Exendin-4 regulates pancreatic ABCA1 transcription via CaMKK/CaMKIV pathway

Abstract

ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and glucose homeostasis. This study investigates whether the long-acting agonist of the glucagon-like peptide 1, namely exendin-4, which mediates stimulatory effects on ABCA1 gene expression, could interfere with the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) cascade. ABCA1 promoter activity was examined by reporter gene assay in rat insulin-secreting INS-1 cells incubated with exendin-4. CaMKIV activity was assessed by detection of activation-loop phosphorylation (Thr(196)) of CaMKIV. We investigated the influence of the constitutively active form (CaMKIVc) or CaMKIV knockdown on ABCA1 expression. Increased abundance of ABCA1 protein was noted in response to rising concentrations of exendin-4 with maximum induction at 10 nM. Exendin-4 also stimulated ABCA1 promoter activity, but failed to do so in the presence of STO-609, a CaMKK inhibitor. Up-regulation of CaMKIV phosphorylation (at Thr(196)) peaked after 10 min. of exposure to exendin-4. CaMKIVc enhanced or up-regulated ABCA1 promoter activity in INS-1 cells. Furthermore, exendin-4 induction of ABCA1 protein expression was significantly suppressed in cells treated with CaMKIV-siRNA. Activation of the CaMKK/CaMKIV cascade by exendin-4 stimulated ABCA1 gene transcription, indicating that exendin-4 plays an important role in insulin secretion and cholesterol ester content in pancreatic beta cells.

Fig 1

Effect of exendin-4 on ABCA1 expression in rat pancreatic islets and INS-1 cells. (A) Total cell lysate was purified from rat pancreatic islets treated with 10 nM of exendin-4 for 24 hrs. Western blot analysis was performed to examine ABCA1 expression. Expression of GAPDH was studied as the control, and the results are shown in the bottom lanes. The plot shows the ratio of ABCA1/GAPDH. Results are represented as mean ± S.E.M. of three experiments for each treatment group. The asterisk denotes a significant difference (P < 0.01). (B) Exendin-4 increases ABCA1 gene transcription. INS-1 cells were transfected with 1 μg pABCA1-LUC and treated with the indicated concentrations of exendin-4 for 24 hrs prior to cell harvesting. All assays were corrected for β-galactosidase activity, and the total amount of protein in each reaction was identical. The results were expressed as relative luciferase activity compared with that in the control cells arbitrarily set at 100. Each data point shows the mean ± S.E. of four separate transfections that were performed on separate days. The ‘*’ denotes the significant difference (P < 0.01). (C) Effects of the phosphatidylinositol 3-kinase inhibitor LY-294002, the PKC inhibitor bisindolylmaleimide I, PKA inhibitor H-89, and the CaMK inhibitor STO-609 on ABCA1 transcriptional activity in INS-1 cells with 10 nM exendin-4. Vehicle: 0.1% dimethyl sulphoxide. Each data point shows the mean ± S.E. of three separate transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01).

Fig 2

CaMKK cascade stimulates the ABCA1 promoter activity in response to exendin-4. (A) Phosphorylation of CaMKIV by exendin-4 in INS-1 cells. INS-1 cells were exposed to 10 nM exendin-4 for 2 min. before harvest at the predetermined time intervals. The total cell extracts were subjected to immunoprecipitation using anti-CaMKIV antibodies and SDS-PAGE, followed by Western blotting analysis using anti-phospho-Thr196 antibodies (upper insert). Total cell lysates were also blotted using anti-CaMKIV antibodies as a control (lower insert). (B) Effect of CaMK cascade on ABCA1 promoter activity. Cells were transfected with pABCA1-LUC and an empty vector, or CaMKIVc expression vectors. The cells were incubated for 24 hrs after transfection. Each data point shows the mean ± S.E. of three separate transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01). (C) Effects of CaMKIV knockdown on ABCA1 expression in INS-1 cells. SiRNA of CaMKIV (siCaMKIV) or scrambled siRNA (siCont) was transfected into INS-1 cells, and then treated with exendin-4 (Ex-4). At 24 hrs after transfection, the abundance of ABCA1 protein level was measured using Western blot analysis (upper panel). The ratio of ABCA1 to GAPDH is shown as the percentage of control. Each data point shows the mean ± S.E. (n= 3) of separate experiments. The asterisk denotes a significant difference (P < 0.05). N.S.; no significant difference.