Glutathione (GSH) conjugating enzymes, glutathione S‐transferases (GSTs), are present in different subcellular compartments including cytosol, mitochondria, endoplasmic reticulum, nucleus and plasma membrane. The regulation and function of GSTs have implications in cell growth, oxidative stress as well as disease progression and prevention. Of the several mitochondria localized forms, GSTK (GST kappa) is mitochondria‐specific since it contains N‐terminal canonical and cleavable mitochondria targeting signals. Other forms like GST alpha, mu and pi purified from mitochondria are similar to the cytosolic molecular forms or ‘echoproteins’. Altered GST expression has been implicated in hepatic, cardiac and neurological diseases. Mitochondria‐specific GSTK has also been implicated in obesity, diabetes and related metabolic disorders. Studies have shown that silencing the GSTA4 (GST alpha) gene resulted in mitochondrial dysfunction, as was also seen in GSTA4 null mice, which could contribute to insulin resistance in type 2 diabetes. This review highlights the significance of the mitochondrial GST pool, particularly the mechanism and significance of dual targeting of GSTA4‐4 under in vitro and in vivo conditions. GSTA4‐4 is targeted in the mitochondria by activation of the internal cryptic signal present at the C‐terminus of the protein by protein‐kinase‐dependent phosphorylation and cytosolic heat shock protein (Hsp70) chaperone. Mitochondrial GST pi, on the other hand, has been shown to have two uncleaved cryptic signals rich in positively charged amino acids at the N‐terminal region. Both physiological and pathophysiological implications of GST translocation to mitochondria are discussed in the review.

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O₂ for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O₂ species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.

Streptozotocin is a pancreatic beta‐cell‐specific cytotoxin and is widely used to induce experimental type 1 diabetes in rodent models. The precise molecular mechanism of STZ cytotoxicity is however not clear. Studies have suggested that STZ is preferably absorbed by insulin‐secretingβ‐cells and induces cytotoxicity by producing reactive oxygen species/reactive nitrogen species (ROS/RNS). In the present study, we have investigated the mechanism of cytotoxicity of STZ in insulin‐secreting pancreatic cancer cells (Rin‐5F) at different doses and time intervals. Cell viability, apoptosis, oxidative stress, and mitochondrial bioenergetics were studied. Our results showed that STZ induces alterations in glutathione homeostasis and inhibited the activities of the respiratory enzymes, resulting in inhibition of ATP synthesis. Apoptosis was observed in a dose‐ and time‐dependent manner. Western blot analysis has also confirmed altered expression of oxidative stress markers (e.g., NOS and Nrf2), cell signaling kinases, apoptotic protein‐like caspase‐3, PARP, and mitochondrial specific proteins. These results suggest that STZ‐induced cytotoxicity in pancreatic cells is mediated by an increase in oxidative stress, alterations in cellular metabolism, and mitochondrial dysfunction. This study may be significant in better understanding the mechanism of STZ‐inducedβ‐cell toxicity/resistance and the etiology of type 1 diabetes induction.

Oxidative stress is an important factor in the etiology and pathogenesis of diabetes. We investigated changes in mitochondrial production of reactive oxygen species (ROS) and mitochondrial antioxidant defense systems in different tissues of streptozotocin (STZ)-induced diabetic rats. Our results show that increased ROS production and oxidative stress differentially affect mitochondrial and cytosolic glutathione (GSH) metabolism. Of the four tissues investigated, the pancreas, kidney, and brain appear to be affected more severely than the liver. We show a five- to eightfold increase of cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase (GST) A4-4 levels in mitochondria from STZ-treated rat tissues compared with those in nondiabetic rat tissues, suggesting possible roles in the disease process. Transient transfection of COS cells with CYP2E1 cDNA caused a similar accumulation of CYP2E1 and GST A4-4 in mitochondria and increased production of mitochondrial ROS. Our results also show an increase in steady-state levels of Hsp70 in the mitochondrial and cytosolic fractions of different tissues of diabetic rats. These results indicate, for the first time, a marked increase in mitochondrial oxidative stress in target tissues of STZ-treated rats and implicate a direct role for mitochondrial CYP2E1 in the generation of intramitochondrial ROS.