Insulin and insulin-like growth factor-1 receptors act as ligand-specific amplitude modulators of a common pathway regulating gene transcription

Abstract

Insulin and insulin-like growth factor-1 (IGF-1) act on highly homologous receptors, yet in vivo elicit distinct effects on metabolism and growth. To investigate how the insulin and IGF-1 receptors exert specificity in their biological responses, we assessed their role in the regulation of gene expression using three experimental paradigms: 1) preadipocytes before and after differentiation into adipocytes that express both receptors, but at different ratios; 2) insulin receptor (IR) or IGF1R knock-out preadipocytes that only express the complimentary receptor; and 3) IR/IGF1R double knock-out (DKO) cells reconstituted with the IR, IGF1R, or both. In wild-type preadipocytes, which express predominantly IGF1R, microarray analysis revealed approximately 500 IGF-1 regulated genes (p < 0.05). The largest of these were confirmed by quantitative PCR, which also revealed that insulin produced a similar effect, but with a smaller magnitude of response. After differentiation, when IR levels increase and IGF1R decrease, insulin became the dominant regulator of each of these genes. Measurement of the 50 most highly regulated genes by quantitative PCR did not reveal a single gene regulated uniquely via the IR or IGF1R using cells expressing exclusively IGF-1 or insulin receptors. Insulin and IGF-1 dose responses from 1 to 100 nm in WT, IRKO, IGFRKO, and DKO cells re-expressing IR, IGF1R, or both showed that insulin and IGF-1 produced effects in proportion to the concentration of ligand and the specific receptor on which they act. Thus, IR and IGF1R act as identical portals to the regulation of gene expression, with differences between insulin and IGF-1 effects due to a modulation of the amplitude of the signal created by the specific ligand-receptor interaction.

FIGURE 1.

Number of probesets significantly regulated by IGF-1. A, fold-change (FC) representation of the 100 probesets most differentially regulated by a 10 nm IGF-1 treatment after 30 min (solid line) or 6 h (dotted line) in brown preadipocytes measured by microarrays. B, total number of probesets significantly up- or down-regulated (p < 0.05) by IGF-1 treatment or significantly regulated more than 2-fold after 30 min or 6 h.

FIGURE 2.

IR and IGF1R expression in WT, IRKO, IGFRKO, and DKO cells. A, WT brown preadipocytes were differentiated for 2, 4, and 8 days as described under “Experimental Procedures” and IR, IGF1R, and β-tubulin protein levels were measured by Western blot analysis. A representative blot from 3 experiments is shown. B, IR and IGF1R immunoblots were performed on confluent WT, IRKO, IGFRKO, or DKO cells. mRNA was extracted from confluent WT, IRKO, IGFRKO, or DKO cells and IR and IGF1R mRNA expression were measured by real time PCR using specific oligonucleotide primers. The data were normalized to levels of TBP mRNA. ND, not detected. Results are mean ± standard error of the mean from 5 independent measurements.

FIGURE 3.

Insulin and IGF-1 regulation of gene expression in nondifferentiated versus differentiated WT cells. WT cells were grown to confluence, and differentiation was induced as described under “Experimental Procedures” for 8 days. Nondifferentiated cells at day 0 (Non Diff) and differentiated cells at day 8 (Diff) were serum starved overnight and stimulated with 10 nm insulin (gray bars) or IGF-1 (black bars) during 30 min or 6 h. Gene expression was measured by real time PCR using specific oligonucleotides. The data were normalized to levels of TBP mRNA. Results are mean ± standard error of the mean from 6 independent experiments for nondifferentiated cells and 3 independent experiments for differentiated adipocytes. * indicates a significant difference compared with the similar treatment in nondifferentiated cells, p value <0.05 by Student's t test.

FIGURE 4.

Insulin and IGF-1 regulation of gene expression in WT, IRKO, and IGFRKO preadipocytes. WT, IRKO, and IGFRKO cells were grown to confluence, serum starved overnight, and stimulated with 10 nm insulin (gray bars) or IGF-1 (black bars) for 30 min or 6 h. Gene expression was measured by real time PCR using specific oligonucleotides. The data were normalized to levels of TBP mRNA. Results are mean ± standard error of the mean from 5 to 6 independent experiments. * indicates a significant difference compared with a similar treatment in WT cells, p value <0.05 by Student's t test.

FIGURE 5.

Insulin and IGF-1 dose response on Egr1, Egr2, and Fos gene expression in WT, IRKO, IGFRKO, and DKO cells. WT, IRKO, and IGFRKO cells were grown to confluence, serum starved overnight, and stimulated with 1, 10, or 100 nm insulin or IGF-1 during 30 min. Egr1, Egr2, and Fos mRNA levels were measured by real time PCR using specific oligonucleotides. The data were normalized to levels of TBP mRNA. Results are mean ± standard error of the mean from 5 independent experiments.

FIGURE 6.

Insulin and IGF-1 dose response on Egr1, Egr2, and Fos gene expression in DKO cells re-expressing IR, IGF1R, or both. DKO cells re-expressing IR, IGF1R, or both were grown to confluence, serum starved overnight, and stimulated with 1, 10, or 100 nm insulin or IGF-1 during 30 min. Egr1, Egr2, and Fos mRNA levels were measured by real time PCR using specific oligonucleotides. The data were normalized to levels of TBP mRNA. Results are mean ± standard error of the mean from 5 independent experiments.

FIGURE 7.

Differentiation of WT and DKO cells or DKO cells re-expressing IR, IGF1R, or both. A, confluent WT and DKO brown preadipocytes were differentiated for 2, 4, and 8 days. PPARγ and aP2 mRNA levels were measured by real time PCR using specific oligonucleotide primers. The data were normalized to levels of TBP mRNA. Results are mean ± standard error of the mean from 3 independent experiments. PPARγ and β-tubulin protein levels were measured by Western blot analysis. A representative blot from 3 experiments is shown. B, oil red O staining from WT and DKO cells differentiated for 8 days. C, DKO cells re-expressing IR, IGF1R, or both were grown to confluence and differentiated with or without the thiazolidinedione (TZD) rosiglitazone (1 μm) for 8 days. PPARγ and aP2 mRNA levels were measured by real time PCR using specific oligonucleotide primers. The data were normalized to levels of TBP mRNA. Results are mean ± standard error of the mean from 3 independent experiments. D, oil red O staining from DKO cells re-expressing IR, IGF1R, or both after 8 days of differentiation in the presence of rosiglitazone.