From Fat to Inflammation

Abstract

It is clear from both cross-sectional and natural history studies that only a minority of patients with risk factors for developing nonalcoholic fatty liver disease (NAFLD) will progress from simple fatty liver (steatosis) to inflammation (steatohepatitis), fibrosis, and ultimately cirrhosis and hepatocellular cancer.1Day C.P. Natural history of NAFLD remarkably benign in the absence of cirrhosis.Gastroenterology. 2005; 129: 375-378Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar A clear understanding of the mechanisms that lead to this progression is urgently required to inform the rational design of treatment and prevention strategies for patients who have developed, or are at risk of developing, advanced disease. Data from several studies published in the last 2 years, largely in the field of insulin resistance, have begun to shed light on the link between hepatic steatosis–hitherto considered a purely “metabolic” disease, hepatic inflammation, and the development of obesity-associated insulin resistance and type 2 diabetes mellitus (T2DM) (Figure 1). Recent studies in genetic- (Zucker rats) and high-fat diet (HFD)–induced obesity in rodents have shown conclusively that hepatic steatosis is associated with a state of chronic hepatic inflammation.2Cai D. Yuan M. Frantz D. Melendez P.A. Hansen L. Lee J. Shoelson S.E. Local and systemic insulin resistance resulting from hepatic activation of IKK-β and NF-κβ.Nat Med. 2005; 11: 183-190Crossref PubMed Scopus (1799) Google Scholar, 3Arkan M.C. Hevener A.L. Greten F.R. Maeda S. Li Z.-W. Lond J.M. Wynshaw-Boris A. Poli G. Olefsky J. Karin M. IKK-β links inflammation to obesity- induced insulin resistance.Nat Med. 2005; 11: 191-198Crossref PubMed Scopus (1481) Google Scholar Both of these models are characterized by an increase in the hepatic activities of nuclear factor κB (NF-κB), and its upstream activator IKK-β NF-κB is a sequence-specific transcription factor that functions as a proinflammatory “master switch” during inflammation, up-regulating the transcription of a wide range of inflammatory mediators. Accordingly, increased NF-κB activity in the livers of HFD-fed mice is associated with the increased hepatic expression of proinflammatory cytokines, including TNFα, IL-6, and IL-1β, and activation of Kupffer cells. Hepatocyte-specific inhibition of NF-κB attenuates the increased inflammatory gene expression and Kupffer cell activation in response to an HFD. The HFD phenotype can be reproduced by hepatocyte-specific expression of constitutively active IKK-β HFD mice; hepatocyte-expressing IKK-β mice also exhibit hepatic and systemic insulin resistance that can be attenuated by hepatocyte-specific NF-κB inhibition, consistent with the known inhibitory effects of TNFα, IL-6, and IL-1β on insulin signalling via the JNK1, PKC, and suppressors of cytokine signalling (SOCS) pathways.4Samuel V.T. Liu Z.-X. Qu X. Elder B.D. Bilz S. Befroy D. Romanelli A.J. Shulman G.I. Mechanism of hepatic insulin resistance in non alcoholic fatty liver disease.J Biol Chem. 2004; 279: 32345-32353Crossref PubMed Scopus (1033) Google Scholar, 5Ueki K. Kondo T. Tseng Y.-H. Kahn C.R. Central role of suppressors of cytokine signaling proteins in hepatic steatosis, insulin resistance, and the metabolic syndrome in the mouse.Proc Natl Acad Sci U S A. 2004; 101: 10422-10427Crossref PubMed Scopus (321) Google Scholar This study shows clearly that obesity-related hepatic steatosis is associated with the increased production of inflammatory cytokines by hepatocytes that results from NF-κB activation and leads to Kupffer cell activation and hepatic and systemic insulin resistance. Although not addressed in this study, the production of inflammatory cytokines by hepatocytes and activated Kupffer cells seems likely to play a major role in the progression from steatosis to steatohepatitis. Cytokines are capable of producing all of the classical histological features of NASH including hepatocyte necrosis/apoptosis (TNFα/TGFβ), neutrophil chemotaxis (IL-8), and activation of hepatic stellate cells (HSC) (TNFα/TGFβ) and Mallory bodies (TGFβ). Moreover, human studies have demonstrated increased cytokine gene expression in the livers of patients with NASH compared to obese controls with normal livers, with the increased expression correlating with histological severity.6Crespo J. Cayon A. Fernandez-Gil P. Hernandez-Guerra M. Mayorga M. Dominguez-Diez A. Fernandez-Escalante J.C. Pons-Romero F. Gene expression of tumour necrosis factor α and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients.Hepatology. 2001; 34: 1158-1163Crossref PubMed Scopus (583) Google Scholar Recent reports that apoptosis is an important mode of cell death in NASH make TNFα a particularly attractive candidate for a role in mediating liver injury given its ability to induce apoptosis in hepatocytes under conditions of oxidative stress known to be present in the livers of both animal models and patients with NAFLD. The production of cytokines by hepatocytes and subsequent Kupffer cell activation may also explain the alterations in the hepatic innate immune system recently reported to occur in HFD-fed mice, which may further contribute to a proinflammatory state.7Li Z. Soloski M.J. Diehl A.M. Dietary factors alter hepatic innate immune system in mice with nonalcoholic fatty liver disease.Hepatology. 2005; 42: 880-885Crossref PubMed Scopus (253) Google Scholar Until recently, the precise mechanisms of hepatocyte activation of NF-κB associated with the development of HFD-induced steatosis remained unclear, with some suggesting a role for lipid peroxidation products arising as a result of increased free fatty acid (FFA) oxidation by mitochondria.3Arkan M.C. Hevener A.L. Greten F.R. Maeda S. Li Z.-W. Lond J.M. Wynshaw-Boris A. Poli G. Olefsky J. Karin M. IKK-β links inflammation to obesity- induced insulin resistance.Nat Med. 2005; 11: 191-198Crossref PubMed Scopus (1481) Google Scholar However, a recent study has clearly demonstrated that FFAs can directly activate the IKK-β/NF-κB pathway in hepatocytes via a lysosomal, cathepsin B–dependent mechanism.8Feldstein A.E. Werneburg N.W. Canbay A. Guicciardi M.E. Bronk S.F. Rydzewski R. Burgart L.J. Gores G.J. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-α expression via a lysosomal pathway.Hepatology. 2004; 40: 185-194Crossref PubMed Scopus (671) Google Scholar This pathway involves the translocation of Bax to lysosomes with subsequent lysosomal destabilization and release of the cysteine protease, cathepsin B, into the cytosol. This subsequently leads to the activation of NF-κB, via IKK-β, and a subsequent increase in the expression of TNFα. Using a sucrose-rich diet-induced model of NAFLD, these investigators were able to show that genetic or pharmacological inactivation of cathepsin B protects against the development of hepatic steatosis, liver injury, and insulin resistance, and they also observed a redistribution of cathepsin B from lysosomes to cytosol in livers from humans with NASH. FFAs are attractive candidates for the role of inflammatory mediators in NAFLD since central obesity, which has been linked to an increased risk of NASH and has recently been shown to be associated with an increased lipolysis of visceral adipose tissue and an increased hepatic supply of FFA.9Nielsen S. Guo Z. Johnson C.M. Hensrud D.D. Jensen M.D. Splanchnic lipolysis in human obesity.J Clin Invest. 2004; 113: 1582-1588Crossref PubMed Scopus (688) Google Scholar Furthermore, a recent study using stable isotope methodology has demonstrated that almost two thirds of fat in the livers of patients with NAFLD is derived from circulating FFA as opposed to dietary sources or de novo lipogenesis.10Donnelly K.L. Smith C.I. Schwarzenberg S.J. Jessurun J. Boldt M.D. Parks E.J. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with non alcoholic fatty liver disease.J Clin Invest. 2005; 115: 1343-1351Crossref PubMed Scopus (2427) Google Scholar A further potential link between steatosis, hepatocyte NF-κB activation, liver injury, and insulin resistance in NAFLD has been suggested by a recent study demonstrating that hepatic endoplasmic reticulum (ER) stress is a feature of HFD- and genetic (Ob/Ob)–induced obesity in rodents.11Ozcan U. Cao Q. Yilmaz E. Lee A.-H. Iwakoshi N.N. Ozdelen E. Tuncman G. Gorgun C. Glimcher L.H. Hotamisligil G.S. Endoplasmic reticulum stress links obesity, insulin action and Type 2 diabetes.Science. 2004; 306;: 457-461Crossref PubMed Scopus (2986) Google Scholar The endoplasmic reticulum (ER) is a membranous network that provides a specialized environment in which membrane and secretory proteins achieve correct folding due to the presence of specialized chaperones. As metabolic demands increase, so does the workload of the ER. Biological stress including excess lipids and hyperinsulinemia, hypoxia, and viral infections can disrupt ER homeostasis and elicit the so-called ER stress response, which results in the activation of a number of transcription factors and kinases.12Ron D. Translational control in the endoplasmic reticulum stress response.J Clin Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (740) Google Scholar This transcriptional program coordinates the unfolded protein response (UPR), which slows down protein synthesis and promotes protein degradation. However, if this response is inadequate, a variety of proteins are activated as part of the ER overload response which can lead to insulin resistance (via IRE1, JNK1), apoptosis (via caspase-12 in mouse cells and caspase-4 in human cells), inflammation (via NF-κB) and mitochondrial dysfunction (via BAP31).13Hidvegi T. Schmidt B. Hale P. Perlmutter D.H. Accumulation of mutant α1ATZ in the ER activates caspases -4 and -12, NFκB and BAP31 but not the unfolded protein response.J Biol Chem. 2005; 280: 39002-39015Crossref PubMed Scopus (209) Google Scholar Evidence that this response plays a role in human NAFLD is at present indirect, although ER stress is increasingly recognized as an important mechanism of alcohol-induced liver disease. In addition to hepatocyte-derived cytokines, at least two other mechanisms have been postulated to play a role in the activation of Kupffer cells in NAFLD and therefore contribute to the pathogenesis of hepatic inflammation and cell injury. First, the clearance of oxidized lipid deposits in lipoproteins is a major function of Kupffer cells via their scavenger receptors. The combination of steatosis and oxidative stress observed in NAFLD may therefore lead to Kupffer cell activation via this route. Second, evidence from animal models14Yang S.Q. Lin H.Z. Lane M.D. Clemens M. Diehl A.M. Obesity increases sensitivity to endotoxin liver injury implications for the pathogenesis of steatohepatitis.Proc Natl Acad Sci U S A. 1997; 94: 2557-2562Crossref PubMed Scopus (691) Google Scholar and humans with NASH suggests that gut-derived portal endotoxin may be a further stimulus for Kupffer cell activation in NAFLD. Small bowel bacterial overgrowth has certainly been reported in obese and diabetic patients and attributed to hyperinsulinemia-induced impaired gut motility. The observation that hepatocyte inhibition of NF-κB is enough to block the increase in proinflammatory gene expression and macrophage activation in the HFD mouse model of obesity does, however, argue against a hepatocyte-independent route of Kupffer cell activation, at least in this animal model. Finally, recent evidence that, as in the liver, adipose tissue in obesity is characterized by macrophage infiltration and associated chronic inflammation15Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr, A.W. Obesity is associated with macrophage accumulation in adipose tissue.J Clin Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7481) Google Scholar suggests a further potential source of inflammatory cytokines that may play an endocrine role in the progression of steatosis to steatohepatitis. The precise stimulus to adipose tissue macrophage activation is unknown, but cytokine release from metabolically “stressed” adipocytes, macrophage scavenging of oxidized lipids, and systemic endotoxemia may all be important. While the production of classical proinflammatory cytokines by these adipose tissue macrophages seems likely to play a role in obesity-related systemic insulin resistance via paracrine effects on adipocytes and muscle,3Arkan M.C. Hevener A.L. Greten F.R. Maeda S. Li Z.-W. Lond J.M. Wynshaw-Boris A. Poli G. Olefsky J. Karin M. IKK-β links inflammation to obesity- induced insulin resistance.Nat Med. 2005; 11: 191-198Crossref PubMed Scopus (1481) Google Scholar this seems unlikely to exert significant endocrine effects on the liver given the potent intrahepatic sources of cytokines discussed previously. However, the increased circulating levels of the adipocyte-derived cytokine (“adipokine”), leptin seen in obesity may play a role in the progression of steatosis to NASH. Leptin stimulates the release of the Th1 proinflammatory cytokine osteopontin from hepatocytes and osteopontin “knockout” mice are protected from hepatic inflammation and fibrosis in the methionine-choline deficient (MCD) diet-induced model of NASH.16Sahai A. Malladi P. Melin-Aldana H. Green R.M. Whitington P.F. Upregulation of osteopontin expression is involved in the development of non alcoholic steatohepatitis in a dietary murine model.Am J Physiol Gastrointest Liver Physiol. 2004; 287: 264-273Crossref Scopus (161) Google Scholar A reduction in circulating levels of the anti-inflammatory adipokine, adiponectin, seen in obesity may also contribute to the progression of NAFLD. The anti-inflammatory effects of adiponectin are most likely related to its ability to suppress the synthesis and action of TNFα.17Maeda N. Shimomura I. Kishida K. Nishizawa H. Matsuda M. Nagaretani H. Furuyama N. Kondo H. Takahashui M. Arita Y. Komuro R. Ouchi N. Kihara S. Tochino Y. Okutomi K. Horie M. Takeda S. Aoyama T. Funahashi T. Matsuzawa Y. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30.Nat Med. 2002; 8: 731-737Crossref PubMed Scopus (1814) Google Scholar Delivery of recombinant adiponectin in the Ob/Ob mouse model of NAFLD alleviates hepatomegaly, steatosis, and abnormal biochemistry.18Xu A. Wang Y. Keshaw H. Xu L.Y. Lam K.S. Cooper G.J. The fat-derived hormone adiponectin alleviates alcoholic and non alcoholic fatty liver disease in mice.J Clin Invest. 2003; 112: 91-100Crossref PubMed Scopus (1137) Google Scholar The reduction in circulating adiponectin levels reported in obesity and T2DM has been attributed to suppression of its synthesis by the paracrine effects of TNFα released by adipose tissue macrophages. Recent studies in humans showing reduced serum levels of adiponectin and reduced hepatic expression of its receptor, RII, in patients with NASH compared with body mass index–matched patients with steatosis,19Hui J.M. Hodge A. Farrell G.C. Kench J.G. Kriketos A. George J. Beyond insulin resistance in NASH TNFα or adiponectin?.Hepatology. 2004; 40: 46-54Crossref PubMed Scopus (891) Google Scholar, 20Kaser S. Moschen A. Cayon A. Kaser A. Crespo J. Pons-Romero F. Ebenbichler C.F. Patsch J.R. Tilg H. Adiponectin and its receptors in non-alcoholic steatohepatitis.Gut. 2005; 54: 117-121Crossref PubMed Scopus (368) Google Scholar provide strong supportive evidence that reduced adipocyte production of adiponectin plays an important role in the pathogenesis of progressive NAFLD. With respect to mechanisms of hepatic fibrosis in NAFLD, it is clear that the mechanisms of inflammation and hepatocyte injury outlined previously will lead to the activation of HSC and deposition of extracellular matrix proteins as part of the normal “healing” response. In addition, an increasing body of evidence supports a role for non-necroinflammatory mediators related to obesity and insulin resistance in the pathogenesis of liver fibrosis in NAFLD. Direct, HSC-activating, profibrogenic roles have been demonstrated for leptin (acting directly and via osteopontin), angiotensin II, and norepinephrine, all of which are secreted by adipose tissue and are raised in the serum of obese patients. A direct fibrogenic role for insulin resistance–associated hyperglycemia and hyperinsulinemia has been suggested by studies showing that the synthesis of the fibrogenic growth factor, connective tissue growth factor (CTGF), by HSC is up-regulated by glucose and insulin. The reduced production of adiponectin associated with obesity may also contribute to the development of liver fibrosis since it appears to exert potent anti-fibrotic effects. Finally, in addition to fibrosis, given the emerging role of inflammation/NF-κB in carcinogenesis,21Pikarsky E. Porat R.M. Stein I. Abramovitch R. Amit S. Kasem S. Gutkovich-Pyest E. Urieli-Shoval S. Galun E. Ben-Neriah Y. NFκB functions as a tumor promoter in inflammation-associated cancer.Nature. 2004; 431: 461-465Crossref PubMed Scopus (2147) Google Scholar it seems likely that the chronic inflammatory state associated with steatosis also contributes to the development of HCC–now a well established complication of advanced NAFLD. In conclusion, these recent studies, largely in the field of metabolic medicine, provide new insights into the link between hepatic steatosis, inflammation, fibrosis, and cancer, and suggest several new research directions in this increasingly common disease. At present, strategies aimed at reducing hepatic FFA supply would seem to hold the most promise, with encouraging results already reported for weight loss and insulin sensitizers. Interestingly, the thiazolidinedione class of insulin sensitizers may exert their action predominantly via anti-inflammatory effects on macrophages, leading to a reduction in the production of insulin resistance–inducing cytokines and a subsequent increase in adiponectin production by adipocytes. For the future, IKK-β/NF-κB, cathepsin B, osteopontin, and the ER stress response appear to be rational treatment targets, although further evidence for their role in the pathogenesis of human NAFLD is required before pilot studies can be justified. However, systemic inhibition of IKK-β with high-dose aspirin has been shown to improve glycemia, insulin sensitivity, and hyperlipidemia in patients with T2DM.22Hundal R.S. Mechanism by which high dose aspirin improves glucose metabolism in type 2 diabetes.J Clin Invest. 2002; 109: 1321-1326Crossref PubMed Scopus (591) Google Scholar These new insights into disease pathogenesis may also lead to a better understanding of the basis for the clearly apparent interindividual variation in susceptibility to advanced NAFLD. Family studies and ethnic variations suggest a role for genetic factors,23Day C.P. The potential role of genes in nonalcoholic fatty liver disease.Clin Liver Dis. 2004; 8: 673-691Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar and these recent studies suggest a number of new candidate genes worthy of study in allelic association studies.