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Objectives: Steatotic liver disease is the most frequent chronic liver disease worldwide. Ultrasonography (US) is commonly employed for the assessment and diagnosis. Few information is available on the possible use of artificial intelligence (AI) to ameliorate the diagnostic accuracy of ultrasonography., Materials and Methods: An AI-based algorithm was developed using a dataset of US images. We prospectively enrolled 134 patients for algorithm validation. Patients underwent abdominal US and Proton Density Fat Fraction MRI scans (MRI-PDFF), assumed as reference technique. The hepatorenal index was manually calculated (HRIM) by 4 operators. An automatic hepatorenal index (HRIA) was obtained by the algorithm. The accuracy of HRIA to discriminate steatosis grades was evaluated by ROC analysis using MRI-PDFF cut-offs., Results: Overweight was 40 % of subjects (BMI 26.4 kg/cm 2 ). The median HRIA was 1.11 (IQR 0.32) and the average of 4 manually calculated HRIM was 1.08 (IQR 0.26), with a 15 % inter-operator variability. Both HRIA (R = 0.79, P < 0.0001) and HRIM (R = 0.69, P < 0.0001) significantly correlated with liver fat percentage (MRI-PDFF). According to MRI-PDFF, 32 % of enrolled subjects had steatosis. Discrimination capacity by AUC between patient with steatosis and patient without steatosis was better for HRIA than HRIM (AUC: 0.87 vs. 0.82, respectively). ROC analysis showed an AUC = 0.98 for HRIA with 1.64 cut-off in distinguishing between mild and moderate/severe groups., Conclusions: The use of AI improves accuracy and speed of ultrasonography in the diagnosis of liver steatosis. Further studies should evaluate the routine use of this technique in the management of liver steatosis at high cardio-metabolic risk., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024. Published by Elsevier B.V.)

The prevalence of overweight, obesity, type 2 diabetes, metabolic syndrome and steatotic liver disease is rapidly increasing worldwide with a huge economic burden in terms of morbidity and mortality. Several genetic and environmental factors are involved in the onset and development of metabolic disorders and related complications. A critical role also exists for the gut microbiota, a complex polymicrobial ecology at the interface of the internal and external environment. The gut microbiota contributes to food digestion and transformation, caloric intake, and immune response of the host, keeping the homeostatic control in health. Mechanisms of disease include enhanced energy extraction from the non-digestible dietary carbohydrates, increased gut permeability and translocation of bacterial metabolites which activate a chronic low-grade systemic inflammation and insulin resistance, as precursors of tangible metabolic disorders involving glucose and lipid homeostasis. The ultimate causative role of gut microbiota in this respect remains to be elucidated, as well as the therapeutic value of manipulating the gut microbiota by diet, pre- and pro- synbiotics, or fecal microbial transplantation., Competing Interests: Declaration of Competing Interest PP received research grants and speaker fees from Institut Allergosan (Graz, Austria)., (Copyright © 2023. Published by Elsevier B.V.)

Background: Carvacrol, a plant phenolic monoterpene, is largely employed as food additive and phytochemical., Objective: We aimed to assess the lipid lowering and protective effects of carvacrol in vitro using cellular models of hepatic steatosis and endothelial dysfunction. We also investigated if and how the binding of carvacrol to albumin, the physiological transporter for small compounds in the blood, might be altered by the presence of high levels of fatty acids (FAs)., Methods: Hepatic FaO cells treated with exogenous FAs mimic hepatosteatosis; endothelial HECV cells exposed to hydrogen peroxide are a model of endothelial dysfunction. In these models, we measured spectrophotometrically lipid accumulation and release, lipoperoxidation, free radical production, and nitric oxide release before and after treatment with carvacrol. The carvacrol binding to albumin in the presence or absence of high levels of FAs was assessed by absorption and emission spectroscopies., Results: Carvacrol counteracted lipid accumulation and oxidative stress in hepatocytes and protected endothelial cells from oxidative stress and dysfunction. Moreover, high levels of FAs reduced the binding of carvacrol to albumin., Conclusion: The results suggest the good potential of carvacrol in ameliorating dysfunction of hepatic and endothelial cells in vitro. High levels of circulating FAs might compete with carvacrol for binding to albumin thus influencing its transport and bio-distribution., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background and Aims: Several chronic multifactorial diseases originate from energy unbalance between food intake and body energy expenditure, including non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiovascular disorders. Vascular endothelium plays a central role in body homeostasis, and NAFLD is often associated with endothelial dysfunction (ED), the first step in atherosclerosis. Both sugars and fatty acids (FAs) are fuel sources for energy production, but their excess leads to liver steatosis which may trigger ED through a network of mechanisms which need to be clarified. Here, we investigated the crosstalk pathways between in vitro cultured steatotic hepatocytes (FaO) and endothelial cells (HECV) being mediated by soluble factors., Methods and Results: We employed the conditioned medium approach to test how different extent and features of hepatic steatosis distinctively affect endothelium leading to ED. The steatogenic media collected from steatotic hepatocytes were characterized by high triglyceride content and led to lipid accumulation and fat-dependent dysfunction in HECV cells. We found a parallelism between (i) extent of hepatocyte steatosis and level of lipid accumulation in HECV cells; (ii) type of hepatocyte steatosis (with macro- or microvesicular LDs) and extent of oxidative stress, lipid peroxidation, nitric oxide release and expression of ED markers in HECV cells., Conclusions: The present findings seem to suggest that, in addition to triglycerides, other soluble mediators should be released by steatotic hepatocytes and may influence lipid accumulation and function of HECV cells. Further studies need to depict the exact profile of soluble factors involved in steatotic hepatocyte-endothelium crosstalk., (Copyright © 2021. Published by Elsevier B.V.)

Intestinal permeability (IP) is essential in maintaining gut-metabolic functions in health. An unequivocal evaluation of IP, as marker of intestinal barrier integrity, however, is missing in health and in several diseases. We aimed to assess IP in the whole gastrointestinal tract according to body mass index (BMI) and liver steatosis. In 120 patients (61F:59M; mean age 45 ± SEM 1.2 years, range: 18-75), IP was distinctively studied by urine recovery of orally administered sucrose (SO, stomach), lactulose/mannitol ratio (LA/MA, small intestine), and sucralose (SA, colon). By triple quadrupole mass-spectrometry and high-performance liquid chromatography, we measured urinary recovery of saccharide probes. Subjects were stratified according to BMI as normal weight, overweight, and obesity, and answered questionnaires regarding dietary habits and adherence to the Mediterranean Diet. Liver steatosis was assessed by ultrasonography. IP at every gastrointestinal tract was similar in both sexes and decreased with age. Stomach and small intestinal permeability did not differ according to BMI. Colonic permeability increased with BMI, waist, neck, and hip circumferences and was significantly higher in obese than in lean subjects. As determined by logistic regression, the odds ratio (OR) of BMI increment was significantly higher in subjects in the highest tertile of sucralose excretion, also after adjusting for age and consumption of junk food. The presence of liver steatosis was associated with increased colonic permeability. Patients with lower score of adherence to Mediterranean diet had a higher score of 'junk food'. Intestinal permeability tended to increase in subjects with a lower adherence to Mediterranean diet. In conclusion, colonic (but not stomach and small intestinal) permeability seems to be linked to obesity and liver steatosis independently from dietary habits, age, and physical activity. The exact role of these last factors, however, requires specific studies focusing on intestinal permeability. Results should pave the way to both primary prevention measures and new therapeutic strategies in metabolic and liver diseases., Competing Interests: The authors declare no conflict of interest.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease often associated with overnutrition. Number and morphometry of lipid droplets (LDs) define micro vs macrovesicular steatosis, influence the morphology and function of hepatocytes and possibly their stiffness. The link between grade and features of steatosis and biomechanical properties of single hepatocytes requires deeper investigations. In vitro NAFLD models with distinct steatosis conditions were set by exposing FaO hepatoma cells to single or combined fructose (Fru), fatty acids (FA), and tumor necrosis factor (TNF)α. Single Cell Force Spectroscopy and Quantitative Phase Microscopy quantified the single cell stiffness and a series of morphometric parameters; the mRNA expression of genes involved in lipid metabolism was quantified by real-time PCR. In our models, LD size and number increased with Fru and FA as single agents, and more with combined Fru/FA (macrovesicular steatosis), while FA/TNFα combination increased LD number with a reduction in their size (microvesicular steatosis). We found that the changes in LD size and number influenced cell stiffness and morphometry as follows: (i) single cell elasticity increased in macrovesicular steatosis (maximally with combined Fru/FA); (ii) FA-induced steatosis resulted in cells thinner and larger, whereas combined FA/TNFα shrunk the hepatocytes. Taken together the data on hepatocyte biomechanics show that, in addition to extent of lipid accumulation, cell stiffness is mainly influenced by LD size, while cell morphometry directly relates to LD number. Our findings suggest that a novel mechanobiology perspective might provide future contributions in NAFLD research., (Copyright © 2019. Published by Elsevier Ltd.)

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.

Nonalcoholic fatty liver disease is one of the most common hepatic disorders worldwide. Given the high-calorie nutrition of children and adults, nonalcoholic fatty liver disease (NAFLD) is expected to become a major cause of cirrhosis and eventually liver transplantation. Familial clustering and ethnic differences indicate that genetic factors contribute to NAFLD. Recently, the common variant p.I148M of the enzyme adiponutrin (PNPLA3) has emerged as a major genetic determinant of hepatic steatosis and nonalcoholic steatohepatitis as well as its pathobiological sequelae fibrosis, cirrhosis, and hepatocellular cancer. PNPLA3 encodes a lipid droplet-associated, carbohydrate-regulated lipogenic and/or lipolytic enzyme. Homozygous carriers of the PNPLA3 variant are prone to develop cirrhosis in the absence of other risk factors such as alcohol or viral hepatitis. Here we review the plethora of studies that unraveled the association between PNPLA3 and NAFLD in children and adults, discuss its distinct effects on liver and metabolic traits, and introduce the term PNPLA3-associated steatohepatitis (PASH) as a novel gene-based liver disease. Given the prevalence of the risk allele in 40 to 50% of Europeans, the authors conclude that PNPLA3 should be considered in the diagnostic workup of fatty liver disease and that homozygous risk allele carriers might benefit from careful cancer surveillance., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD), is a worrisome health problem worldwide characterized by intrahepatic triacylglycerol (TG) overaccumulation. NAFLD is a common feature of metabolic syndrome being often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. The mechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimately resulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic import of glycerol, the other primary source (as glycerol-3-phosphate) of increased TG in hepatocytes. Obese leptin-deficient (ob/ob) mice, an animal model of NAFLD, were used to evaluate the functional involvement of Aquaporin-9 (AQP9), the major pathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obese mice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at the hepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33%) than lean mice, a finding fully confirmed by immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantly lower (53%) than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increased level of plasma glycerol characterizing obese mice. In summary, our results suggest implication of AQP9 in liver steatosis. The reduction of hepatocyte AQP9 and, consequently, glycerol permeability might be a defensive mechanism to counteract further fat infiltration in liver parenchyma.

Accumulation of triacylglycerols within the cytoplasm of hepatocytes to the degree that lipid droplets are visible microscopically is called liver steatosis. Most commonly, it occurs when there is an imbalance between the delivery or synthesis of fatty acids in the liver and their disposal through oxidative pathways or secretion into the blood as a component of triacylglycerols in very low density lipoprotein. This disorder is called nonalcoholic fatty liver disease (NAFLD) in the absence of alcoholic abuse and viral hepatitis, and it is often associated with insulin resistance, obesity and type 2 diabetes. Also, liver steatosis can be induced by many other causes including excessive alcohol consumption, infection with genotype 3 hepatitis C virus and certain medications. Whereas hepatic triacylglycerol accumulation was once considered the ultimate effector of hepatic lipotoxicity, triacylglycerols per se are quite inert and do not induce insulin resistance or cellular injury. Rather, lipotoxic injury in the liver appears to be mediated by the global ongoing fatty acid enrichment in the liver, paralleling the development of insulin resistance. A considerable number of fatty acid metabolites may be responsible for hepatic lipotoxicity and liver injury. Additional key contributors include hepatic cytosolic lipases and the "lipophagy" of lipid droplets, as sources of hepatic fatty acids. The specific origin of the lipids, mainly triacylglycerols, accumulating in liver has been unraveled by recent kinetic studies, and identifying the origin of the accumulated triacylglycerols in the liver of patients with NAFLD may direct the prevention and treatment of this condition., (© 2013 American Physiological Society. Compr Physiol 3:1493-1532, 2013.)

Aim: To investigate the effectiveness of antioxidant compounds in modulating mitochondrial oxidative alterations and lipids accumulation in fatty hepatocytes., Methods: Silybin-phospholipid complex containing vitamin E (Realsil(®)) was daily administered by gavage (one pouch diluted in 3 mL of water and containing 15 mg vitamin E and 47 mg silybin complexed with phospholipids) to rats fed a choline-deprived (CD) or a high fat diet [20% fat, containing 71% total calories as fat, 11% as carbohydrate, and 18% as protein, high fat diet (HFD)] for 30 d and 60 d, respectively. The control group was fed a normal semi-purified diet containing adequate levels of choline (35% total calories as fat, 47% as carbohydrate, and 18% as protein). Circulating and hepatic redox active and nitrogen regulating molecules (thioredoxin, glutathione, glutathione peroxidase), NO metabolites (nitrosothiols, nitrotyrosine), lipid peroxides [malondialdehyde-thiobarbituric (MDA-TBA)], and pro-inflammatory keratins (K-18) were measured on days 0, 7, 14, 30, and 60. Mitochondrial respiratory chain proteins and the extent of hepatic fatty infiltration were evaluated., Results: Both diet regimens produced liver steatosis (50% and 25% of liver slices with CD and HFD, respectively) with no signs of necro-inflammation: fat infiltration ranged from large droplets at day 14 to disseminated and confluent vacuoles resulting in microvesicular steatosis at day 30 (CD) and day 60 (HFD). In plasma, thioredoxin and nitrosothiols were not significantly changed, while MDA-TBA, nitrotyrosine (from 6 ± 1 nmol/L to 14 ± 3 nmol/L day 30 CD, P < 0.001, and 12 ± 2 nmol/L day 60 HFD, P < 0.001), and K-18 (from 198 ± 20 to 289 ± 21 U/L day 30 CD, P < 0.001, and 242 ± 23 U/L day 60 HFD, P < 0.001) levels increased significantly with ongoing steatosis. In the liver, glutathione was decreased (from 34.0 ± 1.3 to 25.3 ± 1.2 nmol/mg prot day 30 CD, P < 0.001, and 22.4 ± 2.4 nmol/mg prot day 60 HFD, P < 0.001), while thioredoxin and glutathione peroxidase were initially increased and then decreased. Nitrosothiols were constantly increased. MDA-TBA levels were five-fold increased from 9.1 ± 1.2 nmol/g to 75.6 ± 5.4 nmol/g on day 30, P < 0.001 (CD) and doubled with HFD on day 60. Realsil administration significantly lowered the extent of fat infiltration, maintained liver glutathione levels during the first half period, and halved its decrease during the second half. Also, Realsil modulated thioredoxin changes and the production of NO derivatives and significantly lowered MDA-TBA levels both in liver (from 73.6 ± 5.4 to 57.2 ± 6.3 nmol/g day 30 CD, P < 0.01 and from 27.3 ± 2.1 nmol/g to 20.5 ± 2.2 nmol/g day 60 HFD, P < 0.01) and in plasma. Changes in mitochondrial respiratory complexes were also attenuated by Realsil in HFD rats with a major protective effect on Complex II subunit CII-30., Conclusion: Realsil administration effectively contrasts hepatocyte fat deposition, NO derivatives formation, and mitochondrial alterations, allowing the liver to maintain a better glutathione and thioredoxin antioxidant activity.

The diagnostic utilities of ultrasonography (US), fatty liver index (FLI) and an algorithm of nine serum markers (Fibromax) were evaluated in family practice to noninvasively characterize patients with nonalcoholic fatty liver disease (NAFLD). A multicenter study was conducted by enrolling 259 consecutively observed patients (age 51 ± 10 years) with clinical and ultrasonographic features of NAFLD . Patients had mild (16.2%), moderate (69.9%), or severe (13.9%) liver steatosis and 60.2% had hypertransaminasemia. The percent of patients with overweight, obesity, diabetes, hypertension, and dyslipidemia were 42.7%, 46.5% (4.2% severe obesity), 24.7%, 40.9%, and 56.4% , respectively. Lean patients (10.8%) had normal transaminases in two/thirds of the cases. A multivariate logistic regression (including age > 50 yrs, BMI > 30 kg/m2, HOMA > 3, and hypertransaminasemia) identified 12.3% of patients at risk for steatohepatitis. With a sensitivity of 50% and specificity of 94.7%, Fibromax identified 34 patients (13.1%) with likely advanced fibrosis and found that over 28% of patients with moderate (ultrasonographic) steatosis were likely to be carrying severe steatosis. Steatotest score was significantly associated with BMI, waist circumference, ALT, triglycerides, and FLI. Fibrotest correlated only with ALT. FLI identified 73.4% of patients as likely to be carrying a fatty liver. In conclusion, NAFLD should be systematically searched and characterized in all patients with metabolic disturbances and cardiovascular risk. Asymptomatic subjects at risk also should be screened for NAFLD. Fibromax is a promising noninvasive diagnostic tool in family medicine for identifying patients at risk for NAFLD who require targeted follow-up.

Objectives: Mitochondria play a major role in cell energy-generating processes and integrate several signalling pathways to control cellular life and death., Design and Methods: Several liver diseases are characterized by mitochondrial alterations which are directly or indirectly dependent on the activation of intracellular stress cascades or receptor-mediated pathways. This article examines the role of mitochondrial dysfunction in critical initiating or propagating events in fatty liver infiltration and nonalcoholic fatty liver disease (NAFLD). Genetic variants and the role of drug-induced toxicity have been considered., Results: Key alterations of mitochondrial physiology associated with hepatocyte fatty changes are described. The value of novel non-invasive diagnostic methods to detect mitochondrial metabolic alterations is also discussed., Conclusions: Mitochondrial metabolic remodeling is a predominant factor in the appearance and perpetuation of hepatocyte fat accumulation. Non-invasive techniques to identify mitochondrial dysfunction and proper mitochondria protection are two necessary clinical steps for an efficient management of NAFLD., (Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The only currently recommended treatment for nonalcoholic fatty liver disease (NAFLD) is lifestyle modification. Preliminary studies of silybin showed beneficial effects on liver function. Realsil (RA) comprises the silybin phytosome complex (silybin plus phosphatidylcholine) coformulated with vitamin E. We report on a multicenter, phase III, double-blind clinical trial to assess RA in patients with histologically documented NAFLD. Patients were randomized 1:1 to RA or placebo (P) orally twice daily for 12 months. Prespecified primary outcomes were improvement over time in clinical condition, normalization of liver enzyme plasma levels, and improvement of ultrasonographic liver steatosis, homeostatic model assessment (HOMA), and quality of life. Secondary outcomes were improvement in liver histologic score and/or decrease in NAFLD score without worsening of fibrosis and plasma changes in cytokines, ferritin, and liver fibrosis markers. We treated 179 patients with NAFLD; 36 were also HCV positive. Forty-one patients were prematurely withdrawn and 138 patients analyzed per protocol (69 per group). Baseline patient characteristics were generally well balanced between groups, except for steatosis, portal infiltration, and fibrosis. Adverse events (AEs) were generally transient and included diarrhea, dysgeusia, and pruritus; no serious AEs were recorded. Patients receiving RA but not P showed significant improvements in liver enzyme plasma levels, HOMA, and liver histology. Body mass index normalized in 15% of RA patients (2.1% with P). HCV-positive patients in the RA but not the P group showed improvements in fibrogenesis markers. This is the first study to systematically assess silybin in NAFLD patients. Treatment with RA but not P for 12 months was associated with improvement in liver enzymes, insulin resistance, and liver histology, without increases in body weight. These findings warrant further investigation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Caveolin-1, the main structural protein of caveolae, is involved in cholesterol homoeostasis, transcytosis, endocytosis and signal transduction and thought to play an important role in lipidogenesis. Little is known about the pathophysiological role of caveolin-1 in nonalcoholic fatty liver disease (NAFLD), a condition frequently associated with the metabolic syndrome and characterized by abnormal accumulation of intrahepatic triglycerides with a potentially harmful risk of evolution to liver fibrosis, cirrhosis and hepatocellular carcinoma., Materials and Methods: Liver steatosis (micro/macrovesicular) was induced in adult rats fed a choline-deficient diet for 14days and compared with a control normal diet. The expression and subcellular distribution of caveolin-1 was assessed using light and electron microscopy by immunohistochemical and immunocytochemical techniques and by Western blotting., Results: Caveolin-1 was mainly associated with the hepatocyte basolateral plasma membrane. Fatty hepatocytes were characterized by a significant increase in the expression of caveolin-1 around and within the lipid droplets as well as in the inner membrane of mitochondria., Conclusions: Our data suggest the involvement of caveolin-1 in the case of abnormal lipogenesis and mitochondrial function typical of steatotic hepatocytes in NAFLD. Addressing the role played by caveolin-1 in liver membranes in NAFLD may help future therapeutic choices in a frequent metabolic liver disease., (© 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.)

Non-alcoholic fatty liver disease (NAFLD), the most common liver disorder in the Western world, is a clinico-histopathological entity in which excessive triglyceride accumulation in the liver occurs. Non-alcoholic steatohepatitis (NASH) represents the necroinflammatory form, which can lead to advanced liver fibrosis, cirrhosis, and hepatocellular carcinoma. The pathogenesis of NAFLD/NASH is complex but increased visceral adiposity plus insulin resistance with increased free fatty acids release play an initial key role for the onset and perpetuation of liver steatosis. Further events in the liver include oxidative stress and lipid peroxidation, decreased antioxidant defences, early mitochondrial dysfunction, iron accumulation, unbalance of adipose-derived adipokines with a chronic proinflammatory status, and gut-derived microbial adducts. New gene polymorphisms increasing the risk of fatty liver, namely APOC3 and PNPLA3, have been lately identified allowing further insights into the pathogenesis of this condition. In our review pathophysiological, genetic, and essential diagnostic and therapeutic aspects of NAFLD are examined with future trends in this field highlighted., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The management of liver steatosis, due to its potential evolution towards severe diseases, requires accurate diagnosis. Fatty infiltration in liver diseases is commonly assessed histologically by semi-quantitative methods, which can be subjective. Automated computerized procedures using commercial software for image analysis have also been recently employed. The aim of the study was to develop an innovative automated computerized procedure to accurately evaluate both the morphometry and degree of lipid accumulation in liver. Fatty infiltration was assessed in paraffin- and resin-embedded samples of steatotic livers from rats undergoing 0, 3, 7, 14, and 30-day choline-deficient diet, and from liver biopsy of a morbidly obese patient undergoing bariatric surgery. Specific software was developed, which works with a morphological operator, in addition to a chromatic one to select lipid droplets. The choline-deficient diet induced steatosis with a gradual shift from micro- to macro-vesicular. In paraffin sections, the macrovesicles-to-microvesicles ratio and the degree of steatosis, when using only the chromatic operator, produced overestimates. Results were consistent in both rat and human samples. An improvement of topographic, morphometric and quantitative estimation of fatty liver infiltration is obtained with our software, working with a morphological operator and using semi-thin sections from resin-embedded samples. This innovative procedure may be applied to human liver samples, offering promising diagnostic and prognostic perspectives.

Background/aims: The epidemic diffusion of nonalcoholic fatty liver disease (NAFLD) represents an emerging problem in family medicine. General Practitioners (GPs) should pay attention to patients with fatty liver, look at associated conditions, identify causal factors and patients at risk of evolution. This study aimed to assess GPs' knowledge and practice and a training project impact about NAFLD:, Methods: 56 GPs filled a questionnaire before and after attending a tailored workshop on NAFLD, and performed a clinical survey in patients with persistent hypertransaminasemia including screening and liver biopsy when indicated. Four months after a training workshop, GPs were questioned again about their practice changes with NAFLD:, Results: At baseline, less than 30% of GPs considered NAFLD as a cause of persistent hypertransaminasemia; over two-thirds thought that NAFLD had a prevalence of 5-10% in the general population; about 50% considered hypertransaminasemia as the main indication for liver biopsy in NAFLD; their main approach included a low lipid-content diet. Comparison of pre/post workshop questionnaires showed significant improvements, despite knowledge on diet composition and steatogenic drugs remained poor. Among screened patients with hypertransaminasemia, NAFLD had a prevalence of 36% and was associated with the metabolic syndrome in more than 50%. Liver biopsy was obtained in 8% of NAFLD: Chronic viral hepatitis was better diagnosed than NAFLD (biopsy performed in 86% of cases). The training workshop resulted in practice changes concerning screening of risk patients, search for NASH and managing NAFLD in chronic viral hepatitis., Conclusions: GPs' knowledge about NAFLD appears barely adequate, thus targeted training is essential to improve their knowledge and practice.

Background: Little is known about nitric oxide (NO) metabolism and redox changes with hepatocyte adipocytic transformation. The aims of this study were to investigate the changes occurring in plasma and hepatic NO metabolites and redox balance in a rat experimental model of simple fatty liver, and to relate plasma with hepatic and mitochondrial changes at different degrees of steatosis., Materials and Methods: Circulating and hepatic redox active and nitrogen regulating molecules thioredoxin, glutathione, protein thiols (PSH), mixed disulfides (PSSG), NO metabolites nitrosothiols, nitrite plus nitrate (NOx), and lipid peroxides (TBARs) were measured in rats fed a choline deprived (CD) diet for 30 days., Results: At histology, the CD diet resulted in hepatocellular steatosis (75% of liver weight at day 30) with no signs of necro-inflammation. In plasma, thioredoxin, nitrosothiols and NOx were unchanged, while TBARs levels increased significantly and were positively related with hepatic TBARs (r = 0.87, P < 0.001) and lipid content (r = 0.90, P < 0.001). In the liver, glutathione initially increased (day 3) and then decreased. From day 14, PSH decreased and NO derivatives increased. Thioredoxin 1 had initially increased (days 7-14) and then decreased. In the mitochondria, on day 14, nitrosothiols were inversely related to thioredoxin 2 (r = 0.988, P < 0.05); on day 30, PSH were decreased by 70%, PSSG were doubled and related with nitrosothiols levels (r = 0.925, P < 0.001)., Conclusion: Adipocytic transformation of hepatocytes is accompanied by major interrelated modifications of redox parameters and NO metabolism especially at mitochondrial level, suggesting an early adaptive protective response but also an increased predisposition towards pro-oxidant insults.

Mitochondrial dysfunction and oxidative stress play a central role in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). This study aimed to elucidate the mechanism(s) responsible for mitochondrial dysfunction in nonalcoholic fatty liver. Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days. We examined the effect of CD diet on various parameters related to mitochondrial function such as complex I activity, oxygen consumption, reactive oxygen species (ROS) generation and cardiolipin content and oxidation. The activity of complex I was reduced by 35% in mitochondria isolated from CD livers compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. Hydrogen peroxide (H(2)O(2)) generation was significantly increased in mitochondria isolated from CD livers. The mitochondrial content of cardiolipin, a phospholipid required for optimal activity of complex I, decreased by 38% as function of CD diet, while there was a significantly increase in the level of peroxidized cardiolipin. The lower complex I activity in mitochondria from CD livers could be completely restored to the level of control livers by exogenously added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is found in individuals who do not drink or abuse alcohol and represents a significant health burden for the general community. NAFLD is often associated with one or more features of the metabolic syndrome and has potential for evolution towards non-alcoholic steatohepatitis (NASH), the necro-inflammatory form of liver steatosis. The most worrisome evolutive events in a subgroup of NASH patients include advanced liver fibrosis, cirrhosis, and hepatocellular carcinoma. Pathophysiology of NAFLD/NASH is complex, but studies point to a pre-eminent role of oxidative stress and lipid peroxidation in the liver, including early mitochondrial dysfunction. Changes follow an insulin resistance status with a background of a chronic pro-inflammatory status due to an excess of visceral adiposity. Although no established therapy exists for NAFLD/NASH, potential therapeutic approaches are discussed in this review.

Objective: To review evidence on the diagnosis and management of nonalcoholic fatty liver disease (NAFLD), the most common cause of chronic liver disease in human beings., Sources of Information: The literature was searched for clinical trials and review articles on NAFLD. Levels I and II evidence indicates the benefit of both lifestyle and pharmacologic interventions for NAFLD and nonalcoholic steatohepatitis (NASH)., Main Message: Scientific evidence does not currently support systematic screening for NAFLD. Both NAFLD and NASH are frequently discovered in overweight and obese patients with asymptomatic elevation of serum aminotransferase levels. Ultrasonography detects the presence of a fatty liver, but is unreliable for detecting and quantifying liver fibrosis. Patients with NAFLD should be monitored for possible progression to NASH, particularly if they have diabetes or metabolic syndrome. Although diet and exercise are the mainstays of treatment, medication might be warranted if an appropriate diet and regular physical activity do not improve biochemical markers and liver morphology. Referral for liver biopsy and further evaluation should be considered for those at higher risk of developing NASH., Conclusion: Although most patients with NAFLD have a benign course, some progress to NASH, liver cirrhosis, and hepatocellular carcinoma. These patients should be carefully monitored for progression of disease and treated for associated metabolic disturbances. An integrated approach to care is essential.

Non-alcoholic fatty liver disease (NAFLD) is often associated with features of the metabolic syndrome, carrying an increased risk to develop non-alcoholic steatohepatitis (NASH), the inflammatory form of liver steatosis. Epidemiological data confirm that obesity, diabetes, hypertension and hyperlipidemia are frequently found in NAFLD and worsen its prognosis because of increased risk of fibrotic evolution, eventually leading to liver cirrhosis. Recent studies confirm the close relationship between the metabolic syndrome and liver steatosis, and further support the detrimental role of oxidative stress and lipid peroxidation, which are pathophysiological processes present in both conditions. Novel diagnostic tools and life style modifications together with targeted therapeutic actions are urgently needed for the management of liver dysfunction in course of metabolic syndrome.

Although evidence suggests the link between chronic inflammation and oxidative stress as the main mechanism responsible for endothelial dysfunction and cardiovascular complications in patients with metabolic syndrome, little is known about the determining role of each metabolic syndrome component in such alterations. This study investigated the relation between systemic oxidative alterations and metabolic syndrome features in 41 patients. Compared with control subjects, serum vitamin C and alpha-tocopherol concentrations were lower and those of lipid peroxides [thiobarbituric acid reactive substances (TBARs)] were higher in metabolic syndrome patients (P < 0.001). A linear relation was observed between visceral fat thickness and serum TBARs:cholesterol ratio (r = 0.541, P < 0.001), whereas negative correlations were found between alpha-tocopherol and BMI (r = -0.212, P < 0.05) and the grade of liver steatosis (r = -0.263, P < 0.02). Patients with metabolic syndrome and liver steatosis had higher serum hyaluronate (HA) concentrations (P < 0.001). Serum HA was positively correlated with serum alanine amino transferase (r = 0.715, P < 0.001) and the homeostasis monitoring assessment index (r = 0.248, P < 0.03). The presence of metabolic syndrome was predicted from a linear combination of visceral fat and all oxidative variables. In metabolic syndrome patients, serum nitrosothiols and vitamin C concentrations, which were lower (P < 0.001) than in control subjects, were inversely related to the grade of hypertension (r = -0.645, P < 0.001 and r = -0.415, P < 0.007, respectively). In conclusion, metabolic syndrome patients exhibited decreased antioxidant protection and increased lipid peroxidation. Our results indicate a strong association between increased abdominal fat storage, liver steatosis, and systemic oxidative alterations in metabolic syndrome patients and diminished nitrosothiols and vitamin C concentrations as important factors associated with hypertension in these patients.

Effectively assessing subtle hepatic metabolic functions by novel non-invasive tests might be of clinical utility in scoring NAFLD (non-alcoholic fatty liver disease) and in identifying altered metabolic pathways. The present study was conducted on 39 (20 lean and 19 obese) hypertransaminasemic patients with histologically proven NAFLD {ranging from simple steatosis to severe steatohepatitis [NASH (non-alcoholic steatohepatitis)] and fibrosis} and 28 (20 lean and eight overweight) healthy controls, who underwent stable isotope breath testing ([(13)C]methacetin and [(13)C]ketoisocaproate) for microsomal and mitochondrial liver function in relation to histology, serum hyaluronate, as a marker of liver fibrosis, and body size. Compared with healthy subjects and patients with simple steatosis, NASH patients had enhanced methacetin demethylation (P=0.001), but decreased (P=0.001) and delayed (P=0.006) ketoisocaproate decarboxylation, which was inversely related (P=0.001) to the degree of histological fibrosis (r=-0.701), serum hyaluronate (r=-0.644) and body size (r=-0.485). Ketoisocaproate decarboxylation was impaired further in obese patients with NASH, but not in patients with simple steatosis and in overweight controls. NASH and insulin resistance were independently associated with an abnormal ketoisocaproate breath test (P=0.001). The cut-off value of 9.6% cumulative expired (13)CO(2) for ketoisocaproate at 60 min was associated with the highest prediction (positive predictive value, 0.90; negative predictive value, 0.73) for NASH, yielding an overall sensitivity of 68% and specificity of 94%. In conclusion, both microsomal and mitochondrial functions are disturbed in NASH. Therefore stable isotope breath tests may usefully contribute to a better and non-invasive characterization of patients with NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a frequent and potentially progressive chronic liver disease that occurs in subjects who do not abuse alcohol. NAFLD is often associated with obesity, metabolic syndrome and insulin resistance and its more aggressive form, nonalcoholic steatohepatitis (NASH) is a major cause of cryptogenic cirrhosis. NAFLD/NASH are commonly detected because of elevated serum aminotransferase levels, ultrasonographic fatty liver and, at liver histology, steatosis, inflammation, and occasionally fibrosis that may progress to cirrhosis. No established treatment exists for this potentially serious disorder. Current management of NAFLD/NASH is largely conservative and includes diet regimen, aerobic exercise, and interventions towards the associated metabolic abnormalities. The main concern is therefore to decrease liver steatosis and its progression toward steatohepatitis and fibrosis, and the risk of "cryptogenic" cirrhosis. Among the most promising medications, weight reducing drugs, insulin sensitizers and lipid-lowering agents, antioxidants, bile salts, co-factors increasing the mitochondrial transport of fatty acids are being considered. Among them, thiazolidinediones are the most promising drug family that act by activating PPARgamma nuclear receptors and by regulating both microsomal and peroxisomal lipid oxidative pathways. Pharmacological treatment of obesity and probiotics should be considered as potential therapeutic options. In this review, after summarizing the general background on fatty liver, the most current and attractive pharmacological approaches to the problem of NAFLD/NASH are discussed.

A condition defined as nonalcoholic fatty liver disease (NAFLD) is frequently found in humans. Deemed as a benign condition until recently, more emphasis is now put on the potential harmful evolution of the inflammatory form, that is, nonalcoholic steatohepatitis (NASH), toward end-stage liver disease. This review highlights the major morphologic and pathophysiological features of NASH. The link between experimental biochemical findings in animal models and clinical and therapeutic approaches in humans is discussed. Once all the other causes of persistent elevation of serum transaminase levels have been excluded, the diagnosis of NASH can be only confirmed by liver histology. Other noninvasive diagnostic tools, however, are being investigated to assess specific subcellular functions and to allow the follow-up of patients at higher risk for major liver dysfunction. A better understanding of various pathogenic aspects of NASH will help in identifying potential therapeutic approaches in these patients.

Nonalcoholic steatohepatitis (NASH) is an increasing recognized form of chronic liver condition affecting both children and adults within the wide spectrum of fatty liver diseases. Recently NASH has been often associated with insulin resistance and has shown potential harmful evolution towards end-stage liver disease. Its incidence and prevalence is increasing, paralleling the rise in obesity and diabetes mellitus in Western Countries. Once all the other causes of persistent elevation of serum transaminase levels are excluded, the diagnosis of NASH can be only confirmed by liver histology. Non-invasive diagnostic tools, however, are awaited to allow the follow-up of patients at higher risk for major liver dysfunction. This article focus on current thoughts on the natural history and clinical presentation of NASH and describes current trends in the diagnosis and treatment of this emerging condition.

The response of fatty liver to stress conditions (t-butyl hydroperoxide [t-BH] or 36 h of fasting) was investigated by assessing intracellular glutathione (GSH) compartmentation and redox status, GSH peroxidase (GSH-Px) and reductase (GSSG-Rx) activities, lipid peroxidation (TBARs) and serum ALT levels in rats on a choline-deficient diet. Baseline cytosolic GSH was similar between fatty and normal livers, while the mitochondrial GSH content was significantly lower in fatty livers. With the except of cytosolic GSH-Px activity, steatosis was associated with significantly higher GSH-related enzymes activities. Liver TBARs and serum ALT levels were also higher. Administration of t-BH significantly decreased the concentration of cytosolic GSH, increased GSSG levels in all the compartments, and increased TBARs levels in cytosol and mitochondria and serum ALT; all these alterations were more marked in rats with fatty liver. Fasting decreased the concentration of GSH in all the compartments both in normal and fatty livers, increased GSSG, TBARs and ALT levels, and decreased by 50% the activities of GSH-related enzymes. Administration of diethylmaleimide (DEM) resulted in cytosolic and microsomal GSH pool depletion. Administration of t-BH to DEM-treated rats further affected cytosolic GSH and enhanced ALT levels, whereas the application of fasting to GSH depleted rats mainly altered the mitochondrial GSH system, especially in fatty livers. This study shows that fatty livers have a weak compensation of hepatic GSH regulation, which fails under stress conditions, thus increasing the fatty liver's susceptibility to oxidative damage. Differences emerge among subcellular compartments which point to differential adaptation of these organelles to fatty degeneration.

Fatty accumulation per se does not appear to affect liver function; however, interest has recently renewed to fatty liver because of the clinical relevance of non alcoholic steato-hepatitis (NASH) and for the increased risk of post-transplant failure in grafted livers with steatosis. Clinical and experimental studies have doubtless demonstrated that oxidative stress ensues in steatotic livers. Mitochondria represent the preferential target of the oxidative injury associated to fatty degeneration and show reduced content of glutathione, higher levels of oxidative products and damages to enzymes involved in the process of ATP synthesis, which become more evident under stressing conditions. Although obese patients with fatty liver are advantaged by weight loss, clinical and experimental observations suggest that fatty livers poorly tolerate excessive food deprivation. These observations represent the rationale for treatment strategies based on the supplementation of antioxidants and energetic substrates rather than solely a diet restriction. This review focuses on data emerging from a series of investigations performed in rats with fatty livers induced by a choline-deficient diet, which resembles human steatosis due to an excessive intake of carbohydrates, and aims to give the cue for the development of therapeutic options able to preserve hepatic function after transplantation of steatotic organs.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in industrialized countries and is associated with increased risk of cardiovascular, hepatic and metabolic diseases. Molecular mechanisms on the root of the disrupted lipid homeostasis in NAFLD and potential therapeutic strategies can benefit of in vivo and in vitro experimental models of fatty liver. Here, we describe the high fat diet (HFD)-fed rat in vivo model, and two in vitro models, the primary cultured rat fatty hepatocytes or the FaO rat hepatoma fatty cells, mimicking human NAFLD. Liver steatosis was invariably associated with increased number/size of lipid droplets (LDs) and modulation of expression of genes coding for key genes of lipid metabolism such as peroxisome proliferator-activated receptors (Ppars) and perilipins (Plins). In these models, we tested the anti-steatotic effects of 3,5-L-diiodothyronine (T2), a metabolite of thyroid hormones. T2 markedly reduced triglyceride content and LD size acting on mRNA expression of both Ppars and Plins. T2 also stimulated mitochondrial oxidative metabolism of fatty acids. We conclude that in vivo and especially in vitro models of NAFLD are valuable tools to screen a large number of compounds counteracting the deleterious effect of liver steatosis. Because of the high and negative impact of liver steatosis on human health, ongoing experimental studies from our group are unravelling the ultimate translational value of such cellular models of NAFLD.

The diagnostic utilities of ultrasonography (US), fatty liver index (FLI) and an algorithm of nine serum markers (Fibromax) were evaluated in family practice to noninvasively characterize patients with nonalcoholic fatty liver disease (NAFLD). A multicenter study was conducted by enrolling 259 consecutively observed patients (age 51 ± 10 years) with clinical and ultrasonographic features of NAFLD. Patients had mild (16.2%), moderate (69.9%), or severe (13.9%) liver steatosis and 60.2% had hypertransaminasemia. The percent of patients with overweight, obesity, diabetes, hypertension, and dyslipidemia were 42.7%, 46.5% (4.2% severe obesity), 24.7%, 40.9%, and 56.4%, respectively. Lean patients (10.8%) had normal transaminases in two/ thirds of the cases. A multivariate logistic regression (including age > 50 yrs, BMI > 30 kg/m2, HOMA > 3, and hypertransaminasemia) identified 12.3% of patients at risk for steatohepatitis. With a sensitivity of 50% and specificity of 94.7%, Fibromax identified 34 patients (13.1%) with likely advanced fibrosis and found that over 28% of patients with moderate (ultrasonographic) steatosis were likely to be carrying severe steatosis. Steatotest score was significantly associated with BMI, waist circumference, ALT, triglycerides, and FLI. Fibrotest correlated only with ALT. FLI identified 73.4% of patients as likely to be carrying a fatty liver. In conclusion, NAFLD should be systematically searched and characterized in all patients with metabolic disturbances and cardiovascular risk. Asymptomatic subjects at risk also should be screened for NAFLD. Fibromax is a promising noninvasive diagnostic tool in family medicine for identifying patients at risk for NAFLD who require targeted follow-up.

Abstract: Background: Despite chronic liver diseases represent an important cause of illness in Italy, data from family practice are poor. Aim: To assess the management of chronic liver diseases by general practitioners in a large area of Southern Italy. Methods: This was a 5-year retrospective analysis from 104 physicians in charge of a population of 143,159 adult subjects. Results: Amongst 6550 patients with chronic liver disease (4.7%, 3400 M, median age 57 years), 1330 (20.3%) had HCV infection, 226 (3.4%) HBV infection, and 293 (4.5%) liver cirrhosis (25 alcohol-related). The prevalence of alcohol consumption, recorded by 90% of physicians, was 20.4%. Hypertransaminasemia and liver steatosis had a prevalence of 6.7% and 2.4%, respectively. Although transaminases were checked 3 times over 5 years in 80% of cases, few patients were investigated for viral infection, and less than 50% underwent ultrasonography and consultation, leaving undefined a consistent number of cases. Alcohol consumption, body mass index and ultrasonography were poorly checked even in hypertransaminasemic patients. Conclusions: This study shows that data recording by general practitioners in chronic liver disease patients lacks homogeneity and can miss important information. One unmet need is therefore the integration between theoretical knowledge and practice to share similar behaviours and improve the management of these patients. [Copyright &y& Elsevier]

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