Obestatin Enhances In Vitro Generation of Pancreatic Islets through Regulation of Developmental Pathways

<div><p>Availability of large amounts of <i>in vitro</i> generated β-cells may support replacement therapy in diabetes. However, methods to obtain β-cells from stem/progenitor cells are limited by inefficient endocrine differentiation. We have recently shown that the ghrelin gene product obestatin displays beneficial effects on pancreatic β-cell survival and function. Obestatin prevents β-cell apoptosis, preserves β-cell mass and stimulates insulin secretion <i>in vitro</i> and <i>in vivo,</i> in both normal and diabetic conditions. In the present study, we investigated whether obestatin may promote <i>in vitro</i> β-cell generation from mouse pancreatic islet-derived precursor cells. Treatment of cultured islets of Langerhans with obestatin (i) enriched cells expressing the mesenchymal/neuronal marker nestin, which is associated with pancreatic precursors; (ii) increased cell survival and reduced apoptosis during precursor selection; (iii) promoted the generation of islet-like cell clusters (ICCs) with increased insulin gene expression and C-peptide secretion. Furthermore, obestatin modulated the expression of fibroblast growth factor receptors (FGFRs), Notch receptors and neurogenin 3 (Ngn3) during islet-derived precursor cell selection and endocrine differentiation. These results indicate that obestatin improves the generation of functional β-cells/ICCs <i>in vitro</i>, suggesting implications for cell-based replacement therapy in diabetes. Moreover, obestatin may play a role in regulating pathways involved in pancreas development and regeneration.</p></div

Obestatin inhibits cell migration and FGF2 binding to MLCs at stage 5.

<p>(A–B) Basal and FGF2-induced cell migration assessed at day 2 (A) and 6 (B) in either untreated cells (Control) or obestatin-treated cultures (–, no FGF2). Neutralizing antibody against FGF2 (Ab) was used to verify the specificity of FGF2 effect. The cells were deprived of FGF2 for 12 h then exposed for 4 h to 100 ng/ml of FGF2 and 100 ng/ml of neutralizing antibody. Results are the mean ± SEM of three independent experiments performed in duplicate (**<i>P</i><0.01, ***<i>P</i><0.001 vs. control; ^#<i>P</i><0.05 vs. –; ^§<i>P</i><0.05, ^§§<i>P</i><0.01; ns, not significant). (C) Expression of FGF2 and FGFRs assessed by RT-PCR at the indicated days in control (c) and obestatin-treated cultures (Obe). Results are representative of three independent experiments. (D) FGF2⁺ MLCs that were cultured in non FGF2-starved conditions, in either absence or presence of obestatin at the indicated days. Results shown in the graph (left) are expressed as percent of total cell number and are the mean ± SEM of three independent experiments performed in duplicate (**<i>P</i><0.01 vs. control, ^#<i>P</i><0.01 vs. obestatin at day 2). Right panel, representative images (X20) of FGF2 staining (green) (n = 3); nuclei are shown in blue (DAPI).</p

Obestatin up-regulates insulin and glucagon gene expression.

<p>INS1 (A), INS2 (B) and glucagon (C) mRNA expression in enriched islets and ICCs at day 9 and day 15 (stage 5) in control (c) and obestatin-treated (Obe) cultures evaluated by Real Time PCR. Gene expression was normalized to β-actin and reported as percent of enriched islets. *<i>P</i><0.05, **<i>P</i><0.01 vs. c; ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs. enriched islets. N = 4 (ns, not significant).</p

Obestatin modulates Notch receptors and Ngn3 gene expression.

<p>(A) RT-PCR analysis showing Notch receptors and Ngn3 mRNA at the indicated times in control (c) and obestatin-treated (Obe) cultures. Results are representative of three independent experiments. (B) Ngn3 mRNA assessed by real-time PCR at day 9 of stage 5 in adherent MLCs. Results are the mean ± SEM of three independent experiments performed in duplicate (*<i>P</i><0.05 vs. control). (C) Immunofluorescent staining for Ngn3 in adherent cells (monolayer) (X20) and clustered cells (X40), in both control and obestatin-treated cultures. Ngn3 is shown in green and nuclei in blue (DAPI). Each image is representative of three independent experiments. (D) Schematic illustration of obestatin mechanisms of action. During pancreatic development FGF2 promotes pancreatic precursor cell proliferation through FGFRs, inducing Notch signaling and preventing Ngn3 expression. Upon serum removal, FGF2 promotes MLC proliferation and Notch receptors expression. Obestatin treatment inhibits FGF2 action by reducing expression of FGFRs. Obestatin also inhibits the expression of selective Notch receptors, leading to increased Ngn3 expression and endocrine differentiation.</p

Obestatin promotes cell survival and decreases serum starvation-induced apoptosis at stage 5.

<p>(A) Protein content in control and obestatin-treated cultures at the indicated days. (B-C) Cell survival (B) and apoptosis (C) assessed by Trypan blue exclusion and Hoechst 33258 staining, respectively, at the indicated days. For all graphs, results are the mean ± SEM of three independent experiments (*<i>P</i><0.05, **<i>P</i><0.01 vs. control; ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. day 0).</p

Enumeration of endocrine hormone-producing cells and BrdU⁺ cells (proliferating MLCs; day 6, stage 4).

<p>Results are the mean ± SEM of three independent experiments (*<i>P</i><0.05; **<i>P</i><0.01; n = 3).</p

Enumeration of endocrine hormone-producing cells and BrdU⁺ cells within the islet (day 6, stage 4).

<p>Results are the mean ± SEM of three independent experiments (*<i>P</i><0.05; **<i>P</i><0.01; n = 3).</p

Overview of culture evolution.

<p>For details also see Materials and Methods. (A) Schematic representation of the protocol used to obtain ICCs from mouse pancreas. Stage 1, pancreas removal; stage 2, pancreas digestion and plating; stage 3, islet enrichment; stage 4, islet adhesion and MLC appearance; stage 5, MLC migration, clustering and ICC formation. (B) Representative photomicrographs comparing the evolution of control and obestatin-treated (Obe) cultures. Islet adhesion to the plate induced generation of MLCs (stage 4). During stage 5 MLCs migrate and assemble to form ICCs. Magnification, 10X. (C) Mean surface area and number of adherent islets (stage 4 day 6) and floating ICCs (stage 5) in either control or obestatin treated conditions. Results are the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 vs. control; ^##<i>P</i><0.01 vs. same condition at stage 4, day 6. N = 6.</p

Association between outcomes at follow-up (dependent variables) and the percentage of total daily caloric intake from dinner in multiple logistic regression models.

<p>Association between outcomes at follow-up (dependent variables) and the percentage of total daily caloric intake from dinner in multiple logistic regression models.</p

Baseline lifestyle and dietary characteristics by the percentage of total daily caloric intake from dinner.

<p>Baseline lifestyle and dietary characteristics by the percentage of total daily caloric intake from dinner.</p