Thermus thermophilus Argonaute Functions in the Completion of DNA Replication.

In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.

Liver circadian clock disruption alters perivascular adipose tissue gene expression and aortic function in mice.

The liver plays a central role that influences cardiovascular disease outcomes through regulation of glucose and lipid metabolism. It is recognized that the local liver molecular clock regulates some liver-derived metabolites. However, it is unknown whether the liver clock may impact cardiovascular function. Perivascular adipose tissue (PVAT) is a specialized type of adipose tissue surrounding blood vessels. Importantly, cross talk between the endothelium and PVAT via vasoactive factors is critical for vascular function. Therefore, we designed studies to test the hypothesis that cardiovascular function, including PVAT function, is impaired in mice with liver-specific circadian clock disruption. Bmal1 is a core circadian clock gene, thus studies were undertaken in male hepatocyte-specific Bmal1 knockout (HBK) mice and littermate controls (i.e., flox mice). HBK mice showed significantly elevated plasma levels of ß-hydroxybutyrate, nonesterified fatty acids/free fatty acids, triglycerides, and insulin-like growth factor 1 compared with flox mice. Thoracic aorta PVAT in HBK mice had increased mRNA expression of several key regulatory and metabolic genes, Ppargc1a, Pparg, Adipoq, Lpl, and Ucp1, suggesting altered PVAT energy metabolism and thermogenesis. Sensitivity to acetylcholine-induced vasorelaxation was significantly decreased in the aortae of HBK mice with PVAT attached compared with aortae of HBK mice with PVAT removed, however, aortic vasorelaxation in flox mice showed no differences with or without attached PVAT. HBK mice had a significantly lower systolic blood pressure during the inactive period of the day. These new findings establish a novel role of the liver circadian clock in regulating PVAT metabolic gene expression and PVAT-mediated aortic vascular function.

The circadian clock and liver function in health and disease.

Each day, all organisms are subjected to changes in light intensity because of the Earth's rotation around its own axis. To anticipate this geo-physical variability, and to appropriately respond biochemically, most species, including mammals, have evolved an approximate 24-hour endogenous timing mechanism known as the circadian clock (CC). The 'clock' is self-sustained, cell autonomous and present in every cell type. At the core of the clock resides the CC-oscillator, an exquisitely crafted transcriptional-translational feedback system. Remarkably, components of the CC-oscillator not only maintain daily rhythmicity of their own synthesis, but also generate temporal variability in the expression levels of numerous target genes through transcriptional, post-transcriptional and post-translational mechanisms, thus, ensuring proper chronological coordination in the functioning of cells, tissues and organs, including the liver. Indeed, a variety of physiologically critical hepatic functions and cellular processes are CC-controlled. Thus, it is not surprising that modern lifestyle factors (e.g. travel and jet lag, night and rotating shift work), which force 'circadian misalignment', have emerged as major contributors to global health problems including obesity, non-alcoholic fatty liver disease and steatohepatitis. Herein, we provide an overview of the CC-dependent pathways which play critical roles in mediating several hepatic functions under physiological conditions, and whose deregulation is implicated in chronic liver diseases including non-alcoholic steatohepatitis and alcohol-related liver disease.

Emerging role of circadian clock disruption in alcohol-induced liver disease.

The detrimental health effects of excessive alcohol consumption are well documented. Alcohol-induced liver disease (ALD) is the leading cause of death from chronic alcohol use. As with many diseases, the etiology of ALD is influenced by how the liver responds to other secondary insults. The molecular circadian clock is an intrinsic cellular timing system that helps organisms adapt and synchronize metabolism to changes in their environment. The clock also influences how tissues respond to toxic, environmental, and metabolic stressors, like alcohol. Consistent with the essential role for clocks in maintaining health, genetic and environmental disruption of the circadian clock contributes to disease. While a large amount of rich literature is available showing that alcohol disrupts circadian-driven behaviors and that circadian clock disruption increases alcohol drinking and preference, very little is known about the role circadian clocks play in alcohol-induced tissue injuries. In this review, recent studies examining the effect alcohol has on the circadian clock in peripheral tissues (liver and intestine) and the impact circadian clock disruption has on development of ALD are presented. This review also highlights some of the rhythmic metabolic processes in the liver that are disrupted by alcohol and potential mechanisms through which alcohol disrupts the liver clock. Improved understanding of the mechanistic links between the circadian clock and alcohol will hopefully lead to the development of new therapeutic approaches for treating ALD and other alcohol-related organ pathologies.

Genetic deletion of the circadian clock transcription factor BMAL1 and chronic alcohol consumption differentially alter hepatic glycogen in mice.

Multiple metabolic pathways exhibit time-of-day-dependent rhythms that are controlled by the molecular circadian clock. We have shown that chronic alcohol is capable of altering the molecular clock and diurnal oscillations in several elements of hepatic glycogen metabolism ( 19 , 44 ). Herein, we sought to determine whether genetic disruption of the hepatocyte clock differentially impacts hepatic glycogen content in chronic alcohol-fed mice. Male hepatocyte-specific BMAL1 knockout (HBK) and littermate controls were fed control or alcohol-containing diets for 5 wk to alter hepatic glycogen content. Glycogen displayed a significant diurnal rhythm in livers of control genotype mice fed the control diet. While rhythmic, alcohol significantly altered the diurnal oscillation of glycogen in livers of control genotype mice. The glycogen rhythm was mildly altered in livers of control-fed HBK mice. Importantly, glycogen content was arrhythmic in livers of alcohol-fed HBK mice. Consistent with these changes in hepatic glycogen content, we observed that some glycogen and glucose metabolism genes were differentially altered by chronic alcohol consumption in livers of HBK and littermate control mice. Diurnal rhythms in glycogen synthase (mRNA and protein) were significantly altered by alcohol feeding and clock disruption. Alcohol consumption significantly altered Gck, Glut2, and Ppp1r3g rhythms in livers of control genotype mice, with diurnal rhythms of Pklr, Glut2, Ppp1r3c, and Ppp1r3g further disrupted (dampened or arrhythmic) in livers of HBK mice. Taken together, these findings show that chronic alcohol consumption and hepatocyte clock disruption differentially influence the diurnal rhythm of glycogen and various key glycogen metabolism-related genes in the liver. NEW & NOTEWORTHY We report that circadian clock disruption exacerbates alcohol-mediated alterations in hepatic glycogen. We observed differential responsiveness in diurnal rhythms of glycogen and glycogen metabolism genes and proteins in livers of hepatocyte-specific BMAL1 knockout and littermate control mice fed alcohol. Our findings provide new insights into potential mechanisms by which alcohol alters glycogen, an important energy source for liver and other organs.

IGFBP6 controls the expansion of chemoresistant glioblastoma through paracrine IGF2/IGF-1R signaling.

BACKGROUND: Glioblastomas (GBMs), the most common and most lethal of the primary brain tumors, are characterized by marked intra-tumor heterogeneity. Several studies have suggested that within these tumors a restricted population of chemoresistant glioma cells is responsible for recurrence. However, the gene expression patterns underlying chemoresistance are largely unknown. Numerous efforts have been made to block IGF-1R signaling pathway in GBM. However, those therapies have been repeatedly unsuccessful. This failure may not only be due to the complexity of IGF receptor signaling, but also due to complex cell-cell interactions in the tumor mass. We hypothesized that differential expression of proteins in the insulin-like growth factor (IGF) system underlie cell-specific differences in the resistance to temozolomide (TMZ) within GBM tumors. METHODS: Expression of IGF-1R was analyzed in cell lines, patient-derived xenograft cell lines and human biopsies by cell surface proteomics, flow cytometry, immunofluorescence and quantitative real time polymerase chain reaction (qRT-PCR). Using gain-of-function and loss-of-function strategies, we dissected the molecular mechanism responsible for IGF-binding protein 6 (IGFBP6) tumor suppressor functions both in in vitro and in vivo. Site direct mutagenesis was used to study IGFBP6-IGF2 interactions. RESULTS: We determined that in human glioma tissue, glioma cell lines, and patient-derived xenograft cell lines, treatment with TMZ enhances the expression of IGF1 receptor (IGF-1R) and IGF2 and decreases the expression of IGFBP6, which sequesters IGF2. Using chemoresistant and chemosensitive wild-type and transgenic glioma cells, we further found that a paracrine mechanism driven by IGFBP6 secreted from TMZ-sensitive cells abrogates the proliferation of IGF-1R-expressing TMZ-resistant cells in vitro and in vivo. In mice bearing intracranial human glioma xenografts, overexpression of IGFBP6 in TMZ-resistant cells increased survival. Finally, elevated expression of IGF-1R and IGF2 in gliomas associated with poor patient survival and tumor expression levels of IGFBP6 directly correlated with overall survival time in patients with GBM. CONCLUSIONS: Our findings support the view that proliferation of chemoresistant tumor cells is controlled within the tumor mass by IGFBP6-producing tumor cells; however, TMZ treatment eliminates this population and enriches the TMZ-resistant cell populationleading to accelerated growth of the entire tumor mass.

Cellular Abnormalities and Emerging Biomarkers in Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (AALD) is the third most common preventable cause for disease burden and mortality in the US. AALD, including alcoholic hepatitis (AH), contributes to half of admissions from decompensated liver disease and 20% of all liver transplants in the US. Peripheral blood cells contribute to systemic inflammation, oxidative stress, mitochondrial dysfunction, and fibrosis in AALD and AH. Alcohol dysregulates function of lymphocytes, neutrophils, monocytes, and tissue macrophages of the innate immune system. These alterations in turn can modulate adaptive immune responses. In this review, we describe these disruptive effects of alcohol on cells of the innate and adaptive immune system and focus on cellular-based emerging biomarkers on diagnosis and prognosis of patients with AALD and AH.

Identification of Small Molecule Inhibitors of Human Cytochrome c Oxidase That Target Chemoresistant Glioma Cells.

The enzyme cytochrome c oxidase (CcO) or complex IV (EC 1.9.3.1) is a large transmembrane protein complex that serves as the last enzyme in the respiratory electron transport chain of eukaryotic mitochondria. CcO promotes the switch from glycolytic to oxidative phosphorylation (OXPHOS) metabolism and has been associated with increased self-renewal characteristics in gliomas. Increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure, and patients with primary glioblastoma multiforme and high tumor CcO activity have worse clinical outcomes than those with low tumor CcO activity. Therefore, CcO is an attractive target for cancer therapy. We report here the characterization of a CcO inhibitor (ADDA 5) that was identified using a high throughput screening paradigm. ADDA 5 demonstrated specificity for CcO, with no inhibition of other mitochondrial complexes or other relevant enzymes, and biochemical characterization showed that this compound is a non-competitive inhibitor of cytochrome c When tested in cellular assays, ADDA 5 dose-dependently inhibited the proliferation of chemosensitive and chemoresistant glioma cells but did not display toxicity against non-cancer cells. Furthermore, treatment with ADDA 5 led to significant inhibition of tumor growth in flank xenograft mouse models. Importantly, ADDA 5 inhibited CcO activity and blocked cell proliferation and neurosphere formation in cultures of glioma stem cells, the cells implicated in tumor recurrence and resistance to therapy in patients with glioblastoma. In summary, we have identified ADDA 5 as a lead CcO inhibitor for further optimization as a novel approach for the treatment of glioblastoma and related cancers.

Altered myocardial metabolic adaptation to increased fatty acid availability in cardiomyocyte-specific CLOCK mutant mice.

A mismatch between fatty acid availability and utilization leads to cellular/organ dysfunction during cardiometabolic disease states (e.g., obesity, diabetes mellitus). This can precipitate cardiac dysfunction. The heart adapts to increased fatty acid availability at transcriptional, translational, post-translational and metabolic levels, thereby attenuating cardiomyopathy development. We have previously reported that the cardiomyocyte circadian clock regulates transcriptional responsiveness of the heart to acute increases in fatty acid availability (e.g., short-term fasting). The purpose of the present study was to investigate whether the cardiomyocyte circadian clock plays a role in adaptation of the heart to chronic elevations in fatty acid availability. Fatty acid availability was increased in cardiomyocyte-specific CLOCK mutant (CCM) and wild-type (WT) littermate mice for 9weeks in time-of-day-independent (streptozotocin (STZ) induced diabetes) and dependent (high fat diet meal feeding) manners. Indices of myocardial metabolic adaptation (e.g., substrate reliance perturbations) to STZ-induced diabetes and high fat meal feeding were found to be dependent on genotype. Various transcriptional and post-translational mechanisms were investigated, revealing that Cte1 mRNA induction in the heart during STZ-induced diabetes is attenuated in CCM hearts. At the functional level, time-of-day-dependent high fat meal feeding tended to influence cardiac function to a greater extent in WT versus CCM mice. Collectively, these data suggest that CLOCK (a circadian clock component) is important for metabolic adaption of the heart to prolonged elevations in fatty acid availability. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Physiological processes underlying organ injury in alcohol abuse.

This review summarizes the American Physiological Society (APS) Presidential Symposium 1 entitled "Physiological Processes Underlying Organ Injury in Alcohol Abuse" at the 2016 Experimental Biology meeting. The symposium was organized by Dr. Patricia Molina, past president of the APS, was held on April 3 at the Convention Center in San Diego, CA, and was funded by the National Institute on Alcohol Abuse and Alcoholism. The "Physiological Processes Underlying Organ Injury in Alcohol Abuse Symposium" assembled experts and leaders in the field and served as a platform to discuss and share knowledge on the latest developments and scientific advances on the mechanisms underlying organ injury in alcohol abuse. This symposium provided unique, interdisciplinary alcohol research, including several organs, liver, muscle, adipose, and brain, affected by excessive alcohol use.

Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism.

Circadian clocks are critical modulators of metabolism. However, mechanistic links between cell autonomous clocks and metabolic processes remain largely unknown. Here, we report that expression of the biotin transporter slc5a6 gene is decreased in hearts of two distinct genetic mouse models of cardiomyocyte-specific circadian clock disruption [i.e., cardiomyocyte-specific CLOCK mutant (CCM) and cardiomyocyte-specific BMAL1 knockout (CBK) mice]. Biotinylation is an obligate posttranslational modification for five mammalian carboxylases: acetyl-CoA carboxylase α (ACCα), ACCß, pyruvate carboxylase (PC), methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). We therefore hypothesized that the cardiomyocyte circadian clock impacts metabolism through biotinylation. Consistent with decreased slc5a6 expression, biotinylation of all carboxylases is significantly decreased (10-46%) in CCM and CBK hearts. In association with decreased biotinylated ACC, oleate oxidation rates are increased in both CCM and CBK hearts. Consistent with decreased biotinylated MCC, leucine oxidation rates are significantly decreased in both CCM and CBK hearts, whereas rates of protein synthesis are increased. Importantly, feeding CBK mice with a biotin-enriched diet for 6 wk normalized myocardial 1) ACC biotinylation and oleate oxidation rates; 2) PCC/MCC biotinylation (and partially restored leucine oxidation rates); and 3) net protein synthesis rates. Furthermore, data suggest that the RRAGD/mTOR/4E-BP1 signaling axis is chronically activated in CBK and CCM hearts. Finally we report that the hepatocyte circadian clock also regulates both slc5a6 expression and protein biotinylation in the liver. Collectively, these findings suggest that biotinylation is a novel mechanism by which cell autonomous circadian clocks influence metabolic pathways.

Chronic ethanol consumption disrupts diurnal rhythms of hepatic glycogen metabolism in mice.

Chronic ethanol consumption has been shown to significantly decrease hepatic glycogen content; however, the mechanisms responsible for this adverse metabolic effect are unknown. In this study, we examined the impact chronic ethanol consumption has on time-of-day-dependent oscillations (rhythms) in glycogen metabolism processes in the liver. For this, male C57BL/6J mice were fed either a control or ethanol-containing liquid diet for 5 wk, and livers were collected every 4 h for 24 h and analyzed for changes in various genes and proteins involved in hepatic glycogen metabolism. Glycogen displayed a robust diurnal rhythm in the livers of mice fed the control diet, with the peak occurring during the active (dark) period of the day. The diurnal glycogen rhythm was significantly altered in livers of ethanol-fed mice, with the glycogen peak shifted into the inactive (light) period and the overall content of glycogen decreased compared with controls. Chronic ethanol consumption further disrupted diurnal rhythms in gene expression (glycogen synthase 1 and 2, glycogenin, glucokinase, protein targeting to glycogen, and pyruvate kinase), total and phosphorylated glycogen synthase protein, and enzyme activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of glycogen metabolism. In summary, these results show for the first time that chronic ethanol consumption disrupts diurnal rhythms in hepatic glycogen metabolism at the gene and protein level. Chronic ethanol-induced disruption in these daily rhythms likely contributes to glycogen depletion and disruption of hepatic energy homeostasis, a recognized risk factor in the etiology of alcoholic liver disease.

Mitochondrial-nuclear genome interactions in non-alcoholic fatty liver disease in mice.

NAFLD (non-alcoholic fatty liver disease) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. In the present study, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, MNX (mitochondrial-nuclear exchange) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared with wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR (respiratory control ratio) when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD.

Involvement of the mitochondrial permeability transition pore in chronic ethanol-mediated liver injury in mice.

Chronic ethanol consumption increases sensitivity of the mitochondrial permeability transition (MPT) pore induction in liver. Ca(2+) promotes MPT pore opening, and genetic ablation of cyclophilin D (CypD) increases the Ca(2+) threshold for the MPT. We used wild-type (WT) and CypD-null (CypD(-/-)) mice fed a control or an ethanol-containing diet to investigate the role of the MPT in ethanol-mediated liver injury. Ca(2+)-mediated induction of the MPT and mitochondrial respiration were measured in isolated liver mitochondria. Steatosis was present in WT and CypD(-/-) mice fed ethanol and accompanied by increased terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-positive nuclei. Autophagy was increased in ethanol-fed WT mice compared with ethanol-fed CypD(-/-) mice, as reflected by an increase in the ratio of microtubule protein 1 light chain 3B II to microtubule protein 1 light chain 3B I. Higher levels of p62 were measured in CypD(-/-) than WT mice. Ethanol decreased mitochondrial respiratory control ratios and select complex activities in WT and CypD(-/-) mice. Ethanol also increased CypD protein in liver of WT mice. Mitochondria from control- and ethanol-fed WT mice were more sensitive to Ca(2+)-mediated MPT pore induction than mitochondria from their CypD(-/-) counterparts. Mitochondria from ethanol-fed CypD(-/-) mice were also more sensitive to Ca(2+)-induced swelling than mitochondria from control-fed CypD(-/-) mice but were less sensitive than mitochondria from ethanol-fed WT mice. In summary, CypD deficiency was associated with impaired autophagy and did not prevent ethanol-mediated steatosis. Furthermore, increased MPT sensitivity was observed in mitochondria from ethanol-fed WT and CypD(-/-) mice. We conclude that chronic ethanol consumption likely lowers the threshold for CypD-regulated and -independent characteristics of the ethanol-mediated MPT pore in liver mitochondria.

The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.

Elucidating nature's solutions to heart, lung, and blood diseases and sleep disorders.

Evolution has provided a number of animal species with extraordinary phenotypes. Several of these phenotypes allow species to survive and thrive in environmental conditions that mimic disease states in humans. The study of evolved mechanisms responsible for these phenotypes may provide insights into the basis of human disease and guide the design of new therapeutic approaches. Examples include species that tolerate acute or chronic hypoxemia like deep-diving mammals and high-altitude inhabitants, as well as those that hibernate and interrupt their development when exposed to adverse environments. The evolved traits exhibited by these animal species involve modifications of common biological pathways that affect metabolic regulation, organ function, antioxidant defenses, and oxygen transport. In 2006, the National Heart, Lung, and Blood Institute released a funding opportunity announcement to support studies that were designed to elucidate the natural molecular and cellular mechanisms of adaptation in species that tolerate extreme environmental conditions. The rationale for this funding opportunity is detailed in this article, and the specific evolved mechanisms examined in the supported research are described. Also highlighted are past medical advances achieved through the study of animal species that have evolved extraordinary phenotypes as well as the expectations for new understanding of nature's solutions to heart, lung, blood, and sleep disorders through future research in this area.

Convergent mechanisms for dysregulation of mitochondrial quality control in metabolic disease: implications for mitochondrial therapeutics.

Mitochondrial dysfunction is associated with a broad range of pathologies including diabetes, ethanol toxicity, metabolic syndrome and cardiac failure. It is now becoming clear that maintaining mitochondrial quality through a balance between biogenesis, reserve capacity and mitophagy is critical in determining the response to metabolic or xenobiotic stress. In diseases associated with metabolic stress, such as Type II diabetes and non-alcoholic and alcoholic steatosis, the mitochondria are subjected to multiple 'hits' such as hypoxia and oxidative and nitrative stress, which can overwhelm the mitochondrial quality control pathways. In addition, the underlying mitochondrial genetics that evolved to accommodate high-energy demand, low-calorie supply environments may now be maladapted to modern lifestyles (low-energy demand, high-calorie environments). The pro-oxidant and pro-inflammatory environment of a sedentary western lifestyle has been associated with modified redox cell signalling pathways such as steatosis, hypoxic signalling, inflammation and fibrosis. These data suggest that loss of mitochondrial quality control is intimately associated with the aberrant activation of redox cell signalling pathways under pathological conditions. In the present short review, we discuss evidence from alcoholic liver disease supporting this concept, the insights obtained from experimental models and the application of bioenergetic-based therapeutics in the context of maintaining mitochondrial quality.

Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes.

Chronic alcohol consumption results in hepatotoxicity, steatosis, hypoxia, increased expression of inducible nitric oxide synthase (iNOS) and decreased activities of mitochondrial respiratory enzymes. The impact of these changes on cellular respiration and their interaction in a cellular setting is not well understood. In the present study we tested the hypothesis that nitric oxide (NO)-dependent modulation of cellular respiration and the sensitivity to hypoxic stress is increased following chronic alcohol consumption. This is important since NO has been shown to regulate mitochondrial function through its interaction with cytochrome c oxidase, although at higher concentrations, and in combination with reactive oxygen species, can result in mitochondrial dysfunction. We found that hepatocytes isolated from alcohol-fed rats had decreased mitochondrial bioenergetic reserve capacity and were more sensitive to NO-dependent inhibition of respiration under room air and hypoxic conditions. We reasoned that this would result in greater hypoxic stress in vivo, and to test this, wild-type and iNOS(-/-) mice were administered alcohol-containing diets. Chronic alcohol consumption resulted in liver hypoxia in the wild-type mice and increased levels of hypoxia-inducible factor 1 α in the peri-venular region of the liver lobule. These effects were attenuated in the alcohol-fed iNOS(-/-) mice suggesting that increased mitochondrial sensitivity to NO and reactive nitrogen species in hepatocytes and iNOS plays a critical role in determining the response to hypoxic stress in vivo. These data support the concept that the combined effects of NO and ethanol contribute to an increased susceptibility to hypoxia and the deleterious effects of alcohol consumption on liver.

What part of NO don't you understand? Some answers to the cardinal questions in nitric oxide biology.

Nitric oxide (NO) regulates biological processes through signaling mechanisms that exploit its unique biochemical properties as a free radical. For the last several decades, the key aspects of the chemical properties of NO relevant to biological systems have been defined, but it has been a challenge to assign these to specific cellular processes. Nevertheless, it is now clear that the high affinity of NO for transition metal centers, particularly iron, and the rapid reaction of NO with oxygen-derived free radicals can explain many of its biological and pathological properties. Emerging studies also highlight a growing importance of the secondary metabolites of NO-dependent reactions in the post-translational modification of key metabolic and signaling proteins. In this minireview, we emphasize the current understanding of the biochemistry of NO and place it in a biological context.

Acquisition of temozolomide chemoresistance in gliomas leads to remodeling of mitochondrial electron transport chain.

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.