Genetic and Diet-Induced Animal Models for Non-Alcoholic Fatty Liver Disease (NAFLD) Research

A rapidly increasing incidence of non-alcoholic fatty liver disease (NAFLD) is noted worldwide due to the adoption of western-type lifestyles and eating habits. This makes the understanding of the molecular mechanisms that drive the pathogenesis of this chronic disease and the development of newly approved treatments of utmost necessity. Animal models are indispensable tools for achieving these ends. Although the ideal mouse model for human NAFLD does not exist yet, several models have arisen with the combination of dietary interventions, genetic manipulations and/or administration of chemical substances. Herein, we present the most common mouse models used in the research of NAFLD, either for the whole disease spectrum or for a particular disease stage (e.g., non-alcoholic steatohepatitis). We also discuss the advantages and disadvantages of each model, along with the challenges facing the researchers who aim to develop and use animal models for translational research in NAFLD. Based on these characteristics and the specific study aims/needs, researchers should select the most appropriate model with caution when translating results from animal to human.     

The Effects of Probiotics,Prebiotics and Synbiotics in Non-Alcoholic Fat Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH): A Systematic Review

Modifications in the microbiota caused by environmental and genetic reasons can unbalance the intestinal homeostasis, deregulating the host’s metabolism and immune system, intensifying the risk factors for the development and aggravation of non-alcoholic fat liver disease (NAFLD). The use of probiotics, prebiotics and synbiotics have been considered a potential and promising strategy to regulate the gut microbiota and produce beneficial effects in patients with liver conditions. For this reason, this review aimed to evaluate the effectiveness of probiotics, prebiotics, and symbiotics in patients with NAFLD and NASH. Pubmed, Embase, and Cochrane databases were consulted, and PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines were followed. The clinical trials used in this study demonstrated that gut microbiota interventions could improve a wide range of markers of inflammation, glycemia, insulin resistance, dyslipidemia, obesity, liver injury (decrease of hepatic enzymes and steatosis and fibrosis). Although microbiota modulators do not play a healing role, they can work as an important adjunct therapy in pathological processes involving NAFLD and its spectrums, either by improving the intestinal barrier or by preventing the formation of toxic metabolites for the liver or by acting on the immune system.     

Significance of Circulating Cell-Free DNA Species in Non-Alcoholic Fatty Liver Disease

The pathogenetic mechanisms involved in the progression of non-alcoholic fatty liver disease (NAFLD) have not been completely elucidated, while the significance of circulating cell-free DNA (cf-DNA) species has been rarely evaluated in NAFLD. Herein, we assessed the serum levels of cf-DNA species in NAFLD patients and investigated their potential associations with patients’ characteristics and severity of liver disease. Forty-nine adult patients with NAFLD of any stage were included in this cohort study. Cf-DNA was isolated from patients’ sera and the levels of several distinct cf-DNA species including total cf-DNA, gene-coding cf-DNA, Alu repeat sequences, mitochondrial DNA copies and 5-methyl-2′-deoxycytidine were determined. Cirrhotic compared to non-cirrhotic patients had significantly lower serum levels of cf-DNA and RNAse P coding DNA as well as higher expression of 5-methyl-2′-deoxycytidine. After adjustment for the significant clinico-epidemiological factors, lower serum levels of cf-DNA or RNAse P were independently associated with the presence of cirrhosis. Serum levels of total and gene-coding DNA are associated with the presence of cirrhosis in NAFLD patients regardless of clinical or epidemiological parameters and may therefore be used as a screening tool for NAFLD progression.     

Non-Alcoholic Fatty Liver Disease Is not Related to the Incidence of Diabetic Nephropathy in Type 2 Diabetes

To analyze the association between non-alcoholic fatty liver disease (NAFLD) and the incidence of diabetic nephropathy in patients with type 2 diabetes, the incidence of diabetic nephropathy was assessed in 413 type 2 diabetic patients, by testing the 24 h urinary albumin excretion rate (UAER). The NAFLD was diagnosed based on patient’s medical history and liver ultrasound. The difference in diabetic nephropathy incidence between patients with and without NAFLD was tested by χ². Multivariate logistic regression analysis was used to assess the factors associated with diabetic nephropathy among type 2 diabetic patients. Total 363 out of 413 type 2 diabetic patients were enrolled in this study. The incidences of NAFLD and diabetic nephropathy in participants were approximately 56% (202/363) and 38% (137/363) respectively, and there was no significant difference in the prevalence of diabetic nephropathy between patients with and without NAFLD (37.1% vs. 38.5%, p = 0.787). The duration of diabetes (odds ratio [OR] 1.065, 95% confidence interval [CI] 1.014–1.120, p = 0.012), waist circumference (OR 1.077, 95% CI 1.040–1.116, p = 0.000), and fasting blood glucose (FBG; OR 1.136, 95% CI 1.023–1.1262, p = 0.017) were significantly associated with diabetic nephropathy, whereas sex, high blood pressure, total cholesterol (TC), triglyceride (TG), and ankle brachial pressure index (ABI) were not significantly associated with the disorder. The present results suggest that NAFLD is not related to the incidence of diabetic nephropathy in type 2 diabetes, but the duration of diabetes, waist circumference, and FBG are important factors for diabetic nephropathy in type 2 diabetes.     

Assessment of Ultra-Early-Stage Liver Fibrosis in Human Non-Alcoholic Fatty Liver Disease by Second-Harmonic Generation Microscopy

Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients.     

Extracellular Vesicles: A New Frontier in Biomarker Discovery for Non-Alcoholic Fatty Liver Disease

In recent years, the global burden of obesity and diabetes has seen a parallel rise in other metabolic complications, such as non-alcoholic fatty liver disease (NAFLD). This condition, once thought to be a benign accumulation of hepatic fat, is now recognized as a serious and prevalent disorder that is conducive to inflammation and fibrosis. Despite the rising incidence of NAFLD, there is currently no reliable method for its diagnosis or staging besides the highly invasive tissue biopsy. This limitation has resulted in the study of novel circulating markers as potential candidates, one of the most popular being extracellular vesicles (EVs). These submicron membrane-bound structures are secreted from stressed and activated cells, or are formed during apoptosis, and are known to be involved in intercellular communication. The cargo of EVs depends upon the parent cell and has been shown to be changed in disease, as is their abundance in the circulation. The role of EVs in immunity and epigenetic regulation is widely attested, and studies showing a correlation with disease severity have made these structures a favorable target for diagnostic as well as therapeutic purposes. This review will highlight the research that is available on EVs in the context of NAFLD, the current limitations, and projections for their future utility in a clinical setting.     

Altered Fatty Acid Metabolism-Related Gene Expression in Liver from Morbidly Obese Women with Non-Alcoholic Fatty Liver Disease

Lipid accumulation in the human liver seems to be a crucial mechanism in the pathogenesis and the progression of non-alcoholic fatty liver disease (NAFLD). We aimed to evaluate gene expression of different fatty acid (FA) metabolism-related genes in morbidly obese (MO) women with NAFLD. Liver expression of key genes related to de novo FA synthesis (LXRα, SREBP1c, ACC1, FAS), FA uptake and transport (PPARγ, CD36, FABP4), FA oxidation (PPARα), and inflammation (IL6, TNFα, CRP, PPARδ) were assessed by RT-qPCR in 127 MO women with normal liver histology (NL, n = 13), simple steatosis (SS, n = 47) and non-alcoholic steatohepatitis (NASH, n = 67). Liver FAS mRNA expression was significantly higher in MO NAFLD women with both SS and NASH compared to those with NL (p = 0.003, p = 0.010, respectively). Hepatic IL6 and TNFα mRNA expression was higher in NASH than in SS subjects (p = 0.033, p = 0.050, respectively). Interestingly, LXRα, ACC1 and FAS expression had an inverse relation with the grade of steatosis. These results were confirmed by western blot analysis. In conclusion, our results indicate that lipogenesis seems to be downregulated in advanced stages of SS, suggesting that, in this type of extreme obesity, the deregulation of the lipogenic pathway might be associated with the severity of steatosis.     

The Natural Course of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease in the world, paralleling the epidemic of obesity and Type 2 diabetes mellitus (T2DM). NAFLD exhibits a histological spectrum, ranging from “bland steatosis” to the more aggressive necro-inflammatory form, non-alcoholic steatohepatitis (NASH) which may accumulate fibrosis to result in cirrhosis. Emerging data suggests fibrosis, rather than NASH per se, to be the most important histological predictor of liver and non-liver related death. Nevertheless, only a small proportion of individuals develop cirrhosis, however the large proportion of the population affected by NAFLD has led to predictions that NAFLD will become a leading cause of end stage liver disease, hepatocellular carcinoma (HCC), and indication for liver transplantation. HCC may arise in non-cirrhotic liver in the setting of NAFLD and is associated with the presence of the metabolic syndrome (MetS) and male gender. The MetS and its components also play a key role in the histological progression of NAFLD, however other genetic and environmental factors may also influence the natural history. The importance of NAFLD in terms of overall survival extends beyond the liver where cardiovascular disease and malignancy represents additional important causes of death.     

Plasma Levels of Homocysteine and Cysteine Increased in Pediatric NAFLD and Strongly Correlated with Severity of Liver Damage

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from simple triglyceride accumulation in the hepatocytes to hepatic steatosis with inflammation, ballooning and fibrosis. It has been demonstrated that the pathogenesis of NAFLD involves increased oxidative stress, with consumption of the major cellular antioxidant, glutathione (GSH). Liver has a fundamental role in sulfur compound metabolism, although the data reported on plasma thiols status in NAFLD are conflicting. We recruited 63 NAFLD patients, and we analyzed all plasma thiols, such as homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly) and GSH, by high-performance liquid chromatography (HPLC) with fluorescence detection. Hcy, Cys and CysGly plasma levels increased in NAFLD patients (p < 0.0001); whereas GSH levels were decreased in NAFLD patients when compared to controls (p < 0.0001). On the contrary, patients with steatohepatitis exhibited lower levels of Hcy and Cys than subjects without. Furthermore, a positive correlation was found between Hcy and Cys and the presence of fibrosis in children with NAFLD. Taken together, these data demonstrated a defective hepatic sulfur metabolism in children with NAFLD, and that high levels of Hcy and Cys probably correlates with a pattern of more severe histological liver damage, due to mechanisms that require further studies.     

Long-Term Adverse Effect of Liver Stiffness on Glycaemic Control in Type 2 Diabetic Patients with Nonalcoholic Fatty Liver Disease: A Pilot Study

Currently, there are limited data regarding the long-term effect of liver stiffness on glycaemic control in patients with type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). We prospectively followed an outpatient sample of 61 consecutive postmenopausal women with T2DM and NAFLD who had baseline data on liver ultrasonography and Fibroscan^®-assessed liver stiffness measurement (LSM) in 2017 and who underwent follow-up in 2022. Haemoglobin A1c (HbA1c) was measured both at baseline and follow-up. At baseline, 52 patients had NAFLD (hepatic steatosis) alone, and 9 had NAFLD with coexisting clinically significant fibrosis (defined as LSM ≥ 7 kPa on Fibroscan^®). At follow-up, 16 patients had a worsening of glycaemic control (arbitrarily defined as HbA1c increase ≥ 0.5% from baseline). The prevalence of NAFLD and coexisting clinically significant fibrosis at baseline was at least three times greater among patients who developed worse glycaemic control at follow-up, compared with those who did not (31.3% vs. 8.9%; p = 0.030). In logistic regression analysis, the presence of NAFLD and clinically significant fibrosis was associated with an approximately 4.5-fold increased likelihood of developing worse glycaemic control at follow-up (odds ratio 4.66, 95% confidence interval 1.07–20.3; p = 0.041), even after adjustment for baseline confounding factors, such as age, body mass index, haemoglobin A1c (or HOMA-estimated insulin resistance) and use of some glucose-lowering agents that may positively affect NAFLD and liver fibrosis. In conclusion, our results suggest that the presence of Fibroscan^®-assessed significant fibrosis was associated with a higher risk of developing worse glycaemic control in postmenopausal women with T2DM and NAFLD.     

The New Therapeutic Approaches in the Treatment of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease which is characterized by extremely complex pathogenetic mechanisms and multifactorial etiology. Some of the many pathophysiological mechanisms involved in the development of NAFLD include oxidative stress, impaired mitochondrial metabolism, inflammation, gut microbiota, and interaction between the brain-liver-axis and the regulation of hepatic lipid metabolism. The new therapeutic approaches in the treatment of NAFLD are targeting some of these milestones along the pathophysiological pathway and include drugs like agonists of peroxisome proliferator-activated receptors (PPARs), glucagon-like peptide-1 (GLP-1) agonists, sodium/glucose transport protein 2 (SGLT2) inhibitors, farnesoid X receptor (FXR) agonists, probiotics, and symbiotics. Further efforts in biomedical sciences should focus on the investigation of the relationship between the microbiome, liver metabolism, and response to inflammation, systemic consequences of metabolic syndrome.     

SGLT2 Inhibitors as the Most Promising Influencers on the Outcome of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD), the most frequent liver disease in the Western world, is a common hepatic manifestation of metabolic syndrome (MetS). A specific cure has not yet been identified, and its treatment is currently based on risk factor therapy. Given that the initial accumulation of triglycerides in the liver parenchyma, in the presence of inflammatory processes, mitochondrial dysfunction, lipotoxicity, glucotoxicity, and oxidative stress, can evolve into non-alcoholic steatohepatitis (NASH). The main goal is to identify the factors contributing to this evolution because, once established, untreated NASH can progress through fibrosis to cirrhosis and, ultimately, be complicated by hepatocellular carcinoma (HCC). Several drugs have been tested in clinical trials for use as specific therapy for NAFLD; most of them are molecules used to cure type 2 diabetes mellitus (T2DM), which is one of the main risk factors for NAFLD. Among the most studied is pioglitazone, either alone or in combination with vitamin E, glucagon-like peptide-1 (GLP-1) receptor agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors. Actually, the most promising category seems to be sodium-glucose cotransporter (SGLT2) inhibitors. Their action is carried out by inhibiting glucose reabsorption in the proximal renal tubule, leading to its increased excretion in urine and decreased levels in plasma. Experimental studies in animal models have suggested that SGLT2 inhibitors may have beneficial modulatory effects on NAFLD/NASH, and several trials in patients have proven their beneficial effects on liver enzymes, BMI, blood lipids, blood glucose, and insulin resistance in NAFLD patients, thus creating strong expectations for their possible use in preventing the evolution of liver damage in these patients. We will review the main pathogenetic mechanisms, diagnostic modalities, and recent therapies of NAFLD, with particular attention to the use of SGLT2 inhibitors.     

Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: Current Issues and Future Perspectives in Preclinical and Clinical Research

Nonalcoholic fatty liver disease (NAFLD) is a continuum of liver abnormalities often starting as simple steatosis and to potentially progress into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Because of its increasing prevalence, NAFLD is becoming a major public health concern, in parallel with a worldwide increase in the recurrence rate of diabetes and metabolic syndrome. It has been estimated that NASH cirrhosis may surpass viral hepatitis C and become the leading indication for liver transplantation in the next decades. The broadening of the knowledge about NASH pathogenesis and progression is of pivotal importance for the discovery of new targeted and more effective therapies; aim of this review is to offer a comprehensive and updated overview on NAFLD and NASH pathogenesis, the most recommended treatments, drugs under development and new drug targets. The most relevant in vitro and in vivo models of NAFLD and NASH will be also reviewed, as well as the main molecular pathways involved in NAFLD and NASH development.     

Nonalcoholic Fatty Liver: A Possible New Target for Type 2 Diabetes Prevention and Treatment

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder worldwide. Several lines of evidence have indicated a pathogenic role of insulin resistance, and a strong association with type 2 diabetes (T2MD) and metabolic syndrome. Importantly, NAFLD appears to enhance the risk for T2MD, as well as worsen glycemic control and cardiovascular disease in diabetic patients. In turn, T2MD may promote NAFLD progression. The opportunity to take into account NAFLD in T2MD prevention and care has stimulated several clinical studies in which antidiabetic drugs, such as metformin, thiazolidinediones, GLP-1 analogues and DPP-4 inhibitors have been evaluated in NAFLD patients. In this review, we provide an overview of preclinical and clinical evidences on the possible efficacy of antidiabetic drugs in NAFLD treatment. Overall, available data suggest that metformin has beneficial effects on body weight reduction and metabolic parameters, with uncertain effects on liver histology, while pioglitazone may improve liver histology. Few data, mostly preclinical, are available on DPP4 inhibitors and GLP-1 analogues. The heterogeneity of these studies and the small number of patients do not allow for firm conclusions about treatment guidelines, and further randomized, controlled studies are needed.     

Development of Thyroid Hormones and Synthetic Thyromimetics in Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the fastest-growing liver disease in the world. Despite targeted agents which are needed to provide permanent benefits for patients with NAFLD, no drugs have been approved to treat NASH. Thyroid hormone is an important signaling molecule to maintain normal metabolism, and in vivo and vitro studies have shown that regulation of the 3,5,3’-triiodothyronine (T3)/ thyroid hormone receptor (TR) axis is beneficial not only for metabolic symptoms but also for the improvement of NAFLD and even for the repair of liver injury. However, the non-selective regulation of T3 to TR subtypes (TRα/TRβ) could cause unacceptable side effects represented by cardiotoxicity. To avoid deleterious effects, TRβ-selective thyromimetics were developed for NASH studies in recent decades. Herein, we will review the development of thyroid hormones and synthetic thyromimetics based on TR selectivity for NAFLD, and analyze the role of TR-targeted drugs for the treatment of NAFLD in the future.     

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

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

Role of Endocrine-Disrupting Chemicals in the Pathogenesis of Non-Alcoholic Fatty Liver Disease: A Comprehensive Review

Non-alcoholic fatty liver disease (NAFLD) is considered the most common liver disorder, affecting around 25% of the population worldwide. It is a complex disease spectrum, closely linked with other conditions such as obesity, insulin resistance, type 2 diabetes mellitus, and metabolic syndrome, which may increase liver-related mortality. In light of this, numerous efforts have been carried out in recent years in order to clarify its pathogenesis and create new prevention strategies. Currently, the essential role of environmental pollutants in NAFLD development is recognized. Particularly, endocrine-disrupting chemicals (EDCs) have a notable influence. EDCs can be classified as natural (phytoestrogens, genistein, and coumestrol) or synthetic, and the latter ones can be further subdivided into industrial (dioxins, polychlorinated biphenyls, and alkylphenols), agricultural (pesticides, insecticides, herbicides, and fungicides), residential (phthalates, polybrominated biphenyls, and bisphenol A), and pharmaceutical (parabens). Several experimental models have proposed a mechanism involving this group of substances with the disruption of hepatic metabolism, which promotes NAFLD. These include an imbalance between lipid influx/efflux in the liver, mitochondrial dysfunction, liver inflammation, and epigenetic reprogramming. It can be concluded that exposure to EDCs might play a crucial role in NAFLD initiation and evolution. However, further investigations supporting these effects in humans are required.