Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity.

BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

The potential role of omentin-1 in obesity-related metabolic dysfunction-associated steatotic liver disease: evidence from translational studies.

BACKGROUND: Obesity, characterized by visceral adipose tissue (VAT) expansion, is closely associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). Recent research has highlighted the crucial role of the adipose tissue-liver axis in the development of MASLD. In this study, we investigated the potential role of omentin-1, a novel adipokine expressed by VAT, in obesity-related MASLD pathogenesis. METHODS: Through in silico analysis of differentially expressed genes in VAT from obese patients with and without MASH, we identified omentin-1 as a significant candidate. To validate our findings, we measured omentin-1 levels in VAT and plasma of lean controls and obese patients with biopsy-proven MASLD. Additionally, we assessed omentin-1 expression in the VAT of diet-induced mice MASLD model. In vitro and ex vivo studies were conducted to investigate the effects of omentin-1 on MASLD-related mechanisms, including steatosis, inflammation, endoplasmic reticulum (ER) stress, and oxidative stress. We also analyzed the impact of D-glucose and insulin on VAT omentin-1 levels ex vivo. RESULTS: Compared to the lean group, the obese groups exhibited significantly lower VAT and plasma levels of omentin-1. Interestingly, within the obese groups, omentin-1 is further decreased in MASH groups, independent of fibrosis. Likewise, VAT of mice fed with high-fat diet, showing histological signs of MASH showed decreased omentin-1 levels as compared to their control diet counterpart. In vitro experiments on fat-laden human hepatocytes revealed that omentin-1 did not affect steatosis but significantly reduced TNF-α levels, ER stress, and oxidative stress. Similar results were obtained using ex vivo VAT explants from obese patients upon omentin-1 supplementation. Furthermore, omentin-1 decreased the mRNA expression of NF-κB and mitogen-activated protein kinases (ERK and JNK). Ex vivo VAT explants showed that D-glucose and insulin significantly reduced omentin-1 mRNA expression and protein levels. CONCLUSIONS: Collectively, our findings suggest that reduced omentin-1 levels contribute to the development of MASLD. Omentin-1 supplementation likely exerts its beneficial effects through the inhibition of the NF-κB and MAPK signaling pathways, and it may additionally play a role in the regulation of glucose and insulin metabolism. Further research is warranted to explore omentin-1 as a potential therapeutic target and/or biomarker for MASLD.

Natural Compounds for Counteracting Nonalcoholic Fatty Liver Disease (NAFLD): Advantages and Limitations of the Suggested Candidates.

The booming prevalence of nonalcoholic fatty liver disease (NAFLD) in adults and children will threaten the health system in the upcoming years. The "multiple hit" hypothesis is the currently accepted explanation of the complex etiology and pathophysiology of the disease. Some of the critical pathological events associated with the development of NAFLD are insulin resistance, steatosis, oxidative stress, inflammation, and fibrosis. Hence, attenuating these events may help prevent or delay the progression of NAFLD. Despite an increasing understanding of the mechanisms involved in NAFLD, no approved standard pharmacological treatment is available. The only currently recommended alternative relies on lifestyle modifications, including diet and physical activity. However, the lack of compliance is still hampering this approach. Thus, there is an evident need to characterize new therapeutic alternatives. Studies of food bioactive compounds became an attractive approach to overcome the reticence toward lifestyle changes. The present study aimed to review some of the reported compounds with beneficial properties in NAFLD; namely, coffee (and its components), tormentic acid, verbascoside, and silymarin. We provide details about their protective effects, their mechanism of action in ameliorating the critical pathological events involved in NAFLD, and their clinical applications.

The Beneficial Effects of Triterpenic Acid and Acteoside in an In Vitro Model of Nonalcoholic Steatohepatitis (NASH).

Triterpenic acid (TA) and acteoside (ACT), the major components of APPLIVER and ACTEOS, respectively, have been reported to exert hepatoprotective effects, but the molecular mechanisms remain elusive, particularly in the NAFLD/NASH context. We assessed their effects in our well-established in vitro model resembling the pathophysiological mechanisms involved in NASH. Human hepatocytes and hepatic stellate cells were exposed to free fatty acids (FFA) alone or in combination with APPLIVER and ACTEOS as a mono- or co-culture. Steatosis, inflammation, generation of reactive oxygen species (ROS), and collagen deposition were determined. ACTEOS reduced both the TNF-α and ROS production, and, most importantly, attenuated collagen deposition elicited by the excess of FFA in the co-culture model. APPLIVER also showed inhibition of both TNF-α production and collagen deposition caused by FFA accumulation. The compounds alone did not induce any cellular effects. The present study showed the efficacy of APPLIVER and ACTEOS on pathophysiological mechanisms related to NASH. These in vitro data suggest that these compounds deserve further investigation for possible use in NASH treatment.

Ficolin-2 Plasma Level Assesses Liver Fibrosis in Non-Alcoholic Fatty Liver Disease.

Fibrosis is the strongest predictor for disease-specific mortality in non-alcoholic fatty liver diseases (NAFLD), but the need for liver biopsy limits its diagnosis. We assessed the performance of plasma ficolin-2 (FCN-2) as a biomarker of fibrosis identified by an in silico discovery strategy. Two hundred and thirty-five morbidly obese (MO) subjects with biopsy-proven NAFLD stratified by fibrosis stage (F0, n = 44; F1, n = 134; F2, n = 46; F3/F4, n = 11) and 40 cirrhotic patients were enrolled. The cohort was subdivided into discovery (n = 76) and validation groups (n = 159). The plasma level of FCN-2 and other candidate markers was determined. FCN-2 was inversely correlated with the stage of liver fibrosis (ρ = −0.49, p < 0.001) independently of steatosis (p = 0.90), inflammation (p = 0.57), and ballooning (p = 0.59). In the global cohort, FCN-2 level decreased significantly in a stepwise fashion from F0/F1 (median 4753 ng/mL) to F2−F3−F4 (2760 ng/mL) and in cirrhotic subjects (1418 ng/mL). The diagnostic performance of FCN-2 in detecting F ≥ 2 was higher than other indexes (APRI, FIB-4) (AUROC 0.82, 0.68, and 0.6, respectively). The accuracy improved when combined with APRI score and HDL values (FCNscore, AUROC 0.85). Overall, the FCN-2 plasma level can accurately discriminate liver fibrosis status (minimal vs. moderate/advanced) significantly improving the fibrosis diagnostic algorithms.

Modifications of IGF2 and EGFR plasma protein concentrations in NAFLD patients after bariatric surgery.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is strictly associated with the epidemic of obesity and is becoming the most prevalent liver disease worldwide. In severe obesity, bariatric surgery (BS) is the most effective treatment not only for obesity but also for the associated metabolic co-morbidities, NAFLD, among others. To date, noninvasive diagnostic/prognostic methods cannot evaluate hepatic improvements following surgery. OBJECTIVES: We aimed to measure plasma level of insulin-growth factor-2 protein (IGF2) and epithermal growth factor receptor (EGFR), and to assess their relationship with clinical and biochemical parameters during the 12 months follow-up. METHODS: Demographic, clinical-biochemical data, and plasma IGF2 and EGFR were measured in 69 patients preoperatively (T0) and 6 and 12 months (T6M and T12M, respectively) after BS. Liver biopsy was performed at T0. Relationships between IGF2, EGFR, and several biochemical parameters were performed using Pearson or Spearman correlation analysis. RESULTS: IGF2 plasma level increases during follow-up, passing from 2.5 (1.8-15.5) at baseline to 13.3 (8.6-19.1) at T12M, p < 0.001. Conversely, EGFR showed a not significant reduction. At T12M, the plasma level of both markers was comparable to those of lean subjects. The clinical-biochemical parameters (BMI, glycated hemoglobin, HOMA-IR) also return to the normal range at T12M. Correlation analysis demonstrated that IGF2 was significantly associated with total bilirubin, direct bilirubin, and albumin at T0 while with blood glucose, ALT, GGT, and AST/ALT ratio at T6M and T12M. CONCLUSIONS: IGF2 plasma levels increase after bariatric surgery, and these changes are associated with the modification of hepatic biochemical parameters, even if other clinic or metabolic improvements cannot be excluded.

Taste perception and expression in stomach of bitter taste receptor tas2r38 in obese and lean subjects.

Differences in taste perception have been related to eating behavior, nutritional status, and diseases. Recently, taste receptors have been identified in several extra-oral tissues, such as the gastrointestinal tract, where they seem to influence processes like digestion, sense of satiety as well as energy balance and intraluminal changes occurring in obesity. Our study aims to analyze differences in taste perception among 42 obese patients (OB) and 41 normal-weight subjects (LEAN). Polymorphisms in the gene codifying for the bitter taste receptor TAS2R38 and its expression on the surface of the gastric mucosa were tested and compared among OB and LEAN. Taste intensity of PROP (6-n-propylthiouracil), quinine, sucrose, citric acid and NaCl were measured on a labeled magnitude scale. DNA from peripheral whole blood was extracted and three polymorphisms in the TAS2R38 gene (rs713598, rs1726866, rs10246939) analyzed. Gastric biopsies were collected during bariatric surgery in OB and during endoscopy in LEAN. RNA was extracted and TAS2R38 gene expression assessed by RT-Real-Time qPCR. Anamnestic and anthropometric data were recorded in all participants during baseline visits. Logistic regression analysis showed that OB perceives sweet (sucrose) and bitter (PROP or 6-n-propylthiouracil) taste more intensely than LEAN (p-value = 0.02 and p-value = 0.005, respectively). While polymorphisms in TAS2R38 gene did not differ among OB and LEAN, we observed a significant increase of TAS2R38 mRNA levels in the stomach of OB compared to LEAN (p = 0.01). Our results provide new evidence of a link between obesity and altered taste perception as well as TAS2R38 expression in the stomach.

Occult hepatitis B virus infection predicts non-alcoholic steatohepatitis in severely obese individuals from Italy.

BACKGROUND & AIMS: Obesity is associated with non-alcoholic fatty liver (NAFL), which may progress towards non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). Occult hepatitis B virus infection (OBI) may contribute to hepatic damage in patients with chronic liver disease of different aetiologies (eg HCV, alcohol). However, information on the prevalence and clinical impact of OBI in obese individuals is lacking. The aims of this study were to investigate NASH prevalence and risk factors in obese people who underwent bariatric surgery. METHODS: Two-hundred and twenty-six subjects (160 females; mean age 42.9 years ±10.8 SD) without evidence of any further cause of liver disease consecutively underwent bariatric surgery in two Italian liver centers. During surgery, all patients underwent liver biopsy for histological evaluation and molecular studies. Liver DNA extracts were tested for PNPLA3, TM6SF2, MBOAT7, IRGM polymorphisms and for OBI. Univariate and multivariate analyses were used to identify predictors of NASH. RESULTS: Histology showed NASH in 115 (50.9%) and NAFL in 111 cases (49.1%). Twenty-nine/226 (12.8%) cases had OBI, 24 (82.8%) of whom had NASH and 5 (17.2%) NAFL, whereas among the 197 OBI-negative cases, 91 (46.2%) had NASH and 106 (53.8%) NAFL (P = .0002). Multivariate analysis showed that older age (P = .03, OR 1.034), alanine aminotransferase values (P = .005, OR 1.023), insulin resistance/diabetes (P = .02, OR 2.257), TM6SF2 polymorphism (P = .04, OR 3.168) and OBI (P = .004, OR 5.503) were independent predictors of NASH. CONCLUSION: NASH is highly prevalent in obese individuals undergoing bariatric surgery. OBI is one of the strongest risk factors of NASH in these patients.

Silybin Modulates Collagen Turnover in an In Vitro Model of NASH.

Silybin has been proposed as a treatment for nonalcoholic steatohepatitis (NASH). In this study, we assessed the effect of Silybin in a well-established in vitro coculture model of early-stage NASH. LX2 and Huh7 cells were exposed to free fatty acid (FFA) and Silybin as mono- or coculture (SCC). Cell viability, LX2 activation, collagen deposition, metalloproteinase 2 and 9 (MMP2-9) activity, and ROS generation were determined at 24, 96, and 144 h. Exposure to FFA induced the activation of LX2 as shown by the increase in cell viability and upregulation of collagen biosynthesis. Interestingly, while cotreatment with Silybin did not affect collagen production in LX2, a significant reduction was observed in SCC. MMP2-9 activity was reduced in FFA-treated Huh7 and SCC and cotreatment with Silybin induced a dose-dependent increase, while no effect was observed in LX2. Silybin also showed antioxidant properties by reducing the FFA-induced production of ROS in all the cell systems. Based on these data, Silybin exerts its beneficial effects by reducing LX2 proliferation and ROS generation. Moreover, MMP2-9 modulation in hepatocytes represents the driving mechanism for the net reduction of collagen in this NASH in vitro model, highlighting the importance of hepatic cells interplay in NASH development and resolution.

Global epidemiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: What we need in the future.

The estimated prevalence of non-alcoholic fatty liver disease (NAFLD) worldwide is approximately 25%. However, the real prevalence of NAFLD and the associated disorders is unknown mainly because reliable and applicable diagnostic tests are lacking. This is further complicated by the lack of consensus on the terminology of different entities such as NAFLD or nonalcoholic steatohepatitis (NASH). Although assessing fatty infiltration in the liver is simple by ultrasound, the gold standard for the assessment of fibrosis, the only marker of progression towards more severe liver disease is still liver biopsy. Although other non-invasive tests have been proposed, they must still be validated in large series. Because NAFL/NAFLD/NASH and related metabolic diseases represent an economic burden, finding an inexpensive method to diagnose and stage fatty liver is a priority. A translational approach with the use of cell and/or animal models could help to reach this goal.

A simple in silico strategy identifies candidate biomarkers for the diagnosis of liver fibrosis in morbidly obese subjects.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disorder, tightly associated with obesity. The histological spectrum of the disease ranges from simple steatosis to steatohepatitis, with different stages of fibrosis, and fibrosis stage is the most significant predictor of mortality in NAFLD. Liver biopsy continues to be the gold standard for its diagnosis and reliable non-invasive diagnostic tools are unavailable. We investigated the accuracy of candidate proteins, identified by an in silico approach, as biomarkers for diagnosis of fibrosis. METHODS: Seventy-one morbidly obese (MO) subjects with biopsy-proven NAFLD were enrolled, and the cohort was subdivided according to minimal (F0/F1) or moderate (F2/F3) fibrosis. The plasmatic level of CD44 antigen (CD44), secreted protein acidic and rich in cysteine (SPARC), epidermal growth factor receptor (EGFR) and insulin-like growth factor 2 (IGF2) were determined by ELISA. Significant associations between plasmatic levels and histological fibrosis were determined by correlation analysis and the diagnostic accuracy by the area under receiver operating characteristic curves (AUROC). RESULTS: Eighty-two percentage of the subjects had F0/F1 and 18% with F2/F3 fibrosis. Plasmatic levels of IGF2, EGFR and their ratio (EGFR/IGF2) were associated with liver fibrosis, correlating inversely for IGF2 (P < .006) and directly (P < .018; P < .0001) for EGFR and EGFR/IGF2 respectively. The IGF2 marker had the best diagnostic accuracy for moderate fibrosis (AUROC 0.83), followed by EGFR/IGF2 ratio (AUROC 0.79) and EGFR (AUROC 0.71). CONCLUSIONS: Our study supports the potential utility of IGF2 and EGFR as non-invasive diagnostic biomarkers for liver fibrosis in morbidly obese subjects.

Hepatocyte-derived macrophage migration inhibitory factor mediates alcohol-induced liver injury in mice and patients.

BACKGROUND & AIMS: Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD. METHODS: MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif-/- bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH). RESULTS: HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to ethanol challenge in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif-/-→WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→Mif-/-) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH. CONCLUSIONS: Taken together, these data provide evidence that hepatocyte-derived MIF is critical in the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Lay summary: Alcoholic liver disease is a major cause of preventable mortality worldwide, and lacks specific pharmacological therapies. Recent studies have recognized that macrophage migration inhibitor factor (MIF) has a critical role in the inflammatory response to liver damage. However, the cells that produce this protein are still unknown. Our present findings reveal that hepatocytes, the main cell type in the liver, are primarily responsible for MIF production in response to alcohol, which promotes liver injury. Our study suggests that drugs inhibiting MIF production could be beneficial in treating patients with liver disease due to excessive alcohol consumption.

The importance of the interaction between hepatocyte and hepatic stellate cells in fibrogenesis induced by fatty accumulation.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease is characterized by an initial accumulation of triglycerides that can progress to non-alcoholic steatohepatitis, which can ultimately evolve to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells play a key role in liver fibrogenesis by an increased activation and an altered profile of genes involved in the turnover of extracellular matrix components. To reproduce in-vitro the functional cell connections observed in vivo it is essential to consider cell-to-cell proximity and interaction. The aim of this study was to determine the response to free fatty acids in a simultaneous co-culture model of hepatocytes and hepatic stellate cells. METHODS: Simultaneous co-culture model and monoculture of each cell type (control) were exposed to FFA for 24 up to 144 h. Quantification of steatosis; stellate cell activation; assessment of fibrogenic response; expression and activity of metalloproteinases as well as collagen biosynthesis were evaluated. RESULTS: Free fatty acids induced comparable steatosis in simultaneous co-culture and monoculture. However, the activation of the stellate cells assessed by alpha-smooth muscle actin expression is greater when cells were in close contact. Furthermore, a time-dependent increment of tissue inhibitor metalloproteinase-2 protein was observed, which was inversely correlated with protein expression and activity of matrix-metalloproteinases, suggesting enhanced collagen biosynthesis. This behavior was absent in cell monoculture. CONCLUSIONS: These data indicate that cell-to-cell proximity between hepatocytes and stellate cells is necessary for the initiation of the fibrotic process.

Liver Bacterial Colonization in Patients with Obesity and Gut Dysbiosis.

PURPOSE: Recently, the link between gut microbiota, liver inflammation, and obesity has become an interesting focus of research. The aim of this study is to show the possible relation between gut microbiota dysbiosis in patients with obesity and the presence of bacterial genomes in their liver biopsies. MATERIALS AND METHODS: A prospective study on patients undergoing bariatric surgery was carried out. Anthropometric and metabolic data, comorbidities, stool samples, and hepatic biopsies were collected and analyzed at the time of surgery. The V3-16S rRNA region was sequenced using the Ion Torrent new-generation sequencing platform. RESULTS: In each of the 23 patients enrolled, the bacterial population was analyzed both in the stools and liver. In eight patients (34.7%), Prevotella (62.5%), Bacteroides (50%), Streptococcus (12.5%), and Dalister (12.5%) were found in both samples, simultaneously; in 15 cases, the liver was free from colonization. The statistically significant difference between groups was a Roseburia intestinalis reduction in fecal samples of patients with liver biopsies colonized by bacteria (1% vs 3%; p = 0.0339). CONCLUSION: To the best of our knowledge, this is the first study reporting the presence of bacterial genome in a liver biopsy on bariatric patients, instead of the microbe-associated molecular patterns. Notably, in literature, the presence of Roseburia intestinalis in stool samples has been shown to prevent intestinal inflammation playing its role in the gut barrier integrity. In our population, the Roseburia reduction was associated with the presence of bacterial genome in the liver, probably related to a greater permeability of the gut and vascular barriers.

Gender Differences in the Impact of a High-Fat, High-Sugar Diet in Skeletal Muscles of Young Female and Male Mice.

In the context of the increasing number of obese individuals, a major problem is represented by obesity and malnutrition in children. This condition is mainly ascribable to unbalanced diets characterized by high intakes of fat and sugar. Childhood obesity and malnutrition are not only associated with concurrent pathologies but potentially compromise adult life. Considering the strict correlation among systemic metabolism, obesity, and skeletal muscle health, we wanted to study the impact of juvenile malnutrition on the adult skeletal muscle. To this aim, 3-week-old C56BL/6 female and male mice were fed for 20 weeks on a high-fat. high-sugar diet, and their muscles were subjected to a histological evaluation. MyHCs expression, glycogen content, intramyocellular lipids, mitochondrial activity, and capillary density were analyzed on serial sections to obtain the metabolic profile. Our observations indicate that a high-fat, high-sugar diet alters the metabolic profile of skeletal muscles in a sex-dependent way and induces the increase in type II fibers, mitochondrial activity, and lipid content in males, while reducing the capillary density in females. These data highlight the sex-dependent response to nutrition, calling for the development of specific strategies and for a systematic inclusion of female subjects in basic and applied research in this field.

Kinetics of the inflammatory response induced by free fatty acid accumulation in hepatocytes.

BACKGROUND. The information available on time and dose effects of the exposure of hepatocytes to free fatty acids (FFA) in vitro is controversial, and very few studies have assessed the hepatocyte inflammatory response in an in vitro model. AIM. To analyze the effect of treatment with FFA on cell viability and on the kinetics of cytokine expression using hepatic cell lines. MATERIAL AND METHODS. Hepatic cell lines, IHH and HuH7, were cultured for 3 h, 6 h, 12 h and 24 h in an enriched medium with palmitic and oleic acids. The cytotoxicity of the FFA was assessed by the MTT test and the intracellular fat content determined cytofluorimetrically and by fluorescence microscopy using Nile Red staining. The expression of mRNA for interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α was assessed by real time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS. Treatment with 600 µM FFA did not affect the viability of either cell line despite a significant increase in the intracellular content of lipid droplets already evident after 3 h of treatment. A time- and dose-dependent upregulation of the expression of IL-6 and IL-8 mRNA was observed during the treatment at 3 and 24 h. In contrast, TNF-α mRNA expression was highly upregulated at 3 h after FFA exposure but returned to control values at 24 h. In conclusion, hepatocytes exposed in vitro for a short time to low FFA concentrations showed a significant upregulation of IL-6 and IL-8 to,and a rapid but transitory elevation of TNF-α.

Effect of Intake of Extra Virgin Olive Oil on Mortality in a South Italian Cohort with and without NAFLD.

BACKGROUND: Extra-virgin olive oil (EVOO) is the main source of seasoning fat in the Mediterranean diet and it is one of the components with known protective factors on chronic-degenerative disease. We aimed to evaluate the effect of a medium-high level of oil consumption on mortality in a cohort with good adherence to the Mediterranean diet. METHODS: A total of 2754 subjects who had completed the food questionnaire in the Multicenter Italian study on Cholelithiasis (MICOL) cohort were included in the study. EVOO consumption was categorized in four levels (<20 g/die, 21-30 g/die, 31-40 g/die, >40 g/die). We performed a flexible parametric survival model to assess mortality by EVOO consumption level adjusted for some covariates. We also performed the analysis on subjects with and without non-alcoholic fatty liver disease (NAFLD) to evaluate the effects of oil in this more fragile sub-cohort. RESULTS: We found a statistically significant negative effect on mortality for the whole sample when EVOO consumption was used, both as a continuous variable and when categorized. The protective effect was stronger in the sub-cohort with NAFLD, especially for the highest levels of EVOO consumption (HR = 0.58 with p < 0.05). CONCLUSIONS: Our study has shown a protective effect of EVOO consumption towards all causes of mortality. Despite the higher caloric intake, the protective power is greater for a consumption >40 g/day in both the overall cohort and the sub-cohorts with and without NAFLD.

Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease.

BACKGROUND: In vitro exposure of liver cells to high concentrations of free fatty acids (FFA) results in fat overload which promotes inflammatory and fibrogenic response similar to those observed in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Since the mechanisms of this event have not been fully characterized, we aimed to analyze the fibrogenic stimuli in a new in vitro model of NASH. METHODS: HuH7 cells were cultured for 24 h in an enriched medium containing bovine serum albumin and increasing concentrations of palmitic and oleic acid at a molar ratio of 1:2 (palmitic and oleic acid, respectively). Cytotoxic effect, apoptosis, oxidative stress, and production of inflammatory and fibrogenic cytokines were measured. RESULTS: FFA induces a significant increment in the intracellular content of lipid droplets. The gene expression of interleukin-6, interleukin-8 and tumor necrosis factor alpha was significantly increased. The protein level of interleukin-8 was also increased. Intracellular lipid accumulation was associated to a significant up-regulation in the gene expression of transforming growth factor beta 1, alpha 2 macroglobulin, vascular endothelial growth factor A, connective tissue growth factor, insulin-like growth factor 2, thrombospondin 1. Flow cytometry analysis demonstrated a significant increment of early apoptosis and production of reactive oxygen species. CONCLUSIONS: The exposure of hepatocytes to fatty acids elicits inflammation, increase of oxidative stress, apoptosis and production of fibrogenic cytokines. These data support a primary role of FFA in the pathogenesis of NAFLD and NASH.

Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma.

BACKGROUND: The Breast Cancer Resistance Protein (BCRP/ABCG2) is one member of ABC transporters proteins super family responsible of drug resistance. Since data on ABCG2 expression in liver malignances are scanty, here we report the expression of ABCG2 in adult human hepatocellular carcinoma (HCC) in both in vivo and in vitro models with different degree of malignancy. METHODS: In cell lines derived from human hepatocellular carcinoma, ABCG2 gene expression was assessed by reverse transcription quantitative real time PCR and function by Hoechst 33342 efflux assay; protein content was assessed by SDS-PAGE Western blot. RESULTS: ABCG2 expression was found to be highest in the most undifferentiated cell lines, and this was related with a higher functional activity. ABCG2 expression was sensitive to antineoplastic drugs since exposure to 5 µM doxorubicin for 24 hours resulted in significant up-regulations of ABCG2 in all cell lines, particularly in those lines with low basal ABCG2 expression (p<0.01). The gene expression was also investigated in 51 adult liver tissues with HCC and related cirrhosis; normal liver tissue was used as control. ABCG2 gene expression was higher in HCC than both cirrhotic paired tissue and normal tissue. This up-regulation was greater (p<0.05) in pathological poorly differentiated grade G3/G4 than in well-differentiated G1/G2 HCC. CONCLUSIONS: Our results suggest a correlation of ABCG2 gene expression and differentiation stage both in human and HCC derived cell lines. The rapid up-regulation of ABCG2 to exposure to doxorubicin emphasizes the importance of this transporter in accounting for drug resistance in liver tumors.

Diagnostic management of nonalcoholic fatty liver disease: a transformational period in the development of diagnostic and predictive tools-a narrative review.

NAFLD is an emerging healthcare epidemic that is causing predictable adverse consequences for healthcare systems, societies and individuals. Whilst NAFLD is recognized as a multi-system disease with compound pathways that are both benign and pernicious in their unfolding; NASH is generally understood as a deleterious follow-on condition with path-specific tendencies that progress to cirrhosis, HCC and liver transplantation. Recent evidence is beginning to challenge this interpretation demanding more attention to the personalized nature of the disease and its pathogenesis across multiple different cohorts. This means that we need better diagnostic and prognostic tools not only to capture those 'at risk' disease phenotypes; but for better stratification and monitoring of patients according to their treatment strategies. With the advent of pipeline therapies for NASH underway, the medical profession looks to adopt more accurate non-invasive diagnostic tools that can help to delineate and eliminate NASH histology. This review looks at the search for the killer application revealing this particular moment in time as a transformational period; one that is pushing the boundaries of technology to integrate diverse panels of species through sensitive profiling and multi-omics approaches that cast wide, yet powerful diagnostic nets that have the potential to elucidate pathway specific biomarkers that are personalized and predictable.