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Hepatitis B Virus X Protein Regulates Hepatic Glucose Homeostasis via Activation of Inducible Nitric Oxide Synthase*

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

gene authentic promoter DNA derived from a pHEX1 vector. HepG2 cells were plated in 6-well culture plates for 24 hr prior to transfection. Cells were transfected with 3 μg of pcDNA3-HA-HBx constructs using a Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After 48 hr, cells were trypsinized and plated in a medium containing 1000 μg/ml G418. Following selection for 2 weeks, total populations of G418-resistant cells were pooled and single-cell sorted into 96-well plates with a growth medium containing 1000 μg/ml G418. Sorted single cells were grown under selection for an additional 2 weeks and expanded into stable cell lines. The candidate clones were analyzed by western blot analysis using a specific HA-antibody (Roche), and by RT-PCR analysis with a specific HBx primer.
Quantitative Real-Time PCR Analysis -Total RNA was prepared from HepG2 cells or mouse tissue using TRIzol (Molecular Research Center) according to the manufacturer's protocol. cDNA synthesized with oligo (dT) primers with the first-strand cDNA Synthesis kit (Fermentas) was mixed with the SYBR Premix Ex Taq (TaKaRa) and sets of gene-specific primers. The resulting mixture was subjected to realtime RT-PCR quantification with the Exicycler TM 96 detection system (Bioneer Corp.). Primers for PCR were designed using the Primer3 software program. The specificity of primers was verified by BLAST analysis of the mouse and human genome, visualized by RT-PCR products after agarose gel electrophoresis, and analyzed by the melting point of the PCR products. Sequences for primers used for this study are provided as supplemental information (Supplemental Table 2).
Metabolic Measurements -The glucose tolerance test, insulin tolerance tests, pyruvate challenge, hepatic glucose production, immunoblotting, and serum metabolic parameter analyses were performed in accordance with standard or published protocols (2). Blood was collected via a tail bleed in mice in the fed state between 8 and 10 a.m. Blood glucose was measured using a GlucheFine glucose meter (KMH Corp.). Serum concentrations of insulin and glucagon were measured using an insulin ELISA kit (Shibayagi) and glucagon EIA kit (Alpco Diagnostics) respectively. Plasma concentrations of triglyceride, total cholesterol, non-esterified fatty acid, alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol were determined enzymatically using reagents from Wako Pure Chemical Industries Ltd. (Japan) on a Hitachi 7600-110 automatic analyzer (Hitachi High-Technologies Co.). For the glucose tolerance tests, mice were fasted overnight, and glucose (2 g/kg of body weight) was administered intraperitoneally. For the insulin tolerance tests, mice were fasted for 5 hr, and human insulin (1 IU/kg of body weight; Humulin R, Lilly) was administered intraperitoneally. For the pyruvate tolerance tests, mice were fasted for 24 hr, and pyruvate (2 g/kg of body weight) was administered intraperitoneally. All metabolic measurements were performed with mice at 6 months of age.
Hepatic Glucose Production Analysis -Mouse primary hepatocytes were isolated using a collagenase perfusion protocol. The viable hepatocyte population was further purified by a Percoll gradient centrifugation. The cells were plated at high density on collagen I-coated Biocoat dishes (BD Biosciences) in Williams' E medium (Invitrogen) supplemented with 10% fetal bovine serum and penicillin-streptomycin-glutamine. Primary hepatocytes were plated at 100,000 cells per well in 96-well tissue culture plates and left to attach for approximately 4 hr. The cells were washed twice with phosphate-buffered saline and incubated for 1 hr in a glucose production medium (glucose-and serumfree Dulbecco's Modified Eagle's Medium, containing 20 mM sodium lactate and 2 mM sodium puruvate). The glucose in the medium was assayed with a colorimetric kit (Invitrogen) according to the manufacturer's instructions.
Hepatic Glycogen Analysis -Glycogen content in the liver was analyzed by a previously described method. Briefly, approximately 50 mg of liver sample was weighted and boiled in 400 μl of 2 N HCl for 2 hr. And then 400 μl of 2 N NaOH and 22 μl of 1 M Tris-HCl (pH 7.4) were added to neutralize the acid.
The glucosyl units from the above glycogen breakdown were measured using an Amplex Red Glucose Assay kit (Invitrogen).
Histology and PAS Staining -Liver tissues from mice were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned with 4 µm in thickness, and stained with hematoxylin-eosin. Periodic acid Schiff (PAS) stain was performed by incubating with 1% periodic acid for 10 minutes, with Schiff solution (Muto) for 10 minutes and with hematoxylin for 3 minutes.