Eleni A. Karavia, Kyriakos E. Kypreos
Pharmacology Department, University of Patras Medical School, Rio, Achaia, Greece
Nonalcoholic fatty liver disease (NAFLD) is a spectrum of metabolic disorders ranging from a simple accumulation of excess triglycerides in the liver (hepatic steatosis) to hepatic steatosis with inflammation, fibrosis, and cirrhosis (steatohepatitis or non-alcoholic steatohepatitis (NASH)). Studies in humans and animal models suggested that alterations in hepatic lipid metabolism, increased generation of reactive oxygen species and consequently oxidative stress, changes in mitochondrial function, DNA damage, microbial infections and release of various cytokines may contribute to the pathogenesis of NAFLD and its progression to NASH. Recent data also suggest an important role of the lipoprotein transport system in hepatic lipid deposition. Currently, no drugs are approved for the treatment of NAFLD and NASH and existing pharmacotherapy aims at the management of intercurrent diseases such as obesity, hyperlipidemia, insulin resistance, and type 2 diabetes mellitus. All guidelines acknowledge that any medicines prescribed for NAFLD treatment should be considered as an off-label treatment and that their efficacy and safety should be carefully monitored. Although current pharmacotherapy may seem limited and of questionable efficacy, there is optimism that innovative safe and effective options for the management of the disease will be made available shortly since specialized drugs such as obeticholic acid, elafibranor and cenicriviroc, are presently tested in clinical trials. Given that patients with NAFLD without steatohepatitis or fibrosis have excellent prognosis if they adopt appropriate therapeutic lifestyle changes, it is generally accepted that pharmacological treatments should be limited to those with established NASH and fibrosis while subjects with early manifestations of NAFLD should resort to therapeutic lifestyle and nutritional changes.
Key words: NAFLD, lipoproteins, dyslipidemia, drugs, new therapeutic modalities
Corresponding author: Kyriakos E. Kypreos, PhD, University of Patras Medical School, Department of Pharmacology, Panepistimioupolis, 26500 Rio, Achaia, Greece, Tel.: +302610969120, Fax: +302610994720, e-mail: firstname.lastname@example.org, web: http://www.kyriakoskypreos.com
Submission: 15.05.2019, Acceptance: 14.06.2019
Nonalcoholic fatty liver disease (NAFLD) is a spectrum of metabolic disorders ranging from a simple accumulation of excess triglycerides in the liver (hepatic steatosis) to hepatic steatosis with inflammation, fibrosis, and cirrhosis (steatohepatitis or non-alcoholic steatohepatitis (NASH)).1,2 NAFLD may develop in four distinct stages which according to the British NHS are classified as: i) steatosis which is characterized by a build-up of fat in the liver cells that does not bear any metabolic consequences and is usually diagnosed circumstantially, ii) NASH which is a more serious form of NAFLD, where the hepatic deposition of fat is associated with inflammation, iii) fibrosis, a stage where persistent inflammation causes excess extracellular connective tissue build-up around the liver and nearby blood vessels, though liver may still be available to function normally, and iv) cirrhosis, the most severe and dangerous stage, occurring after years of inflammation, where the liver shrinks and becomes scarred and lumpy. The hepatic damage during this stage is irreversible and may lead to liver failure and hepatocarcinomas. Therefore, it is important that NAFLD is detected in the early stages when tissue damage is still reversible and treatable. In humans NAFLD is usually associated with obesity and oftentimes with cardiovascular disease, insulin resistance and other metabolic perturbations.1-3 Thus, it is proposed that NAFLD is the hepatic component of metabolic syndrome.4 However, accumulating body of evidence indicates that NAFLD may also be found less frequently in lean subjects5-7 possibly due to different etiological factors, mainly environmental and genetic.8
The molecular etiology of NAFLD is still not well defined and under investigation. Though aging, hormonal imbalance, and genetic predisposition may contribute to NAFLD it appears that excess caloric dietary intake and sedentary lifestyle are the most common contributors.9,10 In terms of the mechanisms, studies in humans and animal models have suggested that alterations in hepatic lipid metabolism, increased generation of reactive oxygen species and consequently oxidative stress, changes in mitochondrial function, DNA damage, microbial infections and release of various cytokines may contribute to the progression of NAFLD to NASH.10-13 As detailed below, recent data also suggest an important role of the lipoprotein transport system in hepatic lipid deposition.
THE LIPOPROTEIN TRANSPORT SYSTEM
It appears that post-prandial management of dietary lipids is an important aspect of NAFLD. Lipoproteins are involved in three major metabolic pathways in plasma: the chylomicron pathway which is responsible for the transport and distribution of dietary lipids, the Very Low Density Lipoprotein (VLDL)/Intermediate density lipoproteins (IDL)/Low Density Lipoprotein (LDL) pathway which is responsible for the transport and distribution of hepatically synthesized lipids, and the HDL pathway which is responsible for transport and redistribution of cholesterol and other lipids among tissues, including liver. Though distinct from each other, these pathways are all metabolically interrelated. Several different proteins, such as apolipoproteins, enzymes, lipid transfer proteins, lipoprotein receptors, and lipid transporters, are required for maintaining plasma lipid homeostasis via these pathways.14
Triglyceride-rich lipoproteins comprise chylomicrons, their remnants and VLDL. Chylomicrons are assembled in the intestine.14 Dietary lipids absorbed by intestinal epithelial cells are transferred onto apolipoprotein B48 (ApoB-48) via intestinal microsomal triglyceride transfer protein (MTTP) to form chylomicrons which are then secreted into circulation via the lymph. In blood, chylomicrons are hydrolyzed by lipoprotein lipase (LPL). This step converts chylomicrons to chylomicron remnants which subsequently acquire apolipoprotein E (ApoE) and are cleared rapidly by members of the LDL receptor (LDLR) superfamily. VLDL are assembled in the liver.14 Hepatically synthesized cholesterol and TG are transferred onto apolipoprotein B100 (ApoB-100) via hepatic MTTP to form nascent VLDL particles which are directly secreted into circulation. Like chylomicrons, VLDL TG are then hydrolyzed by LPL, and converted initially to IDL and then to LDL which are also cleared by members of the LDLR superfamily.
The assembly of HDL takes place exclusively in the circulation.15 HDL biogenesis occurs through a complex pathway that involves the lipid transporter ATP-binding cassette A1 (ABCA1) and the plasma enzyme Lecithin:Cholesterol Acyl Transferase (LCAT).16-18 In the early steps of HDL formation, lipid-free apolipoprotein A-I interacts with the lipid transporter ABCA1 and acquires phospholipids and cholesterol. Through a series of intermediate steps that are poorly understood, minimally lipidated apoA-I gradually forms discoidal HDL particles which are then converted into spherical particles by the action of LCAT.16 ApoA-I on both discoidal and spherical HDL particles interacts functionally with the scavenger receptor class B type I (SR-BI),19-21 also known as HDL receptor. Additional steps in the metabolism of HDL involve the transfer of cholesteryl-esters to VLDL/LDL for eventual catabolism by the LDL receptor through the plasma enzyme cholesteryl-ester transfer protein (CETP), the hydrolysis of phospholipids and residual triglycerides by the various lipases (LpL, hepatic lipase (HL), and endothelial lipase (EL)), and the transfer of phospholipids from VLDL/LDL to HDL by the action of phospholipid transfer protein (PLTP).14
LIPOPROTEINS AND NAFLD
Through a series of experiments in ApoA-I-deficient (apoa1-/-), LCAT-deficient (lcat-/-) and SR-BI-deficient (scarb1-/-) mice it was shown that in addition to the classical role of HDL in plasma cholesterol metabolism, it also plays significant role in hepatic lipid deposition.
Lack of classical ApoA-I containing HDL resulted in increased diet-induced hepatic TG deposition and disturbed hepatic histology in apoa1-/- mice compared to their wild-type counterparts, while they exhibited reduced glucose tolerance and insulin sensitivity.22 This phenotype was reversed by administration of ApoA-IMilano, a gain of function mutant of ApoA-I leading to a significant reduction of hepatic lipid deposition and body weight gain.22 In agreement with this finding, it was shown that expression of wild-type human ApoA-I in C57BL/6 mice, fed methionine and choline-deficient diet for one week, reduces significantly hepatic lipid deposition induced by the diet.23 Along the same lines, administration of varying doses of ApoA-I in New Zealand white rabbits fed a high-fat diet for 20 weeks, led to an ApoA-I dose-dependent reduction in hepatic steatosis.24
Contrasting the role of ApoA-I in diet-induced obesity and hepatic lipid deposition, Apolipoprotein A-II (ApoA-II), the second most abundant protein of HDL25 appears to rather promote the development of obesity and NAFLD.26 Interestingly, the c.-492T>C polymorphism in human ApoA-II gene was associated with increased risk for obesity27 and the T265C ApoA-II polymorphism correlated with increased visceral adipose tissue (VAT) mass.28
Similarly to ApoA-I-deficiency, LCAT-deficiency was also found to be associated with altered plasma TG metabolism in mice.29 Lcat-/- mice were prone to diet-induced obesity and hepatic lipid deposition. Administration of human LCAT by adenovirus mediated gene transfer to lcat-/- mice resulted in a significant improvement of hepatic lipid deposition.29 In contrast, deficiency in the HDL receptor SR-BI prevents hepatic lipid deposition and NAFLD development.30
Even though the relationship between NAFLD and coronary heart disease is unclear31,32 these data strongly support that blood lipoprotein transport is a key link, being as important for NAFLD development22,29,30,33 as it is for heart disease and diabetes. Obviously, the precise contribution of each lipoprotein pathway deems further investigation. Moreover, the role of the lipoprotein system in the management of dietary lipids further reinforces the paramount significance of dietary interventions in NAFLD prevention and treatment, as discussed below.
CURRENT PHARMACOLOGICAL INTERVENTIONS
Currently, no drugs have been officially approved for the treatment of NAFLD and NASH by the US Food and Drug Administration, the European Medicines Agency or other regulatory body. All guidelines for NAFLD treatment acknowledge that any medicines prescribed for the treatment of the disease (Table 1) should be considered as an off-label treatment and that patients should be explicitly informed while critical factors associated with efficacy and safety should be carefully monitored. However, guidelines issued by different medical associations disagree on the degree of benefit of each of the currently prescribed medications.34 The main target in the treatment of NAFLD is the management of intercurrent diseases such as obesity, hyperlipidemia, insulin resistance, and type 2 diabetes mellitus (T2DM).32 While specialized drugs are under development (Table 2) patients with NAFLD without steatohepatitis or any fibrosis have excellent prognosis if they adopt appropriate therapeutic lifestyle changes (TLC). Therefore, it is generally accepted by all guidelines that pharmacological treatments should be limited to those with established NASH and fibrosis while subjects with early manifestations of NAFLD should resort to TLC,35,36 as described below (Table 3).
Metformin, a first-line medicine for the treatment of T2DM, appears to have limited efficacy in improving the histological features of NAFLD.37,38 Therefore, metformin is not recommended by any guidelines to specifically treat NAFLD.39,40 In contrast, pioglitazone (thiazolidinedione), a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist with insulin-sensitizing effects has been shown to improve aminotransferases, steatosis, inflammation, and ballooning in patients with NASH and prediabetes or T2DM41 (https://clinicaltrials.gov/ct2/show/NCT00227110). In another clinical trial42 (PIVENS, Clinical trial NCT00063622, https://clinicaltrials.gov/ct2/show/NCT00063622) pioglitazone failed to show benefit over placebo for the histological features of NASH thought it improved some secondary outcomes (liver enzymes). Due to significant side effects of pioglitazone (weight gain43 and bone fractures in women44) both National Institute for Health and Care Excellence (NICE) and American Association for the Study of Liver Diseases (AASLD) guidelines recommend careful evaluation of risk and benefit and specific administration of pioglitazone to patients with established NASH.40
Glucagon-like peptide-1 (GLP-1) analogues
In a published randomized, placebo-controlled trial consisting of 52 patients with biopsy-proven NASH, treatment with liraglutide administered subcutaneously once-daily for 48 weeks was related to improved NASH and less progression of fibrosis45 (LEAN, Clinical trial NCT01237119, https://clinicaltrials.gov/ct2/show/NCT01237119). Similarly, in another clinical trial (LIRA-NAFLD study, Clinical trial NCT02721888, https://clinicaltrials.gov/ct2/show/NCT02721888), it was reported that six months of treatment with 1.2 mg liraglutide per day significantly reduced liver fat deposits in patients with inadequately controlled type 2 diabetes though there were no data on fibrosis progression.46 Both the AASLD40 and NICE (https://www.nice.org.uk/guidance/ng49) recommendations indicate that there is still little evidence to support the use of GLP-1 analogues in NAFLD treatment. All other guidelines agree on this point; however they also state that further evidence may prove the efficacy of these drugs.34
Lipid lowering strategies
In a post-hoc analysis of The GREek Atorvastatin and Coronary-heart-disease Evaluation (GREACE) study, it was found that use of atorvastatin in coronary heart disease patient (CHD) ameliorates NAFLD and NASH and reduces cardiovascular disease (CVD) events.47,48 Regarding the safety of statin use in the treatment of NAFLD, all guidelines agree that statins are safe, even in patients with compensated cirrhosis. However, routine prescription of a statin is not suggested in patients with decompensated cirrhosis and acute liver failure.49 It should be noted that overall, clinical trials for the use of statins in NASH are limited and somewhat inconsistent, with liver enzymes improving modestly or not at all and variable effects on histology.50 Regarding the effects of fibrates on NAFLD, in a placebo-controlled study in 27 patients with NAFLD, fenofibrate had no effect on hepatic triglyceride content51 while in a larger study in 186 patients with metabolic syndrome and NAFLD the combination of fenofibrate and atorvastatin was not more effective than atorvastatin monotherapy in reducing transaminase levels and liver echogenicity.52 Ezetimibe, another lipid-lowering medication showed promising effects in limited-scale clinical trials.53-56 A recent meta-analysis to evaluate the efficacy of ezetimibe in treating NAFLD and NASH suggested that ezetimibe attenuated serum liver enzymes, hepatic steatosis and ballooning in six studies. Interestingly, hepatocyte ballooning was reduced only in randomized-control trials.57 The study indicated that larger randomized placebo-controlled trials are necessary to determine the effects of ezetimibe on NAFLD and NASH.57
The benefit of vitamin E in the treatment of NASH is attributed to its antioxidant activity. A systematic review and meta-analysis of clinical trials to examine the effects of vitamin E supplementation on liver histology in NASH concluded that vitamin E supplementation had a significant and positive effect in the improvement of steatosis, ballooning degeneration, lobular inflammation and fibrosis in patients with NASH.58 Moreover, Dufour and colleagues showed that two years of treatment with ursodeoxycholic acid in combination with vitamin E improved laboratory values and hepatic steatosis of patients with NASH.59 Similarly, results from a randomized interventional placebo-controlled clinical trial indicated that vitamin E therapy in patients with NASH and without diabetes was associated with a significantly higher rate of improvement in nonalcoholic steatohepatitis42 (PIVENS, Clinical trial NCT00063622, https://clinicaltrials.gov/ct2/show/NCT00063622). It should be noted that there are concerns about the long-term effects of vitamin E on prostate cancer in men over 50 years of age60 and further studies for its overall long-term safety and efficacy are necessary. Vitamin E is recommended by both NICE and AASLD guidelines (limited to established NASH in the latter case40). European Association for the Study of the Liver (EASL) and Italian Association for the Study of the Liver (AISF) guidelines call for more evidence before any recommendation can be made,39,61 while Asia-Pacific guidelines advice against the use of vitamin E which is described as not beneficial by the current evidence.62
DRUGS UNDER DEVELOPMENT
Although current pharmacotherapy for NAFLD may seem limited and of doubtful efficacy, there is optimism that innovative safe and effective options for the management of the disease will be made available shortly. Indeed, specialized drugs are presently tested in clinical trials for the treatment of NAFLD, NASH and hepatic fibrosis. These drugs include obeticholic acid, elafibranor and cenicriviroc.
Bile acid-activated Farnesoid X Receptor (FxR) is a metabolically active nuclear receptor which regulates bile acid, lipid, and glucose metabolism, and contributes to inter-organ communication, in particular the enterohepatic signaling pathway, through bile acids and fibroblast growth factor-15/19 (FGF-15/19).63 Natural ligands for FxR include cafestol, a diterpenoid present in coffee beans, and chenodeoxycholic acid. FxR forms heterodimers with retinoid X receptor (RxR), and upon activation by an appropriate ligand the complex translocates to the cell nucleus, where it binds to FxR response elements (FxREs) on DNA, acting as a transcription factor. The optimal DNA-binding sequence for the FxR/RxR heterodimer is an inverted repeat composed of two AGGTCA half-sites spaced by one nucleotide (IR-1).64 As a result, it increases insulin sensitivity, decreases hepatic gluconeogenesis, and protects against cholestasis-induced liver injury.65 A multicenter, double-blind, placebo-controlled, randomized phase 2b trial of patients with NASH found that patients treated with obeticholic acid presented improved liver histology at 18 months of treatment compared to placebo (relative risk: 2.2; 95% CI: 1.4 to 3.3)65 (FLINT, Clinical trial NCT01265498, https://clinicaltrials.gov/ct2/show/NCT01265498). A serious side effect of the drug in the FLINT trial that may hinder its future development, was a significant deterioration of plasma lipid profile, specifically increased levels of total and LDL cholesterol (LDL-C) and decreased levels of HDL cholesterol (HDL-C), which were not corrected by concomitant use of statins.65 Whether these unfavorable lipid changes are associated with increased cardiovascular risk will be determined in a large phase 3 clinical trial that is currently in progress (REGENERATE, Clinical trial NCT02548351, https://clinicaltrials.gov/ct2/show/NCT02548351).
PPARs (α, γ, δ) are ligand-stimulated nuclear receptors that control gene expression by binding to their respective response elements (PPREs) within promoters in the DNA. PPARs form heterodimers with RxR and, upon binding of specific agonist, the complex translocates to the nucleus where it interact with the transcription machinery, affecting its rate and efficacy.66 PPARα is the pharmacological target of the hypolipidemic drug fenofibrate and PPARγ is the pharmacological target for thiazolidinediones (ciglitazone, pioglitazone, rosiglitazone etc). PPARδ is another member of the PPAR family that is known to shift body’s energy preference from glucose to lipids,67 though no medicine targeting PPARδ is currently approved. Elafibranor, a dual agonist for PPARα and δ which is currently in clinical trials has been shown to improve insulin sensitivity, glucose homeostasis, and lipid metabolism, and attenuates hepatic inflammation. In a multicenter, randomized, placebo-controlled phase 2b trial, elafibranor was shown to be effective in resolving NASH without worsening fibrosis in patients with moderate to severe NASH68 (NASH, Clinical trial NCT01694849, https://clinicaltrials.gov/ct2/show/NCT01694849). This experimental drug is presently in a phase 3 clinical trial in patients with NASH and fibrosis (REGENERATE, Clinical trial NCT02548351, https://clinicaltrials.gov/ct2/show/NCT02548351).
Cenicriviroc is an antagonist of C-C motif chemokine receptor (CCR) types 2 and 5, which promote anti-inflammatory and antifibrotic effects in the liver.69,70 The expected pharmacological effect of this medication is to reduce hepatic fibrosis. A randomized, double-blind, phase 2b study found that cenicriviroc resulted in a significant improvement in fibrosis without worsening NASH after one year of treatment70 (CENTAUR, Clinical trial NCT02217475, https://clinicaltrials.gov/ct2/show/NCT02217475).
THERAPEUTIC LIFESTYLE CHANGES
Pharmacological management of NAFLD should be limited only to those patients with established NASH and fibrosis. For all other subjects with early manifestations of NAFLD the adoption of TLC, such as limited caloric intake, increased physical exercise, and weight-loss produce excellent results and is proposed by many guidelines as a first-line treatment.35,36
Indeed, weight loss has been reported as a key factor in improving hepatic histology.71,72 According to the EASL,39 NICE (https://www.nice.org.uk/guidance/ng49), and AASLD40 guidelines, a 7%-10% weight loss is the target of most lifestyle interventions. Weight loss by a healthy diet such as the Mediterranean diet with a low calorie (1200-1600 kcal/d), low fat (less than 10% of saturated fatty acid) low carbohydrates (<50% of total kcal), and rich in natural antioxidants is recommended. Very low-calorie diets should be avoided as they are considered unsustainable and may pose a challenge to the patient.62 Similarly, in morbidly obese individuals, bariatric surgery improves liver damage, including fibrosis, when significant weight loss is achieved (30% or more).73
Interestingly, natural foods and nutritional supplements have also shown significant benefit in the treatment and reversal of the first stages of NAFLD. Recently, it was reported that coffee consumption may reduce the risk of fibrosis progression and cirrhosis, as it was suggested by a meta-analysis of observational studies.74 Specifically, increasing coffee consumption by two cups per day was associated with a statistically significant reduction in the risk of cirrhosis. Coffee is rich in cafestol, a natural ligand for FxR.
Similarly, a titrated ethanolic extract of Prunus mume (Japanesse Apricot) was shown to have favorable influence on hepatic enzymes both in animals and humans.75,76 The extract coded as MK615 is rich in polyphenols (chlorogenic acid) and triterpanes (oleanolic and ursolic acid) and showed a significant improvement of hepatic histology in tetrachloride-treated rats with concomitant reduction in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels.75 When the same extract was used in patients with liver disorders, a significant reduction in AST, ALT and gamma-glutamyl transferase (γGT) was noted 12 weeks after treatment initiation.75 In another, multicenter randomized, double-blind, parallel, placebo-controlled trial using healthy volunteers of both genders in hospitals from Italy, France and Switzerland, beneficial and statistically significant effects versus placebo were reported on liver function, with decreases in ALT (47%), AST (7%), γ-GT (15%) and glycemia (11%). In addition, MK615 also had a positive of plasma lipids with an increase in HDL cholesterol (13%), and a decrease in LDL/HDL ratio (12%) and triglycerides (8%). As expected MK615 also exhibited antioxidant effects as shown by a decrease in oxidized glutathione, reduced/oxidized cysteine-glycine, oxidized cysteine (intracellular pro-oxidant) and neopterin (inflammation biomarker). The no observed adverse effect level (NOAEL) for the ethanolic prunus mume extract was determined at 3.33 g/kg of body weight in a subacute toxicity study.77
The treatment of NAFLD and most importantly its more severe and life-threatening manifestations of fibrosis and cirrhosis, face significant limitations and challenges. Today, no medicines have been approved to treat NAFLD, though experimental antiofibrotic drugs are in trials. Thus, at the pharmacological level, the disease is treated for its comorbidities with hypolipidemic (statins, ezetimibe) and antidiabetic medications (insulin sensitizers, incretin analogues) and possibly vitamin E. However, the degree of efficacy of each of these medications differs significantly in the different guidelines issued by international medical associations. TLC remains a leading strategy for NAFLD treatment and prevention especially in the early stages. In the context of therapeutic dietary interventions, low calorie diets (1200-1600 kcal/d) containing low fat (less than 10% of saturated fatty acid) low carbohydrates (<50% of total kcal), and natural antioxidants (such as coffee and the MK615 Prunus mume extract) are increasingly recognized to improve liver enzymes (AST, ALT, γGT) and lipid markers (HDL, LDL, triglycerides) associated with early NAFLD development and progression.74-76
Conflict of interest statement
The authors have no conflict of interest to disclose.
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