Obesity, Insulin Resistance, and HCV: Implications for Pathogenesis David S. Heppner, MD, and Stephen A. Harrison, MD*

Address *Brooke Army Medical Center, Division of Gastroenterology and Hepatology, Department of Medicine, 3851 Roger Brooke Drive, Fort Sam Houston, TX 78234, USA. E-mail: [email protected] Current Hepatitis Reports 2005, 4:153–157 Current Science Inc. ISSN 1540-3416 Copyright © 2005 by Current Science Inc.

A significant number of patients with chronic hepatitis C are overweight or obese. Insulin resistance and diabetes mellitus are also frequently encountered, and the association of hepatitis C with hepatic steatosis is well described. Both host and viral factors contribute to insulin resistance, diabetes, and hepatic steatosis. Fibrosis progression is closely linked to insulin resistance and hepatic steatosis. Obesity may contribute to hepatic fibrosis directly through induction of inflammatory pathways or indirectly via development of hepatic steatosis and insulin resistance.

Introduction The past several years have seen a proliferation of data regarding the association of chronic hepatitis C (CHC) with insulin resistance, diabetes mellitus, and hepatic steatosis. The specific pathogenetic mechanisms explaining these associations are not fully understood, but recent data point to a collaboration of both host metabolic and viral factors. The epidemic of obesity and subsequent rise in the prevalence of insulin resistance and diabetes mellitus has fueled the increase in the presence of nonalcoholic fatty liver disease (NAFLD). Because the prevalence of obesity among patients with CHC is also elevated, it is not surprising that the two diseases may occur concomitantly. In fact, most recent data suggest that coexistent steatosis occurs in at least 50% of patients, albeit usually mild in severity [1]. In addition to host metabolic factors, the hepatitis C virus has been implicated in the genesis of hepatic steatosis as well as de novo insulin resistance. Evidence now suggests that the resultant hepatic steatosis and insulin resistance generated through these synergistic processes are associated with fibrosis progression. The ensuing article details the association of obesity, insulin resistance, and hepatic steatosis with CHC, with additional attention focused on the untoward histopathologic consequences of these associations.

Host-mediated Pathways Leading to Insulin Resistance and Hepatic Steatosis Obesity (defined as a body mass index [BMI] of ≥ 30) rose 74% from 1991 to 2001 and is reaching epidemic proportions in the United States [2]. Estimates suggest that the ageadjusted prevalence of obesity is now approximately 30% [3]. The association of obesity with the metabolic syndrome is well known and includes the additional clinical findings of hypertension, elevated fasting glucose, and hyperlipidemia. Concurrent with this increase in obesity, the prevalence of NAFLD (the hepatic manifestation of the metabolic syndrome) is rising. Estimates suggest that 30% of the United States population may now have this condition [4]. The development of NAFLD is thought to be due to both intracellular hepatic and intramyocellular lipid accumulation and subsequent hepatic and peripheral insulin resistance. Although the mechanisms surrounding insulin resistance are not fully defined, the accumulation of free fatty acids (FFAs) within muscle and liver leads to decreased insulin activation (tyrosine phosphorylation) of insulin receptor substrate (IRS)-1 and IRS-2, thereby altering downstream insulin signaling and resulting in decreased glucose transport into cells [5]. Furthermore, due to insulin signaling defects, glycogen synthesis is reduced and there is continued gluconeogenesis. Subsequent inefficient FFA oxidation and altered fat trafficking within the liver contribute further to hepatic steatosis development. Dysregulated adipocytokine production may mediate or enhance these processes. Proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), and IL6 have been shown to be upregulated, further enhancing insulin resistance [6]. Alternatively, adiponectin, an adipocytokine with insulin-sensitizing and anti-inflammatory effects, is diminished in obese patients with NAFLD [7•]. The prevalence of overweight or obesity among those with CHC has been demonstrated to range from 4% in France [8] to 17% to 38% in China, western Europe, and North America [9–12]. Patients with coexistent hepatitis C and hepatic steatosis have an increased prevalence of obesity compared to patients with hepatitis C and no steatosis [9]. Solis-Herruzo et al. [13] compared patients with CHC and no steatosis to patients having both CHC and histopathologic evidence of nonalcoholic steatohepatitis (NASH). They found that 34% of patients having both diseases were obese, compared with no obesity in patients with CHC without NASH [13]. There also

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Hepatitis C: Epidemiology, Natural History, and Pathogenesis Figure 1. Factors associated with hepatic steatosis. The asterisk (*) indicates in vitro models of steatosis that used genotype 1–derived constructs. The dagger (†) indicates other host factors that may contribute to worsening insulin resistance, including race, family history, and male gender. IRS-1—insulin receptor substrate-1; MTP—microsomal transfer protein; PI3K–phosphatidylinositol 3-kinsase; SOC3—suppressor of cytokine signaling 3; TNF-α—tumor necrosis factor-α.

appears to be an incremental increase in the grade of steatosis with rising BMI [11,12,14,15]. In fact, among patients with a BMI ≥ 25, the odds ratio for 30% steatosis or more was 9.74 [12]. Overweight patients with CHC have increased circulating insulin levels [16], suggesting that insulin resistance may be the principle pathogenetic mechanism for development of hepatic steatosis in genotype 1 patients with CHC, similar to patients with NAFLD (Fig. 1). Fartoux et al. [17•] have recently confirmed this by showing that insulin resistance, as measured by the homeostasis model assessment method, is independently associated with hepatic steatosis in genotype 1 patients. Age has also been shown to be independently associated with hepatic steatosis on univariate [17•,18] and multivariate analysis in several studies [18–20]. A plausible explanation for this is the increased prevalence of insulin resistance as patients grow older. Several studies demonstrate an association of age with diabetes mellitus in patients with CHC. It has been suggested that a decline in mitochondrial oxidative and phosphorylation function with age may contribute to progressive insulin resistance [21]. Other host factors that may contribute to worsening insulin resistance and progression to diabetes include race, family history, and male gender.

Virally Mediated Pathways Leading to Insulin Resistance, Diabetes Mellitus, and Hepatic Steatosis Experimental data derived from transgenic mice infected with hepatitis C core protein have recently demonstrated that this protein induces insulin resistance directly, and tends to occur early in the course of infection, prior to development of steatosis or fibrosis [22••]. Among humans with CHC infection, it has been shown that insulin signaling in the liver is altered by defects in IRS-1 tyrosine phosphorylation and phosphatidylinositol 3kinase activation, thus possibly contributing to insulin resistance [23]. The proinflammatory cytokine, TNF-α, may mediate this process. TNF-α is upregulated in patients with

CHC, and this cytokine has been shown to interrupt insulin signaling via reduced tyrosine phosphorylation of IRS-1 and decreased ability of IRS-1 to associate with the insulin receptor (Fig. 1) [24]. Data to support a role for TNF-α in the genesis of insulin resistance are found in insulin-resistant transgenic mice infected with hepatic C core protein. When treated with anti–TNF-α, insulin sensitivity significantly improves [22••]. More recently, evidence suggests that the hepatitis C virus may further alter insulin signaling by upregulating expression of the protein suppressor of cytokine signaling 3, resulting in decreased activation of downstream components of IRS and altered expression of sterol regulatory binding protein-1c [25,26], which is important in de novo lipogenesis. Several cross-sectional and population-based studies have confirmed the increased prevalence of diabetes in the setting of CHC [27–29]. Data from the National Health and Nutrition Examination Survey III survey demonstrated that patients over 40 years of age with CHC were three times more likely to have concomitant diabetes mellitus [30]. However, not all patients with CHC develop diabetes. Transgenic mice infected with hepatitis C core protein develop overt diabetes only if they have concomitant weight gain [22••]. This suggests that host factors may impart a synergistic effect on development of insulin resistance. The hepatitis C virus, specifically genotype 3, has also been shown to be an independent predictor of hepatic steatosis, apart from that occurring as a result of insulin resistance. When risk factors for NASH are excluded, genotype 3 infection has been demonstrated to be the only predictor of hepatic steatosis [31]. Levels of HCV RNA correlate with degree of steatosis [17•,32], regression of steatosis occurs with viral eradication [33], and steatosis returns with viral relapse. Experimental data support a direct steatogenic effect of the hepatitis C virus on hepatocytes via altered lipid metabolism (Fig. 1). Transgenic mice, overexpressing viral core protein, inhibit microsomal triglyceride transfer protein, an enzyme critical to the binding of triglyceride to apolipoprotein B100, which allows for the formation of

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Figure 2. Mechanisms for hepatic fibrosis development.

the transfer molecule by very low density lipoprotein (VLDL) [34]. Without VLDL synthesis, triglycerides are unable to be transferred out of the liver, leading to intracytoplasmic triglyceride storage. Interestingly, in humans with CHC, hypobetalipoproteinemia negatively correlates with both HCV-RNA levels and steatosis [35]. The viral NS5A and core proteins may also be involved in hepatic fat accumulation via mitochondrial dysfunction, enhanced oxidative stress and subsequent lipid peroxidation [36], or through interactions with cytoplasmic lipid structures, specifically apolipoprotein A1 and apolipoprotein A2, resulting in abnormal lipid metabolism [37].

Fibrosis Progression Resulting from Obesity, Insulin Resistance, and Hepatic Steatosis Hepatic fibrosis has been linked to obesity, as well as hostand virally mediated insulin resistance and hepatic steatosis (Fig. 2). Obesity is typically associated with insulin resistance and often hepatic steatosis, confounding the link between obesity and hepatic fibrosis. However, it has been demonstrated that obesity is associated with a state of chronic low-level inflammation, and hepatic inflammation correlates with hepatic fibrosis [17•]. Ortiz et al. [10] have shown that a BMI ≥ 25 was independently associated with fibrosis progression, with an odds ratio of 4.52. Furthermore, among patients with normal alanine aminotransferase levels, only BMI was predictive of advanced disease. In another study, patients with CHC with increased BMI demonstrated an association between steatosis and perisinusoidal fibrosis—the characteristic fibrosis pattern seen in patients with NASH [38]. Similar associations between obesity and progression of fibrosis in patients with CHC and hepatic steatosis have been demonstrated [10,39]. Hepatic steatosis has been found to be an independent predictor of fibrosis in patients with CHC [1], irrespective of genotype, but this is debated [40,41]. However, recent data suggest the probability of fibrosis progression over time increases with increasing amounts of steatosis [12,15,42]. The odds ratio for fibrosis stage III to IV was 5.6 in the setting of more than 30% steatosis, and the cumulative probability of fibrosis progression was found to be 18.1% and 33.2% at 4 and 6 years, respectively, if there was more than 30% steatosis on biopsy [8]. Evidence

suggests that fibrosis progression may occur at a rate that is up to two times faster than those without steatosis [43]. Insulin resistance, occurring either as a result of host metabolic or virally mediated factors, has been associated with increased fibrosis as well [16,17•,44,45•,46,47]. Others have demonstrated that worsening insulin resistance parallels progression of fibrosis [48,49]. Mechanisms for fibrogenesis in the setting of obesity, insulin resistance, and hepatic steatosis appear to be similar to NASH and focus on enhanced lipid peroxidation via generation of reactive oxygen species, dysregulated adipocytokine secretion, and cellular apoptosis, leading to hepatic stellate cell activation. Obesity is associated with increased FFA deposition and subsequent hepatic steatosis in a significant percentage of patients. Steatosis leads to increased production of reactive oxygen species via lipid peroxidation [50] and subsequent further upregulation of proinflammatory cytokines. Hepatic stellate cells undergo a phenotypic transformation from quiescent vitamin A–containing cells to myofibroblasts, resulting in collagen deposition. Interestingly, in patients with NAFLD it is believed that a “second hit” is necessary to allow for progression from simple fatty liver to steatohepatitis and fibrosis. In patients with CHC and hepatic steatosis, the virus may provide the inflammation that results in the “second hit.” Data suggest that just as steatosis is independently associated with fibrosis, necroinflammation may act independently in this setting to perpetuate further fibrogenesis [17•,38,51]. The adipocytokine, leptin, appears to be involved in the genesis of hepatic fibrosis, although specific mechanisms remain to be fully defined. Leptin has been shown to regulate insulin secretion and tissue responsiveness to insulin. This hormone may be important in regulating cytokine activity related to upregulation of inflammatory pathways as well. It has been shown that leptin enhances the secretion of TNF-α, interleukin-6 and -12 from macrophages, and may also upregulate transforming growth factor-β secretion. This hormone is increased in patients with CHC compared with healthy control subjects [52], is inversely associated with obesity and hyperglycemia, and correlates with hepatic steatosis [20,53,54] and fibrosis [54,55]. Hepatic stellate cells exposed to leptin have increased mRNA expression of A2 collagen, thus demonstrating that upregulation of leptin expression may enhance fibrogenesis.

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Apoptosis is enhanced in patients with CHC and hepatic steatosis, suggesting that lipid accumulation in nonadipose tissue may result in cell death [56,57]. Enhanced cellular apoptosis may also contribute to fibrogenesis. In the setting of hepatic steatosis, increasing apoptosis is associated with activation of hepatic stellate cells and increased stage of fibrosis.

Conclusions A significant number of patients with CHC are overweight or obese. Insulin resistance and diabetes mellitus are also frequently encountered, and the association of hepatic steatosis and steatohepatitis with CHC is well described. Among genotype 1 patients, the contribution of host metabolic factors to the development of coexistent fatty liver is thought to be related to pathophysiologic mechanisms that are similar to those found in patients with NAFLD. As the incidence and prevalence of obesity, insulin resistance, and diabetes mellitus increase in our society, these associations are likely to increase. Hepatitis C virus also appears to induce insulin resistance and hepatic steatosis, through mechanisms involving alteration in insulin signaling and lipid metabolism, and appears to be genotype specific. Although virally mediated steatosis may occur independent of host interactions, this predominates in genotype 3 patients. The relative contribution of virally mediated insulin resistance to that of host metabolic factors inducing insulin resistance remains to be fully defined. Our knowledge of this inter-relationship continues to improve and ongoing studies will undoubtedly unravel this conundrum. Fibrosis progression is closely linked to insulin resistance and hepatic steatosis in patients with CHC. Obesity may contribute to hepatic fibrosis directly through induction of inflammatory pathways or indirectly via development of hepatic steatosis and insulin resistance. The presence and severity of steatosis and insulin resistance predict fibrosis progression. Mechanisms for fibrogenesis in the setting of obesity, insulin resistance, and hepatic steatosis have been discussed, and appear to be similar to that seen in NASH.

Disclaimer The opinion or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the view of the U.S. Department of the Army or the U.S. Department of Defense.

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