Abstract
Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and β-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor α, which plays a dominant role in the expression of β-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor α is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced β-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced β-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis.
Highlights
Disturbances in lipid metabolism have long been associated with chronic hepatitis C virus (HCV) infection [41,42,43,44,45,46,47], and the discovery of a specific HCV strain based on genotype 2 (JFH-1; Japanese fulminant hepatitis-1) that efficiently infects and replicates in cultured Huh7.5/7.5.1 hepatoma cells (48 –51) has provided considerable insight regarding the intimate link between host cell lipids and HCV infection, at virtually each stage of the HCV replication cycle [45]
Providing there are sufficient levels of viral replication, an HCV-induced cytopathic effect is readily observed with this system and is characterized by massive cell death due to apoptosis, which strongly correlates with cell cycle arrest and the induction of numerous genes involved in antioxidative stress response [7, 9, 52,53,54]
The attenuated de novo lipogenesis (DNL) and enhanced -oxidation observed during this stage of infection likely reflect AMPK-mediated lowering of both ACC1- and acetyl-CoA carboxylase 2 (ACC2)-derived malonyl-CoA pools [79, 80]
Summary
Materials—Fatty acid-free bovine serum albumin (BSA), oleic acid (OA), glycerol, leupeptin, phenylmethanesulfonyl fluoride (PMSF), protease inhibitor mixture, cholesteryl oleate, L-␣-phosphatidylcholine, heptane, isopropyl ether, GW7647, ␣-tocopherol, N-acetylcysteine (NAC), and digitonin were obtained from Sigma. [1-14C]oleic acid stock (2.5 Ci) bound to 0.4 mM OA, 0.5% BSA in 10 ml DMEM was placed in T150 flasks containing cell cultures and fitted with a center vial with a filter paper moistened with 1 M KOH to absorb CO2. ATP Assay—ATP was measured in cells using the Promega ENLITEN ATP assay system bioluminescence detection kit according to manufacturer’s instructions. For detection of only HCV antigens, the primary antibody solution was replaced with anti-HCV antibodies (core and NS5A) in a PBS solution (containing 1% BSA as carrier, same solution as that provided for anti-HRP mouse monoclonal from Biogenex). Supernatant (cell homogenate) was collected, and lipids were extracted from cell homogenates with chloroform/methanol (2:1, v/v) in the absence (mass determination) or presence (metabolic studies) of nonradioactive lipid carriers (phosphatidylcholine, oleic acid, dioleoylglycerol, trioleoylglycerol, and cholesteryl oleate) as described previously [75]. Other Methods—Protein concentrations were determined using the Pierce micro BCA assay Kit according to the supplied protocol
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