121–125.
  17. Moron M.S., Depierre J.W., Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim. Biophys. Acta (1979) 582:67–78.[Medline]
  18. Slater T.F., Sawyer B.C. The stimulatory effects of carbon tetrachloride and other halogenoalkanes or peroxidative reactions in rat liver fractions in vitro. Biochem. J. (1971) 123:805–814.[Web of Science][Medline]
  19. Guevara I., Iwanejko J., Dembinska-Kiec A., Pankiewicz J., Wanat A., Anna P., et al. Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin. Chim. Acta (1998) 274:177–188.[CrossRef][Web of Science][Medline]
  20. MacLeod K.M. The effect of insulin treatment on changes in vascular reactivity in chronic, experimental diabetes. Diabetes (1985) 34:1160–1167.[Abstract]
  21. Harris K.H., Macleod K.M. Influence of the endothelium on contractile responses of arteries from diabetic rats. Eur. J. Pharmacol. (1988) 153:55–64.[CrossRef][Web of Science][Medline]
  22. Abebe W., Harris K.H., Macleod K.M. Enhanced contractile responses of arteries from diabetic rats to {alpha}1-adrenoceptor stimulation in the absence and presence of extracellular calcium. J. Cardiovasc. Pharmacol. (1990) 16:239–248.[Web of Science][Medline]
  23. Maritim A.C., Sanders R.A., Watkins J.B. Diabetes, oxidative stress, and antioxidants: a review. J. Biochem. Mol. Toxicol. (2003) 17:24–28.[CrossRef][Web of Science][Medline]
  24. Karasu C., Altan V.M. The role of endothelial cells on the alterations in vascular reactivity induced by insulin-dependent diabetes mellitus: effects of insulin treatment. Gen. Pharmacol. (1993) 24:743–755.[Web of Science][Medline]
  25. Chang K.C., Chung S.Y., Chong W.S., Suh J.S., Kim S.H., Noh H.K., et al. Possible superoxide radical-induced alteration of vascular reactivity in aortas from streptozotocin-treated rats. J. Pharmacol. Exp. Ther. (1993) 266:992–1000.[Abstract/Free Full Text]
  26. Oberlet L.W. Free radicals and diabetes. Free Radic. Biol. Med. (1988) 5:112–124.
  27. Baynes J.W. Role of oxidative stress in development of complications in diabetes. Diabetes (1992) 40:405–412.[CrossRef][Web of Science]
  28. Giugliano D., Ceriello A., Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care (1996) 19:257–267.[Abstract]
  29. Tesfamariam B., Jakubowski J.A., Cohen R.A. Contraction of diabetic rabbit aorta caused by endothelium-derived PGH2-TXA2. Am. J. Physiol. (1989) 257:H1327–H1333.[Web of Science][Medline]
  30. Kunisaki M., Bursell S.E., Umeda F., Nawata H., King G.L. Normalization of diacylglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels. Diabetes (1994) 43:1372–1377.[Abstract]
  31. Tesfamariam B. Free radicals in diabetic endothelial cell dysfunction. Free Radic. Biol. Med. (1994) 16:383–391.[CrossRef][Web of Science][Medline]
  32. Zhang F., Sowers J.R., Ram J.L. Effects of pioglitazone in calcium channels in vascular smooth muscle. Hypertension (1994) 24:170–175.[Abstract/Free Full Text]
  33. Buchanan T.A., Meehan W.P., Jeng Y.Y., Yang D., Chan T.M., Nadler J.L., et al. Blood pressure lowering by pioglitazone: evidence for a direct vascular effect. J. Clin. Invest. (1995) 96:354–360.[Web of Science][Medline]
  34. Fujita Y., Yamada Y., Kusama M., Yamauchi T., Kamon J., Kadowaki T., et al. Sex differences in the pharmacokinetics of pioglitazone in rats. Comp. Biochem. Physiol. C. Pharmacol. Toxicol. (2003) 136:85–94.[CrossRef]
  35. Gryglewski R.J., Palmer R.M.J., Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature (1986) 320:454–456.[CrossRef][Medline]
  36. Rubanyi G.M., Vanhoutte P.M. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am. J. Physiol. (1986) 250:H822–H827.[Web of Science][Medline]
  37. Kamata K., Kobayashi T. Changes in superoxide dismutase mRNA expression by streptozotocin-induced diabetes. Br. J. Pharmacol. (1996) 119:583–589.[Web of Science][Medline]
  38. Bagi Z., Koller A., Kaley G. PPAR{gamma} activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with type 2 diabetes. Am. J. Physiol. Heart. Circ. Physiol. (2004) 286:H742–H748.[Abstract/Free Full Text]
  39. Dobrian A.D., Schriver S.D., Kharaibi A.A., Prewitt R.L. Pioglitazone prevents hypertension and reduces oxidative stress in diet-induced obesity. Hypertension (2004) 43:48–56.[Abstract/Free Full Text]
  40. Kanie N., Matsumoto T., Kobayashi T., Kamata K. Relationship between peroxisome proliferator-activated receptors (PPAR{alpha} and PPAR{gamma}) and endothelium-dependent relaxation in streptozotocin-induced diabetic rats. Br. J. Pharmacol. (2003) 140:23–32.[CrossRef][Web of Science][Medline]
  41. Ogihara T., Rakugi H., Ikegami H., Mikami H., Masuo K. Enhancement of insulin sensitivity by troglitazone lowers blood pressure in diabetic hypertensives. Am. J. Hypertens. (1995) 8:316–320.[CrossRef][Web of Science][Medline]
  42. Nolan J.J., Ludvik B., Beerdsen P., Joyce M., Olefsky J.M. Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N. Engl. J. Med. (1994) 331:1188–1193.[Abstract/Free Full Text]
  43. Tack C.J., Ong M.K., Lutterman J.A., Smits P. Insulin-induced vasodilatation and endothelial function in obesity/insulin resistance. Effects of troglitazone. Diabetologia (1998) 41:569–576.[CrossRef][Web of Science][Medline]

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