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Biochimica et Biophysica Acta 1852 (2015) 1765–1778

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Biochimica et Biophysica Acta

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Review Recent insights on the role of cholesterol in non-alcoholic fatty liver disease

Graciela Arguello a,b, Elisa Balboa a, Marco Arrese a,⁎, Silvana Zanlungo a,b,⁎

a Departamento de Gastroenterología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile b FONDAP Center for Genome Regulation (CGR), Santiago, Chile

article info abstract

Article history: Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of hepatic histopathological changes ranging Received 23 March 2015 from non-inflammatory intracellular fat deposition to non-alcoholic steatohepatitis (NASH), which may progress Received in revised form 25 May 2015 into hepatic fibrosis, cirrhosis, or hepatocellular carcinoma. NAFLD hallmark is the excessive hepatic accumula- Accepted 27 May 2015 tion of neutral lipids that result from an imbalance between lipid availability and lipid removal. Recent data sug- Available online 29 May 2015 gest that disturbed hepatic cholesterol homeostasis and liver free cholesterol (FC) accumulation are relevant to

Keywords: the pathogenesis of NAFLD/NASH. Hepatic FC accumulation in NAFLD results from alterations in intracellular cho- NAFLD lesterol transport and from unbalanced cellular cholesterol homeostasis characterized by activation of cholester- NASH ol biosynthetic pathways, increased cholesterol de-esterification and attenuation of cholesterol export and bile Lipotoxicity acid synthesis pathways. FC accumulation leads to liver injury through the activation of intracellular signaling Free cholesterol pathways in Kupffer cells (KCs), Stellate cells (HSCs) and hepatocytes. The activation of KCs and HSCs promotes Cholesterol homeostasis inflammation and fibrogenesis. In addition, FC accumulation in liver mitochondria induces mitochondrial dys- Liver function, which results in increasing production of reactive oxygen species, and triggers the unfolded protein re- sponse in the endoplasmic reticulum (ER) causing ER stress and apoptosis. These events create a vicious circle that contributes to the maintenance of steatosis and promotes ongoing hepatocyte death and liver damage, which in turn may translate into disease progression. In the present review we summarize the current knowl- edge on dysregulated cholesterol homeostasis in NAFLD and examine the cellular mechanisms of hepatic FC tox- icity and its contribution to ongoing liver injury in this disease. The therapeutic implications of this knowledge are also discussed. © 2015 Elsevier B.V. All rights reserved.

1. Introduction of metabolic syndrome, and the hepatic manifestation of this cluster of features linked to an increased cardiometabolic risk [7,8]. A clinically 1.1. Concept and clinical spectrum of non-alcoholic fatty liver disease relevant aspect of NAFLD is that a variable proportion of patients, mainly (NAFLD) those with NASH, exhibit an increased liver-related mortality due to the progression to cirrhosis and its associated complications, including he- The acronym non-alcoholic fatty liver disease (NAFLD) is an umbrel- patocellular carcinoma [9]. In fact, recent data shows that NASH is ex- la term used to describe a clinicopathological entity defined by the pres- pected to become the leading cause of liver transplantation by 2020 ence of a spectrum of hepatic histopathological changes ranging from [10]. Finally, NAFLD has been also linked to an increased risk of incident non-inflammatory intracellular fat deposition (isolated steatosis) to T2DM and cardiovascular disease [9,11]. non-alcoholic steatohepatitis (NASH), with the latter being character- Aspects related to the transition from isolated steatosis to NASH, the ized by steatosis, necro-inflammatory changes and various degrees of progressive form of the disease, are key issues in the study of NAFLD. liver fibrosis [1,2]. Currently, NAFLD is recognized as the most common However, factors involved in the development of more aggressive form of liver disease worldwide affecting between 25 and 30% of the forms of the disease remain only partially unveiled [12]. Recognition general population [3,4]. Notably, NAFLD is highly prevalent among of these factors is important for the identification of potentially useful overweight and obese patients and in subjects with type 2 diabetes therapeutic targets [13,14]. In addition to well-known factors involved (T2DM) [5,6]. Moreover, NAFLD is strongly associated with the features in NASH pathogenesis and progression (i.e., degree of obesity, magni- tude of insulin resistance (IR), adipokine imbalance, excessive dietary fructose intake and ongoing oxidative stress driven by lipotoxic metab- ⁎ Corresponding authors at: Pontificia Universidad Católica de Chile, Departmento de olites), emerging experimental and human data suggest that disturbed Gastroenterología, Facultad de Medicina, Marcoleta 367, 8330024 Santiago, Chile. E-mail addresses: [email protected] (M. Arrese), [email protected] hepatic cholesterol homeostasis and free cholesterol (FC) accumulation (S. Zanlungo). are relevant to the pathogenesis of NASH [15,16]. In the present review,

http://dx.doi.org/10.1016/j.bbadis.2015.05.015 0925-4439/© 2015 Elsevier B.V. All rights reserved. 1766 G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778 we summarize the currently available knowledge about cellular mech- has been estimated that approximately 60% of TGs present in the liver anisms of cholesterol toxicity, and we examine how alterations in cho- in patients with NAFLD originate from WAT [36,37]. Uptake of FAs lesterol homeostasis may promote hepatic cholesterol overload and from plasma in the liver is mediated by fatty acid transport proteins contribute to ongoing liver injury in NAFLD. (FATPs) and the fatty acid translocase FAT/CD36. Both FATP2 and FATP5 are highly expressed in hepatocytes and FATP5 knockout mice 1.2. Pathogenesis of NAFLD/NASH are protected against liver fat accumulation. However, no evidence of a relevant role in humans is available [38]. On the other hand, FAT/CD36 The precise pathophysiology of NAFLD is not fully understood [17]. has been found upregulated in both human and experimental NAFLD Current concepts on disease pathogenesis are evolving as new informa- [39,40], which may contribute to liver fat overload. Once inside the tion is generated from both experimental models and human studies liver FAs are bound by cytosolic fatty acid-binding proteins (FABPs). [12]. Conceptually, NAFLD is generally conceived as a sequential process Mice deficient in FABP are resistant to diet-induced hepatic steatosis consisting first in steatosis development [i.e., pathological accretion of [41] but the role of these proteins in human NAFLD is not known. lipids in the form of triglycerides (TGs)]. In some individuals, steatosis In addition to the overflow of FAs to the liver from WAT, enhanced may promote the occurrence of hepatocyte injury and death and subse- de novo hepatic lipogenesis is also recognized as an important contrib- quently induce localized inflammation and trigger a stereotyped hepatic utor to TG accumulation in liver cells [42,43]. Studies using isotope la- fibrogenic response to injury [18,19]. The latter fuels the progression to beling have shown that this pathway accounts for 25% of hepatic TGs advanced fibrosis and cirrhosis [12,20]. Thus, key aspects of NAFLD path- in NAFLD patients [36,37]. Increased de novo lipogenesis is driven by ogenesis include the mechanisms of fat deposition in the liver cells and compensatory hyperinsulinemia present in IR states [42]. In fact, insulin the determinants of necro-inflammatory and fibrotic changes, that can increases the hepatic activity of critical transcription factors, such as drive disease progression [21]. Steatosis development is clearly related sterol regulatory element-binding protein-1c (SREBP-1c), carbohydrate to the occurrence of IR at the level of the hepatic, muscle and adipose tis- response element-binding protein (ChREBP) and peroxisome sues [22,23]. Although the majority of NAFLD patients exhibit systemic proliferator-activated receptor (PPAR)-γ, which are the major drivers IR it is difficult to determine which of the insulin sensitive tissues is of hepatic de novo lipogenesis [42,44]. Stimulation of this pathway by the primary site of IR in NAFLD [24]. This is a complex issue that faces dietary components, such as fructose and high-fructose corn syrup, methodological problems to be solved since the precise assessment of has been proposed as a contributing factor to the development and se- insulin resistance requires complex techniques such as the euglycemic verity of NAFLD [45]. However, this idea has been disputed by other hyperinsulinemic clamp and the use of stable isotopes, which is cumber- studies showing that the reported associations of fructose with clinical some [25]. Some recent lines of evidence suggest that hepatic IR, second- or histopathological evidence of NAFLD may be more attributable to ex- ary to the accumulation of diacylglycerols (DAGs), may be the primary cess energy than fructose itself [46,47]. pathogenic event [26], although this hypothesis remains controversial The precise role of additional pathways of FA metabolism in NAFLD, [24]. The observation that the disease may recur after transplantation such as impaired hepatic FA oxidation (which would lead to a reduction [27] suggests that peripheral IR is a primary event. Based on this obser- in FA utilization) and/or impaired synthesis or secretion of very low- vation and additional existing data, the predominant view about density lipoproteins (VLDLs) (which would lead to a decrease in the steatosis development is that an initial impaired peripheral insulin ac- export of TG from the liver), is still not well defined. In the case of FA ox- tion in white adipose tissue (WAT) would lead to an uninhibited lipoly- idation although data support the presence of mitochondrial dysfunc- sis that, in turn, produces an increased flux of fatty acids (FAs) to the tion in NAFLD, data on the role of impaired of FA oxidation in this liver and other organs. Visceral WAT is considered an important contrib- setting is inconclusive [48]. With regard to dysregulation of VLDL pro- uting source to the overflow of FA to the liver due to the direct drain of duction and secretion, it has been shown that individuals with NAFLD its circulation to the portal vein [20,28] but the contribution of subcuta- exhibit and increase in VLDL secretion [49] likely as a compensatory re- neous fat is also substantial due to the size of this depot particularly in sponse to intracellular TG accumulation. However, the increase in he- obese subjects [29]. Lipolysis in adipocytes is a complex and tightly reg- patic TG export through this pathway seems to be insufficient to ulated phenomenon that involves neuroendocrine signals resulting in normalize the elevated hepatic TG content. the activation of lipolytic enzymes that drive lipid mobilization. Once steatosis is established, liver cell damage may occur with ac- Among these enzymes, hormone sensitive lipase (HSL) and adipocyte companying inflammation and/or fibrosis consistent with NASH [19, triacylglycerol lipase are the key enzymes for lipolysis initiation [30].De- 20,50]. Multiple factors, including worsening IR, insults from the gut tails of regulatory mechanisms of adipocyte lipolysis are beyond the [bacteria derived products that can activate Toll-like receptors (TLRs)] scope of the present review and can be found elsewhere [31,32].Suffice in both hepatocytes and resident macrophages and/or the adipose tis- to say that insulin by regulating HSL limits the liberation of FA from WAT sue (leading to saturated FA-induced lipotoxicity), promote hepatic and rather induces de novo lipogenesis in this tissue. Thus, if IR is pres- cell injury and death through a variety of mechanisms [51–53].Al- ent lipolysis becomes hyperactivated in adipocytes, resulting in an in- though in some cases steatosis precedes NASH, it is also possible that creased release of FA to plasma. steatosis develops with other features of NASH simultaneously. In fact, The reasons of IR development in WAT are complex and involve a se- it has been proposed that subjects with bland steatosis and NASH quence of events that determine a phenomenon known as adipose tissue could belong to different subsets of individuals with separate histologi- dysfunction that in turn determines local IR and dysregulation of other cal features and pathophysiology [54–56]. physiological functions of WAT such as the secretion of adipokines [33]. Briefly, in conditions of excessive calorie intake, adipocytes become hy- 1.3. Modulators of liver injury in NAFLD perplastic, which is associated to a relative local hypoxia and the devel- opment of WAT stress and autophagy followed by the occurrence of Factors that trigger or modulate liver damage in the setting of NAFLD immune cell infiltration and inflammation [33,34]. This dysfunctional can help to identify potentially useful therapeutic targets [13,14]. In- and injured WAT become resistant to insulin action and uninhibited li- deed, correction and management of known NASH-related factors in- polysis occurs. Dysregulated adipokine secretion is characterized by the volved in pathogenesis and progression, such as the degree of obesity, secretion of a proinflammatory and atherogenic adipokine profile with the severity of IR, the coexistence of T2DM, the occurrence of oxidative decreased adiponectin (an insulin-sensitive adipokine) and increased stress and excessive dietary energy and fructose intake, are the current tumor necrosis factor-α (TNF-α, a cytokine that promotes IR both locally focus of NAFLD/NASH treatment [14,57,58]. In addition, identification of and systemically) [33,35]. Thus, uninhibited lipolysis of triacylglycerol in genetic factors that determine risk of disease occurrence or progression WAT determines an overflow of nonesterified FAs to the liver. In fact, it may help to identify individuals who may experience associated G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778 1767 morbidity [59]. Several genes have been found to be associated to et al. using lipidomic analysis of liver biopsies from NAFLD patients NAFLD but the common variant p.I148M of the enzyme adiponutrin have shown accumulation of FC without an increase in cholesterol es- [also known as Patatin-like phospholipase domain-containing 3 ters (CEs) in isolated steatosis and NASH subgroups [92]. More recent (PNPLA3)] gene has emerged as a one of the major genetic determi- studies have unequivocally demonstrated that hepatic FC accumulation nants of both steatosis and steatohepatitis, and it has been linked to fi- is a prominent feature of NAFLD that correlates with histological sever- brosis, cirrhosis, and hepatocellular cancer [60]. Recently, another ity of the disease [90,94]. Also, epidemiological studies have identified common non-synonymous polymorphism in the TM6SF2 gene dietary cholesterol intake as a factor related to the risk and severity of (rs58542926 c.449 CNT, p.Glu167Lys) has been linked to advanced he- NAFLD [16,101–103]. Animal studies showed that experimental induc- patic fibrosis and cirrhosis [61]. The underlying mechanisms by which tion of hepatic FC accumulation promotes steatohepatitis and liver fi- these gene variants influence liver injury remain to be elucidated. brosis [95,97,98,104,105], and that correction of hepatic FC overload A significant body of work has recently uncovered information on the improves liver disease severity in NASH [99,106–108]. Furthermore, role of novel factors that may modulate liver injury in NAFLD/NASH. the potentially beneficial effects of the lipid-lowering agent ezetimibe Among them the contribution of innate and adaptive immunity with he- in NAFLD/NASH may be mediated by a reduction in hepatic cholesterol patic resident macrophages (Kupffer cells, KCs) sensing danger signals levels [109,110]. Thus, FC accumulation is a reproducible and relevant and recruiting inflammatory cells [62] leading to sterile inflammation, phenomenon in NAFLD that has a significant role in modulating liver in- the occurrence of a disrupted intestinal epithelial barrier in the presence jury and may be amenable to drug therapy. of microbiome dysbiosis [63,64] and activation of TLRs [65].Thesemech- anisms have been suggested to contribute to disease progression in NASH. Importantly, the activation of pattern recognition receptors results in the 2.1. Regulatory mechanisms of cholesterol homeostasis in the liver: activation of the inflammasome, a multimolecular cytosolic protein com- alterations in NAFLD plex, which promotes local production of pro-inflammatory cytokines [66–68] and contributes to inflammation and injury. In addition, path- The liver is a key organ involved in the regulation of cholesterol ways regulating fibrogenesis are important in NASH progression [19].Ac- metabolism and acquires cholesterol from de novo synthesis and tivation of hepatic stellate cells (HSCs) is a key event. HSCs express a from all classes of plasma lipoproteins [111,112]. The synthesis of myriad of receptors that upon stimulation promote differentiation of cholesterol occurs in the endoplasmic reticulum (ER) and is strictly this cell type to cellular matrix-production cells [69,70].Thesereceptor–li- regulated by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A gand interactions and associated intracellular signaling pathways reductase (HMGCR), which catalyzes the first reaction of cholesterol (e.g., the renin–angiotensin–aldosterone system [71] and endothelin-, biosynthesis [111,112]. The uptake of cholesterol from lipoproteins farnesoid X receptor (FXR)- or PPAR-associated pathways) [72–74] are occurs through different surface proteins, including the low density therefore important for modulating fibrosis progression in NASH and lipoprotein (LDL) receptor (LDLR) and the scavenger receptor class may serve as therapeutic targets [75,76]. BtypeI(SR-BI)[112,113]. Information regarding which toxic lipids cause liver damage in NAFLD/ Independent of its origin, hepatic cholesterol has several alternative NASH is limited. The mechanism(s) responsible for the accumulation and metabolic pathways: (1) excretion or efflux into the blood in the form of maintenance of an excessive amount of intrahepatic fat remain only par- VLDL or through ABCA1 to nascent high-density lipoprotein (HDL) par- tially known and, as mentioned above, involve an imbalance between the ticles, respectively; (2) excretion and uptake through bile via ABCG5/G8 hepatic production of TGs (primarily derived from plasma FAs delivered and Niemann–Pick C1-like 1 protein (NPC1L1), respectively; (3) deposi- to the liver from WAT) and the removal of intrahepatic TGs. TGs are pri- tion as CE and (4) substrate for bile acid (BA) synthesis [113]. Under marily exported from the liver within VLDLs [37,49,77,78] but a fraction normal conditions, these pathways interact with each other to maintain of the FAs contained in TGs can be secreted in the form of phospholipids cholesterol levels within a specific range. In addition, cholesterol must through the bile [79]. Observations from experiments inhibiting hepatic be sorted and transported within the hepatocyte toward different re- TG synthesis and patients with hypobetalipoproteinemia, which does gions and organelles [114,115]. These complex and highly regulated not progress to cirrhosis despite massive steatosis, suggest that hepatic metabolic and trafficking pathways involve multiple cholesterol meta- TG accumulation is not toxic per se, but rather protects the liver by buffer- bolic and transport-related genes whose gene expression is regulated ing the accumulation of lipotoxic TG precursors [80–82]. Interestingly, the by the concerted activity of several transcriptional factors, including exposure of cultured cells to unsaturated FA resulted in a significant in- the sterol regulatory element-binding protein 2 (SREBP-2), Liver X re- crease in TG content without an associated decrease in cell viability. In ceptor alpha (LXR-alpha) and FXR [116,117]. contrast, cells incubated with saturated FAs showed a significant increase Intracellular cholesterol synthesis is controlled by an elaborated in apoptotic death in conjunction with an absence of TG accumulation feedback mechanism dependent on the transcription factor SREBP-2, [83]. Saturated FAs can induce hepatocyte cell death by directly activating which is bound to the membrane of the ER [118]. When cholesterol is Jun N-terminal kinase (JNK) and mitochondrial pathways. In addition to abundant, SREBP-2 remains in the ER. Low levels of intracellular choles- saturated FAs, other toxic lipid molecules may be responsible for liver terol cause SREBP-2 to be processed by proteases and its active core damage, including DAGs, ceramide and sphingosine [37,84–89].Inaddi- travels to the nucleus to turn on the expression of genes containing ste- tion to the abovementioned role of direct toxicity of saturated FAs in rol responsive elements in their promoters, such as the LDLR and HMGR NAFLD, recent experimental and human evidence points to a role of al- genes [118]. tered cholesterol homeostasis and hepatic FC accumulation in the patho- BAs are essential for proper absorption of dietary lipids and regulate genesis and progression of NAFLD/NASH [90–100]. The recognition of the transcription of genes that regulate the metabolism of cholesterol intracellular cholesterol accumulation as a modulator of liver injury in and BA [119–123]. The hepatic expression of cholesterol-7α-hydroxy- NASH is relevant as it could be exploited as a therapeutic target. Therefore, lase (CYP7A1), the rate limiting enzyme for the synthesis of BA from in the following sections, we address the underlying mechanisms causing the classical pathway, is suppressed by increased BA levels and this FC accumulation and the relevance of FC accumulation in NAFLD/NASH. feedback mechanism is mediated by FXR [123,124]. Furthermore, the selective uptake of high density lipoprotein CE via SR-BI [125],theintra- 2. Hepatic cholesterol accumulation in NAFLD: relevance cellular CE hydrolysis facilitated by neutral cholesteryl ester hydrolase and mechanisms (nCEH) [126], and the canalicular routing of cholesterol by sterol carrier protein 2 [127] for biliary excretion via adenosine triphosphate binding Different lines of evidence have demonstrated the occurrence of he- cassette subfamily G member 5/8 (ABCG5/8) [128] are positively regu- patic cholesterol accumulation in NAFLD. Seminal studies from Puri lated by FXR [129]. 1768 G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778

Oxysterols are oxygenated derivatives of cholesterol endogenously The role of reduced biliary cholesterol excretion in hepatic cholester- generated during bile acid synthesis that are potent ligands of the LXR ol loading in NAFLD has also been examined. Mice lacking the ABCG5/8 [130,131]. Activation of LXRs induces the expression of several genes in- sterol transporter exhibit reduced biliary cholesterol secretion and are cluding the gene encoding for CYP7A1 and those regulating reverse cho- more susceptible to steatosis, hepatic IR, and loss of glycemic control lesterol transport and cholesterol catabolism [132]. Both FXR and LXR when fed a high-fat diet [146]. Other genetic models of hepatic steatosis must heterodimerize with the 9-cis retinoic acid receptor (RXR) in also exhibit a decreased biliary cholesterol secretion [147,148]. Interest- order to bind DNA and act as transcription factors. Thus, FXR and LXR ingly, promotion of biliary cholesterol secretion using adenoviral co-regulate the synthesis of bile salts and cholesterol homeostasis in he- vectors encoding ABCG5 and ABCG8 restores glycemic control and re- patocytes [132]. duces plasma TG in diabetic mice [149]. However, data from other In addition to the transcription factors described above, another re- models is conflicting as has been also shown that hepatic IR directly pro- cently described regulator, fibroblast growth factor 19 (FGF19), a gut- motes biliary cholesterol secretion [150], which would also explain the derived hormone or enterokine, has been found to play a role in the reg- strong association between NAFLD and cholesterol gallstone disease ulation of hepatic lipid metabolism and glucose and protein homeosta- [151]. However, it is not yet clear whether NAFLD is pathogenetically sis [11]. FGF19 is part of a complex enterohepatic feedback regulatory linked to gallstone formation or whether the presence of gallstones is mechanism involving activation of FXR by BA in the ileum that enhances a marker of the presence of metabolic factors that promote NAFLD de- the gene encoding FGF19. Once produced, FGF19 is secreted into the velopment [152]. Thus, currently available data with regard to biliary portal circulation to bind the FGFR4/b-klotho receptor complex in hepa- cholesterol in NAFLD and its link with gallstone disease from both ani- tocytes [133]. Upon binding to its receptor, this hormone controls he- mal models and epidemiological studies is conflicting and need further patic metabolism by inhibiting BA and glucose synthesis. FGF19 clarification. inhibits BA synthesis through inhibition of CYP7A1 and blunts gluco- Other potential mechanisms for cholesterol accumulation in NAFLD neogenesis through a mechanism involving inactivation of the tran- can be postulated from published data, particularly from the work of scription factor cAMP regulatory element binding protein (CREB) Min et al. These authors extensively assessed the expression of choles- [134]. In addition, FGF19 stimulates protein and glycogen synthesis terol metabolic genes in NAFLD [90]. Decreased expression of the cho- and FA oxidation [133,135]. lesterol efflux gene, ABCG8, in the livers of NAFLD patients supports Existing data support the concept that cholesterol homeostasis is ex- the possibility of decreased FC efflux in fat-laden hepatocytes [90].In tensively dysregulated in NAFLD and that alterations of hepatic choles- addition, the authors reported an increased expression of hepatic neu- terol pathways at different levels do promote FC accumulation in liver tral cholesterol ester hydrolase (nCEH) in NAFLD patients, which may cells (Fig. 1). In fact, hepatic cholesterol synthesis has been shown to contribute to the increase in hepatic FC content by inducing CE hydroly- be increased in NAFLD as shown in studies assessing this pathway sis and returning cholesterol to the intrahepatic FC pool [90] (Fig. 1). In using serum non-cholesterol sterols as surrogate markers of cholesterol this study, patients with NAFLD and NASH had a decreased expression synthesis [136]. Moreover, several studies have shown that the expres- of CYP7A1 and CYP27A proteins, which are both involved in the catab- sion and activity of HMGCR are increased in livers of NAFLD patients olism of cholesterol to BA. Thus, decreased cholesterol biotransforma- which seem to be related to a decrease in HMGCR phosphorylation, tion would also contribute to intrahepatic cholesterol accumulation leading to an increased activity of the enzyme [90,137,138]. In addition, [90]. Interestingly, decreased hepatic expression of CYP7A1 was also ob- increased hepatic levels of active SREBP2, which is the key protein reg- served in a rat model of NASH induced by increasing the cholesterol ulating cholesterol synthesis, may also play a role since increased hepat- content of the diet [153]. Of note, insulin seems to have dual effects on ic FC levels and LDLR expression correlated with SREBP-2 induction in CYP7A1 gene transcription since while physiological concentrations of NASH patients [94]. insulin upregulate the human CYP7A1 gene, prolonged insulin treat- Lipoprotein metabolism is also dysregulated in NAFLD [139,140].A ment induces SREBP-1c, which inhibits CYP7A1 gene transcription pattern of atherogenic dyslipidemia characterized by high concentra- [154]. Thus worsening IR and hyperinsulinemia may contribute to re- tions of plasma TG, low HDL cholesterol and small dense LDL particles duce CYP7A1 activity. is frequently observed in NAFLD patients [141,142], which is related to The accumulation of FC in NASH may be related to increased intesti- increased insulin-induced hepatic lipid synthesis and is associated nal cholesterol absorption. Although indirect assessment of the latter in with disease severity [143]. Liver TG overload is associated to increased humans using surrogate markers has shown that intestinal absorption VLDL secretion toward plasma [49] which may determine an increase in of cholesterol is actually decreased [136], experimental data indicate LDL due to cholesteryl ester transfer protein exchange [139] which is that inhibition of this pathway by NPC1L1 using ezetimibe is beneficial then recaptured in the liver by LDLR-mediated endocytosis. Of note, in NAFLD [155] (Fig. 1) reducing FC accumulation in the liver [99,109, LDLR has been shown to be upregulated in some experimental models 156]. However, a recent trial examining the efficacy of ezetimibe versus of NASFLD/NASH [98], which could contribute to hepatocyte cholesterol placebo in reducing liver fat and improving liver histology in patients overload. However, a recent human study found that the hepatic LDLR with biopsy-proven NASH showed negative results [33]. Further studies expression was decreased and plasma LDL concentrations increased in are needed to confirm this finding. subjects with NAFLD [90]. Thus, further studies are required to deter- Recently, several studies have identified alterations of miRNA pro- mine if LDLR up- or down-regulation is a common and relevant alter- files associated with NAFLD and NASH [32,157–161]. In fact, increased ation in the liver of NASH/NAFLD patients. mRNA levels and enzyme activity of HMGCR in patients with NAFLD Disturbed efflux of cholesterol through ABCA1 to extracellular and NASH [90,162] are related to increased levels of the microRNA apolipoprotein AI could also contribute to increase hepatocyte cho- miR-34a, which inhibits sirtuin-1 expression, a protein with histone lesterol content. In vitro studies in HepG2 cells treated with unsatu- deacetylase activity, and decreases levels of phosphorylated HMGCR rated FA have shown decreased ABCA1 protein levels due to the [90]. Evidence from animal models indicates that NASH development induction of its protein degradation. This correlates with decreased is accompanied by prominent changes in miRNA expression, including hepatic ABCA1 protein levels in experimental NASH [144]. In addi- miR-34a [158,163]. These data suggest that miR-34a changes may con- tion, ABCA1 overexpression in mice resulted in an increase in choles- tribute to the development of NASH. In addition, other microRNA, miR- terol efflux and decreased cellular cholesterol, FA and TG levels 122, the most abundant hepatic miRNA, was shown to be strongly [145]. These data suggest that in NAFLD/NASH a decrease in ABCA1 downregulated in NASH patients [158]. Interestingly, silencing of miR- protein expression may contribute to an increase in lipid storage in 122 led to an initial increase in mRNA levels of its target genes, among hepatocytes although more data is needed to establish the its role them Hmgcr and Srebp-2, by 24 h followed by a decrease by 48 h in in NAFLD/NASH. HepG2 cells. This was correlated with an increase in the protein levels G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778 1769

Fig. 1. Cholesterol homeostasis dysregulation contributes to free cholesterol accumulation in NAFLD. Cholesterol homeostasis is extensively dysregulated at different levels in NAFLD and thus promotes free cholesterol (FC) accumulation. Multi-level changes occur in the liver, including the activation of cholesterol biosynthetic pathways, increased cholesterol de-esterifi- cation, and attenuated pathways in cholesterol export and BA synthesis. These changes may contribute to FC accumulation in the setting of NAFLD. This figure depicts lipoproteins, pro- teins, miRNAs or pathways whose levels are increased or decreased and/or up- or downregulated (arrows) in plasma, hepatocytes and enterocytes. In plasma, NAFLD patients typically exhibit a pattern of atherogenic dyslipidemia characterized by high concentrations of VLDL, and low HDL and apoAI levels. Decreased apoAI levels correlate with a decrease in ABCA1 pro- tein levels. These decreases may contribute to FC accumulation in the liver. In addition, the increased expression and activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) cor- relate with increased levels of miR-34a, and it is a contributing factor to increased FC synthesis. Microsomal sterol 7a hydroxylase (CYP7A1) and mitochondrial sterol 27-hydroxylase (CYP27A) catalyze the synthesis of bile acids (BAs) using classic and acidic pathways, respectively. In NAFLD, both enzymes show decreased levels in the liver, thus limiting cholesterol degradation. Another factor that contributes to hepatic FC accumulation is the increased expression of neutral cholesteryl ester hydrolase (nCEH) through induction of CE hydrolysis and the return of cholesterol to the intrahepatic FC pool. Fibroblast growth factor-19 (FGF-19) is an ileum-derived enterokine that also influences cholesterol metabolism. This hormone signals the liver through the FGFR4/β-klotho receptor to inhibit CYP7A1; furthermore, FGF-19 was found to be decreased in NAFLD patients. The inset shows the relevant regulation gene pathways for cholesterol and BA metabolism in hepatocytes and their dysregulation induced by NAFLD (increased or decreased, shown with arrows). An increase in SREBP-2 induces the expression of the HMGCR gene, promoting synthesis and FC accumulation. In addition, FXR down-regulation correlates with a decreased expression of the Abcg8 gene in the liverofNAFLD patients. This gene encodes for a half-transporter that promotes biliary excretion and limits the intestinal absorption of cholesterol, and the down-regulation of this gene contributes to hepatic cholesterol loading. In the enterocyte, ezetimibe use confers resistance to NAFLD in animal models. This effect is likely mediated by the inhibition of the Niemann–Pick C1-like 1 (NPC1L1) transmembrane protein localized at the apical membrane of enterocytes, which promotes cholesterol absorption. Thus, targeting this pathway may significantly contribute to a decrease in FC accumulation in the liver and potentially reduce liver damage. of these targets at 48 h of treatment [158]. However, there are contra- (Dhcr7) [164]. Similarly, antisense-based silencing of miR-122 in- dictory data for this miRNA. In fact, miR-122 was also found to be upreg- duced a significant decrease in FA and cholesterol biosynthesis and ulated throughout the progression of NAFLD in a rat model [159].In an increase in the FA β-oxidationpathwayinmicefedahighfat addition, evidence in murine studies showed that silencing of miR-122 diet [165]. In conclusion, the effect of miR-122 on cholesterol biosyn- in vivo by antagomirs results in a 25%–30% reduction of plasma choles- thesis is contradictory and it is difficult to establish its relevance and terol levels and decreased expression of several hepatic genes involved role in the progression of liver disease and additional studies are in the biosynthesis of cholesterol, such as 3-hydroxy-3-methylglutaryl- needed to determine if impairment of miR-122 signaling contributes Coa synthase 1 (Hmgcs1), Hmgcr and 7-dehydrocholesterol reductase to NAFLD. 1770 G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778

More recently, a decrease in the serum levels of another microRNA, hepatic cholesterol metabolism and transport to specific cellular miR-21, was found in patients with NAFLD. miR-21 decreased the levels compartments, such as the mitochondria, is further discussed in of TG, FC and total cholesterol in an in vitro model of NAFLD, and this ef- Section 2.3.3. fect was attenuated by Hmgcr overexpression [166]. Collectively, avail- The oxysterol-binding protein (OSBP) family is formed by OSBP and able data suggest the existence of a pathogenic role of miRNAs in various evolutionary conserved OSBP-related proteins (ORPs) and con- NAFLD. However, further studies are necessary to unveil the basis of stitute a conserved family of lipid binding/transfer proteins (LTP) in eu- miRNA dysregulation in NAFLD as well as the exact contribution of spe- karyotes [181]. OSBP is a cytosolic protein that has been implicated in cific microRNAs and the underlying mechanisms that contribute to the regulation of cellular cholesterol, sphingomyelin, and oxysterol NAFLD pathogenesis. Notably, selected miRNAs may be used as bio- transport (reviewed in [182]). Although no direct link has been provid- markers of disease in the near future [32,161]. ed between members of the ORP family and NAFLD, ORP8, has been im- In summary, several metabolic pathways involving hepatic choles- plicated in cholesterol metabolism and IR [181]. Silencing of ORP8 terol homeostasis are dysregulated in NAFLD. A recent detailed molecu- expression in THP-1 macrophages induced the transcription of ABCA1 lar analysis of liver samples from NAFLD patients revealed significant and consequently increased cholesterol efflux [183]. In contrast, ORP8 changes, including activation of cholesterol biosynthetic pathways (in- overexpression in the liver of mice resulted in a reduction of plasma creased SREBP2 processing and HMGCR activity), increased cholesterol and liver tissue lipid levels and down-regulation of the active, nuclear de-esterification (increased nCEH activity), and attenuated pathways in forms of SREBP-1 and -2 [184]. In addition, ORP8 silencing impaired cholesterol export (decreased expression of ABCG8) and BA synthesis insulin's ability to regulate AKT phosphorylation and downstream ki- (decreased expression of CYP7A1) (Fig. 1). These changes resulted in in- nase signaling in liver cells, and its expression was reduced in obesity creased FC levels that may directly promote the hepatocellular injury via a miRNA143-dependent pathway [185]. observed in NAFLD (see Section 2.3) [90]. In summary, multiple pathways of cholesterol intracellular transport may participate in NAFLD pathogenesis and progression. However, the 2.2. Alterations in intracellular cholesterol transport exact contribution of each of these pathways remains unclear.

In addition to the aforementioned changes in cholesterol metabolic 2.3. FC and liver damage in NAFLD: potential mechanisms pathways, alterations in the intracellular cholesterol transport have also been described in NAFLD and NASH. In fact, the study of alterations As detailed above, cholesterol accumulation, and particularly in- of intracellular sterol binding and transfer proteins, FC accumulation creased FC, seems to play an important role in liver injury in the setting and delivery to specific cellular compartments in NAFLD has gained at- of NAFLD. However, although it has been found that FC directly causes tention in the recent years [16,162]. apoptosis and necrosis in hepatocytes [186], the precise mechanisms Caveolin-1, which is the main structural protein of caveolae, exhibits of FC lipotoxicity in NASH are still incompletely delineated. Given the cholesterol-binding and transport activity and has been suggested to fact that inflammation and fibrosis development in the liver involves play a role in the regulation cellular cholesterol homeostasis [167].In- the participation of non-parenchymal cells (i.e. KCs and HSCs) FC accu- terestingly, a significant increase in the expression of caveolin-1 around mulation in these cells may play a role in triggering injury. Also, specific and within the lipid droplets as well as in the inner membrane of mito- subcellular sites of FC accumulation in hepatocytes may also be relevant chondria was found in a murine model of liver steatosis created by feed- to promote either mitochondrial dysfunction or ER stress. Current evi- ing adult rats a choline-deficient diet for 14 days [168]. Moreover, dence on these topics is summarized below. increased levels of caveolin-1 mRNA and protein were found in a NAFLD model after feeding C57BL/6 mice with a high fat and cholesterol diet for 14 weeks [169]. In this study, caveolin-1 localization was al- 2.3.1. FC and activation of Kupffer and stellate cells tered, and the levels of this protein were increased in lipid droplets KCs are the resident macrophages in liver tissue that prevent harm- and mitochondria. These data suggest that the expression and distribu- ful endotoxins present in the portal vein from entering into the circula- tion of caveolin-1 are altered during abnormal hepatic lipogenesis and tion and are responsible for clearance of exogenous particulates and involves the mitochondria of steatotic hepatocytes. immunoreactive material that is perceived as foreign and harmful Niemann–Pick C1 and C2 proteins, NPC1 and NPC2, are involved in [187]. The functions of KC are precisely controlled to avoid an uncon- the trafficking of endocytosed lipoprotein cholesterol from the trolled inflammatory response. endolysosomal compartment to the rest of the cell [170,171].Adeficien- Increased evidence suggests that inflammation mediated by KCs is of cy in NPC1 or NPC2 is responsible for Niemann–Pick type C disease, critical importance in the development of NASH Thus, using chemicals which is a rare neurovisceral disorder characterized by FC accumulation to delete KCs has been demonstrated to alter the release of pro- in most tissues, including the liver [170,171]. Interestingly, decreased inflammatory cytokines and alleviate hepatocellular damage [188, NPC1 in mice is associated with weight gain and metabolic features as- 189]. Additionally, phagocytic dysfunction of KCs can accelerate inflam- sociated with IR, including hepatic steatosis and hyperinsulinemia [172, matory necrosis during hepatocyte fat accumulation [190]. Interesting- 173]. In humans, NPC1 single nucleotide polymorphisms (SNPs) have ly, recent studies have implicated cholesterol accumulation in KCs in been associated with obesity and IR in European populations [174,175]. inflammation animal models of NASH [191,192]. As expected, a high- A family of proteins containing steroidogenic acute regulator fat diet with cholesterol (HFC) induced hepatic inflammation and (StAR)-related lipid transfer (START) domains involved in cholesterol foamy KCs in LDL receptor-deficient and apolipoprotein E2 knock-in trafficking also seem to be particularly relevant [176]. The StAR protein mouse models that was found to be linked to increased plasma VLDL facilitates cholesterol movement from intracellular stores to the mito- cholesterol levels [193]. Interestingly, omitting cholesterol from the chondria of steroidogenic tissues stimulating steroid hormone synthesis HFC diet lowered plasma VLDL cholesterol and prevented the develop- [177]. Interestingly, mRNA levels of StAR were increased 7- and 15-fold ment of hepatic inflammation and foam KCs [193]. C57Bl/6 mice fed a in steatosis and NASH patients [94]. The closest StAR homologue is a high fat high cholesterol (1%) diet developed NASH and showed accu- protein known as MLN64 (STARD3), which is highly expressed in the mulation of cholesterol crystals in KCs and increased inflammasome liver [178] and is localized in the same compartment as NPC1 [179].In- activation [194]. Cholesterol crystals have been implicated in the activa- terestingly, other members of the family, including StARD4 and StARD5 tion of the inflammasome and macrophages in atherosclerotic lesions; are regulated by SREBP-2 and ER stress [180]. Importantly, these factors thus, these findings suggest that a similar process may occur in NASH are also involved in the pathogenesis and progression of NASH. The rel- [195,196]. These findings strongly suggest that cholesterol metabolism evance of the sterol trafficking capacity of StAR and MLN64 in regulating alterations may be relevant to KC activation in NAFLD/NASH. However, G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778 1771 other studies are required to determine the relevance of KC activation attenuated UPR and downstream inflammatory signaling although it inducedbyFCaccumulationinhumanpatients. did not prevent the development of diet-induced steatohepatitis HSCs, also called lipocytes, are one of the inherent liver [212]. These results suggest that JNK is a relevant player in the contribu- nonparenchymal cell types located in the Disse space between hepato- tion of the UPR-ER stress transduction pathway to the pathogenesis of cytes and sinusoidal endothelial cells. In the normal liver, HSCs are in a NAFLD. Supporting this idea, JNK signaling is also activated, as previous- ‘quiescent’ state [197]; however, when the liver is injured by viral infec- ly mentioned by saturated FA, and in conditions such as obesity and al- tion or hepatic toxins, HSCs are activated and acquire features that are tered insulin signaling in liver and adipose tissue [213]. relevant for the development of fibrogenesis [197–199]. Interestingly, Existing data indicate that ER stress induces lipid dysregulation, and recent evidence from animal models indicates that the major cause of hepatic lipid accumulation induces ER stress. Together, these factors accelerated liver fibrosis involved FC accumulation in HSC [200]. Several create a positive feedback loop, which may stimulate the development mechanisms have been proposed to explain the relationship between of hepatic NASH [214,215]. Accordingly, growing evidence suggests FC accumulation and fibrosis in HSC. FC accumulation in HSC increases that chronic ER stress induces numerous intracellular pathways that Toll-like receptor 4 protein (TLR4) levels by suppressing the endosomal- can lead to hepatic steatosis, systemic inflammation, and hepatocyte lysosomal degradation pathway of TLR4 and thereby downregulating cell death. All of these processes are known to be important in the path- bone morphogenetic protein and activin membrane-bound inhibitor ogenesis of NAFLD [210–212,216,217]. Moreover, increasing evidence (Bambi). Consequently, HSCs may become sensitized to transforming indicates that accumulation of cholesterol in the ER membrane induces growth factor β (TGFβ), resulting in HSC activation and progression of ER stress, apoptosis [218,219] and loss of the sarco/ER calcium ATPase liver fibrosis [96]. Recent evidence indicates that ACAT1 mediates liver fi- (SERCA)-mediated calcium homeostasis, which is presumably achieved brosis by regulating FC accumulation in HSC; therefore, the regulation of by promoting the formation of an ordered ER membrane phase that ACAT1 activity in HSCs could be a target for the treatment of liver fibrosis perturbs the SERCA structure [218] (Fig. 2). Other recent study revealed [201]. In summary, KC as well as HSC accumulates FC, which activates cas- that a regular diet enriched in cholesterol (2%) alone is sufficient to in- cades that may contribute to liver inflammation and fibrosis in NAFLD and duce hepatic ER–FC accumulation independent of tissue cholesterol NASH. concentrations [220]. Additionally, ER stress impairs cholesterol efflux and synthesis in human hepatic cells via reduction of HMGCR activity, 2.3.2. The unfolded protein response and may reduce HDL biogenesis due to impaired hepatic ABCA1 func- The ER is the intracellular organelle responsible for the synthesis and tion [221]. Taken together, these data strongly supports the contribu- processing of proteins [202,203]. Moreover, the ER controls cellular cho- tion of the UPR–ER stress to the pathogenesis of NAFLD (Fig. 2). lesterol levels through pathways that sense the level of cholesterol or Interestingly, mitochondria also present an UPR that is activated by cholesterol derivatives within the ER membrane itself and relay signals alterations in mitochondrial proteostasis, such as by the accumulation controlling cholesterol synthesis and clearance [204]. Conditions that of un-assembled or unfolded proteins in the mitochondria [222]. The ac- impair ER protein homeostasis (proteostasis) or change the protein tivation of mitochondrial UPR leads to the expression of nuclear- folding capacity of the ER can lead to ER stress and induce the unfolded encoded genes such as mitochondrial chaperones and proteases that re- protein response (UPR) [205–209]. The UPR is induced through the ac- establish mitochondrial proteostasis and is a key mechanism to main- tivation of three transmembrane ER stress sensing kinases; PKR-like eu- tain mitochondrial health [222]. Of note, the mitochondrial and ER karyotic initiation factor 2 kinase (PERK), activating transcription factor UPR share some of their transduction pathways in mammals, including 6 (ATF6) and inositol requiring 1 α(IRE1α), which aim to restore nor- phosphorylation of eIF2α, which inhibits cytosolic protein translation mal ER function [210]. These 3 proteins induce a signaling cascade [222]. The relationship between cholesterol levels, mitochondrial UPR that briefly consist in: i) PERK mediated phosphorylation of eukaryotic and liver disease has not yet been addressed. However, the hepatic ex- initiation factor 2a (eIF2α) that leads to transient attenuation of transla- pression of genes related with mitochondrial UPR is altered in high fat tion but selective translation of mRNAs containing upstream open read- diet fed mice [223]. ing frames, such as activating transcription factor 4 (ATF4). Increased transcription and translation of GADD34 subsequently lead to de- 2.3.3. Mitochondrial dysfunction phosphorylation of eIF2a and resumption of translation; ii) activation Recent evidence indicates that the accumulation of cholesterol in of IRE1a that leads to the splicing of XBP1 generating the XBP1s tran- mitochondria results in mitochondrial dysfunction and impairment of scription factor; and iii) cleavage and activation of transcription factor specific carriers, including the mitochondrial transport of GSH, through 6 (ATF6), which in conjunction with ATF4 and XBP1s lead to transcrip- alterations in the mitochondrial membrane order [224–226]. tional activation of a number of gene targets related to protein folding Mitochondria contain cholesterol in very low amounts when com- and ER-associated degradation [210]. pared with other cellular compartments, such as the plasma membrane, Interestingly, the feedback inhibitory pathway comprising GADD34 and are highly sensitive to changes in membrane fluidity induced by mediated eIF2-α de-phosphorylation prevents the initiation of apopto- cholesterol enrichment [227]. An increase in cholesterol mitochondrial tic and inflammatory pathways. If the cellular stressor exceeds the ER's content induces decreases in ATP synthesis, which is mediated by ATP ability to restore normal ER function or the feedback inhibitory mecha- synthase, and in the ATP/ADP exchange in the inner and outer mito- nisms are inadequate, inflammatory and apoptotic pathways are initiat- chondrial membranes, which is mediated by the adenine nucleotide ed via the activation of protein kinases such as JNK [210]. translocase and the voltage dependent anion channel, respectively Variable degree of UPR activation in NAFLD and NASH has been re- [228–230]. ported. Livers from NAFLD and NASH were characterized by increased Moreover, mitochondrial cholesterol content is relevant for the trans- phosphorylation of eIF2a, whereas several other markers of ER stress port of mitochondrial-reduced glutathione (mGSH), an essential antioxi- were not increased, including ATF4 mRNA and protein, GADD34 dant that controls reactive oxygen species (ROS) generation and prevents mRNA and unspliced XBP1 mRNA. Livers from NASH subjects were ad- mitochondrial dysfunction and cell death (Fig. 2). mGSH is synthesized de ditionally characterized by a reduction in the amount of XBP1s and in- novo in the cytosol and transported by the mitochondrial membrane by creased phosphorylation of JNK [211]. Dysregulation of the UPR has the 2-oxoglutarate carrier [231]. This carrier is highly sensitive to changes also been observed in db/db mice with diet-induced steatohepatitis in mitochondrial membrane cholesterol content and fluidity. In fact, it has which presented an inadequate activation of recovery pathways been found that cholesterol incorporation in isolated mitochondria de- (GADD34, XBP-1(s)) and accentuated activation of injury pathways re- creases mGSH stores [224]. Decreased levels of the antioxidant mGSH lated to persistent eif2-α phosphorylation (ATF4, JNK, and nuclear fac- sensitize hepatocytes to oxidative stress and inflammatory cytokines. tor kappaB (NF-κB)) [212]. Interestingly, pharmacologic JNK inhibition The survival of the cell is dependent on mGSH because it protects 1772 G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778

Fig. 2. Relevance of free cholesterol accumulation to liver damage in NAFLD/NASH. FC accumulation leads to liver injury in NASH/NAFLD through the activation of intracellular signaling pathways that influence the major hepatic cells: Kupffer cells (KCs), stellate cells (HSCs) and hepatocytes. FC accumulation in KCs and HSCs correlates with their activation. Activation of KCs causes the secretion of pro-inflammatory mediators (e.g., interleukin-6 and 8 and tumor necrosis factor-alpha [TNFα]) and pro-fibrotic factors (i.e., transforming growth factor β [TGFβ]) that influence neighboring cells and induce inflammation. In addition, high FC levels in HSCs upregulate Toll-like receptor 4 (TLR-4), a lipopolysaccharide receptor, and sensitize HSCs to TGFβ. These changes result in HSC activation and increased liver fibrogenesis. Finally, FC accumulation in hepatocytes induces itself lipid peroxidation and lipotoxicity leading to cellular dysfunction and death. Among the specific mechanisms of hepatocyte damage, it has been shown that FC accumulation in the mitochondria causes mitochondrial dysfunction and induces an increase in reactive oxygen species (ROS); furthermore, FC overload in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) and ER stress and apo- ptosis through JNK activation. Notably, ROS and lipid peroxidation products are able to diffuse into the extracellular space affecting both KCs and HSCs. These events lead to a vicious circle that causes continual liver damage, inflammation, and steatosis that ultimately leads to the progression to NAFLD. cardiolipin from oxidation to peroxidized cardiolipin, which determines to the mitochondrial inner membrane in both wild-type and NPC1 defi- mitochondrial membrane permeabilization by proapoptotic bcl-2 family cient cells, demonstrating a significant role for MLN64 in the mitochon- members [227]. These proteins increase cell death and may contribute drial delivery of cholesterol [236]. Interestingly, we found increased to liver injury in fatty liver disease. MLN64 liver expression in NPC1-deficient mice (Balboa et al., unpub- Multiple experimental models showed that FC accumulation in mi- lished observation), which show liver damage, and in mice with a cho- tochondria sensitizes hepatocytes to TNF-α leading to mGSH depletion lestatic hepatocyte injury induced by a diet high in CDCA, which is a [95]. Moreover, recent studies in primary mouse hepatocytes loaded well-characterized model of apoptosis-related liver damage induced with FC and incubated with LDL showed that mitochondrial cholesterol by BA [235]. These data suggest that MLN64 plays a role in the response deposition caused hepatocyte apoptosis and necrosis by activating the of hepatic cells to different insults. JNK1 pathway [232]. These effects were accompanied by activation of By contrast, recent studies in rats have shown that loss of hepatic ex- the mitochondrial membrane pore transition, cytochrome c release, ox- pression of MLN64 induced by genetic obesity may contribute to the idative stress and ATP depletion [233]. pathogenesis of dyslipidemia and steatosis [237]. The authors also The StAR protein has been proposed as a candidate protein mediat- showed that MLN64 overexpression increased lipidation of exogenous ing the increase in cholesterol transport to the mitochondria [16].This apoA-I and facilitated the de novo biosynthetic pathways for neutral protein was originally described for its critical role mediating cholester- lipids in hepatic cells lines. These effects potentiated TG accumulation ol flux toward the mitochondrial inner membranes for steroid synthesis but possibly offered protection against lipotoxicity. The authors pro- in steroidogenic tissues [234]. However, StAR is not only expressed in posed that MLN64 may increase circulating levels of HDL and protect steroidogenic tissues, such as the adrenal cortex and the gonads, but it the liver against lipotoxicity. is also expressed in many other tissues, including the liver, and may Finally, JNK1 activation has been found as a relevant consequence function as a mitochondrial cholesterol transport facilitator. As men- mitochondrial FC deposition in experimental models. Recent work tioned before, mRNA levels of StAR were increased several folds in from Gan et al. [186] demonstrated that JNK1 drives mitochondrial steatosis and NASH patients [94]. cell death pathways leading to release of high mobility group box 1 Another candidate protein mediating cholesterol transport to the (HMGB1) protein which in turn activates TLR4 in neighboring hepato- mitochondria in liver is MLN64. This protein is located in late cytes thus promoting hepatocellular injury. This work is in agreement endosomes [179]. This specific localization suggests that MLN64 may with that of Li et al. examining the role of HMGB1 and TLR4 in the path- transport cholesterol derived from LDL. Furthermore, adenovirus- ogenesis of NASH [238]. Thus, JNK1 and TLR4 seem to be relevant path- mediated overexpression of MLN64 induces an increase in hepatic FC ways in FC lipotoxicity that may be eventually amenable of modulation that is associated with apoptosis and liver damage [235]. Additionally, with specific inhibitors [186]. MLN64 overexpression causes an increase in mitochondrial cholesterol In conclusion, the increase in mitochondrial FC content contributes content in the liver and correlates with decreased ATPase activity and to mitochondrial dysfunction, sensitizes hepatocytes to cytokine signal- decreased maximal respiration rates (Balboa et al., unpublished obser- ing and leads to mGSH depletion contributing to hepatocyte cell death vation). Knockdown of MLN64 by siRNA reduced cholesterol transport by apoptosis and necrosis (Fig. 2) [53,186]. Altered expression of G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778 1773 mitochondrial cholesterol transporters, which may contribute to the in- BA sequestrants (i.e., cholestyramine and colesevelam) are lipid- crease in mitochondrial FC content, and JNK1 activation could be poten- lowering agents that decrease plasma and intrahepatic cholesterol tial therapeutic targets in NAFLD. levels by promoting cholesterol catabolism through BA biosynthetic pathways [251] and improving hepatic insulin sensitivity [252].Inaddi- tion, these agents exert a positive influence on glucose metabolism by 3. Therapeutic perspectives improving glycemic control in diabetic patients. Thus, BA sequestration could be beneficial for NAFLD treatment. However, current experimen- Data discussed above points to a relevant role of disturbed hepatic tal and clinical data do not support this contention [253,254]. cholesterol homeostasis and FC accumulation in NAFLD pathogenesis and progression. In this setting, pharmacological modulation of dysreg- 3.2. FXR agonists ulated cholesterol metabolism pathways could represent an attractive therapeutic aim. Several currently available drugs and new agents that A wealth of information regarding the critical role of FXR in a variety are either in development or being tested in clinical trials may be useful of cellular processes in different tissues, mainly in the liver and intes- to treat NAFLD/NASH due to their effects on cholesterol metabolism. tine, is available. One of the main FXR target genes is the short heterodi- This issue is discussed in the following sections. mer partner SHP, which controls the expression of CYP7A1, the key enzyme in BA synthesis. Through this pathway, along with control of 3.1. Cholesterol lowering agents BA import and export through the regulation of membrane trans- porters, FXR regulates intracellular BA levels. This regulation is critical Reduction of intrahepatic cholesterol content can be achieved using for prevention of hepatocyte injury and death [255]. In addition, FXR in- commonly used drugs, such as statins and ezetimibe, which are consid- tricately controls different pathways in both lipid and carbohydrate me- ered safe in patients with NAFLD [110,239].Therefore,inadditiontoa tabolism, which may be beneficial in NAFLD [256,257]. A recent potential reduction in cardiovascular risk (i.e., coronary artery disease controlled trial showed histological benefits of a FXR agonist remains the most common cause of death in these patients, and the ma- (obeticholic acid) in patients with steatohepatitis [258]. The underlying jority of patients have dyslipidemia) [142], management of dyslipid- mechanisms of the positive effects of this drug in NASH likely occur at emia of NAFLD patients with statins, ezetimibe or a combination of multiple levels, including inhibition of de novo lipogenesis, stimulation both drugs could have beneficial effects on hepatic disease course of lipolysis, reduction of the inflammatory response and stimulation of [240,241]. In experimental models statin administration decreases intestinal pathways involving the secretion of FGF-19. Data on the ef- hepatic cholesterol content in high-fat fed mice [99] and reduces fects of FXR agonists on hepatic cholesterol content is inconclusive steatosis and liver injury in this stetting [242]. In addition to reducing [259,260]; it should be kept in mind that FXR agonism may affect hepatic cholesterol statins have pleiotropic actions including anti- serum lipoprotein concentrations through several mechanisms which inflammatory, immunomodulatory, antioxidative and antithrombotic may determine cardiovascular risks. In addition other side effects such actions that may also be at play in the beneficial effects observed in ex- as pruritus and a modest increase in insulin resistance were observed perimental NAFLD/NASH [241]. With regard to existing clinical data on in patients receiving obeticholic acid. These effects may impact the clin- the effects of statin use on liver histology in NAFLD, studies are very ical utility of this agent [261]. limited and conflicting [240,243,244]. The most recent study by Dongiovanni et al. [245] clearly suggests a protective effect of statins 3.3. Other agents on liver histology in subjects with NASH. This retrospective but well controlled and matched study demonstrated that statin use was associ- Other potential approaches to unload cholesterol from the liver to ated with less intense histological liver damage. Interestingly, individ- reduce hepatic injury in NAFLD/NASH include the use of thyromimetics uals harboring the I148M PNPLA3 risk variant gained limited benefit [56,262] and the activation of the glucagon-like peptide-1 receptor from statin use. Clearly, randomized clinical trials of adequate size and (GLP-1R) that has been shown to reduce lipid accumulation in peripher- duration are needed to confirm these findings. Of note, Dongiovanni al tissues and attenuate hepatic steatosis in preclinical studies [263, and other authors have reported that statins are under-prescribed in pa- 264]. Other novel approaches could be targeting microRNAs that regu- tients with NAFLD [240,245], since only 10% of adults with the disease late hepatic cholesterol synthesis, such as the use of antisense-based si- receive these drugs. This is indeed a low rate considering the cardiovas- lencing of miR-122 [265], treatment with probiotics [266],which cular risk of this patient population and is likely related to a fear to liver indirectly influences cholesterol metabolism in the liver and modula- toxicity, which is a rather rare event [240]. Thus, dyslipidemia manage- tion of JNK1 activation that reduces ongoing liver cell death [186]. Fur- ment with statins should not be withheld in individuals with NAFLD as ther studies are needed to clarify the potential of these approaches to this drug class significantly reduces cardiovascular events in these pa- be exploited therapeutically. tients [142] and might be of benefit for liver disease. With regard to ezetimibe, an agent that reduces intrahepatic choles- 4. Summary, conclusions and perspectives terol by inhibiting cholesterol absorption from the intestine through in- hibition of the NPC1L1 sterol transporter channel and modestly lowers In recent past, a growing body of evidence linking hepatic cholester- serum cholesterol levels [246], both preclinical studies [106,247] and ol accumulation to liver injury in setting of NAFLD/NASH had become clinical data [248,249] suggest that ezetimibe may be beneficial for available. Dietary sources as well as an extensive derangement in NAFLD. Interestingly, the combination of ezetimibe with statins, aiming numerous steps of hepatic cholesterol homeostasis including activation to enhance the hepatic effects of ezetimibe by inhibiting the compensa- of cholesterol biosynthetic pathways, increased cholesterol de- tory up-regulation of hepatic cholesterol synthesis induced by the de- esterification and attenuation of cholesterol export could contribute to crease in hepatic cholesterol content, is markedly efficient in reducing increase levels of FC both in hepatocytes and non-parenchymal hepatic hepatic FC content in experimental models having a significant impact cells (i.e. KC and HSC) and thereby drive NAFLD/NASH disease progres- in ongoing liver injury [99]. This data suggested that ezetimibe has po- sion. In hepatocytes, increased FC in subcellular compartments such as tential for NAFLD/NASH therapy. However, in a recent human trial the ER and mitochondria could trigger ER stress and mitochondrial dys- [250] showed that ezetimibe did not significantly influence either liver function leading to cell death by necrosis and apoptosis. In addition, FC fat or liver histology in patients with NASH. Thus, further well- promotes the activation of KC and HSC which fuels inflammation and designed randomized and fully powered trials are needed to establish fibrogenesis. Thus, hepatic accumulation of cholesterol rather than tri- the role of ezetimibe in NAFLD. glycerides is associated to ongoing hepatocyte death and liver damage 1774 G. Arguello et al. / Biochimica et Biophysica Acta 1852 (2015) 1765–1778 and could play a significant role in NAFLD disease progression from sim- [20] C.C. Duwaerts, J.J. Maher, Mechanisms of liver injury in non-alcoholic steatohepatitis, Curr. Hepatol. Rep. 13 (2014) 119–129. ple steatosis to steatohepatitis. Therefore, targeting cholesterol accumu- [21] M. 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