Stimulation of β-adrenergic receptors plays a protective role via increased expression of RAF-1 and PDX-1 in hyperglycemic rat pancreatic islet (RIN-m5F) cells

Abstract

Introduction: It is a widely held view that a progressive reduction of beta-cell mass occurs in the progression of diabetes. RAF-1 kinase and pancreas duodenal homeobox 1 (PDX-1) are major factors that promote survival of cells and maintain normal insulin functions. In this study we investigated the effect of a β-adrenergic receptor agonist and antagonist on RAF-1 and PDX-1, and their respective effects on apoptosis and insulin release in RIN-m5F cells.

Material and methods: RIN-m5F cells were cultured in normal (5 mM) and high (25 mM) glucose to mimic diabetic conditions, followed by treatment with 5 µM, 10 µM and 20 µM of isoproterenol and isoproterenol + propranolol for 6, 12 and 24 h. Western blotting and reverse transcription analysis were performed to examine the expression of RAF-1 and PDX-1. Annexin-V-FITC and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were used to investigate apoptosis. ELISA was used to measure insulin levels. Reverse transcription polymerase chain reaction was conducted to investigate the expression of genes.

Results: Stimulation of β-adrenergic receptors with isoproterenol significantly induced RAF-1 and PDX-1 genes in a concentration-dependent and time-independent manner. Changes were significant both at protein and mRNA levels. Up-regulation of RAF-1 and PDX-1 was accompanied by improved insulin levels and reduced apoptosis. Concentrations of 10 µM and 20 µM for 12 and 24 h were more effective in achieving significant differences in the experimental and control groups. Propranolol reversed the effect of isoproterenol mostly at maximum concentrations and time periods.

Conclusions: A positive effect of a β-adrenergic agonist on RAF-1 and PDX-1, reduction in β-cell apoptosis and improved insulin contents can help to understand the pathogenesis of diabetes and to develop novel approaches for the β-cell dysfunction in diabetes.

Keywords: apoptosis; hyperglycemia; insulin; pancreas duodenal homeobox 1; v-raf-leukemia viral oncogene 1; β-adrenergic receptors.

Figure 1

Characterization of β1-, β2-, β3-, α1- and α2-adrenergic (AR) receptors in the RIN-m5F cells (A). Cells were grown without glucose, in 10 mM glucose and 20 mM glucose. RIN-m5F cells showed the expression of β1-AR (B), β2-AR (C), β3-AR (D), α1-AR (E) and α2-AR (F). These receptors were not differentially expressed in different concentrations of glucose except α1-AR (p < 0.05)

Figure 2

Shows the glucose-induced apoptosis (B) and the effect of isoproterenol (C) and isoproterenol + propranolol (D) as compared to control (A). Glucose caused increased apoptosis, and treatment of 20 μM isoproterenol for 24 h significantly (p < 0.05) reduced the apoptosis. This effect was reversed by the antagonist propranolol

Figure 3

Stimulation of β-adrenergic receptors with isoproterenol induced the expression of RAF-1 in hyperglycemic RIN-m5F cells (A and B). Isoproterenol treated cells were then subjected to propranolol. Increase in the expression of RAF-1 was dependent on concentration but independent of time periods. The isoproterenol generated effect on RAF-1 protein was reversed when the cells were treated with isoproterenol + propranolol (C and D) Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p < 0.05; ^#p < 0.01).

Figure 4

RIN-m5F cells were treated with glucose alone, glucose + isoproterenol (Iso) (β-adrenergic receptors antagonist) and glucose (Glu) + isoproterenol (Iso) + propranolol (Pro) (β-adrenergic receptor antagonist). Betaadrenergic receptor stimulation by isoproterenol caused increased mRNA level of RAF-1 (A and B) and PDX-1 (C and D) as compared to control and glucose treated cells Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p < 0.05; ^#p < 0.01).

Figure 5

Stimulation of β-adrenergic receptors with isoproterenol induced expression of the PDX-1 gene in hyperglycemic RIN-m5F cells (A and B). Isoproterenol treated cells were then subjected to propranolol. Agonist-induced expression of PDX-1 was dependent on concentration but independent of time periods. The isoproterenol generated effect on PDX-1 protein was reversed when the cells were treated with isoproterenol + propranolol (C and D) Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p < 0.05; ^#p < 0.01).

Figure 6

RIN-m5F cells were treated with glucose alone, glucose + isoproterenol (Iso) (β-adrenergic receptor antagonist) and glucose + isoproterenol (Iso) + propranolol (Pro) (β-adrenergic receptor antagonist). Stimulation with different concentrations (5 μM, 10 μM and 20 μM) of isoproterenol and propranolol for 6 h (A), 12 h (B) and 24 h (C) resulted in increased and decreased insulin release respectively Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p < 0.05; ^#p < 0.01).

Figure 7

RIN-m5F cells were treated with glucose alone, glucose + isoproterenol (β-adrenergic receptors antagonist) and glucose + isoproterenol + propranolol (β-adrenergic receptors antagonist). Stimulation with different concentrations (5 μM, 10 μM and 20 μM) of isoproterenol for 6 h (A), 12 h (B) and 24 h (C) resulted in decreased apoptosis in the cells. Antagonist propranolol was treated with the same concentrations for the same time periods Data are presented as mean ± standard deviation and compare glucose treated vs. glucose + isoproterenol treated vs. glucose + isoproterenol + propranolol treated (*p < 0.05; ^#p < 0.01).