The hormonal action of IGF1 in postnatal mouse growth

Abstract

The mammalian insulin-like growth factor 1 (IGF1), which is a member of a major growth-promoting signaling system, is produced by many tissues and functions throughout embryonic and postnatal development in an autocrine/paracrine fashion. In addition to this local action, IGF1 secreted by the liver and circulating in the plasma presumably acts systemically as a classical hormone. However, an endocrine role of IGF1 in growth control was disputed on the basis of the results of a conditional, liver-specific Igf1 gene knockout in mice, which reduced significantly the level of serum IGF1, but did not affect average body weight. Because alternate interpretations of these negative data were tenable, we addressed genetically the question of hormonal IGF1 action by using a positive experimental strategy based on the features of the cre/loxP recombination system. Thus, we generated bitransgenic mice carrying in an Igf1 null background a dormant Igf1 cDNA placed downstream of a transcriptional "stop" DNA sequence flanked by loxP sites (floxed) and also a cre transgene driven by a liver-specific promoter. The Igf1 cDNA, which was inserted by knock-in into the mutated and inactive Igf1 locus itself to ensure proper transcriptional regulation, was conditionally expressed from cognate promoters exclusively in the liver after Cre-mediated excision of the floxed block. Our genetic study demonstrated that the endocrine IGF1 plays a very significant role in mouse growth, as its action contributes approximately30% of the adult body size and sustains postnatal development, including the reproductive functions of both mouse sexes.

Fig. 1.

Targeting of Igf1 cDNA into the mouse Igf1 locus. (A) Diagrams of the genomic arrangements of exons comprising the predominant alternate mouse Igf1 mRNAs are shown on top. The allele used for knock-in was already targeted (12) by replacement of exon 4 with a neomycin-resistance gene (neo). The locations of a 9.1 kb diagnostic fragment produced by KpnI digestion of genomic DNA that was used for genotyping (panels C and D) and of a 9.6 kb BglII fragment containing exon 3 that was used to generate the arms of a replacement vector are indicated. The sequence in the region of an ATG codon in exon 3 was modified to generate unique XhoI and AscI cloning sites into which we inserted a DNA fragment (3 kb) consisting of a floxed segment that included, in order, the following elements: a loxP site (black triangle); a puromycin (puro) selectable marker (0.6 kb) driven by the Pgk gene promoter (0.5 kb); the Pgk polyadenylation sequence (0.3 kb); a “stop” sequence (SV40 3x-pA; triple polyA; 0.75 kb); a second loxP site; and an Igf1 cDNA (0.44 kb) endowed with the polyadenylation sequence of the bovine growth hormone gene (0.3 kb). (B) The DNA sequence remaining at the beginning of the modified Igf1 exon 3 (the first “ag” is the 3′ splice site of the upstream intron) after Cre-mediated excision of the floxed selectable marker is displayed. The leftover loxP site and the ATG codon are underlined. An asterisk marks a TAG terminator encountered upstream from the ATG (in frame), to indicate that the latter serves by default as a codon of the initiator Met for the peptide that can be translated from the transcript of the cDNA (the presumptive signal peptide is truncated). The WT sequence is shown at the bottom, for comparison (the modified sequence has 86 additional bases). (C) Genotyping by Southern analysis using tail DNA digested with KpnI indicates the sizes in kb of the WT (W) targeted (T) and recombined (R) alleles. (D) Southern analysis of DNA extracted from various tissues of a LIP animal (Liver, Li; Kidney, Ki; Heart, He; Testis, Te; Lung, Lu; Thymus, Th; Salivary gland, SG; Brain, Br; and Spleen, SP) demonstrates that Cre-mediated recombination has occurred only in liver, as expected. (E) Northern analysis of RNA extracted from various tissues of a LIP mouse (lanes 2–10) indicates that the knocked-in Igf1 cDNA is expressed exclusively in the liver (lane 2). The sizes (in kb) of the predominant Igf1 transcripts produced in WT liver are shown in lane 1 (Igf1 cDNA was used as a probe). The same membrane was hybridized to 18S RNA (loading control; bottom). (F) Northern analysis of liver RNA from WT (WT) and LIP mice before and after RNase H treatment (dots indicate the main digestion products; for details, see text).

Fig. 2.

Liver-specific expression of an AT-cre transgene. (A) Diagrams of the genomic arrangements of exons comprising the predominant alternate mRNAs of the human SERPINA1 (AT) gene are shown on top. The position of the promoter fragment (also carrying one of the alternate exons) that was used to drive cre expression is indicated. In the transgenic construct, an intron (IVS) was provided upstream from the cre segment, which was linked to the SV40 polyadenylation sequence (for details, see SI Methods). (B) Comparison between WT (control) and bitransgenic e12.5 embryos carrying AT-cre and a Rosa-lacZ reporter shows that only the transgenic liver exhibits β-galactosidase staining. The same holds for the indicated tissues dissected from 20-day-old mice (in each pair of tissues, the control is on the left).

Fig. 3.

Growth analysis. (A) Growth curves derived by computer-aided regression analysis of average weights of WT (WT; n = 20), 2X (n = 4), 1XL (n = 6), 1X (n = 5), LIP (n = 3), and Null (n = 3) animals. For clarity, standard errors (all of small magnitude) are not displayed. (B) After growth curve fitting, the absolute growth rates (dW/dt) that are shown were calculated by subtracting the value of cumulative weight at a particular day (W_i) from that of the previous day (W_i-1), i.e., each curve point represents weight gain (g/day). (C) The growth curve regression data were also used to calculate relative weights (% of WT) for each genetically-modified mouse and to derive from the resulting steady-state values estimates of the contributions of endocrine (E) and local (L) IGF1 action to total body weight (for details, see Table 3). The distances between curves (arrows), corresponding to pairwise subtraction values between relative weights (Table 3), represent graphically the number of IGF1 “units” of endocrine and local action. Because the calculations were based on relative values, the final estimates of IGF1 contributions would be identical to those shown in Table 3 if, instead of the WT comparison standards, we had used weights relative to the 2X controls.