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Regulation if SUR1 gene by BETA2/NeuroD and AMP-activated protein kinase

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Regulation of SUR1 gene by BETA2/NeuroD

and AMP-activated protein kinase

by

Ji-Won Kim

A Dissertation Submitted to The Graduate School of Ajou University in

Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Supervised by

Haeyoung Suh-Kim, Ph.D.

Department of Medical Sciences

The Graduate School, Ajou University

(3)

- ABSTRACT -

Regulation of SUR1 gene by

BETA2/NeuroD and AMP-activated

protein kinase

ATP-sensitive K+ channels plays a critical role in the regulation of insulin

secretion in the pancreatic β-cell. SUR (Sulfonylurea Receptor)1 constitutes

ATP-sensitive K+ channels in excitable cells and serves as the target for drugs widely used

for the treatment of non- insulin dependent diabetes mellitus. Previo usly we have shown that tissue specific expression of SUR1 is regulated by BETA2/NeuroD, a transcription factor in neuroendocrine cells.

In this study we investigated the molecular mechanisms how BETA2/NeuroD regulates the SUR1 gene expression. Coexpression of a dominant negative mutant of BETA2/NeuroD, BETA2(1-233) repressed the promoter activity of SUR1 gene. BETA2/NeuroD bound specifically to the E3 element located at -141. Mutation of E3 eliminated the stimulatory effect of BETA2/NeuroD. Unlike BETA2/NeuroD, ngn3 could not activate E3 in HeLa cells. Overexpression of ngn3

(4)

concomitantly increased expression of BETA2/NeuroD and SUR1 in HIT-T15 cells but not in HeLa cells. These results indicated that BETA2/NeuroD induces tissue specific expression of the SUR1 gene through the E3 element. These results also suggest that E3 is specific for BETA2/NeuroD and the stimulatory effect of ngn3 in HIT-T15 cells may require factors specifically expressed in HIT-T15 cells.

We also found that changes in the metabolic state of β-cells cause expression

of BETA2/NeuroD and expression of SUR1 gene mediated by BETA2/NeuroD. In particular, expression of NeuroD/BETA2 and SUR1 was upregulated by a high level of glucose in MIN cells and rat pancreatic islets. Interestingly, the stimulatory effect of glucose on BETA2/NeuroD was blocked by the treatment of AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), an activator of AMP kinase (AMPK). AMPK is activated by AMP and known as a metabolic sensor in various tissues. AICAR also reduced expression of SUR1 in MIN cells, probably by repressing BETA2/NeuroD both at the transcription and post-transcription levels.

These results suggest that the plasma glucose level may regulate expression

of ATP sensitive K+ channels by altering expression and transcriptional activity of

(5)

extracellular metabolic signals into the cells and finely tunes the expression of β-cell specific genes in response to glucose level.

Key Words: Sulfonylurea receptor1, BETA2/NeuroD, Pancreatic duodenal homeobox-1, Neurogenin3, AMP-activated protein kinase, 5-Aminoimidazole-4-carboxamide riboside

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TABLE OF CONTENTS

TITLE PAGE --- 1

ABSRACT --- 2

TABLE OF CONTENTS --- 5

LIST OF FIG URES --- 8

LIST OF ABBREVIATIONS --- 10

I . INTRODUCTION --- 13

A. Insulin secretion pathway of pancreatic β-cells --- 13

1. ATP-sensitive K+ channels (KA T P) in insulin secretion --- 13

2. Characterization of the mouse SUR1 gene --- 15

B. Development roles of β-cell specific transcription factors --- 16

1. Pancreatic duodenal homeobox- 1 (Pdx- 1) --- 18

2. Neurogenin (ngn)3 --- 19

3. BETA2/NeuroD--- 20

C. AMP-activated protein kinase--- 21

II . MATERIALS AND METHODS --- 25

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B. Materials --- 26

1. Plasmids --- 26

2. Preparation of recombinant adenovirus expressing NeuroD --- 27

3. Constructions of reporter genes --- 27

4. Cell culture --- 30

5. Isolation of islets and culture of islet cells --- 30

6. Transfection--- 30

7. Chloramphenicol acetyltransferase (CAT) and Luciferase assay--- 31

8. Electrophoretic mobility shift assays --- 32

9. Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) --- 33

10. Preparation of nuclear extracts and Western Blotting --- 34

III. RESULTS --- 36

1. Transactivation of the SUR1 gene by BETA2/NeuroD --- 36

2. Determination of binding sites for BETA2/NeuroD --- 39

3. E3- mediated transactivation by BETA2/NeuroD --- 42

4. Specificity of the E-box-mediated transactivation by BETA2 /NeuroD --- 45

5. Repression of BETA2/NeuroD promoter activity by AICAR in MIN cells --- 49

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

--- 57

7. Expression of BETA2/NeuroD by AMPK activation --- 60

8. Effect of Adenovirus - mediated NeuroD overexpression by AICAR in MIN cells --- 67

VI . DISCUSSION --- 73

V . REFERENCES --- 80

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LIST OF FIGURES

Fig. 1. Cellular events in glucose- induced insulin secretion in pancreatic β-cells ---14

Fig. 2. Transcription factors involved in pancreatic islet development ---17

Fig. 3. Schematic outline of fNeuroD adenovirus generation --- 28

Fig. 4. Repression of the SUR1 promoter by a dominant negative mutant of BETA2/NeuroD --- 37

Fig. 5. Determination of DNA binding ability of three putative E-boxes--- 40

Fig. 6. Binding of BETA2/NeuroD to E3--- 41

Fig. 7. Constructs for E3 box and mutated E3 box of SUR1--- 43

Fig. 8. Mutation of E3 abolishes transactivation by BETA2/NeuroD--- 44

Fig. 9. Specificity of E3 for BETA2/NeuroD--- 46

Fig. 10. Specificity of E3 for BETA2/NeuroD--- 48

Fig. 11. Isolation and culture of rat pancreatic islets--- 51

Fig. 12. Activation of BETA2/NeuroD and SUR1 by high glucose and repression by 400 µM AICAR, an AMPK activator--- 52

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Fig. 13. Activation of BETA2/NeuroD and SUR1 by high glucose and repression by 400 mM AICAR, a AMPK activator (Graph)--- 53

Fig. 14. Repression of the BETA2/NeuroD and SUR1 gene expression by glucose and AICAR--- 54 Fig. 15. Repression of the BETA2/NeuroD and SUR1 gene expression by glucose

and AICAR (Graph)--- 55 Fig. 16. Repression of the BETA2/NeuroD and SUR1 promoter by 400 mM

--- 58 Fig. 17. Repression of the rat insulin II promoter by AICAR--- 59

Fig. 18. Binding of BETA2/NeuroD to E-box in insulin promoter by AICAR--- 61 Fig. 19. Binding of Pdx-1 to A-box in insulin promoter by AICAR--- 62 Fig. 20. Repression of BETA2/NeuroD level by AICAR in MIN cells--- 64

Fig. 21. Repression of BETA2/NeuroD level by Ad-AMPKα.α1312/T172D in MIN

cells --- 66 Fig. 22. Effect of AICAR on the intracellular localization of Pdx-1--- 68 Fig. 23. Overexpression of NeuroD using MIN cells --- 69

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Fig. 24. Map of pAdTrack-CMV-FLAG-NeuroD--- 70 Fig. 25. Repression of BETA2/NeuroD level by AICAR in MIN cells--- 71 Fig. 26. Model for the mechanism by AMPK in high glucose---78

LIST OF ABBREVIATIONS

ACC Acetyl-CoA carboxylase

AICAR 5-Aminoimidazole-4-carboxamide reboside

AMPK AMP-activated protein kinase

AMPKK AMP-activated protein kinase kinase

BETA2 β-cell E-box transactivator 2

bHLH basic helix-loop-helix

bp base pair

β-gal β-galactosidase

CAT Chloramphenicol acetyltransferase

DMEM Dulbecco’s modified Eagle’s medium

DMSO Dimethyl sulfoxide

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EMSA Electrophoretic mobility shift assay

FBS Fetal bovine serum

GLUT-2 Glucose transporter 2

GSK3β Glycogen synthase kinase 3β

HBSS Hank’s balanced salt solution

HIT Hamster insulin tumor

HRP Horseradish peroxidase

IDX-1 Islet duodenum homeobox gene 1

IgG Immunoglobulin G

IPF-1 Insulin amyloid polypeptide

KATP ATP-sensitive K+ channel

Luc Luciferase

MIN Mouse insulinoma

Ngn3 Neurogenin 3

NP-40 Octylphenoxypoluethoxyethanol (Nonidet P-40)

NRE Negative regulatory element

PAGE Polyacrylamide gel electrophoresis

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PDX-1 Pancreatic and duodenal homeobox gene 1

PHHI Persistent hyperinsulinemic hylerglycemia of infancy

PMSF Phenylmethylsulfonyl fluoride

Poly dI/dC polydeoxyinosinicdeoxycytidylic acid

PPI Preproinsulin

RIP Rat insulin II promoter

RIPE3 Rat insulin promoter element 3

RT Reverse transcriptase

S. E. Standard error

SDS Sodium dodecyl sulfate

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I. INTRODUCTION

A. Insulin secretion pathway of pancreatic β -cells

Glucose is transported into β-cell by the glucose transporter GLUT2. It is then

phosphorylated by glucokinase. Further glucose metabolism generates signals that

inhibit the ATP-sensitive K+ channels, regulating in membrane depolarization. This

activates the voltage-gated Ca2+ channels and increases intracellular Ca2+ levels. Ca2+ in

turn triggers the fusion of prestored insulin vesicles with the plasma membrane (Fig.1).1

1. ATP-sensitive K+ channels (KATP) in insulin secretion

ATP-sensitive K+ channels (KATP) play a critical role in converting changes in

the ATP:ADP ratio to differences in electrical activity of the membrane in most excitable cells. KATP is composed of two subunits, the pore- forming unit, KIR6.x, and the regulator

(15)

member of the ATP-binding cassette superfamily with multiple

transmembrane-spanning domains and two nucleotide-binding folds.3

Several isoforms of SUR, SUR1, SUR2A and SUR2B, have been cloned and

K+ insulin glucose mechanism glucose Glut2 ATP:ADP insulin vesicle glucose Ca2+ channel Ca2+ K+ATP channel IR AMPK BETA2 SUR1 E box P Pdx-1 Pdx-1 PI3K IRS2

(16)

Fig. 1. Cellular events in glucose-induced insulin secretion in pancreatic β -cells

combination of different isoforms of SUR and KIR6.x leads to the distinct functional and

phamacological channel profiles in various tissues.4, 5 SUR2A and SUR2B with KIR6.2

constitute the KATP channels of the cardiac and vascular smooth muscle-type whereas

SUR1 with KIR6.2 constitutes the β-cell specific KATP channel.

In pancreatic β-cells, increased glucose metabolism causes an increase in the

ATP:ADP, which blocks KATP channel and leads to activation of voltage-dependent Ca2+

channels. As a result, Ca2+ ions influx into the cell and induce insulin secretion (Fig. 1).

SUR1 is the target of sulfonylurea drugs, for example, tolbutamide and glibenclamide that are widely used to promote insulin secretion in the treatment of non-insulin

dependent diabetes mellitus.3 , 6 Mutations in the human SUR1 gene cause familial

persistent hyperinsulinemic hypoglycemia of infancy (PHHI) that is characterized by

continuous insulin secretion in spite of low concentrations of blood glucose.7, 8

Compared to human, targeted mutations of SUR1 are less effective in mice. SUR1 knockout mice lack the first phase insulin secretion but still exhibit an attenuated

(17)

glucose-stimulated second phase insulin secretion.9

2. Characterization of the mouse SUR1 gene

The promoters of SUR1 have been cloned from human10 and mouse.11

Human and mouse promoters are TATA- less and GC-rich region with several SP1 binding sites around transcription initiation sites. Although human and mouse promoters are relatively similar, sequence analysis does not reveal any particular region of high similarity between two promoters except the transcription initiation

sites.11 Interestingly, while the 1.3 kb-long 5’ flanking sequence of the human

promoter is sufficient to drive the β-cell specific expression,10 the corresponding

region of the mouse SUR1 promoter does not seem to be tissue specific.11

B. Developmental roles of β -cell specific transcription factors

The pancreas forms in the region of the duodenum immediately posterior to the stomach. After the formation of the gut tube, dorsal and ventral pancreatic buds that will later fuse grow from the endodermal epithelium that is surrounded by mesenchyme. Some cells in the bud differentiate into exocrine cells that retain epithelial characteristics and form branched ducts and acini. Endocrine cells emigrate from the epithelium and

(18)

aggregate into islets within the mesenchyme. After bud formation, several transcription factors have been shown to be required for the differentiation of specific cell types (Fig.

2).12 Paired homeodomain proteins Pdx-1, Pax-4, Pax-6, and Prox-1,

LIM-homeodomain, Isl-1, Nkx6.1, and Nkx2.2 as well as the basic helix-loop-helix (bHLH)

ENDODERM duodenum dorsal bud ventral bud Pdx-1 (E8.5) PANCREAS Isl-1, Pax-6, ngn3 (E9.0) Endocrin e

NeuroD NeuorD Pax-4 Nkx2.2

Nkx2.2 Pax-4 (E9.5) (E9.5)

Brn4 Nkx2.2 (E9.5) (E9.5) Nkx6.1 (E10.5) Pdx-1 p48, Prox-1 Exocrine

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Fig. 2. Transcription factors involved in pancreatic islet development.

proteins, BETA2, and neurogenin3 are necessary for the differentiation of all or a subset

of four endocrine cell types. (Fig. 2).13, 14

1. Pancreatic duodenal homeobox-1 (Pdx-1)

Pancreatic duodenal homeobox-1 (Pdx-1) was originally termed in the several literature under several IPF-1 (Insulin Promoter Factor-1) and STF-1 (Somatostatin Transcription Factor-1), and IDX-1 (Islet Duodenum Homeobox gene-1). It was independently discovered by a number of laboratories working on the regulation of hormone gene expression and development in the islets of Langerhans and in the developmental biology of the frog. Pdx-1 belongs to the homeodomain protein family, and it is uniformly expressed in the pancreatic bud at E8.5, marking

the territory of the future pancreas. Later, it becomes restricted to β- and δ-cells.15

Pdx-1 plays a key role in pancreas development, as neonatal mice carrying a null mutation of this gene lack a pancreas, even though the initial pancreatic bud is

(20)

formed. Pdx-1 also has a second role as the β-cell phenotype and diabetes.16 Therefore, Pdx-1 is required for pancreas morphogenesis and maintaining the insulin

production and glucose sensing system in β-cells. Pdx-1 regulates the expression

including insulin gene in response to changes in glucose and insulin concentration. Glucose and insulin regulate Pdx-1 by way of a signaling pathway involving

phosphatidylinositol 3-kinase (PI3K), SAPK2/p38.17 Activation of this pathway

leads to phosphorylation of Pdx-1 and its translocation to nucleus. In low-glucose condition, Pdx-1 localizes predominantly to the nuclear periphery, with some staining in the cytoplasm. After stimulation with glucose, Pdx-1 is present in the nucleoplasm. The translocation of Pdx-1 to the nucleoplasm occurred in high glucose condition.

2 Neurogenin (ngn)3

Ngn3, a member of a family of bHLH family of transcription factors, is involved in the determination of neural precursor cells in the neuroectoderm, and it is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. Expression of Ngn3 detected by in situ hybridization studies starts at E9.0-9.5 in the pancreatic anlage, increases to a peak at E15.5 and decreases

(21)

thereafter with a few ngn3 positive cells at E18.5 and is not found in the adult pancreas. Double immunofluorescence studies fail to observe the co-expression of ngn3 and insulin, glucagons, somatostatin or pancreatic polypeptide. In gain-of-function study of ngn3, overexpression of ngn3 in pancreatic progenitors under the Pdx-1 promoter resulted in their premature differentiation into endocrine cells at the

expense of pancreatic exocrine development.18 Consistently, it is reported that

deletion of ngn3 results in the loss of all endocrine cell lineages as shown by a lack of pancreatic hormones and lack of expression of the early markers, Pax-4, Pax-6,

NeuroD, and Isl-1.19 Thus, ngn3 is required for the specification of a common

precursor for the four pancreatic endocrine cell types.

3. BETA2/NeuroD

BETA2 (Beta cell E-box transactivator-2) also called NeuroD, a bHLH transcription factor, was isolated both as a transcriptional activator of the insulin gene and as a differentiation factor of neurogenesis. Expression of BETA2/NeuroD

starts at E9.5 in the pancreatic bud and can be induced by ngn3.20 Tissue-specific

members of the bHLH transcription factor family heterodimerize with ubiquitous members of this family to control cell type determination and specification in various

(22)

tissues from invertebrates to mammalians. Mice lacking BETA2/NeuroD die within 5 days after birth due to severe diabetes mellitus resulting from the loss of

insulin-producing cells.21 The BETA2/NeuroD deficient mice also have defects in the central

and peripheral nervous system, resulting in epileptic seizure, ataxia, imbalance and

deafness.22 These behavior defects are due to the defects in granule cell

differentiation in dentate gyrus and cerebellum as well as defects in vestibule and cochlear ganglia.

C. AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a sensor for cellular metabolism in response to changes in the energy status of the cells. AMPK has been described to shut down energy-consuming pathways in response to a fall in the ATP/AMP ratio by phosphorylating key enzymes of intermediate metabolism. Depletion of ATP in cells is always accompanied by elevation of AMP, due to displacement of the adenylate

kinase reaction [2ADP ↔ ATP + AMP].23 Elevation of AMP (coupled with

depression of ATP) activates the system by no less than four mechanisms: 1) Binding of AMP causes allosteric activation of the downstream kinase, AMPK; 2) binding of AMP to dephosphorylated AMPK causes it to become a much better substrate for the

(23)

upstream kinase, AMPKK; 3) binding of AMP to phosphorylated AMPK causes it to become a much worse substrate for protein phosphatases, especially protein phosphatase-2C; 4) the upstream kinase, AMPKK, is also allosterically activated by

AMP.24

5-Aminoimidazole-4-carboxamide riboside (AICAR) is taken up into cells and is phosphoryla ted by adenosine kinase to the monophosphorylated form (AICAR or ZMP). Although ZMP is a natural intermediated in purine nucleotide synthesis, in many cells, the formation of ZMP from extracellular AICAR is much more rapid than its subsequent metabolism, so ZMP accumulates. ZMP is an AMP analogue, which mimics all of the effects of AMP on the AMPK.

AMPK is a multisubstrate heterodimeric serine/threonine protein kinase

consisting of a catalytic α-subunit and a regulatory β- and γ-subunits. AMPK

catalytic α-subunit contains the kinase domain, which transfers a phosphate from

ATP to the target proteins. Each subunit has two or three isoforms, designated (α1,

α2, β1, β2, γ1, γ2, γ3. Catalytic α subunit are encoded by different genes; α1

corresponds to the isoform purified from liver. The α1- and β1-subunits are widely

expressed, whereas α2 is expressed at high levels in liver, skeletal and cardiac

(24)

presence of insulin) induces expression of genes encoding glucose transporters and glycolytic and lipogenic enzymes but represses genes of the gluconeogenic pathway.

Likewise, glucose also activates several of the same genes in pancreatic β-cells. In

addition, glucose increases transcription of the preproinsulin (PPI) gene.25 One of the

most extensively studied targets of AMPK is acetyl-CoA carboxylase (ACC), a rate-limiting enzyme of fatty acid synthesis in liver, adipose tissue, and mammary gland and an important regulator of fatty acid oxidation in muscle. AMPK phosphorylates and inactivates ACC, principally through the phosphorylation of serine 79 in the

N-terminal domain of ACC.27

During exercise, AMPK activated, resulting in inhibition of ACC and thereby reduction of the enzyme product malonyl-CoA, which relives inhibition of

CPT allowing an increase in fatty acid oxidation.28 AMPK is known to be involved

in glucose uptake. Perfusion of the isolated rat hindlimb with 200 U/ml AICAR

increases glucose uptake by approximately twofold.29 Taken together, these previous

results suggest that AMPK exerts an important role in coupling metabolic state to expression of genes involved insulin synthesis and secretion. The roles of the AMPK

have been described in the glucose-sensitive pancreatic β-cell lines, HIT- T15 and

(25)

changes in the concentration of AMP and ATP. In HIT-T15 cells of late-passage, both AMPK activity and the AMP/ATP ratio become insensitive to the extracellular glucose concentration and the glucose-dependent insulin secretion response is lost.

The purpose of the study is to characterize the molecular mechanism by

which BETA2/NeuroD regulates the SUR1 gene expression and to investigate if β

-cell specific genes, such as BETA2/NeuroD, SUR1, and insulin, can be regulated by AMPK.

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II. MATERIALS AND METHODS

A. MATERIALS

COS cells as described by Attardi and Tjian. HIT cells were purchased from Korean Cell Line Bank. Rat pancreatic islets were provided by Dr. Yoon KH (Catholic University, Korea). RNAzol B regents were purchased from Tel-Test, INC (Frendwood, TX); LipofectAMINE PLUS reagents from GIBCOBRL (Gland island.

N.Y. 14072 U.S.A); Butyryl-coenzyme A, ONPG (o-NitroPhenyl β

-D-Galactopyranoside) and forskolin from Sigma (St. Louis, MO, U.S.A.); [3H]chloramphenicol from NEB (Boston, MA, U.S.A.); LA PCR kit from Takara (Shuzo, Japen); Dual- Luciferase assay system from Promega (Madison, WI, USA); pcDNA3 from Invitrogen (Sandiego, CA, USA); pGemT plasmid and pGL3-promoter plasmid from Promega (Madison, WI, U.S.A.); anti- BETA2 and anti-PDX-1 antibodies from Santa Cruz Biotechnology (Santa Cruz, U.S.A.); anti-c-myc antibody from Calbiochem (La Jolla, CA, U.S.A.); anti-phospho-AcCoA

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Carboxylase (S79) antibody from Upstate Biotechnology Inc (Lake Placid, NY, U.S.A.). ECL-Plus kit and Hyperfilm from Amersham (Buckinghamshire, U.K.); Immobilon-P membrane from Millipore (Bedford, MA, U.S.A.). A recombinant

adenovirus expressing AMPK (Ad-AMPKα/T172D) and anti-phospho-AcCoA

carboxylase (S79) antibody were provided by Ha JH (Kyung-Hee University, Korea).

B. METHODS

1. Plasmids

pRIPE3(3+) CAT and pINSCAT448- were described previously.30, 31 An

expression vector for pcHA-BETA2(1-233), the 773 bp NcoI fragment was isolated from pGEM-BETA2 and ligated downstream of the 5’ untranslated region of PSD95 and a cDNA sequence for the HA epitope in pcDNA3. To obtain the full length BETA2, the BstI and XbaI fragment of pcHA-BETA2(1-233) was replaced with the

BstI (+641)/XbaI fragment of pGEM-BETA2. These plasmids were designed to

express 26 amino acids encoded by the 5’-untranslated region of the hamster BETA2 (GeneBank accession number: U24679) to the N-terminal of the first methionin of NeuroD. The amino acids of BETA2 peptides were numbered according to the NeuroD amino acids sequence. pCR3.1/ngn3, the expression vector for ngn3 and

(28)

pGL3-BETA2-2.2Luc were given by Dr. Tsai MJ (Baylor College of Medicine, Houston, Texas, U.S.A). pCR3.1/ngn3 and pGL3-BETA2-2.2Luc were described

previously.20

2. Preparation of recombinant adenovirus expressing NeuroD

A recombinant adenovirus expressing NeuroD was prepared using the AdEasy system. The BglII-FLAG-BamHI was inserted to the BamHI of pGEM-BETA2. AdTrack-CMV-FALG-BETA2 was made by ligating the FLAG-BETA2 (BglII/XhoI) to the BglII/XhoI site of a suttle vector pAdTrack-CMV. The resultant is linearized by digesting with restriction endonuclease PmeI, and subsequently cotransformed into E. coli BJ5183 cells with an adenoviral backbone plasmid, e.g., pAdEasy-1. Recombinants are selected for kanamycin resistance, and recombination was confirmed by multiple restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell line, 293 cells. Recombinant adenoviruses typically are generated within 7-10 days. MIN cells were infected with Ad-Track-CMV-FLAG-BETA2, using a 2 h exposure to 5 ml of

adenovirus (1 x 109 PFU/ml). After infection of adenovirus, MIN cells were cultured

for 32 h in 10 ml of DMEM medium. The efficiency of infection could be conveniently followed with GFP.

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- 28 - 3. Constructions of reporter genes

A BamHI/BamHI fragment (-660/+340) from MG10-1 was

subcloned into Amp right arm PacI pAdEasy left arm right arm PmeI fNeuroD -fNeuroD Cotransfection into bacteria Selection with kanamycin Linearize with PmeI pAd -NeuroD Amp GFP fNeuroD PacI PacI PmeI PmeI Linearize with PacI L -LTR GFP fNeuroD R -LTR PacI PacI

Transfect 293 cells Follow

transfection

Harvest virus in 7day

Amp right arm PacI pAdEasy Amp right arm PacI pAdEasy left arm right arm PmeI fNeuroD

-fNeuroD left arm

right arm PmeI fNeuroD -fNeuroD Cotransfection into bacteria Selection with kanamycin Linearize with PmeI pAd -NeuroD Amp GFP fNeuroD PacI PacI PmeI PmeI pAd -NeuroD Amp pAd -NeuroD Amp Amp GFP fNeuroD PacI PacI PmeI PmeI Linearize with PacI L -LTR GFP fNeuroD R -LTR PacI PacI L -LTR GFP fNeuroD R -LTR PacI PacI

Transfect 293 cells Follow

transfection

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exonuclease Ⅲ. The resulting fragments of -138/-20 and -660/-20 base pairs were linked to a CAT reporter plasmid, pCAT3M, to obtain pSUR-138CAT and pSUR-660 CAT, respectively. A KpnI/KpnI fragment (-2432/-627) from MG10-1 was inserted to the KpnI site of pSUR-660CAT to generate pSUR-2432CAT. pSUR-4542CAT was made by replacing the -2432/-1131 fragment of pSUR-2432CAT with an XbaI/NsiI fragment (-4542/-1131) from MG10-1. Reporter genes containing multiple copy of the E-box from the SUR1 promoter were constructed by inserting a double stranded E3 and mutation form of 3Em oligonucleotides into the BglII site of the pGL3-promoter luciferase vector. pSUR-2432/-660 was made by ligating the KpnI/BamHI fragment (-2432/-660) to the pGL3-promoter vector. To introduce a linker scanning mutation at E3, PCR was carried out using MG10-1 as a template and the oligonucleotide 8411 (5’-GGATCCAAGTTCCTCTTCTGGCCTCTA TTGGTA-3’) and 8939R (5’-CCCCCGGGCTCTTGTGGGGCGAGGGTGGG-3’) or the oligonucleotides 8930 (5’-CCCGGGGAAGGGCGGGGGCCAGCGGCA-3’) and

(31)

9052R (5’-CTGCTCTGGCTCCGCGCGCCT-3’). Two PCR products were subcloned into pGEM-T easy vector and subsequently isolated from the vector by digestion with BamH1 and Xma1. The two BamHI/XmaI fragments were inserted to the BglII site of pGL2-basic vector to obtain pSUR-660E3m.

4. Cell culture

HIT, MIN, 293T and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 4500 ㎎/L glucose, supplemented with 4 mM L-glutamine, 10% fetal bovine serum (FBS), 100 unit/ml penicillin, 100 ㎍/ml streptomycin. To measure AMPK’s effects in low glucose, MIN cells were

maintained in MEMα, containing 10 % FBS, 100 unit/ml penicillin, 100 ㎍/ml

streptomycin in a standard humidified atmosphere at 37 oC.

5. Isolation of islets and culture of islet cells

Pancreatic islets were isolated from SD rats (200-250 g) by distending the pancreatic duct with a collagenase P (1.5 mg/ml) in PBS, After digestion, the islet were separated on a Histopaque density gradient (Histopaque-1077) and further purified by handpicking under a microscope. Islet were cultured in RPMI/1640 containing 11.1 mmol/l glucose, 10 % FBS, 100 unit/ml penicillin, 100 ㎍/ml

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streptomycin in uncoated petridish.

6. Transfection

The cells were plated 24 h before transfection. HIT and MIN cells were transfected using LipofectAMINE PLUS. Reporter plasmids (0.5 ㎍), 0.5 ㎍ each expression vector for BETA2/NeuroD, pCMV-BETA2, or ngn3, pCR3.1-ngn3, and 0.07 ㎍ of an expression vector for E12, pSVE-5, or pCR3.1-E47 were used. The total amount of DNA used transfection was kept constant by adding pcDNA3. Transient transfection assay in 293T cells for electrophoretic mobility shift assay were carried out with the reporter plasmid, 12 ㎍ pcDNA3/flag-NeuroD by the

standard calcium phosphate precipitation method as previously described.32 To

evaluate the effect of AICAR, MIN cells and islets were incubated at 400 µM

AICAR. To obtain stably transformed cells with ngn3, pCR3.1-ngn3 was transfected

into HIT-T15 and HeLa cells and selected in the presence of G418 (600㎍/ml) for 2

weeks.

7. Chloramphenicol acetyltransferase (CAT) and luciferase Assay

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repeated cycling of freezing and thawing and heat inactivated at 65 oC for 10 min.

Protein concentration was determined by Bradford assay and 10-20 ㎍ of cell

extracts was assayed for CAT activity using [3H]-chloramphenicol and

butyryl-coenzyme A. Activity was normalized to β-galactosidase activity.

For luciferase assays, cell extracts were prepared according to the manufacturer’s protocol and luciferase activity was determined with 5-20 ㎍ of cell extracts using the Dual- Luciferase assay system. The data are presented as an average + standard error from at least three independent experiments.

8. Electrophoretic mobility shift assays (EMSA).

293T cells were transfected with expression vectors for BETA2/NeuroD and

E47 using calcium phosphate. After 36 h, nuclear extracts of 293T and MIN cells

were prepared from transfected cells as described by Attardi and Tjian.33 Briefly,

cells were lysed in 25 mM Tris-Cl (pH 8.0), 2 mM MgCl2, 0.5 mM dithiothreitol

(DTT), and 0.01 % phenylmethylsulfonyl fluoride (PMSF) for 5 min at room temperature. Nonidet P-40 was then added to a final concentration of 0.05 % and incubated for 2 min. After centrifugation at 1700 ×g for 5 min, the resulting pellet was suspended in 10 mM Tris-Cl (pH 8.0), 400 mM LiCl, 0.5 mM DTT, and 0.01%

(34)

PMSF and kept at room temperature for 5 min. After centrifugation at 12,000 ×g for 2 min, the supernatant was collected and used for EMSA. EMSA was performed using double stranded oligonucleotides as probes containing the E-box of rat insulin (RIPE3) or the E3 of SUR1 promoter. RIPE3, 5'-GATCTGGAACTGCAGCTT CAGCCCCTCTGGCCATCTGCTGATCCA-3' (sense) and 5’-GATCCGGA TCAGCAGATGGCCAGAGGGGCTGAAGCTGCAGTTTCCA-3’ (antisense); RIPA, GATCCCTCTTAAGACTCTAATTACCCTAG-3’ (sense) and 5’-GATCCTAGGGTAATTAGAGTCTTAAGAGG-3’ (antisense) were annealed and

end- labeled with [α-32P] dATP (NEN) and Klenow fragment. The sequences of

putative E-boxes of SUR1 were illustrated in the Fig. 5. The 32P-labeled probe (3 x

104 cpm/lane) and 1 µg of nuclear extracts were incubated in 7 % glycerol, 60 mM

LiCl, 0.5 mM PMSF, 5 mM MgCl2, 2 mM DTT, pH 7.4, with 2 ㎍ of

polyd eoxyinosinicdeoxycytidylic acid (poly dI/dC) for 30 min at room temperature.

To confirm specific binding, the unlabeled probe of 30-100 fold excess or 0.6㎍ of

an anti-NeuroD antibody were added to the reaction mixture. Samples were loaded onto 4% polyacrylamide gels and subject to electrophoresis at 8 V/cm. Gels were

(35)

9. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated using a RNAzolT M B and cDNA was synthesized

using First-strand cDNA synthesis kit and 1 ㎍ of total RNA following the

manufacture’s instructions. The PCR was conducted with 3 µl of the first-strand

cDNA; 98 oC for 1 min, followed by 25 cycles for BETA2 and β-actin, and 30

cycles for SUR1 at 94 oC for 1 min, 55 oC for 30 sec, and 72 oC for 1 min, and finally

72 oC for 7 min. Primers were designed to recognize the separate exons to eliminate

possible DNA contamination. The PCR primers for SUR1 were 5’-GCTCTTCATCACCTTCCCCATCCTC-3’ (forward) and 5’-CACAACCTGCG CTGGATCCTTACC-3’ (reverse); for BETA2 5’-CTCCGGGGTTATGAGAT CGTCAC-3’ (forward) and 5’-GATCTCTGACAGAGCCCA-3’ (reverse); for Pdx-1 5’-GGACACACAGCTCTACAAGGA-3’ (forward) and 5’-CATCACTGCCAG

CTCCACCC-3’ (reverse); for β-actin 5’-CATGTTTGAGACCTTCAACACCCC-3’

(forward) and 5’-GCCATCTCCTGCTCGAAGTCTAG-3’ (reverse). The PCR products were analyzed on a 2 % agarose gel.

10. Nuclear extracts and immunoblotting

(36)

previously described by Attardi and Tjian.33 Briefly, cells were harvested 36 h after

transfection and lysed in 25 mM Tris-Cl (pH 8.0), 2 mM MgCl2, 0.5 mM

dithiothreitol (DTT), and 0.01% phenylmethylsulfonyl fluoride (PMSF) for 5 min at room temperatur e. Nonide P-40 was then added to a final concentration at 1700 ×g for 15 min, the resulting pellet was suspended in 10 mM Tris-Cl (pH 8.0), 400 mM LiCl, 0.5 mM DTT, and 0.01 % PMSF and kept on room temperature for 5 min. After centrifugation at 12000 ×g for 2 min, the supernatant was harvested as nuclear extracts. Usually 30-60 ㎍ of cytosol and nuclear extracts were subjected to 12 % sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The proteins on the gel transferred electrophoretically onto the Immobilon-P Teflon menbrane and western analysis was performed with an specific antibodies. Finally, the membrane was incubated with horseradish peroxidase-conjugated anti- mouse IgG antibody and the proteins were visualized using an enhanced chemiluminescence (ECL) kit following the manufacture’s recommendation.

(37)

III. RESULTS

1. Transactivation of the SUR1 gene by BETA2/NeuroD

Previously, we defined that SUR1 is a target gene of BETA2/NeuroD. To investigate whether the endogenous BETA2/NeuroD confers high activity of pSUR-660CAT in HIT-T15 cells, we determined the effect of a dominant negative form of BETA/NeuroD, BETA2(1-233) (Fig. 4A). BETA2(1-233) functions as a dominant negative mutant because it contains the bHLH domain for heterodimerization with E47 but lacks transactivation domain. Coexpression of BETA2(1-233) reduced the promoter activity of the -660/-20 fragment in a dose

dependent manner (Fig. 4B). Thus, transfection of 0.3 µg pCHA-BETA2(1-233)

decreased the promoter activity of the -660/-20 fragment and the insulin promoter to about 20% and 35% of the reporter alone, respectively. Similarly an insulin reporter gene, pINSCAT448-, which contained the RIPE3 sequence was suppressed by BETA2(1-233). Under the same condition, BETA2(1-233) did not affect the

(38)

pSV2CAT. This result indicates that repression by BETA2(1-233) is specific to β-cell specific genes and high promoter activity of –660/-20 of SUR promoter is due to BETA2/NeuroD-like factors in HIT-T15 cells.

BETA2 (1-233)µg - 0.3 - 0.3 -660 SV2 -138 INS448-- 0.1 0.3 1 - 0.1 0.3 1

Relative CAT Activity

0 20 40 60 80 100 120 ** ** ** BETA2 BETA2(1-233) HA bHLH HA bHLH AD

A

B

(39)

Fig. 4. Repression of the SUR1 promoter by a dominant negative mutant of

BETA2/NeuroD.

A schematic diagram of full- length BETA2 and a truncated form BETA2(1-233) peptides were epitope tagged with hemagglutinin (HA) at the N-terminus. bHLH, basic helix- loop-helix domain; AD, activation domain. B. Reporter genes were cotransfected with pcHA-233), an expression vector for BETA2(1-233). Although pSV2CAT lacking the E-box was not affected by BETA2(1-233), pINSCAT448- containing the E-box of the rat insulin II promoter was repressed by BETA2(1-233). Data are relative values with respect to the CAT activity of

pSV2CAT and presented as averages ± standard errors of three independent

experiments. *p<0.05, **p<0.01, both p values were estimated from t-test compared to the value of the reporter gene alone.

(40)

2. Determination of binding sites for BETA2/NeuroD

Four E-box-like sequences (E1- E4), which are potential sites for BETA2/NeuroD action, were found within the -660/-20 fragment. Since the -138/-20 fragment containing E4 was not synergistically activated by BETA2/NeuroD and E47, E4 alone is insufficient for tissue specific regulation. To determine which of the remaining E-box like sequences was necessary fo r BETA2/NeuroD binding, we carried out electrophoretic mobility shift assays (EMSA) using RIPE3 as a probe and the E-box sequences (E1-E3) of the SUR1 promoter as competitors (Fig. 5). As a source of BETA2/NeuroD, nuclear extracts were prepared from 293T cells transfected with expression vectors for BETA2(1-233) and E47. BETA2(1-233) has been shown to bind RIPE3 stronger than the full length BETA2 when expressed in 293T cells. Several complexes were detected and specific binding of the labeled RIPE3 probe was verified using an excess amount of unlabeled RIPE3 oligonucleotide (data not shown). Interestingly, only E3 was able to compete with RIPE3 for binding to BETA2/NeuroD (lane 6, 7). The binding was specific since the

(41)

same complexes disappeared by addition of an anti-NeuroD antibody (lane 8). This result indicates that the E3 box is necessary for binding of BETA2/NeuroD. To confirm the specificity of binding to E3, we carried out EMSA using E3 as a probe

Fig. 5. Determination of DNA binding ability of three putative E-boxes.

Nuclear extracts were prepared from 293T cells expressing a dominant negative form of BETA2/NeuroD, BETA2(1-233). EMSA was carried out with RIPE3, the E box of

consensus CANNTG

E1 GATCCACATCCAGCTGAGCCTAGA

E2 GATCCCAGCGCACGTGCGCATTGA

E3 GATCCAAGAGCAGCTGGAAGGGCA

(42)

the rat insulin II. Double stranded oligonucleotides containing E1, E2 or E3 of the SUR1 were used as competitors. Only E3 was able to compete with RIPE3 (solid triangle in lanes 6 and 7). The same complex disappeared in the presence of an anti-BETA2 antibody (lane 8).

E3 GATCCAAGAGCAGCTGGAAGGGCA E3m GATCCAAGAGTGGCTGGAAGGGCA

Fig. 6. Binding of BETA2/NeuroD to E3.

Nuclear extracts were prepared from 293T cells expressing a dominant negative form of BETA2/NeuroD, BETA2(1-233). A double stranded oligonucleotide containing E3 of the SUR1 promoter was used as a probe. Specific binding was confirmed by competition

(43)

with an excess amount of the unlabeled E3 (lanes 2 and 3) or E3m containing mutant in E3 (lanes 4 and 5) and RIPE3 (lanes 6 and 7). The specific complexes (solid triangle) disappeared by addition of an anti-BETA2 antibody (lane8).

(Fig. 6). Similar to RIPE3, BETA2/NeuroD bound to E3 specifically. E3 oligonucleotide (lane 2, 3) and RIPE3 (lane 6, 7) were able to compete with E3 of the SUR1 gene (closed triangle). In contrast, the E3m, a mutant oligonucleotide, did not compete with E3 (lane 4, 5). The specific complexes disappeared in the presence of an anti-NeuroD antibody (lane 8). This result indicates that BETA2/NeuroD can bind the E3 element at -141 bp of the SUR1 gene.

3. E3-mediated transactivation by BETA2/NeuroD

To determine whether the E3 box could confer transactivation of the SUR1 gene by BETA2/NeuroD, reporter genes, pSURE3(1+) and pSURE3m(1+), were constructed by ligating one copy of E3 or E3m to a heterologous promoter driving expression of luciferase in the pGL3-promoter vector, respectively (Fig. 7). Coexpression of BETA2/NeuroD and E47 enhanced the luciferase expression by 3.8 fold for pSURE3(1+) while the pGL3-promoter was not affected at all. Mutation of E3 eliminated the stimulatory effect of BETA2/NeuroD (Fig. 8, left panel). To

(44)

confirm that E3 is the only functional E-box element in the -660/-20 fragment, we constructed pSUR-660E3m which contained a linker scanning mutation in E3 of the -660/-20 fragment. Mutation of E3 in the whole promoter context eliminated the

Fig. 7. Construction for E3 box and mutated E3 box of SUR1.

Reporter genes, pSURE3(1+), pSURE3m(1+), pSUR(-2432/-660), were constructed by ligating one copy of E3, E3m, or –2432/-660 to pGL3-promoter. pSUR-660 and pSUR-660E3m were constructed using pGL2-basic (see the Materials

-660 -141 -20 -660 -141 -20 pSURE3(1+)(

1+)

pGL3 pSURE3m(1+) (4

+)

pSUR(-2432/-660) pSUR-660

)

pSUR-660m

)

-2432 -660 TATA Luc TATA Luc TATA Luc TATA Luc Luc Luc E3 E3 E3 E3

(45)

and Methods for details).

Fig. 8. Mutation of E3 abolishes transactivation by BETA2/NeuroD.

Each of reporter genes (0.3 µg) was cotransfected with expression vectors

for BETA2 and E47 into HIT-T15 cells. Note that mutation in E3 caused a loss of

transactivation by BETA2/NeuroD and E47. Data are shown as averages ± standard

errors of three independent experiments with respect to the luciferase activity of pGL3 promoter or pGL2 basic. p values were estimated from t-test compared to the

BETA2/E47 - + - + - + - + - E3 E3m + + - --660 -2432/-660 -660E3m luciferase activity 0 2 4 6 8 10 ** **

(46)

value of reporter genes alone (*p<0.05, **p<0.01).

stimulatory effect of BETA2/NeuroD (Fig. 8, right panel). Sequence analysis revealed nine more E-box- like sequences upstream of –660. To determine whether they might confer higher activity of pSUR-2432CAT than pSUR-660CAT in HIT-15 cells as shown in master’s thesis, we generated a reporter construct, pSUR-2432/-660 by ligating the -2432/-660 fragment to the pGL3-promoter. Coexpression of BETA2/NeuroD with or without E47 did not significantly enhance the -2432/-660 fragment (Fig. 8, left panel). Thus, E3 is essential for transactivation by BETA2/NeuroD.

4. Specificity of the E box-mediated transactivation by BETA2/NeuroD

In addition to BETA2/NeuroD, ngn3 is also a bHLH transcription factor expressed in pancreatic islet cells during their development. Thus, it is possible that ngn3 can also activate the SUR1 gene through the E3 element. We tested this possibility by coexpressing ngn3 and E47 in HIT-T15 cells. In HIT-T15 cells, the stimulatory effect of ngn3 was similar to that of BETA2/NeuroD (compare HIT-T15

(47)

cells in Fig. 9A and 9B). A reporter gene containing one copy of E3 increased 5.7 fold by coexpression of ngn3 and E47 (Fig. 10B). Similar result was obtained with a reporter gene containing the –660/-20 fragment. The stimulatory effect of ngn3 was

Fig. 9. Specificity of E3 for BETA2/NeuroD.

A, Reporter genes (0.3 µg) were cotransfected with expression vectors for

BETA2/NeuroD (0.1 µg) and E47 (0.05 µg) into HIT-T15 (hatched bar) or HeLa

cells (filled bar). B, Reporter genes (0.3 µg) were cotransfected with expression

vectors for ngn3 (0.1 µg) and E47 (0.05 µg) into HIT-T15 or HeLa cells. Data are

shown as averages ± standard errors of three independent experiments with respect to

- + + BETA2/E47 E 3 E3m -660 ** * ** ** - + + - + + B A HIT-T15 HeLa HIT-T15 HeLa - + + ngn3/E47 E 3 E3m -660 ** ** * * - + + - + + Luciferase Activity 0 1 2 3 4 5 6 7 8 Luciferase Activity 0 1 2 3 4 5 6

(48)

the luciferase activity of pSURE3(1+) or pSUR-660 in the absence of BETA2/NeuroD and E47. p values were estimated from t-test compared to the values of pSURE3 or pSUR-660Luc (*p<0.05, **p<0.01).

abrogated when E3 was mutated as shown with E3m (Fig. 9B) or –660E3m (data not shown). In contrast to HIT-T15 cells, transactivation by ngn3 was minimal compared to BETA2/NeuroD in HeLa cells (Fig. 9B). Coexpression of ngn3 and E47 could only activate the promoter activity of pSURE3 by 1.4 fold. In contrast, BETA2/NeuroD enhanced it by 3.1 fold under the same condition. Interestingly, both BETA2/NeuroD and ngn3 could not activate E3 in the homologous context of –660/-20 in HeLa cells. These results indicate that the E3 element is somehow specific for BETA2/NeuroD in HeLa cells. It has been shown that ngn3 is not detectable in

mature β-cells and forced expression of ngn3 causes an increase in the level of

BETA2/NeuroD in HIT-T15 cells.

Thus, it is possible that activation of E3 by ngn3 in HIT-T15 cells may be due to enhanced expression of BETA2/NeuroD, which is absent in HeLa cells. To test this possibility we investigated mRNA levels of SUR1 and BETA2/NeuroD before and after overexpression of ngn3. Stable cells were obtained by transfecting HIT-T15 and HeLa cells with the ngn3 expression vector. Total RNAs were prepared

(49)

from pools of G418-resistant cells and subjected to RT-PCR. Overexpression of ngn3 enhanced expression of both SUR1 and BETA2/NeuroD by 2.3 and 2.4 fold, respectively, in HIT-T15 cells (Fig. 10A and 10B). In contrast, ngn3 could not induce

Fig. 10. Specificity of E3 for BETA2/NeuroD.

RT-PCR products of SUR1 and BETA2/NeuroD with HIT and HeLa cells stably transformed with ngn3. A. BETA2/NeuroD and SUR1 products were detected as 620 bp and 117 bp fragments, respectively. Data present the most representative one from three independent experiments. B, The mRNA levels of SUR1 were

A

SUR1 BETA2 β-Actin HIT-T15 HeLa ngn3 + - + -

mRNA expression (SUR1)

0.0 0.5 1.0 1.5 2.0 2.5 ngn3 + - + -HIT-T15 HeLa ** * 0.0 0.5 1.0 1.5 2.0 2.5 3.0

mRNA expression (BETA2)

ngn3 + - +

-HIT-T15 H e L a

(50)

normalized to the β-actin mRNA and the RT-PCR data from three independent

experiments were summarized as averages ± standard errors with respect to the value

of untreated cells (*p<0.05, **p<0.01).

expression of SUR1 and BETA2/NeuroD in HeLa cells. This result suggests that ngn3 might activate the SUR1 promoter indirectly by inducing the expression of BETA2/NeuroD in HIT-T15 cells but not in HeLa cells.

5. Repression of BETA2/NeuroD promoter activity by AICAR in MIN cells.

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

(51)

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

(52)

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

(53)

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

(54)

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.

AICAR (h)

Low Glucose High Glucose

BETA2 mRNA expression

0 1 2 3 4 5 6 0 6 12 0 6 12 *

SUR1 mRNA expression

0 2 4 6

AICAR (h) 0 6 12 0 6 12

Low Glucose High Glucose

** ** * pdx-1 mRNA expression 1 2 3 4 5 AICAR (h) 0 6 12 0 6 12

Low Glucose High Glucose

(55)

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

(56)

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.

BETA2 mRNA expression

0 1 2 3 4 AICAR

-

+

-

+ Glucose (mM) 5 30

SUR1 mRNA expression

0.0 0.5 1.0 1.5 2.0 2.5 AICAR

-

+

-

+ Glucose (mM) 5 30 Pdx-1 mRNA expression 0 1 2 3 4 5 AICAR

-

+

-

+ Glucose (mM) 5 30

(57)

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

(58)

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.

(59)

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.

Relative Luciferase Activity

0 1 0 0 2 0 0 3 0 0 BETA2 ngn3 AICAR +

-

+

--

+

-

+

-

-

+

-+

-BETA2/NeuroD-2200Luc SURE3Luc * ** ** ** * * ** AICAR

(60)

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

(61)

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.

7. Expression of BETA2/NeuroD by AMPK activation.

We examined the changes of protein level of endogenous BETA2/NeuroD in

MIN cells treated with 400 µM AICAR. Western analysis showed that the protein

level of BETA2/NeuroD in MIN cells was maintained for at least 3 h, but decreased

(62)

(Fig 20). As an internal control, expression of GSK3β (Glycogen Synthase

Kinase-3β) was not affected by AICAR treatment (Fig. 20). Therefore, AMPK activation

decreases BETA2/NeuroD protein level in time-dependent manner.

To determine that repression of BETA2/NeuroD expression by AICAR is

effect of AMPK, we used Ad-AMPKα1312(Τ172) (Ad-AMPKα was provided by Dr.

Fig. 18. Repression of binding activity of BETA2/NeuroD by AICAR.

BETA2/NeuroD RIPE anti-NeuroD AICAR Competitor - - - - - - - - + - - - - - - +

(63)

Nuclear extracts were prepared from MIN cells after treatment with or

without 400 µM AICAR. A double stranded oligonucleotide containing E-box of the

rat insulin promoter was used as a probe. Specific binding to BETA2/NeuroD was confirmed by competition with an excess amount of the unlabeled E-box (lane 2 and 3). The specific complexes (solid triangle) competed by AICAR and shifted by addition of an anti-NeuroD antibody (lane 6).

Fig. 19. Effect of binding activity of Pdx-1 by AICAR.

Pdx-1 RIPA anti-Pdx-1 AICAR Competitor - - - - - - - - + - - - - - - +

(64)

Nuclear extracts were prepared from MIN cells after treatment with or

without 400 µM AICAR. A double stranded oligonucleotide containing A-box of the

rat insulin promoter was used as a probe. Specific binding to Pdx-1 was confirmed by competition with an excess amount of the unlabeled A-box (lane 2 and 3). The specific complexes (solid triangle) competed by AICAR and shifted by addition of an anti-NeuroD antibody (lane 6).

Ha J). Truncation of AMPKα1 at residue 312 yielded a polypeptide that no longer

associated with the β and γ subunits but retained significant kinase activity.35 cDNA

encoding residues 1 to 312 of AMPKα1, containing a mutation that alters threonin

172 to an aspartic acid (T172D), was used to construct the recombinant adenovirus

Ad.α1312 as described previously.36 Hardie DG group showed that mutation of

threonine 172 within the α subunit, the major site phosphorylated by AMPK

kinase,37 to an aspartic acis residue within this truncated protein prevented its

inactivation by protein phosphatase.38 These findings indicated the potential of this

mutant to act as a constitutively active kinase. MIN cells were infected with Ad-AMPKα1312(Τ172)by exposing the adenovirus (1x 108 / cell) for 2 h. At 24 h after infection, the medium was replaced with fresh medium. We showed that the activation of AMPK was measured by one of the downstream targets of AMPK,

(65)

namely acetyl-CoA carboxylase (ACC). Active AMPK is known to inactive ACC by phosphorylating ACC at serine 79. Therefore, western blot analysis was performed using by an antibody specific for phosphorylated S79 of ACC to indirectly examine

the AMPK activity. Following infection with Ad.α1312, MIN cells lysates were

analyzed by western blotting for expression of Ad.α1312. The recombinant α1312

protein contains a Myc epitope tag at the N-terminus, allowing detection with an anti

Fig. 20. Repression of BETA2/NeuroD level by AICAR in MIN cells.

1 2 3 4 5 + AICAR (h) 0 1 3 6 18

BETA2/NeuroD

(66)

MIN cells were incubated in the presence of 400 µM AICAR at 30 mM

glucose. Western blot was carried out with (40 µg for BETA2/NeuroD) and cytosol

(80 µg for GSK3β) fractions after treatment with AICAR.

c-Myc antibody (Fig. 21, Upper). AMPK activation increased Ser79 phosphorylation in acetyl-CoA carboxylase (ACC) (Fig. 21, Middle) and AMPK activation by

expression of Ad.α1312 repressed BETA2/NeuroD (Fig. 21. Lower). This result

suggests that AMPK activation led to the reduced BETA2/NeuroD protein level.

Pancreatic duodenal homeobox1 (Pdx-1) a homeodomain protein is present

in β-cells of islets of Langerhans. It is also expressed in neuroendocrine cells of the

gut. Pdx-1 plays an important role in the development of the pancreas. Pdx-1 plays a role in activating the insulin promoter and increasing insulin mRNA levels in response to glucose. However, Pdx-1 mRNA level was not affected by AICAR in islets and MIN cells (Fig. 12 and 14). It has been shown previously that glucose stimulates the translocation of Pdx-1 form the cytoplasm to the nucleus. We

(67)

investigated whether AMPK activation or AICAR treatment regulates the translocation from the cytoplasm to the nucleus of Pdx-1 in MIN cells. This result suggests that the movement of Pdx-1 from cytosol to nucleus were not been blocked by AMPK and AICAR. Therefore, AMPK did not affected expression or translocation of pdx-1 (Fig. 22). - + BETA2/NeuroD ACC-Pi AMPK AMPKα.α13 1 2 ./ T172D

(68)

Fig. 21. Repression of BETA2/NeuroD level by Ad-AMPKα.α13 1 2/T172D in MIN

cells.

48h after expose to Ad-AMPKα.α1312/T172D, MIN cells were treated with

400 µM AICAR for 16 h. Expression of c- myc tagged AMPKα.α1312/T172D was

verified with anti-c-myc antibody (upper). Activity of AMPK was determined by phosphorylation of Ser79 acetyl-CoA carboxylase (ACC) (middle).

AMPKα.α1312/T172D repressed BETA2/NeuroD (lower).

8. Effect of Adenovirus -mediated NeuroD overexpression by AICAR in MIN

cells.

To determine whether the protein level of exogenous NeuroD is decreased by AICAR, western blotting for NeuroD was performed after infection with Ad-flag-NeuroD (Fig. 23). Ad-flag-Ad-flag-NeuroD was used to construct the recombinant

adenovirus as described previously.36 The recombinant NeuroD protein contains a

flag epitope tag at the C-terminus, allowing detection with an anti flag antibody. MIN

cells was infected with Ad-flag-NeuroD for 32 h, and treated with 400 µM AICAR

in a time-dependent manner. As shown in Fig. 24, GFP expression was visible 32 h after infection in 50-60 % of the MIN cells. Western analysis showed that the protein

(69)

level of BETA2/NeuroD in MIN cells was maintained for at least 6 h, but had

decreased to 80 % at 12 h, and disappeared at 18 h after treatment with 400 µM

AICAR (Fig 26). As an internal control, expression of GSK3β (Glycogen Synthase

Kinase-3β) was not affected by AICAR treatment (Fig. 25). Therefore, AICAR

decreased BETA2/NeuroD protein level in time-dependent manner. This result

Fig. 22. Effect of AICAR on the intracellular localization of Pdx-1.

Pdx-1 (Cytosol)

Pdx-1 (Nucleus)

GSK3β 1 2 3 4

(70)

48 h after exposure to Ad-LacZ or Ad-AMPKα/T172D and 16 h after

treatment with 400 µM AICAR, nuclear and cytosol extracts were obtained and

western blotting was done with an antibody for Pdx-1. GSK3b was used as an

internal control. Lane1; control, lane2; Ad- LacZ, lane3; Ad-AMPKα/T172D, lane4;

400 µM AICAR. Kan LTR PA GFP Pcmv BETA2 FLAG Pcmv Right arm Left arm Ori PA PacI PacI pAdTr-fNeuroD 11.4 kb

(71)

Fig. 23. Map of pAdTrack-CMV-FLAG-NeuroD

(72)

Fig. 24. Overexpression of NeuroD using MIN cells.

MIN cells were infected with recombinant adenovirus, Ad- flag-NeuroD (1 x

108 PFU/ml). The panel shows representative MIN cells 32 h after infected with

Ad-flag-NeuroD. Many cells are infected after exposure to Ad-flag-NeuroD which also contains GFP. Cytosol Nucleus NeuroD GSK3β NeuroD GSK3β AICAR (h) 0 1 3 6 18

(73)

Fig. 25. Repression of BETA2/NeuroD level by AICAR in MIN cells.

32 h after exposure to Ad-flag-NeuroD, MIN cells were treated with 400 µM

AICAR, nuclear and cytosol extracts were obtained. Expression of flag-NeuroD was

verified with anti- flag antibody. An internal control, expression of GSK3β was not

affected by AICAR treatment. AICAR reduced the level of BETA2/NeuroD protein.

suggests that AMPK suppressed the expression of BETA2/NeuroD protein in post-transcription level.

(74)
(75)
(76)
(77)
(78)
(79)
(80)
(81)

수치

Fig. 13. Activation of BETA2/NeuroD and SUR1 by high glucose and repression by  400 mM AICAR, a AMPK activator (Graph)------------------------------------  53
Fig. 24. Map of pAdTrack-CMV-FLAG-NeuroD--------------------------------------- 70  Fig
Fig. 2. Transcription factors involved in pancreatic islet development.
Fig. 4. Repression of the SUR1 promoter by a dominant negative mutant of
+7

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