Nonalcoholic Fatty Liver Disease In Humans Research Articles

Glucose Transporter 8 (GLUT8) Mediates Fructose-induced de Novo Lipogenesis and Macrosteatosis

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.

Expression of toll‐like receptors 1–5 but not TLR 6–10 is elevated in livers of patients with non‐alcoholic fatty liver disease

Animal models of non-alcoholic fatty liver disease (NAFLD) suggest that an increased translocation of bacterial endotoxins, leading to an activation of toll-like receptor-dependent signalling cascades (TLRs) and increased formation of reactive oxygen species, may add to development of insulin resistance and induction of plasminogen activator inhibitor-1 (PAI-1) in the liver. If similar mechanisms are also involved in the development of NAFLD in humans remains to be determined. Toll-like receptor (1-10), myeloid differentiation primary response gene (MyD88), interferon regulatory transcription factor 3 (IRF-3) and insulin receptor substrate 1 (IRS-1) mRNA expression was determined in liver samples of 11 patients with NAFLD and 11 controls. Hepatic PA1-1 and 4-hydroxynonenal protein adducts (4-HNE) levels were determined by immunohistochemistry. Hepatic TLR 1-5 mRNAs expression was significantly higher in livers of NAFLD patients than in controls, whereas expression of TLR 6-10 mRNAs did not differ between groups. Expression of MyD88 but not IRF-3 was also significantly higher in livers of NAFLD patients than in controls. These alterations were associated with significantly higher levels of 4-HNE and PAI-1 protein levels in livers of NAFLD patients than in controls, whereas IRS-1 mRNA expression was ~80% lower in livers of NAFLD patients than in controls. Taken together, these findings add further weight to the hypothesis that alterations at the level of intestine and intestinal barrier function may be critical in the development of NAFLD in humans.

Experimental models of non-alcoholic fatty liver disease in rats.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and it persists at a high prevalence. NAFLD is characterised by the accumulation of triglycerides in the liver and includes a spectrum of histopathological findings, ranging from simple fatty liver through non-alcoholic steatohepatitis (NASH) to fibrosis and ultimately cirrhosis, which may progress to hepatocellular carcinoma. The pathogenesis of NAFLD is closely related to the metabolic syndrome and insulin resistance. Understanding the pathophysiology and treatment of NAFLD in humans has currently been limited by the lack of satisfactory animal models. The ideal animal model for NAFLD should reflect all aspects of the intricate etiopathogenesis of human NAFLD and the typical histological findings of its different stages. Within the past several years, great emphasis has been placed on the development of an appropriate model for human NASH. This paper reviews the widely used experimental models of NAFLD in rats. We discuss nutritional, genetic and combined models of NAFLD and their pros and cons. The choice of a suitable animal model for this disease while respecting its limitations may help to improve the understanding of its complex pathogenesis and to discover appropriate therapeutic strategies. Considering the legislative, ethical, economical and health factors of NAFLD, animal models are essential tools for the research of this disease.

Disruption of JAK2 in Adipocytes Impairs Lipolysis and Improves Fatty Liver in Mice With Elevated GH

Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic expression of the metabolic syndrome, and its prevalence is increasing. The factors that influence the development of fatty liver and its progression to steatohepatitis and cirrhosis are not well understood. The pleiotropic hormone, GH, has been associated with an increased risk of NAFLD in humans and mice. GH is known to have diverse effects on lipid metabolism including decreasing body fat in vivo, presumably through stimulation of lipolysis via an undefined mechanism. Previously we described mice with hepatocyte-specific deletion of the GH signaling mediator, Janus kinase 2 (JAK2L). JAK2L animals have elevated serum GH, reduced body fat, high liver triglyceride content, and increased serum markers of hepatocyte injury (alanine transaminase and aspartate transaminase). We aimed to determine whether the elevation of GH in JAK2L mice contributed to fatty liver by promoting lipolysis directly in adipocytes. We generated mice with adipocyte-specific disruption of JAK2 (JAK2A) and found that GH resistance in adipocytes reduced lipolysis and increased body fat. JAK2A mice were then crossed to JAK2L mice, and the resultant JAK2L/A animals had increased body fat and decreased lipolysis, despite elevated circulating GH. Furthermore, the increased triglyceride content, serum alanine transaminase, and serum aspartate transaminase observed in JAK2L mice were nearly normalized with the additional disruption of JAK2 in adipocytes (JAK2L/A mice). Our results offer novel mechanistic insights into the long-recognized effects of GH on lipid flux and suggest that GH signaling may play an important regulatory role in the development of NAFLD.

Strain‐dependent dysregulation of one‐carbon metabolism in male mice is associated with choline‐ and folate‐deficient diet‐induced liver injury

Dysregulation of one-carbon metabolism-related metabolic processes is a major contributor to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). It is well established that genetic and gender-specific variations in one-carbon metabolism contribute to the vulnerability to NAFLD in humans. To examine the role of one-carbon metabolism dysregulation in the pathogenesis and individual susceptibility to NAFLD, we used a "population-based" mouse model where male mice from 7 inbred were fed a choline- and folate-deficient (CFD) diet for 12 wk. Strain-dependent down-regulation of several key one-carbon metabolism genes, including methionine adenosyltransferase 1α (Mat1a), cystathionine-β-synthase (Cbs), methylenetetrahydrofolate reductase (Mthfr), adenosyl-homocysteinase (Ahcy), and methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), was observed. These changes were strongly associated with interstrain variability in liver injury (steatosis, necrosis, inflammation, and activation of fibrogenesis) and hyperhomocysteinemia. Mechanistically, the decreased expression of Mat1a, Ahcy, and Mthfd1 was linked to a reduced level and promoter binding of transcription factor CCAAT/enhancer binding protein β (CEBPβ), which directly regulates their transcription. The strain specificity of diet-induced dysregulation of one-carbon metabolism suggests that interstrain variation in the regulation of one-carbon metabolism may contribute to the differential vulnerability to NFLD and that correcting the imbalance may be considered as preventive and treatment strategies for NAFLD.

High serum level of fibroblast growth factor 21 is an independent predictor of non-alcoholic fatty liver disease: A 3-year prospective study in China

Fibroblast growth factor 21 (FGF21), a hormone predominantly secreted by the liver, has been shown to be positively associated with the severity of non-alcoholic fatty liver disease (NAFLD) in cross-sectional studies. We investigated the prospective association of FGF21 with NAFLD development in a 3-year prospective study involving a population-based cohort comprising 808 Chinese subjects. Serum FGF21 levels at baseline and follow-up were measured using an enzyme-linked immunosorbent assay. Independent predictors of NAFLD development were identified using multiple logistic regressions. The predicting accuracy of the models was evaluated using area under the receiver-operating characteristic (ROC) curves (AUCs). In subjects who had progressed to NAFLD, the baseline FGF21 concentration (319.12 pg/ml [172.65, 518.78]) was significantly higher than that in subjects who did not develop NAFLD (199.10 pg/ml [123.56, 322.80]) (p <0.001). At follow-up, significant increase of FGF21 level was observed in those subjects who developed NAFLD (p <0.05). Baseline FGF21 was an independent predictor of NAFLD (OR: 7.102 [95% CI 2.488-20.270]; p <0.001), together with body mass index (BMI) (OR: 1.489 [95% CI 1.310-1.691]; p <0.001). The ROC-AUC was 0.816 (95% CI 0.766-0.867) for the FGF21 Model, which was calculated with FGF21 and BMI. FGF21 Model <0.13 can be used to rule out (sensitivity=85.71%, negative likelihood ratio=0.23) and ≥0.30 can be rule in (specificity=86.34%, positive likelihood ratio=3.66) ultrasonography-diagnosed NAFLD after 3 years. High serum FGF21 concentration was an independent predictor of NAFLD in humans. The FGF21 Model and its cut-offs may be useful for early diagnosis and intervention of NAFLD.

Serum levels of pigment epithelium-derived factor (PEDF) are independently associated with procollagen III N-terminal peptide levels in patients with nonalcoholic fatty liver disease

ObjectivesPigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors with complex anti-oxidative, anti-fibrotic, and anti-inflammatory properties, thus being involved in cardiometabolic disorders. Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of the metabolic syndrome as well. However, the pathophysiological role of PEDF in NAFLD remains largely unknown. We studied here the relationship between serum PEDF levels and various clinical markers of NAFLD in humans. Design and methodsThe study involved 194 biopsy-proven NAFLD patients (102 male and 92 female) with a mean age of 51.3±13.8years. We examined which anthropometric, metabolic and inflammatory variables, and liver steatosis and fibrosis markers are independently associated with serum levels of PEDF. ResultsMean serum levels of PEDF were 16.4±5.7μg/mL. Univariate analysis revealed that age (inversely), male, body mass index, waist circumference, numbers of white blood cells and platelets, aspartate aminotransferase, alanine aminotransferase, fasting plasma glucose, glycated hemoglobin, uric acid, procollagen type III N-terminal peptide (P-III-P), subcutaneous fat areas, visceral fat areas and liver to spleen density ratio in computed tomography, the presence of diabetes and medication for hyperlipidemia were significantly associated with serum levels of PEDF. In multiple stepwise regression analysis, age (p<0.01, inversely), male (p<0.05), waist circumference (p<0.01), white blood cell number (p<0.05), P-III-P (p<0.05), and the presence of diabetes (p<0.05) and medication for hyperlipidemia (p<0.01), were independently correlated to serum levels of PEDF (R2=0.285). ConclusionsThe present study reveals that serum levels of PEDF are independently associated with P-III-P levels, suggesting that PEDF level is a novel biomarker of liver fibrosis in patients with NAFLD.

Low-carbohydrate ketogenic diets, glucose homeostasis, and nonalcoholic fatty liver disease.

Obesity-associated nonalcoholic fatty liver disease (NAFLD) is highly prevalent, for which weight loss is the generally recommended clinical management. Low-carbohydrate ketogenic diets have been successful in promoting weight loss, but variations in the range of metabolic responses to these diets indicate that the effects of altering macronutrient content are not completely understood. This review focuses on the most recent findings that reveal the relationship between low-carbohydrate diets and NAFLD in rodent models and humans. Low-carbohydrate diets have been shown to promote weight loss, decrease intrahepatic triglyceride content, and improve metabolic parameters of patients with obesity. These ketogenic diets also provoke weight loss in rodents. However, long-term maintenance on a ketogenic diet stimulates the development of NAFLD and systemic glucose intolerance in mice. The relationship between ketogenic diets and systemic insulin resistance in both humans and rodents remains to be elucidated. Because low-carbohydrate ketogenic diets are increasingly employed for treatment of obesity, NAFLD, and neurological diseases such as epilepsy, understanding the long-term systemic effects of low-carbohydrate diets is crucial to the development of efficacious and safe dietary interventions.

Evaluation of Fbxw7 expression and its correlation with expression of SREBP-1 in a mouse model of NAFLD

The problems of aging, obesity and a number of types of metabolic syndromes, including diabetes, are associated with a higher risk of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms of occurrence and development of NAFLD in humans remain unclear. Sterol regulatory element binding protein (SREBP)-1 is a transcription factor that is important in the development of NAFLD, which regulates the expression of lipogenic genes. SREBP-1 might be degraded through an F-box and WD repeat domain‑containing7 (Fbxw7)-dependent degradation. However, whether or not there is a correlation between Fbxw7 and SREBP-1 in NAFLD remains to be determined. The aim of this study was to investigate whether there was a reduction of Fbxw7 in NAFLD and an induced accumulation of SREBP-1 in a mouse model of NAFLD. Forty C57BL/6J mice were divided into control and HF groups and maintained on normal or high-fat (HF) diets. Following 8weeks of treatment, the mice were sacrificed and assays of blood biomarkers typical of human NAFLD were performed. Liver samples were processed for histological examination. Fbxw7 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and the protein expression of Fbxw7 and SREBP-1 was evaluated by immunohistochemistry and western blot analysis in the mouse liver tissues in the control and HF groups. The mRNA and protein expression of Fbxw7 was significantly decreased in the HF group compared with the control group (P<0.05, for both). In the HF group, the Fbxw7 protein expression was negatively correlated with SREBP-1 (r=-0.584; P<0.05). Fbxw7 was decreased in NAFLD and negatively correlated with SREBP-1, indicating that the Fbxw7-SREBP-1 axis may play a key pathological role in the development of NAFLD.

PNPLA3, the triacylglycerol synthesis/hydrolysis/storage dilemma, and nonalcoholic fatty liver disease

Genome-wide and candidate gene association studies have identified several variants that predispose individuals to developing nonalcoholic fatty liver disease (NAFLD). However, the gene that has been consistently involved in the genetic susceptibility of NAFLD in humans is patatin-like phospholipase domain containing 3 (PNPLA3, also known as adiponutrin). A nonsynonymous single nucleotide polymorphism in PNPLA3 (rs738409 C/G, a coding variant that encodes an amino acid substitution I148M) is significantly associated with fatty liver and histological disease severity, not only in adults but also in children. Nevertheless, how PNPLA3 influences the biology of fatty liver disease is still an open question. A recent article describes new aspects about PNPLA3 gene/protein function and suggests that the I148M variant promotes hepatic lipid synthesis due to a gain of function. We revise here the published data about the role of the I148M variant in lipogenesis/lipolysis, and suggest putative areas of future research. For instance we explored in silico whether the rs738409 C or G alleles have the ability to modify miRNA binding sites and miRNA gene regulation, and we found that prediction of PNPLA3 target miRNAs shows two miRNAs potentially interacting in the 3'UTR region (hsa-miR-769-3p and hsa-miR-516a-3p). In addition, interesting unanswered questions remain to be explored. For example, PNPLA3 lies between two CCCTC-binding factor-bound sites that could be tested for insulator activity, and an intronic histone 3 lysine 4 trimethylation peak predicts an enhancer element, corroborated by the DNase I hypersensitivity site peak. Finally, an interaction between PNPLA3 and glycerol-3-phosphate acyltransferase 2 is suggested by data miming.

Omega-3 fatty acids for the treatment of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) has been recognized as a major health burden. It is the most important cause of chronic liver disease and a major independent cardiovascular risk factor. Lacking a definite treatment for NAFLD, a specific diet and an increase in physical activity represent the most commonly used therapeutic approaches. In this review, major literature data about the use of omega-3 polyunsaturated fatty acids (n-3 PUFAs) as a potential treatment of NAFLD have been described. n-3 PUFAs, besides having a beneficial impact on most of the cardio-metabolic risk factors (hypertension, hyperlipidemia, endothelial dysfunction and atherosclerosis) by regulating gene transcription factors [i.e., peroxisome proliferator-activated receptor (PPAR) α, PPARγ, sterol regulatory element-binding protein-1, carbohydrate responsive element-binding protein], impacts both lipid metabolism and on insulin sensitivity. In addition to an enhancement of hepatic beta oxidation and a decrease of the endogenous lipid production, n-3 PUFAs are able to determine a significant reduction of the expression of pro-inflammatory molecules (tumor necrosis factor-α and interleukin-6) and of oxygen reactive species. Further strengthening the results of the in vitro studies, both animal models and human intervention trials, showed a beneficial effect of n-3 PUFAs on the severity of NAFLD as expressed by laboratory parameters and imaging measurements. Despite available results provided encouraging data about the efficacy of n-3 PUFAs as a treatment of NAFLD in humans, well-designed randomized controlled trials of adequate size and duration, with histological endpoints, are needed to assess the long-term safety and efficacy of PUFA, as well as other therapies, for the treatment of NAFLD and non-alcoholic steatohepatitis patients. It is worthwhile to consider that n-3 PUFAs cannot be synthesized by the human body and must be derived from exogenous sources (fish oil, flaxseeds, olive oil) which are typical foods of the Mediterranean diet, known for its beneficial effects in preventing obesity, diabetes and, in turn, cardiovascular events. According to these data, it is important to consider that most of the beneficial effects of n-3 PUFAs can also be obtained by an equilibrate nutrition program.

Nutrition and physical activity in NAFLD: an overview of the epidemiological evidence.

Nonalcoholic fatty liver disease (NAFLD) has been recognized as a major health burden. The high prevalence of NAFLD is probably due to the contemporary epidemics of obesity, unhealthy dietary pattern, and sedentary lifestyle. The efficacy and safety profile of pharmacotherapy in the treatment of NAFLD remains uncertain and obesity is strongly associated with hepatic steatosis; therefore, the first line of treatment is lifestyle modification. The usual management of NAFLD includes gradual weight reduction and increased physical activity (PA) leading to an improvement in serum liver enzymes, reduced hepatic fatty infiltration, and, in some cases, a reduced degree of hepatic inflammation and fibrosis. Nutrition has been demonstrated to be associated with NAFLD and Non-alcoholic steatohepatitis (NASH) in both animals and humans, and thus serves as a major route of prevention and treatment. However, most human studies are observational and retrospective, allowing limited inference about causal associations. Large prospective studies and clinical trials are now needed to establish a causal relationship. Based on available data, patients should optimally achieve a 5%-10% weight reduction. Setting realistic goals is essential for long-term successful lifestyle modification and more effort must be devoted to informing NAFLD patients of the health benefits of even a modest weight reduction. Furthermore, all NAFLD patients, whether obese or of normal weight, should be informed that a healthy diet has benefits beyond weight reduction. They should be advised to reduce saturated/trans fat and increase polyunsaturated fat, with special emphasize on omega-3 fatty acids. They should reduce added sugar to its minimum, try to avoid soft drinks containing sugar, including fruit juices that contain a lot of fructose, and increase their fiber intake. For the heavy meat eaters, especially those of red and processed meats, less meat and increased fish intake should be recommended. Minimizing fast food intake will also help maintain a healthy diet. PA should be integrated into behavioral therapy in NAFLD, as even small gains in PA and fitness may have significant health benefits. Potentially therapeutic dietary supplements are vitamin E and vitamin D, but both warrant further research. Unbalanced nutrition is not only strongly associated with NAFLD, but is also a risk factor that a large portion of the population is exposed to. Therefore, it is important to identify dietary patterns that will serve as modifiable risk factors for the prevention of NAFLD and its complications.

Induction of microsomal triglyceride transfer protein expression is a candidate mechanism by which ezetimibe therapy might exert beneficial effects in patients with nonalcoholic steatohepatitis

Dear Editor Park and colleagues have reported that ezetimibe therapy improves the histological abnormalities of nonalcoholic fatty liver disease (NAFLD) [1]. In their study, while no significant difference was observed in the fibrosis stage, significant improvements of the steatosis grade, necroinflammatory grade, ballooning score, and NAFLD activity score (NAS) were observed after treatment with ezetimibe (10 mg/day) for 24 months. Ezetimibe, a potent inhibitor of cholesterol absorption, has been shown to inhibit Niemann-Pick C1 Like 1 (NPC1L1)-dependent cholesterol transport at the brush border of the intestine and the liver [2]. Therefore, ezetimibe has been considered as a noteworthy candidate agent for the treatment of metabolic syndrome, including nonalcoholic steatohepatitis (NASH). In several experimental rodent models, drugs antagonizing mechanisms such as sterol regulatory element-binding protein-1c (SREBP-1c) induction, carbohydrate responsive binding protein (ChREBP) induction, and endoplasmic reticulum (ER) stress have been reported as candidate mechanisms for the improvement of hepatic steatosis [3, 4]. However, unlike in rodents, in which NPC1L1 is mainly expressed in the intestine, in humans high levels of expression of NPC1L1 mRNA are observed in both the small intestine and the liver; therefore, ezetimibe would inhibit not only dietary and biliary cholesterol absorption in the small intestine, but also biliary cholesterol absorption in the liver [2]. Therefore, the mechanism by which ezetimibe might improve the changes of NASH in humans remains unknown. We conducted a 6-month pilot study to investigate the effect of ezetimibe in patients with NASH [5]. To investigate the mechanism of action of ezetimibe treatment in human NASH patients, we analyzed the hepatic mRNA expression of SREBP1c, ChREBP, and ER stress in 8 NASH patients before and after ezetimibe treatment. We found no significant changes in SREBP1c expression, ChREBP expression, or ER stress (Glucose-regulated protein 78, C/EBP-homologous protein) in this study. Secondly, we examined microsomal triglyceride transfer protein (MTTP) expression, which is a key factor for triglyceride (TG) transport in the liver, because we had previously found that the hepatic expression of MTTP mRNA was impaired in NASH patients as compared with that in patients with simple steatosis [6]. After ezetimibe treatment, a significant increase of hepatic MTTP mRNA expression was observed as compared with that before ezetimibe treatment (p = 0.0052). We suggest, therefore, that the induction of MTTP expression is an important candidate mechanism by which ezetimibe therapy might exert beneficial effects against NAFLD in humans.

Challenging the Rodent Hegemony: A New Rabbit Model of Nonalcoholic Steatohepatitis

Since its initial description in 1980, nonalcoholic fatty liver disease (NAFLD) has become an increasingly important public health problem.1 It is projected that NAFLD will replace hepatitis C as the most common indication for liver transplantation in the United States within the next 20 years.2 Increasingly, NAFLD is being recognized as a problem not only in the developed countries but worldwide.3 A strong association with metabolic syndrome and the increasing prevalence of NAFLD has paralleled the rising epidemics of obesity, insulin resistance, and diabetes mellitus.4 While important advances have been made in understanding the pathogenesis of NAFLD, the precise molecular events that lead to its development remain elusive. Among the crucial challenges faced is the difficulty in differentiating the presence of fat in the liver (simple steatosis) from fat associated with hepatocellular injury, inflammation, and fibrosis (steatohepatitis, NASH). The latter condition is associated with progressive fibrosis that can ultimately lead to cirrhosis, with further complications of portal hypertension and hepatocellular carcinoma.5 It is very difficult to investigate the molecular pathogenesis of NAFLD and NASH in humans because of the heterogeneity of human populations and wide differences in their diet and lifestyle. More importantly, the inability to obtain multiple liver biopsies from patients with NAFLD or NASH and healthy volunteers adds to the difficulty of these studies. Given these technical issues and associated ethical challenges involved in studying patients with NAFLD, considerable effort has been expended to develop animal models of fatty liver disease. In this issue of the American Journal of Pathology, Ogawa et al6 report another animal model of NAFLD, which utilizes rabbits fed a high-fat diet, resulting in the development of progressive liver fibrosis and hepatic cholesterol accumulation.

The AGEs-RAGE axis and nonalcoholic steatohepatitis: the evidence mounts

I read with great interest the recent article by Kimura et al. [1] about the effects of atorvastatin treatment on advanced glycation end products (AGEs) in patients with nonalcoholic steatohepatitis (NASH). The authors elegantly demonstrated that atorvastatin given at 10 mg daily for 12 months successfully reduced glyceraldehyde-derived AGEs while improving the steatosis grade and nonalcoholic fatty liver disease (NAFLD) activity score. These results suggest a crucial role for AGEs in the pathogenesis of NASH and open an avenue for AGEs-targeted treatment of one of the top three leading causes of cirrhosis. One of the major shortcomings of the study by Kimura, however, was the failure to clarify and discuss the potential role played by the receptor for advanced glycation end products (RAGE) in their findings. Previous studies have shown that interactions between AGEs and RAGE can promote a number of adverse physiologic pathways, such as oxidative stress, inflammation, and insulin resistance [2–4], all of which have been implicated in the development of NASH. Conversely, circulating soluble isoforms of RAGE (sRAGE) can inhibit RAGE-induced activation of these pathways [5, 6], and therefore, sRAGE could conceivably protect against the development of NAFLD and NASH. In fact, we have previously examined the relationship between circulating levels of sRAGE and NAFLD in humans and demonstrated that levels of sRAGE are highly reduced in the setting of NASH and inversely correlated with ALT and AST [7]. Previous studies have also clearly shown that atorvastatin can influence the expression of sRAGE and cell-bound RAGE both in vitro and in vivo [8–11]. Beside RAGE, AGEs bind to other receptors, including macrophage scavenger receptors I and II, including CD36 [12]. Interestingly, Greco et al. [13] have demonstrated that CD36 is one of the most important genes found to be upregulated in subjects with high liver fat content. Taken together, the AGEs-RAGE axis is emerging as a core piece in the pathophysiology of insulin resistance, NAFLD and NASH, and evidence suggests that atorvastatin may influence not only AGEs, but also sRAGE. Kimura et al. are to be congratulated for their important piece of work expanding the current knowledge on the role of AGEs in NASH. Regrettably, however, the paper offers only a partial insight on this issue as the potential mediating role of sRAGE on the authors’ findings was not addressed. Future studies in the field should not ignore this important point.

SIRT1 Transcription Is Decreased in Visceral Adipose Tissue of Morbidly Obese Patients with Severe Hepatic Steatosis

Visceral adipose tissue is known to release greater amounts of adipokines and free fatty acids into the portal vein, being one of the most predictive factors of nonalcoholic fatty liver disease (NAFLD). Our study has the purpose to evaluate sirtuin 1 (SIRT1), adiponectin, Forkhead/winged helix (FOXO1), peroxisome proliferator-activated receptor (PPAR)gamma1-3, and PPARbeta/delta mRNA expression in morbidly obese patients in three different lipid depots: visceral (VAT), subcutaneous (SAT), and retroperitoneal (RAT). Recent studies suggest that SIRT1, a NAD(+)-dependent deacetylase, protects rats from NAFLD. We divided the patients in two groups: those with slight or moderate steatosis (hepatic steatosis, HS) and other comprising individuals with severe steatosis associated or not with necroinflammation and fibrosis (severe hepatic steatosis, SHS). The adipose tissue depots were obtained during bariatric surgery. Total RNAs were extracted using TRIzol. The amount of genes of interest was determined by quantitative real-time polymerase chain reaction. When comparing the two groups of patients, a decrease in SIRT1 was observed in VAT of morbidly obese patients in SHS group (p = 0.006). The mRNA expression of the other genes showed no differences in VAT. No difference was found either in SAT or in RAT for all genes in the study. In addition, the homeostasis model assessment for insulin resistance (HOMA-IR) value was higher in SHS group compared to HS (p = 0.006). Also, our results show that the mRNA expression of SIRT1 and the value of HOMA-IR were positively correlated in VAT of SHS patients (r = 0.654; p = 0.048). Downregulation of SIRT1 mRNA expression in VAT of SHS could be possible impairing mitochondria biogenesis and fatty acid oxidation, promoting severe steatosis in obese patients. Our results provide a possible proof of SIRT1 protective potential in VAT against NAFLD in humans.

High Fat Diet Induced Hepatic Steatosis Establishes a Permissive Microenvironment for Colorectal Metastases and Promotes Primary Dysplasia in a Murine Model

Non-alcoholic fatty liver disease (NAFLD), which includes steatosis and its progression to non-alcoholic steatohepatitis, is a liver disorder of increasing clinical significance. Here we characterize a murine model of high fat diet-induced NAFLD with progression from liver steatosis to histological features compatible with steatohepatitis and more advanced stages of NAFLD in humans, including chronic portal inflammation, pericellular and bridging fibrosis, Mallory body formation, and bile ductular reaction. Chronic changes induced by the prolonged consumption of a high-fat diet alone culminate in the development of primary liver dysplasias. Importantly, we extend these studies to demonstrate that even the early stages of uncomplicated steatosis provide a permissive microenvironment for the growth of colon cancer cells that are metastatic to the liver. High fat diet-induced steatosis, coupled with a splenic injection model of experimental liver metastasis using syngeneic MC38 colon cancer cells, resulted in an increased number of secondary tumor nodules and metastatic burden in steatotic livers. Metastatic nodules were associated with focal peritumoral areas of infiltrating inflammatory cells and associated apoptotic cell populations. These results suggest that the modulation of specific host factors in the steatotic liver contributes to tumor progression in the microenvironment of NAFLD.

Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease†

The role played by the gut in nonalcoholic fatty liver disease (NAFLD) is still a matter of debate, although animal and human studies suggest that gut-derived endotoxin may be important. We investigated intestinal permeability in patients with NAFLD and evaluated the correlations between this phenomenon and the stage of the disease, the integrity of tight junctions within the small intestine, and prevalence of small intestinal bacterial overgrowth (SIBO). We examined 35 consecutive patients with biopsy-proven NAFLD, 27 with untreated celiac disease (as a model of intestinal hyperpermeability) and 24 healthy volunteers. We assessed the presence of SIBO by glucose breath testing (GBT), intestinal permeability by means of urinary excretion of (51)Cr-ethylene diamine tetraacetate ((51)Cr-EDTA) test, and the integrity of tight junctions within the gut by immunohistochemical analysis of zona occludens-1 (ZO-1) expression in duodenal biopsy specimens. Patients with NAFLD had significantly increased gut permeability (compared with healthy subjects; P < 0.001) and a higher prevalence of SIBO, although both were lower than in the untreated celiac patients. In patients with NAFLD, both gut permeability and the prevalence of SIBO correlated with the severity of steatosis but not with presence of NASH. Our results provide the first evidence that NAFLD in humans is associated with increased gut permeability and that this abnormality is related to the increased prevalence of SIBO in these patients. The increased permeability appears to be caused by disruption of intercellular tight junctions in the intestine, and it may play an important role in the pathogenesis of hepatic fat deposition.

Nonalcoholic steatohepatitis and hepatocellular carcinoma in galectin‐3 knockout mice

Nonalcoholic fatty liver disease (NAFLD) represents a growing health concern due to its rapidly increasing prevalence worldwide. Nonalcoholic steatohepatitis (NASH) is a progressing form of NAFLD, and recently many studies have reported that it could eventually develop into hepatocellular carcinoma (HCC). We previously reported that 6-month-old male galectin-3 knockout (gal3(-/-)) mice developed clinicopathological features similar to those of NAFLD in humans. Our aim was to investigate the changes in liver histology in gal3(-/-) mice by long-term observation. We initially investigated three 15-month-old gal3(-/-) mice, of which two developed multiple liver nodules with dysplastic changes. Then, we histopathologically examined the liver specimens of the 15-, 20- and 25-month-old gal3(-/-) mice and attempted to evaluate the liver morphology by contrast enhanced computed tomography (CT) before sacrifice. At the age of 15 months or later, gal3(-/-) mice developed liver nodules with varying degrees of architectural and nuclear atypia based on mild to moderate delicate zone 3 fibrosis. In addition, we successfully confirmed the presence of some of the liver nodules by CT. We report herein that gal3(-/-) mice develop dysplastic liver nodules and HCC. We believe that it would be interesting to use this murine model to investigate liver carcinogenesis based on a natural history of NAFLD. Furthermore, CT scanning might be a useful tool for longitudinal evaluation of morphological changes in vivo.

The effect of metformin on leptin in obese patients with type 2 diabetes mellitus and nonalcoholic fatty liver disease

Insulin resistance is a major feature of type 2 diabetes mellitus, obesity and nonalcoholic fatty liver disease (NAFLD). Several studies pointed out the possible role of increased leptin in NAFLD in humans. The aim of this study is to determine the effect of metformin on plasma leptin levels in obese patients with type 2 diabetes mellitus and NAFLD compared with lifestyle interventions. Thirty-four obese patients with newly diagnosed type 2 diabetes mellitus were prospectively followed for 6 months. All patients had ultrasonographic evidence of NAFLD at baseline. The patients were randomized into two groups: group 1 (n = 15) followed lifestyle changes only and group 2 (n = 19) received metformin (1,700 mg/day). At the end of treatment, BMI, WHR, HbA1c, fasting glucose, leptin, HOMA-IR, alanine aminotransferase values decreased in both groups. No significant difference in the end-points was observed between two groups. Only in group 2, LDL decreased and HDL increased significantly. Liver echogenity decreased significantly at the end of study in both groups. The percentage of patients who no longer had evidence of NAFLD was not significantly different between the groups (20% of patients on lifestyle intervention vs. 16% of patients on metformin). The data demonstrate that, metformin and lifestyle interventions equally affected the plasma leptin levels, BMI and degree of NAFLD in obese patients with type 2 diabetes mellitus. In addition, the effects of metformin on the variables were not found to be mediated by leptin.