Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug 26;286(34):29872-81.
doi: 10.1074/jbc.M111.259978. Epub 2011 Jun 20.

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase

Hye-Jun Shin  1 , Young-Ho Park, Sun-Uk Kim, Hyung-Bae Moon, Do Sim Park, Ying-Hao Han, Chul-Ho Lee, Dong-Seok Lee, In-Sung Song, Dae Ho Lee, Minhye Kim, Nam-Soon Kim, Dae-Ghon Kim, Jin-Man Kim, Sang-Keun Kim, Yo Na Kim, Su Sung Kim, Cheol Soo Choi, Young-Bum Kim, Dae-Yeul Yu
Affiliations

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase

Hye-Jun Shin et al. J Biol Chem. .

Abstract

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.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
HBx regulates the gene expression of glucose metabolism-related molecules in hepatocytes. A, expression profiles of metabolic genes in HepG2 cells. B, expression profiles of metabolic genes of HepG2-HBx cells infected with lentiviruses expressing HBx shRNA. mRNA levels were analyzed by qRT-PCR. C, increased expression of gluconeogenic genes in HBV-infected patient livers. mRNA was purified from the non-tumor regions of patient livers with HBx expression and cancer (n = 15–22). Data are presented as the mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.
FIGURE 2.
FIGURE 2.
HBx expression causes impaired hepatic glucose metabolism in mice. A, growth curves were constructed by plotting mean ± S.E. body weight of WT and HBxTg mice (n = 11–16). B, comparison of liver and epididymal fat weight/body weight between WT (n = 7) and HBxTg (n = 8) mice at 6 months of age. C, glucose concentration in blood derived from mouse tail vein in 12-h fasted and ad libitum-fed mice at 6 months of age. D and E, insulin sensitivity was examined by a glucose tolerance test (D) and insulin tolerance test (E). Mice were 6 months of age (n = 10–13). F, glycogen content was measured in liver of 12-h fasted and ad libitum-fed mice (n = 6). Periodic acid-Schiff stained paraffin-embedded liver sections of HBxTg mice at 6 months of age. The scale bar represents 100 μm. G, glucose amount secreted in media from cultured primary hepatocytes of WT and HBxTg mice. Data are presented as the mean ± S.E. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
FIGURE 3.
FIGURE 3.
Whole body glucose metabolism during hyperinsulinemic-euglycemic clamp in HBxTg mice. A and B, basal and clamp hepatic glucose production (HGP) (A) and basal glucose clearance (B) during the clamp of WT and HBxTg mice. C and D, plasma glucose concentrations (C) and glucose infusion rate (D) during the clamps. E and F, whole body glucose turnover rate, whole body glycolysis rate, whole body glycogen synthesis (E), and insulin-stimulated muscle (gastrocnemius) glucose uptake (F) were determined. 2DG, 2-deoxy-d-[1-14C]glucose. All data are presented as mean ± S.E. (*, p < 0.05, t test, n = 10–11).
FIGURE 4.
FIGURE 4.
Hepatic expression of metabolic genes in HBxTg mice. A, mRNA levels were quantified by qRT-PCR in 6-month-old mice on ad libitum-fed (Ad lib) and 12-h fasted states. Each PCR was carried out in triplicate. B and C, immunoblot analysis of total liver lysates obtained from ad libitum-fed mice. pAkt, phosphorylated Akt; pJNK, phosphorylated JNK; pFOXO1, phosphorylated FOXO1. D, IRS-1 and IRS-2-associated PI 3-kinase activity in the liver from 12-h fasted state. Tissues were collected 6 min after intravenous saline or insulin (10 units/kg (body weight)) injection. E, insulin signaling in liver of WT and HBxTg mice. Liver lysates were subjected to SDS-PAGE and immunoblot with antibodies as indicated. pIR, phospho-insulin receptor; IR, insulin receptor. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control. ##, p < 0.01 versus WT. The bars represent the S.E. (n = 4–7).
FIGURE 5.
FIGURE 5.
Reciprocal activation between HBx and iNOS in HepG2 cells and HBxTg mice. A and B, relative levels of HBx (A) and iNOS (B) mRNAs in HBxTg mice (n = 6–8) in ad libitum-fed (Ad lib), 12-h fasted, and refed states. C, expression of HBx and gluconeogenic genes mRNA in cells treated with SNP (50 μm) at the indicated times. All the data were the mean of three independent experiments and are indicated as mean ± S.E. (*, p < 0.05; ***, p < 0.001 versus control. #, p < 0.05; ###, p < 0.001 versus Mock).
FIGURE 6.
FIGURE 6.
Involvement of JNK1 in induction of gluconeogenic genes by NO in HBx-expressed cells. A, relative JNK1 transcript levels of cells treated with the indicated concentrations of SNP for 48 h (#, p < 0.05 versus 0 μm). B, immunoblot analysis of phosphorylated JNK (pJNK) in cells treated with SNP (50 μm). C, expression profiles of metabolic genes in cells treated with JNK1 siRNA. Levels of JNK1 mRNA 48 h after transfection with siRNAs were evaluated by qRT-PCR and immunoblotting assays. Scr, scrambled. D, HepG2 cells were transfected with JNK1 siRNA at 24 h before treatment with SNP (50 μm) for 48 h, and the transcript levels of JNK1 and gluconeogenic genes were analyzed by qRT-PCR. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Each PCR was carried out in triplicate.
FIGURE 7.
FIGURE 7.
Effect of iNOS on glycemic control in HBxTg mice. A, PCR analysis of genomic DNA from mouse tails (HBxTg, 355 bp; iNOS wild allele, 108 bp; iNOS mutant allele, 275 bp). B, blood glucose levels in 12-h fasted and ad libitum-fed mice. C and D, insulin sensitivity was examined by a glucose tolerance test (C) and an insulin tolerance test (D). Mice were 6 months of age (n = 6–10). E, liver glycogen contents measured in the liver lysates of 12-h fasted and ad libitum-fed mice (n = 6). Periodic acid-Schiff stained paraffin-embedded liver sections at 6 months of age. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for HBxTg versus HBxTg/iNOS−/− mice.
FIGURE 8.
FIGURE 8.
Normalization of the expression of genes related to glucose metabolism by deficiency of iNOS in HBxTg mice. A, HBx mRNA levels were quantified by qRT-PCR in ad libitum-fed (Ad lib) and 12-h fasted states in HBxTg (n = 6) and HBxTg/iNOS−/− (n = 8) mice. B, metabolic gene expression of 6-month-old mice in ad libitum-fed and 12-h fasted states. C, relative levels of hepatic JNK1 gene expression in mice (n = 5–6). *, p < 0.05; ** p < 0.01; ***, p < 0.001 for HBxTg versus HBxTg/iNOS−/− mice. #, p < 0.05 versus WT.
FIGURE 9.
FIGURE 9.
A potential mechanism by which HBx leads to impaired hepatic glucose metabolism. Induction of HBx and iNOS is mutually activated, and HBx participates in activation of the JNK1/FOXO1 signaling pathway. Coupled with PGC1α activation, FOXO1 participates in up-regulation of gluconeogenic genes, such as G6Pase and PEPCK. Thus, blood glucose level is increased, and the risk of glucose intolerance is enhanced by the impaired hepatic glucose metabolism.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nordlie R. C., Foster J. D., Lange A. J. (1999) Annu. Rev. Nutr. 19, 379–406 - PubMed
    1. Pilkis S. J., Granner D. K. (1992) Annu. Rev. Physiol. 54, 885–909 - PubMed
    1. Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (2003) Diabetes Care 26, Suppl. 1, S5–S20 - PubMed
    1. Kruszynska Y. T., Home P. D., McIntyre N. (1991) Hepatology 14, 103–111 - PubMed
    1. La Vecchia C., Negri E., Decarli A., Franceschi S. (1997) Int. J. Cancer 73, 204–207 - PubMed

Publication types

MeSH terms