Hepatic nitrosative stress in experimental diabetes

Abstract

Aim

The effects of the inhibition of nitrosative stress by aminoguanidine in an experimental model of diabetes mellitus (DM) were investigated.

Methods

Twenty-one male Wistar rats were divided into three groups: control (CO), diabetic (DM), and diabetic treated with aminoguanidine (DM + AG). Aminoguanidine (aminoguanidine hemisulfate salt, Sigma Chemical Co., St. Louis, MO, USA) was used at a dose of 50 mg/kg (i.p.) during the last 30 days of the experiment. The expression levels of liver lipoperoxidation (TBARS - nmol/mg protein), inducible oxide nitric synthase (iNOS), nitrotyrosine and the NFκB nuclear transcription factor p65 were examined using western blot analysis.

Results

The DM group demonstrated an increase in lipoperoxidation and in the expression of iNOS, nitrotyrosine and p65. Aminoguanidine reduced hepatic lipid peroxidation and protein expression levels of iNOS, nitrotyrosine and p65.

Conclusion

Aminoguanidine treatment reduces liver oxidative and nitrosative stress in diabetic animals. In addition, aminoguanidine reduced the expression of p65 in the liver.

Introduction

The excessive formation of free radicals in many reactions plays a crucial role in the pathogenesis of chronic diabetic complications (Bonnefont-Rousselot, 2002, Ceriello, 2003). The enhanced level of metabolism in mitochondria leads to increased leakage of superoxide (O₂^−·) from the respiratory chain; furthermore, inducible nitric oxide synthase (iNOS) is induced by many factors, resulting in the production of excess nitric oxide (NO) and O₂^−· (Ha & Lee, 2000, Nishikawa et al., 2000, Pacher et al., 2007). The interaction of the diffusing NO with the O₂ ^−· results in the formation of peroxynitrite (ONOO −) at a diffusion-coefficient-limited rate. Nitrosative stress occurs when the production of the highly reactive nitrogen-containing chemicals exceeds the ability of the human body to neutralize and eliminate them. Nitrosative stress can lead to reactions that alter protein structure, interfering with normal body functions. Oxidative stress causes increased expression of transcription factors that favor increased expression of iNOS and nitrosative stress (Di Naso et al., 2011, Dias et al., 2005). Notably, transcription factors, such as NF-κB, have an important role in the pathogenesis of diabetic complications, and p65 overexpression in the nucleus is related to this activation process.

The liver, which is rich in mitochondria, is an organ that is important to both energy and substance metabolism. Therefore, damage to the liver mitochondria will aggravate the metabolic disorders in diabetes. The liver is the main organ of oxidative and detoxifying processes, as well as free radical reactions. In many diseases, the biomarkers of oxidative stress are elevated in the liver at an early stage (Dey & Swaminathan, 2010, Ren et al., 2011).

Aminoguanidine (AG) is a prototype therapeutic agent for the prevention of advanced glycation end product (AGE) formation. AG reacts rapidly with a,b-dicarbonyl compounds, such as methylglyoxal, glyoxal, and 3-deoxyglucosone, to prevent the formation of AGEs (Thornalley, 2003). The adducts formed are substituted derivatives of 3-amino-1,2,4-triazine. The inhibition of disease mechanisms, particularly vascular complications in experimental diabetes, via AG demonstrates that the accumulation of AGEs is a risk factor for disease progression (Degenhardt et al., 1999). In addition to the effects on advanced glycation, aminoguanidine at low dose can act in a specific manner via the inhibition of nitric oxide synthase (iNOS) activity and nitrosative stress reduction (Szabo et al., 1997). Additionally, AG can inhibit the metabolism of histamine, the catabolism of polyamines and the activity of catalase, and increase the effects of angiotensin in the production of prostacyclin (Nilsson, 1999). Although many inhibitors of NO synthase have been developed to date, few selective inhibitors for the individual isoforms of NOS (inducible, endothelial, neuronal) have been described. Among these, AG has been identified as one of the first iNOS-selective inhibitors (Pacher et al., 2007) (Fig. 1).

Because the liver is subjected to ROS-mediated injury in diabetes, our experiments were performed to investigate the potential protective effects of aminoguanidine treatment on liver oxidative and nitrosative stress in an experimental model of diabetes mellitus.