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. 2011;12(5):3133-47.
doi: 10.3390/ijms12053133. Epub 2011 May 13.

Impaired mitochondrial respiratory functions and oxidative stress in streptozotocin-induced diabetic rats

Haider Raza, Subbuswamy K Prabu, Annie John, Narayan G Avadhani

Impaired mitochondrial respiratory functions and oxidative stress in streptozotocin-induced diabetic rats

Haider Raza et al. Int J Mol Sci. 2011.

Abstract

We have previously shown a tissue-specific increase in oxidative stress in the early stages of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated oxidative stress-related long-term complications and mitochondrial dysfunctions in the different tissues of STZ-induced diabetic rats (>15 mM blood glucose for 8 weeks). These animals showed a persistent increase in reactive oxygen and nitrogen species (ROS and RNS, respectively) production. Oxidative protein carbonylation was also increased with the maximum effect observed in the pancreas of diabetic rats. The activities of mitochondrial respiratory enzymes ubiquinol: cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (Complex IV) were significantly decreased while that of NADH:ubiquinone oxidoreductase (Complex I) and succinate:ubiquinone oxidoreductase (Complex II) were moderately increased in diabetic rats, which was confirmed by the increased expression of the 70 kDa Complex II sub-unit. Mitochondrial matrix aconitase, a ROS sensitive enzyme, was markedly inhibited in the diabetic rat tissues. Increased expression of oxidative stress marker proteins Hsp-70 and HO-1 was also observed along with increased expression of nitric oxide synthase. These results suggest that mitochondrial respiratory complexes may play a critical role in ROS/RNS homeostasis and oxidative stress related changes in type 1 diabetes and may have implications in the etiology of diabetes and its complications.

Keywords: NO; ROS; diabetes; mitochondrial respiration; oxidative stress.

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Figures

Figure 1.
Figure 1.
Reactive oxygen species (ROS) production in streptozotocin (STZ)-induced diabetes. Mitochondria (50 μg protein) (a), microsomes (100 μg) (b) and cytosols (100 μg) (c) from pancreas (P), liver (L), kidney (K) and brain (B) tissues from control and diabetic rats were used for the measurement of ROS by the modified DCFDA method as described before [24]. The values are mean ± S.E.M. of three individual experiments. * indicate significant difference (P < 0.05) from control animals. (□) Control; (▪) Diabetic.
Figure 2.
Figure 2.
Reactive nitrogen species (RNS) production in STZ-induced diabetes. Mitochondrial (a), microsomal (b) and cytosolic (c) proteins (100 μg) from pancreas (P), liver (L), kidney (K) and brain (B) of control and diabetic rats were analyzed for total NO level by Griess Reagent using a kit from Calbiochem as described in the Materials and Methods. The values are mean ± S.E.M. for three determinations. * indicate significant difference (P < 0.05) from control animals. (□) Control; (▪) Diabetic.
Figure 3.
Figure 3.
Protein carbonylation in STZ-induced diabetes. Mitochondrial (a), microsomal (b) and cytosolic (c) proteins (100 μg each) from pancreas (P), liver (L), kidney (K) and brain (B) of control and diabetic rats were incubated with 2 mM DNPH in 1.0 mL assay system for 1 h. The DNPH-coupled carbonylated proteins were then precipitated by 10% ice-cold TCA and washed three times with ethanol: ethyl acetate to remove free DNPH. The DNPH-coupled proteins were then dissolved in 6 M guanidine-HCl and absorption read at 366 nm. Results were calculated based on the molar extinction coefficient of 22,000 as described in the Materials and Methods. The values are mean ± S.E.M. for three determinations. * indicate significant difference (P < 0.05) from the control animals. (□) Control; (▪) Diabetic.
Figure 4.
Figure 4.
Activities of respiratory complexes in STZ-induced diabetes. Mitochondrial protein (10–25 μg) from pancreas, liver, kidney and brain of control and diabetic rats were used for the assays of respiratory chain enzyme complexes as described in the Materials and Methods. (a) Complex I, NADH: ubiquinone oxidoreductase activity; (b) Complex II, succinate:ubiquinone oxidoreductase activity; (c) Complex III, ubiquinol: ferrocytochrome c oxidoreductase activity; and (d) Complex IV, cytochrome c oxidase activity. The values are mean ± S.E.M for three determinations. * indicate significant difference (P < 0.05) from the controls. (□) Control; (▪) Diabetic.
Figure 5.
Figure 5.
Succinate-dependent oxygen uptake by mitochondria from control and diabetic rats. Succinate-dependent oxygen consumption by freshly prepared mitochondria from control and diabetic rats in the presence (State 3) or absence of ADP (State 4) was measured as described in the Materials and Methods (a). Respiratory Control Rate (RCR) was calculated as the ratio of State 3/State 4 respiration (b) and expressed relative to control rats. The values are mean ± S.E.M. of three independent experiments. * indicate significant difference (P < 0.05) from control animals. (□) Control; (▪) Diabetic.
Figure 6.
Figure 6.
Mitochondrial aconitase activity in control and diabetic rats. 100 μg of mitochondrial protein was incubated in a 1.0 mL reaction system containing citrate and NADP coupled with isocitrate dehydrogenase to monitor the rate of NADPH formation as described in the Materials and Methods. The values are mean ± S.E.M. of three independent experiments. * indicate significant difference (P < 0.05) from control animals. (□) Control; (▪) Diabetic.
Figure 7.
Figure 7.
Mitochondrial level of Complex II, HO-1, Hsp-70, and iNOS in STZ-induced diabetic rats. Mitochondrial proteins (50 μg) from pancreas, liver, kidney and brain of control (C) and diabetic rats (D) were resolved by 12% SDS-PAGE and transferred on to nitrocellulose paper by Western blot analysis. The blot was probed with iNOS, Hsp-70, HO-1 and Complex II antibodies. Tim23 was used as a loading control. For relative quantitative comparison, the intensity of the control band in each case was regarded as 1. The blot shown is a typical representation of at least three experiments.
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