Effect of Superoxide Dismutase Mimetic Tempol on Dexamethasone Induced Insulin Resistance-Role of Oxidative Stress.

 

Karnakumar V. Biradar1*, Chandrashekhar B. Patil1, Basavaraj V. Chivde2, M. H. Malipatil3 and Sanjivkumar D. Biradar1

1R.R.K.S College of Pharmacy, Bidar, Karnataka (India).

2S.V.E.T College of Pharmacy, Humnabad, Bidar Karnataka (India).

3Karnataka College of Pharmacy, Bidar Karnataka (India).

ABSTRACT:

Glucocorticoids like Dexamethasone are widely used Immunosuppressant and Anti-inflammatory agent. They have several adverse effects, majorly Insulin Resistance (IR) and Hypertension these are probably attributed to Oxidative Stress. In the present study, effect of SOD Mimetic Tempol (2,2,6,6,-tetramethyl-4-hydroxy-1-piperidinyloxy) was administered (1mmol/L) for Prophylactic as well as Therapeutic regimen in Experimental Induced Insulin Resistance in male Wister rats by using Dexamethasone (20µg/kg) subcutaneously and estimated Plasma Insulin and TBARS were measured in Plasma as well in isolated Aortic tissue as a marker of Oxidative Stress. HOMA-IR index and Mastuda Index were calculated as an Insulin Resistance/ Insulin Sensitivity. From the finding of present study, Tempol failed to produce any effect on IR so in conclusion, Oxidative Stress may be playing the Initiative role in Dexamethasone Induced IR.

 

KEYWORDS: Insulin Resistance (IR), Oxidative Stress, Dexamethasone, Insulin Resistance Index (HOMA- IR) and Insulin Sensitivity Index (MASTUDA INDEX).

 

INTRODUCTION:

Glucocorticoid such as Dexamethasone is widely used Therapeutic agent for Immunosuppressive and Anti-inflammatory action. Its use is associated with several adverse reactions which includes Hypertension, Diabetes and Insulin Resistance due to over production of ROS (Bjelakovic et al., 2007). Insulin Resistance is a cardinal feature of type-2 diabetes and may be an impetus for the development of Hypertension. Dexamethasone induced Insulin Resistance probably due to Oxidative Stress (Xi L et al., 2005). Although, ROS have been proposed to have a causal role in multiple forms of Insulin Resistance. Tempol administration normalized the level of Oxidative Stress and the Insulin Resistance in angiotensin II infused rats (Ogihara T et al., 2002). Hence, in the present study we aimed to Evaluate the effect of SOD mimetic Tempol on Dexamethasone-Induced Experimental Insulin Resistance to explore the role of Oxidative Stress in Insulin Resistance.

 

MATERIAL AND METHODS

Male Wistar rats weighing (240-300g) were used. They were housed in a group of six under environmentally controlled room with 12-h light/dark cycle and had free access to food and water. Dexamethasone injection was procured from Zydus cadila, 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl freies RadiKal (Tempol) was procured from Sigma Aidrich Germany , 2-Thiobarbituric acid, and 1,1,3,3,-Tetramethoxy propane (Malonaldehyde bis) was procured from Himedia Laboratories Ltd, Mumbai, India,  Radio


Immuno Assay Kit for Insulin- RIAK-1 was procured from Board of Radiation and Isotope Technology, BARC, Mumbai, India. Dosage of the Dexamethasone phosphate administered subcutaneously was prepared in the saline (0.9 % sodium chloride saline solution). Dose of the Dexamethasone phosphate (20µg/kg b.w.) for the present study, were chosen based on previous reports (Rhee MS et al., 2004). Dose of the Tempol (1mmol/l) in drinking water for the study, were chosen based on previous report (Britt E et al., 2004). Insulin resistance was induced in rats by using dexamethasone by injecting dexamethasone 20µg/kg, subcutaneously once daily for 14 days. The animals were divided into 4 groups containing six in each as follows.

 

 

(1)  Normal Control (NC): Received normal saline (0.9% NaCl) subcutaneously for 14 days.

(2) Dexamethasone (DEX): Received Dexamethasone 20µg/kg, subcutaneously, once daily for 14 days.

(3) Dexamethasone and Tempol (TEMP-P): Received dexamethasone 20µg/kg, subcutaneously, once daily for 14 days and treated with Tempol (1mmol) given in drinking water from 1stday and continued throughout study.

(4) Dexamethasone and Tempol (TEMP-T): Received Dexamethasone 20µg/kg, subcutaneously, once daily for 14 days and treated with Tempol (1mmol) given in drinking water from 8th day and continued throughout study.

 

During the Experimental period on 13th day of Experimental period Systolic Blood pressure was measured by tail cuff method. At the end of treatment period, Blood samples were collected by retro-orbital plexus puncture under light ether anesthesia after 16 h fasting (on 14th day) and then animals were sacrificed by excess ether anesthesia and thoracic aorta was isolated for estimation of lipid peroxides (TBARS). Systolic Blood pressure was measured indirectly by the tail cuff method using noninvasive blood pressure apparatus. The Insulin estimation was done by using ImmuChem Radioimmunoassay method (Loraine JA et al., 1976). Using a standard kit obtained from BRIT, BARC, Mumbai, India. Triglyceride was estimated by method of (Buccolo G et al., 1976) using a standard kit obtained from ERBA diagnostics Manheim Ltd. Insulin resistance index (Henry RR et al., 2003) and Insulin sensitivity Index (Albareda M et al., 2000) were calculated. The TBARS estimation is based on the reaction between plasma malondialdehyde (MDA), a product of lipid peroxidation and thiobarbituric acid (TBA) and carried out as previously described (Yoshioka T et.al., 1979).

 

Prism 3 graph pad were used for Graphical representation. Statistical analysis was done by using unpaired student’s t-test and One-way ANOVA followed by benferroni multicomparision Test.

 

RESULTS AND DISCUSSION:

There was a significant elevation in fasting Basal Plasma Insulin, Plasma Triglycerides level and concentration of Malondialdehyde in plasma (P<0.01) as well in Aortic tissue (P<0.05) when compared with normal rats injected with dexamethasone for 14 days. There was a significant elevation of HOMA-IR value and decrease in MASTUDA INDEX in rats injected with Dexamethasone for 14 days (P<0.01) when compared with normal rats (Table no 2). Tempol had no significant effect on Dexamethasone induced increase in Blood Pressure and in fasting basal plasma insulin level. Tempol had no effect on Plasma Malondialdehyde level and in Aortic tissue when compared to Dexamethasone injected rats. Tempol had no effect on HOMA-IR index and MASTUDA INDEX (Table no 2) when compared with Dexamethasone injected rats. Elevation of triglycerides level and plasma concentration of Malondialdehyde (P<0.05) induced by Dexamethasone injected rats was significantly reversed by Tempol. In the present study administration of dexamethasone subcutaneously for 14 days lead to development of insulin resistance as indicated by Hyperinsulinemia and Hyppertriglyceridemia surrogate markers of insulin resistance along with increased HOMA-IR index and decrease in MASTUDA INDEX. In addition to these metabolic abnormalities there is an increase in Systolic Blood pressure is also observed in dexamethasone injected rats. Interestingly these abnormalities were associated with increased Oxidative stress as indicated by increased TBARS level in plasma aswell in Aortic tissue. Administaration of Tempol, a SOD Mimetic/ Superoxide scavenger in drinking water prophylactically along with Dexamethasone administration had no significant effect on development of Insulin Resistance as indicated by no effect on Dexamethasone induced Hyperinsulinemia, increased HOMA-IR index and decreased MASTUDA INDEX a measure of Insulin sensitivity and resistance and also had no effect on increased Systolic blood pressure.  Noticeably, however, it tends to lower the TBARS but, had no significant effect on TBARS level. But, Therapeutic administration of Tempol from 8th day onwards of Dexamethasone injection significantly reduced the systemic Oxidative stress. However, it had no reversal effect on Dexamethasone induced Insulin resistance and increased Systolic Blood pressure, both Prophylactic and Therapeutic administration of Tempol significantly reduced the Triglyceride level. These observations, in addition to delayed increase in TBARS level in Dexamethasone injected rats indicates that ROS such as Superoxide may not be playing the initiative role in the development of Insulin Resistance and Hypertension in Dexamethasone injected rats. From the above findings of the present study it appears that Oxidative stress may not be playing an initiative role in the development of Dexamethasone induced Insulin Resistance in rat and Results were expressed in table no. 1and 2 and fig. no. 1 and 2.

 

 

Table 1: Systolic Blood Pressure, Basal insulin level and Plasma Triglycerides.

Groups

Plasma Triglycerides

(mg/dl)

Basal Insulin       level

(mg/dl)

Systolic blood pressure

(mmHg)

Normal (NC)

58.69±12.253

13.92±1.228

130.20±1.594

Dexamethasone (DEX)

131.67±16.038*

25.33±4.128*

197.9 ±5.888*

Dexamethasone and Tempol

(TEMP-P) (Preventive)

60.70±5.654*

19.16±4.989

182.64±15.174

Dexamethasone and Tempol

(TEMP-T) (Therapeutic)

81.79±7.298*

18.66±1.202

190.73 ±4.626

Values are Expressed in Mean ± SEM; n=6.Comparision of mean values between Normal and Dexamethasone groups were performed by unpaired student’s t-test. *P<0.05, **P<0.01 and **P<0.01 when compared with Normal (NC). Dexamethasone and Tempol treated groups were performed by One-way ANOVA followed by benferroni multicomparision Test. *P<0.01 and **P<0.001 when compared with Dexamethasone (DEX).

 

Table 2: Insulin Resistance Index, Insulin Sensitivity Index and Concentration of        Malondialdehyde in Plasma and in Aortic tissue.

 

Groups

Insulin resistance index

(mg/dl)

Insulin sensitivity index

(mg/dl)

Malondialdehyde concentrations in       plasma. (µmol/L)

Malondialdehyde concentrations in            Aorta. (µmol/L)

Normal (NC)

2.967±0.248

0.4600±0.051

0.600±0.06325

0.9500±0.4121

Dexamethasoe (DEX)

8.256±1.843

0.2339±0.036

3.500±0.8062**

3.333±0.8819*

Dexamethasone and Tempol

(TEMP-P) (Preventive)

5.0845±1.552

0.2339±0.036

1.833±0.542

2.783±0.632

Dexamethasone and Tempol

(TEMP-T) (Therapeutic)

4.873±0.2757

0.3099±0.053

1.566±0.454*

2.10±0.513

Values are Expressed in Mean ± SEM; n=6.Comparision of mean values between Normal and Dexamethasone groups were performed by unpaired student’s t-test. *P<0.05, **P<0.01 and **P<0.01 when compared with Normal. Dexamethasone and Tempol treated groups were performed by One-way ANOVA followed by benferroni multicomparision Test. *P<0.01 and **P<0.001 when compared with Dexamethasone.

 

Fig. no 1: Concentration of Malondialdehyde in Aortic tissue and in Plasma.

 

 Fig. no 2: Insulin Resistance Index and Insulin Sensitivity Index.

 

 

CONCLUSION:

In conclusion, Dexamethasone Induced Insulin Resistance and Hypertension accompanied by increased Oxidative Stress. However, it appears that abnormality in Insulin action and elevation of Systolic Blood Pressure occurs earlier than Oxidative Stress. Further, Anti-Oxidant like Tempol does not have influence on markers of Insulin Resistance/ Insulin Sensitivity. Hence, it may be concluded that Oxidative Stress may not playing the Initiative role in Dexamethasone induced Insulin Resistance.

 

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Received on 26.03.2011

Accepted on 10.04.2011     

© A&V Publication all right reserved

Research J. Pharmacology and Pharmacodynamics. 3(3): May –June, 2011, 134-137