Effect of Bariatric Surgery on Plasma Cell-Free Mitochondrial DNA, Insulin Sensitivity and Metabolic Changes in Obese Patients.

While improvement of mitochondrial function after bariatric surgery has been demonstrated, there is limited evidence about the effects of bariatric surgery on circulatory cell-free (cf) mitochondrial DNA (mtDNA) and intracellular mtDNA abundance. Plasma and peripheral blood mononuclear (PBM) cells were isolated from healthy controls (HC) and bariatric surgery patients before surgery and 2 weeks, 3 months, and 6 months after surgery. At baseline, the plasma level of short cf-mtDNA (ND6, ~100 bp) fragments was significantly higher in obese patients compared to HC. But there was no significant variation in mean ND6 values post-surgery. A significant positive correlation was observed between preop plasma ND6 levels and HgbA1c, ND6 and HOMA-IR 2 weeks post-surgery, and mtDNA content 6 months post-surgery. Interestingly, plasma from both HC and obese groups at all time points post-surgery contains long (~8 kb) cf-mtDNA fragments, suggesting the presence of near-intact and/or whole mitochondrial genomes. No significant variation was observed in mtDNA content post-surgery compared to baseline data in both PBM and skeletal muscle samples. Overall, bariatric surgery improved insulin sensitivity and other metabolic parameters without significant changes in plasma short cf-mtDNA levels or cellular mtDNA content. Our study provides novel insights about possible molecular mechanisms underlying the metabolic effects of bariatric surgery and suggests the development of new generalized approaches to characterize cf-mtDNA.

Acute PDE4 Inhibition Induces a Transient Increase in Blood Glucose in Mice.

cAMP-phosphodiesterase 4 (PDE4) inhibitors are currently approved for the treatment of inflammatory diseases. There is interest in expanding the therapeutic application of PDE4 inhibitors to metabolic disorders, as their chronic application induces weight loss in patients and animals and improves glucose handling in mouse models of obesity and diabetes. Unexpectedly, we have found that acute PDE4 inhibitor treatment induces a temporary increase, rather than a decrease, in blood glucose levels in mice. Blood glucose levels in postprandial mice increase rapidly upon drug injection, reaching a maximum after ~45 min, and returning to baseline within ~4 h. This transient blood glucose spike is replicated by several structurally distinct PDE4 inhibitors, suggesting that it is a class effect of PDE4 inhibitors. PDE4 inhibitor treatment does not reduce serum insulin levels, and the subsequent injection of insulin potently reduces PDE4 inhibitor-induced blood glucose levels, suggesting that the glycemic effects of PDE4 inhibition are independent of changes in insulin secretion and/or sensitivity. Conversely, PDE4 inhibitors induce a rapid reduction in skeletal muscle glycogen levels and potently inhibit the uptake of 2-deoxyglucose into muscle tissues. This suggests that reduced glucose uptake into muscle tissue is a significant contributor to the transient glycemic effects of PDE4 inhibitors in mice.

Mitochondrial 8-oxoguanine DNA glycosylase mitigates alveolar epithelial cell PINK1 deficiency, mitochondrial DNA damage, apoptosis, and lung fibrosis.

Alveolar epithelial cell (AEC) apoptosis, arising from mitochondrial dysfunction and mitophagy defects, is important in mediating idiopathic pulmonary fibrosis (IPF). Our group established a role for the mitochondrial (mt) DNA base excision repair enzyme, 8-oxoguanine-DNA glycosylase 1 (mtOGG1), in preventing oxidant-induced AEC mtDNA damage and apoptosis and showed that OGG1-deficient mice have increased lung fibrosis. Herein, we determined whether mice overexpressing the mtOGG1 transgene (mtOgg1tg) are protected against lung fibrosis and whether AEC mtOGG1 preservation of mtDNA integrity mitigates phosphatase and tensin homolog-induced putative kinase 1 (PINK1) deficiency and apoptosis. Compared with wild type (WT), mtOgg1tg mice have diminished asbestos- and bleomycin-induced pulmonary fibrosis that was accompanied by reduced lung and AEC mtDNA damage and apoptosis. Asbestos and H2O2 promote the MLE-12 cell PINK1 deficiency, as assessed by reductions in the expression of PINK1 mRNA and mitochondrial protein expression. Compared with WT, Pink1-knockout (Pink1-KO) mice are more susceptible to asbestos-induced lung fibrosis and have increased lung and alveolar type II (AT2) cell mtDNA damage and apoptosis. AT2 cells from Pink1-KO mice and PINK1-silenced (siRNA) MLE-12 cells have increased mtDNA damage that is augmented by oxidative stress. Interestingly, mtOGG1 overexpression attenuates oxidant-induced MLE-12 cell mtDNA damage and apoptosis despite PINK1 silencing. mtDNA damage is increased in the lungs of patients with IPF as compared with controls. Collectively, these findings suggest that mtOGG1 maintenance of AEC mtDNA is crucial for preventing PINK1 deficiency that promotes apoptosis and lung fibrosis. Given the key role of AEC apoptosis in pulmonary fibrosis, strategies aimed at preserving AT2 cell mtDNA integrity may be an innovative target.

Plasma mitochondrial DNA is elevated in obese type 2 diabetes mellitus patients and correlates positively with insulin resistance.

Cells damaged by mechanical or infectious injury release proinflammatory mitochondrial DNA (mtDNA) fragments into the circulation. We evaluated the relation between plasma levels of mtDNA fragments in obese type 2 diabetes mellitus (T2DM) patients and measures of chronic inflammation and insulin resistance. In 10 obese T2DM patients and 12 healthy control (HC) subjects, we measured levels of plasma cell-free mtDNA with quantitative real-time polymerase chain reaction, and mtDNA damage in skeletal muscle with quantitative alkaline Southern blot. Also, markers of systemic inflammation and oxidative stress in skeletal muscle were measured. Plasma levels of mtDNA fragments, mtDNA damage in skeletal muscle and plasma tumor necrosis factor α levels were greater in obese T2DM patients than HC subjects. Also, the abundance of plasma mtDNA fragments in obese T2DM patients levels positively correlated with insulin resistance. To the best of our knowledge, this is the first published evidence that elevated level of plasma mtDNA fragments is associated with mtDNA damage and oxidative stress in skeletal muscle and correlates with insulin resistance in obese T2DM patients. Plasma mtDNA may be a useful biomarker for predicting and monitoring insulin resistance in obese patients.

8-oxoguanine DNA glycosylase (Ogg1) controls hepatic gluconeogenesis.

Mitochondrial DNA (mtDNA) resides in close proximity to metabolic reactions, and is maintained by the 8-oxoguanine DNA glycosylase (Ogg1) and other members of the base excision repair pathway. Here, we tested the hypothesis that changes in liver metabolism as under fasting/feeding conditions would be sensed by liver mtDNA, and that Ogg1 deficient mice might unravel a metabolic phenotype. Wild type (WT) and ogg1-/- mice were either fed ad libitum or subjected to fasting for 24h, and the corresponding effects on liver gene expression, DNA damage, as well as serum values were analyzed. Ogg1 deficient mice fed ad libitum exhibited hyperglycemia, elevated insulin levels and higher liver glycogen content as well as increased accumulation of 8oxoG in mtDNA compared to age- and gender matched WT mice. Interestingly, these phenotypes were absent in ogg1-/- mice during fasting. Gene expression and functional analyses suggest that the diabetogenic phenotype in the ogg1-/- mice is due to a failure to suppress gluconeogensis in the fed state. The ogg1-/- mice exhibited reduced mitochondrial electron transport chain (ETC) capacity and a combined low activity of the pyruvate dehydrogenase (PDH), alluding to inefficient channeling of glycolytic products into the citric acid cycle. Our data demonstrate a physiological role of base excision repair that goes beyond DNA maintenance, and implies that DNA repair is involved in regulating metabolism.

Mitochondrial DNA Repair through OGG1 Activity Attenuates Breast Cancer Progression and Metastasis.

Production of mitochondrial reactive oxygen species and integrity of mitochondrial DNA (mtDNA) are crucial in breast cancer progression and metastasis. Therefore, we evaluated the role of mtDNA damage in breast cancer by genetically modulating the DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1) in the PyMT transgenic mouse model of mammary tumorigenesis. We generated mice lacking OGG1 (KO), mice overexpressing human OGG1 subunit 1α in mitochondria (Tg), and mice simultaneously lacking OGG1 and overexpressing human OGG1 subunit 1α in mitochondria (KO/Tg). We found that Tg and KO/Tg mice developed significantly smaller tumors than KO and wild-type (WT) mice after 16 weeks. Histologic analysis revealed a roughly 2-fold decrease in the incidence of lung metastases in Tg mice (33.3%) compared to WT mice (62.5%). Furthermore, lungs from Tg mice exhibited nearly a 15-fold decrease in the average number of metastatic foci compared with WT mice (P ≤ 0.05). Primary tumors isolated from Tg mice also demonstrated reduced total and mitochondrial oxidative stress, diminished mtDNA damage, and increased mitochondrial function. Targeting hOGG1 to the mitochondria protected cells from mtDNA damage, resulting in downregulation of HIF1α and attenuated phosphorylation of Akt. Collectively, we demonstrate proof of concept that mtDNA damage results in breast cancer progression and metastasis in vivo. Moreover, our findings offer new therapeutic strategies for modulating the levels of mtDNA repair enzymes to delay or stall metastatic progression.

Free fatty acids and skeletal muscle insulin resistance.

Insulin resistance plays a key role in the development of type 2 diabetes mellitus and is also associated with several other diseases, such as obesity, hypertension, and cardiovascular diseases. Type 2 diabetes and obesity have become epidemic worldwide in the past few decades, and epidemiological and metabolic evidence indicates that the two conditions are linked closely through insulin resistance. The perturbation of free fatty acid (FFA) metabolism is now accepted to be a major factor contributing to whole-body insulin resistance, including that in skeletal muscle. Acute exposure to FFAs and excess dietary lipid intake are strongly associated with the pathogenesis of muscle insulin resistance. Despite an enormous amount of published research and the proposal of numerous hypotheses, however, the mechanisms underlying FFA-induced skeletal muscle insulin resistance have not been fully elucidated. This chapter describes existing hypotheses, recent findings, and debates about the role of FFAs in the development of muscle insulin resistance. Therapeutic options for this condition are also discussed.

Mitochondrial DNA damage via augmented oxidative stress regulates endoplasmic reticulum stress and autophagy: crosstalk, links and signaling.

Saturated free fatty acids (FFAs) have been implicated in the increase of oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, autophagy, and insulin resistance (IR) observed in skeletal muscle. Previously, we have shown that palmitate-induced mitochondrial DNA (mtDNA) damage triggers mitochondrial dysfunction, mitochondrial reactive oxygen species (mtROS) production, apoptosis and IR in L6 myotubes. The present study showed that mitochondrial overexpression of human 8-oxoguanine DNA glycosylase/AP lyase (hOGG1) decreased palmitate-induced carbonylation of proteins in mitochondria. Additionally, we found that protection of mtDNA from palmitate-induced damage significantly diminished markers of both ER stress and autophagy in L6 myotubes. Moreover, we observed that the addition of ROS scavenger, N-acetylcystein (NAC), to palmitate diminished both ER stress and autophagy markers mimicking the effect of mitochondrial overexpression of hOGG1. This is the first study to show that mtDNA damage is upstream of palmitate-induced ER stress and autophagy in skeletal muscle cells.

Alteration of mitochondrial function and insulin sensitivity in primary mouse skeletal muscle cells isolated from transgenic and knockout mice: role of ogg1.

Recent evidence has linked mitochondrial dysfunction and DNA damage, increased oxidative stress in skeletal muscle, and insulin resistance (IR). The purpose of this study was to determine the role of the DNA repair enzyme, human 8-oxoguanine DNA glycosylase/apurinic/apyrimidinic lyase (hOGG1), on palmitate-induced mitochondrial dysfunction and IR in primary cultures of skeletal muscle derived from hind limb of ogg1(-/-) knockout mice and transgenic mice, which overexpress human (hOGG1) in mitochondria (transgenic [Tg]/MTS-hOGG1). Following exposure to palmitate, we evaluated mitochondrial DNA (mtDNA) damage, mitochondrial function, production of mitochondrial reactive oxygen species (mtROS), mitochondrial mass, JNK activation, insulin signaling pathways, and glucose uptake. Palmitate-induced mtDNA damage, mtROS, mitochondrial dysfunction, and activation of JNK were all diminished, whereas ATP levels, mitochondrial mass, insulin-stimulated phosphorylation of Akt (Ser 473), and insulin sensitivity were increased in primary myotubes isolated from Tg/MTS-hOGG1 mice compared to myotubes isolated from either knockout or wild-type mice. In addition, both basal and maximal respiratory rates during mitochondrial oxidation on pyruvate showed a variable response, with some animals displaying an increased respiration in muscle fibers isolated from the transgenic mice. Our results support the model that DNA repair enzyme OGG1 plays a pivotal role in repairing mtDNA damage, and consequently, in mtROS production and regulating downstream events leading to IR in skeletal muscle.

Mitochondrial DNA damage and dysfunction, and oxidative stress are associated with endoplasmic reticulum stress, protein degradation and apoptosis in high fat diet-induced insulin resistance mice.

BACKGROUND: Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver. CONCLUSIONS/SIGNIFICANCE: This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.

Protection from palmitate-induced mitochondrial DNA damage prevents from mitochondrial oxidative stress, mitochondrial dysfunction, apoptosis, and impaired insulin signaling in rat L6 skeletal muscle cells.

Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme human 8-oxoguanine DNA glycosylase/(apurinic/apyrimidinic) lyase (hOGG1) to mitochondria in L6 myotubes. After palmitate exposure, we evaluated mtDNA damage, mitochondrial function, production of mitochondrial reactive oxygen species, apoptosis, insulin signaling pathways, and glucose uptake. Protection of mtDNA from palmitate-induced damage by overexpression of hOGG1 targeted to mitochondria significantly diminished palmitate-induced mitochondrial superoxide production, restored the decline in ATP levels, reduced activation of c-Jun N-terminal kinase (JNK) kinase, prevented cells from entering apoptosis, increased insulin-stimulated phosphorylation of serine-threonine kinase (Akt) (Ser473) and tyrosine phosphorylation of insulin receptor substrate-1, and thereby enhanced glucose transporter 4 translocation to plasma membrane, and restored insulin signaling. Addition of a specific inhibitor of JNK mimicked the effect of mitochondrial overexpression of hOGG1 and partially restored insulin sensitivity, thus confirming the involvement of mtDNA damage and subsequent increase of oxidative stress and JNK activation in insulin signaling in L6 myotubes. Our results are the first to report that mtDNA damage is the proximal cause in palmitate-induced mitochondrial dysfunction and impaired insulin signaling and provide strong evidence that targeting DNA repair enzymes into mitochondria in skeletal muscles could be a potential therapeutic treatment for insulin resistance.

Mitochondrial DNA damage level determines neural stem cell differentiation fate.

The mitochondrial DNA (mtDNA) of neural stem cells (NSCs) is vulnerable to oxidation damage. Subtle manipulations of the cellular redox state affect mtDNA integrity in addition to regulating the NSC differentiation lineage, suggesting a molecular link between mtDNA integrity and regulation of differentiation. Here we show that 8-oxoguanine DNA glycosylase (OGG1) is essential for repair of mtDNA damage and NSC viability during mitochondrial oxidative stress. Differentiating neural cells from ogg1(-/-) knock-out mice spontaneously accumulate mtDNA damage and concomitantly shift their differentiation direction toward an astrocytic lineage, similar to wt NSCs subjected to mtDNA damaging insults. Antioxidant treatments reversed mtDNA damage accumulation and separately increased neurogenesis in ogg1(-/-) cells. NSCs from a transgenic ogg1(-/-) mouse expressing mitochondrially targeted human OGG1 were protected from mtDNA damage during differentiation, and displayed elevated neurogenesis. The underlying mechanisms for this shift in differentiation direction involve the astrogenesis promoting Sirt1 via an increased NAD/NADH ratio in ogg1(-/-) cells. Redox manipulations to alter mtDNA damage level correspondingly activated Sirt1 in both cell types. Our results demonstrate for the first time the interdependence between mtDNA integrity and NSC differentiation fate, suggesting that mtDNA damage is the primary signal for the elevated astrogliosis and lack of neurogenesis seen during repair of neuronal injury.

Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress.

The type of free fatty acids (FFAs), saturated or unsaturated, is critical in the development of insulin resistance (IR), since the degree of saturation correlates with IR. We compared the effects of the saturated FFA palmitate, the unsaturated FFA oleate, and a mixture of each on the production of mitochondrial reactive oxygen species (mtROS), mitochondrial DNA (mtDNA) damage, mitochondrial function, apoptosis, and insulin-signaling pathway in skeletal muscle cells. Only palmitate caused a significant increase of mtROS production, which correlated with concomitant mtDNA damage, mitochondrial dysfunction, induction of JNK, apoptosis, and inhibition of insulin signaling. Blocking de novo synthesis of ceramide abolished the effects of palmitate on mtROS production, viability, and insulin signaling. Oleate alone did not cause mtROS generation and mtDNA damage, and its addition to palmitate prevented palmitate-induced mtDNA damage, increased total ATP levels and cell viability, and prevented palmitate-induced apoptosis and inhibition of insulin-stimulated Akt (Ser(473)) phosphorylation. The peroxisome proliferator activator receptor-γ coactivator 1α (PGC-1α) protein level and promoter activity were decreased at concentrations of palmitate ≥0.5 mM, whereas addition of oleate increased both PGC-1α level and promoter activity. Expression of the mitochondrial transcription factor (TFAM) was significantly diminished after palmitate but not oleate treatment. Addition of the ROS scavenger, N-acetylcystein (NAC), to palmitate restored both the expression and promoter activity of PGC-1α as well as TFAM expression. We propose that 1) mtROS generation is the initial event in the induction of mitochondrial dysfunction and consequent apoptosis and the inhibition of insulin signaling and that 2) oleate ameliorates palmitate-induced mitochondrial dysfunction and thus may contribute to the prevention of palmitate-induced IR.

Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes.

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.

Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress.

Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases.

Role of nitric oxide-induced mtDNA damage in mitochondrial dysfunction and apoptosis.

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.

Protection of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to mitochondria.

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.

Involvement of mtDNA damage in free fatty acid-induced apoptosis.

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.

Contribution of mitochondrial DNA repair to cell resistance from oxidative stress.

Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species.