AMPK enhances the expression of pancreatic duodenal homeobox-1 via PPARalpha, but not PPARgamma, in rat insulinoma cell line INS-1

Abstract

Aim: To investigate whether AMP-activated protein kinase (AMPK) regulates the expression of pancreatic duodenal homeobox-1 (PDX-1), a beta-cell-specific transcription factor and whether PPARalpha/gamma is involved in the regulation of pancreatic beta-cell lines after acute stimulation.

Methods: Rat insulinoma cell line INS-1 was treated with an activator (AICAR) or inhibitor (Compound C) of AMPK as well as inhibitors of PPARs (MK886 to PPARalpha and BADGE to PPARgamma). The mRNA levels of PDX-1, PPARalpha and PPARgamma were measured using real-time RT-PCR, and Western blotting was used to detect the protein expression of these factors.

Results: Activation of AMPK by AICAR induced significantly increased the expression of PDX-1, and this increase was abrogated when AMPK was inactivated by Compound C. Similarly, the expression of PPARalpha and PPARgamma was also increased by AICAR or decreased by Compound C. However AMPK activation did not increase nuclear PDX-1 protein levels when PPARalpha was inhibited. In contrast, AMPK activation still up-regulated PDX-1 protein levels during PPARgamma inhibition. Additionally, PPARalpha activation induced by fenofibrate significantly enhanced nuclear PDX-1 protein expression.

Conclusion: AMPK regulates the expression of PDX-1 at both the transcriptional and protein levels, and PPARalpha may be acutely involved in the regulation of INS-1 cells.

Figure 1

AICAR stimulates the activity of AMPKα. INS-1 cells treated with or without 0.5 mmol/L AICAR in the presence or absence of 10 μmol/L Compound C (Comp C) for 8 h were washed with phosphate-buffered saline and were lysed. For each sample, 60 μg protein was used for Western blotting. (A) A representative blot is shown. (B) Densitometric results from six independent Western blotting experiments are shown. Data were calculated using β-actin as an internal standard and were normalized by setting the control as 1 (mean±SD). ^bP<0.05 compared to the untreated control group; ^eP<0.05 compared to the group treated with AICAR alone.

Figure 2

AMPK activation increases the expression of PDX-1. INS-1 cells were treated with or without 0.5 mmol/l AICAR in the presence or absence of 10 μmol/L Compound C (Comp C) for 8 h, and were then harvested to monitor mRNA levels of PDX-1 (A) by real-time RT-PCR. Data were calculated using GAPDH as an internal standard and were normalized by setting the control as 1 (mean±SD, n=6). PDX-1 protein from nuclear (B) and cytoplasmic (C) fractions were detected by Western blotting. Densitometric results from six independent Western blotting experiments are shown. Data were calculated using β-actin as an internal standard and were normalized by setting the control as 1 (mean±SD, n=6). ^bP<0.05 compared to the untreated control group; ^eP<0.05 compared to the group treated with AICAR alone.

Figure 3

AICAR up-regulates PPARα and PPARγ expression. INS-1 cells were treated with or without 0.5 mmol/L AICAR, in the presence or absence of 10 μmol/L Compound C (Comp C) for 8 h. They were then harvested to monitor mRNA levels of PPARα (A) and PPARγ (B) by real-time RT-PCR. Results from six independent experiments are shown. Data were calculated using GAPDH as an internal standard and were normalized by setting the control as 1 (mean±SD). Nuclear protein fractions were prepared to detect PPARα (C) and PPARγ (D) by immunoprecipitation. Densitometric results from six independent Western blotting experiments are shown. Data were normalized by setting the control as 1 (mean±SD). ^bP<0.05 compared to the untreated control group; ^eP<0.05 compared to the group treated with AICAR alone.

Figure 4

Effects of MK886 and BADGE on the expression of PDX-1. INS-1 cells were pre-incubated with 5 μmol/L MK 886 or 50 μmol/L BADGE for 16 h and in the presence or absence of 0.5 mmol/L AICAR for 8 h. PDX-1 protein from nuclear (A) and cytoplasmic (B) fractions was detected by Western blotting. Densitometric results from six independent Western blotting experiments are shown. Data were calculated using β-actin as an internal standard and were normalized by setting the control as 1 (mean±SD, n=6). ^bP<0.05 compared to the untreated control group; ^eP<0.05 compared to the group treated with AICAR alone.

Figure 5

Effects of fenofibrate on the expression of PDX-1 and PPARα. INS-1 cells were treated with or without 5 μmol/L fenofibrate in the presence or absence of 5 μmol/L MK886 for 8 h. Nuclear PPARα protein (A) was detected by immunoprecipitation, and PDX-1 protein from nuclear (B) and cytoplasmic (C) fractions was detected by Western blotting. Densitometric results from six independent Western blotting experiments are shown. ^bP<0.05 compared to the untreated control group; ^eP<0.05 compared to the group treated with fenofibrate alone.