Original articlesPeroxynitrite-induced protein nitration contributes to liver mitochondrial damage in diabetic rats
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).
References (47)
- et al.
Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization
Journal of Biological Chemistry
(2003) Mitochondrial free radical production and cell signaling
Molecular Aspects of Medicine
(2004)- et al.
Advanced glycosylation end products induce inducible nitric oxide synthase (iNOS) expression via a p38 MAPK-dependent pathway
Kidney International
(2004) - et al.
Nitric oxide and peroxynitrite cause irreversible increases in the K(m) for oxygen of mitochondrial cytochrome oxidase: In vitro and in vivo studies
Biochimica et Biophysica Acta
(2003) - et al.
Aminoguanidine inhibits albuminuria, but not the formation of advanced glycation end-products in skin collagen of diabetic rats
Diabetes Research and Clinical Practice
(1999) - et al.
Hyperglycemia increases mitochondrial superoxide in retina and retinal cells
Free Radical Biology & Medicine
(2003) - et al.
Changes in antioxidant status of lung tissue in experimental diabetes in rabbits
Clinical Biochemistry
(2002) Free radicals, antioxidants, and human disease: Cariosity, cause or consequence
Lancet
(1994)Pathophysiological mechanisms of diabetic angiopathy
Journal of Diabetes and Its Complications
(2003)- et al.
Should minimal blood glucose variability become the gold standard of glycemic control?
Journal of Diabetes and Its Complications
(2005)
Biochemical properties of cytochrome c nitrated by peroxynitrite
Biochimie
Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury
Journal of Biological Chemistry
Oxidative damage to mitochondrial complex I due to peroxynitrite: Identification of reactive tyrosines by mass spectrometry
Journal of Biological Chemistry
Role of peroxynitrite in the pathogenesis of cardiovascular complications of diabetes
Current Opinion in Pharmacology
Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors
Nitric Oxide
Protective effect of melatonin and pinoline on nitric oxide-induced lipid and protein peroxidation in rat brain homogenates
Neuroscience Letters
Inducible nitric oxide synthase catalyzes ethanol oxidation to alpha-hydroxyethyl radical and acetaldehyde
Toxicology
Crystal structure of nitrated human manganese superoxide dismutase: Mechanism of inactivation
Free Radical Biology & Medicine
Peroxynitrite reactions and formation in mitochondria
Free Radical Biology & Medicine
Chronic aminoguanidine attenuates renal dysfunction and injury in aging rats
American Journal of Hypertension: Journal of the American Society of Hypertension
Peroxynitrite causes DNA nicks in plasmid pBR-322
Biochemical and Biophysical Research Communications
Effects of U83836E on nerve functions, hyperalgesia and oxidative stress in experimental diabetic neuropathy
Life Sciences
Factors determining the selectivity of protein tyrosine nitration
Archives of Biochemistry and Biophysics
Cited by (41)
Calcitriol prevents peripheral RSC96 Schwann neural cells from high glucose & methylglyoxal-induced injury through restoration of CBS/H<inf>2</inf>S expression
2016, Neurochemistry InternationalCitation Excerpt :In addition, our results showed that the treatment with HG&MG elevated significant oxidative stress, evidenced by the accumulation O2- in cells. As we know, the excessive NO usually reacts with O2- to produce another toxicant, ONOO−, and the diabetic injury of liver and renal was mainly caused by ONOO− (Liang et al., 2010; Ren et al., 2008). Importantly, we found that before the treatment with HG&MG, the pretreatment of cells with CCT markedly inhibited the iNOS/NO overexpression and O2- accumulation.
Hepatic nitrosative stress in experimental diabetes
2012, Journal of Diabetes and its ComplicationsCitation Excerpt :The diabetic animals showed significant structural damage in the hepatic mitochondria as well as an increased expression of iNOS and nitrotyrosine, which could be related to the nitration of proteins and the damage of mitochondrial structures. In the present study, a reduction in both nitrosative and oxidative stress was seen with the use of aminoguanidine similar to that seen with an iNOS inhibitor (Ren et al., 2008). In the present study, in addition to the role of aminoguanidine in relation to nitrosative stress, benefits regarding the expression of p65 and the activation of nuclear transcription factor NFкB were also demonstrated.
Ferric citrate CYP2E1-independently promotes alcohol-induced apoptosis in HepG2 cells via oxidative/nitrative stress which is attenuated by pretreatment with baicalin
2012, Food and Chemical ToxicologyCitation Excerpt :In addition, chronic iron overload enhances the expression of iNOS in the rat liver (Galleano et al., 2004; Cornejo et al., 2005), and iNOS is shown to be required for ALD (McKim et al., 2003). Moreover, peroxynitrite, derived from the interaction of NO with superoxide, can induce the formation of protein nitration (Langer et al., 2008; Chacko et al., 2011), which has been suggested be a biomarker for liver disease (Ren et al., 2008). However, no study has investigated the combined effect of iron and alcohol on the expression of iNOS, the production of NO or the formation of protein nitration in hepatocytes.
Attenuation of oxidative stress and hepatic damage by some fermented tropical legume condiment diets in streptozotocin-induced diabetes in rats
2012, Asian Pacific Journal of Tropical MedicineRole of inducible nitric oxide synthase in mitochondrial depolarization and graft injury after transplantation of fatty livers
2012, Free Radical Biology and MedicineCitation Excerpt :Stimulation of iNOS expression by proinflammatory cytokines or adenoviral delivery of the iNOS gene causes mitochondrial damage and cell death [42], and iNOS deficiency blocks sepsis-induced mitochondrial RNS production and mitochondrial function inhibition [60,61]. Finally, a high fat diet and diabetes induce iNOS expression, leading to protein nitration, suppression of mitochondrial respiration, and decreased mitochondrial membrane potential in the liver [62,63]. Together, these studies suggest that RNS play a detrimental role in mitochondria.
Hyperglycemia-induced mitochondrial alterations in liver
2010, Life Sciences