Polyphenolic extract attenuates fatty acid-induced steatosis and oxidative stress in hepatic and endothelial cells.

PURPOSE: Phenolic compounds (PC) of virgin olive oil exert several biochemical and pharmacological beneficial effects. Some dietary PC seem to prevent/improve obesity and metabolic-related disorders such as non-alcoholic fatty liver disease (NAFLD). We investigated the possible effects of PC extracted from olive pomace (PEOP) and of the main single molecules present in the extract (tyrosol, apigenin, oleuropein, p-coumaric and caffeic acid) in protecting hepatocytes and endothelial cells against triglyceride accumulation and oxidative stress. METHODS: Rat hepatoma and human endothelial cells were exposed to a mixture of oleate/palmitate to mimic the condition of NAFLD and atherosclerosis, respectively. Then, cells were incubated for 24 h in the absence or in the presence of PC or PEOP. Different parameters were evaluated, such as lipid accumulation and oxidative stress-related markers. RESULTS: In hepatic cells, expression of peroxisome proliferator-activated receptors (PPARs) and of stearoyl-CoA desaturase 1 (SCD-1) were assessed as index of lipid metabolism. In endothelial cells, expression of intercellular adhesion molecule-1 (ICAM-1), activation of nuclear factor kappa-B (NF-kB), release of nitric oxide (NO), and wound-healing rate were assessed as index of inflammation. CONCLUSION: PEOP extract ameliorated hepatic lipid accumulation and lipid-dependent oxidative imbalance thus showing potential applications as therapeutic agent tuning down hepatosteatosis and atherosclerosis.

Excess fructose and fatty acids trigger a model of non­alcoholic fatty liver disease progression in vitro: Protective effect of the flavonoid silybin.

Overconsumption of fats and sugars is a major cause of development of non­alcoholic fatty liver disease (NAFLD). The main objectives of the present study were to explore the pathways sustaining the interfering metabolic effects of excess fructose and fatty acids in hepatocytes, and to clarify the mechanisms through which the nutraceutical silybin rescues the functional and metabolic alterations that are associated with the NALFD progression. Cultured hepatocytes were exposed to fructose and fatty acids, alone or in combination, to induce different grades of steatosis in vitro. Cell viability, apoptosis, free radical production, lipid content, lipid peroxidation and activity of lipogenic enzymes were assessed by spectrophotometric assays. Oxygen consumption and mitochondrial respiration parameters were measured using a Seahorse analyzer. Expression of markers for liver steatosis and dysfunction were also evaluated by reverse transcription­quantitative polymerase chain reaction. The data revealed that fructose and fatty acid combination in vitro had a positive interference on lipogenic pathways, leading to more severe steatosis and liver dysfunction, reduced cell viability, increased apoptosis, oxidative stress and mitochondrial respiration. Hepatic cell abnormalities were almost completely alleviated by silybin treatment. These findings suggest that silybin may serve as a novel and cost­effective dietary supplement for treatment and/or prevention of hepatosteatosis associated with NAFLD progression.

The Nutraceutic Silybin Counteracts Excess Lipid Accumulation and Ongoing Oxidative Stress in an In Vitro Model of Non-Alcoholic Fatty Liver Disease Progression.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidity and mortality. Oxidative stress and release of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), are major consequences of hepatic lipid overload, which can contribute to progression of NAFLD to non-alcoholic steatohepatitis (NASH). Also, mitochondria are involved in the NAFLD pathogenesis for their role in hepatic lipid metabolism. Definitive treatments for NAFLD/NASH are lacking so far. Silybin, the extract of the milk thistle seeds, has previously shown beneficial effects in NAFLD. Sequential exposure of hepatocytes to high concentrations of fatty acids (FAs) and TNFα resulted in fat overload and oxidative stress, which mimic in vitro the progression of NAFLD from simple steatosis (SS) to steatohepatitis (SH). The exposure to 50 µM silybin for 24 h reduced fat accumulation in the model of NAFLD progression. The in vitro progression of NAFLD from SS to SH resulted in reduced hepatocyte viability, increased apoptosis and oxidative stress, reduction in lipid droplet size, and up-regulation of IκB kinase ß-interacting protein and adipose triglyceride lipase expressions. The direct action of silybin on SS or SH cells and the underlying mechanisms were assessed. Beneficial action of silybin was sustained by changes in expression/activity of peroxisome proliferator-activated receptors and enzymes for FA oxidation. Moreover, silybin counteracted the FA-induced mitochondrial damage by acting on complementary pathways: (i) increased the mitochondrial size and improved the mitochondrial cristae organization; (ii) stimulated mitochondrial FA oxidation; (iii) reduced basal and maximal respiration and ATP production in SH cells; (iv) stimulated ATP production in SS cells; and (v) rescued the FA-induced apoptotic signals and oxidative stress in SH cells. We provide new insights about the direct protective effects of the nutraceutic silybin on hepatocytes mimicking in vitro NAFLD progression.

Silybin counteracts lipid excess and oxidative stress in cultured steatotic hepatic cells.

AIM: To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis. METHODS: Rat hepatoma FaO cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations (25 to 100 µmol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays. RESULTS: Lipid-loading resulted in intracellular triglyceride (TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein-1c (SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species (ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B (NF-κB). Incubation of steatotic cells with silybin 50 µmol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1 (CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-κB activation. CONCLUSION: We demonstrated the direct anti-steatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies.

Ethanol and fatty acids impair lipid homeostasis in an in vitro model of hepatic steatosis.

Excess ethanol consumption and fatty acid intake lead to a cumulative effect on liver steatosis through still unclear mechanisms. This study aimed to characterize the lipid homoeostasis alterations under the exposure of hepatocytes to ethanol alone or combined with fatty acids. FaO hepatoma cells were incubated in the absence (C) or in the presence of 100 mM ethanol (EtOH) or 0.35 mM oleate/palmitate (FFA) alone or in the combination (FFA/EtOH). Content of intra- and extra-cellular triglycerides (TAGs) and of lipid droplets (LDs), expression of lipogenic and lipolytic genes, and oxidative stress-related parameters were evaluated. Exposure to either FFAs or EtOH given separately led to steatosis which was augmented when they were combined. Our results show that FFA/EtOH: (i) increased the LD number, but reduced their size compared to separate treatments; (ii) up-regulated PPARγ and SREBP-1c and down-regulated sirtuin-1 (SIRT1); (iii) impaired FFA oxidation; (iv) did not change lipid secretion and oxidative stress. Our findings indicate that one of the major mechanisms of the metabolic interference between ethanol and fat excess is the impairment of FFA oxidation, in addition to lipogenic pathway stimulation. Interestingly, ethanol combined with FFAs led to a shift from macrovesicular to microvesicular steatosis that represents a more dangerous condition.

Triglyceride Mobilization from Lipid Droplets Sustains the Anti-Steatotic Action of Iodothyronines in Cultured Rat Hepatocytes.

Adipose tissue, dietary lipids and de novo lipogenesis are sources of hepatic free fatty acids (FFAs) that are stored in lipid droplets (LDs) as triacylglycerols (TAGs). Destiny of TAGs stored in LDs is determined by LD proteomic equipment. When adipose triglyceride lipase (ATGL) localizes at LD surface the lipid mobilization is stimulated. In this work, an in vitro model of cultured rat hepatocytes mimicking a mild steatosis condition was used to investigate the direct lipid-lowering action of iodothyronines, by focusing, in particular, on LD-associated proteins, FFA oxidation and lipid secretion. Our results demonstrate that in "steatotic" hepatocytes iodothyronines reduced the lipid excess through the recruitment of ATGL on LD surface, and the modulation of the LD-associated proteins Rab18 and TIP47. As an effect of ATGL recruitment, iodothyronines stimulated the lipid mobilization from LDs then followed by the up-regulation of carnitine-palmitoyl-transferase (CPT1) expression and the stimulation of cytochrome-c oxidase (COX) activity that seems to indicate a stimulation of mitochondrial function. The lipid lowering action of iodothyronines did not depend on increased TAG secretion. On the basis of our data, ATGL could be indicated as an early mediator of the lipid-lowering action of iodothyronines able to channel hydrolyzed FFAs toward mitochondrial beta-oxidation rather than secretion.

Altered oxidative stress/antioxidant status in blood of alcoholic subjects is associated with alcoholic liver disease.

BACKGROUND: Oxidative stress is implicated in pathogenesis of alcoholic liver disease (ALD). This study investigated the possible correlation among the erythrocyte indices of oxidative stress, the leukocyte panels of antioxidant proteins (metallothioneins), the serum biochemical parameters and the liver steatosis grade. METHODS: A total of 118 cases including 60 alcoholic subjects and 58 controls were enrolled. All the alcoholic subjects were screened for body mass index (BMI), liver steatosis, and blood chemistry and serology. The level of oxidative stress and oxidative stress-related parameters were measured in the blood and correlated with clinical findings. RESULTS: Alcoholic subjects showed higher BMI, moderate/severe hepatic steatosis, increase in the levels of triglycerides, cholesterol, glucose, γ-glutamyl-transpeptidase (GGT), alanine aminotransferase (ALT), bilirubin, alpha 1 and beta 2 globulins, iron and a decrease in the levels of aspartate aminotransferase (AST) and beta 1 globulin with respect to the reference values. Moreover, alcoholic subjects showed: (i) an increase in Thiobarbituric Acid Reactive Substance (TBARS) content representing a good estimation of global oxidative stress; (ii) a stimulation of the activities of the antioxidant enzymes catalase and SOD; (iii) a modulation of expression of metallothioneins, with a down-regulation of MT-1A and an up-regulation of MT-1E isoforms. CONCLUSIONS: Our data suggest that alcoholism is strongly associated with altered pattern of blood metallothioneins; this parameter combined with the score calculated by an ad hoc implemented algorithm (HePaTest) could offer a non-invasive alternative approach for evaluating alcohol-related damages and could be used in follow-up of alcoholic patients.