Elsevier

Journal of Diabetes and its Complications

Volume 22, Issue 5, September–October 2008, Pages 357-364
Journal of Diabetes and its Complications

Original articles
Peroxynitrite-induced protein nitration contributes to liver mitochondrial damage in diabetic rats

https://doi.org/10.1016/j.jdiacomp.2007.06.013Get rights and content

Abstract

Oxidative stress, especially peroxynitrite (ONOO)-mediated oxidative stress, plays a key role in diabetes. Mitochondria, as the generating source of ONOO, may also be the major damaging target of ONOO, which can cause a series of mitochondrial proteins nitration. Therefore, this study aimed to clarify the relationship between the nitration of entire mitochondrial proteins induced by ONOO and liver mitochondrial structural damage in diabetes. Sprague–Dawley male rats were injected with streptozotocin to induce diabetes. After 10 weeks, transmission electron microscopy was used to observe the ultrastructure of liver mitochondria, and reverse transcription–polymerase chain reaction was used to detect liver inducible nitric oxide synthase (iNOS) mRNA expression. Nitrotyrosine (NT) content and distribution were detected with Western blot analysis and immunohistochemistry. In addition, some biochemical indicators were detected to represent oxidative stress and metabolic disorders. In diabetic rats, increasing levels of iNOS mRNA and NT content (P<.05) were observed, in accord with pathological alterations of the ultrastructure of liver mitochondria. Meanwhile, some alterations in biochemical indicators were observed in diabetes. Treatment with aminoguanidine could significantly attenuate these alterations (P<.01 or P<.05). In conclusion, the nitration of mitochondrial proteins induced by ONOO may be responsible for structural damage to liver mitochondria, and aminoguanidine can reduce ONOO generation and attenuate mitochondrial damage.

Introduction

Oxidative stress plays a crucial role in the pathogenesis of chronic diabetic complications (Du et al., 2003, Gumieniczek et al., 2002, Hirsch & Brownlee, 2005, Mastrocola et al., 2005, Pacher & Szabo, 2006, Sayyed et al., 2006). In diabetes, excessive free radicals are formed in many reactions (Khan et al., 2006, Pacher & Szabo, 2006). Enhanced metabolism in mitochondria leads to more leakage of superoxide (O2−·) from the respiratory chain (Hammes, 2003, Kukidome et al., 2006, Piconi et al., 2006). Meanwhile, inducible nitric oxide synthase (iNOS) is induced by many factors, producing excess nitric oxide (NO) and O2−· (Porasuphatana, Weaver, & Rosen, 2006). Once diffusing NO meets O2−·, peroxynitrite (ONOO) is formed at a diffusion-coefficient-limited rate (Ortega & Amaya, 2000, Pryor & Squadrito, 1995). The oxidative ability of ONOO and its derivative, a species with the strongest oxidation known at present, is 2000-fold stronger than that of H2O2 (Freeman, 1994). ONOO can cause lipid peroxidation, protein tyrosine nitration, and DNA strand breakdown (Szabo, 2003). Although the half-life of ONOO is extremely short, it can make selective 3-nitrotyrosine (3-NT) in proteins; thus, 3-NT is often measured as an indicator for the generation and distribution of ONOO (Kamat, 2006).

Mitochondria are considered to be the major generating source and the main damaging target of ONOO (Cadenas, 2004, Lacza et al., 2006, Palomba et al., 2001). As O2−· is mainly generated from the respiratory chain in mitochondria in diabetes, excessive ONOO is formed at once from excess O2−· and NO in mitochondria. It has been reported that ONOO could cause the nitration of a variety of mitochondrial proteins, such as ATPase (Kocak-Toker et al., 2005, Qayyum et al., 2001), mitochondrial complex I (Araujo et al., 2004, Murray et al., 2003, Pearce et al., 2005), cytochrome c (Jang & Han, 2006), cytochrome oxidase (Cooper et al., 2003), isocitrate dehydrogenase (Lee, Yang, & Park, 2003), aconitases (Han et al., 2005), and manganese superoxide dismutase (SOD; Quint et al., 2006, Xu et al., 2006), which might be responsible for obvious damages to mitochondria. As ONOO causes nitration at random, it will be more significant to evaluate the whole damaging effect of mitochondrial proteins nitration. The relationship between ONOO-induced nitration in entire mitochondrial proteins and liver mitochondrial damage in diabetes has not been reported.

Tricarboxylic acid cycle and β-oxidation of fatty acids occur mainly in mitochondria. The liver, which is richest in mitochondria, is an organ important to both energy and substance metabolism; therefore, damage to liver mitochondria will aggravate metabolic disorders in diabetes.

We hypothesized that excessive ONOO may cause nitration of entire liver mitochondrial proteins, randomly leading to mitochondrial damage. Therefore, the experiment in vivo was designed to investigate the relationship between ONOO-induced nitration of entire mitochondrial proteins and liver mitochondrial structural damage.

It is widely accepted that the beneficial effect of aminoguanidine may be entirely due to its inhibition of iNOS (Degenhardt et al., 1999, Reckelhoff et al., 1999). As a selective inhibitor of iNOS, aminoguanidine can reduce the generation of NO. Decreased NO generation will cause less formation of ONOO, reducing the nitration of mitochondrial proteins induced by ONOO and attenuating mitochondrial structural damage. Thus, aminoguanidine was used in the experiment as a tool drug to reduce ONOO formation.

Diabetes was induced in healthy 4-month-old male Sprague–Dawley (SD) rats by injecting streptozotocin (STZ). After 10 weeks, iNOS expression, nitrotyrosine (NT) content and distribution, mitochondrial structural damage, and metabolic disorders were evaluated.

Section snippets

Animals

The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the Hebei Medical University. Healthy 4-month-old SD male rats were provided by the Department of Experimental Animal of Tongji Medical College, Hubei Province (animal certificate no. 19-20). Rats were divided randomly into three groups: diabetes group, aminoguanidine group, and control group. The first two groups were injected intraperitoneally with STZ (40 mg/kg; dissolved in 10 mg/ml of

Detection of iNOS mRNA in hepatocytes

iNOS expression data determined with RT-PCR are shown in Table 1. iNOS mRNA was highly expressed in the diabetes group but was not found out in the control group. The expression of iNOS mRNA in the aminoguanidine group was lower than that in the diabetes group (P<.05). The result of iNOS mRNA expression determined with RT-PCR is also displayed in Fig. 1.

Detection of NT content in liver mitochondria with Western blot analysis

Western blot analysis data are shown in Table 1. NT content increased in the diabetes group but could not be detected in the control group. NT

Discussion

It is known that there exists obvious oxidative stress in diabetes (Du et al., 2003, Gumieniczek et al., 2002, Mastrocola et al., 2005, Pacher & Szabo, 2006, Sayyed et al., 2006). Similar results were also found in our previous studies (Qi et al., 2005). In diabetes, multipathways can lead to excessive formation of reactive oxygen species (ROS) (Khan et al., 2006, Pacher & Szabo, 2006). It has also been confirmed in the experiment that the level of MDA increased and the activity of SOD

Acknowledgments

This work was supported by the Bureau of Education of Hebei Province (B2004122), the Bureau of Science and Technology of Shijiazhuang City (04146173A), the Bureau of Sanitation of Hebei Province (04062), and Hebei Medical University (040028).

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