Dopamine Modulates Insulin Release and Is Involved in the Survival of Rat Pancreatic Beta Cells

<div><p>The local synthesis of dopamine and its effects on insulin release have been described in isolated islets. Thus, it may be accepted that dopamine exerts an auto-paracrine regulation of insulin secretion from pancreatic beta cells. The aim of the present study is to analyze whether dopamine is a regulator of the proliferation and apoptosis of rat pancreatic beta cells after glucose-stimulated insulin secretion. Glucose stimulated pancreatic islets obtained from male Wistar rats were cultured with 1 or 10 μM dopamine from 1 to 12 h. Insulin secretion was analyzed by RIA. The cellular proliferation rate of pancreatic islets and beta cells was studied with immunocytochemical double labelling for both insulin and PCNA (proliferating cell nuclear antigen), and active caspase-3 was detected to evaluate apoptosis. The secretion of insulin from isolated islets was significantly inhibited (p<0.01), by treatment with 1 and 10 μM dopamine, with no differences between either dose as early as 1 h after treatment. The percentage of insulin-positive cells in the islets decreased significantly (p<0.01) after 1 h of treatment up to 12 h. The proliferation rate of insulin-positive cells in the islets decreased significantly (p<0.01) following treatment with dopamine. Apoptosis in pancreatic islets and beta cells was increased by treatment with 1 and 10 μM dopamine along 12 h. In conclusion, these results suggest that dopamine could modulate the proliferation and apoptosis of pancreatic beta cells and that dopamine may be involved in the maintenance of pancreatic islets.</p></div

Morphometric effect induced by treatment with dopamine on cellular (A) and nuclear (B) area of beta cells.

<p>Histological sections were selected from micrographs of cross sections of each islets (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123197#sec002" target="_blank">methods</a>). In each section the cellular and the nuclear cell profiles from insulin-positive cells were plotted, allowing the surface of the cell and nuclear areas to be calculated after calibration of the Image J application. Cellular and nuclear area from10 islets per study group, and 100cells per islet were measured in 10 mM glucose, 1μM and 10 μM dopamine. (area: μm², **p<0.01).</p

Inmunocytochemical analysis of insulin-positive cells in isolated islets treated with dopamine.

<p><b>(A)</b> Micrographs showing some immunocytochemical staining patterns for insulin (red) in control islets (i), and dopamine-treated islets at 1, 3, 6 and 12 h (ii). <b>(B)</b> Plot showing the decrease induced by dopamine in the percentage of insulin-positive cells at the different time-points assayed; from 1 to 12 hours of treatment a significant decrease (*p<0.05, **p<0.01 with respect to their respective controls) was observed. Scale bar: 50 μm.</p

Effect of dopamine on cellular proliferation rate of insulin-positive beta cells in isolated islets.

<p><b>(A)</b> After double immunocytochemical staining, the nuclei of PCNA-positive cells appear brown (ii). <b>(B)</b> The proliferating insulin-positive cells (i, red) show brown-stained nuclei (ii), and the count of positive cells and calculation of the percentages of insulin-positive cells in proliferation was enabled after combining both images following digital transformation of the brown colour to blue colour (iii). 4000 cells per group were counted to obtain the statistical values. <b>(C)</b> Percentage of PCNA-positive cells out of the total number of cells (alpha, beta and delta cells), and <b>(D)</b> percentage of PCNA- and insulin-positive cells out of the total number of insulin-positive cells in the control pancreatic islets and those treated with 1μM and 10 μM dopamine **p<0.01.</p

Effects of dopamine on insulin content and release.

<p><b>(A)</b> Inhibition of insulin secretion in overnight cultured rat islets followed by treatment in glucose with dopamine 1 μM and 10 μM contrasted with glucose 10 mM as a control. Insulin secretion was then monitored at 1, 3, 6 and 12 h. <b>(B)</b> Absolute values AUC of insulin secretion were calculated for 10 mM glucose, 1μM and 10μM dopamine. <b>(C)</b> The insulin content of the islets used to study insulin secretion was determined at the end of the culture at 1, 3, 6 and 12h. <b>(D)</b> Absolute values of AUC of insulin content were calculated for 10 mM glucose and 1μM and 10μM dopamine. <b>(E)</b> This shows the sum AUC of insulin content (inferior portion) and insulin secretion (superior portion) by the same islets during treatment with dopamine (1 and 10 μM) <i>versus</i> glucose 10 mM. Absolute values are represented as means ± SEM for 15 batches of islets (10 islets per batch) from 2 experiments (*p<0.05 and **p<0.01).</p