Reduction in the levels of CoQ biosynthetic proteins is related to an increase in lifespan without evidence of hepatic mitohormesis

Mitohormesis is an adaptive response induced by a mild mitochondrial stress that promotes longevity and metabolic health in different organisms. This mechanism has been proposed as the cause of the increase in the survival in Coq7+/− (Mclk1+/−) mice, which show hepatic reduction of COQ7, early mitochondrial dysfunction and increased oxidative stress. Our study shows that the lack of COQ9 in Coq9Q95X mice triggers the reduction of COQ7, COQ6 and COQ5, which results in an increase in life expectancy. However, our results reveal that the hepatic CoQ levels are not decreased and, therefore, neither mitochondrial dysfunction or increased oxidative stress are observed in liver of Coq9Q95X mice. These data point out the tissue specific differences in CoQ biosynthesis. Moreover, our results suggest that the effect of reduced levels of COQ7 on the increased survival in Coq9Q95X mice may be due to mitochondrial mechanisms in non-liver tissues or to other unknown mechanisms.This work was supported by grants from Ministerio de Economía Competitividad, Spain, and the ERDF (Grant Number SAF2015-65786-R), from the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (grant number P10-CTS-6133) and from the University of Granada (grant reference “UNETE”, UCE-PP2017-06). AHG is a “FPU fellow” from the Ministerio de Educación Cultura y Deporte, Spain. MLS was a predoctoral fellow from the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía. LCL was supported by the “Ramón y Cajal” National Programme, Ministerio de Economía y Competitividad, Spain (RYC-2011-07643)

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins

Fatty Acid Incubation of Myotubes From Humans With Type 2 Diabetes Leads to Enhanced Release of β-Oxidation Products Because of Impaired Fatty Acid Oxidation: Effects of Tetradecylthioacetic Acid and Eicosapentaenoic Acid

OBJECTIVE—Increased availability of fatty acids is important for accumulation of intracellular lipids and development of insulin resistance in human myotubes. It is unknown whether different types of fatty acids like eicosapentaenoic acid (EPA) or tetradecylthioacetic acid (TTA) influence these processes

Transcriptomic Analysis Reveals Novel Mechanistic Insight into Murine Biological Responses to Multi-Walled Carbon Nanotubes in Lungs and Cultured Lung Epithelial Cells

There is great interest in substituting animal work with in vitro experimentation in human health risk assessment; however, there are only few comparisons of in vitro and in vivo biological responses to engineered nanomaterials. We used high-content genomics tools to compare in vivo pulmonary responses of multiwalled carbon nanotubes (MWCNT) to those in vitro in cultured lung epithelial cells (FE1) at the global transcriptomic level. Primary size, surface area and other properties of MWCNT- XNRI -7 (Mitsui7) were characterized using DLS, SEM and TEM. Mice were exposed via a single intratracheal instillation to 18, 54, or 162 μg of Mitsui7/mouse. FE1 cells were incubated with 12.5, 25 and 100 μg/ml of Mitsui7. Tissue and cell samples were collected at 24 hours post-exposure. DNA microarrays were employed to establish mechanistic differences and similarities between the two models. Microarray results were confirmed using gene-specific RT-qPCR. Bronchoalveolar lavage (BAL) fluid was assessed for indications of inflammation in vivo. A strong dose-dependent activation of acute phase and inflammation response was observed in mouse lungs reflective mainly of an inflammatory response as observed in BAL. In vitro, a wide variety of core cellular functions were affected including transcription, cell cycle, and cellular growth and proliferation. Oxidative stress, fibrosis and inflammation processes were altered in both models. Although there were similarities observed between the two models at the pathway-level, the specific genes altered under these pathways were different, suggesting that the underlying mechanisms of responses are different in cells in culture and the lung tissue. Our results suggest that careful consideration should be given in selecting relevant endpoints when substituting animal with in vitro testing

Mitochondrial Uncoupling Inhibits p53 Mitochondrial Translocation in TPA-Challenged Skin Epidermal JB6 Cells

The tumor suppressor p53 is known to be able to trigger apoptosis in response to DNA damage, oncogene activation, and certain chemotherapeutic drugs. In addition to its transcriptional activation, a fraction of p53 translocates to mitochondria at the very early stage of apoptosis, which eventually contributes to the loss of mitochondrial membrane potential, generation of reactive oxygen species (ROS), cytochrome c release, and caspase activation. However, the mitochondrial events that affect p53 translocation are still unclear. Since mitochondrial uncoupling has been suggested to contribute to cancer development, herein, we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by mitochondrial uncoupling using chemical protonophores, and further verified the results using a siRNA approach in murine skin epidermal JB6 cells. Our results showed that mitochondrial uncoupling blocked p53 mitochondrial translocation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter to induce p53-mediated apoptosis in skin carcinogenesis. This blocking effect, in turn, led to preservation of mitochondrial functions, and eventually suppression of caspase activity and apoptosis. Moreover, uncoupling protein 2 (UCP2), a potential suppressor of ROS in mitochondria, is important for TPA-induced cell transformation in JB6 cells. UCP2 knock down cells showed enhanced p53 mitochondrial translocation, and were less prone to form colonies in soft agar after TPA treatment. Altogether, our data suggest that mitochondrial uncoupling may serve as an important regulator of p53 mitochondrial translocation and p53-mediated apoptosis during early tumor promotion. Therefore, targeting mitochondrial uncoupling may be considered as a novel treatment strategy for cancer

Metabolic State Determines Sensitivity to Cellular Stress in Huntington Disease: Normalization by Activation of PPARγ

Impairments in mitochondria and transcription are important factors in the pathogenesis of Huntington disease (HD), a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. This study investigated the effect of different metabolic states and peroxisome proliferator-activated receptor γ (PPARγ) activation on sensitivity to cellular stressors such as H2O2 or thapsigargin in HD. Striatal precursor cells expressing wild type (STHdhQ7) or mutant huntingtin (STHdhQ111) were prepared in different metabolic conditions (glucose vs. pyruvate). Due to the fact that STHdhQ111 cells exhibit mitochondrial deficits, we expected that in the pyruvate condition, where ATP is generated primarily by the mitochondria, there would be greater differences in cell death between the two cell types compared to the glucose condition. Intriguingly, it was the glucose condition that gave rise to greater differences in cell death. In the glucose condition, thapsigargin treatment resulted in a more rapid loss of mitochondrial membrane potential (ΔΨm), a greater activation of caspases (3, 8, and 9), and a significant increase in superoxide/reactive oxygen species (ROS) in STHdhQ111 compared to STHdhQ7, while both cell types showed similar kinetics of ΔΨm-loss and similar levels of superoxide/ROS in the pyruvate condition. This suggests that bioenergetic deficiencies are not the primary contributor to the enhanced sensitivity of STHdhQ111 cells to stressors compared to the STHdhQ7 cells. PPARγ activation significantly attenuated thapsigargin-induced cell death, concomitant with an inhibition of caspase activation, a delay in ΔΨm loss, and a reduction of superoxide/ROS generation in STHdhQ111 cells. Expression of mutant huntingtin in primary neurons induced superoxide/ROS, an effect that was significantly reduced by constitutively active PPARγ. These results provide significant insight into the bioenergetic disturbances in HD with PPARγ being a potential therapeutic target for HD