Normal growth and development in the absence of hepatic insulin-like growth factor I

Abstract

The somatomedin hypothesis proposed that insulin-like growth factor I (IGF-I) was a hepatically derived circulating mediator of growth hormone and is a crucial factor for postnatal growth and development. To reassess this hypothesis, we have used the Cre/loxP recombination system to delete the igf1 gene exclusively in the liver. igf1 gene deletion in the liver abrogated expression of igf1 mRNA and caused a dramatic reduction in circulating IGF-I levels. However, growth as determined by body weight, body length, and femoral length did not differ from wild-type littermates. Although our model proves that hepatic IGF-I is indeed the major contributor to circulating IGF-I levels in mice it challenges the concept that circulating IGF-I is crucial for normal postnatal growth. Rather, our model provides direct evidence for the importance of the autocrine/paracrine role of IGF-I.

Figure 1

Plasmid constructs. (A) Schematic representation of the albumin-cre construct for production of liver-specific Cre-expressing transgenic mice. p5′cre, the probe used to detect cre positive mice after genomic DNA digestion with BamHI (B) as described (17). MT-1pA, metallothionein poly(A) tail; SV40pA, polyadenylation signal. (B) Schematic representation of the igf1 exon 4 (ex4) locus in the WT allele (W), null (N), and the loxP-flanked allele (L). pSP3, the probe used to detect different igf1 genotypes after DNA digestion with HindIII (H) as described (17); pMI-4, the cDNA probe used to detect igf1 mRNA.

Figure 2

Liver-restricted Cre-mediated recombination. Mice carrying the albumin-cre transgene were crossed with homozygous mice carrying loxP-flanked igf1 allele (L/L). Heterozygous mice carrying one WT (W) allele of igf1 and one loxP-flanked allele that also were positive for Cre recombinase (W/L+) were analyzed. Genomic DNA was prepared from various tissues (liver, Li; heart, H; brain, B; kidney, K; muscle, M; spleen, S; lung, Lu; fat, F) digested with HindIII and subjected to Southern blot analysis. Detection with the pSP3 probe shows a 6.5-kb fragment of WT allele and a 4.9-kb fragment of loxP-flanked allele. In the presence of Cre recombinase the pSP3 probe detects a 2.8-kb fragment of the truncated allele.

Figure 3

igf1 mRNA in liver is abolished in mice expressing Cre recombinase. (A) A representative RNase protection assay (18) using the pMI-4 riboprobe shows that the level of liver igf1 mRNA is abolished in 6-week-old mice positive for Cre (+) and homozygous for the loxP-flanked igf1 allele (L/L+) as well as hemizygous null, loxP-flanked igf1 mice (N/L+) (Top). This reduction correlates with the liver igf1 genotype (Bottom). (B) Quantification of liver igf1 mRNA in mice with various genotypes (L/L−, n = 12; L/L+, n = 15; N/L−, n = 10; N/L+, n = 9). (C) A representative RNase protection assay using the pMI-4 riboprobe showing that igf1 mRNA levels in fat, muscle, kidney, heart, and spleen are not affected by the absence of liver igf1 mRNA in homozygous mice for the loxP-flanked igf1 allele (L/L) in the presence or absence of Cre recombinase. (D) Quantification of fat (L/L−, n = 6; L/L+, n = 10), muscle (L/L−, n = 3; L/L+, n = 5), kidney (L/L−, n = 5; L/L+, n = 9), heart (L/L−, n = 8; L/L+, n = 13), and spleen (L/L−, n = 8; L/L+ n = 14) igf1 mRNA in homozygous mice for the loxP-flanked igf1 allele (L/L) in the presence or absence of Cre recombinase. The protected bands corresponding to igf1 mRNA were corrected to the 18S rRNA by PhosphorImmaging (relative arbitrary units). Statistical analysis of igf1 mRNA gene expression in different tissues was performed by using t test. Values shown are mean ± SD.

Figure 4

Growth of mice with various genotypes was analyzed by measuring total body weight at weekly intervals. Mice were divided into two major groups according to their gene dosage in extra-hepatic tissues: mice that have two copies (Upper) and one copy (Lower) of the igf 1 gene. Statistical analysis done on a weekly basis showed no significant differences (P > 0.01) in both sexes during the first 6 weeks.