Repression of BETA2/NeuroD promoter activity by AICAR in MIN

AICAR is an adenosine analogue, which is taken up into cells and converted by adenosine kinase into a phosphorylated form, AICAR monophosphate (ZMP).

ZMP mimics the effects of AMP on both the allosteric activation and the phosphorylation of AMPK.³⁴

To investigate the effects of glucose and AICAR on β-cells specific gene expression, islets were isolated from pancreas of Sprague-Dawley rats (200-250g male). The average number of islets isolated was ~300 per rat pancreas. The islets showed a regular spherical shape with will-defined smooth borders (Fig. 11). We carried out RT-PCR for the expression of BETA2/NeuroD, SUR1, and Pdx-1 to

investigate the effects of glucose and AICAR on mRNA levels of β-cell specific genes. Islets are incubated in high glucose condition, 30mM Glucose, at 6 h.

Increasing the glucose concentration from 5 to 30 mM significantly elevated the mRNA levels of BETA2/NeuroD in rat pancreatic islets (Fig. 12). Effect of

AICAR on SUR1 mRNA level showed similar results to BETA2/NeuroD (Fig. 12).

Interestingly, in islets treated with 400 µM AICAR for 6 h at 30 mM glucose, expression of BETA2/NeuroD and SUR1 decreased by 4.7 and 7.4 fold (Fig. 13). In contrast, AICAR did not affect the Pdx-1 level. The mRNA levels of each sample were normalized to the β-actin mRNA levels.

We also investigated mRNA levels of β-cell specific genes in MIN cells as a same condition with primary islets and similar results were obtained in MIN cells (Fig. 14). In MIN cells treated with 400 µM AICAR at 30 mM glucose, expression of

BETA2/NeuroD and SUR1 decreased by 3.1 and 2.2 fold, respectively. These results suggest that expression of BETA2/NeuroD and SUR1 were upregulated by a high level of glucose. Interestingly, the stimulatory effect of glucose was blocked in the presence of AICAR, but all genes of β-cells were not affected by AICAR.

To evaluate the effect of AICAR on BETA2/NeuroD gene transcription, we investigated BETA2/NeuroD promoter activity in MIN cells after transient

transfection of BETA2/NeuroD reporter gene with expression vectors, BETA2/NeuroD and ngn3, and treatment with 400 µM AICAR for 16 h before harvest. Coexpression of BETA2/NeuroD or ngn3 increased the promoter activity by

Fig. 11. Isolation and culture of rat pancreatic islets

Islets were isolated from pancreata of 200-250 g male Sprague-Dawley rats with collagenase digestion. Briefly, the common bile duct was cannulated and injected with 6 ml of cold M199 medium containing 1.5 mg/ml collagenase. The islets were collected and separated on Histopaque 1077 density gradient. The washed islets were picked individually under a dissecting microscope and cultured in RPMI medium with 11.1 mmol/l glucose, 10 % FBS, penicillin (100 U/ml), and

streptomycin (100 µg/ml) in a standard humidified culture condition of 5 % CO2 and 95 % air at 37 oC.

Fig. 12. Activation of BETA2/NeuroD and SUR1 by high glucose and repression by 400 µM AICAR, an AMPK activator.

Rat pancreatic islets were treated with 400 µM AICAR for 0-12h at 5mM or

30 mM glucose, and BETA2/NeuroD, SUR1, and Pdx-1 mRNA levels were examined by RT-PCR. BETA2/NeuroD, SUR1, and Pdx-1 products were detected as 390, 117, and 519 bp, respectively. The mRNA levels normalized to the β-actin

400 uM AICAR (h) 0 6 12 0 6 12 Low glucose High glucose

BETA2/NeuroD

SUR1

Pdx-1

β-Actin

mRNA. 12h after treatment with AICAR at 30 mM glucose, islets were suppressed BETA2/NeuroD and SUR1 mRNA level, but was without effect on Pdx-1 mRNA level. Data shown are the most representative of three independent experiments.

Fig. 13. Activation of BETA2/NeuroD and SUR1 by high glucose and repression by 400 mM AICAR, a AMPK activator.

The bar graphs represent quantitation of the results of RT-PCR in primary islets. The mRNA levels of BETA2 /NeuroD, SUR1, and Pdx-1 were normalized to the β-actin mRNA, and the RT-PCR data from three independent experiments were summarized as ± SE with respect of the value of untreated cells (*p<0.05, **p<0.01).

Fig. 14. Repression of the BETA2/NeuroD and SUR1 gene expression by glucose and AICAR.

MIN cells were treated with 400 µM AICAR for 12 h at 5 mM or 30 mM glucose, and RT-PCR were performed. BETA2/NeuroD and SUR1 products were detected as 390 bp and 117 bp fragments, respectively. The mRNA levels normalized

BETA2/NeuroD

SUR1

β-Actin AICAR - + - +

Glucose (mM) 5 5 30 30

Pdx-1

to the β-actin mRNA. BETA2/NeuroD and SUR1 were suppressed by 400 µM AICAR. Data shown are the most representative of three independent experiments.

Fig. 15. Repression of the BETA2/NeuroD and SUR1 gene expression by glucose and AICAR.

The bar graphs represent quantitation of the results of RT-PCR in MIN cells.

The mRNA levels of BETA2/NeuroD and SUR1were normalized to the β-actin mRNA, and the RT-PCR data from three independent experiments were summarized as ± SE with respect of the value of untreated cells.

1.7 and 2.3 fold 48 h after transfection. Importantly, when AICAR was treated, the promoter activity was decreased 4.2 and 5.7 fold, respectively (Fig. 16). MIN cells incubated at 30 mM glucose displayed greater BETA2/NeuroD promoter activity than cells maintained at 5 mM glucose (Data not shown). Addition of 400 µM AICAR completely repressed the promoter activity of the BETA2/NeuroD gene. As a positive control, we used a SUR1 reporter gene, pSURE3, which contained only activated E-box of SUR1 promoter region. In MIN cells at 30 mM glucose, expression of BETA2/NeuroD enhanced the promoter activity of the SURE3 reporter gene by 3.0 fold. After treated with 400 µM AICAR, the SUR1 promoter activity was decreased by 17.8 fold (Fig. 16). BETA2/NeuroD was originally isolated as a transactivator of the insulin gene. We also investigated a rat insulin II promoter activity in MIN cells after transient transfection of insulin reporter gene (RIP) and treatment with 200 and 400 µM AICAR for 16 h before harvest. In MIN cells treated

with 200 and 400 µM AICAR at 30 mM glucose, expression of insulin gene was

decreased by 2.4 fold (Fig. 17). These results suggest that expression of BET2/NeuroD- mediated genes, such as BETA2/NeuroD, SUR1, and insulin, were blocked in the presence of AICAR.

6. Binding of BETA2/NeuroD to E-box of the insulin promoter by AICAR

To verify the DNA binding ability of NeuroD by AICAR, we performed electrophoretic mobility shift assay using a double-stranded oligonucleotide containing the E-box of the rat insulin promoter element 3 (RIPE3). The RIPE3 site

contains the E-box, GCCATCTGC, which is conserved in all characterized mammalian insulin genes. Nuclear extracts were prepared from MIN cells incubated with or without AICAR for 12 h. Several complexes were detected, and specific binding of the labeled RIPE3 probes was verified using an excess amount of unlabeled RIPE3 oligonucleotides (Fig. 18, lane 2, 3). Interestingly, binding of NeuroD was decreased by AICAR (Fig. 18, lane 5). The specificity of the band for BETA2/NeuroD was shifted by BETA2/NeuroD antibody (Fig. 18, lane 6). Pdx-1 as well as BETA2/NeuroD is an important transcription factor for insulin gene. We also investigated DNA binding of Pdx-1 to A-box of the rat insulin promoter by AICAR.

The RIPA site contains the A-box, TAAT, which binds factors belonging to the homeodomain-containing protein family. Nuclear extracts were prepared from MIN cells incubated with or without AICAR for 12 h. Several complexes were detected,

Fig. 16. Repression of the BETA2/NeuroD and SUR1 promoter by 400 mM AICAR.

Reporter genes, pGL3-BETA2-2.2Luc and pGL3-SURE3Luc were cotransfected with pCMV-BETA2, an expression vector for BETA2/NeuroD and pCR3.1-ngn3, an expression vector for ngn3. To evaluate the effect of AICAR, MIN cells were treated with 400 M AICAR for 16h. Addition of AICAR completely repressed the promoter activity in MIN cells at high glucose.

Fig. 17. Repression of the rat insulin II promoter by AICAR.

Relative Luciferase Activity

2 0 4 0 6 0 8 0 100

AICAR (h) 0 200 400 0 200 400

Low Glucose High Glucose

Rat insulin II promoter (RIP), reporter gene, was transfected with AICAR in a dose-dependent for 16 h. Addition of AICAR completely repressed the promoter activity at high glucose.

and specific binding of the labeled RIPA probes was verified using an excess amount of unlabeled RIPA oligonucleotides (Fig. 19, lane 2, 3). DNA binding activity of Pdx-1 was not affected by AICAR (Fig. 19, lane 5). These complexes were supershifted when anti-BETA2 antibody was included in the incubation (Fig. 19, lane 6). These results suggest that the binding of BETA2/NeuroD to the E-box in the RIPE3 element was blocked by AICAR, whereas binding of Pdx-1 to the A-box in the RIPA element was not blocked by AICAR.