Insulin resistance is extremely common; frequently is associated STATE OF THE ART

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2008;6: STATE OF THE ART Insulin Resistance Among Patients With Chronic Hepatitis C: Etiology and Impact on Treatment STEPHEN A. HARRISON Division of Gastroenterology and Hepatology, Brooke Army Medical Center, Fort Sam Houston, Texas Insulin resistance is extremely common and frequently is associated with comorbid conditions such as cardiovascular disease, hypertension, obesity, infertility, and neurodegeneration. In addition, insulin resistance is the driving force for type 2 diabetes mellitus. Interestingly, co-existence of insulin resistance and chronic hepatitis C occurs more often than predicted by chance, with recent estimates indicating that 30% to 70% of patients with chronic hepatitis C display some evidence of insulin resistance. Recent research revealed several molecules, including tumor necrosis factor, suppressor of cytokine signaling 1 and 3 proteins, insulin-receptor substrates 1 and 2, and other adipocytokines, potentially are involved in the development of insulin resistance in patients with chronic hepatitis C. Unfortunately, baseline insulin resistance has a negative impact on treatment outcomes in patients with chronic hepatitis C. However, successfully managing insulin resistance or diabetes mellitus in these patients may improve patients likelihoods of successful outcomes with antiviral therapy. Likewise, eradication of hepatitis C virus in patients with insulin resistance or diabetes mellitus appears to improve glucose metabolism. Although adjunctive therapies such as insulin sensitizers and weight loss often are recommended, their ability to improve antiviral treatment response in patients with chronic hepatitis C is unproven. Studies are under way to determine whether improving insulin sensitivity results in better outcomes in patients receiving pegylated interferon alfa plus ribavirin therapy for chronic hepatitis C. Insulin resistance is extremely common; frequently is associated with comorbid conditions such as cardiovascular disease, hypertension, obesity, infertility, and neurodegeneration; and is the driving force for type 2 diabetes mellitus (DM). A recent report showed that insulin resistance was present in more than 30% of Americans, and its prevalence continues to increase. 1 According to the Bruneck Study a cross-sectional, prospective, population-based survey performed in Bruneck, a small town of approximately 13,500 persons in northeastern Italy the overall prevalence of insulin resistance was approximately 45% and was as high as 95% in patients with 4 metabolic disorders (glucose intolerance, dyslipidemia, hyperuricemia, and hypertension). 2 Evidence from community-based studies indicated that insulin resistance was encountered frequently in patients with chronic hepatitis C (CHC), 3,4 suggesting a close relationship between these 2 clinical conditions. With more than 170 million individuals chronically infected with hepatitis C virus (HCV), the co-existence of insulin resistance and CHC represents a serious global health concern. 5 Insulin Resistance Insulin resistance is defined as an increased requirement for insulin to maintain normal metabolic function, resulting in the compensatory development of hyperinsulinemia. 6,7 In patients with hepatic insulin resistance, suppression of glucose production in the liver is impaired, which largely accounts for the hyperglycemia and glucose intolerance seen in these patients. Insulin resistance is the pathogenic foundation underlying the metabolic syndrome and is a major underlying feature of its phenotypical expressions (including nonalcoholic fatty liver disease) and complications. Insulin Resistance and Liver Disease Initially, the association of DM and CHC was attributed to advanced liver disease (eg, severe fibrosis and cirrhosis). 8 However, more recent reports described an increased incidence of DM in patients with CHC who had not yet developed advanced liver disease. 9,10 In addition, the association was present for only DM and hepatitis C, and not for other causes of liver damage, such as hepatitis B and alcohol abuse. The hypothesized mechanism behind the increased likelihood of DM in patients with CHC is the presence of insulin resistance; however, this likely is not the sole mechanism. The timing of the development of insulin resistance relative to the development of liver disease is not consistent in patients with comorbid DM and CHC, and the association between DM and CHC does not extend to other types of inflammatory liver conditions such as hepatitis B or primary biliary cirrhosis. 9,10 Abbreviations used in this paper: BMI, body mass index; CHC, chronic hepatitis C; DM, diabetes mellitus; EVR, early virologic response; G, genotype; GLUT, glucose transport protein; HCV, hepatitis C virus; HOMA-IR, homeostasis model of assessment for insulin resistance; HOMA-, homeostasis model of assessment for measuring -cell function; IFN, interferon; IRS, insulin-receptor substrate; NAFLD, nonalcoholic fatty liver disease; PEG, pegylated; PI, phosphatidylinositol; PPAR, peroxisome proliferator-activated receptor; RBV, ribavirin; SOCS, suppressor of cytokine signaling; SVR, sustained virologic response; TNF, tumor necrosis factor by the AGA Institute /08/$34.00 doi: /j.cgh

2 August 2008 IULIN RESISTANCE IN CHRONIC HEPATITIS C 865 Figure 1. Insulin resistance, hepatic steatosis, and advanced fibrosis/cirrhosis. Insulin Resistance, Hepatic Steatosis, and Advanced Liver Disease Steatosis occurs more often in patients with CHC than in the general population (45% 65% vs 16% 20% 17,18 ) and is associated independently with high body mass index (BMI), insulin resistance, DM, age older than 40 years, HCV genotype 3 (G3), hepatic inflammation, ongoing alcohol use, and fibrosis. 16 The relationship between insulin resistance and hepatic steatosis appears multifactorial. In some conditions, such as nonalcoholic fatty liver disease (NAFLD), insulin resistance may predispose a patient to hepatic steatosis. Results of recent preclinical studies suggest that accumulation of triglycerides in mouse liver actually may represent a protective mechanism to prevent further liver damage. 19 Conversely, in patients with other conditions, such as CHC, the presence of hepatic steatosis may facilitate the development of insulin resistance. This scenario is complicated in patients with hepatitis C who are subject to genotype-specific influences, including the direct steatogenic effect of HCV G3. In patients with NAFLD, it has been suggested that hepatic steatosis and not the degree of steatohepatitis is associated with insulin resistance. 20 In patients with CHC, steatosis can result from direct cytopathic effects of HCV and/or indirect mechanisms (reviewed by Negro 21 ). For example, HCV G3 directly induces steatosis, whereas insulin resistance plays a major role in the development of steatosis in HCV non-g3 patients. In the latter, insulin resistance can develop independently of HCV infection or be exacerbated by it. Regardless of which develops first, insulin resistance either directly or indirectly through promoting hepatic steatosis is a major risk factor for advanced hepatic fibrosis (Figure 1). At least 1 study of patients with viral steatosis caused by infection with HCV G3 or metabolic steatosis caused by NAFLD showed that insulin resistance was an independent predictor of advanced fibrosis in patients with NAFLD (metabolic steatosis) for which the extent of steatosis contributes to advanced disease and in patients with HCV G3 induced steatosis; however, the latter was not a substantial contributor to advanced disease. 22 Further evidence suggests that the opposite also is true: advanced hepatic fibrosis/cirrhosis is an important predictor of insulin resistance in patients with CHC; those with advanced histologic disease have a greater prevalence of DM than those with early disease. 23 In HCV-infected individuals, severe fibrosis correlates independently with insulin resistance. 24,25 The picture is complicated further by a series of examples in which severe steatosis appears completely unrelated to the development of insulin resistance. Evidence suggests that in G3 patients, mean homeostasis model of assessment insulin resistance (HOMA- IR) scores are similar in patients with and without steatosis. 10,26 A recent preclinical study using a hepatocyte-specific null mutation of phosphatase and tensin homolog (a multifunctional phosphatase involved in the signaling of various cytokines and insulin) in mice also suggests a divergence between insulin resistance and the development of steatosis. 27 In addition, preliminary data suggest that rosiglitazone, a peroxisome proliferator-activated receptor (PPAR- ) insulin-sensitizing agent used to manage DM, significantly reduces the fibrosis stage in patients with CHC compared with untreated patients. 28 Insulin Resistance in Chronic Hepatitis C Prevalence Insulin resistance and CHC occur concomitantly at a greater incidence than predicted solely by chance 10,29 31 : recent estimates indicated that 30% to 70% of patients with CHC displayed some evidence of insulin resistance. Because insulin resistance also plays a primary role in the development of DM, it is not surprising that the incidence of DM in patients with CHC also was significantly greater than that in the general population (14.5% vs 7.8%; P.0008), 23 and it was suggested that the incidence of DM is 2 times greater in patients aged 40 years or older with versus without CHC. 3 Conversely, more recent studies found no relationship between hepatitis C and metabolic syndrome, although a strong association between hepatitis C and insulin resistance was identified. 30 Molecular Pathways Involved in the Development of Insulin Resistance in Chronic Hepatitis C Although the mechanism(s) underlying the development of insulin resistance is not clearly understood, research suggests that several pathologic alterations may be involved (Figure 2). Hyperglycemia and hyperinsulinemia, the formation of advanced glycation end-products, and an increase in the release of nonesterified fatty acids and their metabolites glycerol, hormones, and proinflammatory cytokines from adipose tissue were implicated in contributing to the development of the insulin-resistant state in hepatocytes. 7 Proinflammatory cytokines are believed to play a critical role in insulin resistance. The macrophage infiltrate in white adipose tissue may contribute to the increase in systemic proinflammatory cytokines,

3 866 STEPHEN A. HARRISON CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 8 Figure 2. Proposed molecular pathways involved in the development of insulin resistance in patients with hepatitis C. a Recent evidence indicates that leptin and adiponectin do not account for the increased insulin resistance found in patients with CHC. 53 Additional studies are required to confirm the role of adipocytokines in patients with chronic hepatitis. FAS, fatty acid synthetase; MTP, microsomal triglyceride transfer protein; PP2Ac, protein phosphatase 2Ac; SREBP-1, sterol response element-binding protein-1. representing the molecular link between adipose tissue and hepatic dysfunction. 32 Alternatively, sinusoidal liver cells also may exacerbate insulin resistance in hepatocytes by increasing oxidative stress and secreting proinflammatory cytokines such as tumor necrosis factor (TNF- ) and interleukin-6. Abnormalities in -cell function also play a central role in the development of the insulin-resistant state. 33,34 In addition, other factors, such as obesity, which is characterized by a high BMI, advanced age, and a family history of DM, also are associated with a greater incidence of DM in patients with CHC. Tumor necrosis factor. Overproduction of TNF- is a common pathologic mechanism of insulin resistance and CHC. Several mechanisms were postulated to explain the role of TNF- in the development of insulin resistance (Table 1). 35,36 Insulin resistance significantly correlated with soluble TNF- receptor 1, leptin, and ferritin levels in patients infected with HCV G1. 37 Because TNF- expression also correlated with obesity and degree of insulin resistance, it is conceivable that TNF- plays a role in the development of insulin resistance in patients with CHC. TNF- represents an integral component of the inflammatory response to HCV infection. Serum levels of TNF- and soluble TNF- receptors were increased in HCVinfected patients and correlated with alanine aminotransferase levels and histologic severity of inflammation. 38,39 Various mechanisms linking TNF- with insulin resistance have been proposed, including inhibition of tyrosine phosphorylation of the insulin receptor and insulin-receptor substrate 1 (IRS-1) in adipocytes; stimulation of lipolysis; down-regulation of adipocyte encoding proteins such as IRS-1, glucose transport protein 4 (GLUT-4), PPAR-, and adiponectin; and direct toxicity to -cells (see also section on Insulin-receptor substrate 1). In particular, TNF- induced serine/threonine phosphorylation of both IRS-1 and IRS-2 has been proposed to potentially impede the subsequent interaction of these proteins with insulin receptor, rendering them poorer receptor substrates, which ultimately fail to undergo the appropriate tyrosine phosphorylation. Impaired tyrosine phosphorylation reduces the ability of IRS-1 and IRS-2 to recruit downstream effector molecules such as phosphatidylinositol 3 (PI3) kinase, resulting in impaired insulin signaling. 40 TNF- activity also was involved in the pathogenesis of HCV-related insulin resistance, and TNF- genotype (resulting from promoter polymorphisms) affected TNF- release, insulin sensitivity, and the severity of liver disease in patients with CHC. 41 Suppressor of cytokine signaling 3. The suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 proteins negatively regulate insulin signaling by targeting IRS-1 and IRS-2 for ubiquitin-mediated degradation. In addition, SOCS-1 and SOCS-3 are inhibitors of the Janus kinase/signal transducers and activators of transcription pathway, which control a number of important biological responses, including insulin regulation and interferon (IFN)-mediated biological activities. 42,43 SOCS-1 and SOCS-3 inhibit the Janus kinase/signal transducers and activators of transcription pathway by binding to either Janus kinase or the cytoplasmic tail of cytokine receptors and/or inhibiting the IFN-induced tyrosine phosphorylation and nuclear translocation of signal transducers and activators of transcription 1, a process required for gene activation. As a result of the latter, SOCS-1 and SOCS-3 block IFNmediated antiviral and antiproliferative activities. 44 Many proinflammatory cytokines, including TNF- and interleukin-6, and insulin up-regulate SOCS protein expression. 45 In obese mice, SOCS-3 was shown to reduce insulin-induced tyrosine phosphorylation of IRS-1 and its subsequent association with the p85 regulatory subunit of PI3 kinase. 46 In a study of obese patients with CHC caused by G1 HCV infection, expression of phosphoenolpyruvate carboxy kinase (P.01) and SOCS-3 (P.047), which inhibit IFN signaling, was significantly higher than in lean patients and may be a mechanism by which obesity reduces the biological response to IFN-alfa. 45 In this same study, SOCS-3 immunoreactivity was increased significantly in patients with obesity (P.013) compared with a lean state and in nonresponders compared with responders (P.014). More recently, a study identified a relationship between SOCS-3 expression and virologic outcomes in patients receiving therapy for CHC. 47 Persico et al 47 reported that SOCS-3 expression was significantly greater in patients receiving treatment for CHC who failed to attain sustained virologic response (SVR) than in those who attained SVR or control patients (P.01 in both cases). Moreover, this study indicated that SOCS-3 expression was greater in patients infected with HCV G1b than G2.

4 August 2008 IULIN RESISTANCE IN CHRONIC HEPATITIS C 867 Table 1. Molecules Involved in the Development of Insulin Resistance Molecule TNF- 35,36,40 SOCS-3 42,45 47 IRS-1 and IRS-2 48 Other adipocytokines 51 53,55 Action Inhibits insulin-stimulated tyrosine phosphorylation of insulin receptor and IRS-1 in adipocytes Stimulates lipolysis, resulting in increased serum-free fatty acids, which lead to insulin resistance in muscle and liver Mediates hepatic insulin resistance, leading to increased hepatic glucose production Down-regulates IRS-1, GLUT-4, PPARs, and adiponectin in adipocytes Interferes with pancreatic -cell function directly or by causing insulin resistance in these cells Negatively regulates insulin signaling by targeting IRS-1 and IRS-2 for ubiquitin-mediated degradation Inhibits the cytokine-mediated JAK/STAT pathway Reduces insulin-induced tyrosine phosphorylation of IRS-1 and its subsequent association with the p85 regulatory subunit of PI3 kinase in mice SOCS-3 expression is significantly higher in patients receiving treatment for CHC who fail to attain SVR than in those who attain SVR or control patients Patients with CHC show a 2- to 3-fold increase in insulin-receptor and IRS-1 levels compared with biopsy samples from non HCV-infected individuals Insulin receptor stimulated tyrosine phosphorylation of IRS-1 is reduced in HCV-infected individuals Interaction between IRS-1 and the p85 subunit of PI3 kinase is impacted leading to decreased activation of the PI3 kinase pathway and severely blunted Akt phosphorylation leading to important implications regarding glucose homeostasis SVR is associated with improved insulin resistance, improved -cell function, and a 3-fold increase in IRS-1/-2 expression in hepatocytes Leptin is profibrogenic, facilitating fibrosis and steatosis: adiponectin is anti-inflammatory and restricts fibrosis and steatosis Serum adiponectin levels correlate with insulin resistance and steatosis in patients with G3, but not G1, hepatitis C Leptin levels decrease during PEG IFN-alfa plus RBV treatment, but rebound back to pretherapy levels during follow-up evaluation Adiponectin levels increase during treatment and remain increased during follow-up evaluation Changes in adiponectin and leptin levels during treatment are independent of virologic outcome JAK, Janus kinase; STAT, signal transducers and activators of transcription. Metabolic syndrome and obesity also were higher in patients infected with HCV G1 and were associated with increased SOCS-3 expression, whereas SVR rates were greater in patients infected with HCV G2. Thus, these investigators suggested a G1b-specific induction of SOCS-3, which may account for the lower SVR rates attained in these patients. Furthermore, SOCS-3 overexpression also may explain the high prevalence of metabolic syndrome in patients with hepatitis C and its association with poor treatment outcomes. Insulin-receptor substrate 1. Insulin and insulinlike growth factor exert their effects through receptor-mediated tyrosine phosphorylation of insulin-receptor substrates such as IRS-1 and IRS-2. IRS proteins coordinate signals through the PI3 kinase, Pkd/Akt and Grb2/Sos, and Ras cascades. Liver biopsy samples from patients with CHC showed a 2- to 3-fold increase in insulin receptor and IRS-1 levels compared with biopsy samples from non HCV-infected individuals. 48 In addition, insulin receptor stimulated tyrosine phosphorylation of IRS-1 was decreased in HCV-infected individuals, reducing the association of IRS-1 with the insulin receptor and resulting in reduced coupling efficiency. The interaction between IRS-1 and the p85 subunit of PI3 kinase then is impacted on, resulting in decreased activation of the PI3 kinase pathway and severely blunted Akt phosphorylation in HCV-infected individuals compared with healthy controls. 48 These effects have important implications regarding glucose homeostasis because the PI3 kinase pathway is involved primarily in the control of metabolic actions of insulin (including glucose, lipid, and protein metabolism), transcription of glucose transporter 4, protein synthesis (through mammalian target of rapamycin), and control of cell survival. 7 Evidence suggests that IRS-1 and IRS-2 interfere with insulin signaling in a genotype-specific manner. IRS-1 protein levels were decreased significantly in human hepatoma cells expressing core HCV G1b and 3a sequences, but IRS-2 levels were not. 49 In addition, although HCV G3a promoted IRS-1 degradation through down-regulation of PPAR- and up-regulation of SOCS-7, the actions of HCV G1b appear to be mediated through the activation of mammalian target of rapamycin. Other Adipocytokines Metabolic abnormalities in patients with CHC also were associated with adipocytokines such as adiponectin and leptin. These agents normally exist in a state of equilibrium, with serum leptin showing profibrogenic properties that facilitate fibrosis and steatosis balanced against adiponectin, which is an anti-inflammatory protein that restricts the progression of fibrosis and steatosis. A recent study showed a correlation between insulin resistance and adiponectin in patients infected with HCV G3, but not patients with G1. 50 In addition, 2 studies found an association between adiponectin and steatosis in patients infected with HCV G3. 51,52 Thus, an imbalance in adiponectin/tnf- may underlie the development of steatosis

5 868 STEPHEN A. HARRISON CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 8 Table 2. Influence of Insulin Resistance or Steatosis on (PEG) IFN-alfa Plus RBV Treatment Outcomes in Patients With CHC Study Patients Treatment Mizuta et al 74 HCV G1b and HVL (N 43) PEG IFN-alfa-2b RBV (ND) for 48 wk Huang et al 75 HCV (N 430): no DM (n 389) PEG IFN-alfa-2a (180 g/wk) RBV mg/d for 24 wk Bortoletto et al 76 HCV (N 30) PEG IFN-alfa-2b RBV (ND) Coonjeevaram et al 14,78 HCV G1 (N 399) PEG-IFN alfa-2a 180 g/wk RBV mg/d for 24 wk D Souza et al 71 HCV (N 59) PEG-IFN RBV (ND) Grande et al 72 HCV G1 (N 75) PEG IFN-alfa (ND) RBV ( mg/d) Harrison et al 79 HCV (N 315) IFN or PEG-IFN RBV (ND) Poynard et al 13 HCV (N 1428) IFN-alfa-2b 3 MU 3 times/wk or PEG IFN-alfa-2b 1.5 g/kg/wk for 4 wk followed by 0.5 g/kg/wk for 44 wk RBV mg/d PEG-IFN 1.5 g/kg/wk RBV 800 mg/d Romero-Gomez et al 73 HCV G1 (N 113), non-g1 (N 46) PEG-IFN alfa-2a 180 g/wk (n 85) or PEG-IFN alfa-2b 1.5 g/kg/wk (n 74) RBV 800 mg/d (G3) or mg/d (G1) Thomopoulos et al 12 HCV (N 116) IFN or PEG-IFN RBV (ND) Poustchi 77 HCV G2/3 (N 82) IFN alfa plus RBV (n 59) PEG-IFN alfa plus RBV (n 23) HVL, high viral load; ND, no further details given; ISI, insulin sensitivity index; PPV, positive predictive value; CI, confidence interval. a Not specified whether mean or median value given. in G3 patients. Importantly, it is the high-molecular-weight adiponectins, responsible for hepatic and whole-body insulin sensitivity, that are critically reduced in G3 patients. 50 Interestingly, another study reported conflicting data, finding no difference in adiponectin or leptin levels between patients with hepatitis C and healthy patients and no correlation between adiponectin levels and steatosis in patients with G3 hepatitis C. 53 The relationship between adiponectin and steatosis also may extend to patients with non-g3 infection. Zografos et al 54 recently reported that lower adiponectin levels correlate significantly with liver steatosis in both G3 (P.02) and G1 (P.025) patients. Other studies confirmed that lower adiponectin levels are associated with steatosis in both G3 and non-g3 patients. 52 Zografos et al 54 also reported that lower adiponectin levels were a significant predictor of lack of end-of-treatment response, but not SVR. Therefore, the role of adiponectins in the metabolic abnormalities found in patients with hepatitis C requires confirmation. Leptin levels also decreased significantly during treatment (P.001 for comparison between baseline and end of therapy), but rebounded to pretherapy levels during the follow-up period. Conversely, serum adiponectin levels increased during treatment and remained increased during the follow-up period (P.02). Importantly, these changes were independent of virologic outcome of treatment. These data suggest that the induction of steatosis through insulin resistance may facilitate the progression of fibrosis through an as yet poorly understood mechanism involving inflammatory activity and adipocytokines. 55 Overall, a direct relationship between leptin and insulin resistance in patients with CHC has not been identified. However, independent associations between leptin and steatosis 56 or fibrosis 57,58 have been reported. Last, a recent report showed that protein phosphatase 2A, which is overexpressed in liver biopsy specimens from patients with CHC and inhibits IFN-alfa signaling, 8,59 interferes with insulin and adenosine monophosphate activated protein kinase signaling. HCV-induced protein phosphatase 2A interferes with insulin resistance in hepatocytes and may be one of the mechanisms accounting for the high prevalence of type 2 DM in patients with CHC. 60 Host and Viral Risk Factors for Insulin Resistance in Chronic Hepatitis C Specific risk factors for the development of insulin resistance in patients with CHC are unclear at present. As non HCV-infected individuals, BMI greater than 25 kg/m 2, family history of DM, and age 40 years or older all correlated significantly with the development of type 2 DM in patients with CHC. 3,25 In addition, other factors, such as decreased physical activity, high-fat/high-fructose diet, presence of fibrosis/inflammation, history of smoking, African American ethnicity, and education of 12 years or less, each also may be associated with the development of DM. 3 Visceral fat, in addition to BMI, likely contributes to the development of insulin resistance. 61 However, some studies indicate a direct association between HCV infection and insulin resistance that is independent of visceral fat. 62 Virus-related factors also contribute to the development of insulin resistance. Although advanced hepatic dysfunction likely contributes to insulin resistance in patients with CHC, there is evidence to suggest that insulin resistance may develop independently of the stage of liver disease. Patients with CHC with absent or very early stage hepatic fibrosis show significantly greater values for insulin, C-peptide, and HOMA-IR than healthy controls matched for BMI, waist-hip ratio, and sex. 10 Patients infected with HCV G3 in whom developing steatosis correlates with viral load are at lower risk for developing

6 August 2008 IULIN RESISTANCE IN CHRONIC HEPATITIS C 869 Table 2. Continued Steatosis/IR population Virologic response Comment/associated factors Patients with EVR: HOMA-IR: ISI composite: HOMA-IR, 1.7 EVR, 51% 2 (n 21) 3 (n 15) ISI composite, 5.7 EVR, 76% EVR, 20% Patients without EVR: SVR, 62% SVR, 13% HOMA-IR, 3.0 (P.005 vs patients with EVR) 2 4 (n 19) 3 6 (n 20) ISI composite, 3.3 (P.02 vs patients with SVR) Non-EVR, 49% EVR, 32% EVR, 60% Patients with SVR: SVR, 26% SVR, 45% HOMA-IR, 1.6 SVR, 42% 4 (n 3) 6 9 (n 5) ISI composite, 6.0 EVR, 0% EVR, 100% Patients without SVR: SVR, 0% SVR, 80% HOMA-IR, 2.8 (P.009 vs patients with SVR) Non-SVR, 58% 9 (n 3) ISI composite, 3.4 (P.003 vs patients with SVR) EVR, 67% SVR, 100% PPV for EVR and SVR with ISI 6, 87.5% All patients (n 430) With SVR Baseline HOMA-IR, 2.28 Without SVR Baseline HOMA-IR, 3.40 (P.002 vs patients with SVR) Patients without DM (n 389) Mean baseline insulin, (range, ) Mean baseline HOMA-IR, (range, ) All patients SVR, 78.1% G1 (n 183), 64.5% Other (n 247), 88.3% Patients without DM SVR (baseline HOMA-IR 2.5), 69.1% SVR (baseline HOMA-IR 2.5), 82.7% (P.002 vs baseline HOMA-IR 2.5) Patients with HOMA-IR 4, undetectable HCV RNA during wk 1 12, 0% Patients with HOMA-IR 4, undetectable HCV RNA, 20% (wk 1), 40% (wk 4), and 50% (wk 12) High baseline ( 2) HOMA-IR, significant negative factor associated with SVR in all patients (OD, 2.22; 95% CI, , P.006) Baseline HOMA-IR, significant negative factor associated with SVR G1 patients (OD, 2.91; 95% CI, , P.006) Patients with high HOMA-IR scores at baseline showed significantly slower HCV RNA decay 24 h after administration of PEG IFN-alfa RBV compared with those with lower HOMA-IR Steatosis (n 253) SVR, 37% IR, but not steatosis, was associated independently No steatosis (n 146) SVR, 46% with SVR. Improvements seen in HOMA-IR scores HOMA-IR 2 SVR, 49% during PEG IFN treatment were not sustained on HOMA-IR 2 SVR, 36% cessation of treatment HOMA-IR, 2.45 a SVR IR associated independently with poorer virologic HOMA-IR, 4.25 a No SVR response to therapy HOMA-IR 2 (n 51) EVR, 100% Insulin resistance decreased IFN activity in the early HOMA-IR 2 (n 24) EVR, 61.5% phase of therapy Steatosis ( 33%; n 24) SVR, 28% Steatosis significantly reduced the SVR rate (P Minimal ( 33%) or no steatosis (n 101) SVR, 44%.001) overall, specifically in patients with G2 or G3 infection, but not G1 Steatosis (n 670) No steatosis (n 364) SVR, 50% SVR, 66% Absence of steatosis associated with significantly higher SVR rate in patients with non-g3 infections but did not influence SVR in G3 patients Steatosis disappeared in almost half the G3 patients showing SVR HOMA-IR 2 (n 43) SVR, 60.5% In patients attaining SVR, HOMA-IR scores improved HOMA-IR 2 4 (n 45) SVR, 40% in first 6 mo and continued to decrease over 18- HOMA-IR 4 (n 25) SVR, 20% mo follow-up period Steatosis (grades 1 3; n 52) SVR, 38.5% Steatosis in liver biopsy specimen before antiviral No steatosis (n 64) SVR, 65.6% treatment was a strong independent factor for treatment failure SVR, 77% Compared with patients without SVR, patients with SVR had lower mean serum insulin levels ( U/mL vs ; P.03), fibrosis stage ( vs ; P.007), and insulin resistance (HOMA-IR; vs ; P.03) BMI (P.001) and fibrosis stage (P.001) were associated independently with HOMA-IR After adjusting for fibrosis stage, patients with HOMA-IR of 2 were 6.5 times more likely to attain SVR than those with HOMA-IR of 2

7 870 STEPHEN A. HARRISON CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 8 Table 3. Influence of (PEG) IFN-alfa Plus RBV on Glucose Parameters in Patients With CHC Study Patients Treatment Lo Iacono 55 HCV G1 and G2, 42% with severe steatosis (N 48) PEG IFN-alfa-2a RBV for 24 or 48 wk according to genotype Simo et al 82 HCV and normal fasting glucose (N 234) IFN-alfa-2b RBV for 24 or 48 wk according to genotype at least 3 y before Kawaguchi et al 81 HCV (N 89) IFN-alfa RBV for 24 wk Kawaguchi et al 80 HCV (N 36) IFN (ND) Korenaga et al 83 HCV (N 25), patients with DM (n 12); IGT, (n 5) PEG IFN-alfa-2b RBV for 12 wk ND, no further details given; ISI, insulin sensitivity index; IGT, impaired glucose tolerance; PG, plasma glucose; AUC, area under the curve; IRI, immunoreactive insulin. a Arbitrary units. insulin resistance than non-g3 patients 10 ; however, they are at higher risk for developing steatosis and develop more severe steatosis. 63,64 A recent study by Moucari et al 65 indicated that insulin resistance may be associated more closely with G1 and G4 than G2 or G3, highlighting the variable influence that HCV genotype exerts on the development of insulin resistance. HCV G3 induced steatosis may be related to expression of PPAR-, which is responsible for transport and -oxidation of free fatty acids and is reduced in patients with HCV infection. 66,67 Specifically, impairment of PPAR- and PPAR- expression is greater in patients infected with HCV G3 than in those infected with HCV G1. 67 In addition, evidence suggests that the HCV-3a core protein may influence the promoter activity of fatty acid synthase, a major enzyme involved in de novo lipid synthesis. 68 Both HCV-3a and HCV-1b core proteins up-regulated the fatty acid synthase promoter. However, significantly higher fatty acid synthase promoter activity was induced by HCV-3a core protein compared with HCV-1b, suggesting that the stronger effect of HCV-3a core protein on fatty acid synthase activation could contribute to the higher prevalence and severity of steatosis in HCV-3a infected patients. Other reports provide evidence for a direct association between HCV and insulin resistance. Several studies reported a positive correlation between viral load and insulin resistance. 31,62,69 Kawaguchi et al 69 reported that patients with CHC and high baseline viral load had significantly increased fasting serum insulin levels and HOMA-IR scores compared with patients with low viral load. Similarly, Harrison 31 observed that in vivo, hyperinsulinemia increased HCV replication. However, perhaps the most convincing data were provided recently by Yoneda et al, 62 who reported significant correlations between HCV RNA quantity and fasting plasma insulin level (P.041) and HOMA-IR score (P.029) in patients with no history of DM, obesity, or alcohol consumption. Finally, a preclinical study in rodents found decreased responsiveness to insulin, primarily owing to hepatic insulin resistance, coupled with significantly increased basal insulin levels in mice transgenic for the HCV core gene compared with nontransgenic mice. 70 Levels of TNF- were increased significantly in transgenic mice compared with controls, and administration of anti TNF- antibodies restored insulin sensitivity in transgenic animals. 70 Insulin Resistance and Steatosis: Impact on Treatment for Chronic Hepatitis C Impact of Insulin Resistance Several studies reported that baseline insulin resistance had a negative impact on treatment outcomes in patients with CHC (Table 2) ,71 79 Romero-Gomez et al 73 reported that in patients infected with HCV G1 receiving pegylated (PEG)-IFN alfa-2a (180 g/wk) plus ribavirin (RBV), there was a direct correlation between insulin resistance (measured using HOMA- IR) and SVR (defined as undetectable HCV RNA 24 weeks after completing treatment). In this study, SVR rates of 61%, 40%, and 20% were observed in patients with HOMA-IR scores less than 2, 2 to 4, and higher than 4, respectively. Furthermore, HOMA-IR scores did not change appreciably during treatment in patients who did not respond to treatment, whereas HOMA-IR scores decreased progressively during treatment in patients who attained SVR. In patients who attained SVR, insulin resistance was significantly lower at the end of the follow-up period compared with baseline values (HOMA-IR score, 2.55 vs 1.50; P.05). Interestingly, in relapsers (patients who attained SVR, then regained detectable HCV RNA levels), HOMA-IR scores decreased during treatment, but returned to baseline values during follow-up evaluation, again supporting a virologic basis for insulin resistance in these patients. In another study of obese G2/3 patients in which 59 patients received IFN-alfa plus RBV and 23 patients received PEG IFNalfa plus RBV, the overall SVR rate was 77%. 77 Compared with patients without SVR, patients with SVR had lower mean serum insulin levels ( vs U/mL; P.03), fibrosis stage ( vs ; P.007), and insulin resistance (HOMA-IR, vs ; P.03). Age, sex, ethnicity, alcohol consumption, treatment regimen, viral load, portal activity, and steatosis did not influence SVR rates. According to linear regression analyses, BMI (P.001) and fibrosis stage (P.001) were associated independently with HOMA-IR. After adjusting for fibrosis stage, patients with a HOMA-IR of less than 2 were 6.5 times more likely to attain SVR than those with a HOMA-IR of 2 or greater. Similarly, Kawaguchi et al 80 reported an improvement in HOMA-IR scores in patients attaining SVR, which were paralleled by a 3-fold increase in expression of IRS-1 and IRS-2 in

8 August 2008 IULIN RESISTANCE IN CHRONIC HEPATITIS C 871 Table 3. Continued Response to therapy Patients with SVR (n 30) Patients with SVR (n 96) Patients without SVR (n 138) Sustained responders (n 29) Nonresponders (n 48) Relapsers (n 12) SVR (n 20) No SVR (n 16) Patients with DM and IGT (n 17) EVR (undetectable HCV RNA at wk 12), 64.7% No EVR, 35.3% Assessments (before vs after therapy) All patients Fasting insulin, vs ; P.001 HOMA-IR, vs ; P.001 Serum adiponectin levels, vs ; P.02 Cumulative incidences of glucose abnormalities 14.6% (with SVR), 34.1% (without SVR), P.001 vs patients with SVR HOMA-IR vs (P.05) HOMA-IR PG AUC (wk 0) vs (wk 4; P.05) 35% reduction; P.05 HOMA vs (P.05) ISI composite P.022 IRI AUC (wk 0) vs (wk 12; P.05) 35% reduction; P.05 IRS-1 and IRS-2 a IRS-1, (P.002) IRS-2, (P.001) HOMA- P.047 HOMA-IR (wk 0) vs (wk 12; P ) hepatocytes, and Huang et al 75 reported that high ( 2.5) baseline HOMA-IR scores were significant (P.006) predictors of non-svr. Overall, in the latter study, 78% (336 of 430) of patients attained SVR (64.5% in patients with HCV G1 and 88.3% in patients with non-g1), and baseline HOMA-IR scores were significantly lower in patients who attained SVR compared with those who did not (2.28 and 3.40, respectively; P.002). In another study, Conjeevaram et al 14 showed that the lower SVR rates attained by African American patients with CHC compared with Caucasian American patients correlated with patients levels of insulin resistance. However, although insulin resistance was an important variable between ethnicities, it did not fully account for the differences seen in SVR rates between these groups. Overall, SVR rates of 49% were attained in patients with HOMA-IR scores of 2 or less and 36% in those with HOMA-IR scores higher than 2 (P.001). Three studies examined the impact of insulin resistance on early viral kinetics and SVR rates. 72,74,76 In a study of 75 patients infected with HCV G1, insulin resistance influenced early virologic response (EVR) rates to PEG IFN-alfa plus RBV therapy. 72 All patients with HOMA-IR scores of less than 2 attained EVR (defined as a 2 log 10 decrease in HCV RNA levels at week 12), whereas only 61.5% of those with HOMA-IR scores higher than 2 attained EVR. In another study of 43 patients infected with HCV G1b who had high baseline viral loads and were treated with PEG IFN-alfa-2b and RBV, there were significant differences in HOMA-IR and insulin sensitivity index composite scores between patients who did and did not attain EVR and between patients who did and did not attain SVR. 74 Multivariate analysis showed that scores higher than 6 in insulin sensitivity index composite values was the only factor associated with SVR. Finally, in a study by Bortoletto et al, 76 patients with high HOMA-IR scores at baseline showed significantly slower HCV RNA decay 24 hours after administration of PEG-IFN alfa plus RBV compared with those with lower HOMA-IR scores. In addition, no patient with an HOMA-IR score of 4 or higher attained undetectable HCV RNA levels during the first 12 weeks of treatment, whereas 20%, 40%, and 50% of patients with HOMA-IR scores of less than 4 attained undetectable HCV RNA levels at weeks 1, 4, and 12 of treatment. Impact of Steatosis Steatosis is also an important determinant of treatment outcomes in patients with CHC. Several studies showed that significantly higher SVR rates were achieved in patients with little or no baseline steatosis compared with patients who had a greater degree of steatosis (Table 2). 12,13,79 The impact of HCV genotype on treatment response in the presence of steatosis is unclear. In 1 study, the presence of steatosis (vs no steatosis) was associated with a significant decrease in SVR rates in patients with HCV G2 or G3 (42% vs 78%, respectively), whereas steatosis did not significantly affect SVR rates in patients with HCV G1 (SVR, 23% vs 34%; P.19). 79 Unfortunately, because of the small patient numbers included in this study, a type II error cannot be ruled out; thus, these data require confirmation in a larger patient population. 79 Poynard et al 13 reported that baseline steatosis was associated with a significantly lower SVR rate in patients with non-g3 infections, but that SVR was independent of baseline steatosis in patients with G3 infection, the majority of whom attained SVR. In this study, in G3 patients who attained SVR, baseline steatosis improved by at least 1 stage in 77% of patients and resolved in 46% of patients. In patients infected with non-g3 HCV, baseline steatosis improved by at least 1 stage in 46% and resolved in 29%.

9 872 STEPHEN A. HARRISON CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 6, No. 8 Table 4. Assessing Insulin Resistance Method Homeostasis model assessment (HOMA-IR, HOMA- ) Quantitative insulin sensitivity check index (QUICKI) Whole-body insulin sensitivity index (WBISI) Calculation fasting insulin IU ml fasting glucose mmo1 L HOMA-IR 22.5 Derived using the inverse of the sum of the logarithms of the fasting insulin and fasting glucose 1 QUICKI log fasting insulin U ml log fasting glucose mg dl Based on values of insulin ( U/mL) and glucose (mg/dl) obtained from the oral glucose tolerance test and corresponding fasting values 10, 000 WBISI fasting plasma glucose fasting insulin mean glucose mean insulin Data from Matsuda and DeFronzo, 84 Matthews et al, 85 and Katz et al. 86 Impact of Chronic Hepatitis C Treatment on Insulin Resistance As discussed, successfully managing insulin resistance or DM in patients with CHC improves patients likelihoods of successful outcomes with anti-hcv therapy. IFN-alfa or PEG IFN-alfa plus RBV therapy may influence insulin resistance and adipocytokine levels in patients with hepatitis C, and several studies have evaluated whether eradicating HCV using this therapy in patients with comorbid insulin resistance or DM improves glucose metabolism (Table 3). 55,78,80 83 In a recent study of patients infected primarily with HCV G1 and G2, 42% of whom had severe steatosis, kinetic analysis revealed that fasting insulin levels and insulin resistance (indicated by using HOMA-IR) decreased significantly during antiviral therapy and that these changes were maintained after treatment cessation (P.001 for both comparisons). 55 Simo et al 82 showed that significantly more patients with normal fasting glucose levels and biopsy-proven CHC who did not attain SVR developed DM during follow-up evaluation compared with patients who attained SVR after treatment with IFN-alfa-2b with or without RBV. More recently, a study evaluating the treatment of 89 patients with CHC showed that eradication of HCV improved insulin resistance, -cell function, and hepatic IRS-1/-2 expression. 81 Nonresponders and relapsers experienced no significant changes in HOMA-IR or HOMA for measuring -cell function (HOMA- ) values after treatment with IFN-alfa based therapy; however, HOMA-IR and HOMA- values significantly decreased in sustained responders. Furthermore, the change in insulin resistance was accompanied by a 2-fold increase in hepatocyte expression of IRS-1 and a 3-fold increase in IRS-2 levels, suggesting a direct interaction between HCV and IRS- 1/-2 in the development of insulin resistance. 81 Kawaguchi et al 80 also showed that patients who attained SVR after IFN-alfa based therapy had a significantly higher insulin sensitivity index composite score (liver indicator) and a lower insulinogenic index (whole-body indicator) compared with baseline values than patients who did not attain SVR. In addition, according to a preliminary report by Korenaga et al, 83 insulin resistance and energy metabolism improved when patients with CHC and DM or impaired glucose tolerance were treated with PEG IFN-alfa-2b plus RBV. However, there were no changes in HOMA-IR or HOMA- scores in either group of patients. Patient Assessment and Management Although standard tests exist to assess patients for insulin resistance and HCV infection, who and when to test are unclear. Existing guidelines provide little direction on how to assess or manage patients with CHC with respect to insulin resistance or patients with insulin resistance/dm with respect to HCV infection. A recent study examined whether patients with type 2 DM should undergo screening for HCV and whether HCV-positive patients should be screened for DM. 84 Overall, the investigators concluded against screening patients with DM for HCV for the following reasons: HCV is not directly diabetogenic, the clinical phenotype of HCV-associated type 2 DM might allow for targeted screening of patients with DM, patients with DM are expected to be poor responders to antiviral therapy and evidence that treatment may reverse type 2 DM is insufficient, and no econometric data exist in the specific subset of patients with type 2 DM. However, the investigators suggested that HCV screening may be prudent in (lean) patients with DM with (advanced) liver disease, and glucose tolerance testing should best be performed in those with HCV-related cirrhosis. 84 Assessing Insulin Resistance Although the glucose clamp remains the gold-standard technique for quantitative measurement of insulin resistance, there are a series of simpler and more practical indices based on the measurement of glucose and insulin (Table 4) Managing Patients With Chronic Hepatitis C and Insulin Resistance In addition to genotype, obesity, insulin resistance, and hepatic steatosis impact the success of antiviral therapy and the rate of disease progression. Therefore, determining whether these factors are present at the time of HCV genotyping will assist in establishing an appropriate treatment plan. If present, encouraging weight loss and reducing insulin resistance and steatosis may improve the response to

10 August 2008 IULIN RESISTANCE IN CHRONIC HEPATITIS C 873 Table 5. Potential Adjuvant Therapies for Patients With CHC Therapy Outcome Level of evidence Antioxidants Decrease oxidative stress Minimal (small studies) Decrease TNF- and other inflammatory mediators Lipid-lowering agents Inhibit HCV RNA replication by altering cholesterol biosynthesis Minimal (pilot study) HMG-CoA reductase inhibitors Insulin sensitizers Thiazolidinediones Concomitant HMG-CoA reductase inhibitor (or statin) therapy may improve treatment outcomes in patients receiving PEG IFN-alfa plus RBV Improve insulin sensitivity via activation of PPAR- Increase adiponectin Decrease SOCS-3 Decrease TNF- Reduce hepatic steatosis Minimal (2 retrospective analyses) Good in patients with NASH; none in patients with CHC Metformin Decrease insulin resistance Positive in patients with NASH; none in patients with CHC Weight loss and exercise Decrease insulin resistance Minimal (small studies) Increase adiponectin Decrease SOCS-3 HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; NASH, nonalcoholic steatohepatitis. IFN-based therapies. However, evidence to support this hypothesis is minimal, and prospective randomized controlled trials are needed. Lifestyle modifications. General advice regarding weight control, diet, and exercise is appropriate for overweight patients who are considering a course of treatment with PEG IFN-alfa plus RBV. In a recent review of the benefits of lifestyle modification in patients with NAFLD, Harrison and Day 88 suggested the metabolic profile could be improved by a diet low in carbohydrates or fat combined with moderate exercise. The benefits of diet-induced weight loss, as suggested for patients with NAFLD, potentially would include reduced hepatic free fatty acid supply (resulting in decreased hepatic insulin resistance), improved extrahepatic insulin sensitivity (decreased circulating insulin levels), decreased adipose tissue inflammation resulting from a decrease in proinflammatory cytokines, increased leptin sensitivity (decreased SOCS-1), and increased adiponectin. 88 Whole-body insulin sensitivity (resulting from improved hepatic insulin responsiveness) in patients with NAFLD can be improved by following a low-fat, reduced-calorie diet. Potential adjuvant interventions. Data from small studies indicate that antioxidants such as ursodeoxycholic acid or vitamin E may improve treatment outcomes in patients with CHC receiving IFN-based antiviral therapy (Table 5). 89,90 However, large prospective studies are required to confirm these observations. In vitro data suggest that lipid-lowering drugs possess antiviral therapy 91 ; however, administration of atorvastatin to a small number of patients with CHC had no impact on viral load. 92 Recent retrospective analyses also indicated that concomitant 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (or statin) therapy may improve treatment outcomes in patients with hepatitis C receiving PEG IFN-alfa plus RBV. 93,94 One analysis reported significantly higher SVR rates in patients receiving PEG IFN-alfa plus RBV who were by chance also receiving a statin compared with patients receiving no statin (63% vs 37%; P.009). 95 This effect was particularly marked for G1, with SVR rates of 55% vs 25% (P.014). Insulin sensitizers (eg, thiazolidinediones and metformin) were shown to improve insulin sensitivity and reduce liver fat content in patients with nonalcoholic steatohepatitis 96 and improve fibrosis stage in patients infected with HCV. 28 In patients with CHC, weight loss resulted in decreased mean fasting insulin and serum alanine aminotransferase levels and insulin resistance and improved steatosis. 97 Preliminary data indicate that weight loss before initiating PEG IFN-alfa plus RBV therapy can improve end-of-treatment response in patients infected with HCV (SVR data not available). 98 Ongoing Studies Several prospective trials are ongoing to determine whether improving insulin sensitivity improves treatment outcomes in patients with hepatitis C receiving PEG IFN-alfa plus RBV. TRIC-1 is an ongoing multicenter Spanish trial examining the effect of triple therapy with PEG IFN-alfa-2a plus RBV and metformin versus PEG IFN-alfa plus RBV and placebo in patients with G1 CHC and insulin resistance. Other studies investigating the effect of pioglitazone or rosiglitazone on insulin sensitivity, hepatic steatosis, and SVR when used in combination with PEG IFN-alfa plus RBV in patients with hepatitis C are under way. At least 1 nonresponder study (IPIRED HCV) in patients who previously failed PEG IFN-alfa plus RBV also currently is enrolling patients. Conclusions Patients with CHC have an increased risk for developing insulin resistance. These patients therefore are faced with