Response of arcuate neurons to insulin.

<p>Relative axonal growth in 24 h in whole arcuate neuron explants in the presence of various molar concentrations of insulin (0, 10, 50, 100, 200, 500 nM, and 1μM) was measured by immunohistochemistry against neurofilaments (NF). Arcuate explants were harvested from normally fed (dark circles and plain line) (n = 3–4) or underfed pups (open triangles and dotted line) (n = 6). Illustrative images of explants are presented above the graph for control (0 nM), 100 nM and 1000 nM of insulin. Scale bar indicates 2000 μm. Data are presented as the mean ± SEM. Results were analyzed by two-way ANOVA (see text) with a Bonferroni correction for multiple comparisons. The precise molar concentrations-response effect within each group was further analyzed by one-way ANOVA with a Newman-Keuls multiple comparison test. The results of all comparisons were non-significant.</p

Impact of insulin on primary arcuate neurons culture is dependent on early-postnatal nutritional status and neuronal subpopulation

<div><p>Nutrition plays a critical role in programming and shaping linear growth during early postnatal life through direct action on the development of the neuroendocrine somatotropic (GH/IGF-1) axis. IGF-1 is a key factor in modulating the programming of linear growth during this period. Notably, IGF-1 preferentially stimulates axonal growth of GHRH neurons in the arcuate nucleus of the hypothalamus (Arc), which is crucial for the proliferation of somatotroph progenitors in the pituitary, thus influencing later GH secretory capacity. However, other nutrition-related hormones may also be involved. Among them, insulin shares several structural and functional similarities with IGF-1, as well as downstream signaling effectors. We investigated the role of insulin in the control of Arc axonal growth using an <i>in vitro</i> model of arcuate explants culture and a cell-type specific approach (GHRH-eGFP mice) under both physiological conditions (normally fed pups) and those of dietary restriction (underfed pups). Our data suggest that insulin failed to directly control axonal growth of Arc neurons or influence specific IGF-1-mediated effects on GHRH neurons. Insulin may act on neuronal welfare, which appears to be dependent on neuronal sub-populations and is influenced by the nutritional status of pups in which Arc neurons develop.</p></div

Response of AgRP neurons to insulin.

<p>Relative axonal growth in 24 h in the AgRP neuronal subpopulation of arcuate explants in the presence of various concentrations of insulin (0, 10, 50, 100, 200, 500 nM and 1μM) was measured. Arcuate explants were harvested from normally fed (plain circles and plain line) (n = 3) or underfed pups (open triangles and dotted line) (n = 4). Illustrative images are presented above the graph for control (0 nM), 100 nM and 1000 nM of insulin. Scale bar indicates 2000 μm. Data are presented as the mean ± SEM. Results were analyzed by two-way ANOVA (see text) with a Bonferroni correction for multiple comparisons. The precise molar concentrations-response effect within each group was further analyzed by one-way ANOVA with a Newman-Keuls multiple comparison test. The results of all comparisons were non-significant.</p

Composition of the LasQCdiet Rod18-R chow.

<p>Composition of the LasQCdiet Rod18-R chow.</p

Underfed pups in larger litters have decreased insulin levels and are lighter.

<p>Increasing the litter size of lactating dams from six (normally fed) to 10 pups (underfed) lower the offspring body weight in 7 days-old pups of both sexes. Data are presented as the mean ± SEM, analyzed by the student t-test, *** <i>p</i> < 0.001.</p

Underfeeding during the early postnatal period induces a permanent growth delay.

<p>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 (<b>A</b>). 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>B</b>), accompanied by decreased levels of GH mRNA in the pituitary gland (n = 9 per group) (<b>C</b>). 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 (<b>D</b>, left and middle panels respectively) suggest a somatotrophs pituitary hypoplasia (<b>D</b>, right panel) (n = 3–4 per group). This was preceded by lower plasma levels of IGF-1 (n = 8 per group) (<b>E</b>) and decreased expression of the somatotroph differentiation factor, Pit-1 (n = 5 per group) (<b>F</b>) 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) (<b>G</b>) (n = 5 per group), and of GH (<b>H</b>) (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 (<b>C, F</b>). Comparisons were performed by repeated measure two-way ANOVA analysis (<b>A</b>) or Mann Whitney analysis (<b>B</b>–<b>F</b>), with * p < 0.05 and *** p < 0.001.</p

Primary and secondary antibodies.

<p>Primary and secondary antibodies.</p

The development of GHRH neurons is altered in response to underfeeding.

<p>The numbers of GHRH neurons in the arcuate nucleus of the hypothalamus was estimated by <i>in situ</i> hybridization in 10 day-old normally fed or underfed male pups (n = 3 per group) (<b>A</b>). 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>B</b>). Illustrative immunohistochemistry from <i>in vitro</i> 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 (<b>C</b>). Data are presented as the mean ± SEM. All Comparisons were performed using Mann Whitney analysis, with *: p < 0.05.</p

Alterations of the IGF-IR signaling pathways in arcuate explants from underfed pups.

<p>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) (<b>A</b>). 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>B</b>). Similarly, activation of AKT (fold induction of the pAKT/AKT ratio) (<b>C</b>), ERK1 (<b>D</b>), ERK2 (<b>E</b>) and MEK-1 (<b>F</b>) are presented (left panels) with their respective total protein levels (right panels) (n = 4–6 per group). <i>In vitro</i> 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) (<b>G</b>) and underfed GHRH-eGFP⁺ pups (n = 6 per group) (<b>H</b>). All data are presented as the mean ± SEM with Mann Whitney analysis for Western blot analysis with *: <i>p</i> < 0.05 (<b>V-F</b>); and one way-ANOVA analysis with the Newman Keuls post-test (<b>G</b>, <b>H</b>) with *: <i>p</i> < 0.05 and ***: <i>p</i> < 0.001 for each treatment <i>vs</i>. Control; δδ: <i>p</i> < 0.01 and δδδ: <i>p</i> < 0.001 for each treatment <i>vs</i>. IGF-I; Δ Δ Δ: <i>p</i> < 0.001 IGF-I/PD <i>vs</i>. IGF-I/LY.</p

IGF-I stimulates axon growth of GHRH neurons in arcuate nucleus explants.

<p>Illustrative micrograph of <i>in vitro</i> cultured explants of arcuate nuclei of hypothalamus micro-dissected from 7 day-old normally fed GHRH-eGFP⁺ pups of both sexes are shown (<b>A</b>) 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>B</b>). In a separated set of experiment, a similar experiment was performed on NPY/AgRP neurons labeled by IHC for AgRP (in orange) (<b>C</b>). The relative axon growth of this other subpopulation of arcuate neurons under IGF-I stimulation and/or its inhibitor OSI-906 (OSI) is presented (<b>D</b>). Data are presented as the mean ± SEM calculated from n = 5–6 (<b>B</b>) and n = 4 (<b>D</b>) experiments per group, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170083#sec009" target="_blank">results</a> section for details). Results were compared using a two-way ANOVA analysis with the Bonferonni post-test (<b>B</b>) or one-way ANOVA analysis with the Newman Keuls post-test (<b>C</b>). *: p < 0.05 and **: p < 0.01 for each treatment <i>vs</i>. Control; #: p < 0.05 for GHRH <i>vs</i>. NF.</p