Atorvastatin and flaxseed dietary treatments improve dyslipidemia and liver injuries in a diet-induced rat model of non-alcoholic fatty liver disease.

Without question, nonalcoholic steatohepatitis (NASH) has emerged as a substantial public health problem. The complex and intertwined cellular and molecular mechanisms culminating in the pathophysiology of this disease process remain only partially understood, and therapeutic options remain suboptimal. Lipotoxic hepatocyte injury is a cardinal feature of NASH pathogenesis,1, 2 and ballooned hepatocytes are a prominent histopathological feature of lipotoxic hepatic injury. In fact, the magnitude of ballooned hepatocytes correlates with disease severity,3 and semiquantitation of hepatocyte ballooning is used to calculate the nonalcoholic fatty liver disease activity score (NAS).4 Diehl et al. made a seminal insight when they discovered that ballooned hepatocytes generate sonic hedgehog (Shh), a ligand of the hedgehog-signaling pathway, which promotes hepatic fibrogenesis.5, 6 These data provided mechanistic insight into a mechanism contributing to hepatic fibrogenesis in NASH. However, several relevant questions remain. What is the ballooned hepatocyte, and why does it generate Shh? Does NASH-targeted therapy alter the number of ballooned hepatocytes in NASH? What is the spectrum of Shh signaling in NASH, and is hedgehog signaling inhibition a strategic pharmacologic strategy for NASH? Despite being a hallmark of NASH, little is known about ballooned hepatocytes. They are posited to represent a special form of "cell degeneration" associated with cellular enlargement, loss of cellular polarity, an abundance of intracellular lipids and oxidized phospholipids and are further characterized by loss of keratin 8/18 and accumulation of ubiquitinated proteins.7 However, these latter characteristics have not been extensively validated and are based on immunohistochemistry (IHC), a semiquantitative technique fraught with concerns regarding sensitivity and specificity. Better characterization of these cells is needed. The original observation by Diehl et al. that ballooned cells produce Shh not only shed light on liver injury, but also on the potential pathogenesis of these cells. In Drosophila melanogaster, cells in which the cell death program has been initiated, but cannot be executed, exist as "undead cells" and secrete factors, including Shh, to aid in tissue repair and healing.8 Although ballooned hepatocytes have yet to be generated in vitro, Kakisaka et al. modeled the undead cell concept by treating hepatocytes deficient in caspase 9 (a protease essential for execution of the mitochondrial pathway of cell apoptosis9) with toxic saturated free fatty acids (FFAs).10 Lipotoxicity in these cells was associated with c-Jun N-terminal kinase (JNK) activation, which, in turn, induced Shh expression in the absence of cell death (Fig. 1). Intriguingly, ballooned hepatocytes in a small number of NASH specimens also exhibit reduced expression of caspase 9, perhaps explaining their persistence despite lipotoxic insults. In the Kakisaka et al. study, Shh also served as an autocrine survival factor for the undead cell, raising the testable hypothesis that inhibition of hedgehog signaling would lead to deletion of ballooned hepatocytes. The ballooned hepatocyte may be analogous to the undead cell characterized in D. melanogaster, a concept requiring further investigation as does the role of JNK signaling in promoting Shh expression in this cellular phenotype. Finally, to ultimately solve the conundrum of how important this minor cell population of ballooned hepatocytes and their production of Shh are in the progression of NASH, a specific depletion of the ballooned hepatocytes in vivo by a genetic approach will be required. Schematic overview of hedgehog pathway activation in NASH. Simplified illustration demonstrates that JNK activation by toxic lipids leads to Shh production in ballooned hepatocytes. Released Shh acts through the autocrine pathway as a survival factor for "undead" ballooned hepatocytes and, through the paracrine mechanism, induces fibroblast differentiation into extracellular matrix–producing myofibroblasts. Vitamin E therapy decreases the amount of Shh in NASH liver by an unknown mechanism. The current study by Guy et al. in this issue of Hepatology tested the hypothesis that NASH regression is associated with decreased activity of the hedgehog-signaling pathway. The investigators evaluated liver biopsies and clinical data from a recent National Institute of Diabetes and Digestive and Kidney Diseases–sponsored clinical trial, PIVENS (PIoglitazone, Vitamin E for Non-alcoholic Steatohepatitis). The trial demonstrated that, compared to placebo, therapy with vitamin E, but not pioglitazone, improved steatosis, lobular inflammation, and hepatocellular ballooning, but not fibrosis, in adult patients with aggressive NASH who did not have diabetes or cirrhosis.11 For the current study, the investigators evaluated samples from the vitamin E and placebo treatment group. They unfortunately excluded the pioglitazone-treated group from their analysis, which could have served as an interesting control, because pioglitazone lacked beneficial effects in NASH patients. In both the placebo and vitamin E groups, the investigators were able to demonstrate that a reduction in the number of Shh-positive hepatocytes over time directly correlates with an improvement in serum alanine aminotransferase and aspartate aminotransferase values, biomarkers of liver injury. Moreover, in the whole cohort, responders (patients with an improvement in NAS scores) displayed a greater decrease in Shh-positive cells, as compared to nonresponders. Interestingly, vitamin E therapy decreased the number of Shh-positive hepatocytes in both responders and nonresponders. When comparing both groups of nonresponders, patients from the vitamin E study arm revealed a greater improvement in liver enzymes and lower number of Shh-positive cells. Collectively, improvement in NASH was associated with decreased hedgehog pathway activity, as assessed by number of Shh-positive hepatocytes. One mechanistic interpretation of these data is that vitamin E, as an antioxidant, prevents oxidative stress associated with JNK activation, resulting in inhibition of Shh autocrine survival signaling with deletion of ballooned hepatocytes (Fig. 1). The fact that vitamin E therapy also reduced Shh-positive hepatocytes in nonresponders suggests that other unrelated mechanisms also contribute to tissue injury in this disease. Hedgehog pathway signaling is initiated by its ligands, such as Shh. In the canonical signaling cascade, these ligands bind to the plasma membrane receptor, patched, which leads to disinhibition of another plasma membrane receptor, smoothened. Interruption of this basal smoothened suppression allows for nuclear translocation of glioma-associated oncogene (Gli) transcription factors. This canonical signaling pathway has been best established in mammalian cells with cilia.12 For example, Guy et al. used IHC to identify Gli2-positive cells in the liver, strong evidence for canonical hedgehog-signaling pathway activation.13 Besides the canonical pathway, noncanonical signaling cascades have been described, which do not require cilia. This noncanonical pathway also requires smoothened, but does not involve Gli-mediated transcription responses, rather smoothened functions as a G-protein-coupled receptor in this signaling pathway.14 In animal models of NASH, hedgehog signaling promotes hepatic fibrogenesis (Fig. 1).5 Generally, fibroblasts, which express cilia, are canonical hedgehog pathway-responsive cells. However, the decreased hyperactivation of the hedgehog pathway in the current study was not accompanied by a significant decrease in fibrosis. Rather, the strongest correlation was found between hedgehog activation and hepatocellular injury. This raises the question of hedgehog signaling in hepatocytes. Indeed, it has been previously reported that hepatocytes express smoothened and other components of the hedgehog-signaling cascade.15 Importantly, smoothened inhibition by vismodegib, an U.S. Food and Drug Administration (FDA)-approved drug for the treatment of basal cell carcinoma,16 attenuated liver injury in a mouse model of NASH.15 Smoothened inhibition in this study prevented tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor up-regulation and TRAIL receptor-mediated injury. The effect of smoothened inhibition on TRAIL receptor expression in hepatocytes was reproduced in vitro using FFA treatment. Future research endeavors are required to determine whether hedgehog signaling in hepatocytes by a noncanonical cascade contributes to liver injury in NASH and/or whether the effect of smoothened inhibition on TRAIL receptor expression is a direct or indirect effect. With increasing prevalence of obesity, NASH has become a growing health problem in the Western world. At the moment, there is no effective therapy for NASH, except for lifestyle modifications, which are difficult to obtain and sustain. Therefore, there is a general agreement that pharmacological therapy for NASH will be required for selected individuals, and current research seeks to identify potential therapeutic targets. Given the substantial body of evidence supporting the importance of hedgehog pathway hyperactivation in NASH, clinical trials using hedgehog-signaling pathway inhibitors, several of which have already been approved by the FDA for other indications, might take us a step closer to effective therapy for NASH. Perhaps even intermittent (once-weekly) therapy would be sufficient to delete ballooned hepatocytes, thereby reducing disease progression. Petra Hirsova, Ph.D.Gregory J. Gores, M.D. Division of Gastroenterology and HepatologyMayo ClinicRochester, MN

nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to steatohepatitis (NASH), increasing fibrosis and eventually, cirrhosis ([22][1]). Importantly, NASH accompanied by fibrosis and severe inflammation is the most relevant predictor for disease progression

Review of Clinical Guidelines in the Diagnosis of Pediatric Nonalcoholic Fatty Liver Disease.

Effects of glucagon-like peptide-1 receptor agonists on histopathological and secondary biomarkers of non-alcoholic steatohepatitis: A systematic review and meta-analysis.

Nonalcoholic Fatty Liver Disease and Fibrosis Progression: The Good, the Bad, and the Unknown

Non-alcoholic fatty liver disease (NAFLD) induced by long term high-fat and high-fructose diet has led to serious medical problems such as non-alcoholic steatohepatitis or cirrhosis. NAFLD has related to obesity, insulin resistance or type Ⅱ diabetes induced by western diet. With the increasing the incidence of obesity and NFALD due to western diet consisting of high-fat and high-fructose diets, research of the treatment or prevention of NFALD due to western diets is urgently needed. Atractylodes lancea (AL) is traditional Chinese medicine and has been used for diuretic, sedation or antibacterial effect. To investigate effect of AL on NAFLD rat model induced by high-fat and high fructose diet, present study was performed. To induce NAFLD, wistar rats, which were 9-weeks-old, were fed with 45 kcal% high fat diet and 10% fructose water daily for 16 weeks. The olmesartan and atractylodes lancea were treated by oral administration for 8 weeks every day. The groups were composed of 5 groups; control (CON, n=9) fed with 10 kcal% general diet, non-alcoholic fatty liver disease group (NFD, n=9) induced by 45 kcal% high fat diet and 10% fructose diet, NFD group with 10 mg/kg/day Olmesartan orally (OLM, n=9), NFD group with 100 mg/kg/day AL (ALL, n=9) and NFD group with 200 mg/kg/day AL (ALH, n=9). AL decreased body weight, liver weight and epididymal fat pads weight compared with NFD group induced by high-fat and high fructose diet. In serum biomarkers, glucose level of plasma, triglyceride level and total cholesterol level in plasma were increased in NFD group compared with CON. AL restored the serum biomarkers including glucose, or triglycerides and total cholesterol level in serum. The plasma HDL-Cholesterol level, which was decreased in NFD group, attenuated by AL treatment. The atherogenic coeffient was upregulated by chronic high-fat and high-fructose diet, however, the atherogenic coeffient was significantly restored in AL treatment group. Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvate transaminase (GPT) were significantly deteriorated by long term high-fat and high-fructose diet. However, the GOT and GPT level in serum were alleviated in AL treatment groups. Oil red O staining showed that the lipid accumulation in the liver was increased in NFD group compared with CON. However, the lipid accumulation was restored by AL treatment groups. The fibrosis in liver was deteriorated in NFD induced by chronic high-fat and high fructose diet, however, the AL inhibited collagen deposition in liver. Also, AL suppressed the mRNA expressions of TGF-β, collagen Ⅰ and collagen Ⅲ in liver compared with NFD group. In liver tissue, the LXR and SREBP-1c mRNA expressions were downregulated in ALH group. Taken together, these results showed that AL could be potential supplements for NAFLD induced by long term high-fat and high-fructose diet by regulating fibrosis and LXR/SREBP-1c signaling pathway in liver. This study was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean government (MSIP) (2017R1A5A2015805) (2022R1A6A3A01087272). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

To determine the efficacy of the plant-derived bioflavonoid, quercetin, for treating nonalcoholic fatty liver disease (NAFLD) by using a rat model, and to investigate the molecular mechanism underlying its therapeutic effects. One-hundred Sprague-Dawley rats were randomly assigned into the normal control group (normal group), untreated NAFLD model control group (model group), 75 mg/kg/day quercetin treatment group (low-dose group), and 300 mg/kg/day quercetin treatment group (high-dose group). The NAFLD rat model was established by providing four weeks of a high-fat diet; the normal group received normal rat chow diet. The quercetin treatments were administered for eight weeks after model establishment and control groups received simultaneous gavages of isotonic saline, with continuation of the respective diets. At the end of the eight weeks (experimental week 12), the rats were sacrificed for liver and serum collection. Intergroup differences in liver index, fasting blood glucose (FBG), triglycerides (TG), interleukin (IL)-18, IL-10, malondialdehyde (MDA), and histopathological features were assessed by independent samples t-test (normal vs. model), one-way ANOVA (model vs. treatments), and least significant difference t-test (pairwise comparisons); correlations were assessed by Pearson's correlation coefficient. Compared with the normal group, the model group showed significantly higher liver index (t=-2.327), FBG (t=-3.482), TG (t=-0.302), and serum IL-18 (t=-2.704) (all P less than 0.05), but significantly lower IL-10 (t=2.622, P less than 0.05); the MDA level was also higher in the model group, but the difference was not significant (t=-1.083, P less than 0.05). Livers from the model group showed obvious histological features of inflammation (lymphocyte and neutrophil infiltration) and steatosis (cytoplasmic lipid droplets). Inflammation was positively correlated with IL-18 (P less than 0.05), but negatively correlated with IL-10 (P less than 0.05), while steatosis was negatively correlated with IL-10 (P less than 0.05). Compared to the model group, quercetin treatment (both low- and high-dose) led to significant decreases in the liver index, FBG and IL-18 (all, P less than 0.01), and significant increase in IL-10 (P less than 0.05); however, the changes in liver index, FBG and IL-10 were not significantly different between the low- and high-dose treatment groups, but the high-dose of quercetin did induce a significantly greater decrease in IL-18 than the low-dose (P less than 0.05). NAFLD rats have higher serum levels of IL-18 but lower levels of IL-10 than their healthy counterparts, and these differential cytokine expressions may be related to liver inflammation and steatosis. Quercetin treatment may help to delay the progression of NAFLD, possibly by adjusting the balance of inflammatory cytokines.

Where are we in the search for noninvasive nonalcoholic steatohepatitis biomarkers?

Non-alcoholic fatty liver disease (NAFLD) is a prevalent liver disorder characterized byhepatic fat accumulation unrelated to alcohol consumption, with its prevalence risingalongside obesity rates. The gut-liver axis reveals that gut microbiota and metabolitessignificantly impact NAFLD development and progression. This study aimed to investigatethe effects of probiotic Lactiplantibacillus plantarum Dad-13 on bodyweight, liver function, and histopathological features in a rat model of NAFLD. Theexperimental protocol involved administering probiotic L. plantarumDad-13 at a dose of 3 × 109 CFU/g over six weeks to rats with NAFLD induced bya high-fat and high-fructose (HFFr) diet. The results demonstrated significant reductionsin body and liver weight, improved liver function (serum lipopolysaccharide bindingprotein, aspartate aminotransferase, and alanine aminotransferase levels), and improvedthe non-alcoholic liver activity score in rats fed HFFr diets supplemented withprobiotics. These findings suggest that supplementation with probiotic L.plantarum Dad-13 is a promising therapeutic intervention for NAFLD.

These recommendations are based on the following: (1) a formal review and analysis of the recently published world literature on the topic [Medline search up to June 2011]; (2) the American College of Physicians’ Manual for Assessing Health Practices and Designing Practice Guidelines; (3) guideline policies of the three societies approving this document; and (4) the experience of the authors and independent reviewers with regards to NAFLD. Intended for use by physicians and allied health professionals, these recommendations suggest preferred approaches to the diagnostic, therapeutic and preventive aspects of care. They are intended to be flexible and adjustable for individual patients. Specific recommendations are evidence-based wherever possible, and when such evidence is not available or inconsistent, recommendations are made based on the consensus opinion of the authors. To best characterize the evidence cited in support of the recommendations, the AASLD Practice Guidelines Committee has adopted the classification used by the Grading of Recommendation Assessment, Development, and Evaluation (GRADE) workgroup with minor modifications (Table 1). The strength of recommendations in the GRADE system is classified as strong (1) or weak (2). The quality of evidence supporting strong or weak recommendations is designated by one of three levels: high (A), moderate (B) or low-quality (C). This is a practice guideline for clinicians rather than a review article and interested readers can refer to several comprehensive reviews published recently.

Untargeted metabonomics reveals intervention effects of chicory polysaccharide in a rat model of non-alcoholic fatty liver disease.

Nonalcoholic Fatty Liver Disease in Children: Where Are We?

Diabetes and Nonalcoholic Fatty Liver Disease

Reply

Nonalcoholic fatty liver disease (NAFLD) is already the most common liver disease in the Western world, where overweight or obese adults make up the growing majority.1 Its comparable impact in other regions, such as Asia, is increasingly being recognized.2 As we enter into an era of improving global hepatitis B vaccination coverage and effective therapies to either control or eradiate chronic viral hepatitis, the proportional burden of NAFLD is set to rise dramatically and demand our attention. Accordingly, NAFLD has already become the second-leading indication for liver transplantation in the United States.3 Distinguishing between nonalcoholic fatty liver (NAFL) and the more-progressive nonalcoholic steatohepatitis (NASH) is clinically important given that NASH is currently the target for pharmacological treatment. Routine imaging techniques, such as ultrasound scan, computed tomography, and magnetic resonance imaging, can accurately detect NAFLD and even quantify hepatic steatosis in the case of magnetic resonance spectroscopy. However, these investigations cannot diagnose NASH or liver fibrosis. Currently, the gold standard for diagnosing and differentiating the NAFLD spectrum is a liver biopsy. Its invasiveness, cost, and low patient acceptance make it unfeasible to be used for routine screening, much less for repeated assessments to monitor disease progression or response to treatment. Even when a liver biopsy is successfully performed, its shortcomings attributed to sampling error or inter- and intraobserver variability are well documented.4 Hence, noninvasive tests, such as serum biomarkers, are clearly needed to prioritize "at-risk" patients who require liver biopsy or ideally replace liver biopsy all together. Opportunities exist for biomarkers to make an impact across the whole NAFLD spectrum, such as diagnosing NAFL, diagnosing NASH, and assessing fibrosis (Fig. 1). In this issue of Hepatology, Ajmera et al.5 evaluate 32 potential plasma biomarkers of disease activity and severity in a large, well-characterized cohort of adult patients with biopsy-proven NAFLD. The 648 patients were recruited from two multicenter studies conducted by the NASH Clinical Research Network: the NAFLD database study and the pioglitazone or vitamin E for nonalcoholic steatohepatitis (PIVENS) trial. All participants underwent robust confirmation and phenotyping of NAFLD by baseline liver histology, which was reviewed centrally by a group of nine blinded hepatopathologists. Plasma samples drawn within 6 months of the liver biopsy were then analyzed using a Luminex-based biomarker multiplex assay. Given that all biomarkers evaluated were chosen based on a priori knowledge, the positive findings in this study have largely been demonstrated previously. However, the investigators were seeking to find the most impactful biomarkers in NAFLD and their intercorrelation—a feat only achieved with a large sample size and biomarker panel as in the current study. Diagnosis of definite NASH, which was present in 58% of patients, was the primary outcome. Its individual histological components (steatosis, lobular inflammation, and hepatocyte ballooning) were also studied. On multivariate analysis, only increased activated plasminogen activator inhibitor 1 (PAI-1) had a strong association with definite NASH. This finding confirms previous observations by Verrijken et al. in a study of 273 overweight or obese subjects with biopsy-proven NAFLD.6 PAI-1 is a serine protease inhibitor of tissue plasminogen activator and urokinase, the initiators of fibrinolysis. In relation to the liver, PAI-1 plays a role in fibrogenesis by reducing plasmin-mediated activation of matrix metalloproteinases, which are required for degradation of extracellular matrix. Interestingly, because of its procoagulant properties, PAI-1 has been hypothesized as a potential link between NAFLD and its associated cardiovascular risk.6 For diagnosis of significant fibrosis (F2-4), increased interleukin-8 (IL-8) and soluble IL-2 receptor alpha demonstrated the strongest associations, among others. Whereas the link between these markers of inflammation and advanced fibrosis or cirrhosis has been observed in other chronic liver diseases, their potential value in NASH is confirmed in this study.7, 8 IL-8 is a potent chemokine involved in neutrophil recruitment, degranulation, and liver inflammation. Its association with hepatocyte ballooning (which occurs in the context of steatohepatitis) is also a novel finding, although not entirely unexpected. It should be noted that these associations between biomarkers and their endpoints, although significant, have modest odds ratios (1.2-1.3 per 0.5-SD increment). This highlights that no single biomarker can be used as a stand-alone test in diagnosing NASH. Instead, it is likely that these biomarkers will be used in a panel along with other biomarkers, clinical parameters, and/or imaging techniques. Formal c-statistics analysis to document the reliability and accuracy of the strongest biomarkers, and comparison with more-traditional prediction models such as the NAFLD fibrosis score, would have been informative in this study. Given that this is a study of predominantly white subjects (three quarters of participants), independent validation is needed before one can generalize the findings to other ethnicities. Differences in genetic polymorphisms, which influence lipid metabolism, redox pathways, inflammatory recruitment, fibrogenesis, and therefore downstream biomarkers, have been described between ethnicities.9 Although the investigators performed a cross-sectional study, 90% of subjects in the PIVENS cohort actually underwent an end-of-treatment liver biopsy at 96 weeks. This would have been an opportunity to evaluate the temporal relationship between biomarkers and disease activity and hence their usefulness as a monitoring tool. After all, when applied in the real world, clinicians need biomarkers for not only selecting patients for treatment, but also monitoring treatment response. Along the same line, before new biomarkers can be applied at the clinic, we need to understand their variability over time and clinical confounders. In the current analysis, some of the biomarkers were correlated with different metabolic factors. This by itself may only reflect the mechanism through which the biomarkers were linked to NASH and fibrosis. Further analysis is needed to evaluate whether some clinical factors and concomitant drugs may affect the diagnostic performance of these markers. Aside from limitations in their performance, the utility of biomarkers is somewhat held back by the lack of evidence-based treatment options for NASH. Indeed, lifestyle intervention is the only established therapy and should be encouraged in all individuals regardless of their NAFLD severity. However, even though risk stratification of patients currently would not alter management significantly, this information may help engage and motivate patients to enact lifestyle modifications. Additionally, reliable biomarkers may serve as new tools for patient selection or evaluation of response in randomized, clinical trials. Given that NASH is a complex heterogeneous disease, it is likely that biomarkers of treatment response will be closely related to the mechanism of action of a specific treatment rather than being broadly applicable. With promising new therapies currently being tested, the discovery of noninvasive biomarkers to direct future treatment is becoming increasingly urgent.10 Finally, the importance of biomarkers associated with liver disease severity should be tempered by perspective. Given that fibrosis progression in NASH is slow (mean increase of 0.09 fibrosis stages per year) and not universal (41% of patients), non-liver-related death remains far more common than liver-specific death or hepatocellular carcinoma combined.1 Is there any value in diagnosing NASH or even advanced fibrosis with a biomarker in a patient who will die from cardiovascular disease? The focus and validation of serum biomarkers currently falls in the realm of diagnosis, whereas a shift in focus to the prediction of clinical outcomes is needed. At the end of the day, the most important issue is the ability to predict whether a patient will develop hepatic complications (or even cardiovascular events) in the future. As such, we look forward to further longitudinal studies before serum biomarkers become prime time. Ken Liu, M.B.B.S., F.R.A.C.P.1,2 Weiqi Xu, M.Med.1 Vincent Wai-Sun Wong, M.D.1 1Institute of Digestive Disease The Chinese University of Hong Kong Hong Kong Department of Medicine and Therapeutics The Chinese University of Hong Kong Hong Kong State Key Laboratory of Digestive Disease The Chinese University of Hong Kong Hong Kong 2Faculty of Medicine The University of Sydney Sydney, NSW, Australia