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Identification of differentially expressed mRNA during pancreas regeneration of rat by mRNA differential display
BBRC Biochemical and Biophysical Research Communications 299 (2002) 806–812 www.academicpress.com
Identification of differentially expressed mRNA during pancreas regeneration of rat by mRNA differential display H.W. Lim,a J.E. Lee,b S.J. Shin,a Y.E. Lee,a S.H. Oh,b J.Y. Park,b J.K. Seong,b,1,2 and J.-S. Parka,*,2 a
School of Chemistry & Molecular Engineering, Seoul National University, San 56-1, Shilim-dong, Kwanak-gu, Seoul 151-742, South Korea b Laboratory of Developmental Biology and Genomics, College of Veterinary Medicine, Seoul National University, 103 Seodun-dong, Kwonsun-gu, Suwon, Seoul 441-744, South Korea Received 14 October 2002
Abstract Pancreatectomy (Px) is known to cause islet hypertrophy and is a putative method to mimic hyperglycemia representing type II diabetes mellitus. Therefore, finding new genes related to pancreatectomy will help to understand the molecular mechanism of hypertrophy and hyperglycemia, and may provide new diagnostic markers of type II diabetes. To this end, mRNA differential display was used to isolate genes that show transcriptional changes in pancreas of rat after 90% partial pancreatectomy. Forty-nine candidate pancreas regeneration-associated transcripts were isolated. cDNA sequencing and subsequent database analysis revealed that 15 transcripts showed no significant sequence similarity to previously reported genes, whereas 34 transcripts showed significant similarity with genes deposited in the GenBank. The differential mRNA expression of 49 transcripts was confirmed using screening of slot blots and Northern blot analysis was performed to several genes. It was noteworthy that the Wnt-1 inducible signaling pathway protein-1 (WISP-1), Ras-associated protein 1B (Rap1B), vascular cell adhesion molecule-1 (VCAM-1), and huntingtin interacting protein genes (HIP) were observed to be over-expressed during pancreas regeneration. Several genesÕ expression was modified by pancreatectomy. Profiling of gene expression in response to pancreatectomy may lead to new insights into hypertrophy and hyperglycemia representing type II diabetes, as well as into the identification of novel diagnostic markers of type II diabetes. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Pancreatectomy; mRNA differential display; Diabetes; Hyperglycemia; Pentylenetetrazol-related mRNA; Vascular cell adhesion molecule 1; Wnt-1 inducible signaling pathway protein 1; Huntingtin interacting protein; Ras-associated protein 1B; Carboxypeptidase B
Type II diabetes (NIDDM) is a common disease, which is characterized by disturbance of glucose homeostasis and variable plasma insulin levels. Some diabetic patients show elevated blood insulin level. This finding is totally different from type I diabetes (IDDM) in which less insulin is secreted due to the destruction of insulin producing pancreatic b cells. Also, type II diabetes is characterized by the combination of insulin resistance and hyperinsulinemia [1]. In many type II diabetic patients, the insulin resistance is exacerbated by obesity [2] and hyperinsulinemia is, in part, a result of the deleterious *
Corresponding author. Fax: +82-2-877-5110. E-mail addresses: [email protected] (J.K. Seong), [email protected] (J.-S. Park). 1 Also corresponding author. Fax: 82-2-363-7984. 2 These corresponding authors contributed equally to this work.
effect of chronic hyperglycemia. It has been reported that hyperglycemia triggers loss of pancreatic b cells [3]. A number of studies relating type II diabetes to molecular level have focused on identifying pancreatic b cell specific genes [4–7]; however, these studies had limitations in that they cannot fully characterize the whole profile of gene expression that underlies the pathogenesis of type II diabetes. In this experiment, we have attempted to identify molecular markers for diagnostic usage for type II diabetes, as well as to investigate the whole range of molecular changes that could occur during the onset of chronic hyperglycemia. To identify genes that display transcriptional changes in the rat pancreas after 90% partial pancreatectomy (Px), we performed mRNA differential display [8]. mRNA differential display was developed as a tool to detect and characterize altered gene expression. Unlike the candidate
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gene approach, this technique allows the simultaneous isolation of multiple differentially expressed genes and is not limited solely to known genes. In this study we have used the rat pancreas of shamoperated and 90% partial pancreatectomy, which has been well accepted as a hyperglycemia-linked type II diabetes model, in conjunction with the mRNA differential display technique. Functional characterization of such genes will aid in elucidating the underlying molecular mechanisms that are occurring in the pancreas regeneration, could help to understand the molecular mechanism of type II diabetes related to chronic hyperglycemia, and may ultimately provide molecular markers for diagnostic purposes or therapeutic intervention of diabetes. Here, we expected that most, if not all, differentially expressed genes between Px rat pancreas and shamoperated rat pancreas would probably be involved in pancreas regeneration and hyperglycemia-related function. Materials and methods Ninety percent pancreatectomy. Four to five-week-old male Sprague–Dawley rats (90–200 g) were generally anesthetized (Ketamine hydrochloride 85 mg/kg and xylazine hydrochloride 7 mg/kg), and in addition, were given ether if needed. Ninety percent of their pancreas was removed by the reported technique [9]. For a 90% Px, all of the tail portion and most of the head of the pancreas were removed by cotton applicators; the major blood vessels were left intact so as not to compromise other organs. The remnant pancreas was anatomically well defined, being the tissue within 2 mm of the common bile duct and extending from the duct to the first part of the duodenum. This remnant is the upper portion of the head of the pancreas thought to be embryologically of dorsal anlagen origin. Preparation of total RNA. The frozen rat pancreas tissue was homogenized and the total RNA was isolated following manufacturerÕs suggested protocol using RNAwiz (Ambion, Austin, TX, USA). RNA was collected from the aqueous phase following the addition of 0.2 the starting volume of chloroform, precipitated by adding 0.5 the starting volume of RNase-free water (Promega, Madison, WI, USA), 1 the starting volume of isopropyl alcohol, washed with 75% ethanol, and then air-dried. The dried RNA pellet was then dissolved in RNase-free water. The concentration of RNA was determined by the absorbance at 260 nm with a spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). Differential display of mRNA. To acquire cDNA fragments, we performed reverse transcription (RT). Total RNA (0:2 lg) was mixed with 2:0 ll of 2:0 lM one base-anchored oligo(dT) primer, 9:4 ll RNase-free water was added, and the primer was annealed to the RNA template by incubating at 70 °C for 10 min and quickly cooling. Then, we added 4:0 ll of 5 RT buffer (25 mM Tris–HCl, pH 8.3, 38 mM KCl, 1.5 mM MgCl2 , 5 mM dithiothreitol), 1:6 ll of 250 lM dNTP, and 1:0 ll reverse transcriptase (Promega, Madison, WI, USA). Firststrand cDNA was synthesized by incubation at 37 °C for 1 h. And this reaction was terminated by heating the mixture at 70 °C for 10 min. Amplification of cDNA was performed using Tag DNA polymerase (Promega, Madison, WI, USA). Each PCR mixture contained 2:0 ll of the product from RT, 2:0 ll of 10 Taq-PCR buffer (10 mM Tris–HCl, pH 9.0, 50 mM KCl, and 0.1% Triton X-100), 1:2 ll MgCl2; 1:6 ll of 25 uM dNTP, 2:0 ll of 2 lM 30 one base-anchored oligo(dT) primer which was used in the first-strand synthesis reaction, 2:0 ll of 2:0 lM 50 arbitrary primer (one of eight), 1:0 ll ½a-32 PdCTP (ICN Phamaceuticals, Costa Mesa, CA, USA), and 0:2 ll Tag polymerase, and finally water
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was added up to 40 ll. The PCR cycling parameters were as follows: 94 °C for 30 s, 42 °C for 2 min, and 72 °C for 30 s for 40 cycles, followed by 72 °C for 5 min. The amplified cDNAs were size-fractionated in parallel by 6% polyacrylamide and 8 M urea gel electrophoresis. After electrophoresis, gel was dried onto Whatman 3 mm paper using gel-drier and exposed to X-ray film (Agfa CP-BU film) at )70 °C. Differentially expressed cDNAs were visualized by autoradiography. To acquire differentially expressed cDNA fragments, the regions of dried gel corresponding to the cDNAs were cut. Gel slices were placed in a microtube with 100 ll water. The tube was heated at 100 °C for 15 min and the supernatant was precipitated with ethanol in the presence of 0.3 M sodium acetate (Sigma, St. Louis, MO, USA) and 10 lg glycogen (Sigma, St. Louis, MO, USA). The precipitated cDNA fragments were amplified by PCR. The PCR condition was similar to the above, except in that the dNTP concentration was increased to 20 lM and ½a-32 PdCTP was not used. Amplified cDNA fragments were purified using GENECLEAN Kit (Qiagen, Hilden, Germany). Slot blotting. Three hundred ng cDNA fragments derived from differential display bands were dissolved in 360 ll buffer, respectively, which contained the following components: 120 ll water, 120 ll solution 1 (0.5 M NaOH, 1.5 M NaCl), and 120 ll solution 2 (1.0 M Tris– HCl, pH 7.4). This buffer was heated at 100 °C for 5 min and cooled immediately. The 150 ll buffer was transferred onto a Nytran membrane (Schleicher and Schuell, Dassel, Germany) with a slot-blotter (Schleicher and Schuell, Dassel, Germany) and a cDNA fragment was fixed using a UV-crosslinker (Hoefer, San Francisco, CA, USA). Twenty ng total RNA was radiolabeled with ½a-32 PdCTP through the process of reverse transcription and a labeled probe was purified using a G-50 column. The cpm values of two kinds of radiolabeled cDNAs were counted using a scintillation counter (Wallac Oy, Turku, Finland) and normalized. Actin gene was used as a control. The hybridization was performed with the following procedure: the membrane was prehybridized in Church buffer (1.0 mM EDTA, Na2 HPO4 0.25 M, pH 7.2, 1.0% SDS) at 55 °C for 3 h. After prehybridization, the probe was hybridized in the same Church buffer at 55 °C overnight. The membrane was washed three times at 25 °C with 2 SSC containing 0.1% SDS for 10 min each, followed by three additional washes at 25 °C in 0.1 SSC containing 0.1% SDS of 5 min each. The membrane was then exposed to X-ray film at )70 °C. Subcloning and DNA sequence analysis. Purified cDNA fragments were directly inserted into the TA cloning pGEM-T Easy vector system (Promega, Madison, WI, USA). Competent Escherichia coli cells (JM 109 strain) were transformed with ligated vectors. These subclones were purified using a Qiaprep Spin Kit (Qiagen, Hilden, Germany). DNA inserts were subsequently sequenced using M13 primer with an automatic DNA sequencer (ALF express). The result was compared for homology with BLAST sequence analysis program through the National Center for Biotechnology Information. Northern blotting. Samples of total RNA ð10:0 lgÞ were denatured in 2.2 M formaldehyde, 50% (v/v) formamide buffer and separated by denaturing gel electrophoresis on a 1.0% agarose, 2.2 M formaldehyde gel. The RNA was transferred to Nytran plus membrane and the RNA was fixed using a UV-crosslinker. The membrane was hybridized with 32 P-labeled cDNA fragments. The probe was labeled with ½a-32 PdCTP using a Random Primer DNA labeling Kit (Takara, Otsu, Shiga, Japan). Prehybridization and hybridization solution and methods were the same as those used for slot blotting.
Results Products of mRNA differential display Total RNA concentration was measured by spectrophotometry and then visualized on the 1%
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Table 1 Primers used for mRNA differential display Upstream primer AP-1 2AP-2 AP-3 AP-4 AP-5 AP-6 AP-7 AP-8
50 -AAGCTTGATTGCC-30 50 -AAGCTTCGACTGT-30 50 -AAGCTTTGGTCAG-30 50 -AAGCTTCTCAACG-30 50 -AAGCTTAGTAGGC-30 50 -AAGCTTGCACCAT-30 50 -AAGCTTAACGAGG-30 50 -AAGCTTTTACCGC-30
Downstream primer
T11 C T11 A T11 G
50 -AAGCTT11 C-30 50 -AAGCTT11 A-30 50 -AAGCTT11 G-30
AP means arbitrary primer and numbering 1–8. We used three downstream primers in conjunction with eight upstream primers for mRNA differential display and had 24 combinations.
formamide–aldehyde gel. The quantity and integrity of total RNA were assured by comparing the intensities of 28S and 18S rRNA bands. To identify differentially expressed genes associated with chronic hyperglycemia, twenty-four mRNA differential display reactions were performed using the pancreas of a Px rat and a shamoperated control rat. Three downstream anchored primers were used with the sequence AAGCTT11 X
(where X can be A, G, or C) in conjunction with eight upstream random and arbitrary primers for PCR display (Table 1). As a result of the mRNA differential display, a total of 180 putative differentially expressed cDNAs were found. A representative pattern of mRNA differential display is shown in Fig. 1. These bands are excised from the gel and reamplified by PCR.
Fig. 2. Representative pattern of slot blotting result. Isolated cDNA fragments from the gel were amplified by PCR and purified. These cDNA fragments were transferred to nylon membrane and hybridized with radiolabeled cDNA, which was obtained from pancreas mRNA using by reverse transcription method. Actin was used for control. (A) Sham-operated pancreas and (B) pancreatectomized pancreas. 1, Actin; 2, unknown; 3, Homo sapiens cDNA FLJ13819 clone; 4, HIP gene; 5, rat pancreas triglyceride lipase gene; 6, DRCF-5 gene.
Fig. 1. Representative pattern of mRNA differential display. Two RNA samples from Px rat pancreas and normal rat pancreas were compared by differential display using three different one-baseanchored oligo(dT) primers and eight arbitrary upstream primers. In this figure, AAGCTT11 G and AP-8 primer pair was used. Arrows show differentially expressed cDNA fragments.
Fig. 3. Northern blot analysis. Ten lg total RNA from sham-operated rat pancreas and Px rat pancreas was loaded on to agarose gel, blotted, and hybridized. 18s RNA was used as a control. (A) Carboxypeptidase B gene, (B) Rap 1B gene, (C) pentylenetetrazol-related mRNA; PTZ17 gene, (D) 18s RNA, and (E) 18s RNA gel loading picture. N, Shamoperated pancreas; Px, 90% partial pancreatectomized pancreas.
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Verification of the cDNA fragment by slot blotting and Northern blotting To confirm the expression pattern observed by mRNA differential display, we performed slot blotting (Fig. 2). An equal amount of actin gene was used as an internal control and 49 differentially expressed genes were selected for which the difference in the intensities of bands was most prominent. Some genes like carboxypeptidase B, Rap 1B, and PTZ-17 were subjected to
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Northern blot analysis to confirm the mRNA differential display results. 18s RNA was used as a control to adjust the quantity of sample RNA (Fig. 3). The observed pattern of Northern blotting was consistent with the slot blotting results. Sequence analysis Forty-nine cDNAs as revealed by slot blotting analysis were subjected to DNA sequencing by subcloning
Table 2 cDNA clones over-expressed in normal rat pancreas Clone
Gene homology
N/Px ratio
A1-2 A1-10 A2-1 A2-8 A3-10 A3-12 A5-4 A8-4 G3-2 G6-7 G3-4
Serine protease inhibitor (Kazal type I) Carboxypeptidase B Pancreatic triglyceride lipase Mitochondrial translational elongation factor Diphosphoinositol polyphosphate phosphohydrolase type II (Nudt 4) Similar to actin related protein 2/3 complex, subunit 5 (16 kDa) Pancreatic ribonuclease mRNA Zymogen granule protein; ZG-16p Adult male tongue cDNA Adult male testis cDNA Developmentally regulated cardiac factor (DRCF-5)
2.3 2.5 1.5 1.5 1.6 3.5 2.0 1.5 2.9 2.2 2.3
cDNA bands which were over-expressed in normal rat pancreas detected by mRNA differential display. These bands were confirmed using by slot blotting method, cloned into the pGEM-T Easy vector, and analyzed by automated DNA sequencer. These cDNA bands were identified using the GenBank. N/Px ratio was detected using Sigma plot 4.0 program. A second clone, A1-10 represents carboxypeptidase B gene fragment, which is overexpressed in normal rat pancreas compared to Px rat pancreas as 2.5 times.
Table 3 cDNA clones over-expressed in Px rat pancreas Clone
Gene homology
Px/N ratio
A1-8 A2-2 A2-4 A2-5 A2-6 A2-7 A2-11 A4-9 A5-1 A5-8 A6-8 A6-9 A6-10 A8-1 C2-2 C2-7 C6-2 C6-5 C6-6 C8-2 G2-5 G2-8 G5-15
Human DNA sequence from clone RP1-127B20 on chromosome 22 Mus musculus adult male stomach cDNA Ras-associated protein 1B (Rap 1B) Pentylenetetrazol-related mRNA, PTZ-17 Mus musculus 10-day embryo cDNA Cyclopilin A Ribosomal protein S29, Rps29 Rattus norvegicus mRNA for p47 Surfeit 1 (Surf1) mRNA Sprague–Dawley mRNA for ribosomal protein L24 Rattus norvegicus ATP systhase subunit d Huntingtin interacting protein (HIP) F1-ATPase a subunit Calmodulin Pituitary tumor X2CR1 protein Vascular cell adhesion molecule 1 (VCAM-1) mRNA for endolyn RGC-32 mRNA Rat ribosomal protein S19 Wnt-1 inducible signaling pathway protein 1 (WISP-1) Anaphase-promoting complex subunit 5 Rattus norvegicus mama mRNA 10, 11 days embryo cDNA
1.5 1.8 1.6 1.6 1.6 2.8 1.4 1.4 1.6 1.5 1.8 2.2 2.2 3.0 2.5 2.0 1.8 2.0 2.0 1.9 1.9 1.5 1.5
cDNA bands which were over-expressed in Px rat pancreas detected by mRNA differential display. These bands were confirmed using by slot blotting method, cloned into the pGEM-T Easy vector, and analyzed by automated DNA sequencer. These cDNA bands were identified using the GenBank. Px/N ratio was detected using Sigma plot 4.0 program. A third clone, A2-4, represents Rap 1B gene fragment, which is over-expressed in Px rat pancreas compared to normal rat pancreas as 1.6 times.
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into a pGEM-T Easy vector. The resulting sequences were compared with the GenBank database using the BLAST search program [10]. Thirty-four clones were matched with those of already known genes and the other fifteen genes did not match any of those genes deposited in the GenBank databases. Among the known genes, 11 genes, for instance, serine protease inhibitor (Kazal type I), carboxypeptidase B, and mitochondrial
translational elongation factor were over-expressed in sham-operated rat pancreas. The other 23 genes were over-expressed in the pancreas of Px rat, for instance, human DNA sequence from clone RP1-127B20 on chromosome 22, Rap 1B, and rat ribosomal protein S29. Over-expressed known genes in sham-operated rat pancreas and Px rat pancreas are summarized in Tables 2 and 3, respectively. Unknown genes are listed in Table 4.
Table 4 Unknown cDNA clone sequences
cDNA bands which were differentially expressed. These bands were confirmed using by slot blotting method, cloned into the pGEM-T Easy vector, and analyzed by automated DNA sequencer. These cDNA bands sequence were not found in the GenBank.
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Discussion In the present study, we isolated 49 genes. Thirty-four genes were already known and the other 15 have not previously been reported. Among the known genes, the relation of some of the genes, i.e., Wnt-1 inducible signaling pathway protein1 (WISP-1), Rap1B, VCAM-1, and HIP, to the Px has not been reported to date. At this stage, we did not know the reason why these genes were expressed differentially. However, several genes might play important roles in Px rat pancreas. WISP-1 The adenomatous polyposis coli tumor suppressor gene (APC) plays an important role in the Wnt signaling pathway by regulating b-catenin levels [11–13] and c-myc was identified as a target of the APC pathway [14]. WISP-1 is a downstream target gene of Wnt-1 [15]. And chronic hyperglycemia caused by Px leads to b cell hypertrophy, in parallel with an increase of c-myc [3]. We observed that WISP-1 gene was over-expressed twice as much in the Px rat pancreas as in the sham-operated rat pancreas. Our study, taken together with the previous studies about the Wnt signaling pathway, raises the possibility that b cell hypertrophy caused by Px is mediated by the APC pathway or Wnt signaling pathway. Rap 1B Rap 1B is known as an activator of B-Raf and, subsequently, of the MEK signaling pathway. However, in some cell types, as in fibroblast, Rap 1 antagonizes Rasinduced effects [16,17]. Thus, Rap 1 inhibits MEK signaling, presumably by trapping Raf1 as an inactive complex. Also, it is believed the MEK signaling pathway is related to glycogen synthesis [18]. We found that Rap 1B was over-expressed in the Px rat pancreas. The function of Rap 1B is not known in pancreatic cells. Therefore, we are not sure whether Rap 1B plays an agonistic or antagonistic role in pancreatic cells at this stage. However it is believed Rap 1B is related to MEK-signaling, which is one of the glycogen synthesis pathways. Further characterization of the function of Rap 1B in the pancreas will elucidate its role in the Px rat pancreas. VCAM-1 Vascular cell adhesion molecule-1 (VCAM-1), expressed in the early stages of atherosclerosis, is known to be over-expressed in endothelial cells (EC) of diabetic rabbits [19], and is related to oxidative stress in EC. VCAM-1 gene expression is upregulated when oxidative stress induced by IL-4 is received and the plasma VCAM-1 level is downregulated in an NIDDM patient by the reduction of oxidative stress [20]. Oxidative stress
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by chronic hyperglycemia damages pancreatic b cells [21] and chronic hyperglycemia leads to b cell hypertrophy, which eventually facilitates the apoptosis of b cells [22]. However, the hypertrophic beta cell population was found not to have altered after 4 weeks postPx. This result suggests the expression of protective stress genes [23]. Antioxidant genes, for example, OH-1, Mn-superoxide dismutase, and glutathione peroxidase have shown to be over-expressed in the Px rat pancreas [23]. In addition, oxidative stress damages pancreatic acinar cells and antioxidant protein effectively protects from cell damage [24,25]. In this respect, it is possible that the over-expression of VCAM-1 gene in the Px rat pancreas resulted in the protection of cells from oxidative stress in diabetic rat pancreas. HIP Huntingtin interacting protein (HIP) is known to block the hedgehog signal pathway [26]. Hedgehogs (Hhs) are intercellular signaling molecules consisting of Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Hhs are known to regulate tissue patterning in development. Shh affects cell proliferation in pituitary gland development [26] and the absence of Shh is required in the early stage of development in the pancreas [27,28]. Recent studies revealed that Hh is also necessary for the signaling pathway in differentiated b cells of the endocrine pancreas and activation of Hh signaling increases rat insulin I promoter activation [29]. Therefore, the over-expression of HIP mRNA in the Px rat is likely to be related to the organogenesis of pancreas and HIP may be considered as a potential factor in the pathogenesis of diabetes. The functions of some genes found in our system are not fully characterized yet. However, functions of the genes, such as RGC-32, ZG-16p, clone A1-8, and Kazal type I, are known. RGC-32 is related to cell-cycle activation [30], ZG-16p is incorporated into secretory granules in exocrine cells [31], clone A1-8 may correspond to a novel transcription factor since it possesses a characteristic PHD finger domain [32], and Kazal type I (serine protease inhibitor) is known to be related to pancreatitis [33]. Further functional studies on these newly found clones including unknown genes will lead to elucidation of their roles in the Px rat pancreas and broaden our understanding of the pathogenesis of type II diabetes. Acknowledgments This work was supported by the Korea Research Foundation (DP0344) and the Molecular Therapy Center of KOSEF (R03-200100031). And also, this work was partly supported by the Grants from Ministry of Health, Korea (HMP-00-CO-06-0006) to J.K. Seong.
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Identification of differentially expressed mRNA during pancreas regeneration of rat by mRNA differential display H.W. Lim,a J.E. Lee,b S.J. Shin,a Y.E. Lee,a S.H. Oh,b J.Y. Park,b J.K. Seong,b,1,2 and J.-S. Parka,*,2 a
School of Chemistry & Molecular Engineering, Seoul National University, San 56-1, Shilim-dong, Kwanak-gu, Seoul 151-742, South Korea b Laboratory of Developmental Biology and Genomics, College of Veterinary Medicine, Seoul National University, 103 Seodun-dong, Kwonsun-gu, Suwon, Seoul 441-744, South Korea Received 14 October 2002
Abstract Pancreatectomy (Px) is known to cause islet hypertrophy and is a putative method to mimic hyperglycemia representing type II diabetes mellitus. Therefore, finding new genes related to pancreatectomy will help to understand the molecular mechanism of hypertrophy and hyperglycemia, and may provide new diagnostic markers of type II diabetes. To this end, mRNA differential display was used to isolate genes that show transcriptional changes in pancreas of rat after 90% partial pancreatectomy. Forty-nine candidate pancreas regeneration-associated transcripts were isolated. cDNA sequencing and subsequent database analysis revealed that 15 transcripts showed no significant sequence similarity to previously reported genes, whereas 34 transcripts showed significant similarity with genes deposited in the GenBank. The differential mRNA expression of 49 transcripts was confirmed using screening of slot blots and Northern blot analysis was performed to several genes. It was noteworthy that the Wnt-1 inducible signaling pathway protein-1 (WISP-1), Ras-associated protein 1B (Rap1B), vascular cell adhesion molecule-1 (VCAM-1), and huntingtin interacting protein genes (HIP) were observed to be over-expressed during pancreas regeneration. Several genesÕ expression was modified by pancreatectomy. Profiling of gene expression in response to pancreatectomy may lead to new insights into hypertrophy and hyperglycemia representing type II diabetes, as well as into the identification of novel diagnostic markers of type II diabetes. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Pancreatectomy; mRNA differential display; Diabetes; Hyperglycemia; Pentylenetetrazol-related mRNA; Vascular cell adhesion molecule 1; Wnt-1 inducible signaling pathway protein 1; Huntingtin interacting protein; Ras-associated protein 1B; Carboxypeptidase B
Type II diabetes (NIDDM) is a common disease, which is characterized by disturbance of glucose homeostasis and variable plasma insulin levels. Some diabetic patients show elevated blood insulin level. This finding is totally different from type I diabetes (IDDM) in which less insulin is secreted due to the destruction of insulin producing pancreatic b cells. Also, type II diabetes is characterized by the combination of insulin resistance and hyperinsulinemia [1]. In many type II diabetic patients, the insulin resistance is exacerbated by obesity [2] and hyperinsulinemia is, in part, a result of the deleterious *
Corresponding author. Fax: +82-2-877-5110. E-mail addresses: [email protected] (J.K. Seong), [email protected] (J.-S. Park). 1 Also corresponding author. Fax: 82-2-363-7984. 2 These corresponding authors contributed equally to this work.
effect of chronic hyperglycemia. It has been reported that hyperglycemia triggers loss of pancreatic b cells [3]. A number of studies relating type II diabetes to molecular level have focused on identifying pancreatic b cell specific genes [4–7]; however, these studies had limitations in that they cannot fully characterize the whole profile of gene expression that underlies the pathogenesis of type II diabetes. In this experiment, we have attempted to identify molecular markers for diagnostic usage for type II diabetes, as well as to investigate the whole range of molecular changes that could occur during the onset of chronic hyperglycemia. To identify genes that display transcriptional changes in the rat pancreas after 90% partial pancreatectomy (Px), we performed mRNA differential display [8]. mRNA differential display was developed as a tool to detect and characterize altered gene expression. Unlike the candidate
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 2 7 4 1 - 9
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gene approach, this technique allows the simultaneous isolation of multiple differentially expressed genes and is not limited solely to known genes. In this study we have used the rat pancreas of shamoperated and 90% partial pancreatectomy, which has been well accepted as a hyperglycemia-linked type II diabetes model, in conjunction with the mRNA differential display technique. Functional characterization of such genes will aid in elucidating the underlying molecular mechanisms that are occurring in the pancreas regeneration, could help to understand the molecular mechanism of type II diabetes related to chronic hyperglycemia, and may ultimately provide molecular markers for diagnostic purposes or therapeutic intervention of diabetes. Here, we expected that most, if not all, differentially expressed genes between Px rat pancreas and shamoperated rat pancreas would probably be involved in pancreas regeneration and hyperglycemia-related function. Materials and methods Ninety percent pancreatectomy. Four to five-week-old male Sprague–Dawley rats (90–200 g) were generally anesthetized (Ketamine hydrochloride 85 mg/kg and xylazine hydrochloride 7 mg/kg), and in addition, were given ether if needed. Ninety percent of their pancreas was removed by the reported technique [9]. For a 90% Px, all of the tail portion and most of the head of the pancreas were removed by cotton applicators; the major blood vessels were left intact so as not to compromise other organs. The remnant pancreas was anatomically well defined, being the tissue within 2 mm of the common bile duct and extending from the duct to the first part of the duodenum. This remnant is the upper portion of the head of the pancreas thought to be embryologically of dorsal anlagen origin. Preparation of total RNA. The frozen rat pancreas tissue was homogenized and the total RNA was isolated following manufacturerÕs suggested protocol using RNAwiz (Ambion, Austin, TX, USA). RNA was collected from the aqueous phase following the addition of 0.2 the starting volume of chloroform, precipitated by adding 0.5 the starting volume of RNase-free water (Promega, Madison, WI, USA), 1 the starting volume of isopropyl alcohol, washed with 75% ethanol, and then air-dried. The dried RNA pellet was then dissolved in RNase-free water. The concentration of RNA was determined by the absorbance at 260 nm with a spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). Differential display of mRNA. To acquire cDNA fragments, we performed reverse transcription (RT). Total RNA (0:2 lg) was mixed with 2:0 ll of 2:0 lM one base-anchored oligo(dT) primer, 9:4 ll RNase-free water was added, and the primer was annealed to the RNA template by incubating at 70 °C for 10 min and quickly cooling. Then, we added 4:0 ll of 5 RT buffer (25 mM Tris–HCl, pH 8.3, 38 mM KCl, 1.5 mM MgCl2 , 5 mM dithiothreitol), 1:6 ll of 250 lM dNTP, and 1:0 ll reverse transcriptase (Promega, Madison, WI, USA). Firststrand cDNA was synthesized by incubation at 37 °C for 1 h. And this reaction was terminated by heating the mixture at 70 °C for 10 min. Amplification of cDNA was performed using Tag DNA polymerase (Promega, Madison, WI, USA). Each PCR mixture contained 2:0 ll of the product from RT, 2:0 ll of 10 Taq-PCR buffer (10 mM Tris–HCl, pH 9.0, 50 mM KCl, and 0.1% Triton X-100), 1:2 ll MgCl2; 1:6 ll of 25 uM dNTP, 2:0 ll of 2 lM 30 one base-anchored oligo(dT) primer which was used in the first-strand synthesis reaction, 2:0 ll of 2:0 lM 50 arbitrary primer (one of eight), 1:0 ll ½a-32 PdCTP (ICN Phamaceuticals, Costa Mesa, CA, USA), and 0:2 ll Tag polymerase, and finally water
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was added up to 40 ll. The PCR cycling parameters were as follows: 94 °C for 30 s, 42 °C for 2 min, and 72 °C for 30 s for 40 cycles, followed by 72 °C for 5 min. The amplified cDNAs were size-fractionated in parallel by 6% polyacrylamide and 8 M urea gel electrophoresis. After electrophoresis, gel was dried onto Whatman 3 mm paper using gel-drier and exposed to X-ray film (Agfa CP-BU film) at )70 °C. Differentially expressed cDNAs were visualized by autoradiography. To acquire differentially expressed cDNA fragments, the regions of dried gel corresponding to the cDNAs were cut. Gel slices were placed in a microtube with 100 ll water. The tube was heated at 100 °C for 15 min and the supernatant was precipitated with ethanol in the presence of 0.3 M sodium acetate (Sigma, St. Louis, MO, USA) and 10 lg glycogen (Sigma, St. Louis, MO, USA). The precipitated cDNA fragments were amplified by PCR. The PCR condition was similar to the above, except in that the dNTP concentration was increased to 20 lM and ½a-32 PdCTP was not used. Amplified cDNA fragments were purified using GENECLEAN Kit (Qiagen, Hilden, Germany). Slot blotting. Three hundred ng cDNA fragments derived from differential display bands were dissolved in 360 ll buffer, respectively, which contained the following components: 120 ll water, 120 ll solution 1 (0.5 M NaOH, 1.5 M NaCl), and 120 ll solution 2 (1.0 M Tris– HCl, pH 7.4). This buffer was heated at 100 °C for 5 min and cooled immediately. The 150 ll buffer was transferred onto a Nytran membrane (Schleicher and Schuell, Dassel, Germany) with a slot-blotter (Schleicher and Schuell, Dassel, Germany) and a cDNA fragment was fixed using a UV-crosslinker (Hoefer, San Francisco, CA, USA). Twenty ng total RNA was radiolabeled with ½a-32 PdCTP through the process of reverse transcription and a labeled probe was purified using a G-50 column. The cpm values of two kinds of radiolabeled cDNAs were counted using a scintillation counter (Wallac Oy, Turku, Finland) and normalized. Actin gene was used as a control. The hybridization was performed with the following procedure: the membrane was prehybridized in Church buffer (1.0 mM EDTA, Na2 HPO4 0.25 M, pH 7.2, 1.0% SDS) at 55 °C for 3 h. After prehybridization, the probe was hybridized in the same Church buffer at 55 °C overnight. The membrane was washed three times at 25 °C with 2 SSC containing 0.1% SDS for 10 min each, followed by three additional washes at 25 °C in 0.1 SSC containing 0.1% SDS of 5 min each. The membrane was then exposed to X-ray film at )70 °C. Subcloning and DNA sequence analysis. Purified cDNA fragments were directly inserted into the TA cloning pGEM-T Easy vector system (Promega, Madison, WI, USA). Competent Escherichia coli cells (JM 109 strain) were transformed with ligated vectors. These subclones were purified using a Qiaprep Spin Kit (Qiagen, Hilden, Germany). DNA inserts were subsequently sequenced using M13 primer with an automatic DNA sequencer (ALF express). The result was compared for homology with BLAST sequence analysis program through the National Center for Biotechnology Information. Northern blotting. Samples of total RNA ð10:0 lgÞ were denatured in 2.2 M formaldehyde, 50% (v/v) formamide buffer and separated by denaturing gel electrophoresis on a 1.0% agarose, 2.2 M formaldehyde gel. The RNA was transferred to Nytran plus membrane and the RNA was fixed using a UV-crosslinker. The membrane was hybridized with 32 P-labeled cDNA fragments. The probe was labeled with ½a-32 PdCTP using a Random Primer DNA labeling Kit (Takara, Otsu, Shiga, Japan). Prehybridization and hybridization solution and methods were the same as those used for slot blotting.
Results Products of mRNA differential display Total RNA concentration was measured by spectrophotometry and then visualized on the 1%
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Table 1 Primers used for mRNA differential display Upstream primer AP-1 2AP-2 AP-3 AP-4 AP-5 AP-6 AP-7 AP-8
50 -AAGCTTGATTGCC-30 50 -AAGCTTCGACTGT-30 50 -AAGCTTTGGTCAG-30 50 -AAGCTTCTCAACG-30 50 -AAGCTTAGTAGGC-30 50 -AAGCTTGCACCAT-30 50 -AAGCTTAACGAGG-30 50 -AAGCTTTTACCGC-30
Downstream primer
T11 C T11 A T11 G
50 -AAGCTT11 C-30 50 -AAGCTT11 A-30 50 -AAGCTT11 G-30
AP means arbitrary primer and numbering 1–8. We used three downstream primers in conjunction with eight upstream primers for mRNA differential display and had 24 combinations.
formamide–aldehyde gel. The quantity and integrity of total RNA were assured by comparing the intensities of 28S and 18S rRNA bands. To identify differentially expressed genes associated with chronic hyperglycemia, twenty-four mRNA differential display reactions were performed using the pancreas of a Px rat and a shamoperated control rat. Three downstream anchored primers were used with the sequence AAGCTT11 X
(where X can be A, G, or C) in conjunction with eight upstream random and arbitrary primers for PCR display (Table 1). As a result of the mRNA differential display, a total of 180 putative differentially expressed cDNAs were found. A representative pattern of mRNA differential display is shown in Fig. 1. These bands are excised from the gel and reamplified by PCR.
Fig. 2. Representative pattern of slot blotting result. Isolated cDNA fragments from the gel were amplified by PCR and purified. These cDNA fragments were transferred to nylon membrane and hybridized with radiolabeled cDNA, which was obtained from pancreas mRNA using by reverse transcription method. Actin was used for control. (A) Sham-operated pancreas and (B) pancreatectomized pancreas. 1, Actin; 2, unknown; 3, Homo sapiens cDNA FLJ13819 clone; 4, HIP gene; 5, rat pancreas triglyceride lipase gene; 6, DRCF-5 gene.
Fig. 1. Representative pattern of mRNA differential display. Two RNA samples from Px rat pancreas and normal rat pancreas were compared by differential display using three different one-baseanchored oligo(dT) primers and eight arbitrary upstream primers. In this figure, AAGCTT11 G and AP-8 primer pair was used. Arrows show differentially expressed cDNA fragments.
Fig. 3. Northern blot analysis. Ten lg total RNA from sham-operated rat pancreas and Px rat pancreas was loaded on to agarose gel, blotted, and hybridized. 18s RNA was used as a control. (A) Carboxypeptidase B gene, (B) Rap 1B gene, (C) pentylenetetrazol-related mRNA; PTZ17 gene, (D) 18s RNA, and (E) 18s RNA gel loading picture. N, Shamoperated pancreas; Px, 90% partial pancreatectomized pancreas.
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Verification of the cDNA fragment by slot blotting and Northern blotting To confirm the expression pattern observed by mRNA differential display, we performed slot blotting (Fig. 2). An equal amount of actin gene was used as an internal control and 49 differentially expressed genes were selected for which the difference in the intensities of bands was most prominent. Some genes like carboxypeptidase B, Rap 1B, and PTZ-17 were subjected to
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Northern blot analysis to confirm the mRNA differential display results. 18s RNA was used as a control to adjust the quantity of sample RNA (Fig. 3). The observed pattern of Northern blotting was consistent with the slot blotting results. Sequence analysis Forty-nine cDNAs as revealed by slot blotting analysis were subjected to DNA sequencing by subcloning
Table 2 cDNA clones over-expressed in normal rat pancreas Clone
Gene homology
N/Px ratio
A1-2 A1-10 A2-1 A2-8 A3-10 A3-12 A5-4 A8-4 G3-2 G6-7 G3-4
Serine protease inhibitor (Kazal type I) Carboxypeptidase B Pancreatic triglyceride lipase Mitochondrial translational elongation factor Diphosphoinositol polyphosphate phosphohydrolase type II (Nudt 4) Similar to actin related protein 2/3 complex, subunit 5 (16 kDa) Pancreatic ribonuclease mRNA Zymogen granule protein; ZG-16p Adult male tongue cDNA Adult male testis cDNA Developmentally regulated cardiac factor (DRCF-5)
2.3 2.5 1.5 1.5 1.6 3.5 2.0 1.5 2.9 2.2 2.3
cDNA bands which were over-expressed in normal rat pancreas detected by mRNA differential display. These bands were confirmed using by slot blotting method, cloned into the pGEM-T Easy vector, and analyzed by automated DNA sequencer. These cDNA bands were identified using the GenBank. N/Px ratio was detected using Sigma plot 4.0 program. A second clone, A1-10 represents carboxypeptidase B gene fragment, which is overexpressed in normal rat pancreas compared to Px rat pancreas as 2.5 times.
Table 3 cDNA clones over-expressed in Px rat pancreas Clone
Gene homology
Px/N ratio
A1-8 A2-2 A2-4 A2-5 A2-6 A2-7 A2-11 A4-9 A5-1 A5-8 A6-8 A6-9 A6-10 A8-1 C2-2 C2-7 C6-2 C6-5 C6-6 C8-2 G2-5 G2-8 G5-15
Human DNA sequence from clone RP1-127B20 on chromosome 22 Mus musculus adult male stomach cDNA Ras-associated protein 1B (Rap 1B) Pentylenetetrazol-related mRNA, PTZ-17 Mus musculus 10-day embryo cDNA Cyclopilin A Ribosomal protein S29, Rps29 Rattus norvegicus mRNA for p47 Surfeit 1 (Surf1) mRNA Sprague–Dawley mRNA for ribosomal protein L24 Rattus norvegicus ATP systhase subunit d Huntingtin interacting protein (HIP) F1-ATPase a subunit Calmodulin Pituitary tumor X2CR1 protein Vascular cell adhesion molecule 1 (VCAM-1) mRNA for endolyn RGC-32 mRNA Rat ribosomal protein S19 Wnt-1 inducible signaling pathway protein 1 (WISP-1) Anaphase-promoting complex subunit 5 Rattus norvegicus mama mRNA 10, 11 days embryo cDNA
1.5 1.8 1.6 1.6 1.6 2.8 1.4 1.4 1.6 1.5 1.8 2.2 2.2 3.0 2.5 2.0 1.8 2.0 2.0 1.9 1.9 1.5 1.5
cDNA bands which were over-expressed in Px rat pancreas detected by mRNA differential display. These bands were confirmed using by slot blotting method, cloned into the pGEM-T Easy vector, and analyzed by automated DNA sequencer. These cDNA bands were identified using the GenBank. Px/N ratio was detected using Sigma plot 4.0 program. A third clone, A2-4, represents Rap 1B gene fragment, which is over-expressed in Px rat pancreas compared to normal rat pancreas as 1.6 times.
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into a pGEM-T Easy vector. The resulting sequences were compared with the GenBank database using the BLAST search program [10]. Thirty-four clones were matched with those of already known genes and the other fifteen genes did not match any of those genes deposited in the GenBank databases. Among the known genes, 11 genes, for instance, serine protease inhibitor (Kazal type I), carboxypeptidase B, and mitochondrial
translational elongation factor were over-expressed in sham-operated rat pancreas. The other 23 genes were over-expressed in the pancreas of Px rat, for instance, human DNA sequence from clone RP1-127B20 on chromosome 22, Rap 1B, and rat ribosomal protein S29. Over-expressed known genes in sham-operated rat pancreas and Px rat pancreas are summarized in Tables 2 and 3, respectively. Unknown genes are listed in Table 4.
Table 4 Unknown cDNA clone sequences
cDNA bands which were differentially expressed. These bands were confirmed using by slot blotting method, cloned into the pGEM-T Easy vector, and analyzed by automated DNA sequencer. These cDNA bands sequence were not found in the GenBank.
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Discussion In the present study, we isolated 49 genes. Thirty-four genes were already known and the other 15 have not previously been reported. Among the known genes, the relation of some of the genes, i.e., Wnt-1 inducible signaling pathway protein1 (WISP-1), Rap1B, VCAM-1, and HIP, to the Px has not been reported to date. At this stage, we did not know the reason why these genes were expressed differentially. However, several genes might play important roles in Px rat pancreas. WISP-1 The adenomatous polyposis coli tumor suppressor gene (APC) plays an important role in the Wnt signaling pathway by regulating b-catenin levels [11–13] and c-myc was identified as a target of the APC pathway [14]. WISP-1 is a downstream target gene of Wnt-1 [15]. And chronic hyperglycemia caused by Px leads to b cell hypertrophy, in parallel with an increase of c-myc [3]. We observed that WISP-1 gene was over-expressed twice as much in the Px rat pancreas as in the sham-operated rat pancreas. Our study, taken together with the previous studies about the Wnt signaling pathway, raises the possibility that b cell hypertrophy caused by Px is mediated by the APC pathway or Wnt signaling pathway. Rap 1B Rap 1B is known as an activator of B-Raf and, subsequently, of the MEK signaling pathway. However, in some cell types, as in fibroblast, Rap 1 antagonizes Rasinduced effects [16,17]. Thus, Rap 1 inhibits MEK signaling, presumably by trapping Raf1 as an inactive complex. Also, it is believed the MEK signaling pathway is related to glycogen synthesis [18]. We found that Rap 1B was over-expressed in the Px rat pancreas. The function of Rap 1B is not known in pancreatic cells. Therefore, we are not sure whether Rap 1B plays an agonistic or antagonistic role in pancreatic cells at this stage. However it is believed Rap 1B is related to MEK-signaling, which is one of the glycogen synthesis pathways. Further characterization of the function of Rap 1B in the pancreas will elucidate its role in the Px rat pancreas. VCAM-1 Vascular cell adhesion molecule-1 (VCAM-1), expressed in the early stages of atherosclerosis, is known to be over-expressed in endothelial cells (EC) of diabetic rabbits [19], and is related to oxidative stress in EC. VCAM-1 gene expression is upregulated when oxidative stress induced by IL-4 is received and the plasma VCAM-1 level is downregulated in an NIDDM patient by the reduction of oxidative stress [20]. Oxidative stress
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by chronic hyperglycemia damages pancreatic b cells [21] and chronic hyperglycemia leads to b cell hypertrophy, which eventually facilitates the apoptosis of b cells [22]. However, the hypertrophic beta cell population was found not to have altered after 4 weeks postPx. This result suggests the expression of protective stress genes [23]. Antioxidant genes, for example, OH-1, Mn-superoxide dismutase, and glutathione peroxidase have shown to be over-expressed in the Px rat pancreas [23]. In addition, oxidative stress damages pancreatic acinar cells and antioxidant protein effectively protects from cell damage [24,25]. In this respect, it is possible that the over-expression of VCAM-1 gene in the Px rat pancreas resulted in the protection of cells from oxidative stress in diabetic rat pancreas. HIP Huntingtin interacting protein (HIP) is known to block the hedgehog signal pathway [26]. Hedgehogs (Hhs) are intercellular signaling molecules consisting of Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Hhs are known to regulate tissue patterning in development. Shh affects cell proliferation in pituitary gland development [26] and the absence of Shh is required in the early stage of development in the pancreas [27,28]. Recent studies revealed that Hh is also necessary for the signaling pathway in differentiated b cells of the endocrine pancreas and activation of Hh signaling increases rat insulin I promoter activation [29]. Therefore, the over-expression of HIP mRNA in the Px rat is likely to be related to the organogenesis of pancreas and HIP may be considered as a potential factor in the pathogenesis of diabetes. The functions of some genes found in our system are not fully characterized yet. However, functions of the genes, such as RGC-32, ZG-16p, clone A1-8, and Kazal type I, are known. RGC-32 is related to cell-cycle activation [30], ZG-16p is incorporated into secretory granules in exocrine cells [31], clone A1-8 may correspond to a novel transcription factor since it possesses a characteristic PHD finger domain [32], and Kazal type I (serine protease inhibitor) is known to be related to pancreatitis [33]. Further functional studies on these newly found clones including unknown genes will lead to elucidation of their roles in the Px rat pancreas and broaden our understanding of the pathogenesis of type II diabetes. Acknowledgments This work was supported by the Korea Research Foundation (DP0344) and the Molecular Therapy Center of KOSEF (R03-200100031). And also, this work was partly supported by the Grants from Ministry of Health, Korea (HMP-00-CO-06-0006) to J.K. Seong.
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