Model Of Steatosis Research Articles

Liraglutide Exerts Protective Effects by Downregulation of PPARγ, ACSL1 and SREBP-1c in Huh7 Cell Culture Models of Non-Alcoholic Steatosis and Drug-Induced Steatosis.

(1) Background: With the aging of the population and polypharmacy encountered in the elderly, drug-induced steatosis (DIS) has become frequent cause of non-alcoholic steatosis (NAS). Indeed, NAS and DIS may co-exist, making the ability to distinguish between the entities ever more important. The aim of our study was to study cell culture models of NAS and DIS and determine the effects of liraglutide (LIRA) in those models. (2) Methods: Huh7 cells were treated with oleic acid (OA), or amiodarone (AMD) to establish models of NAS and DIS, respectively. Cells were treated with LIRA and cell viability was assessed by MTT, lipid accumulation by Oil-Red-O staining and triglyceride assay, and intracellular signals involved in hepatosteatosis were quantitated by RT-PCR. (3) Results: After exposure to various OA and AMD concentrations, those that achieved 80% of cells viabilities were used in further experiments to establish NAS and DIS models using 0.5 mM OA and 20 µM AMD, respectively. In both models, LIRA increased cell viability (p < 0.01). Lipid accumulation was increased in both models, with microsteatotic pattern in DIS, and macrosteatotic pattern in NAS which corresponds to greater triglyceride accumulation in latter. LIRA ameliorated these changes (p < 0.001), and downregulated expression of lipogenic ACSL1, PPARγ, and SREBP-1c pathways in the liver (p < 0.01) (4) Conclusions: LIRA ameliorates hepatocyte steatosis in Huh7 cell culture models of NAS and DIS.

Bempedoic acid induces PNPLA3 expression in the liver of female rats fed a high-fat diet supplemented with liquid fructose

Background and Aims : Bempedoic acid (BemA) reduces LDL-cholesterol levels in humans, but plasma triglyceride (TG) levels remains unaffected. We studied BemA effects on plasma TG in a diet-induced rat model of hepatic steatosis, and explored the mechanisms involved.

Role of lysosomal acid lipase in PPAR-mediated reduction of lipid accumulation in hepatocytes

Background and Aims : Lysosomal acid lipase (LAL) catalyzes the hydrolysis of cholesteryl esters and triglycerides in the lysosomal compartment. Recently, it was shown that NAFLD patients can develop an acquired LAL deficiency which is rescued by PPAR-alpha activation. Aim of the study was to investigate the role of other isoform-specific and dual PPAR agonists on lipid accumulation and LAL activity using an in vitro model of steatosis.

Investigating histological changes and gene expression of interleukin-8, interleukin-12, and interferon beta in liver tissue after consumption of Lactobacillus rhamnosus GG probiotic in steatosis model rats

Investigating histological changes and gene expression of interleukin-8, interleukin-12, and interferon beta in liver tissue after consumption of Lactobacillus rhamnosus GG probiotic in steatosis model rats

The aqueous extract of Phragmites rhizome improves hepatic steatosis in obese mice via the AMPK-mediated inhibition of ER stress

Phragmites rhizome has been documented to have antioxidative stress and anti-inflammation properties in several pathogenic models. We aimed to investigate the therapeutic effects and molecular mechanism of the aqueous extract of Phragmites rhizome (AP) in in vitro and in vivo hepatic steatosis models. We found that treatment with AP dose-dependently reduced lipid accumulation and endoplasmic reticulum (ER) stress in palmitate-treated human primary hepatocytes and attenuated hepatic steatosis and ER stress in high-fat diet (HFD)-fed mice. AMPK phosphorylation and autophagy markers in hepatocytes were augmented by AP. siRNA targeting AMPK or 3-methyladenine (3-MA) abolished the impacts of AP on palmitate-treated hepatocytes. These results suggest that AP treatment suppresses hepatic ER stress through AMPK/autophagy signaling, leading to attenuation of hepatic steatosis. The present study may provide a novel therapeutic approach for treating nonalcoholic fatty liver disease (NAFLD) and ER stress-mediated metabolic diseases using natural substances.

Beneficial Effects of Standardized Extracts from Wastes of Red Oranges and Olive Leaves.

The awareness of the large amount of waste produced along the food chain, starting in the agricultural sector and continuing across industrial transformation to the domestic context, has in recent years also aroused strong concern amongst the public, who are ing about the possible consequences that this could have on environmental sustainability, resource waste and human health. The aim of the present research is the recovery of substances with high added value from waste and by-products typical of the Mediterranean area, such as the residue from the industrial processing of red oranges, called pastazzo (peels, pulps and seeds), which is particularly rich in anthocyanins, flavanones and hydroxycinnamic acids, and has numerous nutraceutical properties, as well as the olive leaves coming from olive-tree pruning, which are rich in substances such as oleuropein, elenolic acid, hydroxytyrosol, tyrosol and rutin. The effect of Red Orange Extract (ROE) and Olive Leaf Extract (OLE) on HepG2 fatty storage capacity was assessed performing Oil Red O’ staining, and antioxidant properties of the extracts were evaluated following the steatosis model onset. Based on the results obtained, the preparation of natural extracts that are derived from these waste products can be useful for preventing, counteracting or delaying the onset of the complications of fatty liver disease, such as hepatic steatosis.

Reduction in gut-derived MUFAs via intestinal stearoyl-CoA desaturase 1 deletion drives susceptibility to NAFLD and hepatocarcinoma.

Nonalcoholic fatty liver disease (NAFLD) is defined by a set of hepatic conditions ranging from steatosis to steatohepatitis (NASH), characterized by inflammation and fibrosis, eventually predisposing to hepatocellular carcinoma (HCC). Together with fatty acids (FAs) originated from adipose lipolysis and hepatic lipogenesis, intestinal‐derived FAs are major contributors of steatosis. However, the role of mono‐unsaturated FAs (MUFAs) in NAFLD development is still debated. We previously established the intestinal capacity to produce MUFAs, but its consequences in hepatic functions are still unknown. Here, we aimed to determine the role of the intestinal MUFA‐synthetizing enzyme stearoyl‐CoA desaturase 1 (SCD1) in NAFLD. We used intestinal‐specific Scd1‐KO (iScd1 −/−) mice and studied hepatic dysfunction in different models of steatosis, NASH, and HCC. Intestinal‐specific Scd1 deletion decreased hepatic MUFA proportion. Compared with controls, iScd1 −/− mice displayed increased hepatic triglyceride accumulation and derangement in cholesterol homeostasis when fed a MUFA‐deprived diet. Then, on Western diet feeding, iScd1 −/− mice triggered inflammation and fibrosis compared with their wild‐type littermates. Finally, intestinal‐Scd1 deletion predisposed mice to liver cancer. Conclusions: Collectively, these results highlight the major importance of intestinal MUFA metabolism in maintaining hepatic functions and show that gut‐derived MUFAs are protective from NASH and HCC.

SAT096 - Exposure to the SARS-CoV-2 spike protein in an in vitro model of steatosis enhances hepatocyte oxidative stress: implications for COVID-19 and accelerated disease progression

SAT096 - Exposure to the SARS-CoV-2 spike protein in an in vitro model of steatosis enhances hepatocyte oxidative stress: implications for COVID-19 and accelerated disease progression

Design and Evaluation of Autophagy-Inducing Particles for the Treatment of Abnormal Lipid Accumulation.

Autophagy is a fundamental housekeeping process by which cells degrade their components to maintain homeostasis. Defects in autophagy have been associated with aging, neurodegeneration and metabolic diseases. Non-alcoholic fatty liver diseases (NAFLDs) are characterized by hepatic fat accumulation with or without inflammation. No treatment for NAFLDs is currently available, but autophagy induction has been proposed as a promising therapeutic strategy. Here, we aimed to design autophagy-inducing particles, using the autophagy-inducing peptide (Tat-Beclin), and achieve liver targeting in vivo, taking NAFLD as a model disease. Polylactic acid (PLA) particles were prepared by nanoprecipitation without any surfactant, followed by surface peptide adsorption. The ability of Tat-Beclin nanoparticles (NP T-B) to modulate autophagy and to decrease intracellular lipid was evaluated in vitro by LC3 immunoblot and using a cellular model of steatosis, respectively. The intracellular localization of particles was evaluated by transmission electron microscopy (TEM). Finally, biodistribution of fluorescent NP T-B was evaluated in vivo using tomography in normal and obese mice. The results showed that NP T-B induce autophagy with a long-lasting and enhanced effect compared to the soluble peptide, and at a ten times lower dose. Intracellular lipid also decreased in a cellular model of NAFLD after treatment with T-B and NP T-B under the same dose conditions. Ultrastructural studies revealed that NP T-B are internalized and located in endosomal, endolysosomal and autolysosomal compartments, while in healthy and obese mice, NP T-B could accumulate for several days in the liver. Given the beneficial effects of autophagy-inducing particles in vitro, and their capacity to target the liver of normal and obese mice, NP T-B could be a promising therapeutic tool for NAFLDs, warranting further in vivo investigation.

Bacteroides fight fatty liver.

Sphingolipids from commensal bacteria transit to the liver and normalize lipid accumulation in a mouse model of hepatic steatosis.

Primary Broiler Hepatocytes for Establishment of a Steatosis Model.

Fatty liver hemorrhage syndrome (FLHS) in chickens is characterized by steatosis and bleeding in the liver, which has caused huge losses to the poultry industry. This study aimed to use primary cultured broiler hepatocytes to establish a steatosis model to explore the optimal conditions for inducing steatosis by incubating the cells with a fat emulsion. Primary hepatocytes were isolated from an AA broiler by a modified two-step in situ perfusion method. Hepatocytes were divided into an untreated control group and a fat emulsion group that was incubated with 2.5, 5, 10, or 20% fat emulsion for different times to determine the optimal conditions for inducing steatosis of primary hepatocytes. Incubation of the cells with 10% fat emulsion resulted in cell viability at 48 h of 67%, which was higher than the control group and met the requirements of the model. In the second experiment, steatosis was induced by incubating hepatocytes with 10% fat emulsion for 48 h. In consequence, the apoptosis rate decreased (p &gt; 0.05) and the concentration of ALT (p &lt; 0.001), AST (p &lt; 0.01), and TG (p &lt; 0.05) increased significantly; the expression level of SREBP-1c (p &lt; 0.05) increased, and the expression levels of PPARα (p &lt; 0.001), CPT1 (p &lt; 0.001), and CPT2 (p &lt; 0.05) were lower in the fat emulsion group than in the control group. In conclusion, the induction condition was selected as 10% fat emulsion incubation for 48 h, and we successfully established a fatty liver degeneration model for broilers.

Fatty liver? Microbiome sphingolipids to the rescue

In this issue of Cell Host & Microbe, Le etal. discover that addition of exogenous Bacteroides sphingolipids can reverse lipid accumulation in the liver in a mouse model of hepatic steatosis. An elegant labeling strategy also revealed a unique microbially produced sphingolipid that was able to transit to the liver.

Host hepatic metabolism is modulated by gut microbiota-derived sphingolipids

Microbially-derived gut metabolites are important contributors to host phenotypes, many of which may link microbiome composition to metabolic disease. However, relatively few metabolites with known bioactivity have been traced from specific microbes to host tissues. Here, we use a labeling strategy to characterize and trace bacterial sphingolipids from the gut symbiont Bacteroides thetaiotaomicron to mouse colons and livers. We find that bacterial sphingolipid synthesis rescues excess lipid accumulation in a mouse model of hepatic steatosis and observe the transit of a previously uncharacterized bacterial sphingolipid to the liver. The addition of this sphingolipid to hepatocytes improves respiration in response to fatty-acid overload, suggesting that sphingolipid transfer to the liver could potentially contribute to microbiota-mediated liver function. This work establishes a role for bacterial sphingolipids in modulating hepatic phenotypes and defines a workflow that permits the characterization of other microbial metabolites with undefined functions in host health.

Relationship between Liver Stiffness and Steatosis in Obesity Conditions: In Vivo and In Vitro Studies.

Obesity is a major risk factor for metabolic dysfunction such as non-alcoholic fatty liver disease (NAFLD). The NAFLD spectrum ranges from simple steatosis, to steatohepatitis, fibrosis, and cirrhosis. The aim of this study is to characterize the grade of steatosis being associated with overnutrition and obesity, both at the level of single hepatocyte and whole liver, and to correlate it with the hepatocyte/liver stiffness and dysfunction. For the in vivo study, 60 subjects were enrolled and grouped based on the stage of liver steatosis/fibrosis according to biochemical analyses, liver ultrasonography (USG) and acoustic radiation force impulse shear wave elastography (ARFI-SWE). For single hepatocyte analyses we employed in vitro models of moderate and severe steatosis on which to assess the single cell biomechanics by Single Cell Force Spectroscopy (SCFS) and Quantitative Phase Microscopy (QPM). Results show that in vivo liver stiffness depends mainly on the extent of fat accumulation and not on fibrosis. These results parallel the in vitro observations showing that hepatocyte stiffness and dysfunction increase with increasing fat accumulation and lipid droplet enlargement. Our findings indicate that the extent of steatosis markedly affects the biomechanical properties of both liver and single hepatocytes thus proving insights about the role of modulation of liver/hepatocyte elasticity as a physical mechanism transducing the obesity-dependent excess of plasmatic lipids towards liver steatosis and dysfunction.

Characteristics of Urine Organic Acid Metabolites in Nonalcoholic Fatty Liver Disease Assessed Using Magnetic Resonance Imaging with Elastography in Korean Adults.

The liver is an essential organ that manufactures energy through various metabolic pathways; thus, exploring the intermediate metabolites in nonalcoholic fatty liver disease (NAFLD) may help discover novel parameters in hepatic steatosis or fibrosis. The present study aimed to investigate the traits of urine organic acid metabolites in participants with hepatic steatosis and fibrosis in nonalcoholic Korean adults. Hepatic steatosis and fibrosis, in 68 men and 65 women, were evaluated using quantification by proton density fat fraction with magnetic resonance (MR) imaging and MR elastography, respectively. Urine metabolites were obtained using a high-performance liquid chromatography–mass spectrometry analysis. The candidate metabolites were included in the logistic regression models for hepatic steatosis and fibrosis. The association between high p-hydroxyphenyllactate levels and hepatic steatosis was not independent of body mass index and Homeostatic Model Assessment-insulin resistance. High ethylmalonate, β-hydroxybutyrate, and sulfate levels were significantly related to a low probability of hepatic fibrosis, independent of covariates. In conclusion, urine metabolites were not related to hepatic steatosis independent of obesity and insulin resistance, while several metabolites were specifically associated with hepatic fibrosis. Further study is required to verify the diagnostic value of the metabolites in a population with wide-spectrum NAFLD.

Effect of Vaspin on Fatty acids metabolism in steatosis model: comparison between two-dimensional (2D) and three-dimensional (3D) cultures

Effect of Vaspin on Fatty acids metabolism in steatosis model: comparison between two-dimensional (2D) and three-dimensional (3D) cultures

Methyltransferase SETD2 mediates hepatoprotection of berberine against steatosis.

BackgroundBerberine (BBR) can alleviate nonalcoholic fatty liver (NAFL), but the mechanisms remain uncertain. Mounting evidence has underscored the roles of epigenetic modulation in the development of NAFL. Increased expression of histone methyltransferase SET domain-containing protein 2 (SETD2) is found in the livers of diabetic animals with BBR administration. Whether SETD2 contributes to the protective effects of BBR against NAFL remains to be elucidated.MethodsA C57BL/6 mouse model of NAFL induced by a high-fat high-sucrose diet (HFHS) and a palmitate-treated hepatocyte steatosis model were generated. The effects of BBR were evaluated by Oil Red O staining and the cell viability assay. Quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence staining were used to analyze the expression and activity of SETD2 and its downstream target trimethylation of lysine 36 on histone 3 (H3K36me3).ResultsBBR treatment induced the reduction of lipid droplets in both HFHS mouse livers and HepG2 cells, coincident with the elevation of the mRNA and protein expression of SETD2 and H3K36me3. Knockdown of SETD2 compromised the BBR-mediated inhibition of the accumulation of lipid droplets in the HepG2 cell model of steatosis. Moreover, upregulated SETD2 and H3K36me3 expression was also observed in intact HepG2 cells treated with BBR. The promoter assay indicated that BBR treatment increased the transcriptional activity of SETD2 and H3K36me3.ConclusionsBBR confers hepatoprotection against steatosis through transcriptional regulation of SETD2 activity.

Comparative Transcriptome Analysis Reveals That Exendin-4 Improves Steatosis in HepG2 Cells by Modulating Signaling Pathways Related to Lipid Metabolism.

No therapy exists for non-alcoholic fatty liver disease (NAFLD). However, glucagon-like peptide receptor agonists (GLP-1RAs) showed a beneficial effect on NAFLD, although the underpinning mechanisms remain unclear due to their pleiotropic effects. We examined the implicated signaling pathways using comparative transcriptomics in a cell model of steatosis to overcome pleiotropy. We treated steatotic HepG2 cells with the GLP-1RA Exendin-4 (Ex-4). We compared the transcriptome profiles of untreated steatotic, and Ex-4-treated steatotic cells, and used Ingenuity Pathway Analysis (IPA) to identify the signaling pathways and associated genes involved in the protective effect of Ex-4. Ex-4 treatment significantly reduces steatosis. RNA-seq analysis revealed 209 differentially expressed genes (DEGs) between steatotic and untreated cells, with farnesoid X receptor/retinoid X receptor (FXR/RXR) (p = 8.9 × 10−7) activation being the top regulated canonical pathway identified by IPA. Furthermore, 1644 DEGs were identified between steatotic cells and Ex-4-treated cells, with liver X receptor/retinoid X receptor (LXR/RXR) (p = 2.02 × 10−7) and FXR/RXR (p = 3.28 × 10−7) activation being the two top canonical pathways. The top molecular and cellular functions between untreated and steatotic cells were lipid metabolism, molecular transport, and small molecular biochemistry, while organismal injury and abnormalities, endocrine system disorders, and gastrointestinal disease were the top three molecular and cellular functions between Ex-4-treated and steatotic cells. Genes overlapping steatotic cells and Ex-4-treated cells were associated with several lipid metabolism processes. Unique transcriptomic differences exist between steatotic cells and Ex-4-treated steatotic cells, providing an important resource for understanding the mechanisms that underpin the protective effect of GLP-1RAs on NAFLD and for the identification of novel therapeutic targets for NAFLD.

Overexpression of NAG-1/GDF15 prevents hepatic steatosis through inhibiting oxidative stress-mediated dsDNA release and AIM2 inflammasome activation

Mitochondrial dysfunction and oxidative stress-mediated inflammasome activation play critical roles in the pathogenesis of the non-alcoholic fatty liver disease (NAFLD). Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1), or growth differentiation factor-15 (GDF15), is associated with many biological processes and diseases, including NAFLD. However, the role of NAG-1/GDF15 in regulating oxidative stress and whether this process is associated with absent in melanoma 2 (AIM2) inflammasome activation in NAFLD are unknown. In this study, we revealed that NAG-1/GDF15 is significantly downregulated in liver tissues of patients with steatosis compared to normal livers using the Gene Expression Omnibus (GEO) database, and in free fatty acids (FFA, oleic acid/palmitic acid, 2:1)-induced HepG2 and Huh-7 cellular steatosis models. Overexpression of NAG-1/GDF15 in transgenic (Tg) mice significantly alleviated HFD-induced obesity and hepatic steatosis, improved lipid homeostasis, enhanced fatty acid β-oxidation and lipolysis, inhibited fatty acid synthesis and uptake, and inhibited AIM2 inflammasome activation and the secretion of IL-18 and IL-1β, as compared to their wild-type (WT) littermates without reducing food intake. Furthermore, NAG-1/GDF15 overexpression attenuated FFA-induced triglyceride (TG) accumulation, lipid metabolism deregulation, and AIM2 inflammasome activation in hepatic steatotic cells, while knockdown of NAG-1/GDF15 demonstrated opposite effects. Moreover, NAG-1/GDF15 overexpression inhibited HFD- and FFA-induced oxidative stress and mitochondrial damage which in turn reduced double-strand DNA (dsDNA) release into the cytosol, while NAG-1/GDF15 siRNA showed opposite effects. The reduced ROS production and dsDNA release may be responsible for attenuated AIM2 activation by NAG-1/GDF15 upon fatty acid overload. In conclusion, our results provide evidence that other than regulating lipid homeostasis, NAG-1/GDF15 protects against hepatic steatosis through a novel mechanism via suppressing oxidative stress, mitochondrial damage, dsDNA release, and AIM2 inflammasome activation.

Hepatic PTEN Signaling Regulates Systemic Metabolic Homeostasis through Hepatokines-Mediated Liver-to-Peripheral Organs Crosstalk.

Liver-derived circulating factors deeply affect the metabolism of distal organs. Herein, we took advantage of the hepatocyte-specific PTEN knockout mice (LPTENKO), a model of hepatic steatosis associated with increased muscle insulin sensitivity and decreased adiposity, to identify potential secreted hepatic factors improving metabolic homeostasis. Our results indicated that protein factors, rather than specific metabolites, released by PTEN-deficient hepatocytes trigger an improved muscle insulin sensitivity and a decreased adiposity in LPTENKO. In this regard, a proteomic analysis of conditioned media from PTEN-deficient primary hepatocytes identified seven hepatokines whose expression/secretion was deregulated. Distinct expression patterns of these hepatokines were observed in hepatic tissues from human/mouse with NAFLD. The expression of specific factors was regulated by the PTEN/PI3K, PPAR or AMPK signaling pathways and/or modulated by classical antidiabetic drugs. Finally, loss-of-function studies identified FGF21 and the triad AHSG, ANGPTL4 and LECT2 as key regulators of insulin sensitivity in muscle cells and in adipocytes biogenesis, respectively. These data indicate that hepatic PTEN deficiency and steatosis alter the expression/secretion of hepatokines regulating insulin sensitivity in muscles and the lipid metabolism in adipose tissue. These hepatokines could represent potential therapeutic targets to treat obesity and insulin resistance.