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This study investigated the mechanism of silver nanoparticle (AgNP) cytotoxicity from a mitochondrial perspective. The effect of AgNP on manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, against oxidative stress has not been studied in detail. We demonstrated that AgNP decreased MnSOD mRNA level, protein expression, and activity in human Chang liver cells in a time-dependent manner. AgNP induced the production of mitochondrial reactive oxygen species (mtROS), particularly superoxide anion. AgNP was found to increase mitochondrial calcium level and disrupt mitochondrial function, leading to reduced ATP level, succinate dehydrogenase activity, and mitochondrial permeability. AgNP induced cytochrome c release from the mitochondria into the cytoplasm, attenuated the expression of the anti-apoptotic proteins phospho Bcl-2 and Mcl-1, and induced the expression of the pro-apoptotic proteins Bim and Bax. In addition, c-Jun N-terminal kinase (JNK) phosphorylation was significantly increased by AgNP. Treatment with elamipretide (a mitochondria-targeted antioxidant) and SP600125 (a JNK inhibitor) showed the involvement of MnSOD and JNK in these processes. These results indicated that AgNP damaged human Chang liver cells by destroying mitochondrial function through the accumulation of mtROS.

The primary objective of this study was to assess whether adenine nucleotide translocase (ANT) content could be associated with phylogenetic disparities in mitochondrial coupling efficiency, within liver mitochondria obtained from rats, crocodiles, and ducklings. Our measurements included mitochondrial membrane conductance, ANT content, and oxidative phosphorylation fluxes at various steady-state rates. We observed significant variations in liver mitochondrial coupling efficiency across the three species. These variations correlated with interspecific differences in mitochondrial oxidative capacity and, to a lesser extent, the ANT content of liver mitochondria. These findings expand upon previous research by highlighting the pivotal role of oxidative capacity and ANT in modulating mitochondrial efficiency on an interspecific scale., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Biotin-dependent carboxylases catalyze the MgATP- and bicarbonate-dependent carboxylation of various acceptor substrates through a two-step carboxylation reaction. Biotin-dependent carboxylases play an essential role in the metabolism of key biomolecules and, therefore, they are the subject of ongoing drug discovery efforts, as well as of studies seeking to better characterize their structure and function. It has been an ongoing challenge to obtain high yields of mammalian biotin-dependent carboxylases for in vitro experimentation; these enzymes have not been successfully purified when recombinantly expressed from a bacterial expression host and only low yields of these recombinant, vertebrate enzymes have been obtained through expression in cell culture systems. Here, we describe a revived protocol to isolate mitochondrial biotin-dependent carboxylases (pyruvate carboxylase, propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase) from fresh pig liver. This serves as an inexpensive and effective alternative to using a recombinant expression system. This scalable protocol can be completed in less than 48 h and affords effective separation of mammalian, mitochondrial, biotin-dependent carboxylases from cellular components. The purified, mitochondrial biotin-dependent carboxylases can be used in downstream experiments focused on kinetic and structural characterization, as well as in initial drug discovery experiments., (Copyright © 2024. Published by Elsevier Inc.)

Mitochondrial dysfunction is a major factor in nonalcoholic fatty liver disease (NAFLD), preceding insulin resistance and hepatic steatosis. Carnosol (CAR) is a kind of diterpenoid with antioxidant, anti-inflammatory and antitumor activities. Peroxiredoxin 3 (PRDX3), a mitochondrial H 2 O 2 -eliminating enzyme, undergoes overoxidation and subsequent inactivation under oxidative stress. The purpose of this study was to investigate the protective effect of the natural phenolic compound CAR on NAFLD via PRDX3. Mice fed a high-fat diet (HFD) and AML-12 cells treated with palmitic acid (PA) were used to detect the molecular mechanism of CAR in NAFLD. We found that pharmacological treatment with CAR notably moderated HFD- and PA- induced steatosis and liver injury, as shown by biochemical assays, Oil Red O and Nile Red staining. Further mechanistic investigations revealed that CAR exerted anti-NAFLD effects by inhibiting mitochondrial oxidative stress, perturbation of mitochondrial dynamics, and apoptosis in vivo and in vitro. The decreased protein and mRNA levels of PRDX3 were accompanied by intense oxidative stress after PA intervention. Interestingly, CAR specifically bound PRDX3, as shown by molecular docking assays, and increased the expression of PRDX3. However, the hepatoprotection of CAR in NAFLD was largely abolished by specific PRDX3 siRNA, which increased mitochondrial dysfunction and exacerbated apoptosis in vitro. In conclusion, CAR suppressed lipid accumulation, mitochondrial dysfunction and hepatocyte apoptosis by activating PRDX3, mitigating the progression of NAFLD, and thus, CAR may represent a promising candidate for clinical treatment of steatosis., (Copyright © 2021. Published by Elsevier Inc.)

Background and Aims: Most of the genetic basis of chronic liver disease remains undiscovered., Approach and Results: To identify genetic loci that modulate the risk of liver injury, we performed genome-wide association studies on circulating levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin across 312,671 White British participants in the UK Biobank. We focused on variants associated with elevations in all four liver biochemistries at genome-wide significance (P < 5 × 10 -8 ) and that replicated using Mass General Brigham Biobank in 19,323 European ancestry individuals. We identified a genetic locus in mitochondrial glycerol-3-phosphate acyltransferase (GPAM rs10787429) associated with increased levels of ALT (P = 1.4 × 10 -30 ), AST (P = 3.6 × 10 -10 ), ALP (P = 9.5 × 10 -30 ), and total bilirubin (P = 2.9 × 10 -12 ). This common genetic variant was also associated with an allele dose-dependent risk of alcohol-associated liver disease (odd ratio [OR] = 1.34, P = 2.6 × 10 -5 ) and fatty liver disease (OR = 1.18, P = 5.8 × 10 -4 ) by International Classification of Diseases, 10th Revision codes. We identified significant interactions between GPAM rs10787429 and elevated body mass index in association with ALT and AST (P = 7.1 × 10 -9 and 3.95 × 10 -8 , respectively), as well as between GPAM rs10787429 and weekly alcohol consumption in association with ALT, AST, and alcohol-associated liver disease (P = 4.0 × 10 -2 , 1.6 × 10 -2 , and 1.3 × 10 -2 , respectively). Unlike previously described genetic variants that are associated with an increased risk of liver injury but confer a protective effect on circulating lipids, GPAM rs10787429 was associated with an increase in total cholesterol (P = 2.0 × 10 -17 ), LDL cholesterol (P = 2.0 × 10 -10 ), and HDL cholesterol (P = 6.6 × 10 -37 ). Single-cell RNA-sequencing data demonstrated hepatocyte-predominant expression of GPAM in cells that co-express genes related to VLDL production (P = 9.4 × 10 -103 )., Conclusions: Genetic variation in GPAM is associated with susceptibility to liver injury. GPAM may represent a therapeutic target in chronic liver disease., (© 2021 by the American Association for the Study of Liver Diseases.)

Galactic cosmic rays are primarily composed of protons (85%), helium (14%), and high charge/high energy ions (HZEs) such as 56 Fe, 28 Si, and 16 O. HZE exposure is a major risk factor for astronauts during deep-space travel due to the possibility of HZE-induced cancer. A systems biology integrated omics approach encompassing transcriptomics, proteomics, lipidomics, and functional biochemical assays was used to identify microenvironmental changes induced by HZE exposure. C57BL/6 mice were placed into six treatment groups and received the following irradiation treatments: 600 MeV/n 56 Fe (0.2 Gy), 1 GeV/n 16 O (0.2 Gy), 350 MeV/n 28 Si (0.2 Gy), 137 Cs (1.0 Gy) gamma rays, 137 Cs (3.0 Gy) gamma rays, and sham irradiation. Left liver lobes were collected at 30, 60, 120, 270, and 360 days post-irradiation. Analysis of transcriptomic and proteomic data utilizing ingenuity pathway analysis identified multiple pathways involved in mitochondrial function that were altered after HZE irradiation. Lipids also exhibited changes that were linked to mitochondrial function. Molecular assays for mitochondrial Complex I activity showed significant decreases in activity after HZE exposure. HZE-induced mitochondrial dysfunction suggests an increased risk for deep space travel. Microenvironmental and pathway analysis as performed in this research identified possible targets for countermeasures to mitigate risk.

Aims: Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis., Methods and Results: We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages., Conclusions: We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme gene (GBE1) that lead to the accumulation of aberrant glycogen in affected tissues, mostly in the liver. To determine whether dysfunctional glycogen metabolism in GSD IV affects other components of cellular bioenergetics, we studied mitochondrial function in heterozygous Gbe1 knockout (Gbe1 +/- ) mice. Mitochondria isolated from the livers of Gbe1 +/- mice showed elevated respiratory complex I activity and increased reactive oxygen species production, particularly by respiratory chain complex III. These observations indicate that GBE1 deficiency leads to broader rearrangements in energy metabolism and that the mechanisms underlying GSD IV pathogenesis may include more than merely mechanical cell damage caused by the presence of glycogen aggregates., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Manganese porphyrins are well-known protectors against the deleterious effects of pro-oxidant species such as superoxide ions and hydrogen peroxide. The present study investigated the antioxidant cytochrome c-like activities of Mn(III)TMPyP [meso-tetrakis (4-N-methyl pyridinium) porphyrin] against superoxide ion and hydrogen peroxide that remained unexplored for this porphyrin. The association of TMPyP with a model of the inner mitochondrial membrane, cardiolipin (CL)-containing liposomes, shifted +30 mV vs. NHE (normal hydrogen electrode) redox potential of the Mn (II) /Mn (III) redox couple. In CL-containing liposomes, Mn (III) TMPyP was reduced by superoxide ions and recycled by Fe (III) cytochrome c to the oxidized form. Similarly, isolated rat liver mitoplasts added to a sample of Mn (II) TMPyP promoted immediate porphyrin reoxidation by electron transfer to the respiratory chain. These results show that Mn (III) TMPyP can act as an additional pool of Fe (III) cytochrome c capable of transferring electrons that escape from the IV complex back into the respiratory chain. Unlike Fe (II) cytochrome c, Mn (II) TMPyP was not efficient for hydrogen peroxide clearance. Therefore, by reducing cytochrome c, Mn (II) TMPyP can indirectly contribute to hydrogen peroxide elimination., Competing Interests: Declaration of competing interest Authors declare no conflicts of interest and no competing interests., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Long non-coding RNAs (lncRNAs) are crucial in chronic liver diseases, but the specific molecular mechanism of lncRNAs in alcoholic fatty liver (AFL) remains unclear. In this study, we investigated the in-depth regulatory mechanism of mTOR affected by AIRN non-protein coding RNA (lncRNA-AIRN) in the development of AFL. LncRNA-AIRN was highly expressed in the liver tissues of AFL C57BL/6mice and oleic acid+alcohol (O+A)treated AML-12cells by using quantitative real-timePCR. RNA pull-down and RNA immunoprecipitation experiments demonstrated that there was an interaction between lncRNA-AIRN and mTOR, and that interference with lncRNA-AIRN could promote the mTOR protein level. Results ofcycloheximide-chase assay showed that the proteinlevel of mTOR was decreased with the treatment time after the knockdown of lncRNA-AIRN. Furthermore, the knockdown of lncRNA-AIRN reducedmTOR protein level by promoting the E3 ubiquitin ligase FBXW7-mediated ubiquitination.The lncRNA-AIRN/mTORaxis was involved in the regulation of the mitophagy of O+A treated hepatocytes, which was confirmed by the cell transfection and the MTT assay.SPSS 16.0 was used for analyzing data. The difference between the two groups was analyzed by performing Student's t-test, and ANOVA was used to analyze the difference when more than two groups. P values < 0.05 were considered to be significantly different.Our findings demonstrated that the knockdown of lncRNA-AIRN influencedmitophagy in AFL by promoting mTOR ubiquitination.

We have recently demonstrated that aldose reductase (AR) inhibitor; fidarestat prevents doxorubicin (Dox)-induced cardiotoxic side effects and inflammation in vitro and in vivo. However, the effect of fidarestat and its combination with Dox on immune cell activation and the immunomodulatory effects are not known. In this study, we examined the immunomodulatory effects of fidarestat in combination with Dox in vivo and in vitro. We observed that fidarestat decreased Dox-induced upregulation of CD11b in THP-1 monocytes. Fidarestat further attenuated Dox-induced upregulation of IL-6, IL-1β, and Nos2 in murine BMDM. Fidarestat also attenuated Dox-induced activation and infiltration of multiple subsets of inflammatory immune cells identified by expression of markers CD11b + , CD11b + F4/80 + , Ly6C + CCR2 high , and Ly6C + CD11b + in the mouse spleen and liver. Furthermore, significant upregulation of markers of mitochondrial biogenesis PGC-1α, COX IV, TFAM, and phosphorylation of AMPKα1 (Ser485) was observed in THP-1 cells and livers of mice treated with Dox in combination with fidarestat. Our results suggest that fidarestat by up-regulating mitochondrial biogenesis exerts protection against Dox-induced immune and inflammatory responses in vitro and in vivo, providing further evidence for developing fidarestat as a combination agent with anthracycline drugs to prevent chemotherapy-induced inflammation and toxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and formation of APAP-protein adducts, mitochondrial oxidant stress and activation of the mitogen activated protein (MAP) kinase c-jun N-terminal kinase (JNK) are critical for APAP-induced cell death. However, direct evidence linking these mechanistic features are lacking and were investigated by examining the early temporal course of these changes in mice after 300 mg/kg APAP. Protein adducts were detectable in the liver (0.05-0.1 nmol/mg protein) by 15 and 30 min after APAP, which increased (>500 %) selectively in mitochondria by 60 min. Cytosolic JNK activation was only evident at 60 min, and was significantly attenuated by scavenging superoxide specifically in the cytosol by TEMPO treatment. Treatment of mouse hepatocytes with APAP revealed mitochondrial superoxide generation within 15 min, accompanied by hydrogen peroxide production without change in mitochondrial respiratory function. The oxidant stress preceded JNK activation and its mitochondrial translocation. Inhibitor studies identified the putative source of mitochondrial superoxide as complex III, which released superoxide towards the intermembrane space after APAP resulting in activation of JNK in the cytosol. Our studies provide direct evidence of mechanisms involved in mitochondrial superoxide generation after NAPQI-adduct formation and its activation of the MAP kinase cascade in the cytosol, which are critical features of APAP hepatotoxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.

In recent years, a number of advancements have been made in the study of entire mitochondrial proteomes in both physiological and pathological conditions. Naturally occurring iodothyronines (i.e., T3 and T2) greatly influence mitochondrial oxidative capacity, directly or indirectly affecting the structure and function of the respiratory chain components. Blue native PAGE (BN-PAGE) can be used to isolate enzymatically active oxidative phosphorylation (OXPHOS) complexes in one step, allowing the clinical diagnosis of mitochondrial metabolism by monitoring OXPHOS catalytic and/or structural features. Protocols for isolating mammalian liver mitochondria and subsequent one-dimensional (1D) BN-PAGE will be described in relation to the impact of thyroid hormones on mitochondrial bioenergetics.

The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although the migration of supercomplexes has been demonstrated being real, there are still several concerns about its ability to reveal genuine interactions between respiratory complexes. Moreover, the use of different solubilization conditions generates conflicting interpretations. Here, we thoroughly compare the impact of different digitonin concentrations on the liquid dispersions' physical properties and correlate with the respiratory complexes' migration pattern and supercomplexes. Our results demonstrate that digitonin concentration generates liquid dispersions with specific size and variability critical to distinguish between a real association of complexes from being trapped in the same micelle., (Copyright © 2020. Published by Elsevier B.V.)

Mitochondria possess a genome that codes for proteins, in the same fashion as the nuclear genome. However, the small, circular mitochondrial DNA (mtDNA) molecule has a reduced base pair content, for it can only code for 2 rRNA, 22 tRNA molecules, and 13 proteins, all of them part of the mitochondrial respiratory chain. As such, all of the other mitochondrial components derive from nuclear genome. This separation leads to a requirement for a well-tuned coordination between both genomes, in order to produce fully functional mitochondria. A vast number of pathologies have been demonstrated to involve, to some extent, alterations in mitochondrial function that, no doubt, can be caused by alterations to the respiratory chain activity. As such, several methods and techniques have been developed to assess both content and function of mitochondrial proteins, in order to help understand mitochondrial involvement on the pathogenesis of disease. In this chapter, we will address some of these methods, with the main focus being on isolated mitochondria.

Hepatic ischemia/reperfusion (I/R) injury is a major concern in liver surgery settings. Mitochondria are critical targets or the origin of tissue injury, particularly I/R injury. Mitophagy, a selective form of autophagy, is a fundamental process that removes damaged or unwanted mitochondria for mitochondrial quality control, but its role in hepatic I/R remains unclear. In the present study, we investigated the role of mitophagy in hepatic I/R by focusing on PTEN-induced putative kinase 1 (PINK1). Livers from 10-week-old mice and primary hepatocytes were subjected to in vivo hepatic I/R and in vitro hypoxia-reoxygenation (H/R), respectively. Analyses of oxidative stress, immunoblotting, and ATP generation showed that hepatic I/R leads to mitochondrial damage. Dysfunctional mitochondria promoted reactive oxygen species (ROS) production and apoptosis. Hepatic I/R led to decreases in the mitochondrial proteins COX4 and TOM20 and mitochondrial DNA and increases in the autophagy-related indicators LC3 and P62, which indicates that hepatic I/R promotes mitophagy. We found that I/R also leads to endoplasmic reticulum stress, which has frequent signal communication with mitochondria through the mitochondria-associated membranes (MAMs). We showed that the mitophagy-related proteins Parkin, Beclin, optineurin were enhanced in hepatic I/R. No significant change is in PINK1 but it translocated to MAMs region to initiate mitophagy. The silencing PINK1 by shRNA in cultured primary hepatocytes reduced the level of H/R-induced mitophagy, leading to the accumulation of dysfunctional mitochondria during H/R, increased production of ROS, mitochondria-induced apoptosis, and eventually hepatocyte death. Taken together, these findings indicate that PINK1-mediated mitophagy plays a key role in mitochondrial quality control and liver cell survival during I/R.

NAD(P) + transhydrogenase (NNT) is located in the inner mitochondrial membrane and catalyzes a reversible hydride transfer between NAD(H) and NADP(H) that is coupled to proton translocation between the intermembrane space and mitochondrial matrix. NNT activity has an essential role in maintaining the NADPH supply for antioxidant defense and biosynthetic pathways. In the present report, we evaluated the effects of chemical compounds used as inhibitors of NNT over the last five decades, namely, 4-chloro-7-nitrobenzofurazan (NBD-Cl), N,N'-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-l-carnitine, and rhein, on NNT activity and mitochondrial respiratory function. Concentrations of these compounds that partially inhibited the forward and reverse NNT reactions in detergent-solubilized mouse liver mitochondria significantly impaired mitochondrial respiratory function, as estimated by ADP-stimulated and nonphosphorylating respiration. Among the tested compounds, NBD-Cl showed the best relationship between NNT inhibition and low impact on respiratory function. Despite this, NBD-Cl concentrations that partially inhibited NNT activity impaired mitochondrial respiratory function and significantly decreased the viability of cultured Nnt -/- mouse astrocytes. We conclude that even though the tested compounds indeed presented inhibitory effects on NNT activity, at effective concentrations, they cause important undesirable effects on mitochondrial respiratory function and cell viability., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

Hepatic ischemia/reperfusion (I/R) injury is a leading cause of organ dysfunction and failure in numerous pathological and surgical settings. At the core of this issue lies mitochondrial dysfunction. Hence, strategies that prime mitochondria towards damage resilience might prove applicable in a clinical setting. A promising approach has been to induce a mitohormetic response, removing less capable organelles, and replacing them with more competent ones, in preparation for an insult. Recently, a soluble form of adenylyl cyclase (sAC) has been shown to exist within mitochondria, the activation of which improved mitochondrial function. Here, we sought to understand if inhibiting mitochondrial sAC would elicit mitohormesis and protect the liver from I/R injury. Wistar male rats were pretreated with LRE1, a specific sAC inhibitor, prior to the induction of hepatic I/R injury, after which mitochondria were collected and their metabolic function was assessed. We find LRE1 to be an effective inducer of a mitohormetic response based on all parameters tested, a phenomenon that appears to require the activity of the NAD + -dependent sirtuin deacylase (SirT3) and the subsequent deacetylation of mitochondrial proteins. We conclude that LRE1 pretreatment leads to a mitohormetic response that protects mitochondrial function during I/R injury.

Mitochondrial 2-enoyl-acyl-carrier protein reductase (MECR) is an enzyme in the mitochondrial fatty acid synthase (mtFAS) pathway. MECR activity remains unknown in the mechanism of insulin resistance in the pathogenesis of type 2 diabetes. In the present study, MECR activity was investigated in diet-induced obese (DIO) mice. Mecr mRNA was induced by insulin in cell culture, and was elevated in the liver of DIO mice in the presence hyperinsulinemia. However, MECR protein was decreased in the liver of DIO mice, and the reduction was blocked by treatment of the DIO mice with berberine (BBR). The mechanism of MECR protein regulation was investigated with a focus on ATP. The protein was decreased in the cell lysate and DIO liver by an increase in ATP levels. The ATP protein reduction was blocked in the liver of BBR-treated mice by suppression of ATP elevation. The MECR protein reduction was associated with insulin resistance and the protein restoration was associated with improvement of insulin sensitivity by BBR in the DIO mice. The data suggest that MECR protein is regulated in hepatocytes by ATP in association with insulin resistance. The study provides evidence for a relationship between MECR protein and insulin resistance., (© 2020 The Author(s).)

Aims: β-Estradiol (β-E), one of the chemical forms of female gonad hormone exhibited antioxidant efficacy in biochemical system, in vitro. The aim of the study was to investigate whether any other mechanism of protection by β-E to hepatic mitochondria in presence of stressor agent i.e.,a combination of Cu 2+ and ascorbic acid is involved., Main Methods: Freshly prepared goat liver mitochondria was incubated with stressors and 1 μM β-E and post incubated with the same concentration at 37 °C at pH 7.4. Mitochondrial viability, biomarkers of oxidative stress, activities of Krebs cycle enzymes, mitochondrial membrane potential, Ca 2+ permeability were measured. Mitochondrial morphology and binding pattern of β-E with stressors were also studied., Key Findings: Upon incubation of mitochondria with Cu, ascorbic acid and their combination there is a significant decline in activities of four of Krebs cycle enzymes in an uncompetitive manner with a concomitant increase in Ca 2+ permeability and membrane potential of inner mitochondrial membrane, which is withdrawn during co-incubation with β-E, but was not reversed during post incubation with the β-E. The final studies on mitochondrial membrane morphology using scanning electron microscope also exhibited damage. Isothermal titration calorimetry data also showed the negative heat change in the mixture of β-E with ascorbic acid and also its combination with Cu 2+ ., Significance: Our results for the first time demonstrated that β-E protects againstCu 2+ -ascorbate induced oxidative stress by binding with ascorbic acid. The new mechanism of binding of β-E with stress agents may have a future therapeutic relevance., Competing Interests: Declaration of competing interest Authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

l-Glutaminase has gained an important attention as glutamine-depleting enzyme in treatment of various cancers. Therefore, this study aimed to purify, characterize and investigate antitumor activity of l-glutaminase from camel liver mitochondria (CL-Glu), since no available information about CL-Glu from camel. CL-Glu was purified using cell fractionation, ultrafiltration, DEAE-and CM-cellulose chromatography columns. The purified CL-Glu was a monomer with a molecular weight of 70 ± 3 kDa, isoelectric point of 7.2, optimum temperature of 70 °C and it was active over a broad pH range with a pH optimum at pH 8.0. Its activity had a clear dependence on phosphate ions. The studied enzyme showed sigmoidal kinetics, indicated its allosteric behavior with Km of 36 ± 4 mM and Hill coefficient of 1.5 which suggested a positive cooperatively of active sites. The purified l-glutaminase exerted antitumor activity against different cell lines with the highest cytotoxic activity against Hepatocellular carcinoma cell line (HepG-2) with an IC 50 value of 152 µg/ml. In conclusion, l-glutaminase was purified from camel liver using simple methods and its unique properties such as stability at both wide pH range and at high temperature along with its relatively low molecular weight, facilitated its usage in medical applications as antitumor drug., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are central microdomains of the ER that interact with mitochondria. MAMs provide an essential platform for crosstalk between the ER and mitochondria and play a critical role in the local transfer of calcium (Ca 2+ ) to maintain cellular functions. Despite the potential uses of superparamagnetic iron oxide nanoparticles (SPIO-NPs) in biomedical applications, the hepatotoxicity of these nanoparticles (NPs) is not well characterized and little is known about the involvement of MAMs in ER-mitochondria crosstalk. We studied SPIO-NPs-associated hepatotoxicity in vitro and in vivo . In vitro , human normal hepatic L02 cells were exposed to SPIO-NPs (2.5, 7.5, and 12.5 μg/mL) for 6 h and SPIO-NPs (12.5 μg/mL) was found to induce apoptosis. In vivo , SPIO-NPs induced liver injury when mice were intravenously injected with 20 mg/kg body weight SPIO-NPs for 24 h. Based on both in vitro and in vivo studies, we found that the structure and Ca 2+ transport function of MAMs were perturbated and an accumulation of cyclooxygenase-2 (COX-2) in MAMs fractions was increased upon treatment of SPIO-NPs. The interaction between COX-2 and the components of MAMs, in terms of IP3R-GRP75-VDAC1 complex, was also revealed. Furthermore, the role of COX-2 in SPIO-NPs-associated hepatotoxicity was investigated by modifying the expression of COX-2. We demonstrated that COX-2 increases the structural and functional ER-mitochondria coupling and enhances the efficacy of ER-mitochondria Ca 2+ transfer through the MAMs, thus sensitizing hepatocytes to a mitochondrial Ca 2+ overload-dependent apoptosis. Taken together, our findings link SPIO-NPs-triggered hepatotoxicity with ER-mitochondria Ca 2+ crosstalk which is mediated by COX-2 and provide mechanistic insight into the impact of interorganelle ER-mitochondria communication on hepatic nanotoxicity.

Background & Aims: Oxidative stress-related liver diseases were shown to be associated with elevated serum thyroid stimulating hormone (TSH) levels. Mitochondria are the main source of cellular reactive oxygen species. However, the relationship between TSH and hepatic mitochondrial stress/dysfunction and the underlying mechanisms are largely unknown. Here, we focused on exploring the effects and mechanism of TSH on hepatic mitochondrial stress., Methods: As the function of TSH is mediated through the TSH receptor (TSHR), Tshr -/- mice and liver-specific Tshr -/- mice and liver-specific Tshr -/- mice and liver-specific., Results: A relatively lower degree of mitochondrial stress was observed in the livers of Tshr -/- mice and liver-specific in vitro . Microarray and RT-PCR analyses showed that Tshr -/- mice and liver-specific., Conclusions: TSH stimulates hepatic CypD acetylation through the lncRNA-AK044604/SIRT1/SIRT3 signaling pathway, indicating an essential role for TSH in mitochondrial stress in the liver., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Xiaolei Wang et al.)

Background & Aims: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury., Methods: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif -/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis., Results: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 μmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 μmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif -/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle., Conclusions: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Fatty acid oxidation (FAO)-driven H 2 O 2 has been shown to be a major source of oxidative stress in several tissues and disease states. Here, we established that the mitochondrial flavoprotein long-chain acyl-CoA dehydrogenase (LCAD), which catalyzes a key step in mitochondrial FAO, directly produces H 2 O 2 in vitro by leaking electrons to oxygen. Kinetic analysis of recombinant human LCAD showed that it produces H 2 O 2 15-fold faster than the related mitochondrial enzyme very long-chain acyl-CoA dehydrogenase (VLCAD), but 50-fold slower than a bona fide peroxisomal acyl-CoA oxidase. The rate of H 2 O 2 formation by human LCAD is slow compared to its activity as a dehydrogenase (about 1%). However, expression of hLCAD in HepG2 cells is sufficient to significantly increase H 2 O 2 in the presence of fatty acids. Liver mitochondria from LCAD-/- mice, but not VLCAD-/- mice, produce significantly less H 2 O 2 during incubation with fatty acids. Finally, we observe highest LCAD expression in human liver, followed by kidney, lung, and pancreas. Based on our data, we propose that the presence of LCAD drives H 2 O 2 formation in response to fatty acids in these tissues., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC., (Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Pyridine Nucleotide-Disulphide Oxidoreductase Domain 2 (PYROXD2), a Hepatitis B virus X protein (HBx)-interacting protein, is significantly down-regulated in hepatocellular carcinoma (HCC), however its exact biological function remains unclear. The aim of this study is to investigate the subcellular localization and biological function of PYROXD2 in hepatic cells. The results showed that PYROXD2 was imported to the mitochondrial inner membrane/matrix by Tom40 and Tim23, but not Mia40. PYROXD2 151-230aa might be the mitochondrial targeting sequence. PYROXD2 interacted with complex IV subunit COX5B. Knockout of PYROXD2 decreased MMP, intracellular ROS, complex IV activity, cell proliferation, ATP content and mtDNA copy number, but increased mtROS levels and the number of immature mitochondria. In summary, our data illustrated that PYROXD2 localizes to the mitochondrial inner membrane/matrix, and it plays important roles in regulating mitochondrial function., (Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) is an integral component of the respiratory chain, and recent studies have suggested that it plays an important role in hepatic glucose homeostasis. However, its function in hepatic lipid metabolism is unclear. Here, we identified a role for mGPDH in nonalcoholic fatty liver disease (NAFLD). Specifically, mGPDH expression and activity were lower in fatty livers from patients and mice with NAFLD (ob/ob, high-fat diet [HFD] and db/db). Liver-specific depletion of mGPDH in mice or mGPDH knockdown in cultured hepatocytes exacerbated diet-induced triglyceride accumulation and steatosis through enhanced lipogenesis. RNA-sequencing revealed that mGPDH regulated endoplasmic reticulum (ER)-related proteins and processes. mGPDH deletion exacerbated tunicamycin (ER stress inducer)-induced hepatic steatosis, whereas tauroursodeoxycholic acid (ER stress inhibitor) rescued mGPDH depletion-induced steatosis on an HFD. Moreover, ER stress induced by mGPDH depletion could be abrogated by the intracellular Ca 2+ chelator 1,2-bis (2-aminophenoxy) ethane N,N,N´,N´-tetraacetic acid acetoxymethyl ester, mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A, or cyclophilin-D (Cyp-D) knockdown. mGPDH promoting Cyp-D ubiquitination was also observed. Finally, liver-specific mGPDH overexpression attenuated hepatic steatosis in ob/ob and HFD mice. Conclusion: mGPDH is a pivotal regulator of hepatic lipid metabolism. Its deficiency induces ER stress by suppressing Cyp-D ubiquitination, a key regulator of the mitochondrial Ca 2+ conductance channel mPTP, and results in hepatic steatosis. mGPDH may be a potential therapeutic target for the treatment of NAFLD., (© 2019 by the American Association for the Study of Liver Diseases.)

CoA regulates energy metabolism and exists in separate pools in the cytosol, peroxisomes, and mitochondria. At the whole tissue level, the concentration of CoA changes with the nutritional state by balancing synthesis and degradation; however, it is currently unclear how individual subcellular CoA pools are regulated. Liver and kidney peroxisomes contain Nudt7 and Nudt19, respectively, enzymes that catalyze CoA degradation. We report that Nudt8 is a novel CoA-degrading enzyme that resides in the mitochondria. Nudt8 has a distinctive preference for manganese ions and exhibits a broader tissue distribution than Nudt7 and Nudt19. The existence of CoA-degrading enzymes in both peroxisomes and mitochondria suggests that degradation may be a key regulatory mechanism for modulating the intracellular CoA pools., (© 2019 Federation of European Biochemical Societies.)

Berberine (BBR) shows promising effects in the treatment of nonalcoholic fatty liver disease (NAFLD) by influencing various metabolic aspects. Inhibition of mitochondrial β-oxidation (β-OX) participates in the pathogenesis of NAFLD. Silent mating-type information regulation 2 homolog 3 (SIRT3) has been reported to regulate mitochondrial β-OX by deacetylating its substrate, long-chain acyl-coenzyme A dehydrogenase (LCAD). This study aimed to explore whether BBR can promote mitochondrial β-OX and the role of SIRT3 as well as the mechanisms underlying the effects of BBR on hepatic lipid metabolism in mice fed a high-fat diet (HFD). BBR can significantly improve systematic and hepatic lipid metabolism in HFD-fed mice. Metabolomics analysis revealed that β-OX was inhibited in HFD-induced mice, as indicated by the reduced production of short and medium carbon chain acyl-carnitines, the activated form of free fatty acids, via β-OX, which was reversed by BBR intervention. Exploration of the mechanism found that BBR intervention reversed the down-regulation of SIRT3 and decreased the LCAD hyperacetylation level in HFD-fed mice. SIRT3 knockout (KO) mice were used to identify the role of SIRT3 in the BBR's influence of β-OX. The beneficial effects of BBR on systemic and hepatic metabolism were profoundly attenuated in KO mice. Moreover, the promotive effect of BBR on β-OX in HFD-induced mice was partially abolished in KO mice. These results suggested that BBR alleviates HFD-induced inhibition of fatty acid β-OX partly through SIRT3-mediated LCAD deacetylation, which may provide a novel mechanism and support BBR as a promising therapeutic for NAFLD.-Xu, X., Zhu, X.-P., Bai, J.-Y., Xia, P., Li, Y., Lu, Y., Li, X.-Y., Gao, X. Berberine alleviates nonalcoholic fatty liver induced by a high-fat diet in mice by activating SIRT3.

Aerobic glycolysis and mitochondrial dysfunction are key metabolic features of cancer cells, but their interplay during cancer development remains unclear. We previously reported that human hepatoma cells with mitochondrial defects exhibit down-regulated lactate dehydrogenase subunit B (LDHB) expression. Here, using several molecular and biochemical assays and informatics analyses, we investigated how LDHB suppression regulates mitochondrial respiratory activity and contributes to liver cancer progression. We found that transcriptional LDHB down-regulation is an upstream event during suppressed oxidative phosphorylation. We also observed that LDHB knockdown increases inhibitory phosphorylation of pyruvate dehydrogenase (PDH) via lactate-mediated PDH kinase (PDK) activation and thereby attenuates oxidative phosphorylation activity. Interestingly, monocarboxylate transporter 1 was the major lactate transporter in hepatoma cells, and its expression was essential for PDH phosphorylation by modulating intracellular lactate levels. Finally, bioinformatics analysis of the hepatocellular carcinoma cohort from The Cancer Genome Atlas revealed that a low LDHB/LDHA ratio is statistically significantly associated with poor prognostic outcomes. A low ratio was also associated with a significant enrichment in glycolysis genes and negatively correlated with PDK1 and 2 expression, supporting a close link between LDHB suppression and the PDK-PDH axis. These results suggest that LDHB suppression is a key mechanism that enhances glycolysis and is critically involved in the maintenance and propagation of mitochondrial dysfunction via lactate release in liver cancer progression., (© 2019 Hong et al.)

Rhizoma Paridis, a traditional Chinese medicine, has shown promise in cancer prevention and therapy. Polyphyllin II is one of the most significant saponins in Rhizoma Paridis and it has toxic effects on kinds of cancer cells. However, our results in this study proved that the polyphyllin II has hepatotoxicity in vitro through caspases activation and cell-cycle arrest. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results indicated polyphyllin II inhibited proliferation, induced apoptosis in HepaRG cells and HL-7702 cells and showed a concentration and time-dependent. Then, we selected the innovative cell model-HepaRG cells to explore the mechanism of hepatotoxicity. Our data showed the reactive oxygen species (ROS) increased and the mitochondrial membrane potential decreased in HepaRG cells after administration of polyphyllin II. Besides, with the increase of concentration, the release of lactate dehydrogenase increased and the S phase of the cell cycle was arrested. Nevertheless, when pretreatment with antioxidant N-acetylcysteine, apoptotic cells decreased significantly, inhibited the production of ROS and improved the decrease of membrane potential in HepaRG cells. Moreover, polyphyllin II treatment increased levels of Fas, Bax, cytochrome c, activated caspase-3, -8, -9, cleaved poly(ADP-ribose) polymerase and decreased Bcl-2 expression levels. Finally, we identified two signal pathways of apoptosis induced by polyphyllin II including the death receptor pathway and the mitochondria pathway. This study confirmed the hepatotoxicity of the polyphyllin II in vitro, which has never been discovered and gave a wake-up call for the clinical application of Rhizoma Paridis., (© 2018 Wiley Periodicals, Inc.)

1,2-Dichloropropane (1,2-DCP) is used as an industrial solvent, insecticide fumigant and household dry cleaning product. Carcinogenicity caused by long-term exposure to 1,2-DCP is well established. However, the possible liver damage and related toxic mechanisms associated with acute inhalation exposure to 1,2-DCP are rarely reported. In this study, we investigated the effects of individual and combined exposure to 1,2-DCP and dichloromethane (DCM) on mice liver. The results showed that 1,2-DCP significantly caused liver necrosis, possibly due to 1,2-DCP-induced inhibition of the mitochondrial respiratory chain complex I-IV activities, resulting in mitochondrial dysfunction and extreme ATP consumption. Moreover, 1,2-DCP also decreased mitochondrial defense ability by inhibiting the mitochondrial glutathione S-transferase 1 (MGST1) activity, further aggravating liver damage. Additionally, we found that DCM co-exposure potentially enhanced 1,2-DCP toxicity. Our findings suggest that inhibition of mitochondrial function and MGST1 activity play critical roles in modulating 1,2-DCP-induced liver damage. Furthermore, our results contribute to study the new mechanism of mitochondria-dominated signaling pathways underlying liver injury induced by 1,2-DCP and DCM., (© 2018 John Wiley & Sons, Ltd.)

Glucokinase (GCK) is the principal hexokinase (HK) in the liver, operating as a glucose sensor to regulate glucose metabolism and lipid homeostasis. Recently, we proposed HK domain-containing 1 (HKDC1) to be a fifth HK with expression in the liver. Here, we reveal HKDC1 to have low glucose-phosphorylating ability and demonstrate its association with the mitochondria in hepatocytes. As we have shown previously that genetic deletion of HKDC1 leads to altered hepatic triglyceride levels, we also explored the influence of overexpression of HKDC1 in hepatocytes on cellular metabolism, observing reduced glycolytic capacity and maximal mitochondrial respiration with concurrent reductions in glucose oxidation and mitochondrial membrane potential. Furthermore, we found that acute in vivo overexpression of HKDC1 in the liver induced substantial changes in mitochondrial dynamics. Altogether, these findings suggest that overexpression of HKDC1 causes mitochondrial dysfunction in hepatocytes. However, its overexpression was not enough to alter energy storage in the liver but led to mild improvement in glucose tolerance. We next investigated the conditions necessary to induce HKDC1 expression, observing HKDC1 expression to be elevated in human patients whose livers were at more advanced stages of nonalcoholic fatty liver disease (NAFLD) and similarly, found high liver expression in mice on diets causing high levels of liver inflammation and fibrosis. Overall, our data suggest that HKDC1 expression in hepatocytes results in defective mitochondrial function and altered hepatocellular metabolism and speculate that its expression in the liver may play a role in the development of NAFLD.

Today, nanoparticles (NPs) have been widely used in various fields. Manganese oxide nanoparticles have attracted a lot of attention due to many applications. One of the major concerns regarding the widespread use of various NPs is the exposure and accumulation in human organs and finally toxicity. The generation of reactive oxygen species (ROS) by mitochondria is one of the most important mechanisms of toxicity suggested by published studies induced by other NPs. However, limited studies have been conducted on the mechanism of toxicity of MnO 2 -NPs and MnO 2 -microparticles (MnO 2 -MPs). In this study, we compared the accumulation of MnO 2 -NPs and MnO 2 -MPs in different tissues and evaluated their effects on mitochondrial complexes in isolated mitochondria. Our results showed that intravascular (iv) administration of the MnO 2 -NPs in the same dose compared to the MnO 2 -MPs resulted in more accumulation in the C57 mouse female tissues. The effect of MnO 2 -NPs and MnO 2 -MPs in mitochondria showed that complexes I and III play an important role in increasing ROS generation and this effect is related to type of tissue. Also, our results showed that exposure to MnO 2 -NPs and MnO 2 -MPs reduced the activity of mitochondrial complexes II and IV. Our results suggest that the toxicity of the MnO 2 -NPs is higher than that of the MnO 2 -MPs and can lead to the depletion of antioxidant status, likely induction of apoptosis, cancer, and neurodegenerative disease. Abbreviations: NPs: nanoparticles; ROS: reactive oxygen species; SDH: succinate dehydrogenase; DCFH-DA: dichloro-dihydro-fluorescein diacetate; ELISA: enzyme-linked immunosorbent assay; MnO2-NPs: manganese oxide nanoparticles.

This study aimed to investigate the effects of dietary non-protein energy adjustments on the mitochondrial biosynthesis and function of juvenile Megalobrama amblycephala. Fish (average weight: 37.98 ± 0.07 g) were fed eight diets containing two dietary carbohydrate levels (30% and 43%) and four lipid sources (fish oil, soybean oil, palm oil and the mixed oil) for 11 weeks. Liver mitochondrial respiratory chain complex V activity and ATP (adenosine triphosphate) content both increased significantly with increasing dietary carbohydrate levels, whereas the opposite was true for the AMP (adenosine 5'-monophosphate)/ATP ratio, hepatic transcripts of AMP-activated protein kinase α1 (AMPKα1), AMPKα2, peroxisome proliferators γ-activated receptor coativator-1α (PGC-1α), NADH dehydrogenase 1 and cytochrome c oxidase 1 (COX1) as well as the activities of Na + -K + -ATPase, succinate dehydrogenase (SDH), citrate synthase (CS) and mitochondrial respiratory chain complex I, III and IV. Additionally, hepatic ATP content, the transcripts of AMPKα, COX1 and ATP6 and the activities of Na + -K + -ATPase, SDH, CS and mitochondrial respiratory chain complex III were all significantly affected by lipid sources. Furthermore, an interaction between dietary carbohydrate levels and lipid sources was also observed in the activities of liver mitochondrial Na + -K + -ATPase and respiratory chain complex III as well as the transcripts of ATP6 and PGC-1α. Overall, these findings suggested that dietary carbohydrate levels and lipid sources remarkably affected the mitochondrial biosynthesis and function of M. amblycephala. A diet containing 30% carbohydrate and FO could boost its mitochondrial biosynthesis, while that of 30% carbohydrate and SO could enhance the mitochondrial function., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Visceral lipid accumulation, organ hypertrophy and a reduction in skeletal muscle strength are all signs associated with the severity of obesity-related disease. Intermittent fasting (IF) and high-intensity intermittent exercise (HIIE) are natural strategies that, individually, can prevent and help treat obesity along with metabolic syndrome and its associated diseases. However, the combinatorial effect of IF and HIIE on energetic metabolism is currently not well understood. We hypothesized that their combination could have a potential for more than strictly additive benefits. Here, we show that two months of every-other-day intermittent fasting regimen combined with a high-intensity intermittent exercise protocol (IF/HIIE) produced a synergistic effect, enhancing physical endurance (vs. control, HIIE and IF) and optimizing metabolic pathways of energy production in male Wistar rats. The IF/HIIE group presented enhanced glucose tolerance (vs. control, HIIE and IF), lower levels of plasma insulin (vs. control and HIIE), and a global activation of low Km hexokinases in liver (vs. control, HIIE and IF), heart (vs. control and HIIE) and skeletal muscle (vs. control, HIIE and IF). The IF/HIIE synergism, rather than a simply additive effect, is evidenced by increase in muscle mass and cross-section area, activation of the FoF1 ATP synthase, and the gain of characteristics suggestive of augmented mitochondrial mass and efficiency observed in this group. Finally, important reductions in plasma oxidative stress markers were present preferentially in IF/HIIE group. These findings provide new insights for the implementation of non-pharmaceutical strategies to prevent/treat metabolic syndrome and associated diseases., Competing Interests: The authors have declared that no competing interests exist.

We investigated the standard metabolic rate and liver mitochondria metabolism of the southern catfish when exposed to waterborne cadmium. Juvenile southern catfish were exposed to waterborne cadmium concentrations (0, 62.5, 125, 250 and 500 μg/L, respectively) for 8 weeks, and the final body mass, the standard metabolic rate, the state III respiration rate, the activity of cytochrome C oxidase (CCO) of liver mitochondria, and the hepatosomatic index (HSI) were determined. The results showed that the 62.5 μg/L, 125 μg/L, and 250 μg/L experiment groups had a significantly higher standard metabolic rate than that of the control group. Standard metabolic rate in the 500 μg/L experiment group did not differ from the control group. State III respiration rate of liver mitochondria decreased with an increase in cadmium concentration. The 125 μg/L, 250 μg/L, and 500 μg/L experiment groups had a significantly lower state III respiration rate than that of the control group. The activity of CCO in the 500 μg/L experiment group was significantly lower than that of the control group. These results suggest that at low cadmium concentrations, the southern catfish could continuously improve the standard metabolism to provide extra energy in response to the cadmium stress. Cadmium exposures caused damage to the structure and function of liver mitochondria and decreased the activity of mitochondria enzymes, which results in a decrease in the energy of the liver metabolism. The adjustment of the metabolism of liver mitochondria in southern catfish was inconsistent with the adjustment of individual standard metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Patients with obesity have a high prevalence of non-alcoholic fatty liver disease (NAFLD) and, in parallel, increased susceptibility to fibrosis/cirrhosis/hepatocellular carcinoma (HCC). Herein, we report that a high-fat diet (HFD) can augment glycolysis and then accelerate NAFLD-fibrosis progression by downregulating the expression of geranylgeranyl diphosphate synthase (GGPPS), which is a critical enzyme in the mevalonate pathway. Long-term HFD overloading decreases GGPPS expression in mice, which shifts the fuel preference from fatty acids towards glucose. Liver-specific Ggpps deficiency drives the Warburg effect by impairing mitochondrial function, and then induces hepatic inflammation, thus exacerbating fibrosis. Ggpps deficiency also enhances the hyperfarnesylation of liver kinase B1, and promotes metabolic reprogramming by regulating 5'-AMP-activated protein kinase activity. Clinical data further imply that GGPPS expression can predict the stage of NAFLD and recurrence of NAFLD-associated HCC. We conclude that the level of GGPPS is a susceptibility factor for NAFLD-fibrosis progression, and requires more stringent surveillance to ensure early prediction and precision of treatment of NAFLD-related HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Benzoate (found in milk and widely used as preservative), salicylate (present in fruits and the active component of aspirin), dietary polyphenols produced by gut microbiota, metabolites from organic acidemias, and medium-chain fatty acids (MCFAs) are all metabolised/detoxified by the glycine conjugation pathway. Xenobiotics are first activated to an acyl-CoA by the mitochondrial xenobiotic/medium-chain fatty acid: CoA ligases (ACSMs) and subsequently conjugated to glycine by glycine N-acyltransferase (GLYAT). The MCFAs are activated to acyl-CoA by the ACSMs before entering mitochondrial β-oxidation. This two-step enzymatic pathway has, however, not been thoroughly investigated and the biggest gap in the literature remains the fact that studies continuously characterise the pathway as a one-step reaction. There are no studies available on the interaction/competition of the various substrates involved in the pathway, whilst very little research has been done on the ACSM ligases. To identify variants/haplotypes that should be characterised in future detoxification association studies, this study assessed the naturally observed sequence diversity and protein expression variation of ACSM2A and ACSM2B. The allelic variation, haplotype diversity, Tajima's D values, and phylogenetic analyses indicated that ACSM2A and ACSM2B are highly conserved. This confirmed an earlier hypothesis that the glycine conjugation pathway is highly conserved and essential for life as it maintains the CoA and glycine homeostasis in the liver mitochondria. The protein expression analyses showed that ACSM2A is the predominant transcript in liver. Future studies should investigate the effect of the variants identified in this study on the substrate specificity of these proteins.

Provision of NAD + for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD + , provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1 -/- and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD + to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss, in part through potentiation of fibroblast growth factor 21 (FGF21) secretion. However, FGF21 is only a partial mediator of metabolic actions ensuing from glucagon receptor (GCGR) activation, prompting us to search for additional pathways. Intriguingly, chronic GCGR agonism increases plasma bile acid levels. We hypothesized that GCGR agonism regulates energy metabolism, at least in part, through farnesoid X receptor (FXR). To test this hypothesis, we studied whole-body and liver-specific FXR-knockout ( Fxr ∆liver ) mice. Chronic GCGR agonist (IUB288) administration in diet-induced obese (DIO) Gcgr , Fgf21, and Fxr whole-body or liver-specific knockout ( ∆liver ) mice failed to reduce body weight when compared with wild-type (WT) mice. IUB288 increased energy expenditure and respiration in DIO WT mice, but not Fxr ∆liver mice. GCGR agonism increased [ 14 C]palmitate oxidation in hepatocytes isolated from WT mice in a dose-dependent manner, an effect blunted in hepatocytes from Fxr ∆liver mice. Our data clearly demonstrate that control of whole-body energy expenditure by GCGR agonism requires intact FXR signaling in the liver. This heretofore-unappreciated aspect of glucagon biology has implications for the use of GCGR agonism in the therapy of metabolic disorders., (© 2018 by the American Diabetes Association.)

Polyethyleneimines (PEIs) are used for transfection of cells with nucleic acids. Meanwhile, the interaction of PEI with mitochondria causes cytochrome c release prior to apoptosis; the mechanisms how PEI causes this permeabilization of mitochondrial membranes and the release of cytochrome c remain unclear. To clarify these mechanisms, we examined the effects of branched-type PEI and linear-type PEI, each of which was 25 kDa in size, on mitochondria. The permeabilization potency of mitochondrial membranes by branched PEI was stronger than that by linear PEI. The permeabilization by PEIs were insensitive to permeability-transition inhibitors, indicating that PEI-induced permeabilization was not attributed to permeability transition. Meanwhile, PEIs caused permeabilization of artificial lipid vesicles; again, the permeabilization potency of branched PEI was stronger than that of linear PEI. Such a difference in this potency was close to that in the case of isolated mitochondria, signifying that the PEI-induced permeabilization of mitochondrial membranes could be attributed to PEI's interaction with the phospholipid phase. Furthermore, this PEI-induced permeabilization of the lipid vesicles was observed only in the case of lipid vesicles including negatively charged phospholipids. These results indicate that PEIs interacted with negatively charged phospholipids in the mitochondrial membranes to directly lead to their permeabilization., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Overdose of acetaminophen (APAP) is a common cause of acute liver failure. Oxidative stress and mitochondrial dysfunction are related to APAP-induced hepatotoxicity. This study investigated the protective role of betaine as a methyl donor and S-adenosylmethionine precursor against APAP hepatotoxicity in isolated mice liver mitochondria. We treated male NMRC mice with 125, 250, 500, and 1000 mg/kg betaine for 5 days followed by 300 mg/kg APAP 1 h later. At 24 h after APAP administration, animals were sacrificed and liver mitochondria were isolated by different centrifugation methods. Biochemical, histological and immunohistochemical analyses were then conducted. Pretreatment of mice with 250, 500, and 1000 mg/kg betaine ameliorated hepatotoxicity (serum ALT and AST activity and histopathology finding); reversed mitochondrial complex II activity, glutathione level, superoxide dismutase, glutathione peroxidase and catalase activity, glutamate cysteine ligase catalytic protein expression, and mitochondrial membrane potential; and suppressed mitochondrial lipid peroxidation and reactive oxygen species. A contradictory effect was observed in mice treated with APAP. Betaine (500 mg/kg) was chosen as the most effective dose to rescue APAP hepatotoxicity in mice. These findings confirmed that betaine plays a protective role against APAP hepatotoxicity via protecting mitochondrial complex II and regenerating mitochondrial GSH levels by increasing GCLC expression. Betaine displayed antioxidant actions different from other antioxidants via modifying cysteine supply in the transsulfuration pathway in the liver. These findings suggested the potential of betaine as an alternative agent for the treatment of hepatotoxicity induced by APAP., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis , has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae., Competing Interests: The authors declare no conflict of interest.

Hydroxysteroid (17β) dehydrogenases (HSD17Bs) form an enzyme family characterized by their ability to catalyze reactions in steroid and lipid metabolism. In the present study, we characterized the phenotype of HSD17B13-knockout (HSD17B13KO) mice deficient in Hsd17b13. In these studies, hepatic steatosis was detected in HSD17B13KO male mice, indicated by histologic analysis and by the increased triglyceride concentration in the liver, whereas reproductive performance and serum steroid concentrations were normal in HSD17B13KO mice. In line with these changes, the expression of key proteins in fatty acid synthesis, such as FAS, acetyl-CoA carboxylase 1, and SCD1, was increased in the HSD17B13KO liver. Furthermore, the knockout liver showed an increase in 2 acylcarnitines, suggesting impaired mitochondrial β-oxidation in the presence of unaltered malonyl CoA and AMPK expression. The glucose tolerance did not differ between wild-type and HSD17B13KO mice in the presence of lower levels of glucose 6-phosphatase in HSD17B13KO liver compared with wild-type liver. Furthermore, microgranulomas and increased portal inflammation together with up-regulation of immune response genes were observed in HSD17B13KO mice. Our data indicate that disruption of Hsd17b13 impairs hepatic-lipid metabolism in mice, resulting in liver steatosis and inflammation, but the enzyme does not play a major role in the regulation of reproductive functions.-Adam, M., Heikelä, H., Sobolewski, C., Portius, D., Mäki-Jouppila, J., Mehmood, A., Adhikari, P., Esposito, I., Elo, L. L., Zhang, F.-P., Ruohonen, S. T., Strauss, L., Foti, M., Poutanen, M. Hydroxysteroid (17β) dehydrogenase 13 deficiency triggers hepatic steatosis and inflammation in mice.

We performed a multiple 'omics study by integrating data on epigenomic, transcriptomic, and proteomic perturbations associated with mitochondrial dysfunction in primary human hepatocytes caused by the liver toxicant valproic acid (VPA), to deeper understand downstream events following epigenetic alterations in the mitochondrial genome. Furthermore, we investigated persistence of cross-omics changes after terminating drug treatment. Upon transient methylation changes of mitochondrial genes during VPA-treatment, increasing complexities of gene-interaction networks across time were demonstrated, which normalized during washout. Furthermore, co-expression between genes and their corresponding proteins increased across time. Additionally, in relation to persistently decreased ATP production, we observed decreased expression of mitochondrial complex I and III-V genes. Persistent transcripts and proteins were related to citric acid cycle and β-oxidation. In particular, we identified a potential novel mitochondrial-nuclear signaling axis, MT-CO2-FN1-MYC-CPT1. In summary, this cross-omics study revealed dynamic responses of the mitochondrial epigenome to an impulse toxicant challenge resulting in persistent mitochondrial dysfunctioning. Moreover, this approach allowed for discriminating between the toxic effect of VPA and adaptation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: Heme oxygenase-1 (HO-1), an endogenous cytoprotective enzyme, is reported that can be localized in mitochondria under stress, contributing to preserve mitochondrial function. Mitochondrial quality control (QC) is essential to cellular health and recovery linked with redox homeostasis. Recent studies reported that phosphoglycerate mutase family member (PGAM) 5, a mitochondria-resident phosphatase, plays critical role in mitochondrial homeostasis. Therefore, we aim to investigate cytoprotective mechanisms of HO-1 in I/R-induced hepatic injury focusing on mitochondrial QC associated with PGAM5 signaling., Main Methods: Mice were subjected to 60 min of hepatic ischemia followed by 6 h reperfusion and were pretreated twice with hemin (HO-1 inducer, 30 mg/kg) or zinc protoporphyrin (ZnPP; HO-1 inhibitor, 10 mg/kg) 16 and 3 h before ischemia., Key Findings: I/R increased hepatic and mitochondrial HO activity, which was augmented by hemin. I/R-induced hepatocellular and mitochondrial damages were attenuated by hemin and augmented by ZnPP. Meanwhile, I/R increased mitochondrial biogenesis, as evidenced by increased mitochondrial DNA contents and mitochondrial transcription factor A protein expression. Hemin augmented these results. I/R impaired mitophagy, as indicated by decreases in Parkin protein expression and the number of mitophagic vacuoles. These changes were attenuated by hemin. Hemin attenuated the I/R-induced increase in mitochondrial fission-related protein, dynamin-related protein 1, and the decrease in PGAM5 protein expression. Furthermore, PGAM5 siRNA abolished the effect of HO-1 on mitochondrial QC in HepG2 cells subjected to hypoxia/reoxygenation., Significance: Our findings suggest that HO-1 protects against I/R-induced hepatic injury via regulation of mitochondrial QC by PGAM5 signaling., (Copyright © 2018 Elsevier Inc. All rights reserved.)