Involvement of the PA28gamma-dependent pathway in insulin resistance induced by hepatitis C virus core protein

Abstract

The hepatitis C virus (HCV) core protein is a component of nucleocapsids and a pathogenic factor for hepatitis C. Several epidemiological and experimental studies have suggested that HCV infection is associated with insulin resistance, leading to type 2 diabetes. We have previously reported that HCV core gene-transgenic (PA28gamma(+/+)CoreTg) mice develop marked insulin resistance and that the HCV core protein is degraded in the nucleus through a PA28gamma-dependent pathway. In this study, we examined whether PA28gamma is required for HCV core-induced insulin resistance in vivo. HCV core gene-transgenic mice lacking the PA28gamma gene (PA28gamma(-/-)CoreTg) were prepared by mating of PA28gamma(+/+)CoreTg with PA28gamma-knockout mice. Although there was no significant difference in the glucose tolerance test results among the mice, the insulin sensitivity in PA28gamma(-/-)CoreTg mice was recovered to a normal level in the insulin tolerance test. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), production of IRS2, and phosphorylation of Akt were suppressed in the livers of PA28gamma(+/+)CoreTg mice in response to insulin stimulation, whereas they were restored in the livers of PA28gamma(-/-)CoreTg mice. Furthermore, activation of the tumor necrosis factor alpha promoter in human liver cell lines or mice by the HCV core protein was suppressed by the knockdown or knockout of the PA28gamma gene. These results suggest that the HCV core protein suppresses insulin signaling through a PA28gamma-dependent pathway.

FIG. 1.

Characterization of HCV core gene-transgenic mice deficient in the PA28γ gene. (A) Expression of the HCV core protein and PA28γ in the livers of PA28γ^+/+, PA28γ^+/+CoreTg, PA28γ^−/−, and PA28γ^−/−CoreTg mice. Lysates obtained from liver tissues of the mice (100 μg protein/lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies to the HCV core protein, PA28γ, and β-actin. (B) Body weights of the mice. Body weights of 2-month-old mice were measured (n = 7 in each group). There were no statistically significant differences in body weights among the mice (P > 0.05).

FIG. 2.

Knockout of the PA28γ gene inhibited the hyperinsulinemia induced by HCV core protein. Plasma glucose levels of PA28γ^+/+, PA28γ^+/+CoreTg, PA28γ^−/−CoreTg, and PA28γ^−/− mice under fasting (A) or fed (B) conditions (n = 7 in each group) are shown. Serum insulin levels in fasting (C) or fed (D) mice (n = 7 in each group) are also shown. Values are represented as means ± standard deviations. *P < 0.05; **P < 0.01. NS, not statistically significant.

FIG. 3.

Knockout of the PA28γ gene inhibits the insulin resistance induced by the HCV core protein. (A) Glucose tolerance test. d-Glucose was intraperitoneally administered to mice fasted for more than 16 h at 1 g/kg of body weight. Plasma glucose levels were estimated at the indicated times (n = 5 in each group). There were no significant differences in glucose levels among the mice (P > 0.05). (B) Insulin tolerance test. Human insulin (2 units/kg body weight) was intraperitoneally administered to the mice, and the plasma glucose levels were estimated at the indicated times. Values were normalized to the baseline glucose concentration at the time of insulin administration (n = 5 in each group). The values for the PA28γ^+/+ (open circles), PA28γ^+/+CoreTg (closed circles), PA28γ^−/− (open triangles), and PA28γ^−/−CoreTg (closed triangles) mice are represented as means and ± standard deviations. Significant differences in insulin sensitivity (P < 0.01) in PA28γ^+/+CoreTg mice compared to that in PA28γ^+/+, PA28γ^−/−, or PA28γ^−/−CoreTg mice are indicated by double asterisks (**). There were no significant differences among PA28γ^+/+, PA28γ^−/−, and PA28γ^−/−CoreTg mice (P > 0.05).

FIG. 4.

PA28γ participated in the enlargement of pancreatic islets induced by the HCV core protein. (A) Histological sections prepared from pancreas tissues of PA28γ^+/+, PA28γ^+/+CoreTg, PA28γ^−/−, and PA28γ^−/−CoreTg mice were stained with hematoxylin and eosin. Dotted circles indicate pancreatic islets. (B) The area occupied by pancreatic islets was measured by computer software in three different fields of every six randomly selected sections of 10 mice per genotype and is represented as a percentage of the total pancreatic area. **P < 0.01; ***P < 0.001. The scale bar indicates 100 μm.

FIG. 5.

PA28γ participated in the inhibition of the tyrosine phosphorylation of IRS1 induced by the HCV core protein. Liver tissues from PA28γ^+/+, PA28γ^+/+CoreTg, PA28γ^−/−, and PA28γ^−/−CoreTg mice were prepared after administration of insulin (+) or phosphate-buffered saline (−). The samples (100 μg of total protein) were examined by immunoblotting with antibodies against IRS1 and phospho-Tyr608 of mouse IRS1 (A). Phosphorylated IRS1 was estimated from the density on the immunoblotted membrane by using computer software (B) (n = 5 in each group). The data presented are representative of three independent experiments. *P < 0.05; **P < 0.01.

FIG. 6.

PA28γ participated in the inhibition of the IRS2 expression and Akt phosphorylation induced by HCV core protein. The transcription of IRS1 (A) and IRS2 (B) was estimated by quantitative RT-PCR (n = 5 in each group). (C) The expression levels of IRS1 and IRS2 in the livers of the mice were determined by immunoblotting with specific antibodies. (D) Phosphorylation of Akt in the livers of the mice was examined by immunoblotting with antibodies against Akt and phosphorylated Akt. The ratio of Akt phosphorylation was determined by computer software based on the densities of phosphorylated Akt and a total amount of Akt (n = 3 in each group). The data presented are representative of three independent experiments. *P < 0.05; **P < 0.01. NS, not statistically significant; HPRT, hypoxanthine phosphoribosyl transferase.

FIG. 7.

PA28γ was required for activation of the TNF-α promoter by the HCV core protein. (A) Expression of TNF-α in the livers of mice was determined by ELISA (n = 5 in each group). (B) TNF-α mRNA in the livers of mice was examined by quantitative RT-PCR (n = 5 in each group). (C) Knockdown of the expression of PA28γ in the HepG2 and FLC-4 cell lines by the introduction of a plasmid encoding a short hairpin RNA (shRNA) targeted to the PA28γ gene. The expression levels of PA28γ and β-actin were determined by immunoblotting with specific antibodies. (D) Promoter activity of TNF-α in the presence or absence of the HCV core protein was determined by luciferase assay in the PA28γ-knockdown and control cell lines. The data presented are representative of three independent experiments. HPRT, hypoxanthine phosphoribosyl transferase.