Next Article in Journal
Ethyl Caffeate Can Inhibit Aryl Hydrocarbon Receptor (AhR) Signaling and AhR-Mediated Potentiation of Mast Cell Activation
Next Article in Special Issue
Innovative Therapeutic Approaches in Non-Alcoholic Fatty Liver Disease: When Knowing Your Patient Is Key
Previous Article in Journal
VAX014, an Oncolytic Therapy, Reduces Adenomas and Modifies Colon Microenvironment in Mouse Model of CRC
Previous Article in Special Issue
MCD Diet Modulates HuR and Oxidative Stress-Related HuR Targets in Rats
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 12
10.3390/ijms24129996
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Non-Alcoholic Fatty Liver Disease: Translating Disease Mechanisms into Therapeutics Using Animal Models

by
Amina Basha
1,†,
Sarah C. May
1,†,
Ryan M. Anderson
1,
Niharika Samala
2 and
Raghavendra G. Mirmira
1,*
1
Kovler Diabetes Center, Section of Adult and Pediatric Endocrinology, Diabetes and Metabolism, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA
2
Department of Medicine, Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(12), 9996; https://doi.org/10.3390/ijms24129996
Submission received: 17 May 2023 / Revised: 6 June 2023 / Accepted: 9 June 2023 / Published: 10 June 2023
(This article belongs to the Special Issue Innovative Molecular Target and Therapeutic Approaches in Nonalcoholic Fatty Liver Disease/Nonalcoholic Steatohepatitis (NAFLD/NASH) 3.0)
Browse Figure
Review Reports Versions Notes

Abstract

:
Nonalcoholic fatty liver disease (NAFLD) is a range of pathologies arising from fat accumulation in the liver in the absence of excess alcohol use or other causes of liver disease. Its complications include cirrhosis and liver failure, hepatocellular carcinoma, and eventual death. NAFLD is the most common cause of liver disease globally and is estimated to affect nearly one-third of individuals in the United States. Despite knowledge that the incidence and prevalence of NAFLD are increasing, the pathophysiology of the disease and its progression to cirrhosis remain insufficiently understood. The molecular pathogenesis of NAFLD involves insulin resistance, inflammation, oxidative stress, and endoplasmic reticulum stress. Better insight into these molecular pathways would allow for therapies that target specific stages of NAFLD. Preclinical animal models have aided in defining these mechanisms and have served as platforms for screening and testing of potential therapeutic approaches. In this review, we will discuss the cellular and molecular mechanisms thought to contribute to NAFLD, with a focus on the role of animal models in elucidating these mechanisms and in developing therapies.

1. Introduction

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver pathologies arising from excess fat accumulation in the liver in the absence of excess alcohol use or other causes of liver disease (recently reviewed in [1]). At one end of the spectrum, there is fat accumulation in the liver, or simple steatosis, which progresses to steatosis with inflammation (nonalcoholic steatohepatitis (NASH)), which can further lead to liver scarring (fibrosis). Towards the other end of the spectrum, the disease manifestation is severe, with extensive fibrosis (also known as cirrhosis) and hepatocellular carcinoma. The prevalence of NAFLD is estimated to be 30–40% worldwide and is projected to exceed 50% by 2040 [2]. This steady rise in NAFLD is likely due to an increase in its risk factors, which include obesity, type 2 diabetes mellitus, hyperlipidemia, and metabolic syndrome (MetS). Indeed, in people with type 2 diabetes mellitus and class III obesity (BMI > 40), the prevalence of NAFLD approaches 90% [3,4]. NASH and fibrosis are also more common in individuals with type 2 diabetes mellitus [5,6,7]. Based on these co-morbidities, it is thought that NAFLD is the hepatic manifestation of MetS, with insulin resistance being the main risk factor [8]. Thus, lifestyle modifications with a loss of more than 5–10% of body weight and improvement of underlying conditions can potentially reverse NAFLD before fibrosis occurs. There are currently no U.S. Food and Drug Administration (FDA)-approved drugs to treat NAFLD. Better insight into the molecular pathways that give rise to NAFLD would allow for the development of disease-targeted therapies. By studying animal models of NAFLD, researchers have uncovered the roles of insulin resistance, inflammation, oxidative stress, and endoplasmic reticulum stress in the disease progression. In this review, we discuss the roles of these pathways in NAFLD and assess the utility of various models in replicating NAFLD phenotypes, as summarized in Table 1. Additionally, we highlight treatments that have proven effective in animal models and humans and focus on potential new drug targets and important topics for future research.

2. Pathophysiology of NAFLD

The key drivers of NAFLD progression are insulin resistance and inflammation. In healthy individuals, free fatty acids migrate to the adipose tissue to be stored as triglycerides, and glucose is taken up by skeletal muscle for glycogenesis. However, in states of excess nutrient intake, both adipose tissue and skeletal muscle develop insulin resistance, resulting in reduced lipogenesis and increased lipolysis in adipose tissue and reduced glycogenesis in skeletal muscle, thus diverting excess substrate to the liver to be stored there as triglycerides (Figure 1). At the level of the hepatocyte, selective insulin resistance increases glucose uptake and de novo lipogenesis. Together, increased free fatty acid uptake and lipogenesis result in an overabundance of fat in the liver. In many cases, fatty liver is relatively benign and does not progress beyond this stage. However, hepatic steatosis can progress to NASH upon multiple pro-inflammatory insults from adipose tissue and the gastrointestinal tract [34]. Inflammation leads to oxidative stress and endoplasmic reticulum (ER) stress, which are key mediators of hepatocyte damage and destruction in later stages of liver disease. With this complex pathophysiology in mind, we review models of NAFLD that demonstrate obesity, insulin resistance, and inflammation in the forms of oxidative and ER stress.

3. Obesity and Insulin Resistance

Insulin plays an important role in glucose and lipid homeostasis [35]. In adipose tissue, insulin stimulates glucose uptake and its storage as lipid and inhibits lipolysis, thereby promoting the accumulation of lipids in adipocytes. Meanwhile, in the liver, insulin stimulates glucose storage as glycogen or synthesis into triglycerides that are packaged and exported into circulation. Obesity leads to insulin resistance, thereby disrupting these critical pathways that maintain lipid homeostasis [36]. This is, in part, due to adipose tissue release of pro-inflammatory cytokines that inhibit insulin signaling [37]. Upon insulin resistance, adipocytes release free fatty acids that accumulate in the liver, leading to hepatic steatosis [38]. Thus, obesity and insulin resistance are important hallmarks of NAFLD. Purely genetic causes of NAFLD are rare, but deficiencies in these key genes can serve as suitable animal models of NAFLD. Otherwise, dietary interventions are used to generate animal models of NAFLD.

3.1. Genetic Models of Obesity and Insulin Resistance

Leptin is a satiety hormone released by adipose tissue to suppress appetite [39]. It functions to reduce caloric intake, increase hepatic triglyceride export, and block de novo lipogenesis [40,41]. Therefore, leptin-deficient (ob/ob) mice are an important model of NAFLD. Ob/ob mice become obese and develop high fasting blood glucose with insulin resistance [9,10]. Importantly, ob/ob mice show mild to severe steatosis but do not progress to NASH without additional stimuli, such as a diet containing high cholesterol, trans fat, and fructose (known as the Amylin mouse liver NASH model, or AMLN, diet) [19,42]. Leptin receptor-deficient (db/db) mice are similar to ob/ob mice in terms of obesity, but they show less steatosis than ob/ob mice [11]. Similarly, Zucker (fa/fa) rats have a spontaneous mutation in the leptin receptor that leads to hyperphagia, severe obesity, and insulin resistance [12]. Because human mutations in leptin/leptin receptor are rare [43,44] and because leptin levels increase with NAFLD severity [45], these models may not accurately represent the pathogenesis of NAFLD.
Another gene involved in satiety is Alms1 [46]. Mutations in Alms1 are responsible for Alström syndrome, a rare genetic disorder leading to childhood obesity, severe insulin resistance, and multiple organ failure [47]. Many of these individuals develop an accelerated form of NAFLD, leading to fibrosis and cirrhosis at an unexpectedly young age [48]. In mice, a truncation mutant of Alms1 (foz/foz) leads to increased weight gain, insulin resistance, type 2 diabetes mellitus, and steatosis [13]. On a high-fat diet, foz/foz mice develop NASH due to hepatic cholesterol accumulation [49]. Mutations in Alms1, like mutations in leptin, are rare in humans. Therefore, there may be key differences in the pathogeneses of NAFLD in humans and foz/foz mice.
Insulin signaling activates sterol regulatory-element-binding proteins (SREBPs), which are master transcriptional regulators essential for maintaining lipid homeostasis pathways [50]. During insulin resistance, increased plasma insulin levels may lead to overstimulation of these pathways. SREBP-1c is known to upregulate fatty acid synthesis genes [51]. When SREBP-1c is overexpressed in adipose tissue, mice develop hyperglycemia, insulin resistance, and fatty liver [4]. Insulin also activates SREBP-2, thereby activating genes involved in cholesterol synthesis [52]. SREBP-2 is shown to be upregulated during NASH [53]. Therefore, SREBP-2 may provide a direct link between insulin resistance, cholesterol accumulation, and NAFLD.
It is now recognized that excess hepatic cholesterol, in addition to excess triglycerides, promotes NAFLD progression [54]. Excess hepatic cholesterol may result from increased biosynthesis, impaired very-low-density lipoprotein (VLDL) packaging and export, or impaired biliary excretion. Thus, animal models with defects in cholesterol metabolism are now being used to study NAFLD pathogenesis. Mice deficient in low-density lipoprotein receptor (Ldlr−/−), when fed a high-fat/high-cholesterol diet for 3 months, develop hepatic inflammation, steatosis, and fibrosis [14]. Apolipoprotein E-deficient (Apoe−/−) mice fed a high-fat diet progress to early stages of fibrosis after 8 weeks [55]. In these models, excess free cholesterol accumulation in hepatocytes is likely the main driver of hepatic inflammation.

3.2. Dietary Models of Obesity and Insulin Resistance

Dietary deficiency in choline or defects in methionine metabolism may lead to NAFLD. Choline, an essential nutrient, is required for the synthesis of phosphatidylcholine, which is the main phospholipid on VLDL particles [56]. In the absence of phosphatidylcholine, excess hepatic triglycerides accumulate, as they cannot be packaged into VLDL and exported into circulation. Therefore, in mice, choline-deficient diets augment hepatic lipid accumulation on a high-fat diet [20]. However, their liver disease does not go beyond this stage unless they are fed a diet that is also deficient in methionine. Methionine, an essential amino acid, is another precursor of phosphatidylcholine, acting via methylation of phosphatidylethanolamine. Mice fed a methionine-choline-deficient (MCD) diet show weight gain, insulin resistance, and liver inflammation within 4 weeks [57]. However, this model does not fully mimic the human pathophysiology, as the mice do not develop insulin resistance [58]. Genetic models that interfere with the utilization of methionine—such as an S-adenosylhomocysteine hydrolase mutant in zebrafish (ducttrip) [17,59] or methionine adenosyltransferase 1A (MAT1A) deficiency in mice [18]—have also been useful in studying NAFLD.
A high-fat, high-carbohydrate, and/or high-cholesterol diet effectively induces insulin resistance in animal models, triggering NAFLD without genetic manipulation [60]. Many diet variations exist, but the typical range is 32–60% fat, 30–50% sugar, and 0.2–1% cholesterol [60]. In Sprague–Dawley rats, a high-fat diet (60% fat, 20% carbohydrates) or NASH diet (40% fat, 40% carbohydrates, 2% cholesterol) induces steatosis and inflammation after 16 weeks [22]. On a high-fructose diet (10% fat, 70% carbohydrates), the rats do not show the same phenotypes, suggesting that fat and cholesterol are the main drivers of NAFLD progression [22]. On the American lifestyle-induced obesity syndrome (ALIOS) diet, which consists of 45% fat (of which 30% is trans fat) plus high-fructose corn syrup in drinking water, mice develop obesity, insulin resistance, steatosis, NASH, fibrosis, and hepatic tumors over a 12-month period [23]. Thus, the ALIOS model replicates the human pathophysiology with striking accuracy. Similarly, in the diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND), mice fed a high-fat, high-carbohydrate diet (42% fat, 0.1% cholesterol, high-fructose/glucose water) develop steatosis, NASH, advanced fibrosis, and liver tumors after 52 weeks [24]. When fed a fast-food diet of 40% fat (12% saturated fats) and 0.2% cholesterol plus high-fructose corn syrup in drinking water for 25 weeks, mice show effects that are physiologically comparable to humans, including NASH with fibrosis and markers of cellular stress [25]. However, in this case, liver tumors were not reported [25]. In many of these studies, the length of time to advanced disease is a major limitation. To accelerate disease progression, SMC Laboratories developed a mouse model (STAM™) that progresses to liver tumors in 100% of mice by 20 weeks [26]. STAM™ mice are first injected with the β-cell toxin streptozotocin to induce type 2 diabetes. Subsequently, the mice are fed a high-fat diet to induce liver disease. In conclusion, dietary models show the most phenotypic similarity to human NAFLD, although a longer feeding period may be required to reproduce more severe or advanced stages of liver disease, such as cirrhosis and liver tumors.

4. Inflammation

Obesity and insulin resistance cause hepatic steatosis, but additional pathogenic insults are required to progress to NASH. Inflammation drives the progression of NAFLD from benign fat accumulation to permanent liver damage. Two major sources of inflammation in NAFLD are oxidative stress and ER stress.

4.1. Oxidative Stress

Reactive oxygen species (ROS) are oxygen-containing molecules that can readily react with and damage biomolecules. Whereas low levels of ROS act as key signaling messengers, excess ROS promote oxidative stress. In NAFLD, the accumulation of lipids results in excess ROS, inflammation, and, ultimately, progression to NASH [61,62]. Both increased ROS production and impaired antioxidant capacity contribute to elevated ROS.
Most ROS are produced during mitochondrial oxidative phosphorylation, as electrons escape the electron transport chain, react with oxygen, and form superoxide [63]. During NAFLD, hepatocytes upregulate β-oxidation to break down excess fatty acids [64,65]. With this surge in β-oxidation, more reducing equivalents are shuttled into the electron transport chain, thereby increasing superoxide production and resulting in oxidative stress and inflammation [66,67]. Impaired activity of the electron transport chain complexes further exacerbates ROS production and oxidative stress. In NASH, the activity of all electron transport chain complexes is impaired [68]. Another major source of ROS is the mitochondrial cytochrome P450 (CYP) enzyme CYP2E1, which is elevated in NASH livers [69]. CYP2E1 metabolizes alcohol and, in the process, generates ROS [70]. In mice, CYP2E1 promotes the development of obesity, insulin resistance, NASH, and fibrosis on a high-fat diet [71,72,73]. In some studies, free fatty acids were shown to upregulate CYP2E1 [74,75,76], but other studies contradict these findings [77,78]. NADPH oxidases (NOXs) are another source of ROS. NOXs make superoxide at relatively low levels to support normal physiological functions, such as inflammatory responses and cell signaling [79]. During NAFLD, NOXs may become dysregulated and produce excessive amounts of ROS. In the liver, NOX1, NOX2, and NOX4 are thought to be the most relevant isoforms that contribute to disease pathogenesis. Indeed, NOX1- or NOX4-deficient mice are protected from liver fibrosis induced by carbon tetrachloride [80]. Similarly, NOX2-deficient mice are protected against high-fat diet-induced liver steatosis [81].
In NAFLD, excess ROS can also result from the failure of antioxidant systems. When high amounts of ROS accumulate, they deplete antioxidants and overwhelm the liver’s antioxidant enzymes. A key antioxidant pathway involves the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which binds to antioxidant response elements and upregulates detoxifying enzymes and the antioxidant glutathione [82]. Nrf2 plays an important protective role in NAFLD, as Nrf2-deficient mice fed a high-fat diet are more susceptible to NASH [83]. Conversely, activation of the Nrf2 pathway helps to resolve NASH and fibrosis in mice fed an MCD diet [84]. Nrf2 upregulates detoxifying enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutathione S-transferase (GST), and superoxide dismutase (SOD), and these have been shown to protect against NAFLD progression to varying degrees. In mice, overexpression of NQO1 protects against high-fat diet-induced steatosis [85]. Inducing HO-1 activity protects against MCD diet-induced steatohepatitis [86]. Loss of GST (mu 2 isoform) results in hepatosteatosis and fibrosis in mice fed a high-fat diet [87]. Loss of SOD1 increases hepatic lipids and oxidative damage and eventually leads to hepatocellular carcinoma [88,89]. Nrf2 also upregulates peroxisome proliferator-activated receptor γ (PPARγ) [90,91,92], a transcription factor with anti-inflammatory, antioxidant, and insulin-sensitizing functions [93]. In adipose tissue, PPARγ promotes lipid uptake and storage, as well as insulin sensitivity [92]. Additionally, in hepatic macrophages, PPARγ protects mice against oxidative stress induced by the liver toxin carbon tetrachloride [28]. These potential benefits have led to increased interest in using PPARγ agonists to treat NAFLD. However, because liver PPARγ increases free fatty acid uptake and de novo lipogenesis, its expression in the liver promotes steatosis [94,95,96]. Therefore, cell-specific targeting of PPARγ would be essential. The presence of non-enzymatic antioxidants, including vitamins C and E, has been inversely associated with NAFLD/NASH, suggesting that they also play a role in preventing liver disease [97,98].

Animal Models of Oxidative Stress in NAFLD

To study the role of oxidative stress in NAFLD, mice are commonly fed an MCD diet. This diet appears to induce more inflammation, ROS, mitochondrial DNA damage, and apoptotic cell death than in ob/ob mice [99]. However, there are key differences between the MCD diet model and the human pathophysiology of NAFLD. Most notably, mice on an MCD diet become cachectic with a reduced liver-to-body weight ratio and have elevated liver enzymes, which are not observed in humans with NASH [21]. These key differences lead us to question whether the MCD diet model is suitable for studying NAFLD. Zucker rats (fa/fa leptin-deficient model) also show increased markers of oxidative stress when fed a high-fat diet [100]. However, these fa/fa rats show decreased CYP2E1 with no change on a high-fat diet [100], which is the opposite response of that seen in humans [69]. In zebrafish, overexpression of proinflammatory cytokines (IL-1β, TNFα, and IFNγ) increases inflammation, promotes macrophage infiltration into hepatocytes, and generates excess ROS [29]. Additionally, this model causes lipid accumulation in hepatocytes and signs of both insulin resistance and inflammation, similar to the human pathophysiology of NAFLD [29].

4.2. ER Stress

Hepatocytes are highly secretory cells that produce a variety of proteins to maintain cellular, metabolic, and lipid homeostasis. These proteins must be properly synthesized, processed, and folded in the hepatocyte ER. Anything that disrupts ER function can induce ER stress and the accumulation of unfolded or misfolded proteins. In NAFLD, many factors drive ER stress in hepatocytes, including free cholesterol accumulation in the ER membrane [101]. In response, hepatocytes first attempt to reduce ER stress and promote cell survival, but if unsuccessful they undergo apoptosis. This process is called the unfolded protein response (UPR), a complex signaling pathway with three stress-activated sensors: protein kinase R-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE-1α). Chronic activation of the UPR is thought to contribute to NAFLD.
The master regulator of the UPR is a chaperone protein, glucose-regulated protein 78 (GRP78). Underscoring its protective role in NAFLD, GRP78 overexpression in the liver of ob/ob mice protects against steatosis [102]. Under unstressed conditions, GRP78 binds to the three UPR sensors, maintaining them in an inactive state. During ER stress, GRP78 preferentially binds to the unfolded proteins. Released from GRP78, the UPR sensors enter an active state. Once activated, PERK phosphorylates eukaryotic initiation factor 2α (p-eIF2α), which shuts down global protein translation to ameliorate ER stress but also upregulates activating transcription factor 4 (ATF4). Initially, ATF4 activates genes that promote cell survival, but if ER stress is severe or prolonged ATF4 upregulates pro-apoptotic C/EBP homologous protein (CHOP). Consistent with a pathogenic role in NAFLD, ATF4-deficient mice are protected against high-carbohydrate diet-induced steatosis [103], and ATF4-overexpressing zebrafish developed steatosis [104]. Activated ATF6 is proteolytically cleaved into its nuclear form, which then upregulates genes involved in protein folding and lipid synthesis. It was shown that ATF6 deficiency protects against steatosis in a zebrafish model of chronic hepatic ER stress (i.e., foie gras mutant), whereas it may potentiate steatosis under acute ER stress [32]. IRE1α has endonuclease activity, which it uses to splice Xbp1 mRNA, producing an active transcription factor. Spliced XBP1 activates the transcription of ER chaperones [105] and ER-associated protein degradation components [106] to promote cell survival. Multiple reports have shown that IRE1α protects against hepatic steatosis [107] via UPR-unrelated pathways that regulate lipid homeostasis [108,109]. Under severe stress, IRE1α couples with TNF receptor-associated factor 2 (TRAF2) to activate c-Jun N-terminal kinase (JNK) [110] or IκB kinase (IKK) [111], thereby promoting inflammation. More recently, IRE1α was shown to be involved in NLRP3 inflammasome assembly and production of the pro-inflammatory cytokine IL-1β [112]. Therefore, under the right conditions, IRE1α signaling may promote progression from simple steatosis to NASH.
When the UPR fails to relieve ER stress, it triggers apoptosis through various proteolytic caspases, potentially including a combination of caspases 12, 3, 6, 7, 8, and 9 [113]. Another caspase (caspase 2) may be activated by ER stress, but is not believed to play a role in ER stress-mediated apoptosis [114]. Instead, in the MUP-uPA mouse model of hepatic ER stress, caspase 2 cleaves site-1 protease, thereby activating SREBPs [115]. As discussed, the SREBPs are master regulators of lipid homeostasis and play a role in NAFLD progression. In a fructose-induced mouse model of NAFLD, studies have linked ER stress to SREBP-1c activation and lipid accumulation [116]. Inhibiting SREBP-2 overactivation in vitro improves hepatic autophagy and relieves ER stress in mice fed a high-fat diet [117]. However, activating SREBP-2 in mice with lovastatin/ezetimibe improves autophagy and reduces hepatic triglyceride accumulation [118]. More work is needed to assess the role of SREBP-2 in NAFLD and progression to NASH. Moreover, additional studies must disentangle when the UPR is protective versus when it promotes NAFLD progression, which is an essential first step in developing NAFLD therapeutics to target ER stress.

Animal Models of ER Stress in NAFLD

To that end, we examined select animal models that may prove useful in studying ER stress as a pathogenic step towards NAFLD. As previously mentioned, the major urinary protein urokinase plasminogen activator (MUP-uPA) transgenic mouse is a model of ER stress and NAFLD progression [119]. The protein uPA is delivered to hepatocytes, where it accumulates in the ER and induces ER stress [30]. After 24 weeks on a high-fat diet, the mice develop NASH, and after 32 weeks, they progress to hepatocellular carcinoma [30]. The MUP-uPA mouse model is one of the better models of NAFLD progression owing to its ability to faithfully replicate key human phenotypes throughout the entire disease progression [120]. In zebrafish, deletion of the trafficking protein particle complex subunit 11 (TRAPPC11) analogue (also known as the foie gras gene) disrupts ER and Golgi protein trafficking and induces ER stress [31]. The foie gras model results in hepatic steatosis mediated through the ATF6 branch of the UPR [32]. However, because this mutation can be lethal, the foie gras model progresses neither to liver fibrosis nor to hepatocellular carcinoma. Therefore, this model does not demonstrate full disease pathogenesis. Also in zebrafish, deficiency in CDP-diacylglycerol-inositol 3-phosphatidyltransferase (Cdipt) impairs phosphatidylinositol synthesis, leading to ER stress and hepatic steatosis by an unknown mechanism [33].

5. Treatment Modalities

The mainstay treatment for NAFLD is lifestyle modification. Beyond diet and exercise, there are no current FDA-approved or guideline-approved medications to treat NAFLD. Potential new therapeutics target the underlying causes of NAFLD, which include insulin resistance, imbalances in lipid metabolism, and inflammation.

5.1. Lifestyle Modifications

The American Association for the Study of Liver Diseases (AASLD) recommends a body weight reduction of at least 3–5% to reverse steatosis and >7% to improve histologic features of NASH and fibrosis [121]. The European Association for the Study of the Liver (EASL) guidelines are similar and recommend a weight reduction of 7–10% for treatment of NAFLD [122]. For a healthy eating plan that manages MetS, the American Heart Association (AHA) recommends reducing saturated fat intake to <7% of total calories, minimizing trans fat intake, and maintaining cholesterol intake at <200 mg per day and total fat at 25–35% of total calories [123]. For an exercise plan, individuals should aim for at least 30 min of cardio activity at least 5 days a week [123].
Recent randomized controlled clinical trials have investigated the impacts of diet composition and meal timing on NAFLD. In the REDUCTION trial, subjects with type 2 diabetes mellitus and NAFLD lost weight and improved their glycemic control after 6 months on a low-carbohydrate, high-fat diet compared to patients on a high-carbohydrate, low-fat diet [124]. However, these improvements were not sustained at the 3-month follow-up [124]. Long-term adherence often limits the effectiveness of dietary interventions. Although low-fat diets and Mediterranean diets (low carbohydrate and rich in unsaturated fats) have both been shown to reduce hepatic steatosis in clinical trials, adherence was better for the Mediterranean diet [125], suggesting the longer-term benefits of a Mediterranean diet. Moreover, adherence to a Mediterranean diet has been shown to lower liver inflammatory markers and increase antioxidant pathways [126]. In a US-based study, the effects of alternate-day fasting with and without exercise were tested on subjects with obesity and NAFLD [127]. For 3 months, study participants ate one meal at dinnertime on “fast days” and ate food as desired on “feast days”. At the end of the trial, participants in the group of alternative-day fasting plus aerobic exercise had decreased body weight, reduced steatosis, and improved glycemic control [127]. However, more studies are needed to determine whether exercise provided any benefit over fasting alone. Lastly, the TREATY-FLD trial recently found no additional benefit with time-restricted diets (eating between 8:00 a.m. and 4:00 p.m.) as opposed to a calorie-restricted diet alone [128]. After 12 months, participants in both groups had reduced and comparable intrahepatic fat measurements [128].
Research has clearly shown that exercise improves NAFLD, even without weight loss [129]. Despite NAFLD patients preferring exercise over medication, many are unable to start or maintain exercise programs due to barriers such as fatigue, injury, and shortness of breath [130]. As NAFLD progresses, these barriers are even more difficult to overcome. Thus, there is a need to tailor exercise programs (in terms of exercise type, duration, and intensity) to address these limitations. A comparative analysis of aerobic and resistance exercise found that both forms of exercise improved hepatic steatosis [131]. However, because resistance exercise requires less energy consumption, it may be the better choice for patients who cannot tolerate aerobic exercise [131].

5.2. Drugs Targeting Insulin Resistance

Thiazolidinediones (TZDs) are a class of drugs that improve insulin sensitivity by activating PPARγ [132]. Two TZDs, pioglitazone and rosiglitazone, are FDA approved to treat insulin resistance and improve glycemic control in patients with type 2 diabetes mellitus. Unfortunately, these drugs may lead to heart failure [133,134] and weight gain [135]. Thus, there has been interest in developing substitutes with better safety profiles. Recently, dual PPARα/γ agonists have shown promise in treating NAFLD/NASH, as they not only improve glycemic control (via PPARγ) but also reduce lipid levels (via PPARα). Thus, they could target multiple underlying factors of NAFLD pathogenesis while also preventing side effects such as weight gain. In preclinical studies of Apoe−/− mice on a high-fat/high-cholesterol diet, the dual PPARα/γ agonist aleglitazar improved glucose tolerance and lowered hepatic fat content without an increase in body weight [136]. In the AleCardio trial, aleglitazar reduced hepatic steatosis and fibrosis in subjects with acute coronary syndrome and type 2 diabetes mellitus [137]. Saroglitazar, another dual PPARα/γ agonist, showed similar benefits in mouse models of NAFLD/NASH [138] and in a recent phase 2 clinical trial [139]. However, in the clinical trials, aleglitazar and saroglitazar increased body mass index and body weight, respectively. A third PPAR family member, PPARδ, was recently shown to reduce hepatic lipid content via autophagy [140]. Therefore, it was thought that pan-PPAR agonists (targeting PPARα, PPARγ, and PPARδ) may show additional benefits over more selective agonists. In preclinical studies of mice fed a choline-deficient, amino acid-defined, high-fat diet, the pan-PPAR agonist lanifibranor was more potent than single agonists in improving steatohepatitis and carbon tetrachloride-induced fibrosis [141]. In the NATIVE trial, the pan-PPAR agonist lanifibranor improved advanced fibrosis in subjects with type 2 diabetes mellitus [142]. However, much like the more selective PPAR agonists, lanifibranor also induced weight gain [142]. More studies will be needed to assess the possible benefits of pan-PPAR agonists over dual agonists and weigh the risks of potential side effects.
Sodium-glucose cotransporter 2 (SGLT2) inhibitors prevent glucose from being reabsorbed into circulation by the kidneys, thus improving hyperglycemia [143]. SGLT2 inhibitors result in glucose excretion in urine, and the loss of these calories may be one reason why SGLT2 inhibitors induce weight loss [144]. Various SGLT2 inhibitors have been FDA approved to treat patients with type 2 diabetes mellitus, but they are just now being investigated in the context of NAFLD. In a small clinical trial, the FDA-approved SGLT2 inhibitor dapagliflozin improved glycemic control and hepatic lipid content after 12 weeks of treatment [145]. Similarly, ipragliflozin (a non-FDA-approved drug) helped to resolve NASH and fibrosis in subjects with type 2 diabetes and NAFLD after 72 weeks [146]. Preclinical studies tested the combined effects of tofogliflozin (an SGLT2 inhibitor) and pioglitazone (a PPARγ agonist) on animal models of obesity and type 2 diabetes [147]. Interestingly, the researchers found that tofogliflozin prevented pioglitazone-induced weight gain in mice, and the combination therapy improved hyperglycemia better than monotherapy [147]. These effects were also observed in a clinical trial investigating tofogliflozin/pioglitazone combination therapy, as was a further improvement in steatosis over tofogliflozin monotherapy in patients with type 2 diabetes and NAFLD [148]. These studies used small cohorts, ranging from 20–40 participants, so larger-scale studies will need to be conducted to understand the effects of these medications on NAFLD.
Glucagon-like peptide-1 (GLP-1) analogs are another class of drug commonly used to treat type 2 diabetes. These drugs, like GLP-1, stimulate pancreatic β cells to secrete insulin. The GLP-1 receptor agonist liraglutide has shown some promise in decreasing hepatic fat content in subjects with obesity and/or type 2 diabetes [149,150]. However, a recent meta-analysis concluded that the effects of liraglutide were nonsignificant in individuals with NAFLD [151]. Another GLP-1 receptor agonist, semaglutide, has a similar mechanism of action to that of liraglutide but with additional weight loss and reductions in glycated hemoglobin (HbA1c) [152]. In a phase 2 clinical trial, semaglutide resolved NASH in up to 59% of study participants, but its effects on fibrosis outcomes were less clear [153]. There is ongoing research to understand the mechanisms by which GLP-1 analogs may improve steatosis and fibrosis.

5.3. Drugs Targeting Lipid Metabolism

Cholesterol is now recognized for its role in NAFLD pathogenesis. Many individuals with NAFLD also have dyslipidemia and are prescribed statins to lower their cholesterol levels. Additionally, statins may have anti-inflammatory functions beyond their primary role in lowering cholesterol [154]. Thus, statins could be effective treatments for NAFLD. In the ESSENTIAL trial, a combination of the lipid-lowering agents ezetimibe (a non-statin) and low-dose rosuvastatin was shown to significantly reduce hepatic fat content in participants with NAFLD [155]. However, longer-term studies will be needed to determine whether lipid-lowering therapies can improve NASH and/or fibrosis.
Farnesoid X receptor (FXR) is a nuclear transcription factor that increases bile acid elimination and reduces hepatic triglyceride levels, among various other metabolism-benefiting functions [156]. Therefore, FXR agonists show promise as therapeutics for NAFLD. In recent clinical trials, the FXR agonists MET409 [157], tropifexor [158], vonafexor [159], and cilofexor [160] were shown to reduce hepatic fat content in subjects with NASH. In an ongoing phase 3 clinical trial, the FXR agonist obeticholic acid improved fibrosis in people with NASH and moderate to severe fibrosis [161]. Currently, a new drug application for obeticholic acid is under review by the FDA for treatment of NASH with advanced fibrosis. Common side effects of FXR agonists include itching and/or increased low-density lipoprotein cholesterol levels.
Fibroblast growth factor 21 (FGF21) is another broad metabolic regulator that is being considered as a treatment for NAFLD. In mice fed a high-fat diet, recombinant murine FGF21 upregulates genes involved in fatty acid oxidation and downregulates genes involved in lipogenesis, thus leading to a reduction in hepatic steatosis [162]. Multiple clinical trials have shown that FGF21 analogs, including efruxifermin [163], LLF580 [164], and pegozafermin [165], reduce hepatic fat content and lead to weight loss. Because these FGF21 analogs have such a broad range of functions, long-term safety and efficacy studies will be critical.
More recent studies are investigating thyroid hormone receptor-β (TR-β) agonists as potential NASH treatments. Synthetic thyroid hormone, or thyroxine, has been shown to increase lipid metabolism, leading to weight loss [166]. On the other hand, thyroxine can also lead to heart failure and arrythmias [167]. By targeting the predominant liver isoform (TR-β), hepatic lipid content can be reduced without adverse cardiac effects [168]. Several TR-β agonists have shown efficacy for NAFLD treatment in animal models [169]. In a phase 2 clinical trial, the TR-β agonist resmetirom was shown to reduce hepatic fat in individuals with NASH [170]. Although promising, larger studies are still needed to demonstrate the efficacy and safety of TR-β agonists.

5.4. Drugs Targeting Inflammation

Pentoxyfylline is an FDA-approved anti-inflammatory drug that blocks the production of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) [171]. In MCD diet-induced NASH mice, pentoxyfylline decreases TNFα mRNA expression and markers of hepatic inflammation [172]. However, pentoxyfylline-treated mice have increased hepatic steatosis [172]. Despite this observation, a meta-analysis concluded that pentoxyfylline decreases steatosis, as well as fibrosis and liver inflammation [173]. Larger studies are needed to confirm these effects.
Vitamin E is a key antioxidant with anti-inflammatory functions [174]. Based on its ability to suppress oxidative stress, vitamin E has also been considered as therapy for NAFLD. Indeed, in mice on an MCD diet, vitamin E reduces hepatic steatosis and markers of hepatic inflammation [175]. Additionally, the PIVENS trial showed improvement in steatosis and inflammation in non-diabetic NASH participants treated with vitamin E compared to placebo [176]. There have been some concerns about vitamin E safety in the long term, owing to possible links to hemorrhagic stroke and prostate cancer [121]. Nonetheless, the AASLD guidelines advise that individuals with severe fibrosis and without type 2 diabetes may consider taking a vitamin E supplement [121].

5.5. The Future of Preclinical Testing: Nonhuman Primates as Models of NAFLD

Many drug candidates never succeed in clinical trials because the drug’s effects in mice and other model systems do not translate to humans. Thus, there is a need for model systems that better reflect the human pathogenesis of NAFLD. Nonhuman primates have the potential to more accurately predict how a drug will behave in human studies. NAFLD has been shown to occur in various nonhuman primates spontaneously [177] or as a natural result of aging [178]. However, in these animals, the disease can be accelerated or worsened by feeding special diets. Recently, a new dietary model of NASH in cynomolgus monkeys was developed by feeding a high-fat, high-fructose, high-cholesterol diet for 10 months [27]. In monkeys, this diet induces hepatic steatosis, inflammation, NASH, and fibrosis that is strikingly similar to the human NAFLD pathogenesis at the transcriptional level [27]. In this model system, the tripeptide DT-109 (Gly-Gly-Leu) attenuates NASH and fibrosis by upregulating fatty acid degradation and downregulating inflammation [27]. An important caveat in these studies is their use of only male animals, thereby limiting applicability across the sexes.

6. Conclusions: Translation from Animals to Humans

The demand for animal models to study the pathogenesis of NAFLD is more vital now, as the prevalence of NAFLD is increasing worldwide and therapeutic candidates need to be identified and tested. Given the plethora of research that has been done on animal models to understand the pathophysiology of NAFLD, we now have a better understanding of the various insults that occur in the progression of disease. This has allowed for the implementation of specific guidelines for lifestyle modifications and potential pharmacotherapy. Of all the animal models described here (Table 1), the high-fat/high-fructose diet model has proven to be most like the human pathophysiology of disease. The hypothesized mechanisms describing the causal role of fructose in the development of NAFLD include direct up-regulation of de novo lipogenesis enzymes by fructose breakdown products in hepatocytes and intestinal wall weakening, leading to increased uptake of endotoxins from the gut to the liver and driving inflammation. However, most preclinical studies have used male rodent models, making it is difficult to determine whether sexual dimorphism might preclude applicability to females. Furthermore, these dietary changes alone are usually insufficient for demonstrating severe disease. Although some models, such as fa/fa rats or MCD diet-fed mice, show similar pathophysiology to humans, many of them lack a key clinical feature of obesity that is seen in humans. The MUP-uPA transgenic mouse fed a high-fat diet may be the best animal model of ER stress leading to NAFLD. However, many of the studies discussed earlier were only completed once, leading to the question of reproducibility. Therefore, animal models to date still have limitations that do not fully recapitulate human disease.
Taken together, whereas animal models have provided crucial pathogenic insight into the multiple insults contributing to NAFLD, they only provide a first step towards identifying new therapies. The further development of non-human primate models and/or more rigorous studies in humans will be needed to move the field forward. The most representative animal models must also feature multiple pathogenic insults in order to fully mimic human NAFLD and reveal new therapeutic strategies. In addition to the pathogenic insults described in this review, viral hepatitis may also trigger steatosis and/or accelerate NAFLD progression, but the mechanisms are just beginning to be explored. In the future, a multiple-step treatment program may be necessary to fully treat both the factors that lead to MetS and the inflammatory and oxidative stress pathways that lead to NAFLD.

Author Contributions

Conceptualization, S.C.M. and R.G.M.; writing—original draft preparation, A.B. and S.C.M.; writing—review and editing, A.B., S.C.M., R.M.A., N.S. and R.G.M.; supervision, S.C.M. and R.G.M.; funding acquisition, R.G.M. All authors have read and agreed to the published version of the manuscript.

Funding

The research in the Mirmira lab is funded by NIH grants R01 DK105588, R01 DK060581, R01 DK124906, and U01 DK127786.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Huby, T.; Gautier, E.L. Immune cell-mediated features of non-alcoholic steatohepatitis. Nat. Rev. Immunol. 2022, 22, 429–443. [Google Scholar] [CrossRef]
  2. Le, M.H.; Yeo, Y.H.; Zou, B.; Barnet, S.; Henry, L.; Cheung, R.; Nguyen, M.H. Forecasted 2040 global prevalence of nonalcoholic fatty liver disease using hierarchical bayesian approach. Clin. Mol. Hepatol. 2022, 28, 841–850. [Google Scholar] [CrossRef]
  3. Portillo-Sanchez, P.; Bril, F.; Maximos, M.; Lomonaco, R.; Biernacki, D.; Orsak, B.; Subbarayan, S.; Webb, A.; Hecht, J.; Cusi, K. High Prevalence of Nonalcoholic Fatty Liver Disease in Patients with Type 2 Diabetes Mellitus and Normal Plasma Aminotransferase Levels. J. Clin. Endocrinol. Metab. 2015, 100, 2231–2238. [Google Scholar] [CrossRef]
  4. Shimomura, I.; Hammer, R.E.; Richardson, J.A.; Ikemoto, S.; Bashmakov, Y.; Goldstein, J.L.; Brown, M.S. Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: Model for congenital generalized lipodystrophy. Genes Dev. 1998, 12, 3182–3194. [Google Scholar] [CrossRef] [Green Version]
  5. Casey, S.P.; Kemp, W.W.; McLean, C.A.; Topliss, D.J.; Adams, L.A.; Roberts, S.K. A prospective evaluation of the role of transient elastography for the detection of hepatic fibrosis in type 2 diabetes without overt liver disease. Scand. J. Gastroenterol. 2012, 47, 836–841. [Google Scholar] [CrossRef]
  6. Prashanth, M.; Ganesh, H.K.; Vima, M.V.; John, M.; Bandgar, T.; Joshi, S.R.; Shah, S.R.; Rathi, P.M.; Joshi, A.S.; Thakkar, H.; et al. Prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus. J. Assoc. Physicians India 2009, 57, 205–210. [Google Scholar]
  7. Leite, N.C.; Villela-Nogueira, C.A.; Pannain, V.L.; Bottino, A.C.; Rezende, G.F.; Cardoso, C.R.; Salles, G.F. Histopathological stages of nonalcoholic fatty liver disease in type 2 diabetes: Prevalences and correlated factors. Liver Int. 2011, 31, 700–706. [Google Scholar] [CrossRef]
  8. Kotronen, A.; Yki-Järvinen, H. Fatty liver: A novel component of the metabolic syndrome. Arterioscler. Thromb. Vasc. Biol. 2008, 28, 27–38. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Mayer, J.; Dickie, M.M.; Bates, M.W.; Vitale, J.J. Free selection of nutrients by hereditarily obese mice. Science 1951, 113, 745–746. [Google Scholar] [CrossRef]
  10. Mayer, J.; Bates, M.W.; Dickie, M.M. Hereditary diabetes in genetically obese mice. Science 1951, 113, 746–747. [Google Scholar] [CrossRef]
  11. Suriano, F.; Vieira-Silva, S.; Falony, G.; Roumain, M.; Paquot, A.; Pelicaen, R.; Régnier, M.; Delzenne, N.M.; Raes, J.; Muccioli, G.G.; et al. Novel insights into the genetically obese (ob/ob) and diabetic (db/db) mice: Two sides of the same coin. Microbiome 2021, 9, 147. [Google Scholar] [CrossRef]
  12. Durham, H.A.; Truett, G.E. Development of insulin resistance and hyperphagia in Zucker fatty rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006, 290, R652–R658. [Google Scholar] [CrossRef] [Green Version]
  13. Arsov, T.; Silva, D.G.; O’Bryan, M.K.; Sainsbury, A.; Lee, N.J.; Kennedy, C.; Manji, S.S.; Nelms, K.; Liu, C.; Vinuesa, C.G.; et al. Fat aussie—A new Alström syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis. Mol. Endocrinol. 2006, 20, 1610–1622. [Google Scholar] [CrossRef] [Green Version]
  14. Bieghs, V.; Van Gorp, P.J.; Wouters, K.; Hendrikx, T.; Gijbels, M.J.; van Bilsen, M.; Bakker, J.; Binder, C.J.; Lütjohann, D.; Staels, B.; et al. LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease. PLoS ONE 2012, 7, e30668. [Google Scholar] [CrossRef] [Green Version]
  15. Funke, A.; Schreurs, M.; Aparicio-Vergara, M.; Sheedfar, F.; Gruben, N.; Kloosterhuis, N.J.; Shiri-Sverdlov, R.; Groen, A.K.; van de Sluis, B.; Hofker, M.H.; et al. Cholesterol-induced hepatic inflammation does not contribute to the development of insulin resistance in male LDL receptor knockout mice. Atherosclerosis 2014, 232, 390–396. [Google Scholar] [CrossRef] [Green Version]
  16. Schierwagen, R.; Maybüchen, L.; Zimmer, S.; Hittatiya, K.; Bäck, C.; Klein, S.; Uschner, F.E.; Reul, W.; Boor, P.; Nickenig, G.; et al. Seven weeks of Western diet in apolipoprotein-E-deficient mice induce metabolic syndrome and non-alcoholic steatohepatitis with liver fibrosis. Sci. Rep. 2015, 5, 12931. [Google Scholar] [CrossRef] [Green Version]
  17. Matthews, R.P.; Lorent, K.; Mañoral-Mobias, R.; Huang, Y.; Gong, W.; Murray, I.V.; Blair, I.A.; Pack, M. TNFalpha-dependent hepatic steatosis and liver degeneration caused by mutation of zebrafish S-adenosylhomocysteine hydrolase. Development 2009, 136, 865–875. [Google Scholar] [CrossRef] [Green Version]
  18. Lu, S.C.; Alvarez, L.; Huang, Z.Z.; Chen, L.; An, W.; Corrales, F.J.; Avila, M.A.; Kanel, G.; Mato, J.M. Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation. Proc. Natl. Acad. Sci. USA 2001, 98, 5560–5565. [Google Scholar] [CrossRef] [Green Version]
  19. Trevaskis, J.L.; Griffin, P.S.; Wittmer, C.; Neuschwander-Tetri, B.A.; Brunt, E.M.; Dolman, C.S.; Erickson, M.R.; Napora, J.; Parkes, D.G.; Roth, J.D. Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 2012, 302, G762–G772. [Google Scholar] [CrossRef] [Green Version]
  20. Raubenheimer, P.J.; Nyirenda, M.J.; Walker, B.R. A choline-deficient diet exacerbates fatty liver but attenuates insulin resistance and glucose intolerance in mice fed a high-fat diet. Diabetes 2006, 55, 2015–2020. [Google Scholar] [CrossRef] [Green Version]
  21. Caballero, F.; Fernández, A.; Matías, N.; Martínez, L.; Fucho, R.; Elena, M.; Caballeria, J.; Morales, A.; Fernández-Checa, J.C.; García-Ruiz, C. Specific contribution of methionine and choline in nutritional nonalcoholic steatohepatitis: Impact on mitochondrial S-adenosyl-L-methionine and glutathione. J. Biol. Chem. 2010, 285, 18528–18536. [Google Scholar] [CrossRef] [Green Version]
  22. Jensen, V.S.; Hvid, H.; Damgaard, J.; Nygaard, H.; Ingvorsen, C.; Wulff, E.M.; Lykkesfeldt, J.; Fledelius, C. Dietary fat stimulates development of NAFLD more potently than dietary fructose in Sprague-Dawley rats. Diabetol. Metab. Syndr. 2018, 10, 4. [Google Scholar] [CrossRef]
  23. Harris, S.E.; Poolman, T.M.; Arvaniti, A.; Cox, R.D.; Gathercole, L.L.; Tomlinson, J.W. The American lifestyle-induced obesity syndrome diet in male and female rodents recapitulates the clinical and transcriptomic features of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Am. J. Physiol. Gastrointest. Liver Physiol. 2020, 319, G345–G360. [Google Scholar] [CrossRef]
  24. Asgharpour, A.; Cazanave, S.C.; Pacana, T.; Seneshaw, M.; Vincent, R.; Banini, B.A.; Kumar, D.P.; Daita, K.; Min, H.K.; Mirshahi, F.; et al. A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer. J. Hepatol. 2016, 65, 579–588. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Charlton, M.; Krishnan, A.; Viker, K.; Sanderson, S.; Cazanave, S.; McConico, A.; Masuoko, H.; Gores, G. Fast food diet mouse: Novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition. Am. J. Physiol. Gastrointest. Liver Physiol. 2011, 301, G825–G834. [Google Scholar] [CrossRef] [Green Version]
  26. Fujii, M.; Shibazaki, Y.; Wakamatsu, K.; Honda, Y.; Kawauchi, Y.; Suzuki, K.; Arumugam, S.; Watanabe, K.; Ichida, T.; Asakura, H.; et al. A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma. Med. Mol. Morphol. 2013, 46, 141–152. [Google Scholar] [CrossRef] [PubMed]
  27. Qu, P.; Rom, O.; Li, K.; Jia, L.; Gao, X.; Liu, Z.; Ding, S.; Zhao, M.; Wang, H.; Chen, S.; et al. DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates. Cell Metab. 2023, 35, 742–757.e10. [Google Scholar] [CrossRef]
  28. Morán-Salvador, E.; Titos, E.; Rius, B.; González-Périz, A.; García-Alonso, V.; López-Vicario, C.; Miquel, R.; Barak, Y.; Arroyo, V.; Clària, J. Cell-specific PPARγ deficiency establishes anti-inflammatory and anti-fibrogenic properties for this nuclear receptor in non-parenchymal liver cells. J. Hepatol. 2013, 59, 1045–1053. [Google Scholar] [CrossRef]
  29. Kulkarni, A.; Ibrahim, S.; Haider, I.; Basha, A.; Montgomery, E.; Ermis, E.; Mirmira, R.G.; Anderson, R.M. A Novel 2-Hit Zebrafish Model to Study Early Pathogenesis of Non-Alcoholic Fatty Liver Disease. Biomedicines 2022, 10, 479. [Google Scholar] [CrossRef]
  30. Nakagawa, H.; Umemura, A.; Taniguchi, K.; Font-Burgada, J.; Dhar, D.; Ogata, H.; Zhong, Z.; Valasek, M.A.; Seki, E.; Hidalgo, J.; et al. ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development. Cancer Cell 2014, 26, 331–343. [Google Scholar] [CrossRef] [Green Version]
  31. Sadler, K.C.; Amsterdam, A.; Soroka, C.; Boyer, J.; Hopkins, N. A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease. Development 2005, 132, 3561–3572. [Google Scholar] [CrossRef] [Green Version]
  32. Cinaroglu, A.; Gao, C.; Imrie, D.; Sadler, K.C. Activating transcription factor 6 plays protective and pathological roles in steatosis due to endoplasmic reticulum stress in zebrafish. Hepatology 2011, 54, 495–508. [Google Scholar] [CrossRef] [Green Version]
  33. Thakur, P.C.; Stuckenholz, C.; Rivera, M.R.; Davison, J.M.; Yao, J.K.; Amsterdam, A.; Sadler, K.C.; Bahary, N. Lack of de novo phosphatidylinositol synthesis leads to endoplasmic reticulum stress and hepatic steatosis in cdipt-deficient zebrafish. Hepatology 2011, 54, 452–462. [Google Scholar] [CrossRef] [Green Version]
  34. Tilg, H.; Adolph, T.E.; Moschen, A.R. Multiple Parallel Hits Hypothesis in Nonalcoholic Fatty Liver Disease: Revisited after a Decade. Hepatology 2021, 73, 833–842. [Google Scholar] [CrossRef]
  35. Saltiel, A.R. Insulin Signaling in the Control of Glucose and Lipid Homeostasis. In Metabolic Control; Springer: Cham, Switzerland, 2015; Volume 233, pp. 51–71. [Google Scholar]
  36. Kahn, B.B.; Flier, J.S. Obesity and insulin resistance. J. Clin. Investig. 2000, 106, 473–481. [Google Scholar] [CrossRef] [Green Version]
  37. de Luca, C.; Olefsky, J.M. Inflammation and insulin resistance. FEBS Lett. 2008, 582, 97–105. [Google Scholar] [CrossRef] [Green Version]
  38. McGarry, J.D. What if Minkowski had been ageusic? An alternative angle on diabetes. Science 1992, 258, 766–770. [Google Scholar] [CrossRef]
  39. Pelleymounter, M.A.; Cullen, M.J.; Baker, M.B.; Hecht, R.; Winters, D.; Boone, T.; Collins, F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995, 269, 540–543. [Google Scholar] [CrossRef]
  40. Metz, M.; Beghini, M.; Wolf, P.; Pfleger, L.; Hackl, M.; Bastian, M.; Freudenthaler, A.; Harreiter, J.; Zeyda, M.; Baumgartner-Parzer, S.; et al. Leptin increases hepatic triglyceride export via a vagal mechanism in humans. Cell Metab. 2022, 34, 1719–1731.e5. [Google Scholar] [CrossRef] [PubMed]
  41. Baykal, A.P.; Parks, E.J.; Shamburek, R.; Syed-Abdul, M.M.; Chacko, S.; Cochran, E.; Startzell, M.; Gharib, A.M.; Ouwerkerk, R.; Abd-Elmoniem, K.Z.; et al. Leptin decreases de novo lipogenesis in patients with lipodystrophy. JCI Insight 2020, 5, e137180. [Google Scholar] [CrossRef]
  42. Lindström, P. The physiology of obese-hyperglycemic mice [ob/ob mice]. Sci. World J. 2007, 7, 666–685. [Google Scholar] [CrossRef] [Green Version]
  43. Paz-Filho, G.; Mastronardi, C.; Delibasi, T.; Wong, M.L.; Licinio, J. Congenital leptin deficiency: Diagnosis and effects of leptin replacement therapy. Arq. Bras. Endocrinol. Metabol. 2010, 54, 690–697. [Google Scholar] [CrossRef] [Green Version]
  44. Kleinendorst, L.; Abawi, O.; van der Kamp, H.J.; Alders, M.; Meijers-Heijboer, H.E.J.; van Rossum, E.F.C.; van den Akker, E.L.T.; van Haelst, M.M. Leptin receptor deficiency: A systematic literature review and prevalence estimation based on population genetics. Eur. J. Endocrinol. 2020, 182, 47–56. [Google Scholar] [CrossRef]
  45. Rotundo, L.; Persaud, A.; Feurdean, M.; Ahlawat, S.; Kim, H.S. The Association of leptin with severity of non-alcoholic fatty liver disease: A population-based study. Clin. Mol. Hepatol. 2018, 24, 392–401. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Heydet, D.; Chen, L.X.; Larter, C.Z.; Inglis, C.; Silverman, M.A.; Farrell, G.C.; Leroux, M.R. A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice. Dev. Neurobiol. 2013, 73, 1–13. [Google Scholar] [CrossRef] [PubMed]
  47. Marshall, J.D.; Maffei, P.; Collin, G.B.; Naggert, J.K. Alström syndrome: Genetics and clinical overview. Curr. Genom. 2011, 12, 225–235. [Google Scholar] [CrossRef] [Green Version]
  48. Bettini, S.; Bombonato, G.; Dassie, F.; Favaretto, F.; Piffer, L.; Bizzotto, P.; Busetto, L.; Chemello, L.; Senzolo, M.; Merkel, C.; et al. Liver Fibrosis and Steatosis in Alström Syndrome: A Genetic Model for Metabolic Syndrome. Diagnostics 2021, 11, 797. [Google Scholar] [CrossRef] [PubMed]
  49. Van Rooyen, D.M.; Larter, C.Z.; Haigh, W.G.; Yeh, M.M.; Ioannou, G.; Kuver, R.; Lee, S.P.; Teoh, N.C.; Farrell, G.C. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis. Gastroenterology 2011, 141, 1393–1403, 1403.e1391–1395. [Google Scholar] [CrossRef] [Green Version]
  50. Eberlé, D.; Hegarty, B.; Bossard, P.; Ferré, P.; Foufelle, F. SREBP transcription factors: Master regulators of lipid homeostasis. Biochimie 2004, 86, 839–848. [Google Scholar] [CrossRef]
  51. Shimano, H.; Horton, J.D.; Shimomura, I.; Hammer, R.E.; Brown, M.S.; Goldstein, J.L. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J. Clin. Investig. 1997, 99, 846–854. [Google Scholar] [CrossRef] [Green Version]
  52. Inoue, J.; Sato, R.; Maeda, M. Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase. J. Biochem. 1998, 123, 1191–1198. [Google Scholar] [CrossRef]
  53. Caballero, F.; Fernández, A.; De Lacy, A.M.; Fernández-Checa, J.C.; Caballería, J.; García-Ruiz, C. Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH. J. Hepatol. 2009, 50, 789–796. [Google Scholar] [CrossRef]
  54. Kerr, T.A.; Davidson, N.O. Cholesterol and nonalcoholic fatty liver disease: Renewed focus on an old villain. Hepatology 2012, 56, 1995–1998. [Google Scholar] [CrossRef] [Green Version]
  55. Lu, W.; Mei, J.; Yang, J.; Wu, Z.; Liu, J.; Miao, P.; Chen, Y.; Wen, Z.; Zhao, Z.; Kong, H.; et al. ApoE deficiency promotes non-alcoholic fatty liver disease in mice via impeding AMPK/mTOR mediated autophagy. Life Sci. 2020, 252, 117601. [Google Scholar] [CrossRef]
  56. Yao, Z.M.; Vance, D.E. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J. Biol. Chem. 1988, 263, 2998–3004. [Google Scholar] [CrossRef]
  57. Chiba, T.; Suzuki, S.; Sato, Y.; Itoh, T.; Umegaki, K. Evaluation of Methionine Content in a High-Fat and Choline-Deficient Diet on Body Weight Gain and the Development of Non-Alcoholic Steatohepatitis in Mice. PLoS ONE 2016, 11, e0164191. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Rinella, M.E.; Green, R.M. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance. J. Hepatol. 2004, 40, 47–51. [Google Scholar] [CrossRef]
  59. Yee, N.S.; Lorent, K.; Pack, M. Exocrine pancreas development in zebrafish. Dev. Biol. 2005, 284, 84–101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Eng, J.M.; Estall, J.L. Diet-Induced Models of Non-Alcoholic Fatty Liver Disease: Food for Thought on Sugar, Fat, and Cholesterol. Cells 2021, 10, 1805. [Google Scholar] [CrossRef] [PubMed]
  61. Satapati, S.; Kucejova, B.; Duarte, J.A.; Fletcher, J.A.; Reynolds, L.; Sunny, N.E.; He, T.; Nair, L.A.; Livingston, K.A.; Fu, X.; et al. Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver. J. Clin. Investig. 2015, 125, 4447–4462. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Ore, A.; Akinloye, O.A. Oxidative Stress and Antioxidant Biomarkers in Clinical and Experimental Models of Non-Alcoholic Fatty Liver Disease. Medicina 2019, 55, 26. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Murphy, M.P. How mitochondria produce reactive oxygen species. Biochem. J. 2009, 417, 1–13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Miele, L.; Grieco, A.; Armuzzi, A.; Candelli, M.; Forgione, A.; Gasbarrini, A.; Gasbarrini, G. Hepatic mitochondrial beta-oxidation in patients with nonalcoholic steatohepatitis assessed by 13C-octanoate breath test. Am. J. Gastroenterol. 2003, 98, 2335–2336. [Google Scholar] [CrossRef]
  65. Iozzo, P.; Bucci, M.; Roivainen, A.; Någren, K.; Järvisalo, M.J.; Kiss, J.; Guiducci, L.; Fielding, B.; Naum, A.G.; Borra, R.; et al. Fatty acid metabolism in the liver, measured by positron emission tomography, is increased in obese individuals. Gastroenterology 2010, 139, 846–856.e6. [Google Scholar] [CrossRef]
  66. Lambertucci, R.H.; Hirabara, S.M.; Silveira, L.o.R.; Levada-Pires, A.C.; Curi, R.; Pithon-Curi, T.C. Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells. J. Cell. Physiol. 2008, 216, 796–804. [Google Scholar] [CrossRef] [PubMed]
  67. Nakamura, S.; Takamura, T.; Matsuzawa-Nagata, N.; Takayama, H.; Misu, H.; Noda, H.; Nabemoto, S.; Kurita, S.; Ota, T.; Ando, H.; et al. Palmitate induces insulin resistance in H4IIEC3 hepatocytes through reactive oxygen species produced by mitochondria. J. Biol. Chem. 2009, 284, 14809–14818. [Google Scholar] [CrossRef] [Green Version]
  68. Pérez-Carreras, M.; Del Hoyo, P.; Martín, M.A.; Rubio, J.C.; Martín, A.; Castellano, G.; Colina, F.; Arenas, J.; Solis-Herruzo, J.A. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology 2003, 38, 999–1007. [Google Scholar] [CrossRef]
  69. Weltman, M.D.; Farrell, G.C.; Hall, P.; Ingelman-Sundberg, M.; Liddle, C. Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis. Hepatology 1998, 27, 128–133. [Google Scholar] [CrossRef] [PubMed]
  70. Robertson, G.; Leclercq, I.; Farrell, G.C. Nonalcoholic steatosis and steatohepatitis. II. Cytochrome P-450 enzymes and oxidative stress. Am. J. Physiol. Gastrointest. Liver Physiol. 2001, 281, G1135–G1139. [Google Scholar] [CrossRef] [PubMed]
  71. Abdelmegeed, M.A.; Choi, Y.; Godlewski, G.; Ha, S.K.; Banerjee, A.; Jang, S.; Song, B.J. Cytochrome P450-2E1 promotes fast food-mediated hepatic fibrosis. Sci. Rep. 2017, 7, 39764. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  72. Zong, H.; Armoni, M.; Harel, C.; Karnieli, E.; Pessin, J.E. Cytochrome P-450 CYP2E1 knockout mice are protected against high-fat diet-induced obesity and insulin resistance. Am. J. Physiol. Endocrinol. Metab. 2012, 302, E532–E539. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Correia, M.A.; Kwon, D. Why Hepatic CYP2E1-Elevation by Itself Is Insufficient for Inciting NAFLD/NASH: Inferences from Two Genetic Knockout Mouse Models. Biology 2020, 9, 419. [Google Scholar] [CrossRef] [PubMed]
  74. Hurrell, T.; Kastrinou-Lampou, V.; Fardellas, A.; Hendriks, D.F.G.; Nordling, Å.; Johansson, I.; Baze, A.; Parmentier, C.; Richert, L.; Ingelman-Sundberg, M. Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis. Cells 2020, 9, 964. [Google Scholar] [CrossRef] [Green Version]
  75. Raucy, J.L.; Lasker, J.; Ozaki, K.; Zoleta, V. Regulation of CYP2E1 by ethanol and palmitic acid and CYP4A11 by clofibrate in primary cultures of human hepatocytes. Toxicol. Sci. 2004, 79, 233–241. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  76. Sung, M.; Kim, I.; Park, M.; Whang, Y.; Lee, M. Differential effects of dietary fatty acids on the regulation of CYP2E1 and protein kinase C in human hepatoma HepG2 cells. J. Med. Food 2004, 7, 197–203. [Google Scholar] [CrossRef]
  77. Donato, M.T.; Lahoz, A.; Jiménez, N.; Pérez, G.; Serralta, A.; Mir, J.; Castell, J.V.; Gómez-Lechón, M.J. Potential impact of steatosis on cytochrome P450 enzymes of human hepatocytes isolated from fatty liver grafts. Drug Metab. Dispos. 2006, 34, 1556–1562. [Google Scholar] [CrossRef]
  78. Donato, M.T.; Jiménez, N.; Serralta, A.; Mir, J.; Castell, J.V.; Gómez-Lechón, M.J. Effects of steatosis on drug-metabolizing capability of primary human hepatocytes. Toxicol. Vitr. 2007, 21, 271–276. [Google Scholar] [CrossRef]
  79. Vermot, A.; Petit-Härtlein, I.; Smith, S.M.E.; Fieschi, F. NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology. Antioxidants 2021, 10, 890. [Google Scholar] [CrossRef]
  80. Lan, T.; Kisseleva, T.; Brenner, D.A. Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation. PLoS ONE 2015, 10, e0129743. [Google Scholar] [CrossRef]
  81. Kim, S.Y.; Jeong, J.M.; Kim, S.J.; Seo, W.; Kim, M.H.; Choi, W.M.; Yoo, W.; Lee, J.H.; Shim, Y.R.; Yi, H.S.; et al. Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4-MD2 complex. Nat. Commun. 2017, 8, 2247. [Google Scholar] [CrossRef] [Green Version]
  82. Rojo de la Vega, M.; Chapman, E.; Zhang, D.D. NRF2 and the Hallmarks of Cancer. Cancer Cell 2018, 34, 21–43. [Google Scholar] [CrossRef]
  83. Meakin, P.J.; Chowdhry, S.; Sharma, R.S.; Ashford, F.B.; Walsh, S.V.; McCrimmon, R.J.; Dinkova-Kostova, A.T.; Dillon, J.F.; Hayes, J.D.; Ashford, M.L. Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance. Mol. Cell. Biol. 2014, 34, 3305–3320. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Seedorf, K.; Weber, C.; Vinson, C.; Berger, S.; Vuillard, L.M.; Kiss, A.; Creusot, S.; Broux, O.; Geant, A.; Ilic, C.; et al. Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice. JHEP Rep. 2023, 5, 100651. [Google Scholar] [CrossRef]
  85. Di Francesco, A.; Choi, Y.; Bernier, M.; Zhang, Y.; Diaz-Ruiz, A.; Aon, M.A.; Kalafut, K.; Ehrlich, M.R.; Murt, K.; Ali, A.; et al. NQO1 protects obese mice through improvements in glucose and lipid metabolism. NPJ Aging Mech. Dis. 2020, 6, 13. [Google Scholar] [CrossRef]
  86. Yu, J.; Chu, E.S.; Wang, R.; Wang, S.; Wu, C.W.; Wong, V.W.; Chan, H.L.; Farrell, G.C.; Sung, J.J. Heme oxygenase-1 protects against steatohepatitis in both cultured hepatocytes and mice. Gastroenterology 2010, 138, 694–704, 704.e691. [Google Scholar] [CrossRef]
  87. Lan, T.; Hu, Y.; Hu, F.; Li, H.; Chen, Y.; Zhang, J.; Yu, Y.; Jiang, S.; Weng, Q.; Tian, S.; et al. Hepatocyte glutathione S-transferase mu 2 prevents non-alcoholic steatohepatitis by suppressing ASK1 signaling. J. Hepatol. 2022, 76, 407–419. [Google Scholar] [CrossRef] [PubMed]
  88. Wang, L.; Jiang, Z.; Lei, X.G. Knockout of SOD1 alters murine hepatic glycolysis, gluconeogenesis, and lipogenesis. Free Radic. Biol. Med. 2012, 53, 1689–1696. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  89. Elchuri, S.; Oberley, T.D.; Qi, W.; Eisenstein, R.S.; Jackson Roberts, L.; Van Remmen, H.; Epstein, C.J.; Huang, T.T. CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life. Oncogene 2005, 24, 367–380. [Google Scholar] [CrossRef] [Green Version]
  90. Cho, H.Y.; Gladwell, W.; Wang, X.; Chorley, B.; Bell, D.; Reddy, S.P.; Kleeberger, S.R. Nrf2-regulated PPAR{gamma} expression is critical to protection against acute lung injury in mice. Am. J. Respir. Crit. Care Med. 2010, 182, 170–182. [Google Scholar] [CrossRef] [Green Version]
  91. Huang, J.; Tabbi-Anneni, I.; Gunda, V.; Wang, L. Transcription factor Nrf2 regulates SHP and lipogenic gene expression in hepatic lipid metabolism. Am. J. Physiol. Gastrointest. Liver Physiol. 2010, 299, G1211–G1221. [Google Scholar] [CrossRef] [Green Version]
  92. Leonardini, A.; Laviola, L.; Perrini, S.; Natalicchio, A.; Giorgino, F. Cross-Talk between PPARgamma and Insulin Signaling and Modulation of Insulin Sensitivity. PPAR Res. 2009, 2009, 818945. [Google Scholar] [CrossRef] [Green Version]
  93. Straus, D.S.; Glass, C.K. Anti-inflammatory actions of PPAR ligands: New insights on cellular and molecular mechanisms. Trends Immunol. 2007, 28, 551–558. [Google Scholar] [CrossRef]
  94. Cordoba-Chacon, J. Loss of Hepatocyte-Specific PPAR. PPAR Res. 2020, 2020, 9735083. [Google Scholar] [CrossRef]
  95. Lee, S.M.; Pusec, C.M.; Norris, G.H.; De Jesus, A.; Diaz-Ruiz, A.; Muratalla, J.; Sarmento-Cabral, A.; Guzman, G.; Layden, B.T.; Cordoba-Chacon, J. Hepatocyte-Specific Loss of PPARγ Protects Mice From NASH and Increases the Therapeutic Effects of Rosiglitazone in the Liver. Cell. Mol. Gastroenterol. Hepatol. 2021, 11, 1291–1311. [Google Scholar] [CrossRef] [PubMed]
  96. Pettinelli, P.; Videla, L.A. Up-regulation of PPAR-gamma mRNA expression in the liver of obese patients: An additional reinforcing lipogenic mechanism to SREBP-1c induction. J. Clin. Endocrinol. Metab. 2011, 96, 1424–1430. [Google Scholar] [CrossRef]
  97. Xie, Z.Q.; Li, H.X.; Tan, W.L.; Yang, L.; Ma, X.W.; Li, W.X.; Wang, Q.B.; Shang, C.Z.; Chen, Y.J. Association of Serum Vitamin C With NAFLD and MAFLD Among Adults in the United States. Front. Nutr. 2021, 8, 795391. [Google Scholar] [CrossRef] [PubMed]
  98. Erhardt, A.; Stahl, W.; Sies, H.; Lirussi, F.; Donner, A.; Häussinger, D. Plasma levels of vitamin E and carotenoids are decreased in patients with Nonalcoholic Steatohepatitis (NASH). Eur. J. Med. Res. 2011, 16, 76–78. [Google Scholar] [CrossRef] [PubMed]
  99. Gao, D.; Wei, C.; Chen, L.; Huang, J.; Yang, S.; Diehl, A.M. Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 2004, 287, G1070–G1077. [Google Scholar] [CrossRef] [PubMed]
  100. Carmiel-Haggai, M.; Cederbaum, A.I.; Nieto, N. A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats. FASEB J. 2005, 19, 136–138. [Google Scholar] [CrossRef]
  101. Geng, Y.; Faber, K.N.; de Meijer, V.E.; Blokzijl, H.; Moshage, H. How does hepatic lipid accumulation lead to lipotoxicity in non-alcoholic fatty liver disease? Hepatol. Int. 2021, 15, 21–35. [Google Scholar] [CrossRef]
  102. Kammoun, H.L.; Chabanon, H.; Hainault, I.; Luquet, S.; Magnan, C.; Koike, T.; Ferré, P.; Foufelle, F. GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice. J. Clin. Investig. 2009, 119, 1201–1215. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  103. Li, H.; Meng, Q.; Xiao, F.; Chen, S.; Du, Y.; Yu, J.; Wang, C.; Guo, F. ATF4 deficiency protects mice from high-carbohydrate-diet-induced liver steatosis. Biochem. J. 2011, 438, 283–289. [Google Scholar] [CrossRef] [PubMed]
  104. Yeh, K.Y.; Lai, C.Y.; Lin, C.Y.; Hsu, C.C.; Lo, C.P.; Her, G.M. ATF4 overexpression induces early onset of hyperlipidaemia and hepatic steatosis and enhances adipogenesis in zebrafish. Sci. Rep. 2017, 7, 16362. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  105. Lee, A.H.; Iwakoshi, N.N.; Glimcher, L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol. Cell. Biol. 2003, 23, 7448–7459. [Google Scholar] [CrossRef] [Green Version]
  106. Yamamoto, K.; Suzuki, N.; Wada, T.; Okada, T.; Yoshida, H.; Kaufman, R.J.; Mori, K. Human HRD1 promoter carries a functional unfolded protein response element to which XBP1 but not ATF6 directly binds. J. Biochem. 2008, 144, 477–486. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  107. Zhang, K.; Wang, S.; Malhotra, J.; Hassler, J.R.; Back, S.H.; Wang, G.; Chang, L.; Xu, W.; Miao, H.; Leonardi, R.; et al. The unfolded protein response transducer IRE1α prevents ER stress-induced hepatic steatosis. EMBO J. 2011, 30, 1357–1375. [Google Scholar] [CrossRef] [Green Version]
  108. Lee, A.H.; Scapa, E.F.; Cohen, D.E.; Glimcher, L.H. Regulation of hepatic lipogenesis by the transcription factor XBP1. Science 2008, 320, 1492–1496. [Google Scholar] [CrossRef] [Green Version]
  109. Wang, J.M.; Qiu, Y.; Yang, Z.; Kim, H.; Qian, Q.; Sun, Q.; Zhang, C.; Yin, L.; Fang, D.; Back, S.H.; et al. IRE1α prevents hepatic steatosis by processing and promoting the degradation of select microRNAs. Sci. Signal. 2018, 11, eaao4617. [Google Scholar] [CrossRef] [Green Version]
  110. Urano, F.; Wang, X.; Bertolotti, A.; Zhang, Y.; Chung, P.; Harding, H.P.; Ron, D. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 2000, 287, 664–666. [Google Scholar] [CrossRef] [Green Version]
  111. Tam, A.B.; Mercado, E.L.; Hoffmann, A.; Niwa, M. ER stress activates NF-κB by integrating functions of basal IKK activity, IRE1 and PERK. PLoS ONE 2012, 7, e45078. [Google Scholar] [CrossRef] [Green Version]
  112. Talty, A.; Deegan, S.; Ljujic, M.; Mnich, K.; Naicker, S.D.; Quandt, D.; Zeng, Q.; Patterson, J.B.; Gorman, A.M.; Griffin, M.D.; et al. Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1β. Cell Death Dis. 2019, 10, 622. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  113. Szegezdi, E.; Logue, S.E.; Gorman, A.M.; Samali, A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006, 7, 880–885. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  114. Sandow, J.J.; Dorstyn, L.; O’Reilly, L.A.; Tailler, M.; Kumar, S.; Strasser, A.; Ekert, P.G. ER stress does not cause upregulation and activation of caspase-2 to initiate apoptosis. Cell Death Differ. 2014, 21, 475–480. [Google Scholar] [CrossRef] [Green Version]
  115. Kim, J.Y.; Garcia-Carbonell, R.; Yamachika, S.; Zhao, P.; Dhar, D.; Loomba, R.; Kaufman, R.J.; Saltiel, A.R.; Karin, M. ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P. Cell 2018, 175, 133–145.e15. [Google Scholar] [CrossRef] [Green Version]
  116. Zhang, C.; Chen, X.; Zhu, R.M.; Zhang, Y.; Yu, T.; Wang, H.; Zhao, H.; Zhao, M.; Ji, Y.L.; Chen, Y.H.; et al. Endoplasmic reticulum stress is involved in hepatic SREBP-1c activation and lipid accumulation in fructose-fed mice. Toxicol. Lett. 2012, 212, 229–240. [Google Scholar] [CrossRef] [PubMed]
  117. Deng, X.; Pan, X.; Cheng, C.; Liu, B.; Zhang, H.; Zhang, Y.; Xu, K. Regulation of SREBP-2 intracellular trafficking improves impaired autophagic flux and alleviates endoplasmic reticulum stress in NAFLD. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2017, 1862, 337–350. [Google Scholar] [CrossRef]
  118. Kim, K.Y.; Jang, H.J.; Yang, Y.R.; Park, K.I.; Seo, J.; Shin, I.W.; Jeon, T.I.; Ahn, S.C.; Suh, P.G.; Osborne, T.F.; et al. SREBP-2/PNPLA8 axis improves non-alcoholic fatty liver disease through activation of autophagy. Sci. Rep. 2016, 6, 35732. [Google Scholar] [CrossRef] [Green Version]
  119. Weglarz, T.C.; Degen, J.L.; Sandgren, E.P. Hepatocyte transplantation into diseased mouse liver. Kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes. Am. J. Pathol. 2000, 157, 1963–1974. [Google Scholar] [CrossRef] [PubMed]
  120. Febbraio, M.A.; Reibe, S.; Shalapour, S.; Ooi, G.J.; Watt, M.J.; Karin, M. Preclinical Models for Studying NASH-Driven HCC: How Useful Are They? Cell Metab. 2019, 29, 18–26. [Google Scholar] [CrossRef] [Green Version]
  121. Chalasani, N.; Younossi, Z.; Lavine, J.E.; Charlton, M.; Cusi, K.; Rinella, M.; Harrison, S.A.; Brunt, E.M.; Sanyal, A.J. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018, 67, 328–357. [Google Scholar] [CrossRef] [Green Version]
  122. European Association for the Study of the Liver (EASL); European Association for the Study of Diabetes (EASD); European Association for the Study of Obesity (EASO). EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J. Hepatol. 2016, 64, 1388–1402. [Google Scholar] [CrossRef] [PubMed]
  123. Grundy, S.M.; Cleeman, J.I.; Daniels, S.R.; Donato, K.A.; Eckel, R.H.; Franklin, B.A.; Gordon, D.J.; Krauss, R.M.; Savage, P.J.; Smith, S.C.; et al. Diagnosis and management of the metabolic syndrome: An American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement. Circulation 2005, 112, 2735–2752. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  124. Hansen, C.D.; Gram-Kampmann, E.M.; Hansen, J.K.; Hugger, M.B.; Madsen, B.S.; Jensen, J.M.; Olesen, S.; Torp, N.; Rasmussen, D.N.; Kjærgaard, M.; et al. Effect of Calorie-Unrestricted Low-Carbohydrate, High-Fat Diet Versus High-Carbohydrate, Low-Fat Diet on Type 2 Diabetes and Nonalcoholic Fatty Liver Disease: A Randomized Controlled Trial. Ann. Intern. Med. 2023, 176, 10–21. [Google Scholar] [CrossRef]
  125. Properzi, C.; O’Sullivan, T.A.; Sherriff, J.L.; Ching, H.L.; Jeffrey, G.P.; Buckley, R.F.; Tibballs, J.; MacQuillan, G.C.; Garas, G.; Adams, L.A. Ad Libitum Mediterranean and Low-Fat Diets Both Significantly Reduce Hepatic Steatosis: A Randomized Controlled Trial. Hepatology 2018, 68, 1741–1754. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  126. Monserrat-Mesquida, M.; Quetglas-Llabrés, M.; Bouzas, C.; Montemayor, S.; Mascaró, C.M.; Casares, M.; Llompart, I.; Ugarriza, L.; Martínez, J.A.; Tur, J.A.; et al. Increased Adherence to the Mediterranean Diet after Lifestyle Intervention Improves Oxidative and Inflammatory Status in Patients with Non-Alcoholic Fatty Liver Disease. Antioxidants 2022, 11, 1440. [Google Scholar] [CrossRef]
  127. Ezpeleta, M.; Gabel, K.; Cienfuegos, S.; Kalam, F.; Lin, S.; Pavlou, V.; Song, Z.; Haus, J.M.; Koppe, S.; Alexandria, S.J.; et al. Effect of alternate day fasting combined with aerobic exercise on non-alcoholic fatty liver disease: A randomized controlled trial. Cell Metab. 2023, 35, 56–70.e3. [Google Scholar] [CrossRef]
  128. Wei, X.; Lin, B.; Huang, Y.; Yang, S.; Huang, C.; Shi, L.; Liu, D.; Zhang, P.; Lin, J.; Xu, B.; et al. Effects of Time-Restricted Eating on Nonalcoholic Fatty Liver Disease: The TREATY-FLD Randomized Clinical Trial. JAMA Netw. Open 2023, 6, e233513. [Google Scholar] [CrossRef]
  129. Babu, A.F.; Csader, S.; Lok, J.; Gómez-Gallego, C.; Hanhineva, K.; El-Nezami, H.; Schwab, U. Positive Effects of Exercise Intervention without Weight Loss and Dietary Changes in NAFLD-Related Clinical Parameters: A Systematic Review and Meta-Analysis. Nutrients 2021, 13, 3135. [Google Scholar] [CrossRef]
  130. Glass, O.; Liu, D.; Bechard, E.; Guy, C.D.; Pendergast, J.; Mae Diehl, A.; Abdelmalek, M.F. Perceptions of Exercise and Its Challenges in Patients With Nonalcoholic Fatty Liver Disease: A Survey-Based Study. Hepatol. Commun. 2022, 6, 334–344. [Google Scholar] [CrossRef]
  131. Hashida, R.; Kawaguchi, T.; Bekki, M.; Omoto, M.; Matsuse, H.; Nago, T.; Takano, Y.; Ueno, T.; Koga, H.; George, J.; et al. Aerobic vs. resistance exercise in non-alcoholic fatty liver disease: A systematic review. J. Hepatol. 2017, 66, 142–152. [Google Scholar] [CrossRef]
  132. Lehmann, J.M.; Moore, L.B.; Smith-Oliver, T.A.; Wilkison, W.O.; Willson, T.M.; Kliewer, S.A. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J. Biol. Chem. 1995, 270, 12953–12956. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  133. Delea, T.E.; Edelsberg, J.S.; Hagiwara, M.; Oster, G.; Phillips, L.S. Use of thiazolidinediones and risk of heart failure in people with type 2 diabetes: A retrospective cohort study. Diabetes Care 2003, 26, 2983–2989. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  134. Lago, R.M.; Singh, P.P.; Nesto, R.W. Congestive heart failure and cardiovascular death in patients with prediabetes and type 2 diabetes given thiazolidinediones: A meta-analysis of randomised clinical trials. Lancet 2007, 370, 1129–1136. [Google Scholar] [CrossRef] [PubMed]
  135. Berlie, H.D.; Kalus, J.S.; Jaber, L.A. Thiazolidinediones and the risk of edema: A meta-analysis. Diabetes Res. Clin. Pract. 2007, 76, 279–289. [Google Scholar] [CrossRef]
  136. Werner, C.M.; Schirmer, S.H.; Gensch, C.; Pavlickova, V.; Pöss, J.; Wright, M.B.; Böhm, M.; Laufs, U. The dual PPARα/γ agonist aleglitazar increases the number and function of endothelial progenitor cells: Implications for vascular function and atherogenesis. Br. J. Pharmacol. 2014, 171, 2685–2703. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  137. Grobbee, E.J.; de Jong, V.D.; Schrieks, I.C.; Tushuizen, M.E.; Holleboom, A.G.; Tardif, J.C.; Lincoff, A.M.; Schwartz, G.G.; Castro Cabezas, M.; Grobbee, D.E. Improvement of non-invasive tests of liver steatosis and fibrosis as indicators for non-alcoholic fatty liver disease in type 2 diabetes mellitus patients with elevated cardiovascular risk profile using the PPAR-α/γ agonist aleglitazar. PLoS ONE 2022, 17, e0277706. [Google Scholar] [CrossRef]
  138. Jain, M.R.; Giri, S.R.; Bhoi, B.; Trivedi, C.; Rath, A.; Rathod, R.; Ranvir, R.; Kadam, S.; Patel, H.; Swain, P.; et al. Dual PPARα/γ agonist saroglitazar improves liver histopathology and biochemistry in experimental NASH models. Liver Int. 2018, 38, 1084–1094. [Google Scholar] [CrossRef] [Green Version]
  139. Gawrieh, S.; Noureddin, M.; Loo, N.; Mohseni, R.; Awasty, V.; Cusi, K.; Kowdley, K.V.; Lai, M.; Schiff, E.; Parmar, D.; et al. Saroglitazar, a PPAR-α/γ Agonist, for Treatment of NAFLD: A Randomized Controlled Double-Blind Phase 2 Trial. Hepatology 2021, 74, 1809–1824. [Google Scholar] [CrossRef]
  140. Tong, L.; Wang, L.; Yao, S.; Jin, L.; Yang, J.; Zhang, Y.; Ning, G.; Zhang, Z. PPARδ attenuates hepatic steatosis through autophagy-mediated fatty acid oxidation. Cell Death Dis. 2019, 10, 197. [Google Scholar] [CrossRef] [Green Version]
  141. Lefere, S.; Puengel, T.; Hundertmark, J.; Penners, C.; Frank, A.K.; Guillot, A.; de Muynck, K.; Heymann, F.; Adarbes, V.; Defrêne, E.; et al. Differential effects of selective- and pan-PPAR agonists on experimental steatohepatitis and hepatic macrophages. J. Hepatol. 2020, 73, 757–770. [Google Scholar] [CrossRef]
  142. Francque, S.M.; Bedossa, P.; Ratziu, V.; Anstee, Q.M.; Bugianesi, E.; Sanyal, A.J.; Loomba, R.; Harrison, S.A.; Balabanska, R.; Mateva, L.; et al. A Randomized, Controlled Trial of the Pan-PPAR Agonist Lanifibranor in NASH. N. Engl. J. Med. 2021, 385, 1547–1558. [Google Scholar] [CrossRef] [PubMed]
  143. Scheen, A.J. Pharmacodynamics, efficacy and safety of sodium-glucose co-transporter type 2 (SGLT2) inhibitors for the treatment of type 2 diabetes mellitus. Drugs 2015, 75, 33–59. [Google Scholar] [CrossRef] [PubMed]
  144. Janež, A.; Fioretto, P. SGLT2 Inhibitors and the Clinical Implications of Associated Weight Loss in Type 2 Diabetes: A Narrative Review. Diabetes Ther. 2021, 12, 2249–2261. [Google Scholar] [CrossRef]
  145. Phrueksotsai, S.; Pinyopornpanish, K.; Euathrongchit, J.; Leerapun, A.; Phrommintikul, A.; Buranapin, S.; Chattipakorn, N.; Thongsawat, S. The effects of dapagliflozin on hepatic and visceral fat in type 2 diabetes patients with non-alcoholic fatty liver disease. J. Gastroenterol. Hepatol. 2021, 36, 2952–2959. [Google Scholar] [CrossRef] [PubMed]
  146. Takahashi, H.; Kessoku, T.; Kawanaka, M.; Nonaka, M.; Hyogo, H.; Fujii, H.; Nakajima, T.; Imajo, K.; Tanaka, K.; Kubotsu, Y.; et al. Ipragliflozin Improves the Hepatic Outcomes of Patients With Diabetes with NAFLD. Hepatol. Commun. 2022, 6, 120–132. [Google Scholar] [CrossRef]
  147. Suzuki, M.; Takeda, M.; Kito, A.; Fukazawa, M.; Yata, T.; Yamamoto, M.; Nagata, T.; Fukuzawa, T.; Yamane, M.; Honda, K.; et al. Tofogliflozin, a sodium/glucose cotransporter 2 inhibitor, attenuates body weight gain and fat accumulation in diabetic and obese animal models. Nutr. Diabetes 2014, 4, e125. [Google Scholar] [CrossRef] [Green Version]
  148. Yoneda, M.; Kobayashi, T.; Honda, Y.; Ogawa, Y.; Kessoku, T.; Imajo, K.; Nogami, A.; Taguri, M.; Kirikoshi, H.; Saito, S.; et al. Combination of tofogliflozin and pioglitazone for NAFLD: Extension to the ToPiND randomized controlled trial. Hepatol. Commun. 2022, 6, 2273–2285. [Google Scholar] [CrossRef]
  149. Armstrong, M.J.; Gaunt, P.; Aithal, G.P.; Barton, D.; Hull, D.; Parker, R.; Hazlehurst, J.M.; Guo, K.; Abouda, G.; Aldersley, M.A.; et al. Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): A multicentre, double-blind, randomised, placebo-controlled phase 2 study. Lancet 2016, 387, 679–690. [Google Scholar] [CrossRef] [Green Version]
  150. Petit, J.M.; Cercueil, J.P.; Loffroy, R.; Denimal, D.; Bouillet, B.; Fourmont, C.; Chevallier, O.; Duvillard, L.; Vergès, B. Effect of Liraglutide Therapy on Liver Fat Content in Patients With Inadequately Controlled Type 2 Diabetes: The Lira-NAFLD Study. J. Clin. Endocrinol. Metab. 2017, 102, 407–415. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  151. Kalogirou, M.S.; Patoulias, D.; Haidich, A.B.; Akriviadis, E.; Sinakos, E. Liraglutide in patients with non-alcoholic fatty liver disease: A systematic review and meta-analysis of randomized controlled trials. Clin. Res. Hepatol. Gastroenterol. 2021, 45, 101568. [Google Scholar] [CrossRef] [PubMed]
  152. Capehorn, M.S.; Catarig, A.M.; Furberg, J.K.; Janez, A.; Price, H.C.; Tadayon, S.; Vergès, B.; Marre, M. Efficacy and safety of once-weekly semaglutide 1.0mg vs once-daily liraglutide 1.2mg as add-on to 1-3 oral antidiabetic drugs in subjects with type 2 diabetes (SUSTAIN 10). Diabetes Metab. 2020, 46, 100–109. [Google Scholar] [CrossRef]
  153. Newsome, P.N.; Buchholtz, K.; Cusi, K.; Linder, M.; Okanoue, T.; Ratziu, V.; Sanyal, A.J.; Sejling, A.S.; Harrison, S.A.; the NN9931-4296 Investigator. A Placebo-Controlled Trial of Subcutaneous Semaglutide in Nonalcoholic Steatohepatitis. N. Engl. J. Med. 2021, 384, 1113–1124. [Google Scholar] [CrossRef]
  154. Kim, S.W.; Kang, H.J.; Jhon, M.; Kim, J.W.; Lee, J.Y.; Walker, A.J.; Agustini, B.; Kim, J.M.; Berk, M. Statins and Inflammation: New Therapeutic Opportunities in Psychiatry. Front. Psychiatry 2019, 10, 103. [Google Scholar] [CrossRef] [Green Version]
  155. Cho, Y.; Rhee, H.; Kim, Y.E.; Lee, M.; Lee, B.W.; Kang, E.S.; Cha, B.S.; Choi, J.Y.; Lee, Y.H. Ezetimibe combination therapy with statin for non-alcoholic fatty liver disease: An open-label randomized controlled trial (ESSENTIAL study). BMC Med. 2022, 20, 93. [Google Scholar] [CrossRef]
  156. Panzitt, K.; Wagner, M. FXR in liver physiology: Multiple faces to regulate liver metabolism. Biochim. Biophys. Acta Mol. Basis Dis. 2021, 1867, 166133. [Google Scholar] [CrossRef]
  157. Harrison, S.A.; Bashir, M.R.; Lee, K.J.; Shim-Lopez, J.; Lee, J.; Wagner, B.; Smith, N.D.; Chen, H.C.; Lawitz, E.J. A structurally optimized FXR agonist, MET409, reduced liver fat content over 12 weeks in patients with non-alcoholic steatohepatitis. J. Hepatol. 2021, 75, 25–33. [Google Scholar] [CrossRef] [PubMed]
  158. Sanyal, A.J.; Lopez, P.; Lawitz, E.J.; Lucas, K.J.; Loeffler, J.; Kim, W.; Goh, G.B.B.; Huang, J.F.; Serra, C.; Andreone, P.; et al. Tropifexor for nonalcoholic steatohepatitis: An adaptive, randomized, placebo-controlled phase 2a/b trial. Nat. Med. 2023, 29, 392–400. [Google Scholar] [CrossRef]
  159. Ratziu, V.; Harrison, S.A.; Loustaud-Ratti, V.; Bureau, C.; Lawitz, E.; Abdelmalek, M.; Alkhouri, N.; Francque, S.; Girma, H.; Darteil, R.; et al. Hepatic and renal improvements with FXR agonist vonafexor in individuals with suspected fibrotic NASH. J. Hepatol. 2023, 78, 479–492. [Google Scholar] [CrossRef] [PubMed]
  160. Patel, K.; Harrison, S.A.; Elkhashab, M.; Trotter, J.F.; Herring, R.; Rojter, S.E.; Kayali, Z.; Wong, V.W.; Greenbloom, S.; Jayakumar, S.; et al. Cilofexor, a Nonsteroidal FXR Agonist, in Patients With Noncirrhotic NASH: A Phase 2 Randomized Controlled Trial. Hepatology 2020, 72, 58–71. [Google Scholar] [CrossRef]
  161. Younossi, Z.M.; Ratziu, V.; Loomba, R.; Rinella, M.; Anstee, Q.M.; Goodman, Z.; Bedossa, P.; Geier, A.; Beckebaum, S.; Newsome, P.N.; et al. Obeticholic acid for the treatment of non-alcoholic steatohepatitis: Interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial. Lancet 2019, 394, 2184–2196. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  162. Xu, J.; Lloyd, D.J.; Hale, C.; Stanislaus, S.; Chen, M.; Sivits, G.; Vonderfecht, S.; Hecht, R.; Li, Y.S.; Lindberg, R.A.; et al. Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 2009, 58, 250–259. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  163. Harrison, S.A.; Ruane, P.J.; Freilich, B.L.; Neff, G.; Patil, R.; Behling, C.A.; Hu, C.; Fong, E.; de Temple, B.; Tillman, E.J.; et al. Efruxifermin in non-alcoholic steatohepatitis: A randomized, double-blind, placebo-controlled, phase 2a trial. Nat. Med. 2021, 27, 1262–1271. [Google Scholar] [CrossRef]
  164. Rader, D.J.; Maratos-Flier, E.; Nguyen, A.; Hom, D.; Ferriere, M.; Li, Y.; Kompa, J.; Martic, M.; Hinder, M.; Basson, C.T.; et al. LLF580, an FGF21 Analog, Reduces Triglycerides and Hepatic Fat in Obese Adults With Modest Hypertriglyceridemia. J. Clin. Endocrinol. Metab. 2022, 107, e57–e70. [Google Scholar] [CrossRef] [PubMed]
  165. Loomba, R.; Lawitz, E.J.; Frias, J.P.; Ortiz-Lasanta, G.; Johansson, L.; Franey, B.B.; Morrow, L.; Rosenstock, M.; Hartsfield, C.L.; Chen, C.Y.; et al. Safety, pharmacokinetics, and pharmacodynamics of pegozafermin in patients with non-alcoholic steatohepatitis: A randomised, double-blind, placebo-controlled, phase 1b/2a multiple-ascending-dose study. Lancet Gastroenterol. Hepatol. 2023, 8, 120–132. [Google Scholar] [CrossRef] [PubMed]
  166. Sinha, R.A.; Singh, B.K.; Yen, P.M. Direct effects of thyroid hormones on hepatic lipid metabolism. Nat. Rev. Endocrinol. 2018, 14, 259–269. [Google Scholar] [CrossRef]
  167. Gluvic, Z.; Obradovic, M.; Stewart, A.J.; Essack, M.; Pitt, S.J.; Samardzic, V.; Soskic, S.; Gojobori, T.; Isenovic, E.R. Levothyroxine Treatment and the Risk of Cardiac Arrhythmias—Focus on the Patient Submitted to Thyroid Surgery. Front. Endocrinol. 2021, 12, 758043. [Google Scholar] [CrossRef]
  168. Ribeiro, M.O. Effects of thyroid hormone analogs on lipid metabolism and thermogenesis. Thyroid 2008, 18, 197–203. [Google Scholar] [CrossRef]
  169. Saponaro, F.; Sestito, S.; Runfola, M.; Rapposelli, S.; Chiellini, G. Selective Thyroid Hormone Receptor-Beta (TRβ) Agonists: New Perspectives for the Treatment of Metabolic and Neurodegenerative Disorders. Front. Med. 2020, 7, 331. [Google Scholar] [CrossRef]
  170. Harrison, S.A.; Bashir, M.R.; Guy, C.D.; Zhou, R.; Moylan, C.A.; Frias, J.P.; Alkhouri, N.; Bansal, M.B.; Baum, S.; Neuschwander-Tetri, B.A.; et al. Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: A multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 2019, 394, 2012–2024. [Google Scholar] [CrossRef]
  171. González-Espinoza, L.; Rojas-Campos, E.; Medina-Pérez, M.; Peña-Quintero, P.; Gómez-Navarro, B.; Cueto-Manzano, A.M. Pentoxifylline decreases serum levels of tumor necrosis factor alpha, interleukin 6 and C-reactive protein in hemodialysis patients: Results of a randomized double-blind, controlled clinical trial. Nephrol. Dial. Transplant. 2012, 27, 2023–2028. [Google Scholar] [CrossRef] [Green Version]
  172. Koppe, S.W.; Sahai, A.; Malladi, P.; Whitington, P.F.; Green, R.M. Pentoxifylline attenuates steatohepatitis induced by the methionine choline deficient diet. J. Hepatol. 2004, 41, 592–598. [Google Scholar] [CrossRef] [PubMed]
  173. Zeng, T.; Zhang, C.L.; Zhao, X.L.; Xie, K.Q. Pentoxifylline for the treatment of nonalcoholic fatty liver disease: A meta-analysis of randomized double-blind, placebo-controlled studies. Eur. J. Gastroenterol. Hepatol. 2014, 26, 646–653. [Google Scholar] [CrossRef]
  174. Perumpail, B.J.; Li, A.A.; John, N.; Sallam, S.; Shah, N.D.; Kwong, W.; Cholankeril, G.; Kim, D.; Ahmed, A. The Role of Vitamin E in the Treatment of NAFLD. Diseases 2018, 6, 86. [Google Scholar] [CrossRef] [Green Version]
  175. Nan, Y.M.; Wu, W.J.; Fu, N.; Liang, B.L.; Wang, R.Q.; Li, L.X.; Zhao, S.X.; Zhao, J.M.; Yu, J. Antioxidants vitamin E and 1-aminobenzotriazole prevent experimental non-alcoholic steatohepatitis in mice. Scand. J. Gastroenterol. 2009, 44, 1121–1131. [Google Scholar] [CrossRef]
  176. Sanyal, A.J.; Chalasani, N.; Kowdley, K.V.; McCullough, A.; Diehl, A.M.; Bass, N.M.; Neuschwander-Tetri, B.A.; Lavine, J.E.; Tonascia, J.; Unalp, A.; et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N. Engl. J. Med. 2010, 362, 1675–1685. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  177. Kramer, J.A.; Grindley, J.; Crowell, A.M.; Makaron, L.; Kohli, R.; Kirby, M.; Mansfield, K.G.; Wachtman, L.M. The common marmoset as a model for the study of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Vet. Pathol. 2015, 52, 404–413. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  178. Zheng, W.; Liu, Y.; Shang, H.; Zhang, Y.; Ma, D.; Hou, N.; Wang, J.; Sun, X.; Peng, Y.; Pan, L.; et al. Characterization of spontaneously-developed non-alcoholic fatty liver disease in aged rhesus monkeys. Diabetol. Metab. Syndr. 2018, 10, 68. [Google Scholar] [CrossRef]
Ijms 24 09996 g001 550
Figure 1. Pathogenesis of NAFLD. Insulin resistance stimulates the breakdown of adipose triglycerides into free fatty acids (FFA) that enter the liver and accumulate there as triglycerides. Excess dietary sugars are converted into additional triglycerides via de novo lipogenesis. Pro-inflammatory cytokines from the adipose tissue and lipopolysaccharides (LPS) from the gastrointestinal tract contribute directly or indirectly to hepatic inflammation. Cholesterol accumulates in the liver due to impaired very-low-density lipoprotein (VLDL) secretion and bile formation, as well as increased de novo biosynthesis of cholesterol. These cellular insults trigger the mitochondria to produce toxic levels of reactive oxygen species (ROS). Additionally, cellular insults activate endoplasmic reticulum (ER) stress and the unfolded protein response. Together, these pathways upregulate pro-inflammatory cytokine expression (IFNγ, TNFα, and IL-6), apoptotic mediators (CHOP and JNK), and immune-response mediators (NFκB and NLRP3). Ultimately, this results in hepatic inflammation and cell death, leading to NASH and fibrosis. Image created with Biorender.com.
Figure 1. Pathogenesis of NAFLD. Insulin resistance stimulates the breakdown of adipose triglycerides into free fatty acids (FFA) that enter the liver and accumulate there as triglycerides. Excess dietary sugars are converted into additional triglycerides via de novo lipogenesis. Pro-inflammatory cytokines from the adipose tissue and lipopolysaccharides (LPS) from the gastrointestinal tract contribute directly or indirectly to hepatic inflammation. Cholesterol accumulates in the liver due to impaired very-low-density lipoprotein (VLDL) secretion and bile formation, as well as increased de novo biosynthesis of cholesterol. These cellular insults trigger the mitochondria to produce toxic levels of reactive oxygen species (ROS). Additionally, cellular insults activate endoplasmic reticulum (ER) stress and the unfolded protein response. Together, these pathways upregulate pro-inflammatory cytokine expression (IFNγ, TNFα, and IL-6), apoptotic mediators (CHOP and JNK), and immune-response mediators (NFκB and NLRP3). Ultimately, this results in hepatic inflammation and cell death, leading to NASH and fibrosis. Image created with Biorender.com.
Ijms 24 09996 g001
Table
Table 1. Animal models of nonalcoholic fatty liver disease (NAFLD).
Table 1. Animal models of nonalcoholic fatty liver disease (NAFLD).
BackgroundModelDescriptionPhenotypeLimitations
Genetic models of obesity and insulin resistance
Mouseob/ob
[9,10]		Spontaneous mutation in leptin gene (ob)Obesity
Insulin resistance
Steatosis		Leptin mutations rare in humans, require second stimulus (high-fat diet) to progress beyond steatosis
Mousedb/db
[11] 		Spontaneous mutation in leptin receptor gene (db)Obesity
Insulin resistance
Steatosis		Leptin receptor mutations rare in humans, require second stimulus (high-fat diet) to progress beyond steatosis
Rat
(Zucker)		fa/fa
[12]		Spontaneous mutation in leptin receptor gene (fa)Obesity
Insulin resistance
Steatosis		Leptin mutations rare in humans, require second stimulus (high-fat diet) to progress beyond steatosis
Mouse
(NOD)		foz/foz
[13]		Mutation in Alms1 geneObesity
Insulin resistance
High cholesterol
Steatosis		Requires second stimulus (high-fat diet) to develop NASH, fibrosis
Mouse
(B6SJLF1/J)		aP2-nSREBP-1c transgenic [4]Overexpression of SREBP-1c in adipose tissueInsulin resistance
Steatosis
Lipodystrophy		No evidence of fibrosis, liver tumors
Mouse
(C57BL/6J)		Ldlr−/−
[14,15]		Targeted mutation in Ldlr gene encoding low-density lipoprotein receptor + high-fat/high-cholesterol diet (3 months)Steatosis
Liver inflammation
Fibrosis
High cholesterol		Mutation does not induce insulin resistance
Mouse
(C57BL/6J)		Apoe−/−
[16]		Targeted mutation in Apoe gene encoding apolipoprotein E + high-fat/high-cholesterol diet (7 weeks)Mild obesity
Insulin resistance
Steatosis
NASH
Fibrosis		Less pronounced obesity
ZebrafishDucttrip (dtp)
[17] 		Mutation in ahcy gene encoding S-adenosylhomocysteine hydrolaseIncreased pro-inflammatory cytokines
Steatosis		Recessive lethal, does not induce NASH, fibrosis, or liver tumors
Mouse
(C57BL/6J)		Mat1a−/−
[18]		Mutation in Mat1a gene encoding methionine adenosyltransferase 1ASteatosis
Liver inflammation		No evidence of metabolic syndrome (MetS) or fibrosis
Dietary models of obesity and insulin resistance
Mouse
(C57BL/6 or ob/ob)		Amylin liver NASH (AMLN) [19]High-fat/high-fructose diet (40% fat, 22% fructose, 2% cholesterol; 8 weeks)Obesity
Steatosis
NASH
Fibrosis		No evidence of liver tumors
Mouse
(C57BL/6)		Choline-deficient + high-fat diet
[20]		Choline-deficient/high-fat diet (45% fat; 8 weeks)Obesity
Steatosis		Choline-deficient diet improves high-fat diet-induced insulin sensitivity, no evidence of NASH or more advanced stages
Mouse
(C57BL/6)		Methionine–choline-deficient (MCD) diet
[21]		Standard chow diet deficient in methionine and choline (15 days)Weight loss
Steatosis
NASH
Fibrosis		Some phenotypes are the opposite of humans with NAFLD (i.e., model induces weight loss, insulin sensitivity)
Rat (Sprague–Dawley)NASH diet [22]High-fat/high-fructose/high-cholesterol diet (40% fat, 40% carbohydrate, 2% cholesterol; 16 weeks)Obesity
Steatosis
Fibrosis		No evidence of liver tumors
Mouse (C57BL/6N)American lifestyle-induced obesity syndrome (ALIOS)
[23]		High-fat/high-carbohydrate diet (45% fat, 55% fructose/45% glucose in drinking water; 26–52 weeks)Obesity
Insulin resistance
Steatosis
NASH
Fibrosis
Liver tumors		Lengthy feeding time required to progress to fibrosis and liver tumors
Mouse (B6/129)Diet-induced animal model of NAFLD (DIAMOND)
[24]		High-fat/high-carbohydrate diet (42% fat, 0.1% cholesterol, high fructose/glucose in drinking water; 8–52 weeks)Obesity
Insulin resistance
Steatosis
NASH
Fibrosis
Cirrhosis
Liver tumors		Lengthy feeding time required to progress to cirrhosis and liver tumors
Mouse (C57BL/6)Fast-food diet
[25]		High-fat/high-carbohydrate diet (40% fat, 2% cholesterol, high fructose in drinking water; 25 weeks)Obesity
Insulin resistance
Steatosis
NASH
Fibrosis		Liver tumors not reported in this model
Mouse (C57BL/6)STAM™ (streptozotocin + high-fat diet)
[26]		Perinatal injection of streptozotocin followed by high-fat diet (6–20 weeks)Obesity
Insulin resistance
Steatosis
NASH
Fibrosis
Liver tumors		Only male mice developed liver tumors
Monkey
(Cynomolgus)		NASH diet
[27]		High-fat/high-carbohydrate/high-cholesterol diet (47% fat, 37% carbohydrates, 1% cholesterol; 10 months)Obesity
Hyperglycemia
Hyperlipidemia
Steatosis
NASH
Fibrosis		Studies were only performed in male monkeys
Models of oxidative stress
MousePPARγ deletion in hepatic macrophages + carbon tetrachloride
[28]		Hepatic macrophage-specific deletion of peroxisome proliferator-activated receptor γ, liver fibrosis induced by carbon tetrachlorideOxidative stress
Hepatic inflammation
Fibrosis		No evidence of MetS
Zebrafishfabp10-CETI-PIC3 + high-fat diet
[29]		Over-expression of pro-inflammatory cytokinesInsulin resistance
Oxidative stress
Steatosis		No evidence of fibrosis or liver tumors
Models of endoplasmic reticulum (ER) stress
Mouse
(C57BL/6)		MUP-uPA transgenic + high-fat diet
[30]		Transgenic mice expressing urokinase-type plasminogen activator (uPA) under a hepatocyte-specific promoter for major urinary protein (MUP) fed a high-fat diet (16–40 weeks)Obesity
ER stress
Steatosis
NASH
Fibrosis
Liver tumors		Lengthy feeding time required for disease progression
ZebrafishFoie gras (fgr)
[31,32]		Mutation in gene encoding an analog of trafficking protein particle complex 11 (TRAPPC11)ER stress
Steatosis
NASH		Recessive lethal, does not show full pathogenesis of fibrosis or liver tumors
Zebrafishhi559
[33]		Mutation in cdipt gene encoding phosphatidylinositol synthaseER stress
Steatosis		No evidence of NASH, fibrosis, or liver tumors
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Basha, A.; May, S.C.; Anderson, R.M.; Samala, N.; Mirmira, R.G. Non-Alcoholic Fatty Liver Disease: Translating Disease Mechanisms into Therapeutics Using Animal Models. Int. J. Mol. Sci. 2023, 24, 9996. https://doi.org/10.3390/ijms24129996

AMA Style

Basha A, May SC, Anderson RM, Samala N, Mirmira RG. Non-Alcoholic Fatty Liver Disease: Translating Disease Mechanisms into Therapeutics Using Animal Models. International Journal of Molecular Sciences. 2023; 24(12):9996. https://doi.org/10.3390/ijms24129996

Chicago/Turabian Style

Basha, Amina, Sarah C. May, Ryan M. Anderson, Niharika Samala, and Raghavendra G. Mirmira. 2023. "Non-Alcoholic Fatty Liver Disease: Translating Disease Mechanisms into Therapeutics Using Animal Models" International Journal of Molecular Sciences 24, no. 12: 9996. https://doi.org/10.3390/ijms24129996

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop