Mosaic analysis of insulin receptor function
Tadahiro Kitamura, Yukari Kitamura, Jun Nakae, Antonio Giordano, Saverio Cinti, C. Ronald Kahn, Argiris Efstratiadis, Domenico Accili
Tadahiro Kitamura, Yukari Kitamura, Jun Nakae, Antonio Giordano, Saverio Cinti, C. Ronald Kahn, Argiris Efstratiadis, Domenico Accili
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Article Endocrinology

Mosaic analysis of insulin receptor function

Abstract

Insulin promotes both metabolism and growth. However, it is unclear whether insulin-dependent growth is merely a result of its metabolic actions. Targeted ablation of insulin receptor (Insr) has not clarified this issue, because of early postnatal lethality. To examine this question, we generated mice with variable cellular mosaicism for null Insr alleles. Insr ablation in approximately 80% of cells caused extreme growth retardation, lipoatrophy, and hypoglycemia, a clinical constellation that resembles the human syndrome of leprechaunism. Insr ablation in 98% of cells, while resulting in similar growth retardation and lipoatrophy, caused diabetes without β-cell hyperplasia. The growth retardation was associated with a greater than 60-fold increase in the expression of hepatic insulin-like growth factor binding protein-1. These findings indicate that insulin regulates growth independently of metabolism and that the number of insulin receptors is an important determinant of the specificity of insulin action.

Authors

Tadahiro Kitamura, Yukari Kitamura, Jun Nakae, Antonio Giordano, Saverio Cinti, C. Ronald Kahn, Argiris Efstratiadis, Domenico Accili

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Growth retardation in Insr mosaics. (a) Appearance of 3-week-old mice. (...
Growth retardation in Insr mosaics. (a) Appearance of 3-week-old mice. (b) Average growth curves of WT and mosaic mice. The curves of the two subsets of mosaics are superimposable. The analysis was terminated at the time of death of Δ80 mosaics (n = 19 for WT, 18 for Δ80, and 15 for Δ98). (c) Δ98 survivors were followed for up to 13 weeks. The data represent the mean ± SEM (n = 28 for WT and 19 for Δ98 survivors). The insets present weight ratios of mosaic mice to WT control per time point examined. The decline of these ratios is indicative of lower than normal growth rate for the mosaics throughout the period of observation. (d) Real-time RT-PCR analysis of Igfbp1 expression in liver. We isolated total RNA from WT (n = 20), Δ80 (n = 17), and Δ98 (n = 20) mice and subjected it to RT-PCR using a Light Cycler instrument (Roche Perkin-Elmer). We normalized mRNA values using β-actin as a control. **P < 0.01. (e) Northern blot analysis of Igfbp1 expression. We pooled mRNA samples from four different mice for each set and analyzed them by hybridization with an Igfbp1 probe (upper panel), followed by a β-actin probe as gel loading control. (f) 125I-IGF2 ligand blotting. We obtained serum (5 μl for each animal) from WT (n = 10) and Δ98 mice (n = 10). In the panel on the left, we used whole serum to determine the presence of IGF-binding proteins. As shown, in the middle and right panels, we subjected serum to immunoprecipitation (IP) with the indicated antibodies (Bp1, Bp2) prior to gel electrophoresis and transfer to nylon membranes. We show the region of the gel in the 20- to 40-kDa region.