Cholesterol is a significant risk factor for non-alcoholic steatohepatitis

Abstract

Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation (steatosis) and inflammation (steatohepatitis). Currently, the exact underlying mechanisms leading to hepatic inflammation remain incompletely understood and therefore therapy options are poor. Analogous to the predominant metabolic risk factor for the metabolic syndrome, NASH patients often display diet-induced dyslipidemia and are therefore also at high risk for cardiovascular disease. Higher lipid levels, in general, are also widely associated with the production of reactive oxygen species during oxidation. However, the exact contribution of the specific type of lipids to hepatic inflammation still remains unclear. In this editorial, we aim to show that cholesterol, in addition to triglycerides and free fatty acids, is an important risk factor in NASH disease pathogenesis. Developing a better understanding of the contribution of lipids underlying NASH pathogenesis is essential for creating effective therapies against this prevalent disease.

Keywords:

• cholesterol
• inflammation
• lipids
• liver
• non-alcoholic steatohepatitis

In parallel to the continuously increasing obesity prevalence, non-alcoholic steatohepatitis (NASH) is becoming an emerging health problem worldwide. When left untreated, NASH is expected to be the primary indication for a liver transplantation in the near future. Of particularly high concern is the strong rise in cases of children with NASH, which has been associated with severe liver disease later in life and even a shorter lifespan. As approved treatment regimens against NASH do not exist, elucidating the molecular mechanisms underlying NASH may provide new bases for the treatment of hepatic inflammation. In addition to the disturbed lipid transport across the circulation, adipose tissue, skeletal muscle, gut and liver, NASH is typically associated with impairments in de novo lipogenesis and beta-oxidation. Consequently, these events lead to lipid accumulation and inflammation in the liver [1]. A dyslipidemic blood profile in NASH patients also increases their risk for the development of atherosclerosis and other severe cardiovascular events and is underlined by the fact that a close relationship exists between NASH and atherosclerosis [2].

Lipid alterations during NASH pathogenesis

Comparative liver and plasma lipid analysis of human subjects with NASH demonstrated modified levels of triglycerides (TGs), cholesterol and free fatty acids compared to subjects without NASH [3,4]. Genes related to lipid metabolism were found to be differentially expressed in NASH patients compared to subjects without NASH. Thus, similar to the significant protein changes on lipid metabolism in NASH patients compared to patients without NASH, significant changes on lipid metabolism also occur at gene expression level [5]. Yet, the central question remains whether all type of lipids contribute equally to the development of hepatic inflammation during NASH. According to the initial view – the ‘two-hit’ model – it was hepatic steatosis (i.e. excess TG accumulation) that was considered to be the primary harmful step for further advanced liver disease, such as NASH [6]. In contrast to the ‘two-hit’ model, recent work demonstrated a dissociation between steatosis and NASH, suggesting a minor role for liver TGs during NASH progression [7]. This view is in line with findings that suggest TG synthesis may not be harmful and even protects hepatocytes from lipotoxicity by providing a useful mechanism to buffer free fatty acid accumulation [8]. Thus, rather than being hepatotoxic, TGs may possess hepatoprotective properties. Of note, while TGs may possess hepatoprotective properties within the context of NASH, it should not be considered completely harmless, as steatosis was shown to be associated with obesity, dyslipidemia, insulin resistance and hypertension [9]. Whereas these conclusions were drawn from mouse models, it is difficult to translate this knowledge to NASH patients. For example, there is currently no evidence that diacyglycerol O-acyltransferases (DGATs), enzymes that catalyze diacylglycerol to form TGs are differentially expressed between healthy individuals and NASH patients. In fact, DGATs were shown to be associated more with liver fibrosis and liver cancer [10]. Additionally, functional polymorphisms in DGAT genes, which would lead to reduced TG formation, have not been shown to promote hepatic inflammation and insulin resistance. Fatty acids are essential for TG formation and are derived from the diet, de novo synthesis and adipose tissue. In case of NASH, there is an imbalance toward increased free fatty acid influx through excess dietary intake, elevated de novo lipogenesis and increased lipolysis, followed by a release of free fatty acids [11]. Beneficial effects have been attributed to long-chain omega-3 polyunsaturated fatty acids, a distinct form of unsaturated fatty acids. However, so far, the efficacy of PUFA supplementation on NASH is still controversial [12,13]. While unsaturated fatty acids are available for hepatic uptake and are incorporated into TG particles, saturated fatty acids (SFAs) activate several intracellular signaling cascades of apoptosis and inflammation and are therefore considered more harmful than the unsaturated form [14]. In line, the total number of SFAs in the liver was shown to be increased in NASH patients compared to healthy controls. In addition to SFAs, numerous other specific free fatty acid classes have been shown to be affected in human NASH [3,4]. However, despite these clear findings about the role of SFAs in NASH, high supplementation of SFAs to the diet failed to induce NASH in rats [15]. Altogether, these findings provide an indication that the contribution of fatty acids to NASH depends on the specific type of fatty acids and warrants further investigation.

Cholesterol is a significant risk factor for NASH

In an elegant study by Marí et al., it was demonstrated that free cholesterol, rather than TGs and free fatty acid accumulation, sensitized the liver to TNF-α and Fas-induced murine steatohepatitis [16]. Further experimental evidence evaluated that free cholesterol, rather than steatosis, is an important risk factor for the transition from steatosis to NASH in hyperlipidemic mice. Omitting dietary cholesterol improved NASH, while adding cholesterol to the diet caused hepatic inflammation [7] and even inflammation in extrahepatic tissues [17]. Hepatic gene expression analysis of cholesterol metabolic genes, involving cholesterol efflux, cholesterol and bile acid synthesis, shows that there is increased hepatic cholesterol synthesis and a dysregulation of the cholesterol metabolism in NASH patients compared to lean and obese controls [18]. In line, a progressive increase of hepatic free cholesterol was observed in patients with NASH compared to patients without hepatic inflammation [4,19]. Whereas in human NASH livers numerous cholesterol-containing droplets and cholesterol crystals were demonstrated inside Kupffer cells, these were not observed in patients with steatosis [20]. Thus, in addition to TGs and free fatty acids, cholesterol is a significant risk factor for NASH.

Besides the direct effect of cholesterol on the liver, cholesterol can contribute to the development of NASH by affecting extrahepatic tissues. Cholesterol elicits a strong macrophage-driven inflammatory response in the arterial wall and adipose tissue [21,22]. Recent evidence now also point to an association between the gut and cholesterol metabolism [23]. Thus, besides the liver, cholesterol may induce overall systemic inflammation by affecting extrahepatic tissues. This follows the same line of the more recently developed ‘multi-hit’ hypothesis, which proposes that various parallel events (i.e., adipose tissue- and gut-derived signals) may contribute to the development of NASH [24].

Underlying cellular mechanisms to cholesterol-induced hepatic inflammation

For cholesterol to be transported through the circulation, it is packaged into so-called lipoproteins. Under normal circumstances, low-density lipoprotein (LDL) cholesterol is internalized into macrophages by LDL receptor-mediated endocytosis. However, under fat-rich conditions and thus high plasma LDL cholesterol levels, LDL cholesterol is taken up via the scavenger receptor-mediated pathway in an unlimited manner. In contrast to the LDL receptor, scavenger-receptor mediated uptake is not downregulated in the presence of high LDL cholesterol concentrations. As such, the uncontrolled scavenger receptor-mediated uptake of LDL cholesterol into the liver is responsible for the generation of lipid-laden (foamy) Kupffer cells and cholesterol crystals, which both are considered to be crucial events for the development of NASH [1,2]. The formation of cholesterol crystals is the consequence of long-term intracellular accumulation of free cholesterol, which in turn may act as a direct activator of the inflammasome and trigger inflammation [2]. Additionally, LDL cholesterol is highly susceptible for oxidative damage and transforms the LDL particle into a harmful oxidized LDL (oxLDL) lipid, which was found to be elevated in plasma of NASH patients [25]. In fact, it has been shown that it is particularly oxLDL, rather than native and acetylated LDL, that drives the hepatic inflammatory response by transforming Kupffer cells into foamy cells. Further research demonstrated that oxLDL specifically has the tendency to accumulate inside lysosomes of Kupffer cells and hereby increase the release of pro-inflammatory cytokines [1,2]. Other modified forms of cholesterol such as aggregated LDL (aggLDL) and cholesteryl-ester rich lipid dispersions can also trigger foam cell formation and could contribute to hepatic inflammation as well [26]. Besides an important role for Kupffer cells in NASH, cholesterol accumulation inside hepatocytes was increased in NASH and further sensitized these cells to TGF-β-induced activation in a vicious cycle, resulting in enhanced cholesterol accumulation, NASH development and subsequent liver damage [27]. Recently, an association was found between impaired lysosomal acid lipase (LAL) activity and human NASH [28]. LAL is a lysosomal enzyme essential for correct intracellular cholesterol trafficking. As such, a reduction in LAL activity results in increased lysosomal cholesterol storage and suggests LAL as a potential target for the treatment of this disease.

Cholesterol-lowering strategies useful as NASH therapy

Promising therapeutic strategies to reduce human NASH are primarily based on lowering cholesterol levels and are currently under investigation. One of these most well-known strategies to reduce cholesterol is the use of statins. Statins or ezetimibe have been shown to inhibit intestinal cholesterol absorption and significantly lower LDL cholesterol levels in humans [29]. Statin therapy was tested in several human preliminary studies and demonstrated improvements in serum aminotransferase levels and ultrasound imaging. However, currently, convincing histological evidence is still lacking [30]. Thus, the beneficial effects of statins on NASH are still under debate. Future adequate randomized trials are needed with a larger cohort size and the inclusion of histological assessment procedures, before statins can be proposed as an effective treatment for NASH. As such, clinicians are cautious with prescribing statins to NASH patients, also because of potential liver damage, particularly in the case of elevated liver enzymes [30]. Experimental evidence further demonstrated that therapy aimed at blocking uptake of oxLDL was successful in preventing the development of NASH in livers of mice [31]. Similar observations were found for a therapy aimed at reducing lysosomal cholesterol in hyperlipidemic mice [32]. A popular way to lower cholesterol is the use of plant sterol and stanol-containing foods. Administration of such a food additive to hyperlipidemic mice resulted in a marked improvement of NASH and holds promise for future NASH therapy [33]. In spite of cholesterol-lowering treatment showing promising results, it should be taken into account that most clinicians fail to detect high cholesterol levels in NASH patients. The lack of an association between high levels of cholesterol and NASH in patients could be explained by the fact that, in case elevated cholesterol is detected in subjects visiting the clinic, these patients receive immediate cholesterol-lowering treatment before the diagnosis of NASH could have been made.

Conclusions

In conclusion, the effect of cholesterol on hepatic inflammation is superior to TGs and free fatty acids. As such, cholesterol should be viewed as a substantial risk factor for the development of NASH and should be paid more attention to in future research. Targeted approaches should be developed to minimize the burden of (modified) cholesterol in order to collectively alleviate hepatic inflammation and other related inflammatory metabolic diseases, such as atherosclerosis.

Financial & competing interests disclosure

The authors were supported by funding from Maag Lever Darm Stichting (MLDS) (grant numbers: WO08-16 and WO11-35) and the Netherlands Organisation for Scientific Research (NWO) (vidi grant: 016.126.327). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.