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

Raza H, Prabu SK, John A, Avadhani NG - Int J Mol Sci (2011)

Bottom Line: These animals showed a persistent increase in reactive oxygen and nitrogen species (ROS and RNS, respectively) production.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.

View Article: PubMed Central - PubMed

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.

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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.
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f3-ijms-12-03133: 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.

Mentions: Tissues from diabetic rats exhibited a modest but significant increase (20–35%; P < 0.05) in oxidative carbonylation of proteins (Figure 3). Mitochondria from pancreas and kidney (Figure 3a) appear to be more affected (30–35% increase) than liver and brain (∼20% increase). On the other hand, a marked increase in microsomal protein carbonylation in pancreas, liver and kidney (about 2–3 fold) was observed, while brain exhibited only about a 20% increase in carbonylated proteins (Figure 3b). Cytosolic fraction also showed a significant increase in protein carbonylation in pancreas and kidney from diabetic tissues while brain and liver exhibited a marginal increase (15–20%) (Figure 3c).


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

Raza H, Prabu SK, John A, Avadhani NG - Int J Mol Sci (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116180&req=5

f3-ijms-12-03133: 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.
Mentions: Tissues from diabetic rats exhibited a modest but significant increase (20–35%; P < 0.05) in oxidative carbonylation of proteins (Figure 3). Mitochondria from pancreas and kidney (Figure 3a) appear to be more affected (30–35% increase) than liver and brain (∼20% increase). On the other hand, a marked increase in microsomal protein carbonylation in pancreas, liver and kidney (about 2–3 fold) was observed, while brain exhibited only about a 20% increase in carbonylated proteins (Figure 3b). Cytosolic fraction also showed a significant increase in protein carbonylation in pancreas and kidney from diabetic tissues while brain and liver exhibited a marginal increase (15–20%) (Figure 3c).

Bottom Line: These animals showed a persistent increase in reactive oxygen and nitrogen species (ROS and RNS, respectively) production.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.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus