Division of Gastroenterology and Hepatology, University of California San Francisco (UCSF) Fresno Education Program, Community Regional Medical Center, Fresno, CA
Associate Professor of Clinical Medicine and Chief, Division of Gastroenterology and Hepatology, UCSF Fresno, Community Regional Medical Center, 2823 Fresno Street, 1st Floor, Endoscopy, Fresno, CA 93721
Potential conflict of interest: Nothing to report.
Chronic infection with hepatitis C virus (HCV) can induce insulin resistance (IR) in a genotype-dependent fashion, thus contributing to steatosis, progression of fibrosis and resistance to interferon therapy. The molecular mechanisms in genotype 1 patients that lead to metabolic syndrome are still ambiguous. Based on our current understanding, HCV proteins associate with mitochondria and endoplasmic reticulum and promote oxidative stress. The latter mediates signals involving the p38 mitogen-activated protein kinase and activates nuclear factor kappa B. This transcription factor plays a key role in the expression of cytokines, tumor necrosis factor alpha (TNF-α), interleukin 6, interleukin 8, tumor growth factor beta, and Fas ligand. TNF-α inhibits the function of insulin receptor substrates and decreases the expression of the glucose transporter and lipoprotein lipase in peripheral tissues, which is responsible for the promotion of insulin resistance. Furthermore, reduced adiponectin levels, loss of adiponectin receptors, and decreased anti-inflammatory peroxisome proliferator-activated receptor alpha in the liver of HCV patients may contribute to reduced fatty acid oxidation, inflammation, and eventually lipotoxicity. This chain of events may be initiated by HCV-associated IR and provides a direction for future research in the areas of therapeutic intervention. (HEPATOLOGY 2008.)
Hepatitis C virus (HCV) and nonalcoholic fatty liver disease (NAFLD) are the two most common causes of chronic liver disease in North America. HCV affects approximately 2%, NAFLD 6 to 14%, and nonalcoholic steatohepatitis (NASH) 3% to 5% of the general population in the United States. The concomitant presence of obesity, NAFLD, and HCV has significant consequences on the liver histology.1 The interaction of these various factors in HCV patients predisposes them to steatosis, thus increasing the risk of fibrosis and liver cancer.2, 3 Recently HCV infection is noted to be an independent predictor of diabetes mellitus, and the latter as a predictor of treatment failure in patients with hepatitis C.4
HCV induces several complex mechanisms that lead to inflammation, insulin resistance, steatosis, fibrosis, apoptosis, altered gene expression, and hepatocellular carcinoma (HCC).2, 3, 5, 6 Increased oxidative stress is now proposed as a major initiator of HCV selected pathogenesis.7 With emerging insight into the pathogenic mechanism leading to insulin resistance, HCV is now viewed to cause a metabolic syndrome, as opposed to simple viral infection. Significant attention is presently being drawn toward the HCV molecular pathways that lead to insulin resistance. By treating HCV as a metabolic disease, novel approaches toward understanding the pathogenesis of hepatitis C as a virus-associated steatohepatitis could be developed in the future. This review summarizes the current understanding and potential molecular pathways by which HCV, particularly genotype 1, contributes to insulin resistance. Risk factors affecting these patients and future perspectives for research in this field are also outlined.
HCV Genome and Oxidative Stress
HCV genome is composed of structural (core, E1 and E2) and nonstructural genes (NS2-NS5B), arranged in the following order: NH2-Core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. Of the HCV nonstructural proteins, NS3 and NS5A act as key mediators in the induction of oxidative stress and inflammation. The association of NS5A with the endoplasmic reticulum (ER) has been suggested to stimulate mitochondrial reactive oxygen species (ROS) production by releasing calcium from the ER.8 In addition, NS3 has been shown to activate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2) that generates ROS.9 Nox2 can nitrosylate proteins within cells and can lead to a large number of pathological processes. Consequently, a role for NOX enzymes in hepatic fibrosis, characterized by hepatic stellate cell (HSC) proliferation and accumulation of extracellular matrix proteins, has been suggested.10, 11
The HCV core protein has been found to localize to the outer mitochondrial membrane. It facilitates the uptake of Ca2+ into the mitochondria and induces mitochondrial permeability transition.12 Following calcium accumulation, there is a stimulation of the electron transport, which increases the production of ROS. Such an environment stimulates the activity of nitric oxide (NO) synthase and displaces cytochrome C (cyt C). NO synthase generates nitric oxide and its derivatives, which can induce inflammation, DNA damage, and cell death.13 The net depletion of glutathione (GSH) and the release of cytochrome C impair mitochondrial function and further elevate ROS in hepatocytes.12, 14
During HCV replication, core also associates with the ER.15 Under normal conditions, various secretory and membrane proteins are correctly folded and assembled by chaperones in the ER. HCV has been shown to replicate in lipid rafts/Golgi membranes and on the cytosolic side of the ER membrane and drives the ER to produce and process viral proteins.16 This replication may then cause the chaperones to become overloaded. The ER fails to export synthesized proteins properly; leading to an accumulation of misfolded proteins.17 These misfolded proteins generate an unfolded protein response (UPR) that eventually causes ER dysfunction and cell death.18 It has been demonstrated that once the functional capacity of the ER is jeopardized, the resulting ER stress can lead to the activation of inflammatory signaling pathways.18 A summary of the sequential molecular effects of HCV-core proteins is described in Fig. 1.
Role of the HCV NS-Proteins
NS5A-induced mitochondrial ROS production8, 15, 17 has been suggested to activate nuclear factor-κB (NF-κB). NS5A can also activate NF-κB by binding to Toll-like receptor 4 (TLR-4) found on the plasma membranes of hepatocytes and B cells.19 Activation of NF-κB by oxidative stress may involve p38 mitogen activated protein kinase (MAPK).20 In nonstimulated cells, NF-κB resides in a sequestered form bound to its inhibitor (IκBα) in the cytoplasm. When stimulated, IκBα releases its inhibition, allowing NF-κB to enter the nucleus. NF-κB would then bind to IκB elements and up-regulate genes involved in cytokine production, thus increasing the levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8). Such activation of NF-κB and Stat by NS5A may play an important role in inflammation, immune responses, alterations in Fas-mediated apoptotic pathways, and tumor formation.8
NS3-induced ER and oxidative stress may also activate NF-κB and increase the risk of inflammation, insulin resistance and HCC in a similar way as NS5A.9, 22 Effects of HCV-NS proteins on the ER are summarized in Fig. 2.
Role of the HCV Core Protein
Peroxisome proliferator-activated receptor (PPAR) α induces gene transcription of an array of enzymes involved in mitochondrial and peroxisomal β-oxidation and microsomal ω-oxidation, required for proper metabolism of triglycerides (TG) and lipids, respectively.23 HCV core protein has been shown to inhibit PPAR-α and PPAR-γ, expressed in macrophages, adipocytes, and hepatocytes.24 Accumulation of hepatic TG is associated with loss of adiponectin receptors in the liver, and together with reduction in circulating adiponectin contributes to systemic insulin resistance and various other metabolic anomalies.25
Role of Cytokines in Insulin Resistance and Fibrosis
During HCV infection, several antioxidant enzymes may be up- or down-regulated, suggesting chronic oxidative stress with possible dysfunction of the cellular antioxidant defense. Immune response against HCV releases ROS from sequestered phagocytes and activated Kupffer cells in the liver.26 Increased oxidative stress caused by HCV may result in the activation of Kupffer cells.26 The change in H+ concentration alters the balance in the Na+/H+ exchanger, causing the Kupffer cell to swell and eventually burst. This releases ROS and an arsenal of inflammatory mediators such as, TNF-α, TGF-β, IL-6, IL-8. The rising concentrations of ROS induce lipid peroxidation and damage triglycerides. The process of lipid peroxidation disrupts cellular membranes and can induce mitochondrial dysfunction.13
TNF-α is released from Kupffer cells, B cells, adipocytes, and hepatocytes. Once produced, this can modulate adipocytes and bring about changes in the production of cytokines, adiponectin and leptin.27, 28 It is known that decreased adiponectin levels ultimately lead to insulin resistance.28 TNF-α has also been shown to directly impair insulin signaling through serine phosphorylation of IRS-1 and 2, thus down-regulating glucose transporter (GLUT2/GLUT4) gene expression.29 By preventing the uptake of glucose into hepatocytes and adipocytes, TNF-α promotes a state of hyperinsulinemia and hyperglycemia.30 Hyperinsulinemia decreases apolipoprotein B-100, thus preventing the formation of hepatic VLDL and accumulating triglycerides31 TNF-α further enhances triglyceride levels by inhibiting the transcription of lipoprotein lipase (LPL).32 Decreased lipoprotein lipase in peripheral tissues and reduced apolipoprotein B-100 levels in liver can enhance steatosis in liver, increasing the risk for NAFLD and insulin resistance.31 Excessive TNF-α response in HCV-infected patients has recently been suggested as a possible mechanism of increased development of diabetes in this patient population.33 Pro-fibrogenic leptin and anti-fibrogenic adiponectin can also be unregulated and down regulated, respectively, by TNF-α.
Interleukin-6 is a cytokine that is secreted from Kupffer cells, adipocytes, B cells, and hepatocytes. HCV-infected patients are known to have elevated levels of IL-6 due to the virus-induced inflammatory state.34 Increased IL-6 derived from adipocytes leads to an ongoing acute-phase response that acts on hepatocytes and promotes hepatic insulin resistance. IL-6 is able to inhibit the expression of LPL in mice.35 Unlike TNF-α, IL-6 circulates at high levels in plasma, perhaps representing a hormonal role of IL-6 that may induce insulin resistance in other tissues besides liver.
Transforming growth factor-beta (TGF-β), released from Kupffer cells activates hepatic stellate cells (HSC) and regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans.36 TGF-β activates SMAD (mothers against decapentaplegic homolog) proteins through two signaling pathways. The SMAD proteins further increase the production of TGF-β by promoting its transcription.37 SMAD proteins are also involved in the synthesis and deposition of collagen. This increase in collagen by both TGF-β and TNF-α enhances the risks of fibrosis and development of HCC.
Role of Adiponectin in Insulin Resistance
Adiponectin is a soluble matrix protein produced by adipocytes that has anti-atherogenic and anti-inflammatory properties. It enhances the insulin sensitivity of hepatocytes and thus plays an important role in energy homeostasis and fatty acid metabolism. Decreased levels of adiponectin are associated with insulin resistance, obesity and other symptoms of the metabolic syndrome.25 Adiponectin modulates its effects by binding to adiponectin receptors, Adipo R-1 and R-2. Adipo R2 is a seven transmembrane domain receptor that is most abundant in the liver. Genetic variations in Adipo R1 and Adipo R2 have been implicated in Amish people with impaired glucose tolerance and type-2 diabetes.38 The synthesis and secretion of adiponectin is increased by activation of the nuclear receptors, PPAR-γ and PPAR-α.25, 39 Adiponectin is also reduced by caloric excess, presumably associated with obesity, and leptin deficiency or resistance.25, 39 There is also a loss of adiponectin receptors described in the livers of HCV patients. In combination with decreased circulating adiponectin and reduced hepatic PPARα, this may contribute to reduced fatty acid oxidation, increased inflammation, and eventually lipotoxicity. Experiments using a liver-specific PPARα deletion in ob/ob mice resulted in hypoadiponectinemia, hypoglycemia, hyperlipidemia, and fatty liver suggesting a mechanistic link between PPARα, adiponectin and metabolic abnormalities. The sequential molecular pathways that may lead to HCV-induced insulin resistance, fibrosis, apoptosis, and fatty liver are summarized in Fig. 3.
Other Factors Affecting Insulin Resistance
Viral and host factors may also play a crucial role in compounding the onset of insulin resistance in patients with chronic hepatitis C.
HCV may promote insulin resistance irrespective of the severity of liver disease and this effect appears to be genotype specific.40 Steatosis in HCV appears to be due to viral induced cytopathic or metabolic effects. Cytopathic steatosis occurs in genotype 3 and is due to blockade of lipoprotein secretion during viral replication and disappears with antiviral therapy. Metabolic steatosis occurs in non-genotype 3 and is predominantly due to virus induced insulin resistance41; correlates with the body mass index (BMI) and plays a role in the progression of fibrosis and decreased response to antiviral therapy.42
Obesity is viewed as a low-grade generalized systemic inflammation. It is an independent risk factor for insulin resistance in HCV, NASH, alcoholic liver disease and HCC.1, 25 Abdominal adipose tissue is an important source of free fatty acids and inflammatory factors that cause insulin resistance. Elevated BMI plays a role in the pathogenesis of steatosis and thus contributing to fibrosis in HCV patients.43 The combination of IR, steatosis, and fibrosis has significant impact on prognosis and therapeutic response in HCV.
Resistin is mainly an adipocyte derived hormone44 but it is associated with the production of proinflammatory cytokines (TNF-, IL-1, IL-6 and IL-12) through an NF-κB-dependent pathway. It up-regulates the expression of adhesion molecules (VCAM1 and ICAM1) and promotes the release of endothelin-1 in human endothelial cells. Elevated serum levels are associated with insulin resistance, disease severity, clinical complications, and prognosis in patients with chronic liver diseases.45
Several host genes have been found to be up-or-down regulated due to HCV core protein expression and 14 newly identified cellular proteins were found to bind the HCV core protein. Understanding the functional implications of these interactions using genomics and proteomics approaches will enhance our understanding of HCV pathogenesis and may shed light on functional associations with insulin resistance.
To date, several studies have demonstrated the potential of adiponectin to improve insulin sensitivity, lower lipid deposition, and activate anti-inflammatory pathways. Future studies are required to clearly define the role of adiponectin therapy in humans.
The pathways leading to HCV-induced insulin resistance are shown to be a risk for HCC development. Insulin resistance markers need to be further investigated to prove their associations with HCC pathogenesis. The implementation of drugs to improve glucose metabolism and curb insulin resistance may prove to be useful in the prevention of fibrosis and HCC.
Resistin has been shown to induce severe hepatic insulin resistance. At present, there is no study establishing a relationship between HCV infections and resistin. Additional studies are needed to assess the biological mechanisms of resistin and potential use of resistin antibodies in humans as a potential mode of therapy to reduce insulin resistance.
It is presently known that the HCV core protein affects intracellular calcium concentrations that generate ROS from within the mitochondrial electron transport chain. However, the secondary effect (s) of core protein on oxidative phosphorylation should be studied further.
There are presently ongoing studies exploring the role of anti-TNFα therapy in the management of HCV infection.
Viral and metabolic factors are now known to cause steatosis in genotype 3 and 1 patients respectively. Future studies are now needed to assess the role of different genotypes in the development of insulin resistance and steatosis.
Thiazolidinediones are receptor agonists for PPAR-γ that activate the transcription of LPL and adiponectin. Two members of this class, pioglitazone and rosiglitazone have been shown to improve insulin resistance in combination with metformin. The role of thiazolidinediones in improving sustained virologic response (SVR) is presently being evaluated in combination with interferon based combination therapy in chronic hepatitis C (CHC) genotype 1 patients.
Lifestyle modification is the cornerstone of treatment for metabolic syndrome. Formal studies are presently needed to evaluate the efficacy of exercise therapy along with pharmacologic therapy in achieving SVR in CHC patients.