Mitochondrial Dysfunction Plays Central Role in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a global pandemic that affects one-quarter of the world’s population. NAFLD includes a spectrum of progressive liver disease from steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis and can be complicated by hepatocellular carcinoma. It is strongly associated with metabolic syndromes, obesity, and type 2 diabetes, and it has been shown that metabolic dysregulation is central to its pathogenesis. Recently, it has been suggested that metabolic- (dysfunction) associated fatty liver disease (MAFLD) is a more appropriate term to describe the disease than NAFLD, which puts increased emphasis on the important role of metabolic dysfunction in its pathogenesis. There is strong evidence that mitochondrial dysfunction plays a significant role in the development and progression of NAFLD. Impaired mitochondrial fatty acid oxidation and, more recently, a reduction in mitochondrial quality, have been suggested to play a major role in NAFLD development and progression. In this review, we provide an overview of our current understanding of NAFLD and highlight how mitochondrial dysfunction contributes to its pathogenesis in both animal models and human subjects. Further we discuss evidence that the modification of mitochondrial function modulates NAFLD and that targeting mitochondria is a promising new avenue for drug development to treat NAFLD/NASH.     

The Role of l-Carnitine in Mitochondria,Prevention of Metabolic Inflexibility and Disease Initiation

Mitochondria control cellular fate by various mechanisms and are key drivers of cellular metabolism. Although the main function of mitochondria is energy production, they are also involved in cellular detoxification, cellular stabilization, as well as control of ketogenesis and glucogenesis. Conditions like neurodegenerative disease, insulin resistance, endocrine imbalances, liver and kidney disease are intimately linked to metabolic disorders or inflexibility and to mitochondrial dysfunction. Mitochondrial dysfunction due to a relative lack of micronutrients and substrates is implicated in the development of many chronic diseases. l-carnitine is one of the key nutrients for proper mitochondrial function and is notable for its role in fatty acid oxidation. l-carnitine also plays a major part in protecting cellular membranes, preventing fatty acid accumulation, modulating ketogenesis and glucogenesis and in the elimination of toxic metabolites. l-carnitine deficiency has been observed in many diseases including organic acidurias, inborn errors of metabolism, endocrine imbalances, liver and kidney disease. The protective effects of micronutrients targeting mitochondria hold considerable promise for the management of age and metabolic related diseases. Preventing nutrient deficiencies like l-carnitine can be beneficial in maintaining metabolic flexibility via the optimization of mitochondrial function. This paper reviews the critical role of l-carnitine in mitochondrial function, metabolic flexibility and in other pathophysiological cellular mechanisms.     

P2Y2R Deficiency Ameliorates Hepatic Steatosis by Reducing Lipogenesis and Enhancing Fatty Acid β-Oxidation through AMPK and PGC-1α Induction in High-Fat Diet-Fed Mice

Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease associated with obesity and insulin resistance. Activation of the purinergic receptor P2Y2R has been reported to promote adipogenesis, inflammation and dyslipidemia in adipose tissues in obese mice. However, the role of P2Y2R and its mechanisms in NAFLD remain unknown. We hypothesized that P2Y2R deficiency may play a protective role in NAFLD by modulating lipid metabolism in the liver. In this study, we fed wild type and P2Y2R knockout mice with a high-fat diet (HFD) for 12 weeks and analyzed metabolic phenotypes. First, P2Y2R deficiency effectively improved insulin resistance with a reduction in body weight and plasma insulin. Second, P2Y2R deficiency attenuated hepatic lipid accumulation and injury with reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Third, P2Y2R deficiency decreased the expression of fatty acid synthesis mediators (cluster of differentiation (CD36), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1)); and increased the expression of adipose triglyceride lipase (ATGL), a lipolytic enzyme. Mechanistically, P2Y2R deficiency increased the AMP-activated protein kinase (AMPK) activity to improve mitochondrial fatty acid β-oxidation (FAO) by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase 1A (CPT1A)-mediated FAO pathway. In addition, P2Y2R deficiency increased peroxisome proliferator-activated gamma co-activator-1α (PGC-1α)-mediated mitochondrial biogenesis. Conclusively, P2Y2R deficiency ameliorated HFD-induced hepatic steatosis by enhancing FAO through AMPK signaling and PGC-1α pathway, suggesting P2Y2R as a promising therapeutic target for NAFLD.     

Mitochondria Matter: Systemic Aspects of Nonalcoholic Fatty Liver Disease (NAFLD) and Diagnostic Assessment of Liver Function by Stable Isotope Dynamic Breath Tests

The liver plays a key role in systemic metabolic processes, which include detoxification, synthesis, storage, and export of carbohydrates, lipids, and proteins. The raising trends of obesity and metabolic disorders worldwide is often associated with the nonalcoholic fatty liver disease (NAFLD), which has become the most frequent type of chronic liver disorder with risk of progression to cirrhosis and hepatocellular carcinoma. Liver mitochondria play a key role in degrading the pathways of carbohydrates, proteins, lipids, and xenobiotics, and to provide energy for the body cells. The morphological and functional integrity of mitochondria guarantee the proper functioning of β-oxidation of free fatty acids and of the tricarboxylic acid cycle. Evaluation of the liver in clinical medicine needs to be accurate in NAFLD patients and includes history, physical exam, imaging, and laboratory assays. Evaluation of mitochondrial function in chronic liver disease and NAFLD is now possible by novel diagnostic tools. “Dynamic” liver function tests include the breath test (BT) based on the use of substrates marked with the non-radioactive, naturally occurring stable isotope ¹³C. Hepatocellular metabolization of the substrate will generate ¹³CO₂, which is excreted in breath and measured by mass spectrometry or infrared spectroscopy. Breath levels of ¹³CO₂ are biomarkers of specific metabolic processes occurring in the hepatocyte cytosol, microsomes, and mitochondria. ¹³C-BTs explore distinct chronic liver diseases including simple liver steatosis, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, hepatocellular carcinoma, drug, and alcohol effects. In NAFLD, ¹³C-BT use substrates such as α-ketoisocaproic acid, methionine, and octanoic acid to assess mitochondrial oxidation capacity which can be impaired at an early stage of disease. ¹³C-BTs represent an indirect, cost-effective, and easy method to evaluate dynamic liver function. Further applications are expected in clinical medicine. In this review, we discuss the involvement of liver mitochondria in the progression of NAFLD, together with the role of ¹³C-BT in assessing mitochondrial function and its potential use in the prevention and management of NAFLD.     

Maternal Fructose Intake Causes Developmental Reprogramming of Hepatic Mitochondrial Catalytic Activity and Lipid Metabolism in Weanling and Young Adult Offspring

Excess dietary fructose is a major public health concern, yet little is known about its influence on offspring development and later-life disease when consumed in excess during pregnancy. To determine whether increased maternal fructose intake could have long-term consequences on offspring health, we investigated the effects of 10% w/v fructose water intake during preconception and pregnancy in guinea pigs. Female Dunkin Hartley guinea pigs were fed a control diet (CD) or fructose diet (FD; providing 16% of total daily caloric intake) ad libitum 60 days prior to mating and throughout gestation. Dietary interventions ceased at day of delivery. Offspring were culled at day 21 (D21) (weaning) and at 4 months (4 M) (young adult). Fetal exposure to excess maternal fructose intake significantly increased male and female triglycerides at D21 and 4 M and circulating palmitoleic acid and total omega-7 through day 0 (D0) to 4 M. Proteomic and functional analysis of significantly differentially expressed proteins revealed that FD offspring (D21 and 4 M) had significantly increased mitochondrial metabolic activities of β-oxidation, electron transport chain (ETC) and oxidative phosphorylation and reactive oxygen species production compared to the CD offspring. Western blotting analysis of both FD offspring validated the increased protein abundances of mitochondrial ETC complex II and IV, SREBP-1c and FAS, whereas VDAC1 expression was higher at D21 but lower at 4 M. We provide evidence demonstrating offspring programmed hepatic mitochondrial metabolism and de novo lipogenesis following excess maternal fructose exposure. These underlying asymptomatic programmed pathways may lead to a predisposition to metabolic dysfunction later in life.     

Adiponectin Deficiency Alters Placenta Function but Does Not Affect Fetal Growth in Mice

Adiponectin administration to pregnant mice decreases nutrient transport and fetal growth. An adiponectin deficiency, on the other hand, as seen in obese women during pregnancy, alters fetal growth; however, the mechanism is unclear. To determine the role of adiponectin on placenta function and fetal growth, we used adiponectin knockout, adiponectin heterozygote that displays reduced adiponectin levels, and wild-type mice on a control diet or high fat/high sucrose (HF/HS) diet. Triglycerides (TGs) in the serum, liver, and placenta were measured using colorimetric assays. Gene expression was measured using quantitative RT-PCR. Adiponectin levels did not affect fetal weight, but it reduced adiponectin levels, increased fetal serum and placenta TG content. Wildtype dams on a HF/HS diet protected the fetuses from fatty acid overload as judged by increased liver TGs in dams and normal serum and liver TG levels in fetuses, while low adiponectin was associated with increased fetal liver TGs. Low maternal adiponectin increased the expression of genes involved in fatty acid transport; Lpl and Cd36 in the placenta. Adiponectin deficiency does not affect fetal growth but induces placental dysfunction and increases fetal TG load, which is enhanced with obesity. This could lead to imprinting effects on the fetus and the development of metabolic dysfunction in the offspring.     

The Influence of Maternal Early to Mid-Gestation Nutrient Restriction on Long Chain Polyunsaturated Fatty Acids in Fetal Sheep

The early to mid-gestational period (days 28-78) in sheep is the period of most rapid placental development. Maternal nutrient restriction (MNR) in this phase has negative consequences on fetal growth and development, predisposing the fetus to disease in adult life. The influence of MNR on fetal tissue fatty acids has not been reported. Ewes were fed to 50% (MNR) or 100% (control fed) of total digestible nutrients from days 28 to 78 of gestation. At 78 days, fetuses were sacrificed and the fatty acids in fetal liver, lung and muscle as well as maternal and fetal plasma were analyzed. Most fatty acids were not influenced by MNR. The n-3 long chain PUFA eicosapentaenoic acid (20:5n-3, EPA) concentration (microg/mg) was low and more than doubled in the MNR sheep. Similarly, docosapentaenoic acid (22:5n-3, DPA) increased by 60, 19, and 38% in liver, lung, and muscle, respectively. Neither docosahexaenoic acid (22:6n-3, DHA) nor any of n-6 PUFA changed. Arachidonic acid (20:4n-6; ARA) increased in MNR maternal plasma as a percent of total fatty acids only, while in MNR fetal plasma only EPA increased. These results provide the first indication that MNR in early to mid-gestation influences the profiles of LCPUFA in fetal tissues, and suggest that metabolic processes involving LCPUFA should be considered in evaluations of the impact of maternal nutriture on perinatal health.     

Role of Endocrine-Disrupting Chemicals in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: A Comprehensive Review

Non-alcoholic fatty liver disease (NAFLD) is considered the most common liver disorder, affecting around 25% of the population worldwide. It is a complex disease spectrum, closely linked with other conditions such as obesity, insulin resistance, type 2 diabetes mellitus, and metabolic syndrome, which may increase liver-related mortality. In light of this, numerous efforts have been carried out in recent years in order to clarify its pathogenesis and create new prevention strategies. Currently, the essential role of environmental pollutants in NAFLD development is recognized. Particularly, endocrine-disrupting chemicals (EDCs) have a notable influence. EDCs can be classified as natural (phytoestrogens, genistein, and coumestrol) or synthetic, and the latter ones can be further subdivided into industrial (dioxins, polychlorinated biphenyls, and alkylphenols), agricultural (pesticides, insecticides, herbicides, and fungicides), residential (phthalates, polybrominated biphenyls, and bisphenol A), and pharmaceutical (parabens). Several experimental models have proposed a mechanism involving this group of substances with the disruption of hepatic metabolism, which promotes NAFLD. These include an imbalance between lipid influx/efflux in the liver, mitochondrial dysfunction, liver inflammation, and epigenetic reprogramming. It can be concluded that exposure to EDCs might play a crucial role in NAFLD initiation and evolution. However, further investigations supporting these effects in humans are required.     

In Vivo Two-Photon Imaging Analysis of Dynamic Degradation of Hepatic Lipid Droplets in MS-275-Treated Mouse Liver

The accumulation of hepatic lipid droplets (LDs) is a hallmark of non-alcoholic fatty liver disease (NAFLD). Appropriate degradation of hepatic LDs and oxidation of complete free fatty acids (FFAs) are important for preventing the development of NAFLD. Histone deacetylase (HDAC) is involved in the impaired lipid metabolism seen in high-fat diet (HFD)-induced obese mice. Here, we evaluated the effect of MS-275, an inhibitor of HDAC1/3, on the degradation of hepatic LDs and FFA oxidation in HFD-induced NAFLD mice. To assess the dynamic degradation of hepatic LDs and FFA oxidation in fatty livers of MS-275-treated HFD C57BL/6J mice, an intravital two-photon imaging system was used and biochemical analysis was performed. The MS-275 improved hepatic metabolic alterations in HFD-induced fatty liver by increasing the dynamic degradation of hepatic LDs and the interaction between LDs and lysozyme in the fatty liver. Numerous peri-droplet mitochondria, lipolysis, and lipophagy were observed in the MS-275-treated mouse fatty liver. Biochemical analysis revealed that the lipolysis and autophagy pathways were activated in MS-275 treated mouse liver. In addition, MS-275 reduced the de novo lipogenesis, but increased the mitochondrial oxidation and the expression levels of oxidation-related genes, such as PPARa, MCAD, CPT1b, and FGF21. Taken together, these results suggest that MS-275 stimulates the degradation of hepatic LDs and mitochondrial free fatty acid oxidation, thus protecting against HFD-induced NAFLD.     

The Mechanism of Mitochondrial Injury in Alpha-1 Antitrypsin Deficiency Mediated Liver Disease

Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.     

One Week of CDAHFD Induces Steatohepatitis and Mitochondrial Dysfunction with Oxidative Stress in Liver

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rapidly increasing worldwide. A choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) has been used to create a mouse model of nonalcoholic steatohepatitis (NASH). There are some reports on the effects on mice of being fed a CDAHFD for long periods of 1 to 3 months. However, the effect of this diet over a short period is unknown. Therefore, we examined the effect of 1-week CDAHFD feeding on the mouse liver. Feeding a CDAHFD diet for only 1-week induced lipid droplet deposition in the liver with increasing activity of liver-derived enzymes in the plasma. On the other hand, it did not induce fibrosis or cirrhosis. Additionally, it was demonstrated that CDAHFD significantly impaired mitochondrial respiration with severe oxidative stress to the liver, which is associated with a decreasing mitochondrial DNA copy number and complex proteins. In the gene expression analysis of the liver, inflammatory and oxidative stress markers were significantly increased by CDAHFD. These results demonstrated that 1 week of feeding CDAHFD to mice induces steatohepatitis with mitochondrial dysfunction and severe oxidative stress, without fibrosis, which can partially mimic the early stage of NASH in humans.     

Maternal Metformin Treatment Reprograms Maternal High-Fat Diet-Induced Hepatic Steatosis in Offspring Associated with Placental Glucose Transporter Modifications

Maternal high-fat (HF) diet exposure in utero may affect fetal development and cause metabolic problems throughout life due to lipid dysmetabolism and oxidative damage. Metformin has been suggested as a potential treatment for body weight reduction and nonalcoholic fatty liver disease, but its reprogramming effect on offspring is undetermined. This study assesses the effects of maternal metformin treatment on hepatic steatosis in offspring caused by maternal HF diet. Female rats were fed either a control or an HF diet before conception, with or without metformin treatment during gestation, and placenta and fetal liver tissues were collected. In another experiment, the offspring were fed a control diet until 120 d (adult stage). Metformin treatment during pregnancy ameliorates placental oxidative stress and enhances placental glucose transporter 1 (GLUT1), GLUT3, and GLUT4 expression levels through 5’ adenosine monophosphate-activated protein kinase (AMPK) activation. Maternal metformin treatment was shown to reprogram maternal HF diet-induced changes in offspring fatty liver with the effects observed in adulthood as well. Further validation is required to develop maternal metformin therapy for clinical applications.     

The Alterations of Mitochondrial Function during NAFLD Progression—An Independent Effect of Mitochondrial ROS Production

The progression of non-alcoholic fatty liver (NAFL) into non-alcoholic steatohepatitis implicates multiple mechanisms, chief of which is mitochondrial dysfunction. However, the sequence of events underlying mitochondrial failure are still poorly clarified. In this work, male C57BL/6J mice were fed with a high-fat plus high-sucrose diet for 16, 20, 22, and 24 weeks to induce NAFL. Up to the 20th week, an early mitochondrial remodeling with increased OXPHOS subunits levels and higher mitochondrial respiration occurred. Interestingly, a progressive loss of mitochondrial respiration along “Western diet” feeding was identified, accompanied by higher susceptibility to mitochondrial permeability transition pore opening. Importantly, our findings prove that mitochondrial alterations and subsequent impairment are independent of an excessive mitochondrial reactive oxygen species (ROS) generation, which was found to be progressively diminished along with disease progression. Instead, increased peroxisomal abundance and peroxisomal fatty acid oxidation-related pathway suggest that peroxisomes may contribute to hepatic ROS generation and oxidative damage, which may accelerate hepatic injury and disease progression. We show here for the first time the sequential events of mitochondrial alterations involved in non-alcoholic fatty liver disease (NAFLD) progression and demonstrate that mitochondrial ROS are not one of the first hits that cause NAFLD progression.     

Mitochondrial Mutations and Genetic Factors Determining NAFLD Risk

NAFLD (non-alcoholic fatty liver disease) is a widespread liver disease that is often linked with other life-threatening ailments (metabolic syndrome, insulin resistance, diabetes, cardiovascular disease, atherosclerosis, obesity, and others) and canprogress to more severe forms, such as NASH (non-alcoholic steatohepatitis), cirrhosis, and HCC (hepatocellular carcinoma). In this review, we summarized and analyzed data about single nucleotide polymorphism sites, identified in genes related to NAFLD development and progression. Additionally, the causative role of mitochondrial mutations and mitophagy malfunctions in NAFLD is discussed. The role of mitochondria-related metabolites of the urea cycle as a new non-invasive NAFLD biomarker is discussed. While mitochondria DNA mutations and SNPs (single nucleotide polymorphisms) canbe used as effective diagnostic markers and target for treatments, age and ethnic specificity should be taken into account.     

Mitochondrial Molecular Pathophysiology of Nonalcoholic Fatty Liver Disease: A Proteomics Approach

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver condition that can progress to nonalcoholic steatohepatitis, cirrhosis and cancer. It is considered an emerging health problem due to malnourishment or a high-fat diet (HFD) intake, which is observed worldwide. It is well known that the hepatocytes’ apoptosis phenomenon is one of the most important features of NAFLD. Thus, this review focuses on revealing, through a proteomics approach, the complex network of protein interactions that promote fibrosis, liver cell stress, and apoptosis. According to different types of in vitro and murine models, it has been found that oxidative/nitrative protein stress leads to mitochondrial dysfunction, which plays a major role in stimulating NAFLD damage. Human studies have revealed the importance of novel biomarkers, such as retinol-binding protein 4, lumican, transgelin 2 and hemoglobin, which have a significant role in the disease. The post-genome era has brought proteomics technology, which allows the determination of molecular pathogenesis in NAFLD. This has led to the search for biomarkers which improve early diagnosis and optimal treatment and which may effectively prevent fatal consequences such as cirrhosis or cancer.     

The Association between Hepatic Encephalopathy and Diabetic Encephalopathy: The Brain-Liver Axis

Hepatic encephalopathy (HE) is one of the main consequences of liver disease and is observed in severe liver failure and cirrhosis. Recent studies have provided significant evidence that HE shows several neurological symptoms including depressive mood, cognitive dysfunction, impaired circadian rhythm, and attention deficits as well as motor disturbance. Liver disease is also a risk factor for the development of diabetes mellitus. Diabetic encephalopathy (DE) is characterized by cognitive dysfunction and motor impairment. Recent research investigated the relationship between metabolic changes and the pathogenesis of neurological disease, indicating the importance between metabolic organs and the brain. Given that a diverse number of metabolites and changes in the brain contribute to neurologic dysfunction, HE and DE are emerging types of neurologic disease. Here, we review significant evidence of the association between HE and DE, and summarise the common risk factors. This review may provide promising therapeutic information and help to design a future metabolic organ-related study in relation to HE and DE.     

Mitochondrial Mechanisms of Necroptosis in Liver Diseases

Cell death represents a basic biological paradigm that governs outcomes and long-term sequelae in almost every hepatic disease. Necroptosis is a common form of programmed cell death in the liver. Necroptosis can be activated by ligands of death receptors, which then interact with receptor-interactive protein kinases 1 (RIPK1). RIPK1 mediates receptor interacting receptor-interactive protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) and necrosome formation. Regarding the molecular mechanisms of mitochondrial-mediated necroptosis, the RIPK1/RIPK3/MLKL necrosome complex can enhance oxidative respiration and generate reactive oxygen species, which can be a crucial factor in the susceptibility of cells to necroptosis. The necrosome complex is also linked to mitochondrial components such as phosphoglycerate mutase family member 5 (PGAM5), metabolic enzymes in the mitochondrial matrix, mitochondrial permeability protein, and cyclophilin D. In this review, we focus on the role of mitochondria-mediated cell necroptosis in acute liver injury, chronic liver diseases, and hepatocellular carcinoma, and its possible translation into clinical applications.     

Role of Impaired Mitochondrial Dynamics Processes in the Pathogenesis of Alzheimer’s Disease

Mitochondrial dysfunction is now recognized as a contributing factor to neurodegenerative diseases, including Alzheimer’s disease (AD). Mitochondria are signaling organelles with a variety of functions ranging from energy production to the regulation of cellular metabolism, energy homeostasis, and response to stress. The successful functioning of these complex processes is critically dependent on the accuracy of mitochondrial dynamics, which includes the ability of mitochondria to change shape and position in the cell, which is necessary to maintain proper function and quality control, especially in polarized cells such as neurons. There has been much evidence to suggest that the disruption of mitochondrial dynamics may play a critical role in the pathogenesis of AD. This review highlights aspects of altered mitochondrial dynamics in AD that may contribute to the etiology of this debilitating condition. We also discuss therapeutic strategies to improve mitochondrial dynamics and function that may provide an alternative treatment approach.