Glucose-dependent insulinotropic polypeptide is expressed in adult hippocampus and induces progenitor cell proliferation - PubMed (original) (raw)

. 2005 Feb 16;25(7):1816-25.

doi: 10.1523/JNEUROSCI.4920-04.2005.

Michelle F Anderson, Björn Meister, Ann-Marie Alborn, Anna-Karin Ström, Anke Brederlau, Ann-Christin Illerskog, Ola Nilsson, Timothy J Kieffer, Max Albert Hietala, Anne Ricksten, Peter S Eriksson

Affiliations

Glucose-dependent insulinotropic polypeptide is expressed in adult hippocampus and induces progenitor cell proliferation

Jenny Nyberg et al. J Neurosci. 2005.

Abstract

The hippocampal dentate gyrus (DG) is an area of active proliferation and neurogenesis within the adult brain. The molecular events controlling adult cell genesis in the hippocampus essentially remain unknown. It has been reported previously that adult male and female rats from the strains Sprague Dawley (SD) and spontaneously hypertensive (SHR) have a marked difference in proliferation rates of cells in the hippocampal DG. To exploit this natural variability and identify potential regulators of cell genesis in the hippocampus, hippocampal gene expression from male SHR as well as male and female SD rats was analyzed using a cDNA array strategy. Hippocampal expression of the gene-encoding glucose-dependent insulinotropic polypeptide (GIP) varied strongly in parallel with cell-proliferation rates in the adult rat DG. Moreover, robust GIP immunoreactivity could be detected in the DG. The GIP receptor is expressed by cultured adult hippocampal progenitors and throughout the granule cell layer of the DG, including progenitor cells. Thus, these cells have the ability to respond to GIP. Indeed, exogenously delivered GIP induced proliferation of adult-derived hippocampal progenitors in vivo as well as in vitro, and adult GIP receptor knock-out mice exhibit a significantly lower number of newborn cells in the hippocampal DG compared with wild-type mice. This investigation demonstrates the presence of GIP in the brain for the first time and provides evidence for a regulatory function for GIP in progenitor cell proliferation.

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Figures

Figure 1.

Figure 1.

The Gip gene is expressed in adult rat hippocampus. A schematic picture of our microarray strategy is viewed in A, where the results obtained were analyzed with regard to variation in parallel with in vivo proliferation levels of cells in rat DG. Arrows indicate the gene dots representing the Gip gene on arrays from the three groups, and the corresponding gene dots are enlarged in the bottom right panel. PCR analysis confirmed the Gip gene expression (B) in the hippocampus (lane 2) and small intestine (positive control; lane 4). Negative controls are without cDNA (lane 1) or hippocampal RNA without RT (lane 3). As an internal standard, PCR primers detecting RNA for ribosomal protein L 27A (Rpl) were used. C, Semiquantitative PCR comparisons of hippocampal RNA from SHR males, SD males, and SD females. D, In situ hybridization for Gip mRNA showed a weak but specific localization in the GCL as well as the CA1-CA3 region. E, Quantitative real-time PCR using a different set of primers confirmed the expression of the Gip gene in hippocampus of the three groups. As an internal standard, PCR primers detecting RNA for GAPDH (Gapdh) were used. SHR males showed the highest expression, viewed here as exhibiting a lower _C_T value (threshold cycle; cycle number when the system begins to detect the increase in the signal associated with an exponential growth of PCR product during the log-linear phase) and starting the amplification at a lower cycle number. However, the expression level was similar in both male and female SD rats.

Figure 2.

Figure 2.

GIP immunoreactivity is present in the adult rat hippocampus. A, Apart from the GCL, GIP immunoreactivity was also observed in the CA1-CA3 region and in scattered cells in the hilus. GIP immunoreactivity (IR) in brain slices from SHR males, SD males, and SD females (B) was measured, and the immunoreactive area of the GCL was compared with the total GCL area; *p < 0.05; ***p < 0.001. GIP immunoreactivity (C; red) was colocalized with NeuN (D; green) but not with GFAP (E; blue) in the hippocampal GCL (F; merged). Developing neuroblasts in the GCL expressing doublecortin (G; green) and GIP (H; red) are shown as merged in I. Arrows in G-I indicate a doublecortin-positive neuroblast also expressing GIP. The GCL contains Sox-2-immunoreactive progenitor cells (J, K; green) also exhibiting GIP immunoreactivity (J, K; red). The arrow in J indicates the enlarged cell shown in K. Scale bars: A, 100 μm; C-F, 50 μm; G-J, 20 μm.

Figure 3.

Figure 3.

The GIP receptor is present in both mature neurons and progenitor cells in the adult rat DG. In situ hybridization for the Gipr gene showed weak but specific expression in the DG, particularly in the GCL (A). GIPR immunoreactivity (B; red) was observed in progenitor cells also expressing Sox-2 (B; green). GFAP-immunopositive cells (B; blue) also expressing Sox-2 did not express the GIPR. The arrow in B indicates a GIPR-expressing progenitor cell without GFAP expression. GIPR immunoreactivity (C, E; red) was also observed in developing neuroblasts expressing doublecortin (D, E; green). The arrows in C-E indicate a doublecortin/GIPR-immunopositive cell. In addition to being produced by progenitor cells, GIPR (F, H; red) was also present in mature granule cells in the GCL together with NeuN (G, H; green). Scale bars: B, 20 μm; C-E, 8 μm; F-H, 20 μm.

Figure 6.

Figure 6.

The GIP receptor is present in cultured AHPs. A, PCR analysis demonstrated expression of the Gipr gene in cultured AHPs. Lane 1 (+) shows RNA from proliferating cell cultures (+FGF-2). Cells were also allowed to differentiate for 6 and 10 d (lanes 2 and 3). Lane H, Hippocampus (positive control); lane S, spleen (negative control). B, Western blot analysis demonstrated the presence of the GIPR in protein from cultured AHPs and the hippocampus. The GIPR (C-E; red) was also observed in cultured mitotic AHPs expressing Ki-67 (C; green) and in cells expressing Nestin (D; green) as well as cells expressing NeuN (E; green). Scale bars: C, D, 20 μm; E, 25 μm.

Figure 4.

Figure 4.

GIP increases cell proliferation in the adult GCL. The density of BrdU-positive cells in the GCL was determined in rats that had been given intracerebroventricular PBS (A) or GIP (B). Quantification of BrdU-positive and Ki-67-positive cells (C) showed that GIP-treated animals exhibited 86% more BrdU-positive cells and 105% more Ki-67-positive cells than animals treated with PBS (control). Quantification of BrdU-positive cells in the GCL of Gipr_-/- and wild-type (Wt+/+) mice (D) demonstrated that Gipr_-/- mice only have ∼40% of the number of newborn cells compared with Wt+/+ mice. Means ± SEM are given. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars: A, B, 100 μm.

Figure 5.

Figure 5.

GIP is present in cultured AHPs. A, Cultured AHPs express the Gip gene, as demonstrated by PCR. As an internal standard, PCR primers detecting Rpl were used. The lanes marked + and - represent RNA from cells cultured with and without FGF-2, respectively. The progenitor cells in culture were also immunoreactive for GIP (C; red) in combination with Map2ab (D; green), as shown merged in E. Mitotic cells expressing Ki-67 (G; yellow) also expressed GIP (H; red), shown merged in I. Nuclei in B and F are marked with Hoechst (blue). Scale bars, 25 μm.

Figure 7.

Figure 7.

GIP induces proliferation of cultured AHPs. A, GIP increased proliferation in a dose-dependent manner. B, Treatment of the cells with a combination of FGF-2 and GIP (1 n

m

) resulted in a greater DNA content than treatment with the two substances on their own. Values were calculated as a percentage of DNA content obtained from cells grown without FGF-2 and are shown as means ± SEM. C, The proliferative effect of GIP was confirmed using a methyl-[3H]thymidine incorporation assay, in which 1 n

m

GIP significantly increased thymidine incorporation. The proliferative effect of GIP was abolished when the cells were pretreated with 1 μ

m

ANTGIP. ANTGIP also decreased cell proliferation to ∼50% when used alone. Cell-death analyses, including Apop Tag labeling of cells (black bars) and lactate dehydrogenase measurements (gray bars) of cultured AHPs after GIP treatment, showed no difference from control (D; AHPs cultured without FGF-2 or GIP). *p < 0.05; **p < 0.01; ***p < 0.001.

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