Table 1.
Primary and secondary antibodies.
Fig 1.
Underfeeding during the early postnatal period induces a permanent growth delay.
Increasing litter size from 6 (Normally fed) to 10 (Underfed) pups per dam permanently delayed postnatal growth of male pups, as observed with postnatal body weight gain (n = 23–25 per group) that persisted into adulthood (A). This was associated with low circulating plasma levels of IGF-I in 3 month-old male mice previously underfed during lactation (n = 7–9 per group) (B), accompanied by decreased levels of GH mRNA in the pituitary gland (n = 9 per group) (C). Representative micrograph of GH-producing somatotroph cells (labeled in red by immunohistochemistry, counterstaining with DAPI) from normally fed and underfed 20 day-old male pups (D, left and middle panels respectively) suggest a somatotrophs pituitary hypoplasia (D, right panel) (n = 3–4 per group). This was preceded by lower plasma levels of IGF-1 (n = 8 per group) (E) and decreased expression of the somatotroph differentiation factor, Pit-1 (n = 5 per group) (F) in 10 day-old underfed male pups. Moreover, this was associated with a non—significant tendency of decreased expression levels of GHRH receptor (GHRH-R) (G) (n = 5 per group), and of GH (H) (n = 5–7 per group) in 10 day-old underfed male pups. All data are presented as the mean ± SEM. Gene expression determinations are normalized against the histone H3 gene (C, F). Comparisons were performed by repeated measure two-way ANOVA analysis (A) or Mann Whitney analysis (B–F), with * p < 0.05 and *** p < 0.001.
Fig 2.
The development of GHRH neurons is altered in response to underfeeding.
The numbers of GHRH neurons in the arcuate nucleus of the hypothalamus was estimated by in situ hybridization in 10 day-old normally fed or underfed male pups (n = 3 per group) (A). Concomitantly, the area of innervation of the median eminence (highlighted with the arrow) by axons of GHRH neurons was lower in 10 day-old underfed male pups (n = 4 per group) (B). Illustrative immunohistochemistry from in vitro cultured arcuate nucleus explants from 7 day-old normally fed pups show that IGF-1R (in red, middle panel) is preferentially enriched in the distal part of growing GHRH axon (in green, left panel). Merged picture is in the right panel (C). Data are presented as the mean ± SEM. All Comparisons were performed using Mann Whitney analysis, with *: p < 0.05.
Fig 3.
IGF-I stimulates axon growth of GHRH neurons in arcuate nucleus explants.
Illustrative micrograph of in vitro cultured explants of arcuate nuclei of hypothalamus micro-dissected from 7 day-old normally fed GHRH-eGFP+ pups of both sexes are shown (A) in basal condition, under IGF-I stimulation or in presence of OSI-906 inhibitor (alone or in combination). Axons from whole arcuate (NF) and GHRH neurons are labeled by a dual-IHC for neurofilament (NF, top panels in red) and eGFP (GHRH-eGFP, lower panels in green), respectively. Quantification of relative axon growth stimulated by IGF-I and/or its inhibitor OSI-906 (OSI) in 24 h for NF (plain bars) and GHRH-eGFP (GHRH, dashed bars) are presented (B). In a separated set of experiment, a similar experiment was performed on NPY/AgRP neurons labeled by IHC for AgRP (in orange) (C). The relative axon growth of this other subpopulation of arcuate neurons under IGF-I stimulation and/or its inhibitor OSI-906 (OSI) is presented (D). Data are presented as the mean ± SEM calculated from n = 5–6 (B) and n = 4 (D) experiments per group, respectively (see results section for details). Results were compared using a two-way ANOVA analysis with the Bonferonni post-test (B) or one-way ANOVA analysis with the Newman Keuls post-test (C). *: p < 0.05 and **: p < 0.01 for each treatment vs. Control; #: p < 0.05 for GHRH vs. NF.
Fig 4.
GHRH neurons from underfed pups do not respond to IGF-I stimulation.
Illustrative micrograph of in vitro cultured explants of arcuate nuclei of hypothalamus micro-dissected from 7 day-old underfed GHRH-eGFP+ pups of both sexes are shown (A) in basal condition, under IGF-I stimulation or in presence of OSI-906 inhibitor (alone or in combination, with axons from whole arcuate (NF) and GHRH neurons labeled by a dual-IHC for neurofilament (NF, top panels in red) and eGFP (GHRH-eGFP, lower panels in green), respectively. Quantification of relative axon growth stimulated by IGF-I and/or its inhibitor OSI-906 (OSI) in 24 h for NF (plain bars) and GHRH-eGFP (GHRH, dashed bars) are presented (B). In a separated set of experiment, a similar experiment was performed on NPY/AgRP neurons labeled by IHC for AgRP (in orange) (C). The relative axon growth of this other subpopulation of arcuate neurons under IGF-I stimulation and/or its inhibitor OSI-906 (OSI) is presented (D). Data are presented as the mean ± SEM calculated from n = 4 (B) and 5 (D) experiments per group (see results section for details). Results were compared using a two-way ANOVA analysis with the Bonferonni post-test (B) or one-way ANOVA analysis with the Newman Keuls post-test (C). *: p < 0.05 and **: p < 0.01 for each treatment vs. Control; δ: p < 0.05, and δδδ: p < 0.001 for each treatment vs. IGF-I.
Fig 5.
Alterations of the IGF-IR signaling pathways in arcuate explants from underfed pups.
Activation of key elements of IGF-1R signaling pathways were measured by Western blot analysis as illustrated with representative blots in basal condition (-) or 15 min after IGF-I stimulation (IGF-I) (A). Quantification indicates activation of the IGF-1R (fold induction of the p-IGF-1R/ IGF-1R ratio) by IGF-I stimulation relative to basal levels (left panel), and total IGF-1R protein levels (right panel) in arcuate explants harvested from normally fed and underfed pups of both sexes (B). Similarly, activation of AKT (fold induction of the pAKT/AKT ratio) (C), ERK1 (D), ERK2 (E) and MEK-1 (F) are presented (left panels) with their respective total protein levels (right panels) (n = 4–6 per group). In vitro explant cultures indicate that specific inhibition of PI3K by LY294002 (LY) and of MEK by PD0325901 (PD) impaired axon growth of GHRH neurons in normally fed (n = 4 per group) (G) and underfed GHRH-eGFP+ pups (n = 6 per group) (H). All data are presented as the mean ± SEM with Mann Whitney analysis for Western blot analysis with *: p < 0.05 (V-F); and one way-ANOVA analysis with the Newman Keuls post-test (G, H) with *: p < 0.05 and ***: p < 0.001 for each treatment vs. Control; δδ: p < 0.01 and δδδ: p < 0.001 for each treatment vs. IGF-I; Δ Δ Δ: p < 0.001 IGF-I/PD vs. IGF-I/LY.