Hepatocyte-specific perturbation of NAD+ biosynthetic pathways in mice induces reversible nonalcoholic steatohepatitis-like phenotypes.

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to NAD+. As low hepatic NAD+ levels have been linked to the development of nonalcoholic fatty liver disease, we hypothesized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine and choline-free 60% high-fat diet, hepatocyte-specific Nampt knockout (HNKO) mice accumulated less triglyceride than WT littermates but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet. This HNKO phenotype was associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in purified control diet-fed HNKO liver. In addition, fibrotic area in HNKO liver sections correlated negatively with hepatic NAD+, and liver injury was prevented by supplementation with NAD+ precursors nicotinamide riboside and nicotinic acid. MS-based proteomic analysis revealed that nicotinamide riboside supplementation rescued hepatic levels of oxidoreductase and OXPHOS proteins. Finally, single-nucleus RNA-Seq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis because of low NAD+ levels. Our data suggest a critical threshold level of hepatic NAD+ that determines the predisposition to liver injury and supports that NAD+ precursor supplementation can prevent liver injury and nonalcoholic fatty liver disease progression.

The Mitochondrial Trigger in an Animal Model of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is the leading liver chronic disease featuring hepatic steatosis. Mitochondrial ß-oxidation participates in the derangement of lipid metabolism at the basis of NAFLD, and mitochondrial oxidative stress contributes to the onset of the disease. We evaluated the presence and effects of mitochondrial oxidative stress in the liver from rats fed a high-fat plus fructose (HF-F) diet inducing NAFLD. Supplementation with dehydroepiandrosterone (DHEA), a multitarget antioxidant, was tested for efficacy in delaying NAFLD. A marked mitochondrial oxidative stress was originated by all diets, as demonstrated by the decrease in Superoxide Dismutase 2 (SOD2) and Peroxiredoxin III (PrxIII) amounts. All diets induced a decrease in mitochondrial DNA content and an increase in its oxidative damage. The diets negatively affected mitochondrial biogenesis as shown by decreased peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), mitochondrial transcription factor A (TFAM), and the COX-IV subunit from the cytochrome c oxidase complex. The reduced amounts of Beclin-1 and lipidated LC3 II form of the microtubule-associated protein 1 light chain 3 (LC3) unveiled the diet-related autophagy's decrease. The DHEA supplementation did not prevent the diet-induced changes. These results demonstrate the relevance of mitochondrial oxidative stress and the sequential dysfunction of the organelles in an obesogenic diet animal model of NAFLD.

Supplementation of pyrroloquinoline quinone with atorvastatin augments mitochondrial biogenesis and attenuates low grade inflammation in obese rats.

Mitochondrial dysfunction and Inflammation play a significant role in the manifestation of the co-morbidities of obesity. The study deciphered the impact of Pyrroloquinoline quinone (PQQ) per se and with Atorvastatin (ATS) on high fat, 10% fructose diet (HFFD) induced obese rats expressing low-grade inflammation, dyslipidemia, and mitochondrial dysfunction. HFFD was fed for 10 weeks followed by treatment for 5 weeks with ATS 10 or 20 mg/kg, PQQ 10 or 20 mg/kg, p.o. per se or their combinations. The impact on blood glucose, lipid profile and serum insulin, TNF-α, IL-1ß, IL-18, IL-6 was estimated. Gene and protein expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC 1α), Sirtuin 1 (SIRT1), Mitochondrial transcriptional factor A (TFAM) and augmented mitochondrial DNA (mtDNA), NOD like receptor protein 3 (NLRP3) and Caspase 1 was assessed. Rats receiving PQQ and ATS revealed significant decrease in body weights, anthropometric parameter, and adipose tissue vis-à-vis positive control. PQQ alone and with ATS improved glucose tolerance, lipid profile, insulin indices and lowered serum levels of inflammatory cytokines IL-18, IL-1ß, TNF-α and IL-6 along with a rise in adiponectin. PQQ supplementation with ATS upregulated the mRNA expression of PGC 1α, SIRT1, TFAM and augmented mtDNA while downregulating inflammatory markers NLRP3 and Caspase 1. PQQ supplementation with atorvastatin holds therapeutic promise to effectively combat mitochondrial dysfunction and chronic low-grade inflammation in obesity.

Dihydromyricetin Ameliorates Nonalcoholic Fatty Liver Disease by Improving Mitochondrial Respiratory Capacity and Redox Homeostasis Through Modulation of SIRT3 Signaling.

Aims: Our previous clinical trial indicated that the flavonoid dihydromyricetin (DHM) could improve hepatic steatosis in patients with nonalcoholic fatty liver disease (NAFLD), altough the potential mechanisms of these effects remained elusive. Here, we investigated the hepatoprotective role of DHM on high-fat diet (HFD)-induced NAFLD. Results: DHM supplementation could effectively ameliorate the development of NAFLD by inhibiting hepatic lipid accumulation both in HFD-fed wild-type mice and in palmitic acid-induced hepatocytes. We reveal for the first time that mitochondrial dysfunction characterized by ATP depletion and augmented oxidative stress could be reversed by DHM treatment. Moreover, DHM enhanced the mitochondrial respiratory capacity by increasing the expression and enzymatic activities of mitochondrial complexes and increased mitochondrial reactive oxygen species scavenging by restoring manganese superoxide dismutase (SOD2) activity. Interestingly, the benefits of DHM were abrogated in SIRT3 knockout (SIRT3KO) mice and in hepatocytes transfected with SIRT3 siRNA or treated with an SIRT3-specific inhibitor. We further showed that DHM could increase SIRT3 expression by activating the adenosine monophosphate-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC1α)/estrogen-related receptor-α (ERRα) signaling pathway. Innovation: Our work indicates that SIRT3 plays a critical role in the DHM-mediated beneficial effects that include ameliorating mitochondrial dysfunction and oxidative stress in a nutritional NAFLD model both in vivo and in vitro.Conclusion: Our results suggest that DHM prevents NAFLD by improving mitochondrial respiratory capacity and redox homeostasis in hepatocytes through a SIRT3-dependent mechanism. These results could provide a foundation to identify new DHM-based preventive and therapeutic strategies for NAFLD.

Gene pathways associated with mitochondrial function, oxidative stress and telomere length are differentially expressed in the liver of rats fed lifelong on virgin olive, sunflower or fish oils.

This study investigates the effect of lifelong intake of different fat sources rich in monounsaturated (virgin olive oil), n6 polyunsaturated (sunflower oil) or n3 polyunsaturated (fish oil) fatty acids in the aged liver. Male Wistar rats fed lifelong on diets differing in the fat source were killed at 6 and at 24 months of age. Liver histopathology, mitochondrial ultrastructure, biogenesis, oxidative stress, mitochondrial electron transport chain, relative telomere length and gene expression profiles were studied. Aging led to lipid accumulation in the liver. Virgin olive oil led to the lowest oxidation and ultrastructural alterations. Sunflower oil induced fibrosis, ultrastructural alterations and high oxidation. Fish oil intensified oxidation associated with age, lowered electron transport chain activity and enhanced the relative telomere length. Gene expression changes associated with age in animals fed virgin olive oil and fish oil were related mostly to mitochondrial function and oxidative stress pathways, followed by cell cycle and telomere length control. Sunflower oil avoided gene expression changes related to age. According to the results, virgin olive oil might be considered the dietary fat source that best preserves the liver during the aging process.

Ephedrine-induced mitophagy via oxidative stress in human hepatic stellate cells.

The herb Ephedra sinica (also known as Chinese ephedra or Ma Huang), used in traditional Chinese medicine, contains alkaloids identical to ephedrine and pseudoephedrine as its principal active constituents. Recent studies have reported that ephedrine has various side effects in the cardiovascular and nervous systems. In addition, herbal Ephedra, a plant containing many pharmacologically active alkaloids, principally ephedrine, has been reported to cause acute hepatitis. Many studies reported clinical cases, however, the cellular mechanism of liver toxicity by ephedrine remains unknown. In this study, we investigated hepatotoxicity and key regulation of mitophagy in ephedrine-treated LX-2 cells. Ephedrine triggered mitochondrial oxidative stress and depolarization. Mitochondrial swelling and autolysosome were observed in ephedrine-treated cells. Ephedrine also inhibited mitochondrial biogenesis, and the mitochondrial copy number was decreased. Parkin siRNA recovered the ephedrine-induced mitochondrial damage. Excessive mitophagy lead to cell death through imbalance of autophagic flux. Moreover, antioxidants and reducing Parkin level could serve as therapeutic targets for ephedrine-induced hepatotoxicity.

Celastrol induces mitochondria-mediated apoptosis in hepatocellular carcinoma Bel-7402 cells.

Celastrol is a natural terpenoid isolated from Tripterygium wilfordii, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. Here, we investigated whether celastrol induces apoptosis on hepatocellular carcinoma Bel-7402 cells and further explored the underlying molecular mechanisms. Celastrol caused a dose- and time-dependent growth inhibition and apoptosis of Bel-7402 cells. It increased apoptosis through the up-regulation of Bax and the down-regulation of Bcl-2 in Bel-7402 cells. Moreover, celastrol induced the release of cytochrome c and increased the activation of caspase-3 and caspase-9, suggesting that celastrol-induced apoptosis was related to the mitochondrial pathway. These results indicated that celastrol could induce apoptosis in Bel-7402 cells, which may be associated with the activation of the mitochondria-mediated pathway.

Mitochondrial protective effects of Myrica rubra extract against acetaminophen-induced toxicity.

The present study investigates the hepatoprotective activity of Myrica rubra Sieb. et Zucc. extract (MCE) against acetaminophen (AAP)-induced liver damage and elucidates the possible mechanisms behind the hepatoprotection observed. Serum alanine aminotransferase and serum aspartate aminotransferase activities were detected and liver histopathology was observed. Mitochondrial swelling, mitochondrial membrane potential, and voltage-dependent anion channel (VDAC) gene transcription were also investigated. The results showed that 50, 150, and 450 mg/kg MCE could restore AAP-induced changes in mice liver in a dose-dependent manner. The mechanisms behind the hepatoprotective effects of MCE may be related to the mitochondrial protection of liver cells, especially of VDAC, an important protein on the outer membrane of the mitochondria.

Mitochondrial bioenergetic profile and responses to metabolic inhibition in human hepatocarcinoma cell lines with distinct differentiation characteristics.

The classical view of tumour cell bioenergetics has been recently revised. Then, the definition of the mitochondrial profile is considered of fundamental importance for the development of anti-cancer therapies, but it still needs to be clarified. We investigated two human hepatocellular carcinoma cell lines: the partially differentiated HepG2 and the undifferentiated JHH-6. High resolution respirometry revealed a marked impairment/uncoupling of OXPHOS in JHH-6 compared with HepG2, with the phosphorylation system limiting the capacity for electron transport much more in JHH-6. Blocking glycolysis or mitochondrial ATP synthase we demonstrated that in JHH-6 ATP synthase functions in reverse and consumes glycolytic ATP, thereby sustaining ΔΨm. A higher expression level of ATP synthase Inhibitor Factor 1 (IF1), a higher extent of IF1 bound to ATP synthase and a lower ATPase/synthase capacity were documented in JHH-6. Thus, here IF1 appears to down-regulate the reverse mode of ATPsynthase activity, thereby playing a crucial role in controlling energy waste and ΔΨm. These results, while confirming the over-expression of IF1 in cancer cells, are the first to indicate an inverse link between cell differentiation status and IF1 (expression level and regulatory function).

Aromatase deficiency inhibits the permeability transition in mouse liver mitochondria.

Lack of estrogens affects male physiology in a number of ways, including severe changes in liver metabolism that result in lipid accumulation and massive hepatic steatosis. Here we investigated whether estrogen deficiency may alter the functionality and permeability properties of liver mitochondria using, as an experimental model, aromatase knockout (ArKO) male mice, which cannot synthesize endogenous estrogens due to a disruption of the Cyp19 gene. Liver mitochondria isolated from ArKO mice displayed increased activity of the mitochondrial respiratory complex IV compared with wild-type mice and were less prone to undergo cyclosporin A-sensitive mitochondrial permeability transition (MPT) induced by calcium loading. The altered permeability properties of the mitochondrial membranes were not due to changes in reactive oxygen species, ATP levels, or mitochondrial membrane potential but were associated with increased content of the phospholipid cardiolipin, structural component of the mitochondrial membranes and regulator of the MPT pore, and with increased mitochondrial protein levels of Bcl-2 and the adenine nucleotide translocator (ANT), regulator and component of the MPT pore, respectively. Real-time RT-PCR demonstrated increased mRNA levels for Bcl-2 and ANT2 but not for the ANT1 isoform in ArKO livers. Supplementation of 17beta-estradiol retrieved ArKO mice from massive hepatic steatosis and restored mitochondrial permeability properties, cardiolipin, Bcl-2, and ANT2 levels. Overall, our findings demonstrate an important role of estrogens in the modulation of hepatic mitochondrial function and permeability properties in males and suggest that estrogen deficiency may represent a novel positive regulator of Bcl-2 and ANT2 proteins, two inhibitors of MPT occurrence and powerful antiapoptotic molecules.

Gene expression profiling in male B6C3F1 mouse livers exposed to kava identifies--changes in drug metabolizing genes and potential mechanisms linked to kava toxicity.

The association of kava products with liver-related health risks has prompted regulatory action in many countries. We used a genome-wide gene expression approach to generate global gene expression profiles from the livers of male B6C3F1 mice administered kava extract by gavage for 14 weeks, and identified the differentially expressed drug metabolizing genes in response to kava treatments. Analyses of gene functions and pathways reveal that the levels of significant numbers of genes involving drug metabolism were changed and that the pathways involving xenobiotics metabolism, Nrf2-mediated oxidative stress response, mitochondrial functions and others, were altered. Our results indicate that kava extract can significantly modulate drug metabolizing enzymes, potentially leading to herb-drug interactions and hepatotoxicity.

Dietary modulation of mitochondrial DNA deletions and copy number after chemotherapy in rats.

Mitochondrial DNA (mtDNA) is particularly susceptible to mutation by alkylating agents, and mitochondrial damage may contribute to the efficacy and toxicity of these agents. We found that folate supplementation decreased the frequency of the "common deletion" (4.8kb, bases 8103-12,936) in liver from untreated rats and from animals treated with cyclophosphamide but not 5-fluorouracil (5-FU). The relative abundance of mitochondrial DNA was greater after chemotherapy but there was no effect of diet. Rats fed with a purified diet had fewer mitochondrial deletions than those maintained on a cereal-based diet after chemotherapy. These results indicate that diet can modulate the extent of mitochondrial damage after cancer chemotherapy, and that folic acid supplementation may be protective against mitochondrial DNA deletions.