Activity of serum paraoxonase 1 , lipid profile and atherogenic indexes in diabetic induced rats treated with alpha lipoic acid

1Student Research Committee, Lorestan University of Medical Sciences, Khorramabad, Iran 2Razi Herbal Researches Center, Lorestan University of Medical Sciences, Khorramabad, Iran 3Department of Biochemistry, Lorestan University of Medical Sciences, Khorramabad, Iran 4Department of Anesthesiology, Faculty of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran 5Department of Pharmaceutical biotechnology, Tabriz University of Medical Sciences, Tabriz, Iran


Background
Hyperlipidemia is a key risk factor in cardiovascular diseases caused by diabetes.Diabetes increases the levels of total cholesterol (TC), triglyceride (TG), and low density lipoprotein (LDL-C) and decreases the high density lipoprotein (HDL-C) level which can cause the formation of small dense LDL-C particles with high potential for oxidation (1).Therefore, it increases the ability of HDL-C to protect against lipid oxidation (2).Since the PON1 is correlated to HDL-C, under the condition of diabetes it/HDL-C can reduce the level of the antioxidant enzyme.The process that is postulated to play a central role in endothelial cell damage (3).Hence diabetes is involved in the elevation of microvascular and macrovascular abnormalities in coronary heart diseases (1,3).Human serum paraoxonase 1 (aryl dialkyl phosphatase EC 3.1.8.1) has serum esterase activity in the liver, kidney and small intestine.High amount of this enzyme in the serum is contained in HDL-C particles.Paraoxonase 1 (PON1) is firmly connected with N-terminal domain of apolipoprotein A1 in HDL-C and very low in other lipoprotein such as chylomicrons and VLDL (4).PON1 does the enzymatic hydrolysis of organophosphorus compounds such as oxidized cholesteryl esters and phosphatidylcholine core aldehydes (5).It is postulated that PON1 has two active sites: one for hydrolysis of oxygen analog and anti-atherogenic enzyme that is known to attenuate the formation of plaques.The antiatherogenic of ability PON1 causes protection of lipoprotein particle against oxidation-free radicals because of the hydrolysis of the cholesteryl ester and steroid nucleus phosphatidylcholine (6,7).Different studies have shown that PON1 and HDL-C prevent oxidative LDL-C in vitro and in vivo.Oxidized forms of LDL aggregate in the subendothelial space of arteria starting the initial steps of atherosclerosis (8).Thus, there is inverse relationship between oxidized LDL-C and PON 1 activity.Hence, decreasing levels of PON 1 activity in people with high risk of developing atherosclerosis have been detectable (8).PON1 activity, inhibits the lipid oxidation on macrophage and erythrocyte and increase the expression of monocyte chemotactic protein 1 (MCP-1) due to the induction of inflammatory responses in endothelial cells preventing the complication of atherosclerosis (9).PON 1, Hydrolyze hydrogen peroxide as a major active oxygen radical and thereby is effective in reduction of oxidative stress (10).Alpha lipoic acid is a supplement with high antioxidant activity that contracts in mitochondria membrane as a main co-factor.Previous studies have proven alpha lipoic acid antioxidant effects on improving diabetic nephropathy and neurologic diseases (11).Thus, alpha lipoic acid is an antioxidant compound with antidiabetic properties that improves diabetes complications such as hyperlipidemia and atherosclerosis (12).

Objectives
The aim of current study is to investigate the effects of alpha lipoic acid on PON1 activity, lipid profile, atherogenic index and correlation between HDL-C and atherogenic index in alloxan monohydrate-induced diabetic rats.

Experimental design
The study was carried out on thirty adult male Sprague Dawley rats (180±20 g).The rats were purchased from the animal house of the Shahid Beheshti Medical University, Tehran, Iran.The animals were kept for 1 week in animals laboratory to adapt themselves with conditions.established standard laboratory conditions (temperature of 22°C and humidity of 50±10%), and 12-hour light/ dark cycles.The animals were randomly divided into three groups of tens.control (group I), non-treated diabetics (group II) and diabetic rats treated with alpha lipoic acid (group III) groups.Diabetes was inducted with injection of 100 mg/kg alloxan monohydrate (SC) for animals in II and III groups (Sigma, St. Louis, MO, USA) (13).FBS was measured after three days.The animals with glucose level of ≥250 mg/dL were recognized diabetics and separated for the study (9).Alpha lipoic acid powder (Sigma, St. Louis, MO) 100 mg/kg dissolved in 0.9% NaCl and ethanol (96%) was injected to animals in group III for 6 weeks daily (14).then the animals were anesthetized with ketamine (87 mg/kg intraperitoneally) and xylazine (13 mg/kg intraperitoneally) and blood samples were obtained from animals in all groups.The blood samples were placed in laboratory temperature for 20 minute so as to clot and then centrifuged at 3000 rpm for 15 minutes for serum separation.

Determination of PON1 activity
PON1 activity in serum was determined with spectrophotometry at 412 nm according to our previous study (7) with some modifications.Determination of PON1 activity was made using paraoxon.The activity was measured by adding 10 µL of serum to 1 mL Tris-HCl buffer (100 mM at pH 8) containing 2 mM CaCl2 and 5 mM of paraoxon.The production of 4-nitrophenol and detected with spectrophotometer at 412 nm (Caution XS-D5520M, France) after 3 minutes.PON1 activity was expressed with unit/mg-pr.

Ethical approval
All of the experimental protocols were conducted in accordance with the manuals of the Animal Ethics Committee of the Lorestan University of Medical Sciences.Additionally, all experimental protocols and steps of the tests were conducted in compliance with the regulations of the Research Ethics Committee of our university and Iranian Ethical Guidelines for the use of animals in research.Additionally All animal experiments were in accordance with protocols approved by the United States National Institutes of Health (NIH, 1978).This research was supported by Lorestan University of Medical Sciences (Grant# 21/91).

Statistical analysis
All values are expressed as mean± SD.The data were compared between groups by Mann-Whitney U test.Statistical analysis was conducted using the SPSS 22 (Chicago, IL) for windows software.A P value of ≤0.05 was considered statistically significant.

The effects of alpha lipoic acid on FBG and lipid profile
The levels of FBS and lipid profile are shown in Table 1.The level of serum FBS was significantly (P < 0.05) increased in non-treated diabetic group compared to control group.Treatment with alpha lipoic acid significantly (P < 0.05) inhibited the increase of FBS (39.31%) in treated diabetic group compared to untreated diabetic group.The levels of TC, TG, VLDL and LDL-C were significantly (P < 0.05) increased in non-treated diabetic group compared to control group.Treatment with alpha lipoic acid significantly (P < 0.05) inhibited the increase of TC, TG, VLDL and LDL-C in treated diabetic group with alpha lipoic acid compared to diabetic group.The level of HDL-C was significantly (P < 0.05) decreased in non-treated diabetic group compared to control group.Treatment with alpha lipoic acid significantly (P < 0.05) increased HDL-C in treated diabetic group compared to diabetic group.

The effects of alpha lipoic acid on atherogenic index, AC and CRR
The levels of atherogenic index, ACand CRR are shown in Table 1.The level of atherogenic index ([unite] [log TG/HDL-C]) and AC (TC-HDL-C/HDL-C) were significantly (P < 0.05) increased in non-treated diabetic group compared to control group.Treatment with alpha lipoic acid significantly (P < 0.05) inhibited the increase of atherogenic index and AC in diabetic group treated with alpha lipoic acid compared to diabetic group.The levels of CRR1 (TC/HDL-C) and CRR2 (LDL/HDL-C) were significantly (P < 0.05) increased in non-treated diabetic group compared to control group.Treatment with alpha lipoic acid significantly (P < 0.05) inhibited the increase of CRR1 and CRR2 levels in diabetic group treated with alpha lipoic acid compared to untreated diabetic group.

The effects of alpha lipoic acid on PON 1 activity and correlation with HDL-C, AC and CRR
The activity of PON1 and its correlation with HDL-C, AC and CRR are shown in Table 1 and Figures 1, 2, 3 and 4. activity of PON1 was significantly (P < 0.05) decreased in non-treated diabetic group compared to control group.

Effect of alpha lipoic acid on serum level of PON1 activity and correlation between PON 1 activity with HDL-C and atherogenic index
All findings of the present study support a role of PON1 in cardiovascular diseases causes by diabetes.Hyperglycemia, hyperinsulinemia and dyslipidemia are associated with oxidative stress and dysfunctional endothelium (17).Diabetes significantly decreased the serum PON1 activity and HDL-C concentration in comparison to control group.Treatment with alpha lipoic acid inhibited the reduction in PON1 activity and HDL-C concentration compared to non-treated diabetic group.There is strong evidences that show PON1 has beneficial effect against atherosclerosis by protection of HDL-C from peroxidation and plasma membrane from injury thereof.In clinical studies on patients with type 1 diabetes have demonstrated the reduced level of PON1 activity compared to healthy group (15).Different studies have shown that antioxidant therapy is one of the most important treatment strategies for diabetic patients (18).
All results of the current study obviously demonstrated that the activity of PON1 correlated positively with HDL-C and negatively with AC, CRR1 and CRR2 in treated diabetic rats.PON1 has an antioxidant enzyme that decreased LDL-oxidation (19).HDL-C can inhibit LDL-C oxidation via the transition of metals and the prevention of lipid hydro peroxides and ester hydro peroxides formation (20,21).Several study showed HDL-C has anti-atherogenic and anti-inflammatory activities by inhibiting the expression of adhesion molecules on the endothelial cells and the transmigration of monocytes (22).PON1 activity was positivity correlated with HDL-C level.In return, PON1 activity was inversely correlated with atherogenic index.Therefore, it may be suggested that reduced PON 1 activity is probably due to the expenditure of PON1 for the protection of oxidation (10,11).All the results of the present study were in accordance with the previous reports (10,11).Therefore, alpha lipoic acid has an antioxidant properties that may cause an increase in PON1 activity and has a positive correlation with HDL-C and negative correlation with the atherogenic index.Numerous studies have indicated that oxidative stress has an important role in coronary artery diseases, diabetes and complications such as dyslipidemia and hyperinsulinemia (3,22).Therefore the previous studies and the results of our study demonstrated the efficacy of oxidative supplements in improving such complications.Administration of antioxidants has improving impact on the each stage of diabetes and its complications.

Effect of alpha lipoic acid on serum lipid profile and atherogenic index
Hyperlipidemia occurs when the amount of lipoproteins in the serum is abnormal.It has been detected that the concentration of cholesterol in HDL-C has inverse relationship while serum TC, TG and LDL-C levels are positively related to atherosclerosis (23).In the current study, there was significantly increased serum FBS, TG, TC, VLDL-C and LDL-C concentrations in diabetic group comparison with the control group.Treatment of diabetic rats with alpha lipoic acid significantly inhibited the increase of serum FBS, TG, TC, VLDL and LDL-C concentrations in comparison with diabetic group.Additionally, atherogenic index, AC, CRP1 and CRP2 as risk factors in cardiovascular diseases, were decreased on diabetic rats when treated with alpha lipoic acid.In addition, various studies on rabbits, indicated that atherogenic index and the levels of LDL-C were increased in diabetes condition when treated with alpha lipoic acid (24,25).Findings of the recent researches on patients with chronic kidney diseases shown that decreased levels of PON1 activity, were accompanied by the increased levels of lipid profile and atherogenic index that was in accordance with our results (24,25).The HDL-C serum concentration were significantly decreased in diabetic rats in comparison to control.
Treatment with alpha lipoic acid increased HDL-C concentration in diabetic rats.Several studies demonstrated increased concentrations of lipid profile in diabetics (24,25).Therefore, antioxidant therapy may be one of the modalities for improvement of lipid and glucose levels.Natural antioxidants such as vitamin E, vitamin C and Q10 show hypoglycemic and hypolipidemic effects in diabetes conditions (7).Other studies have demonstrated beneficial effects of alpha lipoic acid on lipid profile in insulin resistance (24,25).
Findings of our study are consistent with other studies.The mechanism by which alpha lipoic acid can decrease TC and LDL-C concentration is unknown.However, it may be conducted through lipoprotein lipase (LPL) activity or through cholesterol metabolism by the liver.Antioxidant effects of alpha lipoic acid to reduce lipid profile in diabetes condition may be due to the ability of alpha lipoic acid to initiate LDL-C receptor synthesis in liver.Thus, alpha lipoic acid in return augments the uptake of cholesterol back to the hepatocyte and raise the synthesis of apolipoprotein that is present in HDL-C particles to inverse cholesterol transport (12,26).Therefore, natural antioxidants such as olive and Satureja khozestanica essential oil improve LDL-C oxidation invitro and hyperlipidemia, glycation and peroxidation in diabetes condition.In addition, Alpha lipoic acid as a natural antioxidant can have beneficial effects to decrease oxidative stress (27,28).

Conclusions
The results of our study showed that alpha lipoic acid has modulatory effects on lipid profile and atherogenic index.Alpha lipoic acid increased PON1 activity and serum HDL-C levels in diabetic rats.Alpha lipoic acid therapy is correlated positively with HDL-C and negatively with AC, CRR1, and CRR2 in rats.