Nonalcoholic Fatty Liver Disease: Cytokine-Adipokine Interplay and Regulation of Insulin Resistance

Abstract

Nonalcoholic fatty liver disease (NAFLD), the major reason for abnormal liver function in the Western world, is associated with obesity and diabetes and is characterized by insulin resistance (IR). IR is regulated by mediators released from cells of the immune system or adipocytes and proinflammatory cytokines such as tumor necrosis factor-α (TNFα). The importance of TNFα in human and animal fatty liver diseases, both caused by genetic manipulation and overnutrition, has been shown convincingly. Furthermore, neutralization of TNFα activity improves IR and fatty liver disease in animals. Adiponectin is a potent TNFα-neutralizing and anti-inflammatory adipokine and in vitro and experimental animal studies have proven the importance of this mediator in counteracting inflammation and IR. Anti-inflammatory effects of adiponectin are exerted both by suppressing TNFα synthesis and by induction of anti-inflammatory cytokines such as interleukin-10 or interleukin-1–receptor antagonist. Therefore, the balance between various mediators, either derived from the immune system or adipose tissue, appears to play an important role in hepatic and systemic insulin action and in the development of fatty liver disease. Nonalcoholic fatty liver disease (NAFLD), the major reason for abnormal liver function in the Western world, is associated with obesity and diabetes and is characterized by insulin resistance (IR). IR is regulated by mediators released from cells of the immune system or adipocytes and proinflammatory cytokines such as tumor necrosis factor-α (TNFα). The importance of TNFα in human and animal fatty liver diseases, both caused by genetic manipulation and overnutrition, has been shown convincingly. Furthermore, neutralization of TNFα activity improves IR and fatty liver disease in animals. Adiponectin is a potent TNFα-neutralizing and anti-inflammatory adipokine and in vitro and experimental animal studies have proven the importance of this mediator in counteracting inflammation and IR. Anti-inflammatory effects of adiponectin are exerted both by suppressing TNFα synthesis and by induction of anti-inflammatory cytokines such as interleukin-10 or interleukin-1–receptor antagonist. Therefore, the balance between various mediators, either derived from the immune system or adipose tissue, appears to play an important role in hepatic and systemic insulin action and in the development of fatty liver disease. The number of obese and overweight individuals has increased dramatically over the past 2 decades. Obesity is associated not only with the development of type 2 diabetes and hypertension, but also has negative effects on liver function. There is now convincing evidence that nonalcoholic fatty liver disease (NAFLD) is a component of the metabolic syndrome.1Angulo P. 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Insulin acts in all cells through binding to its specific receptor and thereby activating a cascade of intracellular signaling events. After binding, the insulin receptor phosphorylates itself and several members of the insulin-receptor substrate (IRS) family. IRS-1 and IRS-2 are the main mediators of insulin signaling in the liver, controlling insulin sensitivity. Therefore, the primary mechanism of IR induced by inflammatory mediators is exerted by interference at this level of signaling.8Wellen K.E. Hotamisligil G.S. Inflammation, stress, and diabetes.J Clin Invest. 2005; 115: 1111-1119Crossref PubMed Scopus (1526) Google Scholar The importance of visceral fat in the pathogenesis of hepatic IR and steatosis has been shown in many animal models including fa/fa rats. In these animals with inherited leptin resistance, surgical resection of intra-abdominal fat depots reverses both conditions.9Gabriely I. Barzilai N. Surgical removal of visceral adipose tissue: effects on insulin action.Curr Diab Rep. 2003; 3: 201-206Crossref PubMed Google Scholar The details of insulin receptor signaling pathways are not covered here but can be found in excellent recent reviews.10Malbon C.C. Insulin signalling: putting the “G-” in protein-protein interactions.Biochem J. 2004; 380: e11-e12Crossref PubMed Google Scholar, 11Pirola L. Johnston A.M. Van Obberghen E. Modulation of insulin action.Diabetologia. 2004; 47: 170-184Crossref PubMed Scopus (167) Google Scholar This article summarizes the current knowledge, highlighting the inflammatory/cytokine view of this disease, with a detailed discussion on the role of cytokines and adipokines in NAFLD and their contribution to IR. Identification of the mechanisms that cause and mediate obesity-related fatty liver disease are awaited and progress in the past few years has been substantial.6Tilg H. Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis.N Engl J Med. 2000; 343: 1467-1476Crossref PubMed Scopus (593) Google Scholar, 7Sanyal A.J. Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease.Nat Clin Pract Gastroenterol Hepatol. 2005; 2: 46-53Crossref PubMed Scopus (93) Google Scholar Cytokines are critically involved in the physiology of a healthy liver and in the pathophysiology of many acute and chronic liver diseases. These mediators are released by almost all cell types in the liver and play a fundamental role in liver function and regeneration. Cytokines are key mediators of hepatic inflammation, liver cell death, cholestasis, and fibrosis,12Mizuhara H. O’Neill E. Seki N. Ogawa T. Kusunoki C. Otsuka K. Satoh S. Niwa M. Senoh H. Fujiwara H. T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6.J Exp Med. 1994; 179: 1529-1537Crossref PubMed Google Scholar, 13Friedman S.L. 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Fernandez-Escalante J.C. Pons-Romero F. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients.Hepatology. 2001; 34: 1158-1163Crossref PubMed Scopus (345) Google Scholar showed increased expression of TNFα and its type 1 receptor in patients with NASH compared with patients with simple steatosis. Interestingly, more advanced fibrosis also was accompanied by increased TNFα expression. Apoptosis is a common mechanism of liver injury. Patients with NASH compared with simple steatosis show increased numbers of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling–positive cells in the liver being accompanied by enhanced expression of caspases 3 and 7, confirming higher rates of apoptosis in NASH.19Feldstein A.E. Canbay A. Angulo P. Taniai M. Burgart L.J. Lindor K.D. Gores G.J. 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Furthermore, certain TNFα polymorphisms are associated with susceptibility for IR, highlighting the importance of this cytokine in the interaction between fat accumulation, insulin action, and inflammation in human beings.22Valenti L. Fracanzani A.L. Dongiovanni P. Santorelli G. Branchi A. Taioli E. Fiorelli G. Fargion S. Tumor necrosis factor alpha promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease.Gastroenterology. 2002; 122: 274-280Abstract Full Text Full Text PDF PubMed Google Scholar Additional studies to assess local cytokine production/expression and to understand detailed mechanisms of action in regulating liver function would be necessary for the development of effective therapeutic interventions. Free fatty acids (FFAs) are important mediators of lipotoxicity. When lipids accumulate in various nonadipose tissues (as in obesity), they may enter nonoxidative pathways leading to cell injury and death. FFAs have been shown to circulate at higher concentrations in patients with NAFLD, and even more importantly their levels correlate with disease severity.23Nehra V. Angulo P. Buchman A.L. Lindor K.D. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis.Dig Dis Sci. 2001; 46: 2347-2352Crossref PubMed Scopus (69) Google Scholar Accumulation of FFA affects lysosomal permeabilization, which also is observed after exposure to proinflammatory cytokines such as TNFα. Feldstein et al24Feldstein 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-alpha expression via a lysosomal pathway.Hepatology. 2004; 40: 185-194Crossref PubMed Scopus (346) Google Scholar recently provided evidence regarding the role of FFAs in mediating hepatic lipotoxicity. FFAs show dramatic lipotoxicity in the liver and induce TNFα expression. Liver injury triggers translocation of bax, a proapoptotic Bcl2 family member, to lysosomes and subsequent lysosomal destabilization with release of the cysteine protease cathepsin B (Figure 1). Lysosomal destabilization leads to activation of NF-κB and generation of more TNFα, initiating a vicious cycle. Importantly, genetic or pharmacologic inactivation of cathepsin B protects against the development of hepatic steatosis, liver injury, and IR associated with the metabolic syndrome.24Feldstein 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-alpha expression via a lysosomal pathway.Hepatology. 2004; 40: 185-194Crossref PubMed Scopus (346) Google Scholar In the cytosol, cathepsin B activates cytokine signaling cascades, thereby also facilitating triglyceride accumulation in the liver and aggravating steatosis and inflammation. This also could reflect a pathway where hepatic steatosis develops and worsens under conditions in which inflammation is dominant irrespective of the extent of obesity. For example, TNFα could promote IR by triggering I κ B kinase-β (IKKβ), the upstream activator of NF-κB, and/or other critical intracellular kinases such as c-Jun N-terminal kinase (JNK) activation and consequently block insulin receptor signaling (Figure 2).8Wellen K.E. Hotamisligil G.S. Inflammation, stress, and diabetes.J Clin Invest. 2005; 115: 1111-1119Crossref PubMed Scopus (1526) Google Scholar All these potential loops and mechanisms are supported by the fact that in a murine model of steatohepatitis: (1) antibody-mediated neutralization of TNFα improves liver disease25Li Z. Yang S. Lin H. Huang J. Watkins P.A. Moser A.B. Desimone C. Song X.Y. Diehl A.M. Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease.Hepatology. 2003; 37: 343-350Crossref PubMed Scopus (451) Google Scholar and (2) development of fatty liver disease requires the presence of intact TNFR1 and is dependent on cathepsin B activity.24Feldstein 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-alpha expression via a lysosomal pathway.Hepatology. 2004; 40: 185-194Crossref PubMed Scopus (346) Google Scholar Therefore, evidence is increasing that inflammation in general and proinflammatory cytokines such as TNFα in particular could be involved in the development of NASH and IR at various stages, either earlier or later in the disease process.Figure 2Regulation of NASH and IR: involved mediators and pathways. TNFα and other cytokines such as IL-6 are involved in the generation of IR. Other factors of the immune system such as SOCS-proteins (1, 2, and 7) also are involved in these processes. SOCS-1 and SOCS-3 can link IRSs to ubiquitin-mediated degradation pathways and also to an increase in the key regulator of fatty acid synthesis in liver, the transcription factor SREBP-1c. In recent years, critical intracellular pathways have been identified that are involved in the molecular pathogenesis of IR. Endoplasmic reticulum (ER) stress and various kinases such as JNK and IKKβ, are of critical importance because they not only activate inflammatory pathways but also attenuate insulin signaling. ER stress leads to suppression of insulin-receptor signaling through activation of JNK and the subsequent serine phosphorylation of IRS-1.View Large Image Figure ViewerDownload (PPT) Obesity is associated with production of increased inflammatory cytokines, particularly from visceral adipose tissue.8Wellen K.E. Hotamisligil G.S. Inflammation, stress, and diabetes.J Clin Invest. 2005; 115: 1111-1119Crossref PubMed Scopus (1526) Google Scholar Definitive links also have been established between TNFα action and suppression of insulin action in cells, whole animals, and human beings (see later).8Wellen K.E. Hotamisligil G.S. Inflammation, stress, and diabetes.J Clin Invest. 2005; 115: 1111-1119Crossref PubMed Scopus (1526) Google Scholar A high-fat diet causes an increase in soluble and membrane-bound TNFα both in fat, liver, and muscle tissues in experimental animals and human beings, whereas TNFα remains mostly at low levels in serum.26Xu H. Hirosumi J. Uysal K.T. Guler A.D. Hotamisligil G.S. Exclusive action of transmembrane TNF alpha in adipose tissue leads to reduced adipose mass and local but not systemic insulin resistance.Endocrinology. 2002; 143: 1502-1511Crossref PubMed Scopus (55) Google Scholar, 27Xu H. Uysal K.T. Becherer J.D. Arner P. Hotamisligil G.S. 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The importance of these negative regulators (ie, anti-inflammatory mediators) is best illustrated by data showing that SOCS-1 (−/−) mice die by 3 weeks of age with inflammation and fatty necrosis of the liver.39Alexander W.S. Suppressors of cytokine signalling (SOCS) in the immune system.Nat Rev Immunol. 2002; 2: 410-416Crossref PubMed Google Scholar It has been shown recently that mice with SOCS-1 deficiency in myeloid and lymphoid cells also develop disease, although at 50–250 days of age, with uncontrolled inflammation.40Chong M.M. Metcalf D. Jamieson E. Alexander W.S. Kay T.W. Suppressor of cytokine signaling-1 in T cells and macrophages is critical for preventing lethal inflammation.Blood. 2005; 106: 1668-1675Crossref PubMed Scopus (46) Google Scholar These animals also showed liver inflammation with infiltration by CD4+ and CD8+ T cells, and macrophages localized mainly to portal tracts or in isolated foci. More interestingly, SOCS-1 and SOCS-3 have been shown to block insulin signaling by ubiquitin-mediated degradation of IRS-1 and IRS-2 (Figure 2).41Rui L. Yuan M. Frantz D. Shoelson S. White M.F. SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2.J Biol Chem. 2002; 277: 42394-42398Crossref PubMed Scopus (424) Google Scholar, 42Emanuelli B. Peraldi P. Filloux C. Chavey C. Freidinger K. Hilton D.J. Hotamisligil G.S. Van Obberghen E. SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice.J Biol Chem. 2001; 276: 47944-47949Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Several SOCS proteins are regulated by cytokines that could modulate insulin action.43Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. SOCS3 negatively regulates IL-6 signaling in vivo.Nat Immunol. 2003; 4: 540-545Crossref PubMed Scopus (390) Google Scholar For example, SOCS-3 seems to be involved in IL-6–dependent IR in hepatocytes.44Senn J.J. Klover P.J. Nowak I.A. Zimmers T.A. Koniaris L.G. Furlanetto R.W. Mooney R.A. Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator of interleukin-6-dependent insulin resistance in hepatocytes.J Biol Chem. 2003; 278: 13740-13746Crossref PubMed Scopus (340) Google Scholar The overexpression of SOCS-1 and SOCS-3 in liver also causes IR and an increase in the key regulator of fatty acid synthesis in the liver, the transcription factor sterol regulatory element-binding protein (SREBP)-1c. Conversely, inhibition of SOCS-1 and SOCS-3 in obese diabetic mice improves insulin sensitivity, normalizes increased expression of SREBP-1c, and improves hepatic steatosis and hypertriglyceridemia.45Ueki K. Kondo T. Tseng Y.H. Kahn C.R. 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Deletion of SOCS7 leads to enhanced insulin action and enlarged islets of Langerhans.J Clin Invest. 2005; 115: 2462-2471Crossref PubMed Scopus (38) Google Scholar A similar SOCS-mediated mechanism might be involved in hepatitis C–associated IR because it has been shown that hepatitis C virus core protein up-regulates SOCS-3 and promotes proteosomal degradation of IRS-1 and IRS-2 through ubiquitination.48Kawaguchi T. Yoshida T. Harada M. Hisamoto T. Nagao Y. Ide T. Taniguchi E. Kumemura H. Hanada S. Maeyama M. Baba S. Koga H. Kumashiro R. Ueno T. Ogata H. Yoshimura A. Sata M. Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3.Am J Pathol. 2004; 165: 1499-1508Abstract Full Text Full Text PDF PubMed Google Scholar SOCS proteins therefore reflect good candidates as a key link between liver inflammation, steatosis, and IR. Studies of genetically obese ob/ob mice and fa/fa rats have provided information about the pathogenesis of obesity-related fatty liver disease. Both of these rodent strains have spontaneous mutations that either diminish production of the appetite-suppressing hormone, leptin (in ob/ob mice), or that inactivate the leptin receptor (in fa/fa rats).6Tilg H. Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis.N Engl J Med. 2000; 343: 1467-1476Crossref PubMed Scopus (593) Google Scholar, 7Sanyal A.J. Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease.Nat Clin Pract Gastroenterol Hepatol. 2005; 2: 46-53Crossref PubMed Scopus (93) Google Scholar, 49Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice.Science. 1995; 269: 540-543Crossref PubMed Google Scholar Similar to obese human beings, ob/ob mice and fa/fa rats have IR, hyperglycemia, hyperlipidemia, and fatty livers.6Tilg H. Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis.N Engl J Med. 2000; 343: 1467-1476Crossref PubMed Scopus (593) Google Scholar, 7Sanyal A.J. Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease.Nat Clin Pract Gastroenterol Hepatol. 2005; 2: 46-53Crossref PubMed Scopus (93) Google Scholar, 8Wellen K.E. Hotamisligil G.S. Inflammation, stress, and diabetes.J Clin Invest. 2005; 115: 1111-1119Crossref PubMed Scopus (1526) Google Scholar These rodent models also show several immunologic defects including phagocyte dysfunction and altered cytokine gene transcription including enhanced TNFα expression.6Tilg H. Diehl A.M. Cytokines in alcoholic and nonalcoholic steatohepatitis.N Engl J Med. 2000; 343: 1467-1476Crossref PubMed Scopus (593) Google Scholar, 7Sanyal A.J. Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease.Nat Clin Pract Gastroenterol Hepatol. 2005; 2: 46-53Crossref PubMed Scopus (93) Google Scholar, 50Diehl A.M. Fatty liver, hypertension, and the metabolic syndrome.Gut. 2004; 53: 923-924Crossref PubMed Scopus (32) Google Scholar Furthermore, murine leptin deficiency influences production of other cytokines such as IL-12 or IL-15, thereby promoting hepatic CD4-positive natural killer cell depletion in ob/ob livers.51Li Z. Lin H. Yang S. Diehl A.M. Murine leptin deficiency alters Kupffer cell production of cytokines that regulate the innate immune system.Gastroenterology. 2002; 123: 1304-1310Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Therefore, in fatty liver disease, cytokine imbalance and dysregulation might involve not only inflammatory but also other immunoregulatory cytokines. It has to be mentioned that these animal models might have certain limitations. Obese human beings almost always have high serum levels of leptin.52Chitturi S. Farrell G. Frost L. Kriketos A. Lin R. Fung C. Liddle C. Samarasinghe D. George J. Serum leptin in NASH correlates with hepatic steatosis but not fibrosis: a manifestation of lipotoxicity?.Hepatology. 2002; 36: 403-409Crossref PubMed Scopus (213) Google Scholar Leptin-deficient ob/ob mice show no or only mild chronic steatohepatitis and do not develop fibrosis, which can be overcome by exogenous administration of leptin,53Leclercq I.A. Farrell G.C. Schriemer R. 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Despite the shortcomings of several animal models including the ob/ob mouse and their limited relation to human NAFLD, they are helpful in studying and elucidating several pathophysiologic aspects of fatty liver diseases. Considering that gut-derived flora and proinflammatory cytokines play a key role in fatty liver diseases, interference at one of these steps might be beneficial. Similarities in the histopathology of alcohol-induced steatohepatitis and obesity-related NASH suggest that common mechanisms may mediate both diseases. Various treatments, at least in experimental animal systems, that inhibit TNFα activity prevent both diseases. Several anti-TNF agents such as anti-TNF antibodies protect against fatty liver diseases.25Li Z. Yang S. Lin H. Huang J. Watkins P.A. Moser A.B. Desimone C. Song X.Y. Diehl A.M. Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease.Hepatology. 2003; 37: 343-350Crossref PubMed Scopus (451) Google Scholar, 55Yin M. Wheeler M.D. Kono H. Bradford B.U. Gallucci R.M. Luster M.I. Thurman R.G. Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.Gastroenterology. 1999; 117: 942-952Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar It also has been shown that metformin improves liver disease in ob/ob mice via suppression of TNFα.56Lin H.Z. Yang S.Q. Chuckaree C. Kuhajda F. Ronnet G. Diehl A.M. Metformin reverses fatty liver disease in obese, leptin-deficient mice.Nat Med. 2000; 6: 998-1003Crossref PubMed Scopus (433) Google Scholar Furthermore, peroxisome proliferator activating receptor γ (PPARγ) ligands such as pioglitazone, which can suppress TNFα function, also positively affect fatty liver diseases.57Solomon S.S. Mishra S.K. Cwik C. Rajanna B. Postlethwaite A.E. Pioglitazone and metformin reverse insulin resistance induced by tumor necrosis factor-alpha in liver cells.Horm Metab Res. 1997; 29: 379-382Crossref PubMed Google Scholar, 58Tomita K. Azuma T. Kitamura N. Nishida J. Tamiya G. Oka A. Inokuchi S. Nishimura T. Suematsu M. Ishii H. Pioglitazone prevents alcohol-induced fatty liver in rats through up-regulation of c-Met.Gastroenterology. 2004; 126: 873-885Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 59Ohata M. Suzuki H. Sakamoto K. Hashimoto K. Nakajima H. Yamauchi M. Hokkyo K. Yamada H. Toda G. Pioglitazone prevents acute liver injury induced by ethanol and lipopolysaccharide through the suppression of tumor necrosis factor-alpha.Alcohol Clin Exp Res. 2004; 28: 139S-144SPubMed Google Scholar Li et al25Li Z. Yang S. Lin H. Huang J. Watkins P.A. Moser A.B. Desimone C. Song X.Y. Diehl A.M. Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease.Hepatology. 2003; 37: 343-350Crossref PubMed Scopus (451) Google Scholar treated ob/ob mice with either a probiotic (VSL#3, ie, lyophilized bifidobacteria, lactobacilli, and Streptococcus thermophilus) or an anti-TNFα antibody for 4 weeks. Treatment with both agents improved liver histology, reduced hepatic total fatty acid content, and decreased enhanced liver function tests. These benefits were paralleled by decreased hepatic expression of TNFα, especially after treatment with the anti-TNFα antibody. Furthermore, these treatments reduced activity of JNK and NF-κB pathways, both of which are downstream of TNFα and promote IR. Therefore, intestinal bacteria might induce endogenous signals that play a role in hepatic IR and NAFLD and suggest that either interfering at this stage or disrupting the proinflammatory cytokine cascades might be beneficial in fatty liver diseases. There is now compelling evidence that (1) enhanced liver TNFα expression is observed in animal models and human beings with NASH/NAFLD, (2) this cytokine, released by many cells in the body including various cell types in liver and adipocytes, is crucially involved in the pathogenesis of IR (as discussed later), and (3) neutralization of TNFα, at least in experimental animal models, improves IR, hepatic steatosis, and liver inflammation.