Role of mitochondrial dysfunction and PINK1/Parkin-mediated mitophagy in Cd-induced hepatic lipid accumulation in chicken embryos.

The present study was performed to investigate the effects of Cd exposure on lipid metabolism and mitochondrial dysfunction and to explore the role of mitophagy in Cd-induced dysregulation of lipid metabolism in chicken embryo liver tissues and hepatocytes. To this end, seven-day-old chicken embryos were exposed to different concentrations of Cd for 7 days, and primary chicken embryo hepatocytes were treated with Cd at four different concentrations for 6 h. Furthermore, the mitophagy inhibitor cyclosporine A (CsA) was used to investigate the role of mitophagy in Cd-induced disruption of lipid metabolism. Lipid accumulation, the expression levels of genes involved in lipid metabolism, mitochondrial dysfunction, and mitophagy were measured. The results demonstrated that Cd exposure increases hepatic triglyceride (TG) accumulation and the expression levels of lipogenic genes while decreasing those of lipolytic genes. Furthermore, Cd exposure was observed to alter mitochondrial morphology in terms of reduced size, excessive mitochondrial damage, and the formation of mitophagosomes. The co-localization of lysosome-associated membrane glycoprotein 2 and LC3 puncta was significantly increased in primary chicken embryo hepatocytes after Cd exposure. Moreover, Cd exposure increased LC3, PINK1, and Parkin protein expression levels. CsA effectively alleviated Cd-induced mitochondrial dysfunction, blocked mitochondrial membrane potential collapse, and suppressed PINK1/Parkin-mediated mitophagy. Furthermore, CsA treatment reversed the Cd-induced TG accumulation in liver tissues but further increased it in hepatocytes. Taken together, our findings demonstrate (for the first time) the importance of mitochondrial dysfunction and mitophagy via the PINK1/Parkin pathway in Cd-induced disruption of lipid metabolism.

Elucidation of SIRT-1/PGC-1α-associated mitochondrial dysfunction and autophagy in nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) can lead to chronic liver diseases associated with mitochondrial damages. However, the exact mechanisms involved in the etiology of the disease are not clear. METHODS: To gain new insights, the changes affecting sirtuin 1 (SIRT-1) during liver fat accumulation was investigated in a NAFLD mouse model. In addition, the in vitro research investigated the regulation operated by SIRT-1 on mitochondrial structures, biogenesis, functions, and autophagy. RESULTS: In mice NAFLD, high-fat-diet (HFD) increased body weight gain, upregulated serum total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, blood glucose, insulin levels, and liver malondialdehyde, and decreased liver superoxide dismutase activity. In liver, the levels of SIRT-1 and peroxisome proliferator-activated receptor-gamma coactivator -1α (PGC-1α) decreased. The expression of peroxisome proliferator-activated receptor-α and Beclin-1 proteins was also reduced, while p62/SQSTM1 expression increased. These results demonstrated SIRT-1 impairment in mouse NAFLD. In a well-established NAFLD cell model, exposure of the HepG2 hepatocyte cell line to oleic acid (OA) for 48 h caused viability reduction, apoptosis, lipid accumulation, and reactive oxygen species production. Disturbance of SIRT-1 expression affected mitochondria. Pre-treatment with Tenovin-6, a SIRT-1 inhibitor, aggravated the effect of OA on hepG2, while this effect was reversed by CAY10602, a SIRT-1 activator. Further investigation demonstrated that SIRT-1 activity was involved in mitochondrial biogenesis through PGC-1α and participated to the balance of autophagy regulatory proteins. CONCLUSION: In conclusion, in high-fat conditions, SIRT-1 regulates multiple cellular properties by influencing on mitochondrial physiology and lipid autophagy via the PGC-1α pathway. The SIRT-1/PGC-1α pathway could be targeted to develop new NAFLD therapeutic strategies.

Hepatic mitochondria in diet-induced obese mice (stereology and molecular analysis) / Mitocondrias hepáticas en ratones obesos inducidos por la dieta (estereología y análisis molecular)

SUMMARY: The world population is going through an obesity epidemic that has severe consequences for the health system. This study focused on studying hepatic mitochondria in obese animals induced by a high-fat (HF) diet and used the model-based stereology in electron micrographs for the quantitative study. Besides, the gene expressions of molecular markers of mitochondrial biogenesis carnitine palmitoyltransferase 1a (Cpt 1α), mitochondrial transcription factor a (Tfam), uncoupling protein 3 (Ucp 3), and nuclear respiratory factor 1 (Nrf 1) were analyzed. The HF diet caused a weight gain of +1820 % comparing the control group (C) with the HF group (from 0.32±0.31 g to 5.5±0.39 g, P<0.001). The HF group showed fat droplets in the hepatocyte cytoplasm (steatosis) and less dense and large mitochondria in transmission electron microscopy. The mitochondria size (cross-section) did not show a significant difference between the groups C and HF. However, the mitochondria numerical density per area was 30 % less, the mitochondrial surface density (outer membrane) was 20 % less, and the mitochondrial volume density was 22 % less in the HF group than the C group. The gene expressions of molecular markers of mitochondrial biogenesis Cpt 1α, Tfam, Ucp 3, and Nrf 1 decreased in the HF group compared to the C group. The quantitative results match perfectly with the molecular ones of mitochondrial biogenesis markers. In the future, it will be crucial to verify if and how these data recover with the reduction of obesity, which would be of significant interest given the current obesity epidemic that affects the world population.

RESUMEN: La población mundial atraviesa una epidemia de obesidad que tiene graves consecuencias para el sistema de salud. Este estudio se centró en el análisis de las mitocondrias hepáticas en animales obesos inducidos por una dieta alta en grasas (HF) y utilizó la estereología basada en modelos en micrografías electrónicas para el estudio cuantitativo. Además, se analizaron las expresiones génicas de los marcadores moleculares de la biogénesis mitocondrial carnitina palmitoiltransferasa 1a (Cpt 1α), factor de transcripción mitocondrial a (Tfam), proteína desacoplante 3 (Ucp 3) y factor respiratorio nuclear 1 (Nrf 1). La dieta HF provocó un aumento de peso de +1820 % comparando el grupo de control (C) con el grupo HF (de 0,32 ± 0,31 g a 5,5 ± 0,39 g, P <0,001). El grupo HF mostró gotas de grasa en el citoplasma de los hepatocitos (esteatosis) y mitocondrias menos densas y grandes en la microscopía electrónica de transmisión. El tamaño de las mitocondrias (sección transversal) no mostró una diferencia significativa entre los grupos C y HF. Sin embargo, la densidad numérica de mitocondrias por área fue 30% menor, la densidad de superficie mitocondrial (membrana externa) fue 20 % menor y la densidad de volumen mitocondrial fue 22 % menor en el grupo HF que en el grupo C. Las expresiones génicas de los marcadores moleculares de la biogénesis mitocondrial Cpt 1α, Tfam, Ucp 3 y Nrf 1 disminuyeron en el grupo HF en comparación con el grupo C. Los resultados cuantitativos coinciden perfectamente con los moleculares de los marcadores de biogénesis mitocondrial. En el futuro, será crucial verificar si estos datos se recuperan y cómo se recuperan con la reducción de la obesidad, lo que sería de gran interés dada la actual epidemia de obesidad que afecta a la población mundial.

Plant sterol ester of α-linolenic acid ameliorates high-fat diet-induced nonalcoholic fatty liver disease in mice: association with regulating mitochondrial dysfunction and oxidative stress via activating AMPK signaling.

The present study was designed to explore the beneficial mitochondrial effects and anti-oxidative activities of plant sterol ester of α-linolenic acid (PS-ALA) through AMP-activated protein kinase (AMPK) signaling in the treatment of nonalcoholic fatty liver disease (NAFLD) using in vivo and in vitro models. The mitochondrial function was evaluated and the oxidative stress index was measured. The protein expression was analyzed by immunohistochemical, immunofluorescence, and western blotting methods. The results showed that PS-ALA significantly suppressed NAFLD and alleviated steatosis in HepG2 cells induced by oleic acid (OA). In addition, PS-ALA promoted mitochondrial biogenesis, enhanced mitochondrial fatty acid oxidation capacity, improved mitochondrial dynamics, and restored mitochondrial membrane potential. Moreover, PS-ALA reduced reactive oxygen species production both in the liver tissue of HFD-fed mice and OA-loaded HepG2 cells. At the molecular level, PS-ALA accelerated the phosphorylation of AMPK and increased the protein expression of peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and nuclear NF-E2-related factor 2 (Nrf2). Furthermore, the stimulating effects of PS-ALA on the PGC-1α/Nrf1/Tfam pathway and Nrf2/HO-1 pathway as well as its mitochondrial biogenesis promotion effects and anti-oxidative activities were abrogated by the AMPK inhibitor in OA-treated HepG2 cells. In conclusion, the protective effects of PS-ALA on NAFLD appear to be associated with improving mitochondrial function and oxidative stress via activating AMPK signaling.

Three-dimensional ultrastructure of giant mitochondria in human non-alcoholic fatty liver disease.

Giant mitochondria are peculiarly shaped, extremely large mitochondria in hepatic parenchymal cells, the internal structure of which is characterised by atypically arranged cristae, enlarged matrix granules and crystalline inclusions. The presence of giant mitochondria in human tissue biopsies is often linked with cellular adversity, caused by toxins such as alcohol, xenobiotics, anti-cancer drugs, free-radicals, nutritional deficiencies or as a consequence of high fat Western diets. To date, non-alcoholic fatty liver disease is the most prevalent liver disease in lipid dysmetabolism, in which mitochondrial dysfunction plays a crucial role. It is not well understood whether the morphologic characteristics of giant mitochondria are an adaption or caused by such dysfunction. In the present study, we employ a complementary multimodal imaging approach involving array tomography and transmission electron tomography in order to comparatively analyse the structure and morphometric parameters of thousands of normal- and giant mitochondria in four patients diagnosed with non-alcoholic fatty liver disease. In so doing, we reveal functional alterations associated with mitochondrial gigantism and propose a mechanism for their formation based on our ultrastructural findings.

Transkingdom interactions between Lactobacilli and hepatic mitochondria attenuate western diet-induced diabetes.

Western diet (WD) is one of the major culprits of metabolic disease including type 2 diabetes (T2D) with gut microbiota playing an important role in modulating effects of the diet. Herein, we use a data-driven approach (Transkingdom Network analysis) to model host-microbiome interactions under WD to infer which members of microbiota contribute to the altered host metabolism. Interrogation of this network pointed to taxa with potential beneficial or harmful effects on host's metabolism. We then validate the functional role of the predicted bacteria in regulating metabolism and show that they act via different host pathways. Our gene expression and electron microscopy studies show that two species from Lactobacillus genus act upon mitochondria in the liver leading to the improvement of lipid metabolism. Metabolomics analyses revealed that reduced glutathione may mediate these effects. Our study identifies potential probiotic strains for T2D and provides important insights into mechanisms of their action.

Overexpression of the Lias gene attenuates hepatic steatosis in Leprdb/db mice.

Oxidative stress is proposed to be involved in nonalcoholic fatty liver disease (NAFLD). However, antioxidant therapy results in controversial outcomes. Therefore, we generated a new antioxidant/NAFLD mouse model, LiasHigh/HighLeprdb/db mice, by crossbreeding Leprdb/db mice, an obesity mouse model, with LiasHigh/High mice, generated by overexpression of lipoic acid synthase gene (Lias) and having increased endogenous antioxidant capacity, to investigate whether the new model could block the development of NAFLD. We have systemically characterized the novel model based on the main features of human NAFLD, determined the impact of enhanced endogenous antioxidant capacity on the retardation of NAFLD and elucidated the underlying mechanisms using various biological and pathological methods. We found that LiasHigh/HighLeprdb/db mice ameliorated many pathological changes of NAFLD compared with the control. In particular, LiasHigh/HighLeprdb/db mice displayed the improved liver mitochondrial function, reflecting the decline of mitochondrial microvesicular steatosis, and reduced oxidative stress, which mainly contributes to the alleviation of pathologic alterations of the NAFLD progression. Our new model shows that mitochondrial dysfunction is a major pathogenesis for liver steatosis. Overexpression of Lias gene effectively reduces oxidative stress and protects mitochondria, and consequently attenuates NAFLD/NASH.

Acute Elevated Resistin Exacerbates Mitochondrial Damage and Aggravates Liver Steatosis Through AMPK/PGC-1α Signaling Pathway in Male NAFLD Mice.

Resistin was identified as a link between obesity and insulin resistance and is associated with many diseases in mice. Deciphering the related development and molecular mechanism is necessary for the treatment of these diseases. Previous studies have revealed that increased resistin levels are correlated with lipid accumulation and play a role in non-alcoholic fatty liver disease (NAFLD) development. However, the exact mechanisms underlying these processes remain unclear. To further clarify whether acute elevated resistin level exacerbated liver steatosis, a high-fat diet-induced NAFLD animal model was used and treated with or without resistin for 6 days. We discovered that resistin altered mitochondrial morphology, decreased mitochondrial content, and increased lipid accumulation in HFD mice. qRT-PCR and western blot analysis showed that acute elevated resistin significantly altered the gene expression of mitochondrial biogenesis and liver lipid metabolism molecules in HFD mice. Consequently, in vitro experiments verified that resistin reduced the mitochondrial content, impaired the mitochondrial function and increased the lipid accumulation of palmitate-treated HepG2 cells. Additionally, we demonstrated that resistin upregulated proinflammatory factors, which confirmed that resistin promoted the development of inflammation in NAFLD mice and palmitate-treated HepG2 cells. Signaling-transduction analysis demonstrated that acute elevated resistin aggravated liver steatosis through AMPK/PGC-1α pathway in male mice. This reveals a novel pathway through which lipogenesis is induced by resistin and suggests that maintaining mitochondrial homeostasis may be key to treatments for preventing resistin-induced NAFLD aggravation.

Post-treatment with glycyrrhizin can attenuate hepatic mitochondrial damage induced by acetaminophen in mice.

Overdose of acetaminophen (APAP) is responsible for the most cases of acute liver failure worldwide. Hepatic mitochondrial damage mediated by neuronal nitric oxide synthase- (nNOS) induced liver protein tyrosine nitration plays a critical role in the pathophysiology of APAP hepatotoxicity. It has been reported that pre-treatment or co-treatment with glycyrrhizin can protect against hepatotoxicity through prevention of hepatocellular apoptosis. However, the majority of APAP-induced acute liver failure cases are people intentionally taking the drug to commit suicide. Any preventive treatment is of little value in practice. In addition, the hepatocellular damage induced by APAP is considered to be oncotic necrosis rather than apoptosis. In the present study, our aim is to investigate if glycyrrhizin can be used therapeutically and the underlying mechanisms of APAP hepatotoxicity protection. Hepatic damage was induced by 300 mg/kg APAP in balb/c mice, followed with administration of 40, 80, or 160 mg/kg glycyrrhizin 90 min later. Mice were euthanized and harvested at 6 h post-APAP. Compared with model controls, glycyrrhizin post-treatment attenuated hepatic mitochondrial and hepatocellular damages, as indicated by decreased serum glutamate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities as well as ameliorated mitochondrial swollen, distortion, and hepatocellular necrosis. Notably, 80 mg/kg glycyrrhizin inhibited hepatic nNOS activity and its mRNA and protein expression levels by 16.9, 14.9, and 28.3%, respectively. These results were consistent with the decreased liver nitric oxide content and liver protein tyrosine nitration indicated by 3-nitrotyrosine staining. Moreover, glycyrrhizin did not affect the APAP metabolic activation, and the survival rate of ALF mice was increased by glycyrrhizin. The present study indicates that post-treatment with glycyrrhizin can dose-dependently attenuate hepatic mitochondrial damage and inhibit the up-regulation of hepatic nNOS induced by APAP. Glycyrrhizin shows promise as drug for the treatment of APAP hepatotoxicity.

Mitochondrial UQCC3 Modulates Hypoxia Adaptation by Orchestrating OXPHOS and Glycolysis in Hepatocellular Carcinoma.

Bioenergetic reprogramming during hypoxia adaption is critical to promote hepatocellular carcinoma (HCC) growth and progression. However, the mechanism underlying the orchestration of mitochondrial OXPHOS (oxidative phosphorylation) and glycolysis in hypoxia is not fully understood. Here, we report that mitochondrial UQCC3 (C11orf83) expression increases in hypoxia and correlates with the poor prognosis of HCC patients. Loss of UQCC3 impairs HCC cell proliferation in hypoxia in vitro and in vivo. Mechanistically, UQCC3 forms a positive feedback loop with mitochondrial reactive oxygen species (ROS) to sustain UQCC3 expression and ROS generation in hypoxic HCC cells and subsequently maintains mitochondrial structure and function and stabilizes HIF-1α expression to enhance glycolysis under hypoxia. Thus, UQCC3 plays an indispensable role for bioenergetic reprogramming of HCC cells during hypoxia adaption by simultaneously regulating OXPHOS and glycolysis. The positive feedback between UQCC3 and ROS indicates a self-modulating model within mitochondria that initiates the adaptation of HCC to hypoxic stress.

Lycopene alleviates sulfamethoxazole-induced hepatotoxicity in grass carp (Ctenopharyngodon idellus) via suppression of oxidative stress, inflammation and apoptosis.

Antibiotics are used worldwide to treat diseases in humans and other animals; most of them and their secondary metabolites are discharged into the aquatic environment, posing a serious threat to human health. However, the toxicity of antibiotics on aquatic organisms, especially the effects on the detoxification system and immune system, has not been thoroughly studied. Lycopene (LYC) is a naturally occurring hydrocarbon carotenoid, which has received extensive attention as a potential antioxidant. The aim of this study was to investigate whether LYC alleviates exogenous toxicity in carp induced by sulfamethoxazole (SMZ) and the underlying molecular mechanisms. The grass carp were treated with SMZ (0.3 µg L-1) and/or LYC (10 mg per kg body weight) for 30 days. Indexes, such as hepatic function-related including histopathological changes and biochemical parameters, detoxification system-related including the cytochrome P450 enzyme system and antioxidant system, and immune system-related including inflammatory and apoptosis processes were detected. The results showed that SMZ stress leads to significant pathological damage of the liver and induction of oxidative stress. LYC coadministration recovered the cytochrome p450-1A1 homeostasis and decreased SMZ-induced accumulation of intracellular reactive oxygen species (ROS). Mechanistically, indicators in the innate immune system (such as toll like receptors (TLRs), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and IL-8) and the apoptosis pathway (p53, PUMA, B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax), and Caspase-9/3) disclosed adaptive activation under SMZ exposure; these anomalies returned to normal or close-to-normal levels after LYC coadministration. Therefore, LYC dietary supplement possesses liver protective function against exogenous toxic compounds like SMZ, making LYC a functional aquatic feed ingredient for aquiculture.

The effect of Ginsenoside Rg1 in hepatic ischemia reperfusion (I/R) injury ameliorates ischemia-reperfusion-induced liver injury by inhibiting apoptosis.

Hepatic ischemia reperfusion (I/R) injury (HIRI) HIRI is a complex, multifactorial pathophysiological process and in liver surgery has been known to significantly affect disease prognosis, surgical success rates, and patient survival. Ginsenoside Rgl (Rgl) monomer is one of the main active ingredients of ginseng. Previous studies have demonstrated that Rgl exerts various pharmacological effects through several mechanisms including suppression of apoptosis-related proteins levels, downregulation of inflammatory mediators and as well as antioxidant, which effectively exerts an organ protective effect I/R-induced damage. However, the exact mechanisms of Rg1 on HIRI remain to be elucidated. In the present study, we investigated the protective effect of Rg1 on hepatic ischemia-reperfusion (I/R) injury (HIRI) and explored its underlying molecular mechanism. A rat warm I/R injury model in vivo and an oxygen-glucose deprivation/reperfusion (OGD/R)-treated BRL-3A cell model in vitro were established after pretreating with Rg1(20 mg/kg). The results showed that Rg1 reduced the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). TUNEL staining showed that pretreated with Rg1 inhibited the apoptosis rate compared with the I/R group. Moreover, pretreated with Rg1 significantly reduced the expression of Cyt-C, Caspase-9 and Caspase-3 to inhibit the cell apoptosis. Flow cytometry analysis showed the MMP in the I/R group was significantly increased, whereas pretreated with Rg1 effectively stabilized the MMP compared with the I/R group. in vitro, the proliferation of BRL-3A cells was significantly decreased by the OGD/R treatment, while Rg1 effectively reversed this phenomenon. In addition, western blotting showed that the increase of Cyt-C, Caspase-9 and Caspase-3 was inhibited by H2O2. These observations suggest that Rg1 exerts the protective effect by inhibiting the CypD protein-mediated mitochondrial apoptotic pathway.

Deletion of Perilipin 5 Protects Against Hepatic Injury in Nonalcoholic Fatty Liver Disease via Missing Inflammasome Activation.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver diseases with an increasing prevalence due to rising rates of obesity, metabolic syndrome and type II diabetes. Untreated NAFLD may progress to steatohepatitis (NASH) and ultimately liver cirrhosis. NAFLD is characterized by lipid accumulation, and when sufficient excess lipids are obtained, irreversible liver injury may follow. Perilipin 5 (PLIN5), a known lipid droplet coating protein and triglyceride metabolism regulator, is highly expressed in oxidatively modified tissues but it is still unclear how it affects NAFLD/NASH progress. We here studied how PLIN5 affects NAFLD development induced by a 30-week high-fat diet (HFD) administration in wild type and PLIN5 knock out (Plin5-/-) mice. The disruption of PLIN5 induced differences in lipid metabolism during HFD feeding and was associated with reduced hepatic fat accumulation. Surprisingly, Plin5-/- mice showed mitigated activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome, leading to minor hepatic damage. We conclude that PLIN5 is a pleiotropic regulator of hepatic homeostasis in NASH development. Targeting the PLIN5 expression appears critical for protecting the liver from inflammatory activation during chronic NAFLD.

Changes to Liver Structure and Energy Metabolism During Cold Storage of Transplanted Liver in Mice.

OBJECTIVES: In this study, we aimed to investigate the pathologic and ultrastructural changes in transplanted mouse livers after different durations of cold storage by testing indicators of liver function and energy metabolism. We aimed to describe the effects of cold storage on liver function and the mechanisms of cold storage damage. MATERIALS AND METHODS: We randomly placed 8-weekold male C57BL/6 mice into the following 4 groups to establish a cold-preserved mouse model of liver transplant: a normal control group and 3 cold storage groups, in which livers were stored for 4, 12, and 24 hours. Hepatic morphology, ultrastructural changes, and glycogenolysis were observed by hematoxylin and eosin staining, periodic acid-Schiff staining, and transmission electron microscopy. After different durations of cold storage, livers were reperfused with 4°C University of Wisconsin solution to obtain perfusion fluid, and alanine and aspartate aminotransferase levels were measured. Glycogen synthase, hypoxia-inducible factor-1α, Krüppel-like factor 2, and endothelial nitric oxide synthase mRNA expression levels in liver tissues were detected by real-time polymerase chain reaction, and aquaporin 8 protein expression levels in liver tissues were detected by Western blot. RESULTS: Hematoxylin and eosin staining and electron microscopy ofliver showed signs ofinjury after 12 hours of cold storage, which included mainly cytoplasmic edema characterized by loose liver cell arrangement, increased hepatic sinus fissure, mitochondrial swelling, and nuclear pyknosis. Periodic acid-Schiff staining showed that glycogen content was significantly reduced, with glycogen synthase levels also reduced. Alanine aminotransferase and aspartate aminotransferase levels gradually increasedwith cold storage. Glycogen synthase, Krüppel-like factor 2, endothelial nitric oxide synthase, and aquaporin 8 expression levels also gradually increased in liver tissue. These levels gradually decreased, but hypoxia-inducible factor-1α increased. CONCLUSIONS: Mouse livers showed progressive damage to structure and function during cold storage, with mitochondrial damage perhaps showing the earliest damage.

Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK.

Impaired mitochondrial respiratory activity contributes to the development of insulin resistance in type 2 diabetes. Metformin, a first-line antidiabetic drug, functions mainly by improving patients' hyperglycemia and insulin resistance. However, its mechanism of action is still not well understood. We show here that pharmacological metformin concentration increases mitochondrial respiration, membrane potential, and ATP levels in hepatocytes and a clinically relevant metformin dose increases liver mitochondrial density and complex 1 activity along with improved hyperglycemia in high-fat- diet (HFD)-fed mice. Metformin, functioning through 5' AMP-activated protein kinase (AMPK), promotes mitochondrial fission to improve mitochondrial respiration and restore the mitochondrial life cycle. Furthermore, HFD-fed-mice with liver-specific knockout of AMPKα1/2 subunits exhibit higher blood glucose levels when treated with metformin. Our results demonstrate that activation of AMPK by metformin improves mitochondrial respiration and hyperglycemia in obesity. We also found that supra-pharmacological metformin concentrations reduce adenine nucleotides, resulting in the halt of mitochondrial respiration. These findings suggest a mechanism for metformin's anti-tumor effects.

Astaxanthin attenuates the increase in mitochondrial respiration during the activation of hepatic stellate cells.

Upon liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate to myofibroblast-like activated HSCs (aHSCs), which are primarily responsible for the accumulation of extracellular matrix proteins during the development of liver fibrosis. Therefore, aHSCs may exhibit different energy metabolism from that of qHSCs to meet their high energy demand. We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, prevents the activation of HSCs. The objective of this study was to determine if ASTX can exert its antifibrogenic effect by attenuating any changes in energy metabolism during HSC activation. To characterize the energy metabolism of qHSCs and aHSCs, mouse primary HSCs were cultured on uncoated plastic dishes for 7 days for spontaneous activation in the presence or absence of 25 µM ASTX. qHSCs (1 day after isolation) and aHSCs treated with or without ASTX for 7 days were used to determine parameters related to mitochondrial respiration using a Seahorse XFe24 Extracellular Flux analyzer. aHSCs had significantly higher basal respiration, maximal respiration, ATP production, spare respiratory capacity and proton leak than those of qHSCs. However, ASTX prevented most of the changes occurring during HSC activation and improved mitochondrial cristae structure with decreased cristae junction width, lumen width and the area in primary mouse aHSCs. Furthermore, qHSCs isolated from ASTX-fed mice had lower mitochondrial respiration and glycolysis than control qHSCs. Our findings suggest that ASTX may exert its antifibrogenic effect by attenuating the changes in energy metabolism during HSC activation.

Single organelle analysis to characterize mitochondrial function and crosstalk during viral infection.

Mitochondria are key for cellular metabolism and signalling processes during viral infection. We report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. Resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. Thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies.

Efficiently Capturing Mitochondria-Targeted Constituents with Hepatoprotective Activity from Medicinal Herbs.

Targeting mitochondria as a hepatic-protective strategy has gained attention, because of their important roles in energy production, adjustment of apoptosis, and generation of reactive oxygen species. To promote the discovery of natural mitochondria-targeted hepatic-protectants, we established a hepatocellular mitochondria-based capturing method by coupling affinity ultrafiltration with liquid chromatography/mass spectrometry (LC/MS), which is suitable for identifying mitochondrial ligands from medicinal herbs (MHs). After evaluating the feasibility of the method, it was applied for capturing mitochondria-targeting constituents from Peucedani Radix extract. A total of 10 active compounds were identified by LC/MS, all of which were newly identified mitochondrial ligands. The mitochondria-remedying activity of 4 of the 10 hits was confirmed by pharmacological tests in vitro. Additionally, the hepatic-protective abilities of 4 hits were verified in both carbon tetrachloride-damaged liver L02 cells and mice. These results indicated that the method could be used for identifying hepatic mitochondria-targeting constituents in MHs, which might be beneficial for hepatic-protective development.

Cholesterol enrichment in liver mitochondria impairs oxidative phosphorylation and disrupts the assembly of respiratory supercomplexes.

Mitochondrial cholesterol accumulation is a hallmark of alcoholic and non-alcoholic fatty liver diseases and impairs the function of specific solute carriers through changes in membrane physical properties. However, its impact on mitochondrial respiration and organization of respiratory supercomplexes has not been determined so far. Here we fed mice a cholesterol-enriched diet (HC) supplemented with sodium cholate to examine the effect of cholesterol in mitochondrial function. HC feeding increased liver cholesterol content, which downregulated Srebp2 and Hmgcr expression, while sodium cholate administration decreased Cyp7a1 and Cyp8b1 mRNA levels, suggesting the downregulation of bile acid synthesis through the classical pathway. HC-fed mice exhibited increased expression of Stard1 and Mln64 and enhanced mitochondrial free cholesterol levels (2-3 fold), leading to decreased membrane fluidity. Mitochondria from HC-fed mice displayed increased cholesterol loading in both outer and inner mitochondrial membranes. Cholesterol loading decreased complex I and complex II-driven state 3 respiration and mitochondrial membrane potential. Decreased respiratory and uncoupling control ratio from complex I was also observed after in situ enrichment of mouse liver mitochondria with cholesterol or enantiomer cholesterol, the mirror image of natural cholesterol. Moreover, in vivo cholesterol loading decreased the level of complex III2 and the assembly of respiratory supercomplexes I1+III2+IV and I1+III2. Moreover, HC feeding caused oxidative stress and mitochondrial GSH (mGSH) depletion, which translated in hepatic steatosis and liver injury, effects that were rescued by replenishing mGSH with GSH ethyl ester. Overall, mitochondrial cholesterol accumulation disrupts mitochondrial functional performance and the organization of respiratory supercomplexes assembly, which can contribute to oxidative stress and liver injury.