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

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

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

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

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

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

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 muscles, and β2 in skeletal and cardiac muscle.²⁶ In hepatocytes, glucose (in the

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 INS-1. AMPK activation in response to glucose depletion appears dependent on

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.

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

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

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.

- 28 -

3. Constructions of reporter genes

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

fNeuroD -fNeuroD Cotransfectioninto bacteria Selection with kanamycin

Linearizewith PmeI pAd-NeuroD

L-LTRGFPfNeuroDR-LTR PacIPacI Transfect 293 cells Follow transfection Harvest virus in 7day

Amp

fNeuroD -fNeuroD Cotransfectioninto bacteria Selection with kanamycin

Linearizewith PmeI pAd-NeuroD

L-LTRGFPfNeuroDR-LTR PacIPacI Transfect 293 cells Follow transfection Harvest virus in 7day

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

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

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

For CAT assays, cell extracts were prepared 48 h after transfection by

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%

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 dried and exposed to X-ray film for 1-2 day at –70 ^oC.

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

Total RNA was isolated using a RNAzol^{T 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

Nuclear extracts were prepared from transfected COS and 293T cells as

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.

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

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

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

bHLH, basic helix- loop-helix domain; AD, activation domain. B. Reporter genes