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Biochem. J. (2008) 416, e15–e17 (Printed in Great Britain) e15 doi:10.1042/BJ20081916 COMMENTARY Viral hepatitis and fatty liver disease: how an unwelcome guest makes pâté of the host Andrew J. BROWN1 BABS, School of Biotechnology and Biomolecular Sciences, Biosciences Building D26, University of New South Wales, Sydney, NSW 2052, Australia HBV and HCV (hepatitis B and C viruses respectively) affect hundreds of millions of people globally, and are a major cause of chronic liver disease, including NAFLD (non-alcoholic fatty liver disease). Previous work on HCV-associated fatty liver disease has implicated two transcription factors that are important in lipid metabolism, SREBP1c (sterol-regulatory-element-binding protein 1c) and the LXRα (liver X receptor α). HBV-associated fatty liver disease has been less well-studied. New work from Kim and colleagues in this issue of the Biochemical Journal has provided new insight into how HBV causes fatty liver disease. Investigating HBV’s so-called X gene product (HBx), they report that this viral protein directly binds to LXRα in the host liver cells to up-regulate the lipogenic transcription factor, SREBP1c. Also discussed in this commentary is another way that viruses such as HBV and HCV could induce SREBP1c-mediated lipogenesis, via the PI3K (phosphoinositide 3-kinase)–Akt signalling pathway. It is a curious thought that a virus can commandeer the lipid production machinery in the host’s liver to make their own version of pâté de foie gras. Fatty liver disease (hepatic steatosis) is a common consequence of infection by HBV or HCV (hepatitis B or C viruses respectively), occurring in roughly a quarter to a half of cases. These viruses are a major global cause of morbidity and mortality, chronically infecting more than half a billion people around the world. From fatty liver disease, a subset of infected individuals will go on to develop one of the most prevalent forms of human cancer, hepatocellular carcinoma [1,2]. Although causing similar disease outcomes, HBV and HCV are very different viruses, belonging to distinct families (Hepadnaviridae and Flaviviridae respectively). Whereas HCV is a positive-sense single-stranded RNA virus, HBV is made of circular partially double-stranded DNA. The genome of HBV encodes four overlapping open reading frames that are translated to make the viral core protein, the surface proteins, a reverse transcriptase, and the so-called X gene product of the human hepatitis B virus (HBx). Although the precise function of HBx remains elusive, it appears to be a multifunctional regulator that modulates many cellular processes by directly or indirectly interacting with host factors [1]. In this issue of the Biochemical Journal, Kim and colleagues [2] reveal novel molecular insights into how HBx may contribute to fatty liver disease induced by HBV infection. A fatty liver could be created in a number of ways, including decreased secretion of lipoproteins, reduced fat catabolism and increased fat synthesis (steatosis). There is some evidence that HBV and HCV may affect all three processes. However, the focus of the paper by Kim and colleagues [2] is on the steatosis side of the equation. The chief controllers of fat manufacture in animal cells are the SREBP (sterol-regulatory-element-binding protein) family of transcription factors. SREBP2 is principally in charge of regulating cholesterol synthesis and uptake, whereas SREBP1c’s primary responsibility is controlling the synthesis of fatty acids and their constituent lipids. As the storage form of fatty acids, triacylglycerol is the major lipid found in fatty liver disease, accumulating in vacuoles in the hepatocyte. Targets of SREBP1c include genes encoding key enzymes involved in fatty acid synthesis, ACC1 (acetyl-CoA carboxylase 1) and FAS (fatty acid synthase). SREBP1c also binds to its own promoter in a feed-forward loop. In addition, both SREBP1c and FAS gene expression are also positively regulated via LXR (liver X receptor) [3]. LXR is a nuclear receptor which complexes with the RXR (retinoid X receptor) to form a heterodimer. Two LXR isoforms have been characterized. LXRα is abundantly expressed in the liver and is also detected in other tissues (intestine, adipose tissue, spleen and macrophages), whereas LXRβ is ubiquitously expressed. Oxygenated forms of cholesterol, so-called oxysterols such as (24S),25-epoxycholesterol, are the natural ligands that activate LXR [4]. It is known that certain synthetic LXR agonists, such as T0901317, induce dramatic liver steatosis akin to that seen in viral hepatitis. Another impetus for the research by Kim et al. [2] was their previous findings that HBx induces hepatic steatosis via transcriptional activation of SREBP1c and PPARγ (peroxisome-proliferator-activated receptor γ ), a nuclear receptor best known for its role in adipogenesis [5]. In their study [2], Kim and colleagues employed a combination of molecular, cellular and animal models to investigate the influence of HBx on LXRα-mediated SREBP1c induction. Using a luciferase reporter assay in a variety of human liver cell lines, they showed that HBx increased LXRα transcriptional activity. This effect was most pronounced when cells were also treated with an LXR agonist (T0901317 or an oxysterol). Another clear indication for the involvement of LXRα was that the induction by HBx of SREBP1c and FAS gene transcription was abolished by small-interfering RNA directed against LXRα. Moreover, Kim et al. [2] provide evidence that HBx and LXRα co-localize within the nucleus and form a transcriptional protein complex on the LXREs (LXR-response elements) in the promoter of SREBP1c. 1 Key words: complex 1 of the mammalian target of rapamycin (mTORC1), hepatic steatosis, hepatitis B virus (HBV), oxysterol, statin, steatohepatitis. email [email protected] c The Authors Journal compilation c 2008 Biochemical Society e16 A. J. Brown Figure 1 A schematic representation of how HBV induces lipogenic gene expression via its protein HBx The promoters of both lipogenic genes, SREBP1c and FAS , contain at least one LXRE and SRE. Mechanism 1: as featured in the article by Kim et al. [2], HBx up-regulates lipogenic genes by binding directly to the ligand-binding domain of LXRα. The star symbolizes the LXR ligand which can either be an endogenously produced oxysterol or an administered synthetic agonist. The transcriptional co-activator, ASC2, enhances the effect of HBx on LXRα-mediated transactivation of lipogenic genes. Mechanism 2: HBx may also induce lipogenesis by increasing activation of SREBP1c through stimulation of the PI3K–Akt signalling pathway. This may occur by one or more different ways: (2a) stimulated activation of SREBP1c by Akt; or (2b) a downstream target via mTORC1; and/or (2c) increased stability of the nuclear SREBP1c via a GSK3β-mediated pathway. HBx is a small protein of 154 amino acids and is depicted as a spiky ball to signify its viral origin. See the text for more details. Specifically, HBx appears to interact with the ligand-binding domain of LXRα. The ability of HBx to up-regulate lipogenic genes (SREBP1c or FAS) via LXRα was potentiated by ASC2 (activating signal co-integrator-2), a transcriptional co-activator that mediates the transactivation of nuclear receptors including LXRα [6] (see Figure 1). Kim et al. [2] also presented data from an HBx-transgenic mouse model, where they demonstrated increased levels of SREBP1 and FAS protein in response to HBx. However, definitive proof for the involvement of LXRα in HBx-mediated lipogenesis in vivo would require additional control experiments. For example, is the lipogenic effect abolished if HBx-transgenic mice are crossed with LXRα-null animals? Overall, the studies of Kim et al. [2] firmly place HBx, LXRα and SREBP1c in the frame for the hepatic lipid accumulation often observed with HBV infection. This work also adds LXRα to a growing list of transcription factors with which HBx has been shown to interact [1]. The link between LXR, SREBP1c and steatosis has been noted before with HCV infection. HCV ‘non-structural protein2’ activated SREBP1c and FAS transcription which involved the LXREs and SREs (sterol-response elements) in the SREBP1c promoter [7]. In addition, HCV core protein and non-structural protein-4b also activated SREBPs [8]. In the light of Kim et al.’s study on HBx [2], it would be interesting to determine whether any of these HCV proteins also act through binding to LXRα. Therefore different viruses may cause lipid accumulation by common or at least similar pathways, although a particular virus may harness more than one strategy to do so. Besides inducing lipogenic genes, LXRα also represses various inflammatory genes in macrophages. Kim et al. [2] make the point that it is difficult to predict whether the inflammatory nature of chronic HBV infection might be offset to some extent by the antiinflammatory effect of LXRα activation by HBx. This may well be another cunning trick by the virus to weaken the host’s ability to fight it. LXRα also induces an array of genes involved in other aspects of lipid and lipoprotein metabolism, such as cholesterol export from the cell (ATP-binding cassette proteins ABCA1 and c The Authors Journal compilation c 2008 Biochemical Society ABCG1), apolipoproteins (apoE, apoD and apoAIV), transporters and enzymes involved in lipoprotein metabolism (cholesteryl ester and phospholipid transfer proteins, and lipoprotein lipase), as well as genes involved in enhancing bile acid transport and metabolism. Since HBV infection is associated with general liver pathology, and given the complexity of LXRα responses, it is difficult to extrapolate how HBx-mediated activation of LXRα in the liver might have an impact on overall lipoprotein metabolism. Another way that viruses such as HBV and HCV could induce lipogenesis is via the PI3K (phosphoinositide 3-kinase)–Akt signalling pathway. The PI3K–Akt pathway is commonly activated in response to growth factors and hormones such as insulin that act through cell-surface receptors. Virtually all mammalian viruses, both DNA and RNA, activate this pathway at some point in their life cycle to benefit from the growth, metabolic, survival and translational functions that the pathway controls [9]. Indeed, HBx protects against apoptotic cell death by exploiting PI3K–Akt signalling in the host’s liver cells [10]. Importantly, PI3K– Akt signalling also activates SREBPs ([11,12] and references therein), suggesting another mechanism through which viruses can up-regulate lipogenesis. Activation of SREBPs occurs via some elegant cell biology, involving regulated transport of the inactive membrane-bound precursor from the ER (endoplasmic reticulum) to the Golgi apparatus, where proteolytic processing occurs to release the truncated active transcription factor. The best documented regulation is via feedback inhibition of ERto-Golgi transport of precursor SREBP by sterols (cholesterol and oxysterols) [3]. Beyond the familiar lipid end-products, it is now becoming increasingly clear that PI3K–Akt signalling represents another important input into the regulation of SREBP activation. Thus we have shown that the PI3K–Akt pathway is involved in the ER-to-Golgi trafficking of SREBP2 [11]. Others have shown that PI3K–Akt signalling stabilizes nuclear SREBP1c [via phosphorylation by the Akt substrate GSK3β (glycogen synthase kinase-3β)], and stimulates lipogenesis via activation of an Akt downstream target, mTORC1 (complex 1 of the mammalian target of rapamycin) ([12] and references therein) (Figure 1). However, it should be noted that SREBP2 transport and processing are unaffected by rapamycin treatment [13], indicating that PI3K– Akt signalling may activate SREBP1c and SREBP2 via different means. Irrespective of the precise mechanism(s) involved, it is likely that many viruses, including HBV and HCV, will also stimulate lipid synthesis via PI3K–Akt signalling. In terms of clinical implications for the paper by Kim et al. [2], there is ongoing interest in LXR agonists as a potential drug treatment for various human diseases, notably cardiovascular disease. First-generation synthetic LXR agonists have shown beneficial effects in various animal models of human disease, and yet have not progressed to the clinic owing to problems with hepatic steatosis. Second-generation agonists are under investigation and hold promise in avoiding these deleterious lipogenic side-effects. Kim et al. [2] found that HBx enhanced the transcriptional activity of LXRα in the absence of added LXR agonist, suggesting that there may be sufficient endogenous ligand available to activate LXRα. It should be noted that endogenous oxysterol ligands have an advantage over their synthetic non-steroidal counterparts: they suppress the proteolytic activation of SREBP1c which helps to counteract the stimulation of SREBP1c gene expression via LXR. (24S),25-Epoxycholesterol is a potent LXR ligand produced in human liver by the same pathway that synthesizes cholesterol, and can thus be inhibited by the statin class of drugs [4]. It would be interesting to determine whether a statin would abolish the effect of HBx on LXRα-mediated SREBP1c induction in liver cells. Conversely, statin treatment may run the risk of increasing local production of other oxysterol ligands for LXRα, derived Commentary from cholesterol, by increasing hepatic clearance of circulating lipoproteins. In conclusion, the study by Kim et al. [2] has shed new light on the mechanism by which a prevalent human pathogen, HBV, causes lipid accumulation in the liver. LXRα represents a vital link between the viral protein, HBx, and the host’s lipogenic transcription factor, SREBP1c, and may be the missing ingredient in HBV’s recipe for fatty liver disease. REFERENCES 1 Nguyen, D. H., Ludgate, L. and Hu, J. (2008) Hepatitis B virus–cell interactions and pathogenesis. J. Cell. Physiol. 216, 289–294 2 Kim, K., Kim, K. H., Kim, H. H. and Cheong, J. (2008) Hepatitis B virus X protein induces lipogenic transcription factor SREBP1 and fatty acid synthase through the activation of nuclear receptor LXRα. Biochem. J. 416, xxxx–xxxx 3 Horton, J. D., Goldstein, J. L. and Brown, M. S. (2002) SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest. 109, 1125–1131 4 Brown, A. J. (2008) 24(S ),25-Epoxycholesterol: a messenger for cholesterol homeostasis. Int. J. Biochem, Cell Biol., doi:10.1016/j.biocel.2008.05.029 5 Kim, K. H., Shin, H. J., Kim, K., Choi, H. M., Rhee, S. H., Moon, H. B., Kim, H. 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Commun. 373, 670–674 Received 22 September 2008; accepted 30 September 2008 Published on the Internet 12 November 2008, doi:10.1042/BJ20081916 c The Authors Journal compilation c 2008 Biochemical Society