SIRT5 rs12216101 T>G variant is associated with liver damage and mitochondrial dysfunction in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Sirtuin 5, encoded by the SIRT5 gene, is a NAD+-dependent deacylase that modulates mitochondrial metabolic processes through post-translational modifications. In this study, we aimed to examine the impact of the SIRT5 rs12216101 T>G non-coding single nucleotide polymorphism on disease severity in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: The rs12216101 variant was genotyped in 2,606 consecutive European patients with biopsy-proven NAFLD. Transcriptomic analysis, expression of mitochondrial complexes and oxidative stress levels were measured in liver samples from a subset of bariatric patients. Effects of SIRT5 pharmacological inhibition were evaluated in HepG2 cells exposed to excess free fatty acids. Mitochondrial energetics in vitro were investigated by high-performance liquid chromatography. RESULTS: In the whole cohort, the frequency distribution of SIRT5 rs12216101 TT, TG and GG genotypes was 47.0%, 42.3% and 10.7%, respectively. At multivariate logistic regression analysis adjusted for sex, age >50 years, diabetes, and PNPLA3 rs738409 status, the SIRT5 rs12216101 T>G variant was associated with the presence of non-alcoholic steatohepatitis (odds ratio 1.20, 95% CI 1.03-1.40) and F2-F4 fibrosis (odds ratio 1.18; 95% CI 1.00-1.37). Transcriptomic analysis showed that the SIRT5 rs12216101 T>G variant was associated with upregulation of transcripts involved in mitochondrial metabolic pathways, including the oxidative phosphorylation system. In patients carrying the G allele, western blot analysis confirmed an upregulation of oxidative phosphorylation complexes III, IV, V and consistently higher levels of reactive oxygen species, reactive nitrogen species and malondialdehyde, and lower ATP levels. Administration of a pharmacological SIRT5 inhibitor preserved mitochondrial energetic homeostasis in HepG2 cells, as evidenced by restored ATP/ADP, NAD+/NADH, NADP+/NADPH ratios and glutathione levels. CONCLUSIONS: The SIRT5 rs12216101 T>G variant, heightening SIRT5 activity, is associated with liver damage, mitochondrial dysfunction, and oxidative stress in patients with NAFLD. IMPACT AND IMPLICATIONS: In this study we discovered that the SIRT5 rs12216101 T>G variant is associated with higher disease severity in patients with non-alcoholic fatty liver disease (NAFLD). This risk variant leads to a SIRT5 gain-of-function, enhancing mitochondrial oxidative phosphorylation and thus leading to oxidative stress. SIRT5 may represent a novel disease modulator in NAFLD.

(+)-Lipoic acid reduces mitochondrial unfolded protein response and attenuates oxidative stress and aging in an in vitro model of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a liver disorder characterized by the ac-cumulation of fat in hepatocytes without alcohol consumption. Mitochondrial dysfunction and endoplasmic reticulum (ER) stress play significant roles in NAFLD pathogenesis. The unfolded protein response in mitochondria (UPRmt) is an adaptive mechanism that aims to restore mitochondrial protein homeostasis and mitigate cellular stress. This study aimed to investigate the effects of ( +)-Lipoic acid (ALA) on UPRmt, inflammation, and oxidative stress in an in vitro model of NAFLD using HepG2 cells treated with palmitic acid and oleic acid to induce steatosis. RESULTS: Treatment with palmitic and oleic acids increased UPRmt-related proteins HSP90 and HSP60 (heat shock protein), and decreased CLPP (caseinolytic protease P), indicating ER stress activation. ALA treatment at 1 µM and 5 µM restored UPRmt-related protein levels. PA:OA (palmitic acid:oleic acid)-induced ER stress markers IRE1α (Inositol requiring enzyme-1), CHOP (C/EBP Homologous Protein), BIP (Binding Immunoglobulin Protein), and BAX (Bcl-2-associated X protein) were significantly reduced by ALA treatment. ALA also enhanced ER-mediated protein glycosylation and reduced oxidative stress, as evidenced by decreased GPX1 (Glutathione peroxidase 1), GSTP1 (glutathione S-transferase pi 1), and GSR (glutathione-disulfide reductase) expression and increased GSH (Glutathione) levels, and improved cellular senescence as shown by the markers ß-galactosidase, γH2Ax and Klotho-beta. CONCLUSIONS: In conclusion, ALA ameliorated ER stress, oxidative stress, and inflammation in HepG2 cells treated with palmitic and oleic acids, potentially offering therapeutic benefits for NAFLD providing a possible biochemical mechanism underlying ALA beneficial effects.

(+)-Lipoic Acid Reduces Lipotoxicity and Regulates Mitochondrial Homeostasis and Energy Balance in an In Vitro Model of Liver Steatosis.

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids within hepatocytes, which compromises liver functionality following mitochondrial dysfunction and increased production of reactive oxygen species (ROS). Lipoic acid is one of the prosthetic groups of the pyruvate dehydrogenase complex also known for its ability to confer protection from oxidative damage because of its antioxidant properties. In this study, we aimed to investigate the effects of lipoic acid on lipotoxicity and mitochondrial dynamics in an in vitro model of liver steatosis. HepG2 cells were treated with palmitic acid and oleic acid (1:2) to induce steatosis, without and with 1 and 5 µM lipoic acid. Following treatments, cell proliferation and lipid droplets accumulation were evaluated. Mitochondrial functions were assessed through the evaluation of membrane potential, MitoTracker Red staining, expression of genes of the mitochondrial quality control, and analysis of energy metabolism by HPLC and Seahorse. We showed that lipoic acid treatment restored membrane potential to values comparable to control cells, as well as protected cells from mitochondrial fragmentation following PA:OA treatment. Furthermore, our data showed that lipoic acid was able to determine an increase in the expression of mitochondrial fusion genes and a decrease in mitochondrial fission genes, as well as to restore the bioenergetics of cells after treatment with palmitic acid and oleic acid. In conclusion, our data suggest that lipoic acid reduces lipotoxicity and improves mitochondrial functions in an in vitro model of steatosis, thus providing a potentially valuable pharmacological tool for NAFLD treatment.

Effects of intragastric balloon in patients with nonalcoholic fatty liver disease and advanced fibrosis.

BACKGROUND AND AIMS: Effective therapy for clinically significant fibrosis in nonalcoholic fatty liver disease (NAFLD) is an unmet need. Data on the effectiveness of endoscopic placement of intragastric balloon (IGB) in patients with NAFLD are limited. In this study, we evaluated the impact of IGB placement in NAFLD patients with advanced fibrosis. METHODS: We retrospectively assessed the effects of the Orbera™ fluid-filled IGB in a cohort of obese patients with liver stiffness ≥9.7 kPa (corresponding to F3-F4). Patients with endoscopic signs of portal hypertension were excluded. Changes in metabolic and liver parameters from baseline to follow-up (6 mo) were assessed. RESULTS: A total of 26 obese patients, aged 53 [44 - 62] years, with BMI 35.1 ± 4.7 kg/m2 were included. All patients achieved a significant body weight loss (106 ± 19.7 vs. 92 ± 18.3 kg, P < .001) and waist circumference reduction (116 ± 13.3 vs. 104 ± 13.4 kg, P < .001) at 6-month follow-up after IGB placement. Weight loss induced by IGB lowered blood glucose (140 [112; 169] vs. 118 [94; 144] mg/dl, P < .01), glycated hemoglobin (7.5 ± 1.3 vs. 6.6 ± 1.2%, P < .001), FIB-4 (3.2 ± 0.7 vs. 2.7 ± 0.8, P < .001), liver stiffness (13.3 ± 3.2 vs. 11.3 ± 2.8 kPa, P < .001) and controlled attenuation parameter (355 [298-400] vs. 296 [255-352] dB/m, P < .01). Gastroesophageal reflux symptoms were common, but no severe adverse event was observed. CONCLUSION: Obese patients with advanced liver fibrosis, treated with 6-month IGB, can achieve regression of fibrosis as assessed by reduction of liver stiffness and FIB-4. Randomized controlled trials are needed to confirm these findings.

Endoscopic bariatric and metabolic therapies for non-alcoholic fatty liver disease: Evidence and perspectives.

Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in industrialized countries because of the worldwide epidemic of obesity. Beyond metabolic complications, a subset of patients with NAFLD develop non-alcoholic steatohepatitis (NASH) with fibrosis, which is emerging as a leading cause of liver transplantation due to progression to cirrhosis and cancer. For these reasons, NAFLD is considered a public health burden. In recent years endoscopic bariatric and metabolic therapies (EBMT) have emerged as safe and effective for the treatment of obesity and type 2 diabetes mellitus. EBMT include gastric and duodenal devices and techniques such as intragastric balloons, endoscopic sleeve gastroplasty, endoscopic small bowel by-pass and duodenal mucosal resurfacing. Observational studies and pilot trials have revealed beneficial effects of EBMT on NAFLD as assessed by non-invasive parameters or histology. In this review we summarise current evidence for the efficacy and safety of EBMT in obese patients with NAFLD and examine future clinical applications.

Hepatitis C virus eradication by direct antiviral agents abates oxidative stress in patients with advanced liver fibrosis.

BACKGROUND AND AIMS: HCV eradication improves non-hepatic outcomes such as cardiovascular diseases, although without clearly defined mechanisms. In this study we aimed to assess whether improvement of carotid atherosclerosis may be linked to a reduction in systemic oxidative stress after viral clearance. METHODS: We studied a retrospective cohort of 105 patients (age 62.4 ± 11.2 years; 62 men) with F3/F4 fibrosis, characterized by carotid ultrasonography at baseline and at sustained virologic response (SVR) follow-up. Levels of 8-iso-prostaglandin F2α (F2 -isoprostanes) and other oxidative stress markers were measured on frozen sera. Association between change (denoted as Δ) in oxidative stress markers (exposures) and change in carotid intima-media thickness (cIMT) (outcome) was examined using multiple linear regression. RESULTS: Subclinical atherosclerosis, defined as the presence of carotid plaque and/or cIMT ≥ 0.9, was present in 72% of the cohort. All patients achieved SVR that led to reduction in cIMT (0.92 ± 0.20 vs 0.83 ± 0.21 mm, P < .001). HCV eradication markedly decreased serum levels of F2 -isoprostanes (620.5 [143.2; 1904.1] vs 119.51 [63.2; 400.6] pg/mL, P < .0001), lipid hydroperoxides (13.8 [6.3; 20.7] vs 4.9 [2.3; 9.6] nmol/µl, P < .0001) and 8-hydroxy-2'-deoxyguanosine (558.9 [321.0; 6301.2] vs 294.51 [215.31; 408.95] pg/mL, P < .0001), whereas increased serum GPx activity (10.44 [4.6; 16.3] vs 13.75 [9.42; 20.63] nmol/min/mL, P = .001). By multiple linear regression analysis ΔcIMT was independently associated with ΔF2 -isoprostanes (ß: 1.746 [0.948; 2.543]; P < .0001) after adjustment for age, baseline F2 -isoprostanes and baseline IMT. CONCLUSIONS: Besides association of lipid peroxidation with severity of liver disease, the reduction in F2 -isoprostanes may be involved in the improvement of atherosclerosis after HCV eradication.

Crosstalk between adipose tissue insulin resistance and liver macrophages in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is likely due to the interaction between a deranged metabolic milieu and local mediators of hepatic inflammation and fibrosis. We undertook this study to elucidate the interplay between macrophage activation, insulin resistance (IR) in target organs/tissues and hepatic damage. METHODS: In 40 non-diabetic patients with biopsy-proven NAFLD we assessed: i) endogenous glucose production (EGP), glucose clearance and indexes of IR in the adipose tissue (Adipo-IR and Lipo-IR) and in the liver (Hep-IR) by tracer infusion ([6,6-2H2]glucose and [2H5]glycerol); ii) macrophage activity (by soluble sCD163) and iii) hepatic expression of CD163 (hCD163). RESULTS: We found that sCD163 levels paralleled both the plasma free fatty acid (FFA) levels and lipolysis from adipose tissue. Consistently, sCD163 significantly correlated with adipose tissue IR (Adipo-IR: r = 0.32, p = 0.042; Lipo-IR: r = 0.39, p = 0.012). At multiple regression analysis, sCD163 levels were associated with FFA levels (rp = 0.35, p = 0.026). In vitro exposure of human monocyte-derived macrophages to palmitate enhanced sCD163 secretion. Conversely, sCD163 did not correlate with EGP or with Hep-IR. In the liver, hCD163 positively correlated with sCD163 (r = 0.58, p = 0.007) and the degree of steatosis (r = 0.34, p = 0.048), but not with EGP or Hep-IR (r = -0.27 and r = 0.11, respectively, p >0.10, both). CONCLUSIONS: Our findings suggest a link between deranged metabolism in the adipose tissue and activation of hepatic macrophages in patients with NAFLD, possibly in response to FFA overflow and independent of obesity and diabetes. Conversely, our findings do not support a link between activated hepatic macrophages and glucose metabolism (EGP or Hep-IR). The relationship between adipose tissue IR and hepatic macrophages should be considered to define therapeutic targets for NAFLD. LAY SUMMARY: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is likely due to the interaction between a deranged metabolic milieu and local mediators of hepatic inflammation and fibrosis in the insulin resistant state. This study provides in vivo support for a possible link between deranged metabolism in the adipose tissue and activation of hepatic macrophages in patients with NAFLD, most likely in response to free fatty acid overflow and independent of obesity and diabetes.

Hepatitis C virus eradication by direct antiviral agents improves glucose tolerance and reduces post-load insulin resistance in nondiabetic patients with genotype 1.

BACKGROUND AND AIMS: Genotype 1 chronic hepatitis C is associated with an impairment of glucose homoeostasis, especially in the advanced stages of the disease. Glucose tolerance is an independent predictor of liver-related mortality in patients with cirrhosis because of chronic hepatitis C. However, no study has demonstrated so far weather hepatitis C virus clearance affects glucose tolerance. METHODS: To this aim, we performed a prospective study assessing the effects of direct antiviral agents treatment in nondiabetic cirrhotic patients with genotypes 1a/1b and impaired glucose tolerance based on a 75-g oral glucose tolerance test. Impaired glucose tolerance was diagnosed by a 2-hour plasma glucose between 140 and 199 mg/dL. Insulin resistance was estimated by the oral glucose insulin sensitivity index, an oral glucose tolerance test-derived measure. RESULTS: After meeting the inclusion criteria, the study population included 32 outpatients (26/6 genotypes 1b/1a; age 62 ± 7.4 years; 18 males) with compensated Child-A cirrhosis. All patients achieved a sustained virological response following direct antiviral agents treatment. After viral eradication, we did not observe change in fasting plasma glucose (103.5 ± 7.1 vs 102.8 ± 7.2 mg/dL, P = .15) but 2-hour plasma glucose was reduced (165.2 ± 22.7 vs 138.5 ± 21.3 mg/dL, P < .001). Hepatitis C virus eradication led also to a significant reduction in HbA1c (6.1 ± 0.2% vs 5.7 ± 0.3%, P < .001) and post-load insulin resistance as assessed by the oral glucose insulin sensitivity index (6.92 ± 1.56 vs 9.52 ± 1.39 mg/kg/min, P < .001). These effects were observed despite no change in body mass index from baseline to follow-up (25.6 ± 4.3 vs 25.8 ± 4.4, P > .5). CONCLUSIONS: Our results indicate that hepatitis C virus eradication may early improve glucose tolerance in patients with hepatitis C virus-related cirrhosis.

Peripheral insulin resistance predicts liver damage in nondiabetic subjects with nonalcoholic fatty liver disease.

UNLABELLED: Surrogate indexes of insulin resistance and insulin sensitivity are widely used in nonalcoholic fatty liver disease (NAFLD), although they have never been validated in this population. We aimed to validate the available indexes in NAFLD subjects and to test their ability to predict liver damage also in comparison with the NAFLD fibrosis score. Surrogate indexes were validated by the tracer technique (6,6-D2 -glucose and U-(13) C-glucose) in the basal state and during an oral glucose tolerance test. The best-performing indexes were used in an independent cohort of 145 nondiabetic NAFLD subjects to identify liver damage (fibrosis and nonalcoholic steatohepatitis). In the validation NAFLD cohort, homeostasis model assessment of insulin resistance, insulin to glucose ratio, and insulin sensitivity index Stumvoll had the best association with hepatic insulin resistance, while peripheral insulin sensitivity was most significantly related to oral glucose insulin sensitivity index (OGIS), insulin sensitivity index Stumvoll, and metabolic clearance rate estimation without demographic parameters. In the independent cohort, only oral glucose tolerance test-derived indexes were associated with liver damage and OGIS was the best predictor of significant (≥F2) fibrosis (odds ratio = 0.76, 95% confidence interval 0.61-0.96, P = 0.0233) and of nonalcoholic steatohepatitis (odds ratio = 0.75, 95% confidence interval 0.63-0.90, P = 0.0021). Both OGIS and NAFLD fibrosis score identified advanced (F3/F4) fibrosis, but OGIS predicted it better than NAFLD fibrosis score (odds ratio = 0.57, 95% confidence interval 0.45-0.72, P < 0.001) and was also able to discriminate F2 from F3/F4 (P < 0.003). CONCLUSION: OGIS is associated with peripheral insulin sensitivity in NAFLD and inversely associated with an increased risk of significant/advanced liver damage in nondiabetic subjects with NAFLD.

The Mediterranean dietary pattern as the diet of choice for non-alcoholic fatty liver disease: Evidence and plausible mechanisms.

Non-alcoholic fatty liver disease (NAFLD) has become a major global health burden, leading to increased risk for cirrhosis, hepatocellular carcinoma, type-2 diabetes and cardiovascular disease. Lifestyle intervention aiming at weight reduction is the most established treatment. However, changing the dietary composition even without weight loss can also reduce steatosis and improve metabolic alterations as insulin resistance and lipid profile. The Mediterranean diet (MD) pattern has been proposed as appropriate for this goal, and was recommended as the diet of choice for the treatment of NAFLD by the EASL-EASD-EASO Clinical Practice Guidelines. The MD has an established superiority in long term weight reduction over low fat diet, but it improves metabolic status and steatosis even without it. However, the effect on liver inflammation and fibrosis was tested only in few observational studies with positive results. Furthermore, considering the strong association between NAFLD and diabetes and CVD, the MD has a highly established advantage in prevention of these diseases, demonstrated in randomized clinical trials. The individual components of the MD such as olive oil, fish, nuts, whole grains, fruits, and vegetables, have been shown to beneficially effect or negatively correlate with NAFLD, while consumption of components that characterize a Western dietary pattern as soft drinks, fructose, meat and saturated fatty acids have been shown to have detrimental association with NAFLD. In this review we will cover the epidemiological evidence and the plausible molecular mechanisms by which the MD as a whole and each of its components can be of benefit in NAFLD.

Natural antioxidants for non-alcoholic fatty liver disease: molecular targets and clinical perspectives.

Non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease (NAFLD), is emerging as a main health problem in industrialized countries. Lifestyle modifications are effective in the treatment of NAFLD; however, the long-term compliance is low. Therefore, several pharmacological treatments have been proposed but none has shown significant efficacy or long-term safety. Natural polyphenols are a heterogeneous class of polyphenolic compounds contained in vegetables, which are being proposed for the treatment of different metabolic disorders. Although the beneficial effect of these compounds has traditionally related to their antioxidant properties, they also exert several beneficial effects on hepatic and extra-hepatic glucose and lipid homeostasis. Furthermore, natural polyphenols exert antifibrogenic and antitumoural effects in animal models, which appear relevant from a clinical point of view because of the association of NASH with cirrhosis and hepatocellular carcinoma. Several polyphenols, such anthocyanins, curcumin and resveratrol and those present in coffee, tea, soy are available in the diet and their consumption can be proposed as part of a healthy diet for the treatment of NAFLD. Other phenolic compounds, such as silymarin, are commonly consumed worldwide as nutraceuticals or food supplements. Natural antioxidants are reported to have beneficial effects in preclinical models of NAFLD and in pilot clinical trials, and thus need clinical evaluation. In this review, we summarize the existing evidence regarding the potential role of natural antioxidants in the treatment of NAFLD and examine possible future clinical applications.

Non-high-density lipoprotein cholesterol independently predicts new onset of non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is associated with increased cardiovascular disease (CVD) risk. Non-high-density lipoprotein cholesterol (non-HDL-C), i.e. total cholesterol minus HDL, is a well-established risk factor for CVD; however, its association with NAFLD development has not been established. Our aim was to test whether non-HDL-C is an independent predictor of new onset of NAFLD. METHODS: A prospective cohort study of 213 subjects from the general population, without liver disease, was studied. Evaluation of medical history, dietary and physical activity habits, fasting blood tests and ultrasonographic evidence of NAFLD was performed at baseline and after a 7-year follow-up by identical protocols. RESULTS: From 147 patients that did not have NAFLD at baseline, 28 (19%) developed NAFLD at the 7-year follow-up. The baseline levels of non-HDL-C were higher among subjects who developed NAFLD (179.5 ± 37.1 vs. 157.3 ± 35.1 mg/dl, P = 0.003). Non-HDL-C independently predicted new onset of NAFLD adjusting for age, gender, BMI or waist circumference, lifestyle and serum insulin (OR = 1.02 for every mg/dl increment, 1.01-1.04 95% CI, P = 0.008). Non-HDL-C was a stronger predictor for NAFLD than total cholesterol, low-density lipoprotein cholesterol, triglycerides and HDL. No patients with non-HDL-C < 130 mg/dl developed NAFLD, whereas 20.8% of those with values between 130 to 160 and 24.6% of those with values >160 mg/dl developed NAFLD (P for trend = 0.015). CONCLUSIONS: Non-HDL-C is an independent predictor for NAFLD and a stronger predictor than other lipoproteins. This association may stem from the combined hepato-toxic effect of non-HDL-C and may explain the association between NAFLD and CVD.

Effects of IL28B rs12979860 CC genotype on metabolic profile and sustained virologic response in patients with genotype 1 chronic hepatitis C.

BACKGROUND & AIMS: Patients with genotype 1 chronic hepatitis C (G1 CHC) frequently develop steatosis and insulin resistance (IR), caused by metabolic and viral factors. These accelerate the progression of liver disease and reduce the response to therapy. A sustained virologic response (SVR) to therapy in patients with G1 CHC is associated strongly with polymorphisms near the interleukin-28B (IL28B) gene, but the interaction between IL28B genotype and IR, and their combined effects on SVR, have not been defined. We tested the association between the IL28B rs12979860 single-nucleotide polymorphism and metabolic features, including IR, and evaluated their effects on SVR. METHODS: We performed genotype analysis of IL28B rs12979860 for 434 white G1 CHC patients who underwent consecutive biopsy analysis at 3 tertiary centers. Metabolic profile analyses included assessments of lipid levels and IR by the homeostasis model assessment. RESULTS: Patients with the CC polymorphism in IL28B had higher levels of total and low-density lipoprotein cholesterol, lower levels of triglycerides, and a lower prevalence of IR and moderate-severe steatosis (P < .05) than patients without this genotype. By multiple logistic regression analysis, body mass index (odds ratio [OR], 1.223; P < .001), level of triglycerides (OR, 1.007; P = .006), the CC polymorphism in IL28B (OR, 0.378; P = .001), and levels of HCV RNA greater than 850,000 IU/mL (OR, 1.803; P = .01) were associated with IR. The CC polymorphism in IL28B (OR, 8.350; P < .001) and IR (OR, 0.432; P = .005), but not steatosis (OR, 0.582; P = 0.25), was associated with an SVR. CONCLUSIONS: In white patients with G1 CHC, the IL28B rs12979860 CC genotype is associated with reduced IR. IL28B rs12979860 genotype and IR by the homeostasis model assessment strongly affect the outcome of antiviral therapy.

Unconjugated bilirubin, a potent endogenous antioxidant, is decreased in patients with non-alcoholic steatohepatitis and advanced fibrosis.

BACKGROUND AND AIM: Oxidative stress is considered a key element in the progression of non-alcoholic fatty liver to non-alcoholic steatohepatitis (NASH). Unconjugated bilirubin is the main endogenous lipid antioxidant and is cytoprotective in different tissues and organs. In this study, it was evaluated if unconjugated bilirubin levels are associated with the degree of liver injury in patients with non-alcoholic fatty liver disease. METHODS: Two hundred and eighty-five patients were retrospectively evaluated with biopsy-confirmed non-alcoholic fatty liver disease. Multiple logistic regression models were used to assess the relationship of steatosis, inflammation, and fibrosis levels to the features of patients. RESULTS: Unconjugated bilirubin levels differed significantly according to inflammation and fibrosis scores. Unconjugated bilirubin was lower in patients with moderate-severe inflammation compared with those with absent-mild (P = 0.001) and in patients with moderate-severe fibrosis compared with those with absent-mild (P < 0.001), whereas no difference was observed for steatosis grades. At logistic regression analysis, low unconjugated bilirubin levels were associated with moderate-severe inflammation (odds ratio, 0.11; 95% confidence interval 0.02-0.76; P = 0.025) and moderate-severe fibrosis (odds ratio, 0.013; 95% confidence interval 0.001-0.253; P = 0.004). CONCLUSIONS: Low unconjugated bilirubin levels are independent predictors of advanced inflammation and fibrosis in patients with steatohepatitis, indicating the lack of antioxidant protection as a possible molecular determinant for the progression of liver injury.

Resistin-like beta reduction is associated to low survival rate and is downregulated by adjuvant therapy in colorectal cancer patients.

Colorectal Cancer (CRC) is one of the most common cancers accounting for 1.8 million new cases worldwide every year. Therefore, the identification of new potential therapeutic targets represents a continuous challenge to improve survival and quality of CRC patient's life. We performed a microarray analysis dataset consisting of colon biopsies of healthy subjects (HS) and CRC patients. These results were further confirmed in a clinical setting evaluating a series of CRC patients to assess the expression of Resistin-Like Beta (RETNLB) and to correlate it with their clinical data. Our results showed a significant reduction of RETNLB expression in CRC biopsies compared to the HS mucosa. Furthermore, such reduction was significantly associated with the TNM grade and patients' age. Furthermore, a significantly positive correlation was found within mutated subjects for KRAS, TP53, and BRAF. In particular, patients with poor prognosis at 5 years exhibited RETNLB lower levels. In-silico analysis data were confirmed by histochemical analysis in a series of CRC patients recruited by our group. The results obtained provided that RETNLB low levels are associated with an unfavorable prognosis in CRC patients and its expression is also dependent on adjuvant therapy. Further studies are warranted in order to evaluate the molecular mechanisms underlying the role of RETNLB in CRC progression.

Beneficial Effects of Silybin Treatment After Viral Eradication in Patients With HCV-Related Advanced Chronic Liver Disease: A Pilot Study.

Introduction and Aims: HCV eradication by direct-acting antivirals (DAAs) improves liver outcomes and reduces overall liver mortality. However, patients with advanced chronic liver disease (ACLD) may experience a progression of liver disease despite viral clearance. Silybin has shown hepatoprotective effects in experimental models, but clinical data are limited. The aim of this study is to evaluate the effect of a highly bioavailable form of silybin on liver fibrosis in patients with HCV-related ACLD after viral eradication with DAAs, in comparison with the standard of care. Methods: In this multicenter and prospective study, HCV patients with ACLD achieving SVR12 were treated with the combination of silybinphospholipid complex with vitamin D and vitamin E (Realsil 100D®, Ibi Lorenzini S.p.A., Aprilia, Italy) for 12 months (R group) compared to controls (C group). Patients were submitted to transient elastography (TE) and to the enhanced liver fibrosis (ELF) test at baseline, week 24, and week 48. Results: One hundred sixteen patients were enrolled, 56 in the R group and 60 in the C group. The median age was 68 years, and 53% were male, with no differences between groups. In both groups, liver stiffness improved at 6 and 12 months compared to baseline. However, patients in the R group compared to those in the C group showed a higher reduction of liver stiffness after 6 months (-2.05, 95% CI -3.89 to -0.22, p < 0.05) and 12 months of treatment (-2.79, 95% CI -4.5 to -1.09, p < 0.01) in comparison with baseline. No significant difference in the reduction of ELF was observed between the two groups. During the follow-up, four patients developed HCC, all in the C group. Conclusions: In HCV-related ACLD, the hepatoprotective effects of silybin may represent a tool to counteract liver disease progression.

High Meat Consumption Is Prospectively Associated with the Risk of Non-Alcoholic Fatty Liver Disease and Presumed Significant Fibrosis.

Non-alcoholic fatty liver disease (NAFLD) has been associated with meat consumption in cross-sectional studies. However, only a few prospective studies have been conducted, and they did not test for liver fibrosis. We aimed to assess the association between meat consumption changes and the incidence and remission of NAFLD and significant liver fibrosis. We used a prospective cohort study design, including 316 subjects aged 40-70 years, participating in baseline and follow-up evaluations at Tel-Aviv Medical Center. NAFLD was determined by liver ultrasound or controlled attenuation parameter (CAP), and liver fibrosis was determined by FibroScan. Meat consumption (g/day) was assessed by a food frequency questionnaire (FFQ). In multivariable-adjusted analyses, high consumption of red and/or processed meat (≥gender-specific median) was associated with a higher risk of NAFLD with elevated alanine aminotransferase (ALT) (OR = 3.75, 1.21-11.62, p = 0.022). Consistently high (in both baseline and follow-up evaluations) total meat consumption was associated with 2.55-fold (95% CI 1.27-5.12, p = 0.009) greater odds for new onset and/or persistence of NAFLD compared to consistently low meat consumption. A similar association was shown for consistently high consumption of red and/or processed meat (OR = 2.12, 95% CI 1.11-4.05, p = 0.022). Consistently high red and/or processed meat consumption was associated with 4.77-fold (95% CI 1.36-16.69, p = 0.014) greater odds for significant fibrosis compared to consistently low consumption. Minimizing the consumption of red and/or processed meat may help prevent NAFLD and significant fibrosis.

Coffee Restores Expression of lncRNAs Involved in Steatosis and Fibrosis in a Mouse Model of NAFLD.

BACKGROUND AND AIM: Coffee intake exerts protective effects against non-alcoholic fatty liver disease (NAFLD), although without fully cleared mechanisms. In this study we aimed to assess whether coffee consumption may influence the expression of long non-coding RNAs (lncRNAs) in the liver. METHODS: C57BL/6J mice were fed a 12-week standard diet (SD), high-fat diet (HFD) or HFD plus decaffeinated coffee solution (HFD + coffee). Expression of specific lncRNAs involved in NAFLD was analyzed by real-time PCR. For the most differentially expressed lncRNAs, the analysis was also extended to their mRNA targets. RESULTS: Decaffeinated coffee intake reduced body weight gain, prevented NAFLD, lowered hyperglycemia and hypercholesterolemia. NAFLD was associated with lower hepatic expression of Gm16551, a lncRNA inhibiting de novo lipogenesis, and higher expression of H19, a lncRNA promoting fibrogenesis. Coffee intake restored Gm16551 to levels observed in lean mice and downregulated gene expression of its targets acetyl coenzyme A carboxylase 1 and stearoyl coenzyme A desaturase 1. Furthermore, coffee consumption markedly decreased hepatic expression of H19 and of its target gene collagen alpha-1(I) chain; consistently, in mice fed HFD + coffee liver expression of αSMA protein returned to levels of mice fed SD. Expression of lncRNA involved in circadian clock such as fatty liver-related lncRNA 1 (FLRL1) and fatty liver-related lncRNA 2 (FLRL2) were upregulated by HFD and were also modulated by coffee intake. CONCLUSION: Hepatoprotective effects of coffee may be depending on the modulation of lncRNAs involved in key pathways of NAFLD onset and progression.