Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Provides information on a study determining the feasibility of gene transfer in Rickettsia prowazekii. Methodology and materials used to conduct the study; Information on the electroporation of R. prowazekii; Results of the study; Discussion on the results.

Characterizes the induction of Escherichia coli ereB gene in Rickettsia prowazekii. Capability of the gene of encoding an erythromycin esterase; Relevance of the availability of the positive selection for rickettsial transformants; Electroporation conditions used in rifampin selection transformations; Use of transforming plasmid with the plasmid pMOB.

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]

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. [ABSTRACT FROM AUTHOR]

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 1a in mitochondria (Tg), and mice simultaneously lacking OGG1 and overexpressing human OGG1 subunit 1a 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 HIF1a 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. [ABSTRACT FROM AUTHOR]

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., (© 2014 Elsevier Inc. All rights reserved.)

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.