Insulin-like growth factor 2 and the insulin receptor, but not insulin, regulate fetal hepatic glycogen synthesis

Abstract

Whether insulin or IGFs regulate glycogen synthesis in the fetal liver remains to be determined. In this study, we used several knockout mouse strains, including those lacking Pdx-1 (pancreatic duodenal homeobox-1), Insr (insulin receptor), and Igf2 (IGF-II) to determine the role of these genes in the regulation of fetal hepatic glycogen synthesis. Our data show that insulin deficiency does not alter hepatic glycogen stores, whereas Insr and Igf2 deficiency do. We found that both insulin receptor isoforms (IR-A and IR-B) are present in the fetal liver, and their expression is gestationally regulated. IR-B is highly expressed in the fetal liver; nonetheless, the percentage of hepatic IR-A isoform, which binds Igf2, was significantly higher in the fetus than the adult. In vitro experiments demonstrate that Igf2 increases phosphorylation of hepatic Insr, insulin receptor substrate-2, and Akt proteins and also the activity of glycogen synthase. Igf2 ultimately increased glycogen synthesis in fetal hepatocytes. This increase could be blocked by the phosphoinositide 3-kinase inhibitor LY294008. Taken together, we propose Igf2 as a major regulator of fetal hepatic glycogen metabolism, the insulin receptor as its target receptor, and phosphoinositide 3-kinase as the signaling pathway leading to glycogen formation in the fetal liver.

Figure 1

Glycogen concentrations in livers of Pdx-1^−/−, Igf1r^−/−, Insr^−/−, and Igf2^−/− fetuses on d 18 of gestation expressed as a percentage of their respective WT controls (n = 3–5/genotype from three different litters). Notice that there are significant differences in glycogen levels in livers of Insr^−/− and Insr^+/+ fetuses and Igf2^−/− and Igf2^+/+ controls. Data are expressed as means ± sem. *, P < 0.001.

Figure 2

Developmental expression of the IR-A and IR-B in livers from embryos collected on d 13, 15, and 18 of gestation and adult mice. A, Representative RT-PCR results from both Igf2^−/− and Igf2^+/+ livers (n = 3/genotype per age group). B, IR-A and IR-B values are expressed as percentage of total IR in both Igf2^+/+ (top) and Igf2^−/− (bottom) fetal livers. *, P < 0.01, differences between IR-A and IR-B percentages; #, P < 0.01, differences between IR-A percentage in e15 and e13, e18, and adult; &, P < 0.01, differences between adult IR-A percentage and fetal.

Figure 3

Igf2 treatment significantly increases glycogen concentrations (A) and GS activity (B) in Igf2^−/− fetal hepatocytes (black bars) when compared with those treated with only vehicle (hatched bars). Data are expressed as mean ± sem (n = 3–5/genotype). *, Significance (P < 0.01).

Figure 4

Igf2 significantly increases the levels of insulin receptor tyrosine phosphorylation [Tyr(P)] in fetal hepatocytes. A, Insr phosphorylation levels determined by immunoprecipitation. B, Densitometric quantification of bands was carried out using ImageJ software. Igf2^−/− hepatocytes were cultured for 2 d and treated with 100 nm Igf2 or vehicle for 5 min. After hepatocytes were lysed, 600 μg of total protein were immunoprecipitated (IP) with anti-Insr antibodies and analyzed by Western blot (WB) with antityrosine phosphate and Insr antibodies. Data shown are representative of three experiments (n = 3). Data are expressed as means ± sem. *, P < 0.01.

Figure 5

Igf2 significantly increases Irs-2 (A) and Akt (B) phosphorylation (p-Akt) levels in fetal hepatocytes. IP, Immunoprecipitation; WB, Western blot. C, LY294008, a PI3K inhibitor, blocks (**) the glycogenic effect of Igf2 (*) in fetal hepatocytes. Fetal hepatocytes from e18 Igf2^−/− fetus were pretreated with 25 μm LY294008 for 1 h and then treated with either vehicle or 100 nm Igf2 for 16 h. Inhibitors remained in the media for the entire period of treatment. Data shown are representative of three experiments (n = 3). Data are expressed as means ± sem. * and **, P < 0.05.