# The Global Transcriptional Response of Isolated Human Islets of Langerhans to Glucagon-Like Peptide-1 Receptor Agonist Liraglutide

Authors: Xiaoning Zhao; Yongming G. Tang; S. Vincent Wu; Charles Wang; Ricardo Perfetti; Nasif Khoury; Dehong Cai; Fang He; Xiaogang Su; Vay Liang W. Go; Hongxiang Hui
Journal: ISRN Endocrinology
 (2012)

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## Abstract

GLP-1 and its analog have been used in diabetes treatment; however, the direct alteration of gene expression profile in human islets induced by GLP-1 has not been reported. In present study, transcriptional gene expression in the liraglutide-treated human islets was analyzed with 12 human U133A chips including 23000 probe sets. The data compared between liraglutide and control groups showed a significant difference on glucose-induced insulin secretion, rather than viability. Microarray analysis identified 7000 genes expressed in human islets. Eighty genes were found to be modulated by liraglutide treatment. Furthermore, the products of these genes are proteins involved in binding capability, enzyme activity, transporter function, signal transduction, cell proliferation, apoptosis, and cell differentiation. Our data provides a set of information in the complex events, following the activation of the GLP-1 receptor in the islets of Langerhans.


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## Body

## 1. Introduction

GLP-1 is synthesized by the intestinal L-cells and it has been known to stimulate insulin release in the postprandial state, to inhibit glucagon release, and to slow down the rate of gastric emptying,  along  with  acid  secretion [1].  In  addition  to  these  well-known  functions,  novel properties of GLP-1 on maintaining beta-cell  mass have been identified recently. In glucose-intolerant  Wistar  rats,  GLP-1  reverses  an  age-dependent  β-cell  abnormalities,  and  this  is associated with an expansion of β-cell mass via islet cell neogenesis [2]. Similarly, Exendin-4, a GLP-1  receptor  agonist,  stimulates  both  beta-cell  replication  and  neogenesis,  resulting  in increased  beta-cell mass  and  improved  glucose  tolerance  in  diabetic  rat created  by partial subpancreatectomy  [3].  A GLP-1-dependent differentiation of pancreatic precursor cells into mature β-cells has also been proposed [4, 5]. Finally an inhibitory effect on islet cell apoptosis has been observed in the pancreas of animal models of diabetes, as well as in β-cell lines and isolated human  islets  in  culture [6–8].  All these data suggested that treatment of human islets with exogenous GLP-1 could improve function and survival of pancreatic islets. Indeed, it has been demonstrated by several groups that GLP-1 enhances β-cell mass by both promoting islet cell neogenesis and inhibiting beta-cell apoptosis [8, 13]. However, the elucidation of the molecular and cellular mechanisms regulating this complex set of diverse biological actions is still very marginally understood.To evaluate GLP-1 efficacy on human islets and to identify GLP-1 induced gene profile as well as the related signal pathways, we cultured human islets isolated from cadaveric donors with liraglutide [9], a long-lasting GLP-1 analog and investigated the gene expression profile after the exposure of human islets to GLP-1 by microarray analysis.   We demonstrated that in response to activation of the GLP-1 receptor, a coordinated expression of factors regulating biological process  took  place.  This  paper  provides  a  preliminary  map  on  the  intracellular responses of islet cells to GLP-1.


## 2. Materials and Methods

### 2.1. Materials

Liberase-HI purified enzyme blend was purchased from Boehringer Mannheim (Indianapolis, IN); dithizone was purchased from Sigma (St. Louis, MO). Human U133A C hips were obtained from Affymetrix, INC (Santa Clara, CA). Medium and RNA extraction kit were purchased from Qiagen (QIAGEN Inc., Valencia, C A). Live/Dead Viability/Cytotoxicity Kit and streptavidin-phycoerythrin  were from Molecular  Probe (Eugene, OR).  GLP-1 was purchased from American Peptide Co. (Sunnyvale, CA). Ficoll Cobe 2991  was purchased  from COBE BCT,  Inc. (Lakewood, CO). Tissue culture plastic ware was obtained  from Corning Coaster Corporation (Cambridge, MA). Fetal calf serum and SuperScript Choice system were from Life Technologies,  Inc.  BRL  (Rockville,  MD).  M199  medium  was  obtained  from  Gibco-BRL (Gaithersburg,  MD).  Bradford  reagents  for  protein  detection  were  obtained  from  Bio-Rad (Richmond, CA). Oligo-(dT)24   anchored  T7 primer was obtained  from Amersham Pharmacia Biotech  (Piscataway,  NJ).  Phase  Lock  Gels   were  purchased  from  5-prime-3-prime,  Inc. (Boulder, CO). The BioArray high yield RNA transcript labeling kit was purchased from Enzo Diagnostics, Inc. (Farmingdale, NY).  Biotinylated antistreptavidin was purchased from Vector Laboratories, Inc. (Burlingame, CA). PCR reagents, the software for the design of primers and TaqMan probes (Express V1.5 software), and the ABI PRISM 7700 Sequence Detector were from Applied Biosystems Inc. (Foster City, CA).


### 2.2. Islet Isolation, Purification, and Quantification

Human pancreases were recovered from cadaveric donors. Pancreas were placed in cold UW (University of Wisconsin) solution on ice and immediately transported to the islet isolation laboratory for processing and selected with criteria of having at least 10 h of cold storage in UW. All organs were processed  using  identical  isolation techniques of collagenase digestion and Ficoll purification. Briefly, human pancreases were placed in a customized perfusion apparatus and injected  with  liberase-HI.  They  were  then  transferred  to  a continuous  digestion  device  for mechanical disrupt and  enzymatic digestion  until the  majority of the  islets  were  free  from exocrine tissue. Samples of pancreatic tissue were evaluated for purity during the digestion after staining with dithizone. Pancreatic digest containing endocrine and exocrine tissue was purified by continuous  gradients  centrifugation of Ficoll Cobe 2991.  Islet-enriched  layers  were then selected and collected. Islet recovery after purification was assessed in duplication by counts of dithizone-stained aliquots of the final suspension of tissue. The purity of the preparations was assessed by comparing the relative quantity of dithizone-stained endocrine tissue divided by the unstained tissue. The viability of isolated cells was 87± 6%, and their purity was  ≥57±4%.


### 2.3. Living/Dead Cell Determination

Cell  viability  was  determined  using  a  Living/Dead  Viability/Cytotoxicity  Kit.  Briefly, isolated islets were washed twice with P BS and 50μL aliquots were placed in the 96-well plate. Then 10 μL of solution component A (Calcein AM) and component B (Ethidium homodimer-1) in  Live/Dead  Viability/Cytotoxicity K it  were added to each well.  After 30 min,  islets were examined under a microscope.   Living cells were identified by a green staining (Calcein AM staining), while dead cells showed a brown nuclear staining (ethidium homodimer-1 staining).


### 2.4. Incubation of Isolated Islets with GLP-1

Isolated islets were cultured, in 75-mL flasks, in the presence of M199  medium with 100μg/mL penicillin, 50 μg/mL streptomycin, and 10% of fetal calf serum at 37°C under humidified conditions with 5% CO2. After 4 h, the islets were washed twice with M199 without FBS and antibiotics. They were then treated with 10 nM of GLP-1 in M199 medium containing 16 mM glucose (without F BS) for 22 h. Islet pellets were collected by centrifugation at 1000 rpm for 3 min. RNA was isolated by using the Qiagen RN easy Mini kit and immediately stored at −80°C.


### 2.5. Detection of Insulin Secretion

After 22 h of culturing in the presence of GLP-1, or vehicle,  insulin released  into the medium was measured by RIA (radioimmunoassay). Total cellular protein content was measured using the method of Bradford  Assay. The amount of proteins measured served as a correction factor for determining the relative amount of insulin released into the culture medium by each individual culture condition.


### 2.6. Microarray Analysis

Affymetrix   arrays   (Human   U133A)   were used for mRNA expression profiling. Experimental procedures for gene chips were performed according to the Affymetrix Gene Chip Expression  Analysis  Technical Manual. Briefly:  double-stranded  cDNAs  were  synthesized using the  SuperScript Choice system and an oligo-(dT)24  anchored T7  primer.  Two samples (duplicate) of 5 μg of total RNA from each sample were used to start the synthesis of cDNA. Double-stranded cDNA products were purified by phenol:chloroform:isoamyl alcohol (25 : 24 : 1 saturated with 10 mM Tris-HC l, pH8.0/1 mM EDTA), followed by extraction, phase separation with Phase Lock Gels, and ethanol precipitation.  Biotinylated RNA was synthesized using the BioArray high yield RNA transcript labeling kit. Biotinylated RNA products were purified using Qiagen RNeasy columns and  fragmented to a size of 30 to 200 nucleotides.   A total of 15 ug biotinylated fragmented RNA was then hybridized with Affymetrix GeneChip arrays (Human, U133A). After  washing,  the  arrays  were  stained  with  streptavidin-phycoerythrin,  signal amplified by biotinylated antistreptavidin, and then scanned on an Agilent Gene array scanner. The intensity for each signal of the array was captured with the Affymetrix GeneChip Software (MAS  5.0),  according  to   standard   Affymetrix  procedures.   The   mRNA  abundance  was determined  based  on the average  of the differences between  perfect  match  and  intentional mismatch intensities  for each probe family.   Gene induction or downregulation was evaluated for statistical significance  using the software provided by Silicon Genetics'  GeneSpring 5.0, Affymetrix DMT 3.0.


### 2.7. Data Conversion and Statistics

All array assays were used at least in duplicate and the results represented an average of duplicates  to  minimize  the  assay  variation.  Expression  data  obtained  from  image  files  by Affymetrix Microarray Suite  5.0  were scaled  to  200  expression  units as the  median.  Raw expression values were normalized within each chip by dividing the median expression value of each individual chip. For each gene, the expression values of the  treated samples were further normalized  across  chips  by  dividing  the  mean  expression  values  of  the  control  samples. Statistical analysis was performed with MATLAB software and data was transformed by natural log format. Differentially expressed genes were selected by three methods: (1)t-test (P<0.05); (2) a density score greater than 100 for positively identified genes; (3) more than twofold differences from control. All identified genes were annotated based on NetAffy database and functionally classified by gene ontology.


### 2.8. Real-Time PCR

The  expression  of selected  genes  identified  by  Affymetrix  GeneChip  analysis  was  further verified  by  real-time  PCR  (with  TaqMan  technology)  on  an  ABI  Prism 7700  Sequence Detection System. PCR primers and TaqMan probes were designed with Primer Express V1.5 software, based on gene sequences downloaded from the GenBank or NetAffx web sites. PCR primers used for the real time PCR are listed in Table1.  TaqMan probes were labeled with 6-carboxy-fluorecein (6-FAM) as the reporter dye and 6-carboxy-tetramethyl-rhodamine (TAMR) as the quencher dye.   Real-time PCR was performed in a two-step process.   In the first step, sample  RNA  (0.1 μg)  or  reference  RNA  was  reverse  transcribed  in  a  volume  of 100 μL containing  TaqMan RT  buffer,  5.5 mM MgCl2, 500 μM of each dNTP,  2.5 μM random hexamers, 0.4 U/μL RNase inhibitor, and 1.25 U/μL MultiScribe Reverse Tanscripatase at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min.  In the second step, real-time PCR was carried out  in a  MicroAmp  Optical 96-well plate  using  TaqMan  Gold  PCR reagents.  Each well contained 5 μL of reverse-transcribed cDNA, TaqMan buffer A, 5.5 mM MgCl2, 200 μM each of dATP/dC TP/dGTP, 400 μM dUTP, 900 nM each of forward and reverse primers, 250 nM TaqMan probe, 0.01 U/μL AmpErase UNG, and 0.025 U/μL AmpliTaq Gold DNA polymerase in a total volume of 50 μL.  The thermal cycling conditions for real-time PCR were: a) 50°C for 2 min, b) 95°C for 10 min, and c) 40 cycles of melting (95°C, 15 sec) and annealing/extension (60°C,  60 sec). PCR reactions  were  monitored  in  real time  using  the  ABI P RISM 7700 Sequence Detector.  A standard curve for each target gene was generated with reference RNA. Relative quantification of gene expression was determined using the standard curve method as described in ABI's User Bulleting #2.Table 1
Primers sequences used for RT-PCR.
Genes
Primers sequence (5′-3′)
U133A accession number and primer location on cDNA
BarH-like homeobox 1
219845_at
TGGGC TCTAACC TGGGAGACT
21F
GAGCTCAGGGTAGAGACTGTAGCTTC
83R
Chondroitin sulfate proteoglycan 2 (versican)
211571_s_at
TTTAAAAATTCCTCATCAGCAAAGG
67F
TCATGTTTGGATGTATTTATTGAATTGTC
19R
Growth arrest-specific 2
205848_at
TGC TATGCTTTCAAGTAAAGTAAATTCAC
64F
CAGCCCTGTCCCAGGTATAC AA
148R
Zinc finger protein 185 (LIM domain)
203585_at
CGTTGGTGAGAAGGTGCCTATC
11F
TCCACTGGTCCTGCACTGAG
62R
NK6 transcription factor related, locus 1 (Drosophila)
221366_at
AGAAGCAGGACTCGGAGACAGA
1F
TGCTGGACTTGTGCTTCTTCAA
138R


## 2.1. Materials

Liberase-HI purified enzyme blend was purchased from Boehringer Mannheim (Indianapolis, IN); dithizone was purchased from Sigma (St. Louis, MO). Human U133A C hips were obtained from Affymetrix, INC (Santa Clara, CA). Medium and RNA extraction kit were purchased from Qiagen (QIAGEN Inc., Valencia, C A). Live/Dead Viability/Cytotoxicity Kit and streptavidin-phycoerythrin  were from Molecular  Probe (Eugene, OR).  GLP-1 was purchased from American Peptide Co. (Sunnyvale, CA). Ficoll Cobe 2991  was purchased  from COBE BCT,  Inc. (Lakewood, CO). Tissue culture plastic ware was obtained  from Corning Coaster Corporation (Cambridge, MA). Fetal calf serum and SuperScript Choice system were from Life Technologies,  Inc.  BRL  (Rockville,  MD).  M199  medium  was  obtained  from  Gibco-BRL (Gaithersburg,  MD).  Bradford  reagents  for  protein  detection  were  obtained  from  Bio-Rad (Richmond, CA). Oligo-(dT)24   anchored  T7 primer was obtained  from Amersham Pharmacia Biotech  (Piscataway,  NJ).  Phase  Lock  Gels   were  purchased  from  5-prime-3-prime,  Inc. (Boulder, CO). The BioArray high yield RNA transcript labeling kit was purchased from Enzo Diagnostics, Inc. (Farmingdale, NY).  Biotinylated antistreptavidin was purchased from Vector Laboratories, Inc. (Burlingame, CA). PCR reagents, the software for the design of primers and TaqMan probes (Express V1.5 software), and the ABI PRISM 7700 Sequence Detector were from Applied Biosystems Inc. (Foster City, CA).


## 2.2. Islet Isolation, Purification, and Quantification

Human pancreases were recovered from cadaveric donors. Pancreas were placed in cold UW (University of Wisconsin) solution on ice and immediately transported to the islet isolation laboratory for processing and selected with criteria of having at least 10 h of cold storage in UW. All organs were processed  using  identical  isolation techniques of collagenase digestion and Ficoll purification. Briefly, human pancreases were placed in a customized perfusion apparatus and injected  with  liberase-HI.  They  were  then  transferred  to  a continuous  digestion  device  for mechanical disrupt and  enzymatic digestion  until the  majority of the  islets  were  free  from exocrine tissue. Samples of pancreatic tissue were evaluated for purity during the digestion after staining with dithizone. Pancreatic digest containing endocrine and exocrine tissue was purified by continuous  gradients  centrifugation of Ficoll Cobe 2991.  Islet-enriched  layers  were then selected and collected. Islet recovery after purification was assessed in duplication by counts of dithizone-stained aliquots of the final suspension of tissue. The purity of the preparations was assessed by comparing the relative quantity of dithizone-stained endocrine tissue divided by the unstained tissue. The viability of isolated cells was 87± 6%, and their purity was  ≥57±4%.


## 2.3. Living/Dead Cell Determination

Cell  viability  was  determined  using  a  Living/Dead  Viability/Cytotoxicity  Kit.  Briefly, isolated islets were washed twice with P BS and 50μL aliquots were placed in the 96-well plate. Then 10 μL of solution component A (Calcein AM) and component B (Ethidium homodimer-1) in  Live/Dead  Viability/Cytotoxicity K it  were added to each well.  After 30 min,  islets were examined under a microscope.   Living cells were identified by a green staining (Calcein AM staining), while dead cells showed a brown nuclear staining (ethidium homodimer-1 staining).


## 2.4. Incubation of Isolated Islets with GLP-1

Isolated islets were cultured, in 75-mL flasks, in the presence of M199  medium with 100μg/mL penicillin, 50 μg/mL streptomycin, and 10% of fetal calf serum at 37°C under humidified conditions with 5% CO2. After 4 h, the islets were washed twice with M199 without FBS and antibiotics. They were then treated with 10 nM of GLP-1 in M199 medium containing 16 mM glucose (without F BS) for 22 h. Islet pellets were collected by centrifugation at 1000 rpm for 3 min. RNA was isolated by using the Qiagen RN easy Mini kit and immediately stored at −80°C.


## 2.5. Detection of Insulin Secretion

After 22 h of culturing in the presence of GLP-1, or vehicle,  insulin released  into the medium was measured by RIA (radioimmunoassay). Total cellular protein content was measured using the method of Bradford  Assay. The amount of proteins measured served as a correction factor for determining the relative amount of insulin released into the culture medium by each individual culture condition.


## 2.6. Microarray Analysis

Affymetrix   arrays   (Human   U133A)   were used for mRNA expression profiling. Experimental procedures for gene chips were performed according to the Affymetrix Gene Chip Expression  Analysis  Technical Manual. Briefly:  double-stranded  cDNAs  were  synthesized using the  SuperScript Choice system and an oligo-(dT)24  anchored T7  primer.  Two samples (duplicate) of 5 μg of total RNA from each sample were used to start the synthesis of cDNA. Double-stranded cDNA products were purified by phenol:chloroform:isoamyl alcohol (25 : 24 : 1 saturated with 10 mM Tris-HC l, pH8.0/1 mM EDTA), followed by extraction, phase separation with Phase Lock Gels, and ethanol precipitation.  Biotinylated RNA was synthesized using the BioArray high yield RNA transcript labeling kit. Biotinylated RNA products were purified using Qiagen RNeasy columns and  fragmented to a size of 30 to 200 nucleotides.   A total of 15 ug biotinylated fragmented RNA was then hybridized with Affymetrix GeneChip arrays (Human, U133A). After  washing,  the  arrays  were  stained  with  streptavidin-phycoerythrin,  signal amplified by biotinylated antistreptavidin, and then scanned on an Agilent Gene array scanner. The intensity for each signal of the array was captured with the Affymetrix GeneChip Software (MAS  5.0),  according  to   standard   Affymetrix  procedures.   The   mRNA  abundance  was determined  based  on the average  of the differences between  perfect  match  and  intentional mismatch intensities  for each probe family.   Gene induction or downregulation was evaluated for statistical significance  using the software provided by Silicon Genetics'  GeneSpring 5.0, Affymetrix DMT 3.0.


## 2.7. Data Conversion and Statistics

All array assays were used at least in duplicate and the results represented an average of duplicates  to  minimize  the  assay  variation.  Expression  data  obtained  from  image  files  by Affymetrix Microarray Suite  5.0  were scaled  to  200  expression  units as the  median.  Raw expression values were normalized within each chip by dividing the median expression value of each individual chip. For each gene, the expression values of the  treated samples were further normalized  across  chips  by  dividing  the  mean  expression  values  of  the  control  samples. Statistical analysis was performed with MATLAB software and data was transformed by natural log format. Differentially expressed genes were selected by three methods: (1)t-test (P<0.05); (2) a density score greater than 100 for positively identified genes; (3) more than twofold differences from control. All identified genes were annotated based on NetAffy database and functionally classified by gene ontology.


## 2.8. Real-Time PCR

The  expression  of selected  genes  identified  by  Affymetrix  GeneChip  analysis  was  further verified  by  real-time  PCR  (with  TaqMan  technology)  on  an  ABI  Prism 7700  Sequence Detection System. PCR primers and TaqMan probes were designed with Primer Express V1.5 software, based on gene sequences downloaded from the GenBank or NetAffx web sites. PCR primers used for the real time PCR are listed in Table1.  TaqMan probes were labeled with 6-carboxy-fluorecein (6-FAM) as the reporter dye and 6-carboxy-tetramethyl-rhodamine (TAMR) as the quencher dye.   Real-time PCR was performed in a two-step process.   In the first step, sample  RNA  (0.1 μg)  or  reference  RNA  was  reverse  transcribed  in  a  volume  of 100 μL containing  TaqMan RT  buffer,  5.5 mM MgCl2, 500 μM of each dNTP,  2.5 μM random hexamers, 0.4 U/μL RNase inhibitor, and 1.25 U/μL MultiScribe Reverse Tanscripatase at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min.  In the second step, real-time PCR was carried out  in a  MicroAmp  Optical 96-well plate  using  TaqMan  Gold  PCR reagents.  Each well contained 5 μL of reverse-transcribed cDNA, TaqMan buffer A, 5.5 mM MgCl2, 200 μM each of dATP/dC TP/dGTP, 400 μM dUTP, 900 nM each of forward and reverse primers, 250 nM TaqMan probe, 0.01 U/μL AmpErase UNG, and 0.025 U/μL AmpliTaq Gold DNA polymerase in a total volume of 50 μL.  The thermal cycling conditions for real-time PCR were: a) 50°C for 2 min, b) 95°C for 10 min, and c) 40 cycles of melting (95°C, 15 sec) and annealing/extension (60°C,  60 sec). PCR reactions  were  monitored  in  real time  using  the  ABI P RISM 7700 Sequence Detector.  A standard curve for each target gene was generated with reference RNA. Relative quantification of gene expression was determined using the standard curve method as described in ABI's User Bulleting #2.Table 1
Primers sequences used for RT-PCR.
Genes
Primers sequence (5′-3′)
U133A accession number and primer location on cDNA
BarH-like homeobox 1
219845_at
TGGGC TCTAACC TGGGAGACT
21F
GAGCTCAGGGTAGAGACTGTAGCTTC
83R
Chondroitin sulfate proteoglycan 2 (versican)
211571_s_at
TTTAAAAATTCCTCATCAGCAAAGG
67F
TCATGTTTGGATGTATTTATTGAATTGTC
19R
Growth arrest-specific 2
205848_at
TGC TATGCTTTCAAGTAAAGTAAATTCAC
64F
CAGCCCTGTCCCAGGTATAC AA
148R
Zinc finger protein 185 (LIM domain)
203585_at
CGTTGGTGAGAAGGTGCCTATC
11F
TCCACTGGTCCTGCACTGAG
62R
NK6 transcription factor related, locus 1 (Drosophila)
221366_at
AGAAGCAGGACTCGGAGACAGA
1F
TGCTGGACTTGTGCTTCTTCAA
138R


## 3. Results

### 3.1. Islet Viability and Responsiveness to GLP-1

Islets viability was determined by the Living/Dead Viability/Cytotoxicity assay. Living cells were identified by a green staining (Calcein AM staining), while a brown nuclear staining distinguished the dead cells (Ethidium homodimer-1 staining).  We observed a majority (>90%) of islet cells viable, with no  significant difference in the proportion of live versus dead cells between islets cultured in the presence of GLP-1, and control groups (Figure1(a)).Viability and function of isolated human islets. Human islets were cultured in medium containing GLP-1 (10 nM), or vehicle for 22 h. (a) Cell viability was  determined  using the Living/Dead  Viability/Cytotoxicity Kit.  Living cells were identified by a green staining (Calcein AM staining), while dead cells were identified by a brown nuclear staining (Ethidium homodimer-1 staining).  (b) The insulin accumulation into the culture medium was determined by RIA, and normalized to the total protein content in the cell pellet. Statistical significance of the data was evaluated by Student’st-test. *= P<0.01.
(a)
(b)The  concentration  of  insulin  in  the  medium,  measured  by  RIA,  and  its  abundance, normalized  for  by  the total concentration of proteins in each individual culture are listed  in Figure1(b).   There was 28.87 ±  3.29 ng insulin/mg of protein in the culture medium of islets grown in the presence of GLP-1 versus a 15.43 ±  3.6 ng insulin/mg of protein in the culture medium of islets  grown  in the control culture (no  GLP-1). Statistic  analysis  indicates the difference between two status is significant (P<0.01).


### 3.2. Genes Regulated by GLP-1

A  total  of  24  microarrays  were  employed  to  evaluate  the  gene  expression  profile associated  with  the exposure of human  islets to  GLP-1.  After 22 h  induction  in a medium containing 10 nM GLP-1, more than 7000 gene transcripts were detected. Among them, the 80 genes (1%) with differential expression by 2-fold or more (up- or downregulation) are listed in Tables1 and 2.  The top 5 genes found upregulated by GLP-1 are: S EMA3C (sema domain, immunoglobulin  domain  (Ig),  short  basic domain,  secreted,  (semaphorin) 3C),  3.172-folds; RBBP6 (retinoblastoma binding protein 6], 3.147-folds; BARX1 (BarH-like homeobox 1), 3.122-folds; S100A9 (S100 calcium binding protein A9 (calgranulin B)), 2.940-folds; and DNAM-1 (adhesion glycoprotein), 2.935-folds,  respectively.  The top 5  genes found down-regulated by GLP-1 are: KCNJ15 (potassium inwardly-rectifying channel, subfamily J,  member 15), 0.306-folds; UTS2 (urotensin 2), 0.299-folds; S LC7A6 (solute carrier family 7  (cationic amino acid transporter, y+ system), member 6), 0.260-folds; HSA9947 (putative ATPase), 0.231-folds; and ARHGEF9 (Cdc42 guanine nucleotide exchange factor (GEF) 9), 0.169-folds.Table 2
(a) Upregulated genes selected from GLP-1 treated islet, (b) Downregulated genes selected from GLP-1 treated islet.
(a)
Symbol
Name
Chromosomelocation
FC
P (t-test)
Homo sapiens cDNA FLJ39067 fis,   cloneNT2RP7014910.
3.240
0.027
SEM A3C
Sema domain, immunoglobulin domain (Ig),   short basic domain, secreted, (semaphorin) 3C
7q21-q31
3.172
0.031
RBBP6
Retinoblastoma binding protein 6
16p 12-p11.2
3.147
0.032
BARX1
BarH-like homeobox 1
9q12
3.122
0.020
S100A9
S100 calcium binding protein A9 (calgranulin B)
1q21
2.940
0.006
DNAM-1
Adhesion glycoprotein
18q22.3
2.935
0.007
Homo sapiens cDNA FLJ14046 fis, cloneHEMBA1006461.
2.907
0.006
Homo sapiens cDNA FLJ10906 fis, cloneOVARC1000035.
2.829
0.016
SEC15B
Sec15B protein
2p12
2.776
0.026
HIST1H3I
Histone 1, H3i
6p22-p21.3
2.773
0.001
PRLR
Prolactin receptor
5p14-p13
2.698
0.039
CYLD
Cylindromatosis (turban tumor syndrome)
16q11.2
2.672
0.031
SULT1C1
Sulfotransferase family, cytosolic, 1C, member 1
2q11.1-q11.2
2.586
0.034
Homo sapiens, clone IMAGE: 4702418, mRNA
2.522
0.016
PTPN7
Protein tyrosine phosphatase, non-receptor type 7
1q32.1
2.504
0.017
CCL3
Chemokine (C–C motif) ligand 3
17q11-q21
2.500
0.000
S100A4
S100 calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog)
1q21
2.398
0.026
Homo sapiens, clone IMAGE: 4471726, mRNA
2.364
0.020
KIAA1076
KIAA1076 protein
12q24.31
2.310
0.020
APOBEC3G
Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G
22q13.1-q13.2
2.301
0.043
TRPM3
Transient receptor potential cation channel, subfamily M, member 3
9q21.11
2.265
0.027
Homo sapiens mRNA; cDNA DKFZp434E2423 (from clone DKFZp434E2423)
2.225
0.040
PNUTL2
Peanut-like 2 (Drosophila)
17q22-q23
2.201
0.020
CSPG2
Chondroitin sulfate proteoglycan 2 (versican)
5q14.3
2.198
0.037
COL14A1
Collagen, type XIV, alpha 1 (undulin)
8q23
2.189
0.025
MGC27165
Hypothetical protein MGC27165
14
2.111
0.021
C16orf5
Chromosome 16 open reading frame 5
16p13.3
2.106
0.016
FLJ23342
Hypothetical protein FLJ23342
11q24.2
2.089
0.024
SBBI31
SBBI31 protein
5q23.2
2.085
0.029
PAX4
Paired box gene 4
7q32
2.082
0.040
FLJ10140
Hypothetical protein FLJ10140
22q13
2.077
0.029
KIAA0999
KIAA0999 protein
11q23.3
2.048
0.015
Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 362780.
2.008
0.036
(b)
Symbol
Name
Chromosomelocation
FC
P (t-test)
GAS2
Growth arrest-specific 2
11p14.3-15.2
0.498
0.045
SUOX
Sulfite oxidase
12q13.13
0.496
0.034
KIAA0469
KIAA0469 gene product
1p36.23
0.496
0.026
DNAJB6
DnaJ (Hsp40) homolog, subfamily B, member 6
7q36.3
0.495
0.022
RBM 9
RNA binding motif protein 9
22q13.1
0.490
0.034
MYB
v-myb myeloblastosis viral oncogene homolog (avian)
6q22-q23
0.488
0.030
CLASP2
Cytoplasmic linker associated protein 2
3p 22.2
0.487
0.047
MGC5297
Hypothetical protein M GC5297
5p15.3-p15.2
0.486
0.039
PPP2R4
Protein phosphatase 2A, regulatory subunit B′ (PR 53)
9q34
0.484
0.031
PIP5K1C
Phosphatidylinositol-4-phosphate 5-kinase, type I, gamma
19p13.3
0.482
0.022
ZFHX1B
Zinc finger homeobox 1b
2q22
0.479
0.028
ADCY9
Adenylate cyclase 9
16p13.3
0.463
0.043
FLJ12788
Hypothetical protein FLJ12788
2p12
0.458
0.015
TM 6SF1
Trans membrane 6 superfamily member 1
15q24-q26
0.447
0.043
TBCE
Tubulin-specific chaperone e
1q42.3
0.446
0.035
GTF2H2
General transcription factor IIH, polypeptide 2, 44k Da
5q12.2-q13.3
0.438
0.007
PRO1331
Hypothetical protein PRO1331
5q33.3
0.437
0.024
ZNF185
Zinc finger protein 185 (LIM domain)
Xq28
0.428
0.021
KIAA0460
KIAA0460 protein
1q21.2
0.427
0.021
DKFZP434B168
DKFZP434B168 protein
1p36.13-q42.3
0.426
0.042
FCMD
Fukuyama type congenital muscular dystrophy (fukutin)
9q31-q33
0.415
0.045
ABCF2
ATP-binding cassette, subfamily F (GCN20), member 2
7q36
0.414
0.012
LM O7
LIM domain only 7
13q21.33
0.413
0.039
SULF1
Sulfatase 1
8q13.1
0.412
0.033
KIAA0090
KIAA0090 protein
1p36.13
0.411
0.003
POR
P450 (cytochrome) oxidoreductase
7q11.2
0.407
0.015
SE70-2
Cutaneous T-cell lymphoma tumor antigen se70-2
13q22.1
0.406
0.030
NKX6-1
NK6 transcription factor related, locus 1 (Drosophila)
4q21.2-q22
0.403
0.006
LOC51231
VRK3 for vaccinia related kinase 3
19q13
0.403
0.002
BYSL
Bystin-like
6p21.1
0.401
0.032
HIP1R
Huntingtin interacting protein-1-related
12q24
0.396
0.012
PPP2R4
Protein phosphatase 2A, regulatory subunit B′ (PR 53)
9q34
0.387
0.046
WASL
Wiskott-Aldrich syndrome-like
7q31.3
0.383
0.032
TIMM 17A
Translocase of inner mitochondrial membrane 17 homolog A (yeast)
1q32.1
0.378
0.032
ARL4
ADP-ribosylation factor-like 4
7p21-p15.3
0.375
0.002
LOC51204
Clone HQ0477 PRO0477p
17q24.1
0.363
0.023
KIAA1023
KIAA1023 protein
7p22.3
0.357
0.017
CPT1A
carnitine palmitoyl transferase 1A (liver)
11q13.1-q13.2
0.351
0.021
RAD51C
RAD51 homolog C (S. cerevisiae)
17q22-q23
0.347
0.022
ITGB3
integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
17q21.32
0.345
0.008
MGC4309
hypothetical protein M GC4309
1q32.1
0.336
0.007
ESTs, weakly similar to  neuronal thread protein (Homo sapiens)
0.334
0.030
KCNJ15
potassium inwardly-rectifying channel, subfamily J, member 15
21q22.2
0.306
0.006
UTS2
urotensin 2
1p36
0.299
0.032
SLC7A6
solute carrier family 7 (cationic amino acid transporter, y+ system), member 6
16q22.1
0.260
0.016
HSA9947
putative ATPase
1p36
0.231
0.050
ARHGEF9
Cdc42 guanine nucleotide exchange factor (GEF) 9
Xq11.1
0.169
0.019


### 3.3. Validation of Microarray Data by Real-Time PCR

The alteration of gene expression by GLP-1 induction, as observed by microarray analysis, was further  validated by real-time PCR. RNA samples from the same islet preparations were split and used for both microarray analysis and real-time PCR.  We verified mRNA expression of 5 genes, two upregulated genes (BarH-like homeobox 1 and chondroitin sulfate proteoglycan) and  three  downregulated  genes (growth arrest-specific 2,  zinc  finger protein 185, and NK6 transcription factor), responding GLP-1 induction. We were able to confirm the consistent results from real-time PCR and from microarray analysis (Figure2).Figure 2
Results comparison between microarray and real-time-PCR. Five selected transcripts among those identified by microarray analysis were validated by the real-time-PCR analysis. The three transcripts selected were: BarH-like homeobox 1 (BARH); chondroitin sulfate proteoglycan (CSP); growth arrest-specific 2 (GAS); zinc finger protein 185 (LIM domain) (ZFP); NK6 transcription factor (NK6T). The graph shows the folds of induction or suppression as derived by the two methods. The data are expressed as the means± SE of three independent analyses.


### 3.4. Clustering of Genes Regulated by GLP-1

Among the 80 genes regulated by GLP-1, 66 were with known molecular functions, 34 were linked to cellular structural components; 73 were involved in various biological processes. Further analysis demonstrated  that  genes encoding for proteins, with protein-DNA or protein-protein binding capacity (n=25) and enzyme activity (n=17) were in two main subgroups (Figure 3). Genes encoding proteins with intracellular  structure (n=30) represented a  main class  for cellular  component  synthesis  (Figure 4).  Genes  involved  in  physiological process  (n=35), including metabolism, response to stress, and response to external stimuli and the genes involved in cellular processes (n=22), as cell growth and cell-cell interaction, represented the genes with known  biological  processes   (Figure 5).  Functional  analysis  showed  that  GLP-1  widely modulated the expression of genes  linked  with cell adhesion/extracellular matrix, cell cycle, cytoskeleton/structural, enzymatic activity and metabolism, growth factors/hormones/cytokines, nucleotide processing, protein processing, receptors, signal transduction,  transcription  factors, and transporter proteins.Figure 3
Functional clustering of islet genes regulated by GLP-1.Figure 4
Cellular distribution of genes regulated by GLP-1.Figure 5
Biological function of genes regulated by GLP-1.


## 3.1. Islet Viability and Responsiveness to GLP-1

Islets viability was determined by the Living/Dead Viability/Cytotoxicity assay. Living cells were identified by a green staining (Calcein AM staining), while a brown nuclear staining distinguished the dead cells (Ethidium homodimer-1 staining).  We observed a majority (>90%) of islet cells viable, with no  significant difference in the proportion of live versus dead cells between islets cultured in the presence of GLP-1, and control groups (Figure1(a)).Viability and function of isolated human islets. Human islets were cultured in medium containing GLP-1 (10 nM), or vehicle for 22 h. (a) Cell viability was  determined  using the Living/Dead  Viability/Cytotoxicity Kit.  Living cells were identified by a green staining (Calcein AM staining), while dead cells were identified by a brown nuclear staining (Ethidium homodimer-1 staining).  (b) The insulin accumulation into the culture medium was determined by RIA, and normalized to the total protein content in the cell pellet. Statistical significance of the data was evaluated by Student’st-test. *= P<0.01.
(a)
(b)The  concentration  of  insulin  in  the  medium,  measured  by  RIA,  and  its  abundance, normalized  for  by  the total concentration of proteins in each individual culture are listed  in Figure1(b).   There was 28.87 ±  3.29 ng insulin/mg of protein in the culture medium of islets grown in the presence of GLP-1 versus a 15.43 ±  3.6 ng insulin/mg of protein in the culture medium of islets  grown  in the control culture (no  GLP-1). Statistic  analysis  indicates the difference between two status is significant (P<0.01).


## 3.2. Genes Regulated by GLP-1

A  total  of  24  microarrays  were  employed  to  evaluate  the  gene  expression  profile associated  with  the exposure of human  islets to  GLP-1.  After 22 h  induction  in a medium containing 10 nM GLP-1, more than 7000 gene transcripts were detected. Among them, the 80 genes (1%) with differential expression by 2-fold or more (up- or downregulation) are listed in Tables1 and 2.  The top 5 genes found upregulated by GLP-1 are: S EMA3C (sema domain, immunoglobulin  domain  (Ig),  short  basic domain,  secreted,  (semaphorin) 3C),  3.172-folds; RBBP6 (retinoblastoma binding protein 6], 3.147-folds; BARX1 (BarH-like homeobox 1), 3.122-folds; S100A9 (S100 calcium binding protein A9 (calgranulin B)), 2.940-folds; and DNAM-1 (adhesion glycoprotein), 2.935-folds,  respectively.  The top 5  genes found down-regulated by GLP-1 are: KCNJ15 (potassium inwardly-rectifying channel, subfamily J,  member 15), 0.306-folds; UTS2 (urotensin 2), 0.299-folds; S LC7A6 (solute carrier family 7  (cationic amino acid transporter, y+ system), member 6), 0.260-folds; HSA9947 (putative ATPase), 0.231-folds; and ARHGEF9 (Cdc42 guanine nucleotide exchange factor (GEF) 9), 0.169-folds.Table 2
(a) Upregulated genes selected from GLP-1 treated islet, (b) Downregulated genes selected from GLP-1 treated islet.
(a)
Symbol
Name
Chromosomelocation
FC
P (t-test)
Homo sapiens cDNA FLJ39067 fis,   cloneNT2RP7014910.
3.240
0.027
SEM A3C
Sema domain, immunoglobulin domain (Ig),   short basic domain, secreted, (semaphorin) 3C
7q21-q31
3.172
0.031
RBBP6
Retinoblastoma binding protein 6
16p 12-p11.2
3.147
0.032
BARX1
BarH-like homeobox 1
9q12
3.122
0.020
S100A9
S100 calcium binding protein A9 (calgranulin B)
1q21
2.940
0.006
DNAM-1
Adhesion glycoprotein
18q22.3
2.935
0.007
Homo sapiens cDNA FLJ14046 fis, cloneHEMBA1006461.
2.907
0.006
Homo sapiens cDNA FLJ10906 fis, cloneOVARC1000035.
2.829
0.016
SEC15B
Sec15B protein
2p12
2.776
0.026
HIST1H3I
Histone 1, H3i
6p22-p21.3
2.773
0.001
PRLR
Prolactin receptor
5p14-p13
2.698
0.039
CYLD
Cylindromatosis (turban tumor syndrome)
16q11.2
2.672
0.031
SULT1C1
Sulfotransferase family, cytosolic, 1C, member 1
2q11.1-q11.2
2.586
0.034
Homo sapiens, clone IMAGE: 4702418, mRNA
2.522
0.016
PTPN7
Protein tyrosine phosphatase, non-receptor type 7
1q32.1
2.504
0.017
CCL3
Chemokine (C–C motif) ligand 3
17q11-q21
2.500
0.000
S100A4
S100 calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog)
1q21
2.398
0.026
Homo sapiens, clone IMAGE: 4471726, mRNA
2.364
0.020
KIAA1076
KIAA1076 protein
12q24.31
2.310
0.020
APOBEC3G
Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G
22q13.1-q13.2
2.301
0.043
TRPM3
Transient receptor potential cation channel, subfamily M, member 3
9q21.11
2.265
0.027
Homo sapiens mRNA; cDNA DKFZp434E2423 (from clone DKFZp434E2423)
2.225
0.040
PNUTL2
Peanut-like 2 (Drosophila)
17q22-q23
2.201
0.020
CSPG2
Chondroitin sulfate proteoglycan 2 (versican)
5q14.3
2.198
0.037
COL14A1
Collagen, type XIV, alpha 1 (undulin)
8q23
2.189
0.025
MGC27165
Hypothetical protein MGC27165
14
2.111
0.021
C16orf5
Chromosome 16 open reading frame 5
16p13.3
2.106
0.016
FLJ23342
Hypothetical protein FLJ23342
11q24.2
2.089
0.024
SBBI31
SBBI31 protein
5q23.2
2.085
0.029
PAX4
Paired box gene 4
7q32
2.082
0.040
FLJ10140
Hypothetical protein FLJ10140
22q13
2.077
0.029
KIAA0999
KIAA0999 protein
11q23.3
2.048
0.015
Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 362780.
2.008
0.036
(b)
Symbol
Name
Chromosomelocation
FC
P (t-test)
GAS2
Growth arrest-specific 2
11p14.3-15.2
0.498
0.045
SUOX
Sulfite oxidase
12q13.13
0.496
0.034
KIAA0469
KIAA0469 gene product
1p36.23
0.496
0.026
DNAJB6
DnaJ (Hsp40) homolog, subfamily B, member 6
7q36.3
0.495
0.022
RBM 9
RNA binding motif protein 9
22q13.1
0.490
0.034
MYB
v-myb myeloblastosis viral oncogene homolog (avian)
6q22-q23
0.488
0.030
CLASP2
Cytoplasmic linker associated protein 2
3p 22.2
0.487
0.047
MGC5297
Hypothetical protein M GC5297
5p15.3-p15.2
0.486
0.039
PPP2R4
Protein phosphatase 2A, regulatory subunit B′ (PR 53)
9q34
0.484
0.031
PIP5K1C
Phosphatidylinositol-4-phosphate 5-kinase, type I, gamma
19p13.3
0.482
0.022
ZFHX1B
Zinc finger homeobox 1b
2q22
0.479
0.028
ADCY9
Adenylate cyclase 9
16p13.3
0.463
0.043
FLJ12788
Hypothetical protein FLJ12788
2p12
0.458
0.015
TM 6SF1
Trans membrane 6 superfamily member 1
15q24-q26
0.447
0.043
TBCE
Tubulin-specific chaperone e
1q42.3
0.446
0.035
GTF2H2
General transcription factor IIH, polypeptide 2, 44k Da
5q12.2-q13.3
0.438
0.007
PRO1331
Hypothetical protein PRO1331
5q33.3
0.437
0.024
ZNF185
Zinc finger protein 185 (LIM domain)
Xq28
0.428
0.021
KIAA0460
KIAA0460 protein
1q21.2
0.427
0.021
DKFZP434B168
DKFZP434B168 protein
1p36.13-q42.3
0.426
0.042
FCMD
Fukuyama type congenital muscular dystrophy (fukutin)
9q31-q33
0.415
0.045
ABCF2
ATP-binding cassette, subfamily F (GCN20), member 2
7q36
0.414
0.012
LM O7
LIM domain only 7
13q21.33
0.413
0.039
SULF1
Sulfatase 1
8q13.1
0.412
0.033
KIAA0090
KIAA0090 protein
1p36.13
0.411
0.003
POR
P450 (cytochrome) oxidoreductase
7q11.2
0.407
0.015
SE70-2
Cutaneous T-cell lymphoma tumor antigen se70-2
13q22.1
0.406
0.030
NKX6-1
NK6 transcription factor related, locus 1 (Drosophila)
4q21.2-q22
0.403
0.006
LOC51231
VRK3 for vaccinia related kinase 3
19q13
0.403
0.002
BYSL
Bystin-like
6p21.1
0.401
0.032
HIP1R
Huntingtin interacting protein-1-related
12q24
0.396
0.012
PPP2R4
Protein phosphatase 2A, regulatory subunit B′ (PR 53)
9q34
0.387
0.046
WASL
Wiskott-Aldrich syndrome-like
7q31.3
0.383
0.032
TIMM 17A
Translocase of inner mitochondrial membrane 17 homolog A (yeast)
1q32.1
0.378
0.032
ARL4
ADP-ribosylation factor-like 4
7p21-p15.3
0.375
0.002
LOC51204
Clone HQ0477 PRO0477p
17q24.1
0.363
0.023
KIAA1023
KIAA1023 protein
7p22.3
0.357
0.017
CPT1A
carnitine palmitoyl transferase 1A (liver)
11q13.1-q13.2
0.351
0.021
RAD51C
RAD51 homolog C (S. cerevisiae)
17q22-q23
0.347
0.022
ITGB3
integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
17q21.32
0.345
0.008
MGC4309
hypothetical protein M GC4309
1q32.1
0.336
0.007
ESTs, weakly similar to  neuronal thread protein (Homo sapiens)
0.334
0.030
KCNJ15
potassium inwardly-rectifying channel, subfamily J, member 15
21q22.2
0.306
0.006
UTS2
urotensin 2
1p36
0.299
0.032
SLC7A6
solute carrier family 7 (cationic amino acid transporter, y+ system), member 6
16q22.1
0.260
0.016
HSA9947
putative ATPase
1p36
0.231
0.050
ARHGEF9
Cdc42 guanine nucleotide exchange factor (GEF) 9
Xq11.1
0.169
0.019


## 3.3. Validation of Microarray Data by Real-Time PCR

The alteration of gene expression by GLP-1 induction, as observed by microarray analysis, was further  validated by real-time PCR. RNA samples from the same islet preparations were split and used for both microarray analysis and real-time PCR.  We verified mRNA expression of 5 genes, two upregulated genes (BarH-like homeobox 1 and chondroitin sulfate proteoglycan) and  three  downregulated  genes (growth arrest-specific 2,  zinc  finger protein 185, and NK6 transcription factor), responding GLP-1 induction. We were able to confirm the consistent results from real-time PCR and from microarray analysis (Figure2).Figure 2
Results comparison between microarray and real-time-PCR. Five selected transcripts among those identified by microarray analysis were validated by the real-time-PCR analysis. The three transcripts selected were: BarH-like homeobox 1 (BARH); chondroitin sulfate proteoglycan (CSP); growth arrest-specific 2 (GAS); zinc finger protein 185 (LIM domain) (ZFP); NK6 transcription factor (NK6T). The graph shows the folds of induction or suppression as derived by the two methods. The data are expressed as the means± SE of three independent analyses.


## 3.4. Clustering of Genes Regulated by GLP-1

Among the 80 genes regulated by GLP-1, 66 were with known molecular functions, 34 were linked to cellular structural components; 73 were involved in various biological processes. Further analysis demonstrated  that  genes encoding for proteins, with protein-DNA or protein-protein binding capacity (n=25) and enzyme activity (n=17) were in two main subgroups (Figure 3). Genes encoding proteins with intracellular  structure (n=30) represented a  main class  for cellular  component  synthesis  (Figure 4).  Genes  involved  in  physiological process  (n=35), including metabolism, response to stress, and response to external stimuli and the genes involved in cellular processes (n=22), as cell growth and cell-cell interaction, represented the genes with known  biological  processes   (Figure 5).  Functional  analysis  showed  that  GLP-1  widely modulated the expression of genes  linked  with cell adhesion/extracellular matrix, cell cycle, cytoskeleton/structural, enzymatic activity and metabolism, growth factors/hormones/cytokines, nucleotide processing, protein processing, receptors, signal transduction,  transcription  factors, and transporter proteins.Figure 3
Functional clustering of islet genes regulated by GLP-1.Figure 4
Cellular distribution of genes regulated by GLP-1.Figure 5
Biological function of genes regulated by GLP-1.


## 4. Discussion

We report the identification of a large set of genes whose expression is regulated by the binding of GLP-1 to its receptor localized on human islets. The elucidation of this complex gene-regulatory network is essential for a better understanding  of  the physiological  and pharmacological effects of GLP-1, as well as for deciphering the mechanism(s) by which this peptide has such a diverse repertoire of actions on islet cells. The effect of GLP-1 on islet cells has been investigated with rodent cell lines, as well as murine and human islets [10, 11]. From these researches, most data suggested that GLP-1 promoted beta-cell mass by increasing beta-cell proliferation, inhibiting beta-cell apoptosis, and inducing the differentiation of beta-cell from progenitor cells.  While the  in vivoapplication of GLP-1 on the diabetic and  aging animals suggested that multifactors might be involved and sounded complex on the mechanism, the in vitrotreatment of GLP-1 on the  isolated islets provided more direct evidence of GLP-1 effect on islets. Farrilaetc demonstrated that GLP-1 added to freshly isolated human islets preserved the cell morphology and function and was able to inhibit cell apoptosis [12].  This effect is associated with a higher expression of Bcl-2  and  a  lower expression of active Caspase 3. Similarly, Mancosuetc found that high glucose concentration and glucagon-like peptide 1 (GLP-1) were associated  with  the  maintenance of either  the  insulin  secretary patterns  from the  incubated monolayer  cells,  or  the   transcriptional  marker  expression  associated  with  beta-cell  like phenotypes [13]. Upregulated expression of PDX-1, PAX4, Glut-2, and GK was also detected in their long cultured islets, in the presence of GLP-1.In  this study,  we observed  that  the expression of most proliferation-related  genes  was associated with GLP-1 induction. These GLP-1 upregulated, proliferation-related genes include: the  prolactin  receptor,  which  has  been  proposed  to  regulate  islet  mass  expansion  during pregnancy  [14];  retinoblastoma  binding   protein-6   (RBBP6)  is  a  multifunctional  protein  found ostensibly in all eukaryotes but not in bacteria, which is  implicated in a diverse set of cellular functions including  mRNA  metabolism, regulation  of  the  cell  cycle,  tumour igenesis  and  development.   [15]; CSPG2, which regulates cell motility and growth under P53 activation [16]; S100A4 can act as a novel  cardiac  growth  and  survival  factor  and  may  have  regenerative  effects  in   injured myocardium [17]; CYLD, which acts as tumor suppressor gene [18]; SEMA3a, which inhibits the binding of the VEGF  with 165-amino acids (VEGF-165) to np1 and  was reported  to  inhibit angiogenesis [19].  We were unable to observe the significant alteration of bcl-2, P ik3, and Irs2 reported by others groups with cell line and/or animal models [20, 21].Antiapoptosis  is another  important  biological event  modulated  by  GLP-1  [22]. Our microarray analyses observed 3  apoptosis-related  genes (CYLD,  RBBP6, and  PNUTL2) upregulated,  and   one  (GAS2)  downregulated.  CYLD  (the   familial  cylindromatosis  tumor suppressor  gene) enhances the  activation of the transcription factor NFkapa-B [23],  RBBP6, (which binds to the retinoblastoma gene product pRB) [24], and PNUTL2 (which is an apoptosis-related  protein  in  the  TGF-beta signaling  pathway) [25].  GAS2  plays  an  important role  in apoptosis by acting as a cell-death gene substrate for caspases [26]. These data suggest a conflict function of GLP-1 on apoptosis regulation.  Increasing CYLD promotes apoptosis and increasing PNUTL enhances cell death via TGF. On the other hand, increased expression of RBBP6 and suppressed expression of GAS2 revealed an antiapoptosis effect of GLP-1.These  observations  contradictory  on  apoptosis  and   differentiation   might  indicate complicated, but balanced functions between gene groups (pro- and antiapoptosis), rather than a simple and one direction to lead cell-death. They also indicate that cellular function regulation, such as apoptosis, differentiation, and so forth. are more complicated than what we speculated previously. To understand such a complicated regulation and balance, more advanced methodologies, such as gene expression profiling,  may provide  unique capacities or  advantages  to  overview the cellular response to  specific stimuli. Most of islet differentiation  factors are  transcriptional factors, and several transcripts of these factors in GLP-1 treated islets have be identified in this study.  GLP-1  increased  the expression of BARX1,  PAX4,  and  decreased  the expression of MYB,  NKx6-1,  FCMD,  and  ZNF185. The  homoebox  gene  Barx1  is  highly  expressed  in prospective stomach mesenchyme and is required to specify this organ [27], while PAX4 plays a role in the differentiation and development of pancreatic islet cells [28]. This is consistent with Brun’s  observation of that Pax4 expresses in human pancreatic islets and  is activated during mitoss and by GLP-1  treatment [29].  MYB is essential for  mammary tumorigenesis and  its upregulation is associated with estrogen receptor (ER)-positive breast cancer [30]; NKx6-1 is a popular transcription factor involved in differentiation and  development of pancreatic islet β-cells [31].  Significantly elevated expression of INSM1 was reported to be associated with both the AR42J cell line and  the primary cultured  mouse acinar cells differentiation into  insulin-positive cells [32]. However in our observations, the expression of NKx6-1 was downregulated in GLP-1 treated islets.  Similarly, the reduced expression of FCMD, which was reported to be involved  in brain development [33], and ZNF185, a tumor-suppressor protein associated  with actin-cytoskeleton  [34] and reported in prostate cancer, was observed in our studies.  Beyond our expectation, we were unable to detect the expression of PDX-1, although it is among the most extensively studied pancreatic transcriptional factor [35], responding to GLP-1 induction.This study provides, the first time, the gene expression profile of human islets regulated by GLP-1 induction. The data presented here further supports the complex and diversity effects of GLP-1  in  the regulation of protein  governing,  beta-cell  mass control, and  metabolism.  The elucidation  of  the  signal pathway  triggered  by  GLP-1  may  provide  a  scientific  basis  for molecular target identification, new drugs design, and diabetes disorder treatment.



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*Source: 608672-2012-09-29.xml*