Elevated Mitochondrial Oxidative Stress Impairs Metabolic Adaptations to Exercise in Skeletal Muscle

Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in Sod2 ^+/- mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training Sod2 ^+/- mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised Sod2 ^+/- mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.     

Superoxide dismutase 1 knockdown induces oxidative stress and DNA methylation loss in the prostate

Increased oxidative stress and concordant DNA methylation changes are found during aging and in many malignant processes including prostate cancer. Increased oxidative stress has been shown to inhibit DNA methyltransferase in in vitro assays, but whether this occurs in vivo is unknown. To generate increased oxidative stress we utilized mice containing mutations in the CuZnSOD (Sod1) gene, a major superoxide dismutase in mammals. Increased 8-hydroxy-2'-deoxyguanosine, an adduct indicating oxidative damage, was found in liver and prostate tissues at 2 and 12 mo Sod1^+/- mice compared to controls. Prostate tissues from Sod1^+/- mice demonstrated decreased weight at 2 mo compared to controls, but this difference was not significant at 12 mo. Histologic changes were not seen. Global DNA methylation was significantly decreased at 2 mo in the prostate in Sod1^+/- mice. 11p15 containing the epigenetically modulated insulin-like growth factor 2 (Igf2) and H19 genes, both which display oncogenic functions, may be particularly sensitive to oxidative stress. CpG island methylation at an intergenic CTCF binding site and the Igf2 P3 promoter was decreased in Sod1 mutants compared to controls. This is the first in vivo study to show that a deficiency of Sod1 leads to a decrease in DNA methylation. These studies indicate that increased oxidative stress, a factor implicated in neoplasia, can induce DNA hypomethylation in prostate tissues.     

Cytochrome P4502E1, oxidative stress, JNK, and autophagy in acute alcohol-induced fatty liver

Binge alcohol drinking induces hepatic steatosis. Recent studies showed that chronic ethanol-induced fatty liver was, at least in part, CYP2E1 dependent. The mechanism of acute alcohol-induced steatosis and whether CYP2E1 plays any role are still unclear. Increasing oxidative stress by alcohol can activate the JNK MAP kinase signaling pathway, suggesting that JNK might be a target for prevention of alcohol-induced steatosis. We used CYP2E1 knockout (KO) mice, a JNK inhibitor, and JNK1 or JNK2 knockout mice to test the role of CYP2E1, JNK, and the individual role of JNK1 and JNK2 in acute alcohol-induced steatosis. In wild-type (WT) mice, acute alcohol activates CYP2E1 and increases oxidative stress, which reciprocally increases activation of the JNK signaling pathway. Acute alcohol-induced fatty liver and oxidative stress were blunted in CYP2E1 KO mice and by the JNK inhibitor in WT mice. The antioxidant N-acetylcysteine decreased the acute alcohol-induced oxidative stress, the activation of JNK, and the steatosis but not the activation of CYP2E1. Acute alcohol decreased autophagy and increased expression of SREBP, effects blocked by the JNK inhibitor. Acute alcohol-induced fatty liver was the same in JNK1 and JNK2 KO mice as in WT mice; thus either JNK1 or JNK2 per se is sufficient for induction of steatosis by acute alcohol. The results show that acute alcohol elevation of CYP2E1, oxidative stress, and activation of JNK interact to lower autophagy and increase lipogenic SREBP resulting in fatty liver.     

Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1^+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1^+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE^-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.     

Mitochondrial alterations in livers of Sod1-/- mice fed alcohol

Chronic alcohol consumption induced liver injury in Cu,Zn-superoxide dismutase-deficient mice (Sod1-/-), with extensive centrilobular necrosis and inflammation and a reduction in hepatic ATP content. Mechanisms by which ethanol decreased ATP in these mice remain unclear. We investigated alterations in mitochondria of Sod1-/- mice produced by chronic ethanol treatment. These mitochondria had an increase in State 4 oxygen consumption with succinate and especially with glutamate plus malate compared to mitochondria from pair-fed Sod1-/- mice or mitochondria from wild-type mice fed dextrose or ethanol. This uncoupling was associated with a decrease in ADP/O and respiratory control ratios, a decline in mitochondrial membrane potential, enhanced mitochondrial permeability transition, and decreased aconitase activity. Total thiols and uncoupling protein 2 levels were elevated in the pair-fed Sod1-/- mitochondria, perhaps an adaptive response to oxidant stress. However, no such increases were found with the ethanol-fed Sod1-/- mitochondria, suggesting a failure to develop these adaptations. The mitochondria from the ethanol-fed Sod1-/- mice had elevated levels of cleaved Bax, Bak, Bcl-xl, and adenine nucleotide translocator. Immunoprecipitation studies revealed increased association of Bax and Bak with the adenine nucleotide translocator. ADP-ATP exchange was very low in the ethanol-fed Sod1-/- mitochondria. These results suggest that ethanol treatment of Sod1-/- mice produces uncoupling and a decline in Deltapsi, swelling, increased association of proapoptotic proteins involved in the permeability transition, and decreased adenine nucleotide translocator activity, which may be responsible for the decline in ATP levels and development of necrosis in this model of alcohol-induced liver injury.     

Heme Oxygenase-1 Regulates the Progression of K/BxN Serum Transfer Arthritis

Background

Heme oxygenase-1 (HO-1) is induced in many cell types as a defense mechanism against stress. We have investigated the possible role of endogenous HO-1 in the effector phase of arthritis using the K/BxN serum transfer model of arthritis in HO-1 heterozygous and homozygous knock-out mice.

Methodology/Principal Findings

Arthritis was induced in C57/Black-6 xFVB (HO-1^+/+, HO-1^+/− and HO-1^−/−) mice by intraperitoneal injection of 150 µl serum from arthritic K/BxN mice at days 0 and 2. Blood was collected and animals were sacrificed at day 10. Histological analysis was performed in ankle sections. The levels of inflammatory mediators were measured in serum and paw homogenates by enzyme-linked immunosorbent assay or Multiplex technology. The incidence of arthritis was higher in HO-1^+/− and HO-1^−/− groups compared with HO-1^+/+. The inflammatory response was aggravated in HO-1^+/− mice as shown by arthritic score and the migration of inflammatory cells that could be related to the enhancement of CXCL-1 production. In addition, the HO-1^+/− group showed proteoglycan depletion significantly higher than HO-1^+/+ mice. Serum levels of matrix metalloproteinase-3, monocyte chemotactic protein-1, plasminogen activator inhibitor-1, E-selectin and intercellular adhesion molecule-1 were increased in arthritic HO-1^−/− mice, whereas vascular endothelial growth factor and some cytokines such as interferon-γ showed a reduction compared to HO-1^+/+ or HO-1^+/− mice. In addition, down-regulated gene expression of ferritin, glutathione S-reductase A1 and superoxide dismutase-2 was observed in the livers of arthritic HO-1^+/− animals.

Conclusion/Significance

Endogenous HO-1 regulates the production of systemic and local inflammatory mediators and plays a protective role in K/BxN serum transfer arthritis.     

Alcohol-induced One-carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr^+/− mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.     

Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron

In many models, a protective role for heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, has been demonstrated. Also, HO-1 null mice (KO) are more susceptible to inflammation and hypoxia and transplant rejection. Nonetheless, their response to hyperoxia (> 95% O(2)) has not yet been evaluated. Surprisingly, after acute hyperoxic exposure, KO had significantly decreased markers of lung oxidative injury and survived chronic hyperoxia as well as wild-type (WT) controls. Disrupted HO-1 expression was associated with decreased lung reactive iron and iron-associated proteins, decreased NADPH cytochrome cp450 reductase activity, and decreased lung peroxidase activity compared to WT. Injection of tin protoporphyrin, an inhibitor of HO, in the WT decreased acute hyperoxic lung injury, whereas transduction of human HO-1 in the KO reversed the relative protection of the KO to acute injury and worsened hyperoxic survival. This suggests that disruption of HO-1 protects against hyperoxia by diminishing the generation of toxic reactive intermediates in the lung via iron and H(2)O(2).     

CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice

Elevated hepatic reactive oxygen species play an important role in pathogenesis of liver diseases, such as alcohol-induced liver injury, hepatitis C virus infection, and nonalcoholic steatohepatitis. In the present study, we investigated and compared the hepatic lipid metabolisms of liver-specific Sod2 (superoxide dismutase 2) knock-out (Sod2 KO), Sod1 knock-out (Sod1 KO), and Sod1/liver-specific Sod2 double knock-out mice (double KO). We observed significant increases in lipid peroxidation and triglyceride (TG) in the liver of Sod1 KO and double KO mice but not in the liver of Sod2 KO mice. We also found that high fat diet enhanced fatty changes of the liver in Sod1 KO and double KO mice but not in Sod2 KO mice. These data indicated that CuZn-SOD deficiency caused lipid accumulation in the liver. To investigate the molecular mechanism of hepatic lipid accumulation in CuZn-SOD-deficient mice, we measured TG secretion rate from liver using Triton WR1339. We found significant decrease of TG secretion in CuZn-SOD-deficient mice. Furthermore, we observed marked degradation of apolipoprotein B (apoB) in the liver and plasma of CuZn-SOD-deficient mice, indicating that degradation of apoB impairs secretion of lipoprotein from the liver. Our data suggest that oxidative stress enhances hepatic lipid accumulation by impaired lipoprotein secretion due to the degradation of apoB in liver.     

Heme oxygenase activity in fetal and adult sheep is not altered by acclimatization to high altitude hypoxia

Hypoxic stress has been reported to induce the expression of stress proteins such as heme oxygenase (HO), which catalyze the breakdown of heme to generate biliverdin, ferrous iron, and carbon monoxide. These degradation products play a role in the regulation of a variety of processes such as vascular tone, inflammation, and central nervous system function. In mammals, there are 2 catalytically functional HO isozymes, HO-1 (inducible) and HO-2 (constitutive). HO-1 expression is regulated by an array of nonphysiological and physiological stimuli including acute hypoxemia. As relatively little is known of the HO response to prolonged hypoxia in whole animals other than small laboratory rodents, the aim of this work was to examine the effect of long-term hypoxia on total HO activity in fetal and adult ovine tissue. Sheep were maintained at high altitude (3820 m), after which the following tissues were harvested from near-term fetal and non-pregnant ewes for in vitro measurement of HO activity: left ventricle, renal papilla, lung apex, pulmonary artery, carotid artery, mesenteric artery, placental cotyledon, spleen, and brain frontal cortex. There were no significant differences between HO activities in tissues from hypoxic fetal and adult sheep compared with their normoxic controls. Fetal heart HO activities were higher than those of adult tissue (p < 0.05), whereas adult spleen HO activity was significantly higher than that of fetal tissue (p < 0.05). In conclusion, these data indicate that long-term exposure to high altitude hypoxia does not have a persistent effect on HO activity in ovine tissues. Also, except for the spleen where there is a high expression of HO-1 under normal conditions, tissue HO activity is correlated with the expression of HO-2, the constitutive isozyme.     

Protoporphyrin Treatment Modulates Susceptibility to Experimental Autoimmune Encephalomyelitis in miR-155-Deficient Mice

We previously identified heme oxygenase 1 (HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155 ^-/- CD4⁺ T cells to promote antigen-driven inflammation after adoptive transfer in antigen-expressing recipients. Protoporphyrins are molecules recognized for their modulatory effect on HO-1 expression and function. In the present study, we investigated the effect of protoporphyrin treatment on the development of autoimmunity in miR-155-deficient mice. MiR-155-mediated control of HO-1 expression in promoting T cell-driven chronic autoimmunity was confirmed since HO-1 inhibition restored susceptibility to experimental autoimmune encephalomyelitis (EAE) in miR-155-deficient mice. The increased severity of the disease was accompanied by an enhanced T cell infiltration into the brain. Taken together, these results underline the importance of miR-155-mediated control of HO-1 expression in regulating the function of chronically-stimulated T cells in EAE.     

Role of mitochondrial superoxide dismutase in contraction-induced generation of reactive oxygen species in skeletal muscle extracellular space

Contractions of skeletal muscles produce increases in concentrations of superoxide anions and activity of hydroxyl radicals in the extracellular space. The sources of these reactive oxygen species are not clear. We tested the hypothesis that, after a demanding isometric contraction protocol, the major source of superoxide and hydroxyl radical activity in the extracellular space of muscles is mitochondrial generation of superoxide anions and that, with a reduction in MnSOD activity, concentration of superoxide anions in the extracellular space is unchanged but concentration of hydroxyl radicals is decreased. For gastrocnemius muscles from adult (6–8 mo old) wild-type (Sod2^+/+) mice and knockout mice heterozygous for the MnSOD gene (Sod2^+/-), concentrations of superoxide anions and hydroxyl radical activity were measured in the extracellular space by microdialysis. A 15-min protocol of 180 isometric contractions induced a rapid, equivalent increase in reduction of cytochrome c as an index of superoxide anion concentrations in the extracellular space of Sod2^+/+ and Sod2^+/- mice, whereas hydroxyl radical activity measured by formation of 2,3-dihydroxybenzoate from salicylate increased only in the extracellular space of muscles of Sod2^+/+ mice. The lack of a difference in increase in superoxide anion concentration in the extracellular space of Sod2^+/+ and Sod2^+/- mice after the contraction protocol supported the hypothesis that superoxide anions were not directly derived from mitochondria. In contrast, the data obtained suggest that the increase in hydroxyl radical concentration in the extracellular space of muscles from wild-type mice after the contraction protocol most likely results from degradation of hydrogen peroxide generated by MnSOD activity. hydroxyl radicals; microdialysis     

Role of Hypoxia Inducing Factor-1β in Alcohol-Induced Autophagy,Steatosis and Liver Injury in Mice

Chronic alcohol causes liver hypoxia and steatosis, which eventually develops into alcoholic liver disease (ALD). While it has been known that alcohol consumption activates hepatic hypoxia inducing factor-1α (HIF-1α), conflicting results regarding the role of HIF-1α in alcohol-induced liver injury and steatosis in mice have been reported. In the present study, we aimed to use hepatocyte-specific HIF-1β knockout mice to eliminate the possible compensatory effects of the single knockout of the 1α subunit of HIF to study the role of HIFs in ALD. C57BL/6 wild type mice were treated with acute ethanol to mimic human binge drinking. Matched wild-type and hepatocyte specific HIF-1β knockout mice were also subjected to a recently established Gao-binge alcohol model to mimic chronic plus binge conditions, which is quite common in human alcoholics. We found that acute alcohol treatment increased BNIP3 and BNIP3L/NIX expression in primary cultured hepatocytes and in mouse livers, suggesting that HIF may be activated in these models. We further found that hepatocyte-specific HIF-1β knockout mice developed less steatosis and liver injury following the Gao-binge model or acute ethanol treatment compared with their matched wild type mice. Mechanistically, protection against Gao-binge treatment-induced steatosis and liver injury was likely associated with increased FoxO3a activation and subsequent induction of autophagy in hepatocyte-specific HIF-1β knockout mice.     

Reduction in Tcf7l2 expression decreases diabetic susceptibility in mice

Objective: The WNT signaling pathway effector gene TCF7L2 has been associated with an increased risk of type 2 diabetes. However, it remains unclear how this gene affects diabetic pathogenesis. The goal of this study was to investigate the effects of Tcf7l2 haploinsufficiency on metabolic phenotypes in mice.Experimental Design: Tcf7l2 knockout (Tcf7l^-/-) mice were generated. Because of the early mortality of Tcf7l2^-/- mice, we characterized the metabolic phenotypes of heterozygous Tcf7l2^+/- mice in comparison to the wild-type controls. The mice were fed a normal chow diet or a high fat diet (HFD) for 9 weeks.Results: The Tcf7l2^+/- mice showed significant differences from the wild-type mice with regards to body weight, fasting glucose and insulin levels. Tcf7l2^+/- mice displayed improved glucose tolerance. In the liver of Tcf7l2^+/- mice fed on the HFD, reduced lipogenesis and hepatic triglyceride levels were observed when compared with those of wild-type mice. Furthermore, the Tcf7l2^+/- mice fed on the HFD exhibited decreased peripheral fat deposition. Immunohistochemistry in mouse pancreatic islets showed that endogenous expression of Tcf7l2 was upregulated in the wild-type mice, but not in the Tcf7l2^+/- mice, after feeding with the HFD. However, the haploinsufficiency of Tcf7l2 in mouse pancreatic islets resulted in little changes in glucose-stimulated insulin secretion.Conclusion: These results suggest that decreased expression of Tcf7l2 confers reduction of diabetic susceptibility in mice via regulation on the metabolism of glucose and lipid.     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1^+/-) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1^+/- mice relative to wild-type mice. Endothelial cells from Becn1^+/- mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1^+/- cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1^+/- endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.