Transgenic overexpression of insulin receptor substrate 1 in hepatocytes enhances hepatocellular proliferation in young mice only

Abstract

Aim: The insulin receptor substrate-1 (IRS-1) is a multisite docking protein which plays a central role in the signal transduction of growth factors such as insulin and insulin-like growth factors (IGF-1 and IGF-2). It is found to be frequently overexpressed in human hepatocellular carcinoma (HCC).

Methods: To study IRS-1 overexpression in hepatocytes in vivo, transgenic mice overexpressing IRS-1 exclusively in hepatocytes were created, showing enhanced hepatocyte proliferation in young animals. In the present study, the phenotype of IRS-1 transgenic animals was characterized over a period of two years. The livers of transgenic and control mice were analyzed for IRS-1 expression and phosphorylation, activation of the downstream mitogen-activated protein kinase (MAPK) cascade and phosphatidylinositol 3' kinase (PI3'K) and macroscopical and histological abnormalities.

Results: The enhanced hepatocyte proliferation observed in young IRS-1 transgenic animals was no longer detectable in adult mice. Despite constitutive overexpression and phosphorylation of IRS-1, MAPK- and IRS-1-associated PI3'K activity were significantly reduced in older transgenic mice. Furthermore, no premalignant lesions or HCC were detected in IRS-1 transgenic animals up to the age of 24 months.

Conclusions: Therefore, additional mechanisms such as enhanced growth factor expression or impaired negative feedback control mechanisms may augment IRS-1 overexpression in human hepatocarcinogenesis.

Fig. 1. Hepatocyte proliferation

(A) Liver DNA synthesis was measured by the incorporation of ³[H]-thymidine into total liver DNA following tail vein injection of 10 μCi ³[H]-thymidine. At 3 months of age, tritium incorporation was found to be significantly (p = 0.005) enhanced in IRS-1 transgenic mice, whereas no difference was found at the age of 12 months. Data are expressed as mean + / - SD. (B) Immunohistochemical analysis of BrdU incorporation into hepatocyte nuclei showed a significantly higher number of S-phase hepatocyte nuclei in IRS-1 transgenic mice at the age of 6 weeks and 3 months (p < 0.01), whereas no difference was found in older mice.

Fig. 2. A: IRS-1 expression and tyrosyl phosphorylation in transgenic mice and control littermates

(A) Marked overexpression of the tyrosyl phosphorylated transgene in the livers of IRS-1 transgenic mice could be demonstrated by Western blot in mice of all age groups analyzed as compared to the low levels of IRS-1 expression in control littermates. B: MAPK activation. Enhanced MAPK phosphorylation could be detected by Western blot using monoclonal antibodies specific for the phosphorylated MAP-kinases ERK1 and ERK2 in 6 weeks old animals (left panel), but significant difference in MAPK phosphorylation could be detected in adult animals at the age of 9 months (right panel). C: IRS-1 associated PI3′K activity. Equal expression levels of PI3′K in liver tissue in 6 weeks and 9-month old animals was demonstrated by Western blot using antibodies specific against the p85 subunit of PI3′K (upper left panel), but the amount of p85 subunit of PI3′K associated with IRS-1 as determined by Western blot following immunoprecipitation of IRS-1 was enhanced in transgenic mice, especially in 6 week old animals (upper right panel). Enzymatic PI3′K activity associated with IRS-1 as measured by an enzymatic assay in liver lysates of 6 weeks old animals and 9-month old animals. The IRS-1 associated PI3′K activity was significantly enhanced in comparison to control littermates in 6 weeks old mice (p = 0,004), whereas in 9 months old animals the difference was reduced and did not reach statistical significance (lower panel).