Submitted to: Endocrine Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/1/2004
Publication Date: 5/1/2004
Citation: Hadsell, D.L., George, J., Lawrence, N., Torres, D., Fiorotto, M. 2004. Differential ability of insulin and long-r3-IGF-I to induce Akt phosphorylation in mammary gland of the virgin mouse is accounted for by cellular heterogeneity [abstract]. In: Endocrine Society Meeting Proceedings, June 16-19, 2004, New Orleans, Louisiana. Abstract P3-177. p. 507-508. Interpretive Summary: Interpretive Summary not needed for this 115.
Technical Abstract: Postnatal mammary ductal development requires IGF-I and its receptor. To test the hypothesis that the responses of the postnatal mammary gland to IGF-I stimulation are mediated via activation of Akt, dose-response studies comparing insulin and Long-R3-IGF-I (LR3) were conducted in 5 wk old virgin mice. Fasted mice were given a single tail-vein injection of either insulin or LR3 at a dose of 0.025, 0.05, 0.25, 0.5 or 2.5 mg/kg bodyweight. After 5 min, mammary gland, liver and kidney were collected for Western blot analysis or immunofluorescent staining. Liver or kidney served as positive control organs being highly sensitive to insulin or IGF-I, respectively. Western blotting with an antibody to the phosphorylated beta-subunits of the insulin receptor (IR beta) and IGF-I receptor (IGF-IR beta) demonstrated increased phosphorylation of IR beta in liver extracts by the lowest dose of insulin and increased phosphorylation of IGF-IRb in kidney extracts by the lowest dose of LR3, respectively. Western blotting with an antibody to phospho-Akt (Thr308) failed to detect Akt phosphorylation in either kidney or liver extracts despite the detection of Akt by a pan Akt antibody. In whole mammary gland tissue extracts, phosphorylation of IRbeta was increased 6-fold by 0.025 mg/kg of insulin and was maximal (16-fold) at 0.05 mg/kg. In contrast, 0.025 mg/kg of LR3 induced only a twofold increase in IGF-IR beta phosphorylation and required a dose of 0.5 mg/kg to produce a maximal, 11-fold induction. The lowest dose of insulin induced a 7-fold increase in phospho-Akt while an induction of similar magnitude by LR3 required 0.25 mg/kg. Analysis of the mammary stromal adipocyte population for phospho-Akt by immunofluorescent staining revealed a significant induction with as little as 0.05 mg/kg insulin. LR3 also induced Akt phosphorylation within the stromal adipocyte compartment, but this induction was only 63% of that observed with insulin. In contrast, induction of phospho-Akt in the mammary ductal epithelium was observed with as little as 0.05 mg/kg LR3, but required 5-fold more insulin. These data indicate that with respect to Akt activation, the stromal adipocytes within the mammary gland are primarily insulin responsive, whereas the ductal epithelium responds preferentially to IGF-I. These data also demonstrate that the interpretation of Western blots produced from homogenates of tissues composed of a heterogeneous cell population can be misleading. This work is supported by a grant from the NIDDK (DK52197).