Identification of the differential and synergic lipotoxic patterns of oleic acid, palmitic acid, and their mixture in 3D HepG2/C3A tissue using liver-on-chip technology.

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.

Changing Nomenclature from Nonalcoholic Fatty Liver Disease to Metabolic Dysfunction-Associated Fatty Liver Disease – Not Only Premature But Also Confusing

Purpose: To establish a free fatty acid (FFA)-induced non-alcoholic fatty liver disease (NAFLD) model in HepG2 cells.Methods: HepG2 cells were incubated with 0.1, 1, or 5 mM oleic acid (OA) or palmitic acid (PA) for 24 h. Histological features were examined by oil-red-O staining. Expression levels of metabolic genes (peroxisome proliferator activated receptors α/γ, sterol regulatory element binding proteins 1a/1c, acetyl-CoA carboxylase, acyl-CoA oxidase, and fatty acid synthase), antioxidative genes (catalase and superoxide dismutases 1/2), and cytochrome P450 genes (CYP1A2, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11) were determined by reverse transcription-real time polymerase chain reaction (RT-qPCR).Results: Intracellular lipid storage was observed in cells treated with 1 mM OA or PA while cell shrinkage was present at 5 mM concentrations of both. Expression of all metabolic genes were elevated by 1 mM PA and 5 mM OA and PA. Expression of all antioxidative genes were diminished by 5 mM OA whereas 5 mM PA only reduced superoxide dismutase-2 expression. Expression of CYP1A2, CYP2D6, and CYP3A4 genes were down-regulated by both FFAs, CYP2C19 was induced by PA, while CYP2E1 and CYP4A11 were up-regulated in a concentration-dependent manner.Conclusion: PA was the more potent steatogenic agent in an OA- or PA- induced NAFLD model in HepG2 cells. Increase in intracellular hepatic lipid and expression of metabolic genes, suppression of antioxidative genes, suppression of CYP1A2, CYP2D6, and CYP3A4, and induction of CYP2E1 andCYP4A11 correlated with the multiple-hit pathogenesis model of NAFLD. These findings suggest that PA-induced NAFLD model in HepG2 cells is a suitable in vitro model for studying novel therapeutic approaches to NAFLD treatment.
 Keywords: NAFLD, Multiple-hit pathogenesis, Free fatty acid, Oleic acid, Palmitic acid

Many controversies regarding the association of liver miRNAs with obesity and nonalcoholic fatty liver diseases (NAFLD) call for additional validations. This study sought to investigate variations in genes and hepatic miRNAs in a sample of obese patients with or without NAFLD and human hepatocytes (HH). A total of 60 non-consecutive obese women following bariatric surgery were recruited. Subjects were classified as NAFLD (n=17), borderline (n=24) and controls (n=19) with normal enzymatic profile, liver histology and ultrasound assessments. Profiling of 744 miRNAs was performed in 8 obese women with no sign of hepatic disease and 11 NAFLD patients. Additional validation and expression of genes related to de novo fatty acid (FA) biosynthesis, uptake, transport and β-oxidation; glucose metabolism, and inflammation was tested in the extended sample. Induction of NAFLD-related genes and miRNAs was examined in HepG2 cells and primary HH treated with palmitic acid (PA), a combination of palmitate and oleic acid, or high glucose, and insulin (HG) mimicking insulin resistance in NAFLD. In the discovery sample, 14 miRNAs were associated with NAFLD. Analyses in the extended sample confirmed decreased miR-139-5p, miR-30b-5p, miR-122-5p and miR-422a, and increased miR-146b-5p in obese subjects with NAFLD. Multiple linear regression analyses disclosed that NAFLD contributed independently to explain miR-139-5p (P=0.005), miR-30b-5p (P=0.005), miR-122-5p (P=0.021), miR-422a (P=0.007) and miR-146a (P=0.033) expression variance after controlling for confounders. Decreased miR-122-5p in liver was associated with impaired FA usage. Expression of inflammatory and macrophage-related genes was opposite to decreased miR-30b-5p, miR-139-5p and miR-422a, whereas increased miR-146b-5p was associated with FABP4 and decreased glucose metabolism and FA mobilization. In partial agreement, PA (but not HG) led to decreased miR-139-5p, miR-30b-5p, miR-422a and miR-146a in vitro, in parallel with increased lipogenesis and FA transport, decreased glucose metabolism and diminished FA oxidation. This study confirms decreased liver glucose and lipid metabolism but increased FA biosynthesis coupled with changes in five unique miRNAs in obese patients with NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic accumulation of lipids. Antisteatotic effects of cerium oxide nanoparticles (CeO2NPs) have recently been shown in animal models of liver disease. However, it is unclear whether the activity of CeO2NPs is related solely to the decrease in oxidative stress or, in addition, they directly decrease liver fatty acid accumulation. To address this question, in this work, we used an in vitro model of hepatocellular steatosis, exposing HepG2 cells to oleic and palmitic acid. Cell uptake of CeO2NPs and their effect on oxidative stress and viability of hepatic cells cultured with H2O2 were also evaluated. Results show that CeO2NPs were uptaken by HepG2 cells and reduced oxidative stress and improved cell viability. Treatment with oleic and palmitic acid increased lipogenesis and the content of different fatty acids. CeO2NPs reduced palmitic and stearic acid and most fatty acids consisting of more than 18 carbon atoms. These effects were associated with significant changes in elongase and desaturase activity. In conclusion, CeO2NPs directly protected HepG2 cells from cell injury in oxidative stress conditions and reduced fatty acid content in steatotic conditions by inducing specific changes in fatty acid metabolism, thus showing potential in the treatment of NAFLD.

Anandamide (arachidonylethanolamide) is known as an endogenous agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed anandamide, was solubilized from the microsomal fraction of porcine brain with 1% Triton X-100. The enzyme was partially purified by Phenyl-5PW hydrophobic chromatography to a specific activity of approximately 0.37 mumol/min/mg of protein at 37 degrees C. As assayed with 14C-labeled substrates, the apparent Km value for anandamide was 60 microM, and anandamide was more active than ethanolamides of linoleic, oleic, and palmitic acids. Ceramidase and protease activities were not detected in our enzyme preparation. The purified enzyme also synthesized anandamide from free arachidonic acid in the presence of a high concentration of ethanolamine with a specific activity of about 0.16 mumol/min/mg of protein at 37 degrees C. On the basis of cochromatographies, pH dependence, heat inactivation, and effects of inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride, it was suggested that the anandamide amidohydrolase and synthase activities were attributable to a single enzyme protein.

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.

Metabolic dysfunction-associated fatty liver disease: from basic research to clinical application.

The Liver and Heart: How the Two Most Beloved “Babies” in the Human Body Communicate

Oleic acid improves hepatic lipotoxicity injury by alleviating autophagy dysfunction

The objective of this study was to compare the long-term effects of oleic (cis 18:1), elaidic (trans 18:1), and palmitic (16:0) acids on hepatic lipoprotein production, using HepG2 cells as an experimental model. The net accumulation in the medium of apolipoprotein A-I (apoA-I) was not significantly altered by fatty acids, whereas that of apoB was increased with oleic and elaidic acids. Oleic acid, and to a lesser extent elaidic and palmitic acids, increased the mass of triglycerides in the medium and the incorporation of [(3)H]glycerol into secreted triglycerides. The incorporation of [(14)C]acetate into cellular and secreted total cholesterol was stimulated by 96% and 83%, respectively, with elaidic acid but was not significantly modified by oleic or palmitic acid. Relative to oleic acid, the secretion of (14)C-labeled phospholipids and triglycerides was decreased 28% to 31% with elaidic and palmitic acids whereas that of free cholesterol and cholesteryl esters was enhanced 93% and 73%, respectively, with elaidic acid but remained unchanged with palmitic acid. Compared with oleic acid, elaidic acid stimulated the secretion of very low density lipoprotein cholesterol (VLDL-Chol), low density lipoprotein cholesterol (LDL-Chol), and high density lipoprotein cholesterol (HDL-Chol) by 43%, 70%, and 34%, respectively, whereas palmitic acid decreased VLDL-Chol but had no significant effect on LDL-Chol and HDL-Chol. The ratios of total cholesterol to HDL-Chol were 3.17, 3.60, and 3.25 with oleic, elaidic, and palmitic acids, respectively; the corresponding ratios of LDL-Chol to HDL-Chol were 0.87, 1.10, and 0.93, respectively. Compared with oleic and palmitic acids, the LDL and HDL particles secreted in the presence of elaidic acid contained higher levels of free cholesterol and cholesteryl esters and a lower content of phospholipids. The phospholipid-to-total cholesterol ratios of HDL were 1.05, 0.40, and 0.76 with oleic, elaidic, and palmitic acids, respectively. Our results indicate that in comparison with cis monounsaturated and saturated fatty acids, trans fatty acids have more adverse effects on the concentration and composition of lipoproteins secreted by HepG2 cells.

Objective To analyze the serum levels of medium-and long-chain free fatty acids (FFAs)in patients with hyperlipidemic non-alcoholic fatty liver disease (NAFLD) in order to shed some light on prevention and treatment of NAFLD.Methods The clinical data of 125 patients with high triglyceride (TG)levels who were treated in Hebei General Hospital from January 2011 to May 2011 were analyzed in this study.They were further divided into HF group (n =64) and H group (n =61) based on the presence of NAFLD or not.In addition,63 healthy individuals were recruited from the Central Hospital of Handan during the same period as the control group (N group).Serum medium-and long-chain FFAs were detected by gas chromatography.The body mass index (BMI),abdominal circumference,blood pressure,fasting blood glucose (FBG),and serum lipids including TG,total cholesterol (TC),high-density lipoprotein cholesterol (HDL-C),and low-density lipoprotein cholesterol (LDL-C) were measured.Results Compared with the N group,the H group had significantly higher BMI [(25.24 ± 1.41) kg/m2 vs.(24.32 ± 1.12) kg/m2,P =0.004],abdominal circumference [(84.72 ± 1.34) cm vs.(77.33 ±0.89) cm,P =0.010],and diastolic blood pressure [(77.35±1.21) mmHgvs.(75.21 ±1.61) mmHg,P=0.014]; also,the serum TG [(2.86±0.55) mmol/Lvs.(0.93±0.27) mmol/L,P=0.000] andTC levels [(4.56±0.66) mmol/Lvs.(4.36±0.47) mmol/L,P=0.000],serum myristic acid (C14∶0) [(0.49±0.04)％ vs.(0.36±0.01)％,P=0.011],palmitic acid (C16 ∶ 0) [(18.36 ± 0.47) ％ vs.(15.97 ± 0.30) ％,P =0.000],palmitoleic acid (C16∶ 1) [(1.00±0.12)％ vs.(0.58±0.02)％,P=0.001],and oleic acid (C18 ∶ 1) [(18.20±0.70) ％ vs.(12.23 ± 0.37) ％,P =0.000] all significantly increased,while stearic acid (C18 ∶ 0) [(7.52 ±0.22)％ vs.(8.15 ±0.28)％,P=0.012],eicosadienoic acid (C20 ∶ 2) [(0.61 ±0.07)％ vs.(1.03 ±0.17) ％,P =0.000],eicosatrienoic acid (C20 ∶ 3) [(1.77 ± 0.15) ％ vs.(2.49 ± 0.18) ％,P =0.002],and docosahexenoic acid (C22 ∶ 6) [(1.44 ± 0.08) ％ vs.(1.67 ± 0.09) ％,P =0.014] significantly decreased.Compared with the H group,the HF group had significantly higher weight [(85.76 ± 3.10) kg vs.(71.45 ±2.88) kg,P =0.003],abdominal circumference [(96.30 ±2.05) cm vs.(84.72 ± 1.34) cm,P=0.000],systolic blood pressure [(117.12 ±1.15) mmHg vs.(113.23 ±1.25) mmHg,P=0.009],and diastolic blood pressure [(79.54 ± 1.42) mmHg vs.(77.35 ± 1.21) mmHg,P =0.016],whereas the sreum FGB,TG,TC,HDL-C,and LDL-C showed no significant differentces; serum palmitic acid (C16 ∶ 0)[(19.54 ± 0.30) ％ vs.(18.36 ± 0.47) ％,P =0.000] also significantly increased.The serum level of eicosadienoic acid (C20 ∶ 2) in HF group was between that in N group and H group [(0.78 ± 0.09) ％ vs.(1.03 ±0.17)％,(0.78±0.09)％ vs.(0.61 ±0.07)％,both P=0.000].Conclusions The increased serum level of palmitic acid may be a risk factor for NAFLD.Reducing saturated fatty acids and increasing unsaturated acids in diets may be helpful for preventing NAFLD. Key words: Hyperlipidemia; Non-alcoholic fatty liver disease; Free fatty acid; Gas chromatography

(1) Background: Cadmium (Cd) is a potentially toxic element with a long half-life in the human body (20-40 years). Cytotoxicity mechanisms of Cd include increased levels of oxidative stress and apoptotic signaling, and recent studies have suggested that these aspects of Cd toxicity contribute a role in the pathobiology of non-alcoholic fatty liver disease (NAFLD), a highly prevalent ailment associated with hepatic lipotoxicity and an increased generation of reactive oxygen species (ROS). In this study, Cd toxicity and its interplay with fatty acid (FA)-induced lipotoxicity have been studied in intestinal epithelium and liver cells; the cytoprotective function of melatonin (MLT) has been also evaluated. (2) Methods: human liver cells (HepaRG), primary murine hepatocytes and Caco-2 intestinal epithelial cells were exposed to CdCl2 before and after induction of lipotoxicity with oleic acid (OA) and/or palmitic acid (PA), and in some experiments, FA was combined with MLT (50 nM) treatment. (3) Results: CdCl2 toxicity was associated with ROS induction and reduced cell viability in both the hepatic and intestinal cells. Cd and FA synergized to induce lipid droplet formation and ROS production; the latter was higher for PA compared to OA in liver cells, resulting in a higher reduction in cell viability, especially in HepaRG and primary hepatocytes, whereas CACO-2 cells showed higher resistance to Cd/PA-induced lipotoxicity compared to liver cells. MLT showed significant protection against Cd toxicity either considered alone or combined with FFA-induced lipotoxicity in primary liver cells. (4) Conclusions: Cd and PA combine their pro-oxidant activity to induce lipotoxicity in cellular populations of the gut-liver axis. MLT can be used to lessen the synergistic effect of Cd-PA on cellular ROS formation.

Objectives: Nonalcoholic fatty liver disease (NAFLD) is characterized by increased fatty acid levels in serum and liver. The mechanism of NAFLD is unclear. The role of endoplasmic reticulum (ER) stress attracts attention. First aim of this study was to design an in vitro NAFLD model. The effects of palmitic acid (PA) alone or combination with oleic acid (OA) on intracellular reactive oxygen species (ROS) production and ER stress in liver cells were investigated as a second aim. Materials and Methods: AML12 cells were exposed to PA and/or OA with different concentrations and combinations. Intracellular lipids and cell viability were detected with Oil red O staining and WST-1 assay respectively. Intracellular ROS accumulation was measured by flow cytometry analysis. E xpression of ER stress proteins, BiP and IRE1, were evaluated with western blot analysis. Results: Intracellular lipid content was increased in all treated groups. Cell viability was decreased whereas ROS generation and expression of the ER stress proteins were increased in cells treated with PA. However these effects were not observed in the cells treated with OA+PA combination. Conclusion: PA induces ROS generation and the ER stress pathway that is mediated by IRE1 in liver cells. Addition of OA enhances these effects.

Fertility in high-producing dairy cows has declined over the last decades. An increased serum and follicular fluid concentration of non-esterified fatty acids (NEFAs), due to body fat mobilization in the early post partum period, has been postulated as a cause for this fertility decline. NEFA concentrations and composition may change in the environment of the oocyte and thus might affect the storage depots of esterified NEFAs in the oocyte. We exposed COCs to unsaturated (oleic acid) or saturated (palmitic acid) NEFAs during maturation and subsequently examined lipid droplets and developmental competence of the oocytes. COCs from 3-8 mm follicles of slaughterhouse ovaries were cultured in control maturation medium (TCM-199) and medium containing 100, 250, or 500 μM oleic and/or palmitic acid (10 mM fatty acid was bound to 10% BSA fatty acid free). These concentrations were based on in vivo measured NEFA concentrations in follicular fluid in the early post partum period (Leroy et al. 2005 Reproduction 130, 485-495). After 23 h of maturation, COCs were fertilized (450 per group) and cultured till the blastocyst stage, or fixed (80 per group) for lipid droplet staining with C1-BODIPY® 500/510 C12. Confocal microscopy was performed to determine lipid droplet size in (im(mean) and the number of lipid droplets per oocyte. Lipid droplet number and the log of size were analyzed using analysis of variances with condition as fixed factor. Variation was described as the standard error of the mean. Similar concentrations of palmitic or oleic acid had an opposite effect on the size of lipid droplets in oocytes. The number of lipid droplets dramatically decreased in oocytes exposed to 500 μM palmitic acid (178 ± 20), whereas the number increased after exposure to 500 μM oleic acid (554 ± 15). The number of lipid droplets of oocytes exposed to a combination of 250 μM palmitic acid and 250 μM oleic acid (421 ± 23) was comparable with the control and lower oleic and palmitic acid concentrations. Exposure of COCs to palmitic acid during maturation resulted in reduced blastocyst development in a dose-dependent manner (from 18 ± 1.4%, 13 ± 2.4% to 2.8 ± 1.3% after exposure to 500 μM) when compared to control (20 ± 2.2%) or oocytes exposed to oleic acid (from 23 ± 1.6%, 23 ± 3.3% till 28 ± 3.3%). Negative effects of palmitic acid were counteracted by simultaneous exposure to oleic acid during in vitro oocyte maturation (26 ± 5.5%). We conclude that palmitic acid elicited negative effects on early embryonic development, possibly because it induces a reduction in the number of lipid droplets. These adverse effects can be offset by oleic acid during maturation. Moreover a high oleic acid concentration increased the number and size of lipid droplets of oocytes. The regulatory pathways involved in the noted differences in lipid storage features of in vitro-matured oocytes as well as the adverse effects of palmitic acid on early embryonic development are currently under research.