Aquaporin 9 Represents a Novel Target of Chronic Liver Injury That May Antagonize Its Progression by Reducing Lipotoxicity

In recent years, chronic liver injury has become a common disease that harms human health. Its clinical manifestations are hepatic steatosis and secondary chronic steatohepatitis, which can quickly transform into liver fibrosis and cirrhosis if not treated in time. Therefore, this study is aimed at searching for new therapeutic targets of chronic liver injury and clarifying the molecular mechanisms of the new targets involved in chronic liver injury. After aquaporin 9 was identified as a target by proteomics, Aqp9-/- mice were constructed using the CRISPR/Cas9 system. Biochemical and morphological tests were used to verify the effect of Aqp9 knockout on early chronic liver injury. Proteomics, molecular biology, and morphology experiments were used to screen and verify the effects of Aqp9 knockout on its downstream pathway. Through the above experiments, we demonstrated that aquaporin 9 could be used as an intervention target for antagonizing the development of early chronic liver injury and its gene knockout affected downstream inflammation, oxidative stress, apoptosis, and pyroptosis by alleviating hepatic lipotoxicity.

Nicotinamide N-methyltransferase (NNMT) upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes

Defined as the dysfunction and/or cell death caused by toxic lipids accumulation in hepatocytes, hepatic lipotoxicity plays a pathological role in non-alcoholic fatty liver disease. The cellular and molecular mechanisms underlying lipotoxicity remain to be elucidated. In this study, using AML12 cells, a non-transformed murine hepatocyte cell line, exposed to palmitate (a 16-C saturated fatty acid) as an experimental model, we investigated the role and mechanisms of nicotinamide N-methyltransferase (NNMT), a methyltransferase catalyzing nicotinamide methylation and degradation, in hepatic lipotoxicity. We initially identified activating transcription factor 4 (ATF4) as a major transcription factor for hepatic NNMT expression. Here, we demonstrated that palmitate upregulates NNMT expression via activating ATF4 in a mechanistic target of rapamycin complex 1 (mTORC1)-dependent mechanism in that mTORC1 inhibition by both Torin1 and rapamycin attenuated ATF4 activation and NNMT upregulation. We further demonstrated that the mTORC1-dependent ATF4 activation is an integral signaling event of unfolded protein response (UPR) as both ATF4 activation and NNMT upregulation by tunicamycin, a well-documented endoplasmic reticulum (ER) stress inducer, are blunted when hepatocytes were pretreated with Torin1. Importantly, our data uncovered that NNMT upregulation contributes to palmitate-induced hepatotoxicity as NNMT inhibition, via either pharmacological (NNMT inhibitors) or genetic approach (siRNA transfection), provided protection against palmitate lipotoxicity. Our further mechanistic exploration identified protein kinase A (PKA) activation to contribute, at least, partially to the protective effect of NNMT inhibition against lipotoxicity. Collectively, our data demonstrated that NNMT upregulation by the mTORC1-ATF4 pathway activation contributes to the development of lipotoxicity in hepatocytes.

Repetitive amiodarone administration causes liver damage via adipose tissue ER-stress dependent lipolysis, leading to hepatotoxic free fatty acid accumulation

Drug-induced liver injury is an emerging form of acute and chronic liver disease that may manifest as fatty liver. Amiodarone (AMD), a widely used anti-arrhythmic drug, can cause hepatic injury and steatosis by a variety of mechanisms, not all completely understood. We hypothesized that repetitive AMD administration may induce hepatic lipotoxicity not only via effects on the liver, but also via effects on adipose tissue. Indeed, repetitive AMD administration induced endoplasmic reticulum (ER) stress in both liver and adipose tissue. In adipose tissue, AMD reduced lipogenesis and increased lipolysis. Moreover, AMD treatment induced ER stress and ER stress-dependent lipolysis in 3T3L1 adipocytes in vitro. In the liver, AMD caused increased expression of genes encoding proteins involved in fatty acid (FA) uptake and transfer (Cd36, Fabp1 and Fabp4) and resulted in increased hepatic accumulation of free FAs, but not of triacylglycerols. In line with this, there was increased expression of hepatic de novo FA synthesis genes. However, AMD significantly reduced the expression of the desaturase Scd1 and elongase Elovl6, detected at mRNA and protein levels. Accordingly, the FA profile of hepatic total lipids revealed increased accumulation of palmitate, a SCD1 and ELOVL6 substrate, and reduced levels of palmitoleate and cis-vaccenate, products of the enzymes. In addition, AMD-treated mice displayed increased hepatic apoptosis. The studies show that repetitive AMD induces ER stress and aggravates lipolysis in adipose tissue, while inducing a lipotoxic hepatic lipid environment, suggesting that AMD-induced liver damage is due to compound insult to liver and adipose tissue.

Magnesium Isoglycyrrhizinate Reduces Hepatic Lipotoxicity through Regulating Metabolic Abnormalities

The excessive accumulation of lipids in hepatocytes induces a type of cytotoxicity called hepatic lipotoxicity, which is a fundamental contributor to liver metabolic diseases (such as NAFLD). Magnesium isoglycyrrhizinate (MGIG), a magnesium salt of the stereoisomer of natural glycyrrhizic acid, is widely used as a safe and effective liver protectant. However, the mechanism by which MGIG protects against NAFLD remains unknown. Based on the significant correlation between NAFLD and the reprogramming of liver metabolism, we aimed to explore the beneficial effects of MGIG from a metabolic viewpoint in this paper. We treated HepaRG cells with palmitic acid (PA, a saturated fatty acid of C16:0) to induce lipotoxicity and then evaluated the antagonistic effect of MGIG on lipotoxicity by investigating the cell survival rate, DNA proliferation rate, organelle damage, and endoplasmic reticulum stress (ERS). Metabolomics, lipidomics, and isotope tracing were used to investigate changes in the metabolite profile, lipid profile, and lipid flux in HepaRG cells under different intervention conditions. The results showed that MGIG can indeed protect hepatocytes against PA-induced cytotoxicity and ERS. In response to the metabolic abnormality of lipotoxicity, MGIG curtailed the metabolic activation of lipids induced by PA. The content of total lipids and saturated lipids containing C16:0 chains increased significantly after PA stimulation and then decreased significantly or even returned to normal levels after MGIG intervention. Lipidomic data show that glycerides and glycerophospholipids were the two most affected lipids. For excessive lipid accumulation in hepatocytes, MGIG can downregulate the expression of the metabolic enzymes (GPATs and DAGTs) involved in triglyceride biosynthesis. In conclusion, MGIG has a positive regulatory effect on the metabolic disorders that occur in hepatocytes under lipotoxicity, and the main mechanisms of this effect are in lipid metabolism, including reducing the total lipid content, reducing lipid saturation, inhibiting glyceride and glycerophospholipid metabolism, and downregulating the expression of metabolic enzymes in lipid synthesis.

Fucoidan and Fucoxanthin Attenuate Hepatic Steatosis and Inflammation of NAFLD through Modulation of Leptin/Adiponectin Axis

Non-alcoholic fatty liver disease (NAFLD) is the emerging cause of chronic liver disease globally and lack of approved therapies. Here, we investigated the feasibility of combinatorial effects of low molecular weight fucoidan and high stability fucoxanthin (LMF-HSFx) as a therapeutic approach against NAFLD. We evaluated the inhibitory effects of LMF-HSFx or placebo in 42 NAFLD patients for 24 weeks and related mechanism in high fat diet (HFD) mice model and HepaRGTM cell line. We found that LMF-HSFx reduces the relative values of alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceride, fasting blood glucose and hemoglobin A1c in NAFLD patients. For lipid metabolism, LMF-HSFx reduces the scores of controlled attenuation parameter (CAP) and increases adiponectin and leptin expression. Interestingly, it reduces liver fibrosis in NAFLD patients, either. The proinflammatory cytokines interleukin (IL)-6 and interferon-γ are reduced in LMF-HSFx group. In HFD mice, LMF-HSFx attenuates hepatic lipotoxicity and modulates adipogenesis. Additionally, LMF-HSFx modulates SIRI-PGC-1 pathway in HepaRG cells under palmitic acid-induced lipotoxicity environment. Here, we describe that LMF-HSFx ameliorated hepatic steatosis, inflammation, fibrosis and insulin resistance in NAFLD patients. LMF-HSFx may modulate leptin-adiponectin axis in adipocytes and hepatocytes, then regulate lipid and glycogen metabolism, decrease insulin resistance and is against NAFLD.

Lipotoxic stress alters the membrane lipid profile of extracellular vesicles released by Huh-7 hepatocarcinoma cells

Extracellular vesicles (EVs) are well-known mediators in intercellular communication playing pivotal roles in promoting liver inflammation and fibrosis, events associated to hepatic lipotoxicity caused by saturated free fatty acid overloading. However, despite the importance of lipids in EV membrane architecture which, in turn, affects EV biophysical and biological properties, little is known about the lipid asset of EVs released under these conditions. Here, we analyzed phospholipid profile alterations of EVs released by hepatocarcinoma Huh-7 cells under increased membrane lipid saturation induced by supplementation with saturated fatty acid palmitate or Δ9 desaturase inhibition, using oleate, a nontoxic monounsaturated fatty acid, as control. As an increase of membrane lipid saturation induces endoplasmic reticulum (ER) stress, we also analyzed phospholipid rearrangements in EVs released by Huh-7 cells treated with thapsigargin, a conventional ER stress inducer. Results demonstrate that lipotoxic and/or ER stress conditions induced rearrangements not only into cell membrane phospholipids but also into the released EVs. Thus, cell membrane saturation level and/or ER stress are crucial to determine which lipids are discarded via EVs and EV lipid cargos might be useful to discriminate hepatic lipid overloading and ER stress.

Ferulic acid alleviates lipotoxicity-induced hepatocellular death through the SIRT1-regulated autophagy pathway and independently of AMPK and Akt in AML-12 hepatocytes

Background Lipotoxicity-induced cell death plays a detrimental role in the pathogenesis of metabolic diseases. Ferulic acid, widespread in plant-based food, is a radical scavenger with multiple bioactivities. However, the benefits of ferulic acid against hepatic lipotoxicity are largely unclear. Here, we investigated the protective effect of ferulic acid against palmitate-induced lipotoxicity and clarified its potential mechanisms in AML-12 hepatocytes. Methods AML-12 mouse hepatocytes were exposed to palmitate to mimic lipotoxicity. Different doses (25, 50, and 100 μM) of ferulic acid were added 2 h before palmitate treatment. Cell viability was detected by measuring lactate dehydrogenase release, nuclear staining, and the expression of cleaved-caspase-3. Intracellular reactive oxygen species content and mitochondrial membrane potential were analysed by fluorescent probes. The potential mechanisms were explored by molecular biological methods, including Western blotting and quantitative real-time PCR, and were further verified by siRNA interference. Results Our data showed that ferulic acid significantly inhibited palmitate-induced cell death, rescued mitochondrial membrane potential, reduced reactive oxygen species accumulation, and decreased inflammatory factor activation, including IL-6 and IL-1beta. Ferulic acid significantly stimulated autophagy in hepatocytes, whereas autophagy suppression blocked the protective effect of ferulic acid against lipotoxicity. Ferulic acid-activated autophagy, which was triggered by SIRT1 upregulation, was mechanistically involved in its anti-lipotoxicity effects. SIRT1 silencing blocked most beneficial changes induced by ferulic acid. Conclusions We demonstrated that the phytochemical ferulic acid, which is found in plant-based food, protected against hepatic lipotoxicity, through the SIRT1/autophagy pathway. Increased intake of ferulic acid-enriched food is a potential strategy to prevent and/or improve metabolic diseases with lipotoxicity as a typical pathological feature.