Non-Alcoholic Fatty Liver Disease: Pathogenesis and the Significance of High-Density Lipoprotein as a Molecular Modifier

Abstract

The pathophysiology of non-alcoholic fatty liver disease (NAFLD) can be identified by modifications in lifestyle, diet and inflammation, all of which have significant implications for the severity of the clinicopathologic outcome of the disease. Prolonged accumulation of hepatic lipid may result in hepatic dysfunction, inflammation and advanced forms of NAFLD. NAFLD describes the presence of hepatic steatosis in the absence of alcohol use and other causes of liver disease. It covers a broad spectrum of hepatic histopathological alterations, from a non-inflammatory intracellular accumulation of fat to non-alcoholic steatohepatitis (NASH), which may progress to hepatic fibrosis, cirrhosis, or hepatocellular carcinoma (HCC). Previous evidence has shown that NAFLD is associated with a range of metabolic syndromes, including obesity, hyperlipidaemia, insulin resistance and diabetes. Hepatic fibrosis and cirrhosis are more common in people with NAFLD, which is partly associated with hyperlipidaemia and low high-density lipoprotein-cholesterol (HDL-C) levels. The ability of HDL to facilitate cholesterol efflux, as determined by cholesterol efflux capacity (CEC), has been linked to its hepatoprotective functions in the body. Findings have demonstrated that NAFLD patients have suppressed HDL CEC. This chapter summarizes the molecular mechanisms and pathogenesis involved in NAFLD. The role of HDL as a molecular modulator of NAFLD, clinical implications and the therapeutic targets to prevent NAFLD have also been discussed.

1. Introduction

Non-alcoholic fatty liver disease is defined as a broad spectrum of hepatic histopathological changes, from a non-inflammatory intracellular accumulation of lipid to NASH, which can develop into hepatic fibrosis, cirrhosis, or HCC [1]. NAFLD is the excessive hepatic deposition of neutral lipids, initiated by an imbalance between lipid availability and clearance. Hepatic lipid accumulation in NAFLD is caused by changes in intracellular cholesterol transport and imbalanced cellular cholesterol homeostasis, characterized by the activations of cholesterol biosynthetic pathways. Enhanced cholesterol de-esterification, modulations of bile acid synthetic pathways and cholesterol export [2]. Through the activation of intracellular signaling pathways in Kupffer cells (KCs), hepatic Stellate cells (HSCs), and hepatocytes, the hepatic lipids accumulation causes liver damage, inflammation and fibrogenesis. Additionally, the mitochondrial dysfunction in the liver may cause an increase in the formation of reactive oxygen species (ROS), which could in turn causes endoplasmic reticulum (ER) stress and death by triggering the unfolded protein response [2]. These actions result in a vicious cycle that supports the progress of steatosis, liver damage and hepatocyte death, which may eventually result in disease progression. Triglycerides (TG) and HDL-C frequently undergo distinctive alterations in atherogenic dyslipidaemia, which is intimately associated with NAFLD. Indeed, atherogenic dyslipidaemia is closely linked to NAFLD, attributed to modifications in TG and HDL-C, hence, constant monitoring of atherosclerotic lipids is essential to evaluate the risk of NAFLD [1, 3, 4].

The incidence of NAFLD is currently around 25% worldwide, with a significant regional variation in the Middle East (32%), South America (31%) as well as the United States (24.1%) and Africa (14%). The prevalence also varies with metabolic disorders, which indicated that approximately 90% of obese individuals, 65% of overweight people, and up to 70% of diabetes mellitus (DM) patients have NAFLD. NAFLD has also been investigated in most ethnic groups but with a lower prevalence in African Americans compared with European-Americans and Hispanics [2, 5]. HDL-C has been considered to modulate NAFLD through several pathways that promote cholesterol efflux in the system, including the reverse cholesterol transport pathway [2, 5], however, essential information to fully understand the impact of HDL-C in NAFLD are limited. In this review, progress on the existing knowledge of the dysregulated cholesterol homeostasis in NAFLD and the cellular mechanisms underlying hepatic lipids toxicity and its role in liver injury were elaborated. The contribution of HDL-C as a molecular modulator and the therapeutic implications of this knowledge were also discussed.

2. Molecular mechanisms and pathogenesis of NAFLD

It is essential to understand the processes that lead to NAFLD and NASH development. Even with the current advancement, our understanding of the pathogenesis of NAFLD is still lacking. The initial theory for the pathophysiology of NAFLD was based on two hypotheses. The first hypothesis described the accumulation of hepatic triglycerides which makes the liver more vulnerable to injury, mediated by the second hypothesis, such as inflammatory cytokines and adipokines, mitochondrial dysfunction, and oxidative stress, which in turn cause steatohepatitis and fibrosis. Also, an increased influx of free fatty acids (FFA) to the liver has been observed in IR and obesity. This concept, however, has been altered as FFA is increasingly understood to play a direct role in triggering liver injury [4, 6, 7]. The FFA can also promote hepatic lipid accumulation either through β-oxidation or esterified with glycerol to form triglycerides. Since convincing evidence has suggested that FFA can trigger inflammatory pathways by facilitating oxidative stress, hepatic triglyceride formation may serve as a defensive mechanism to counteract the harmful effects of unesterified FFA. In a healthy liver, apoptotic cells prompt mature hepatocytes to multiply, replacing the dead cells and re-establishing normal tissue function [4]. However, oxidative stress, a key factor in the development of NAFLD, prevents mature hepatocytes from replicating, which causes the population of hepatic progenitor cells to increase. The hepatic progenitor cells can differentiate into hepatocyte-like cells, and together with intermediate hepatocyte-like cells, can have numbers that are strongly correlated with the fibrosis stage, signifying that cumulative hepatocyte loss stimulates both the deposition of progenitor cells and their differentiation into hepatocytes. Hepatocellular carcinogenesis has also been linked to the activation of these cells. Since the effectiveness of hepatocyte regeneration is required for the fibrosis and cirrhosis that results from chronic liver injury, cell death with impaired hepatocyte progenitor proliferation is thought to be the “third hypothesis” in the pathogenesis of NAFLD [4]. Several factors, including oxidative stress, insulin resistance, steatohepatitis, endoplasmic reticulum stress, bacterial overgrowth, fibrosis, genetic implications, immune system, and beverages consumption, have been implicated in the progress of NAFLD (Figure 1) [4].

Figure 1.

Molecular mechanisms of NAFLD progression. Lipid accumulation and and high cholesterol levels can lead to alterations in intestinal flora, insulin resistance, and adipocyte proliferation. Free fatty acid and free cholesterol consumption leads to ER stress, oxidative stress, hepatic inflammation, and fibrogenesis, which promotes the development of NAFLD. Adipocytes secrete adipokines including IL-6 and TNF, which have an impact on the liver inflammatory environment and hepatocyte fat accumulation. Macrophages are crucial in the development of inflammation and insulin resistance. Recent study has recognized the gut microbiome as being associated with the development of NAFLD. The pattern of microbiome diversity can facilitate intestinal mucosal permeability and lead to lipopolysaccharidaemia, which is correlated with the development of NAFLD and NASH. Lipoprotein lipase (LPL) activity and triglyceride accumulation are both increased when enteric bacteria inhibit the secretion of adipocyte factor.

2.1 Contribution of oxidative stress and mitochondrial dysfunction in NAFLD formation

The influences of oxidative stress and mitochondrial dysfunction in NAFLD and NASH are well-recognized. Within the normal liver, β-oxidation occurs in the mitochondria but in the setting of NAFLD, this process can become overwhelmed due to elevated FFA load, leading to the generation of ROS. ROS stimulate oxidative stress with a progressive activation of mitochondrial damage and inflammatory pathways [8, 9]. Oxidative stress defines the imbalance between the production of ROS and the scavenging capacity of the antioxidant system to counteract the effect of the ROS produced. A high concentration of ROS intensifies the modifications of cellular macromolecules, such as DNA, proteins and lipids, which could lead to the deposition of damaged macromolecules and subsequently induce liver injury. Hence, the mechanism by which ROS contribute to NAFLD development may be associated with deregulated redox signaling and undifferentiating oxidative biomolecular injury (Figure 2) [9]. While the hepatic detoxification process in the liver serves as the main cause of oxidative stress, the biotransformation responses and physiologically generate intermediate ROS to permit the oxidation of toxins and promote their detoxification and excretion. Thus, under normal situations, the levels of ROS generated are the actual amounts needed for the normal body detoxification process and the body system syntheses many antioxidant cofactors that are essentially needed to counterbalance the generation of ROS [4, 6, 7]. On the other hand, insufficient production of endogenous antioxidant molecules and overloading of toxins may facilitate oxidative stress, which in turn may enhance tissue injury and promote the inflammation process [2, 5]. Furthermore, oxidative stress has progressively shown to be one of the major essential pathological processes in the development of NAFLD and the relationship between NASH manifestation and simple steatosis. Oxidative stress has been attributed to various chronic disorders, particularly those associated with low-grade inflammation, including DM, obesity and other metabolic syndromes [2, 5]. Oxidative stress has also been investigated as the major factor associated with the pathophysiology and development of CVDs and was suggested to be a possible mechanism, that links NAFLD to CVDs. While CVDs represent the leading cause of global death and morbidity, in this respect, only a few NAFLD patients may have chronic liver disease. Another important cellular source of oxidative stress is NADPH oxidase (Nox) and its stimulation has been linked with the possible progress of liver injury. The NADPH oxidase family, such as Nox1, Nox2, and Nox4, were suggested to control the activation of hepatic apoptosis and HSCs, which are essential in the fibrogenic process [2, 4, 5].

Figure 2.

Mechanisms of endoplasmic reticulum stress induced-NAFLD. NAFLD is associated with deposition of lipids in the liver, on which lipids promote several cellular stress pathways, such as ER stress and oxidative stress. ER stress enhances UPR which is facilitated by the activation of ER proteins, including ATF6, IRE1 and PERK. Through the phosphorylation of Nrf2, which controls the transcription of antioxidant genes, PERK activation enhances the defense against oxidative stress. Additionally, PERK promotes ATF4, which in turn triggers the transcription of CHOP and controls apoptosis through the Bax protein. Apoptosis results through an interaction between activated IRE1 and TRAF2 and complex recruits Caspase-12. When ER stress is maintained by increasing FFA, PERK and ATF-6 sensors can also activate NF-kB. Additionally, hyperlipidaemia, hypercholesterolemia and obesity can raise the amount of ROS that trigger apoptosis through oxidative stress pathways. Oxidative stress and ER stress are correlated in a bidirectional manner. ATF: Activating transcription factor, CHOP: C/EBP-homologous protein, FFA: Free fatty acid, IRE1: Inositol requiring enzyme-1, NF-κB: Nuclear factor κ-light-chain-enhancer of activated B cells, Nrf2: Nuclear factor 2 erythroid 2-related factor, PERK: Protein kinase RNA-like ER kinase, ROS; reactive oxygen species, TRAF2: TNF-α receptor-associated factor 2.

2.2 Endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is another pathway associated with the pathophysiology of NAFLD and NASH [10]. Unfolded proteins can accumulate within the ER, due to the increased protein synthesis input, the dysfunctional ER or a lack of ATP, which can activate the so-called “unfolded protein response (UPR),” an adaptive response designed to alleviate ER stress [11]. To identify the protein-folding defect that would otherwise result in the onset of apoptosis, UPR activation involves adaptive mechanisms such as reduction of protein synthesis, increased capacity for protein transit through the ER (Figure 2), increased protein folding and transport, and activation of pathways for protein degradation. The ER stress has been explained by various biological stresses, including hyperlipidaemia, hyperinsulinemia, high blood sugar, hypercholesterolaemia, oxidative stress, mitochondrial damage that depletes ATP, and low phosphatidylcholine levels. These can facilitate various pathways that could lead to mitochondrial dysfunction, IR, inflammation and apoptosis, which have been considered as the major factors that cause UPR in NAFLD [6, 8]. UPR has been identified to stimulate c-junk terminal kinase (JNK), a potent enhancer of inflammation and apoptosis. Although the activity of JNK was suggested to differentiate between patients with NASH from those with simple steatosis, its silencing in animal models suppresses both steatosis and steatohepatitis. JNK activity is also linked with decreased insulin signaling, which could initiate the episode of DM. Future research on the consequence of ER stress in NAFLD and NASH is important because it was investigated to have a significant implication on alcohol-induced steatohepatitis [6].

2.3 Insulin resistance

In healthy individuals, insulin receptor substrates (IRS), among other substrates, are phosphorylated when it binds to its receptor, which transmits the insulin signal [4]. Insulin resistance is among the major causes of NAFLD, which increases hepatic lipogenesis and inhibits adipose tissue lipolysis, resulting in an enhanced influx of fatty acids into the liver. Hepatocytes store fat mostly in the form of triglycerides generated by the esterification of glycerol and FFAs. Hepatic accumulation of triglyceride functions as a defense mechanism to balance off the excess FFAs in the plasma rather than as a hepatotoxic event. However, diacylglycerol (DAG) and other bioactive intermediates, such as ceramides, can cause lipotoxicity, which may progress to inflammation, necrosis, and hepatic fibrosis [12]. When the mechanisms protecting hepatocytes against lipotoxicity are depleted, NAFLD develops into NASH. This causes necrosis, secondary repair processes, and accumulation of scar collagen tissue, which are controlled by hepatic stellate cells, leading to the progress and development of hepatic fibrosis. Insulin resistance is also associated with hyperinsulinemia, which can result in the upregulation of transcription factor sterol regulatory element binding protein-1c (SREBP-1c), a major transcriptional regulator of genes involved in DNL and the inhibition of FFA’s β-oxidation, which could further promote the deposition of hepatic lipids. Several anomalies investigated in NAFLD inhibit the insulin signaling pathway, which in turn causes IR. Elevated lipid metabolites, including diacylglycerol (DAG), have been linked to a protein kinase C (PKC) dependent process that blocks insulin receptor activation and insulin signaling modifies IRS-2 phosphorylation [13].

2.4 Inflammation and steatohepatitis

Hepatic steatosis in NAFLD is primarily caused by systemic insulin resistance, while NASH is majorly caused by lipotoxicity of accumulating lipids and innate immune system activation. Inflammatory mechanisms, such as the production of proinflammatory extracellular vesicles and cell death, are activated due to lipid-induced sub-lethal and lethal stress [1, 14]. Steatosis and chronic hepatic inflammation are strongly linked to NAFLD, and the Iκκ-β/NF-κB signaling pathway is partially responsible for this association. FFA can directly activate the Ikk-/BNF-kB pathway in hepatocytes, providing another mechanism through which central obesity and the resultant increase in hepatic FFA supply promote inflammation (Figures 1 and 2). Additionally, the transformation of FFA into hepatic triglyceride might act to protect against the direct toxicity of lipoproteins to the liver. Existing evidence has demonstrated that the suppression of DGAT2, the enzyme that catalyzes the last stage in triglyceride synthesis, improved hepatic steatosis and IR while exacerbating damage and fibrosis in a mouse model of NAFLD [15].

2.5 Fibrosis

Hepatocellular carcinoma, fibrosis and its more severe form, hepatic cirrhosis represent a final common pathway of most chronic liver diseases, such as NAFLD and NASH. Fibrosis is caused by excessive secretion of extracellular matrix (ECM) that is not sufficiently balanced by degradation, leading to a net accumulation. In the models of toxic, biliary liver disease and NAFLD, hepatic stellate cells (HSC) are the primary source of ECM-producing fibroblasts [16]. Twenty genetically distinct types of fibrillary and non-fibrillar collagen, as well as non-collagenous glycoproteins-like elastin, laminin, and fibronectin, as well as glycosaminoglycans-like hyaluronan and proteoglycans, including aggrecan, fibromodulin, decorin, biglycan, glypicans, and syndecans, are all contained in the complex network of the ECM proteins. Together with increased amounts, the composition of the ECM proteins is also modified in fibrosis, leading to an increase in embryonic or wound-healing associated ECM and an increase in crosslinks that make the ECM more resistant to degradation, contributing to delay and incomplete reversibility of severe fibrosis. A more progressive pattern of liver damage may be caused by an inability to develop a ductular response, as seen in patients with denervated liver, who have undergone liver transplantation for NASH [16].

2.6 Bacterial overgrowth

Existing evidence points to the involvement of bacterial overgrowth in the pathogenesis of NAFLD and NASH. The gut microbiome is implicated in the pathogenesis and progression of NAFLD through the so-called gut-liver axis, investigating that the gut microbiome could be considered a metabolic organ in the host, which can affect human metabolism in health and disease [13]. Bacterial overgrowth results in the secretion of bacterial lipopolysaccharides (LPS), which can activate the production of TNF-α, and ethanol. Bacterial LPS are produced when Gram-negative bacteria proliferate excessively and are mostly transported due to increased intestinal permeability. Furthermore, the interaction between LPS and the Toll-like receptors (TLRs4) system increases oxidative stress in NAFLD because of the excessive ROS generation and deficiency in endogenous antioxidant molecules [7]. Oxidative stress has also been reported to be overexpressed in CVDs, which may be a link that connects LPS to the elevated cardiovascular risk in NAFLD patients. The elevated LPS levels in the circulation may result from various factors. In the intestine, the absorption of LPS together with chylomicrons intensifies the chance of NAFLD development, which is activated by hepatic inflammatory cells. Additionally, intestinal bacteria stimulate lipoprotein lipase activity and triglyceride accumulation by inhibiting the synthesis of fasting-induced adipocyte factor (FIAF) [7]. Furthermore, the gut microbiota syntheses enzymes that facilitate the transformation of dietary choline into toxic substances, such as methylamines, which can be utilized by the liver, transformed into trimethylamine-N-oxide, and subsequently promote inflammation and liver damage. Also, bacterial endotoxins have damaging effects on hepatocytes and can stimulate Kupffer cells to generate inflammatory cytokines, which would then cause the waterfall effect and the generation of oxygen radicals [6, 7]. TLRs on hepatocytes, HSCs, and Kupfer cells detect bacterial endotoxins. Signaling of bacterial LPS through TLR4 activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) (Figures 1 and 2) and subsequent inflammasome activation. Increased secretion of bile acids (Bas) facilitated by a high-fat diet is another mechanism that may result in gut hyperpermeability in individuals with NAFLD. Gut permeability is compromised because of BAs, which increase epidermal growth receptor (EGFR) activity. Generally, these pathways could partially account for the “leaky gut” phenomenon seen in the majority of NAFLD patients [6, 7, 13].

2.7 Glucocorticoids (GCs)

GCs sources from both exogenous and endogenous have been recognized to be implicated in NAFLD development. Individuals with Cushing’s syndrome, who have elevated GCs levels are associated with characteristic metabolic phenotype, including IR, central obesity, and DM, and many of these patients will have hepatic steatosis. Inhibition of fatty acid β-oxidation and activation of hepatocyte DNL have been reported as the potential mechanisms through which GCs stimulate hepatic lipid accumulation. Still, several individuals will have normal cortisol levels, indicating that tissue-specific pathways are involved in this metabolic dysfunction [4].

2.8 Involvement of the immune system

Innate immune cells play a crucial role in the pathogenesis of NAFLD [5]. Although the innate immune system is activated and proinflammatory monocytes are recruited into the liver in NASH, the precise signals that result from this are still poorly understood [17, 18]. Increased FFA levels, which result in lipotoxicity, insulin resistance, dysfunctional peripheral adipose tissue, and endotoxins originating from the gut, contribute to activating and maintaining the synthesis and release of pro-inflammatory cytokines in the liver at both local and systemic levels. JNK-AP-1 and IKK-NF-kB are two major inflammatory pathways that play a crucial role in the emergence of the chronic inflammatory state in NAFLD [6]. In vitro experiments employing cultured HepG2 cells and primary mouse, hepatocytes show that the release of damage-associated molecular patterns (DAMPs) from hepatocytes activates innate immune cells, especially macrophages (Kupffer cells). Additionally, several specific DAMPs, including high-mobility-group protein box 1 (HMGB1), have been demonstrated to activate TLR4 in NASH and NAFLD, playing a crucial role in the initial setting of NAFLD. In humans, other DAMPs, such as sonic hedgehog (SHH) ligands, have also been linked to the development of NAFLD and fibrosis. Another important component of NAFLD and NASH is neutrophils. The protease neutrophil elastase (NE), which is synthesized by neutrophils, secretes cellular IR, while deletion of NE results in less tissue inflammation [5]. Increased concentration of blood from the gut to the portal vein exposed the liver to gut-derived endotoxin, leading to endotoxemia, which activates Kupffer cells through the TLR4 complex on the cell surface. The Toll-like family of pattern recognition receptors are crucial for host defense against invading pathogens. When endotoxin interacts with TLR-4, a variety of proinflammatory mediators are released, leading to hepatic fibrosis and damage. Furthermore, cytokines have a significant impact on lipid metabolism [19]. Chitotriosidase (CHIT), an enzyme from the glycosylhydrolase family, is one of the mechanisms associated with the immune response in NASH and NAFLD. The CHIT gene spans around 20 kb of genomic DNA and is found on chromosome 1q31–q32 and has 12 exons. The majority of freshly generated 50 kDa CHIT is secreted by tissue macrophages, and a subsequent step cleaves the enzyme to secrete the active form of CHIT (39 kDa). Pathological tissue macrophages massively express CHIT in many conditions. In humans, NASH patients have much greater levels of CHIT expression than NAFLD patients or control subjects, which indicates a direct relationship between CHIT expression and the severity of NASH. Hence, patients with NASH had greater plasma levels of CHIT activity than those with NAFLD [20].

2.9 Genetic implication

Identification of genetic factors to determine the risk of disease progression may assist to evaluate individuals who may have associated morbidity. Various genes associated with NAFLD have been investigated but the most frequent variant; p.I148M of the enzyme adiponectin gene is one of the major genetic determinants of steatosis and steatohepatitis, fibrosis, cirrhosis, and hepatocellular cancer [2]. Furthermore, polymorphism in the TM6SF2 gene (rs58542926c.449 C > T, p.Glu167Lys) has been associated with severe hepatic fibrosis and cirrhosis, but the underlying mechanisms responsible for these gene variants to influence liver damage are still lacking [2]. In addition to environmental factors, genes also affect NAFLD and through the genome-wide association analyses, several genes have been discovered, on which transmembrane 6 superfamily member 2 (TM6SF2) and patatin-like phospholipase domain containing 3 (PNPLA3) appear to be more implicated. Triacylglycerol hydrolysis is mediated by a 481 amino acid protein that is encoded by the PNPLA3 gene, which is found on chromosome 22. PNPLA3’s I148M variant (rs738409), is substantially linked to NAFLD in adults, as well as obese kids and teenagers, although the precise mechanism is still unknown. The TM6SF2 gene, which is found on chromosome 19, contributes to the development of NAFLD. A single nucleotide polymorphism (rs58542926) that replaces the position 167 of cytosine to thymine has been associated with an elevated hepatic triglyceride level. The development of fibrosis has also been linked to this gene variant and the effects of PNLPA3 and TM6SF2 on NASH and severe fibrosis are cumulative [21]. Only a small percentage of people with obesity and IR develop NASH and cirrhosis, even though hepatic steatosis is widespread in these patients, indicating an essential interaction between genetic predisposition and environmental factors. NAFLD and NASH formations may be made more susceptible by polymorphisms in genes involved in lipid metabolism, IR, oxidative stress, cytokines, and fibrogenesis. Single nucleotide polymorphisms (SNPs) that affect fibrosis development in various liver diseases, including chronic hepatitis C, have been found in several reports. Existing evidence has shown that angiotensinogen and TGF-1 gene polymorphisms have been linked to progressive liver fibrosis in obese patients with NAFLD and NASH. Also, NAFLD and NAFLD-related fibrosis are linked to SNPs in the angiotensin II type 1 receptor [4].

2.10 Influences of beverages in non-alcoholic fatty liver disease

The most popular types of alcoholic drinks include wines, beers, and spirits, whereas non-alcoholic drinks include juices, carbonated and non-carbonated sweetened drinks, and hot beverages like tea and coffee. Even though certain drinks provide fundamental health advantages, beverages are regarded as functional foods because they include well-known macro- or micro-molecules that support optimal health. Independent of the metabolic syndrome, soft drink use can raise the prevalence of NAFLD. Regular soft drink consumption causes the primary effect of fructose, which increases lipogenesis. The additional contribution of aspartame sweetener and caramel colorant, which are rich in advanced glycation end products, may increase insulin resistance and inflammation. Hence, lipids accumulation in the liver can result from regular soft drink consumption [22, 23]. Consuming sugar-sweetened drinks (SSBs) is also linked to increased triglyceride levels, abdominal fat, blood pressure, IR and lower HDL cholesterol levels, which may facilitate the development of NAFLD and obesity. Despite the negative effects of sugar-sweetened beverages and energy drinks, there is some evidence that drinking tea, coffee, and alcoholic beverages may have some positive effects on liver disease. SSBs increase hepatic de novo lipogenesis while reducing fatty acid β-oxidation, which in turn promotes NAFLD. Despite the lifestyle factors, obesity, CVDs and metabolic syndrome, increased deposition of visceral and hepatic fat are the main risk factors linked to daily consumption of sugar-sweetened beverages [24, 25]. Several mechanisms have been investigated to show how fructose might participate in the production of lipids in the liver. Fructose has also been investigated to facilitate hepatic lipid deposition through the dysfunctional mitochondria and mitigate β-oxidation of fatty acids. Another significant component of SSB is glucose, which can either directly or indirectly stimulate hepatic lipid storage by converting into fructose through the polyol pathway in the liver [22]. Moreover, fructose may activate the lipogenic transcription factors sterol receptor element binding protein 1c (SREBP-1c) and carbohydrate response element binding protein (ChREBP). Fructose may limit the breakdown of fatty acids by lowering the activity of β-oxidation in the liver as another mechanism. Numerous studies have demonstrated that SSB consumption may raise the risk of hyperuricemia by depleting adenosine triphosphate (ATP), which may then increase alanine aminotransferase (ALT) levels [25].

3. Modulation of liver injury in NAFLD by HDL

The hepato-protective activity of HDL in the body is tightly related to its function in facilitating cholesterol efflux, determined by CEC through various pathways [26].

3.1 Lipid metabolism and significant of HDL

Lipid metabolism involves several key enzymes and subtypes of lipid fractions and lipoproteins. These include lipoprotein lipase (LPL), TGs, TC, LDL, VLDL, HDL, and chylomicrons (Figure 3). Lipid metabolism formed the cornerstone for understanding the mechanisms involved in the atherothrombotic formation. Lipid metabolism occurs through three essential pathways, including exogenous and endogenously produced lipids, and finally the reverse cholesterol transport pathway (Figure 3) [27, 28]. The exogenous (dietary) pathway begins with chylomicron synthesis and secretion by the intestine. Dietary fat and cholesterol are absorbed by the duodenum and proximal jejunum. In the intestinal duodenum, dietary lipids undergo emulsification and then hydrolysed by the pancreatic and intestinal lipases. Hydrolysis products, such as free fatty acids and monoglycerides are then transferred to the intestinal epithelial cell, where they diffuse through the epithelial cell membranes into the intestinal mucosal cells. In the small intestinal mucosal cells, free fatty acids and monoglycerides reassemble to form triglycerides, which then combine with proteins, phospholipids, free and esterified cholesterol to form Chylomicrons [29]. Chylomicrons are the lipoprotein class responsible for dietary lipids transport. After their formation in the enterocytes, chylomicrons, which mainly contain triglycerides, are secreted into the lacteals and enter into the blood circulation through the lymphatic system. Chylomicrons contain apolipoproteins (Apos) B-48, C-II, and E. The Apo C-II is an essential co-factor of lipoprotein lipase (LPL) during the transportation of fatty acids to adipose tissue. After LPL activity, the chylomicron remnant is relatively enriched in cholesterol due to the loss of triacylglycerol and absorbed into the liver by Apo E [30]. Lipoprotein, which is exposed on the chylomicron surface, activates the lipoprotein lipase attached to the capillary beds in adipose and skeletal muscle tissues, which then hydrolyses triglycerides into free fatty acids (FFAs) and glycerol. The FFAs enter the muscle cells, where they are used for energy production and to the adipocytes, where they would be re-esterified into triglycerides for storage. The chylomicron remnants return to HDL to be recycled by the liver and are recognized by specific hepatic receptors that rapidly remove them from the circulation by endocytosis. The cholesterol found in chylomicron remnants can be used for VLDL, bile acid formation, or stored as cholesteryl esters [28]. While the chylomicrons are responsible for the transport of dietary lipids, endogenously synthesized triglycerides, cholesterols and cholesteryl esters, including VLDL, LDL and HDL are mainly involved in the endogenous lipid metabolism pathway. The endogenous pathway starts with the synthesis of VLDL particles, which are triglyceride-rich and contain Apo B-100, C-II, and E. After the removal of the triglycerides in adipose tissue, a portion of VLDL remnants is metabolized to LDL particles [30]. Thus, VLDL remnants are either removed from the circulation by the liver or undergo further transformation by lipoprotein lipase or hepatic lipase to form LDL. As LPL cleaves TGs, the cholesterol concentration within the lipoprotein increases and becomes a smaller denser lipoprotein named “intermediate-density lipoproteins” (IDL) [27]. The IDL can be taken up by the liver through an apoE-dependent process, while LDL is taken up by the liver through the binding of apoB100 to LDL receptors. The LDL which mainly contains cholesteryl esters and phospholipids circulates in the blood and binds to specific receptors that are widely distributed throughout the tissues. The small VLDL, IDL, and LDL particles may be taken up by peripheral tissues to deliver nutrients, cholesterol, and fat-soluble vitamins, to be used for the synthesis of steroid hormones and cell membranes as well as for hepatic metabolism [31].

Figure 3.

Mechanism of reverse cholesterol transport (RCT): From the exogenous pathway, LPL acts on chylomicrons to generate FFAs, while the chylomicron remnant are transported to the liver. In the endogenous pathway, the LPL cleaves VLDL to form IDLs, which are utilized by the liver. The reverse cholesterol transport pathway involves the action of HDL, which picks up the peripheral cholesterols and returns them to the liver for recycling. LPL; lipoprotein lipase, FFAs; free fatty acids, HDL; high-density lipoproteins, VLDL; very low-density lipoprotein, LDL; low-density lipoprotein, IDL; intermediate lipoprotein.

HDL is a mixture of lipoproteins associated with various minor lipids and proteins that stimulates the function of HDL. Most of the HDL particles arise from lipid-free or poorly lipidated apoA-I secreted by hepatocytes and the intestinal mucosa or dissociated from lipolyzed chylomicrons and VLDL as well as from interconverting mature HDL particles [32]. The interaction between the lipid-free or poorly lipidated apoA-I, also known as the pre-β1-HDL with the ATP-binding cassette transporter A1 (ABCA1) leads to efflux of phospholipids and unesterified cholesterol from various cells, such as hepatocytes, enterocytes, and macrophages, which progress to the formation of small discoidal HDL particles, known as α₄-HDL. The α₄-HDL precursors can further facilitate the lipid efflux from cells, basically from scavenger receptor BI (SR-BI) or ATP-binding cassette transporter G1 (ABCG1) [2]. The effluxed cholesterol and phosphatidylcholine function as substrates of lecithin-cholesterol acyltransferase (LCAT) to generate water-insoluble cholesteryl esters, which transform to the core mature spherical HDL. The initial small α-HDL3 particles develop into larger α-HDL2 particles obtained from phospholipids and cholesterol from both cells (SR-BI or ABCG1), which is involved with the activity of phospholipid transfer protein, and apoB-containing lipoproteins fused with other HDL particles. The breaking down of HDL varies from that of LDL since only a minor proportion of HDL would be removed by holo-particle uptake into cells. In this poorly understood pathway, a high-affinity interaction of apoA-I with ectopic F0F1–ATPase leads to the generation of ADP, which stimulates purinergic receptors to facilitate the uptake of HDL by an as-yet-unidentified low-affinity HDL receptor [2, 32]. These pathways (Figure 3) promote the elimination of lipids from HDL irrespective of their protein content. The cholesteryl ester transfer protein (CETP) converts triglycerides of apoB-containing lipoproteins for cholesteryl esters of HDL, which are finally removed through the LDL receptor pathway, while SR-BI coordinates the uptake of HDL lipids into the liver and steroidogenic organs [8]. The elimination of cholesteryl esters by CETP and SR-BI, and the lipolysis of triglycerides and phospholipids by hepatic lipase and endothelial lipase, respectively, promote the transformation of HDL2 to HDL3, and the ultimate formation of pre-β1-HDL. This lipid-poor apoA-I either transforms to a mature HDL by activating ABCA1-mediated lipid efflux from several cells or is filtrated by the renal glomeruli through the proximal tubule of the kidneys. The apoA-I is endocytosed by the cubilin and megalin receptors and targeted for lysosomal degradation [32].

3.2 Reverse cholesterol transport (RCT)

Existing studies have investigated that extracellular levels, HDL molecular content and the activities of ABC transporter determine the cholesterol efflux. Also, previous reports have indicated that cellular cholesterol homeostasis, HDL-mediated efflux together with ABCG1 and cholesterol efflux by apoA-I/ ABCA1 plays a significant regulatory step in several cellular activities, such as proliferation, differentiation and mobilization of haemopoietic cells [26]. The RCT described a mechanism by which the body removes excess cholesterol from peripheral cells and tissues and delivers it to the liver after conversion to bile acids. The cholesterol will then be redistributed to other tissues or excreted from the body through the gallbladder, or to adrenals, testes, and ovaries for the synthesis of steroid hormones. Because cholesterol could not be metabolized by peripheral tissues, it must be transported back to the liver for removal through a pathway known as “reverse cholesterol transport” (Figure 3) determined by the HDL and its precursors [29]. The HDL-C is the main lipoprotein involved in this process, followed by the intestine, while the liver produces the protein Apo A-1 (70% of the protein content of HDL-c), which passes through the bloodstream and goes to peripheral tissues, such as the heart. In the circulation, Apo A-1 interacts with receptors in several cell types, including hepatocytes, enterocytes, and macrophages, known as ATP-Binding Cassette, Sub-Family A, Member 1 (ABCA1) [33]. In macrophages, the immune system specialized in phagocytosing particles, the interaction with this protein forces the cholesterols and some phospholipids to move toward the molecule Apo A-1. This interaction leads to the formation of nascent HDL-c particles (pre-b HDL), which can subsequently interact with scavenger receptor class B member 1 (SR-B1) and ATP-binding cassette, sub-family G, member 1 (ABCG1), and then incorporate more cholesterol to form a mature molecule of HDL-C (α-HDL), catalyzed by the enzyme LCAT. Cholesterols are delivered to the liver in both direct and indirect ways. In a direct way, mature molecules of HDL-C interact with SR-B1 in the liver, which permits the transfer of its cholesterol content, and the resulting HDL-C molecule can enter circulation and initiate another RCT process. The mature molecules of HDL-C can indirectly transfer its cholesterol content to apolipoproteins B-100 (Apo B-100), particularly to the LDL, in exchange for triacylglycerol molecules, a process catalyzed by the enzyme CETP, and hence, these lipoproteins can be linked with their liver receptors and deliver their cholesterol content. The CETP was also identified to catalyze the reverse transference, i.e., triacylglycerol from HDL-C in exchange for Apo B-100 cholesterol. The reduction in the synthesis of hepatic cholesterol leads to increased hepatic LDL-receptors, which bind and reduces the synthesis of circulating LDL and its precursors; IDL and VLDL [34]. The HDL cholesterol content in plasma may therefore be a crucial modulator to treat and prevent NAFLD since this molecule exerts anti-inflammatory functions as well as positive effects on CRT. Furthermore, recent studies have suggested that the functionality of HDL-c shows a much greater potential in some conditions, including dyslipidaemia and NAFLD [32, 35].

4. Current therapeutic target

The information above pointed out the various pathways associated with NAFLD pathogenesis and the role of HDL as a molecular modifier of NAFLD. Finding the most effective therapeutic targets is now much easier, due to our growing understanding of the pathophysiology of NAFLD. Indeed, correction and management of established NAFLD and NASH-associated factors implicated with the pathogenesis and progression, including excessive dietary energy and fructose intake, the extent of obesity, hyperlipidaemia, degree of IR, DM and oxidative stress are the current therapeutic targets of NAFLD and NASH treatment [2]. The current therapies available are projected toward improving the factors that suppress the disease pathogenesis, including exercise, weight loss, modification of lifestyle, decreasing IR and promoting DM control. At end-stage cirrhosis, liver transplant appears to be the only treatment option. Therapies that can cure or prevent fibrosis are essential in this regard because it is known that the existence of fibrosis in NAFLD is linked with other liver-associated complications [4]. Antioxidants, such as vitamins C, E, and betaine, iron depletion, statins, and pentoxifylline are some of the current treatments being evaluated in NAFLD and NASH. Others, including Glucagon-like peptide-1 (GLP-1)-based therapy, may be an advanced therapeutic alternative to inhibit the development of NAFLD. Drugs like exenatide, have been demonstrated to boost insulin secretion, inhibit glucagon secretion, suppress gastric emptying, and promote satiety with weight loss, demonstrated in both animal models and DM individuals [2]. Since angiotensin has been demonstrated to encourage myofibroblast survival and liver fibrosis, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) are anticipated to have significant effects, such as antifibrotic. The proliferative, contractile, and fibrogenic activities of HSCs are closely regulated by a wide number of cytokines, whose antagonistic effects constitute another possible target for antifibrotic treatments [27]. Platelet-derived growth factor (PDGF), transforming growth factor beta-1 (TGF-β1), connective tissue growth factor (CTGF), endothelin-1 (ET-1), thrombin, vascular endothelial growth factor (VEGF), fibroblast growth factor, and insulin-like growth factor are potential candidates, that exert their effects through tyrosine-kinase receptors [4]. Additionally, inhibiting ER stress and modifying the gut-liver axis utilizing pre- and probiotics are two other possible targets. A Mediterranean diet has been shown to minimize oxidative stress, and high daily doses of vitamin E have been shown to cause the resolution of NASH in 36% of treated individuals. Silymarin reduced transaminase levels in NAFLD patients, and long-term use of the drug may help lessen fibrosis and halt the progression of liver disease in NAFLD and NASH patients [4].

4.1 Common HDL-C-raising drugs

Even though HDL is considered a promising biomarker and potential therapeutic target based on its epidemiological data and the effects of healthy HDL in vitro in endothelial cells and macrophages, as well as based on infusion studies of reconstituted HDL in patients with hypercholesterolemia [32], it would be assumed that HDL-C–raising drugs will become part of preventive armamentarium in the future. Therefore, it will be important to demonstrate that novel drugs not only increase HDL-C plasma levels but also improve HDL functions. Based on the inverse epidemiological association that linked HDL-C plasma levels with several adverse effects of CVDs, HDL-C has been recognized as a potential therapeutic target. As such, many drugs to increase HDL-C levels have been established and examined at basic and clinical levels [8, 32]. Various advanced agents and drugs associated with cholesterol metabolism have been established in clinical trials that may be used to treat NAFLD and NASH. The CETP inhibitors.is the most potent novel category of HDL-C-increasing drugs. CETP is a plasma protein that suppresses the movement of cholesterol esters from HDL to LDL, leading to a marked and consistent increase in the plasma HDL-C levels. The most common is the fibrates, which significantly decreased plasma triglyceride levels and elevate the HDL-C levels [8]. Niacin has been used to treat individuals with hyperlipidaemia by raising the HDL-C levels to about 15% and 30% while reducing the concentration of LDL-C and triglycerides ApoA-I contains 243 amino acids were identified to promote HDL anti-inflammatory activities in various animal models of atherosclerosis and NAFLD. Oral administration of apoA-I mimetic peptide of 4.3 and 7.14 mg/kg doses significantly enhances HDL functionality and the HDL inflammatory index [2, 8, 32].

4.2 Other evidence-based treatment alternatives

4.2.1 Coffee consumption and NAFLD

Coffee and other caffeinated beverages enhance intestinal barrier performance, promote hepatic autophagy, and prevent the activation of HSCs. Due to the antioxidant and antifibrotic properties of several physiologically active chemicals it possesses, coffee appears to be protective against many hepatic conditions, including liver fibrosis and cirrhosis. Coffee is also protective in people with NAFLD; due to the number of antioxidants as well as the caffeine itself, which has anti-inflammatory characteristics [6, 13]. The beneficial effect of decaffeinated coffee on the development of NASH was mediated through attenuating intestinal nitric oxide synthase (NOS) protein and restoration of intestinal barrier functioning, whereas the antifibrotic effect of coffee was exerted by caffeine-mediated antagonism of adenosine receptor which further leads to hepatic stellate cells inactivation. Chlorogenic acid, a key component of regular coffee, lowers the frequency of NAFLD perhaps by facilitating hepatic autophagy, enhancing gut barrier function, and reducing hepatic inflammation through the TLR4 pathway [22].

4.2.2 Tea consumption and NAFLD

Tea intake modulates NAFLD by suppressing inflammation and lipogenesis while promoting fatty acid β-oxidation. Camellia sinensis leaves and buds are used to make green and black tea, however, due to variations in post-harvest processing, their polyphenol content varies. Catechins (flavan-3-ols), which make up about 20% of the total flavonoids in green tea leaves are the main polyphenols and EGCG (Epigallocatechin gallate) [2, 22]. In humans, between the ages of 10 and 16, drinking green tea has been shown to lower plasma levels of aminotransferases, triglycerides, and improve BMI, explaining that drinking tea helps protect against NAFLD is supported by the tight association between these factors and NAFLD. In-vivo research has previously demonstrated that green tea has antioxidant capabilities, it reduces the accumulation of lipids in the liver and adipose tissue and prevents intestinal absorption of dietary lipids. In the mouse model of NAFLD, green tea extract (GTE) has been discovered to protect against hepatic steatosis and related liver damage. Recent research suggests that GTE therapy reduces pro-inflammatory signals through TLR4 and TNFR1, which in turn reduces inflammation in steatohepatitis [22]. Green tea catechins have also been demonstrated to support hepatic lipid metabolism. It was suggested that oxidative degradation of fatty acids by catechins acts as a protective mechanism against NAFLD and also functioning as a natural iron chelator. It has been found that giving patients EGCG reduces non-heme iron absorption by 27%. Since elevated hepatic iron levels have been linked to NASH in patients, while catechins that block iron absorption may be a useful treatment for NAFLD. Similar to this, theaflavin from black tea reduced liver steatosis, oxidative stress, inflammation, and apoptosis in mice with NAFLD-induced ischemia-reperfusion injury [22].

5. Conclusion

The mechanisms associated with the deposition and maintenance of excess hepatic lipids define an imbalance between the hepatic production and removal of TGs, which are majorly transferred from the liver within VLDLs. Data concerning which toxic lipids induce liver injury in NAFLD and NASH are limited. Agents or factors that stimulate or modulate liver damage in NAFLD can assist to identify potential therapeutic targets. HDLs have been recognized as good cholesterol, essential to the body, which functions to ameliorate various metabolic conditions, including CVDs and NAFLD. Correction and management of the factors involved in the pathophysiology and progression of NAFLD, including hyperlipidaemia, obesity, IR, DM, oxidative stress, lifestyle, inactivity and poor dietary control are the current therapeutic targets for NAFLD. To minimize liver damage in NAFLD, new approaches that target HDL-induced drugs and cholesterol metabolism pathways may be helpful in lowering hepatic cholesterol content.