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Thiazolidinediones (TZDs), such as troglitazone (TRO) and rosiglitazone (ROSI), improve insulin resistance by acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma). TRO was withdrawn from the market because of reports of serious hepatotoxicity. A growing body of evidence suggests that TRO caused mitochondrial dysfunction and induction of apoptosis in human hepatocytes but its mechanisms of action remain unclear. We hypothesized that damage to mitochondrial DNA (mtDNA) is an initiating event involved in TRO-induced mitochondrial dysfunction and hepatotoxicity. Primary human hepatocytes were exposed to TRO and ROSI. The results obtained revealed that TRO, but not ROSI at equimolar concentrations, caused a substantial increase in mtDNA damage and decreased ATP production and cellular viability. The reactive oxygen species (ROS) scavenger, N-acetyl cystein (NAC), significantly diminished the TRO-induced cytotoxicity, suggesting involvement of ROS in TRO-induced hepatocyte cytotoxicity. The PPARgamma antagonist (GW9662) did not block the TRO-induced decrease in cell viability, indicating that the TRO-induced hepatotoxicity is PPARgamma-independent. Furthermore, TRO induced hepatocyte apoptosis, caspase-3 cleavage and cytochrome c release. Targeting of a DNA repair protein to mitochondria by protein transduction using a fusion protein containing the DNA repair enzyme Endonuclease III (EndoIII) from Escherichia coli, a mitochondrial translocation sequence (MTS) and the protein transduction domain (PTD) from HIV-1 TAT protein protected hepatocytes against TRO-induced toxicity. Overall, our results indicate that significant mtDNA damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug.

Chronic exposure to elevated levels of free fatty acids (FFAs) impairs pancreatic beta-cell function and contributes to the decline of insulin secretion in type 2 diabetes. Previously, we reported that FFAs caused increased nitric oxide (NO) production, which damaged mitochondrial DNA (mtDNA) and ultimately led to apoptosis in INS-1 cells. To firmly establish the link between FFA-generated mtDNA damage and apoptosis, we stably transfected INS-1 cells with an expression vector containing the gene for the DNA repair enzyme human 8-oxoguanine DNA glycosylase/apurinic lyase (hOGG1) downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase. Successful integration of MTS-OGG1 into the INS-1 cellular genome was confirmed by Southern blot analysis. Western blots and enzyme activity assays revealed that hOGG1 was targeted to mitochondria and the recombinant enzyme was active. MTS-OGG1 cells showed a significant decrease in FFA-induced mtDNA damage compared with vector-only transfectants. Additionally, hOGG1 overexpression in mitochondria decreased FFA-induced inhibition of ATP production and protected INS-1 cells from apoptosis. These results indicate that mtDNA damage plays a pivotal role in FFA-induced beta-cell dysfunction and apoptosis. Therefore, targeting DNA repair enzymes into beta-cell mitochondria could be a potential therapeutic strategy for preventing or delaying the onset of type 2 diabetes symptoms.

Abstract: An increasing body of evidence suggests that nitric oxide (NO) can be cytotoxic and induce apoptosis. NO can also be genotoxic and cause DNA damage and mutations. It has been shown that NO damages mitochondrial DNA (mtDNA) to a greater extent than nuclear DNA. Previously, we reported that conditional targeting of the DNA repair protein hOGG1 into mitochondria using a mitochondria targeting sequence (MTS) augmented mtDNA repair of oxidative damage and enhanced cellular survival. To determine whether enhanced repair resulting from augmented expression of hOGG1 could also protect against the deleterious effects of NO, we used HeLa TetOff/MTS-OGG1-transfected cells to conditionally express hOGG1 in mitochondria. The effects of additional hOGG1 expression on repair of NO-induced mtDNA damage and cell survival were evaluated. These cells, along with vector transfectants, in either the presence or absence of doxycycline (Dox), were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate). Functional studies revealed that cells expressing recombinant hOGG1 were more proficient at repairing NO-induced mtDNA damage, which led to increased cellular survival following NO exposure. Moreover, the results described here show that conditional expression of hOGG1 in mitochondria decreases NO-induced inhibition of ATP production and protects cells from NO-induced apoptosis. [Copyright &y& Elsevier]

Recent studies suggested that the accumulation of muscular free fatty acids (FFA) might be responsible for the mitochondrial dysfunction and insulin resistance (IR) seen in type 2 diabetes mellitus (T2DM), and that the type of FFA is critical in this process. Additionally oxidative stress, which results from increased production of reactive oxygen/nitrogen species (ROS/RNS) leads to 1R and T2DM. We hypothesized that oxidative damage to mitochondrial DNA (mtDNA) is an early step involved in mitochondrial dysfunction and IR following exposure of skeletal muscle to FFA. Damage to mtDNA initiates processes which heighten production of ROS causing progressive mitochondrial and cellular dysfunction which ultimately leads to IR. To test this hypothesis, rat L6 myotubes were exposed to palmitate, which is the most prevalent saturated FFA; oleate, which makes up 80% of the circulating monounsaturated pool; and a mixture (oleate/palmitate; 2/1 ). Previously palmitate has been shown to induce IR in L6 myotubes whereas oleate improves insulin sensitivity. Moreover, palmitate-induced IR and apoptosis were positively related, while oleate did not induce apoptosis in L6 myotubes. The current studies were undertaken to investigate whether FFA-induced apoptosis and IR correlate with mtDNA damage and mitochondrial dysfunction in L6 myotubes. The results showed that only palmitate caused production of nitric oxide (NO), ROS, mtDNA damage and apoptosis in L6 myotubes. Incubation of L6 myotubes with similar concentrations of either oleate alone or oleate combined with palmitate did not stimulate production of NO. Oleate did not cause mtDNA damage in L6 myotubes and the addition of oleate to palmitate prevented palmitate-induced mtDNA damage, and palmitate-induced decrease in total ATP levels and cell viability. Addition of oleate to palmitate prevented palmitate-induced apoptosis. CONCLUSION: FFA-induced mtDNA damage is a key component contributing to IR in skeletal muscle probably by triggering mitochondrial dysfunction which leads to apoptosis. [ABSTRACT FROM AUTHOR]