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Keywords:

  • PREB;
  • glucokinase;
  • β-cells;
  • cAMP;
  • glucose;
  • promoter;
  • transcription

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Prolactin regulatory element binding (PREB) is a transcription factor that regulates prolactin promoter activity in rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the pancreas. We have recently reported that in pancreatic β-cells, PREB regulates the transcription of the insulin gene in response to glucose stimulation. In the current study, we have examined the role of PREB in regulating glucokinase (GK) in pancreatic β-cells. To analyse the effects of PREB on GK gene transcription, we employed a reporter gene assay. In the cells expressing or with knocked down PREB, GK expression was determined. GK expression was regulated by glucose and cAMP, and both glucose and cAMP stimulated the expression of PREB in a dose-dependent manner. Conversely, overexpression of PREB using a PREB-expressing adenovirus increased the expression of the GK protein. GK enzymatic activity was also significantly increased in the cells that stably expressed PREB. In addition, PREB induced GK promoter activity. Chromatin immunoprecipitation (ChIP) analyses showed that PREB mediated its transcriptional effect by binding to the PREB-responsive cis-element of the GK promoter. Finally, we used siRNA to inhibit PREB expression in cells and demonstrated that the knockdown of PREB attenuated the effects of glucose and cAMP on GK expression. Our data show that in pancreatic β-cells, PREB regulates the transcription of the GK gene in response to glucose and cAMP.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The enzyme glucokinase (GK) acts as a glucose sensor in the pancreatic β-cells [1, 2]. This tissue-specific enzyme is present in the hepatocytes, selected neuroendocrine cells in the brain and gut, and pancreatic β-cells [3–7]. Small changes in the GK activity have large effects on insulin secretion from the pancreatic β-cells [8]. In humans, mutations in the GK gene lead to the maturity-onset diabetes in youth [9] or hyperinsulinaemia [10]. Even though glucose seemed to be responsible for the GK induction, either directly or through its enhanced metabolism, the question of whether the process might be subjected to modification by physiological factors other than the substrate of this reaction has been examined by various researchers [11–14]. For example, placental lactogen, cAMP, biotin and retinoic acid enhanced the GK induction in the pancreatic β-cells. A recent study showed that the GK expression was stimulated and inhibited by cAMP in the pancreas and liver, respectively. These findings support the idea that the regulation of GK transcription in these two tissues is different [12].

The prolactin regulatory element binding (PREB) gene encodes a 1.9-kb mRNA that translates into a transcription factor, which binds to and activates the basal prolactin promoter activity. The PREB protein also mediates the protein kinase A (PKA) action in the pituitary gland [15, 16]. The primary sequence of the PREB protein contains two potential transregulatory PQ-rich domains and three regions of high similarity to the WD-repeat, thus making it a member of a eukaryotic family of WD-repeat proteins. Members of this ever-growing family are involved in multiple cellular functions that include signal transduction, RNA processing, cytoskeletal assembly and vesicle trafficking [17]. The PREB has similarities to a subset of this family that act as gene regulators.

Although PREB transcripts are present in the pancreas (in addition to pituitary, heart and skeletal muscle) [15], its role in the pancreas is not known. Recently, we reported that the PREB regulates the transcription of the insulin gene by binding to the glucose response element of the insulin promoter. PREB expression induces the gene expression and secretion of insulin [18]. PREB also participates in the PKA stimulation of the prolactin promoter activity, suggesting a role for this protein in the cAMP-mediated responses [15]. The differing roles of PREB in connection with the glucose- and cAMP-mediated regulation of the insulin and prolactin genes fit with the known actions of the WD-repeat protein family members. Because PREB is present in the pancreas and is known to mediate the actions of glucose or cAMP, we wondered whether the PREB might participate in controlling the GK gene expression by glucose and cAMP. Therefore, we examined the effect of PREB on the transcription of the pancreatic GK gene. Our findings show that the PREB binds to the glucose response element of the GK promoter and also regulates the GK gene activity in response to cAMP. These results suggest that the PREB is an important transcriptional factor that regulates the GK gene in the pancreas.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell culture

The insulin-1 cells were originated from a rat insulinoma cell line developed and propagated at the Division of Biochimie Cliniqe (courtesy of Dr. C.B. Wollheim, Geneva, Switzerland). We isolated pancreatic islets from adult Wister rats as described previously [18]. Animal protocols employed in this experiment were reviewed and approved by the Kagawa University Institutional Animal Care and Use Committee. These cells were cultured in RPMI-1640 media (GIBCO BRL, Tokyo, Japan) containing 11.2 mM glucose (unless otherwise stated) and supplemented with 10% heat-inactivated foetal bovine serum (Dainippon Pharmaceutical Co., Ltd, Tokyo, Japan), 50 μmol/l 2-mercaptoethanol, 100 units/ml penicillin and 0.1 mg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37°C.

Generation of adenovirus and adenovirus treatment

The full-length rat PREB cDNA was inserted into the pShattle vector plasmid as previously described [18]. Adenovirus expressing the PREB (Ad-PREB) was constructed according to the instructions for the Adeno-X Expression System kit (CLONTECH laboratories, Inc., Palo Alto, CA, USA). As a control, Adeno-X-lacZ adenovirus (Ad-LacZ) was generated. Adenoviruses were amplified in HEK 293 cells, and purified and concentrated to 1012 plaque-forming units per ml (pfu/ml) by CsCl ultracentrifugation. Expression of PREB was transduced by incubation with the Ad-PREB adenoviruses for 3 hrs at a multiplicity of infection of 1000 pfu/cells.

Transfection of siRNA

The siRNAs were designed to target the following cDNA sequences: scrambled, 5′-CCGTTCTGTACAGGGAGTACT-3′ and PREB-siRNA, 5′-AATGGCGTGCACTTTCTGCAG-3′[18]. Transfection of PREB-siRNA was performed with siPORT Amine (Ambion, CA, USA). GK protein expression was examined using Western blot analysis 3 days after transfection.

Stable transfection

The PREB expression vector was transfected into the cultured INS-1 cells by the conventional cationic liposome transfection method (Lipofectamine, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The transfected cells were selected by addition of G418 (100 μg/ml) to the media, and clones showing high PREB expression were identified using Western blot analysis as described previously [18]. Mock-transfected cells were transfected with only empty vector of pcDNA3.1(+).

GK activity

INS-1 cells transfected with/without PREB were pre-incubated for 90 min. in 2.8 mM glucose, homogenized, and then centrifuged for 10 min. at 12,000 ×g to remove mitochondrial-bound hexokinase [19]. DNA content was measured using 10 μl aliquots of the extract [20]. Glucose phosphorylation was measured by the conversion of nicotinamide adenine dinucleotide (NAD+) to NADH using exogenous glucose-6-phosphate dehydrogenase [21]. Briefly, 5 μl of extract was added to 80 μl of reaction buffer (50 mM Hepes/HCl pH 7.60, 100 mM KCl, 7.4 mM MgCl2, 0.05% BSA, 5 mM ATP, 0.5 mM NAD+, 15 mM β-mercaptoethanol, 0.7 U/ml glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Boehringer Mannheim, Indianapolis, IN, USA), glucose (6–100 mM) and incubated 90 min. at 30°C. The reaction was stopped with 1 ml of 500 mM NaHCO3 (pH 9.4). Triplicate samples were measured per glucose concentration (excitation 350 nm/emission 460/nm), and the mean value was used as a single observation. For the standard curve, 0.3–3.0 nmol of glucose-6-phosphate was used in the reaction buffer containing 100 mM glucose. The GK Vmax and Km were obtained by Lineweaver-Burk plot. In INS-1 cells, we could not detect the activity of low Km Hexokinase (data not shown).

Chromatin immunoprecipitation (ChIP) assays

INS-1 cells were grown to 80–90% confluence in 10-cm culture plates. After cross-linking for 10 min. with 1% formaldehyde in serum-free medium, phosphate-glycine buffer was added to a final concentration of 0.125 M and cells were washed twice with ice-cold phosphate-buffered saline (PBS). The chromatin lysate was sonicated on ice to an average DNA length of 600 bp. Chromatin was pre-cleared with blocked Sepharose A, and ChIP assays were performed as described by Spencer et al. [22], using 8 μg of anti-PREB antibody as the negative control. The final PCR step was performed to amplify the fragment spanning the nucleotides from −445 to −245 of the promoter sequence using the primers 5′-GGAGTGGAAACAAGAAGTCACTCAT-3′ and 5′-CTGAGTAGGCCCCCAAGGACC-3′. Following an initial 5-min. denaturation at 95°C, the PCR amplification involved 32 cycles of the following steps: denaturation for 1 min. at 94°C, annealing for 30 sec. at 59°C, extension for 30 sec. at 72°C. The reaction products were analysed on a 1.5% agarose-tris-borate-EDTA (TBE) gel stained with ethidium bromide and visualized under ultraviolet light.

Western blot analysis

Cells were washed, scraped in PBS and lysed as described previously [23]. The proteins (15 μg) were separated on a 7.5% SDS-polyacrylamide gel under reducing conditions and transferred to polyvinylidene difluoride membranes for an immunoblot assay. The membranes were incubated for 1 hr at 4°C with 0.2% Tween 20 in PBS (PBS-T) containing anti-PREB antiserum (dilution 1:250) or anti-GK antiserum (dilution 1:2000) as described previously [18]. Each antibody binding was visualized using a chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK).

Transfection of INS-1 cells and luciferase reporter gene assay

To confirm the transcriptional regulation of the GK promoter by PREB, we used a plasmid construct containing the rat pancreatic GK promoter, which was obtained by PCR amplification, cloned in front of the luciferase reporter gene as previously described [24, 25]. The wild-type plasmid construct [p-1000 WT luciferase (LUC)] contains the rat GK DNA upstream promoter β–cell-specific promoter gene sequences, spanning the region from −1000 to +14 linked to the luciferase reporter gene. The mutant plasmid constructs (p-1000 mt 1-LUC, p-1000 mt 2-LUC) containing mutations in the consensus binding sequence for PREB were generated as described previously [18]. Mutagenesis within the first 1000 bp of the rat GK upstream promoter gene was performed according to the manufacture’s instruction (Stratagene, La Jolla, CA, USA). Purified reporter plasmid (wild-type or mutant) was cotransfected with a PREB expressing plasmid or an empty vector and a β-galactosidase expression plasmid (for determining transfection efficiency into the INS-1 cells (at 80% confluence) using Lipofectamine (Life Technologies). Transfected cells were maintained in control media for 24 hrs as previously described [18]. Transfected cells were harvested, and β-galactosidase activity was measured in an aliquot of the cytoplasmic preparation. The luciferase assay was performed with 20 μL aliquots, according to the manufacturer’s instructions (ToyoInk, Tokyo, Japan).

Real time PCR

Template cDNA were prepared from the INS-1 cells as previously described [26]. The PREB cDNA was detected by PCR using a LightCycler (Roche Diagnostic, GmbH, Mannheim, Germany). The sequences of the PCR primers for the rat PREB were as follows: sense primer, 5′-GTCATTTCCTGCCTCACT-3′ and antisense primer, 5′-GTCACATCTGTCACCACA-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the control housekeeping gene, and was amplified and analysed under identical conditions by using previously described primers. The level of PREB cDNA was determined as the relative ratio of the levels of PREB and GAPDH in the same sample.

Statistical analysis

Statistical comparisons were made by one-way anova and Student’s t-test, with P < 0.05 considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

PREB induces GK protein expression and GK activity in pancreatic cells

To examine whether PREB affected the GK expression, we measured the GK protein levels in rat pancreatic islet and INS-1 cells infected with the Ad-PREB adenovirus. As shown in Fig. 1, infection with the Ad-PREB adenovirus increased GK protein expression in both the rat pancreatic islet (Fig. 1A) and INS-1 cells (Fig. 1B); in contrast, the expression of GAPDH was not affected (Fig. 1A and B).

imageimage

Figure 1. Effect of PREB overexpression on the expression of GK. (A) Western blot analysis of the effect of Ad-PREB on the GK protein expression. Pancreatic islets were infected with the Ad-PREB or Ad-LacZ for 48 hrs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a control and the results are shown in the bottom lanes. A plot showing the results for each treatment group is shown on the lower panel. The asterisk denotes a significant difference (P < 0.01). Control: without adenovirus, LacZ: infected with Ad-LacZ, PREB: infected with Ad-PREB. (B) Western blot analysis of the effect of Ad-PREB on the GK protein expression. INS-1 cells were infected with the Ad-PREB or Ad-LacZ for 48 hrs. GAPDH served as a control and its expression is shown in the bottom lanes. LacZ: infected with Ad-LacZ, PREB: infected with Ad-PREB. An identical experiment independently performed gave similar results.

Previously, we established permanent INS-1 cells expressing high levels of PREB [18]. In these cells, not only the expression of insulin but also the expression of the GK protein was enhanced (data not shown). To determine whether PREB also affected the catalytic activity of GK, we measured both the Vmax and Km of the enzyme (Table 1) in PREB-overexpressing INS-1 cells. The results are shown in Fig. 2. The Vmax of GK in the PREB-overexpressing INS-1 cells was 22.2% that in the control cells; however, the Km remained unchanged when compared to that of the control (Table 1). These findings suggest that in the INS-1 cells, the overexpression of PREB increases the expression of the pancreatic GK protein and its catalytic activity.

Table 1.  Km and Vmax of glucokinase in INS-1 cells stably expressing PREB
  V max mol glucose/kg DNA/60minK m mM glucose
  1. Glucokinase activity was measured by following the formation of glucose-6-phosphate at various glucose concentrations and results were graphically shown in Fig. 2. The Km and Vmax values for the glucokinase were determined by analyzing these results as described in Materials and methods. The data was expressed as mean ± SEM for six experiments.

mock 8.193 ± 0.27425.067 ± 3.829
PREB 10.040 ± 0.87319.963 ± 3.702
image

Figure 2. GK activity in PREB-transfected cells. The phosphorylation of glucose to glucose-6-phosphate in transfected INS-1 cells was measured after 90-min. incubation at the glucose concentrations shown (6–100 mM). INS-1 cells were cultured for 24 hrs in 11.2 mM glucose containing medium; PREB-transfected cells (○—○n= 3) or mock-transfected cells (▪—▪n= 3).

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PREB siRNA inhibits GK expression and attenuates glucose-induced stimulation

GK participates in pancreatic β-cell sensing of ambient glucose for the purpose of regulating the insulin secretion. We have shown that glucose increases PREB protein expression in a dose-dependent manner [18]. To determine whether PREB played a role in the glucose-induced effects on GK, INS-1 cells were treated with a specific or scrambled PREB siRNA for 48 hrs and then exposed to glucose at either low (2.8 mM) or high (11.2 mM) concentrations. Initial studies showed that the expression of PREB was inhibited by the PREB siRNA-treatment but not by the scrambled PREB siRNA treatment (data not shown). The results showed that the GK protein levels increased in the cells treated with the scrambled siRNA and high glucose (Fig. 3, comparing lanes 1 and 2) but not in the cells treated with the PREB-specific siRNA (Fig. 3, lane 3 compared to lane 4). These findings are in keeping with the idea that the glucose-mediated induction of the GK expression requires PREB.

image

Figure 3. Effect of knockdown of PREB on glucose-induced GK expression. PREB siRNA (lanes 3, 4) or scrambled siRNA (lanes 1, 2) was transfected into INS-1 cells. At 48 hrs after transfection, western blot analysis was performed to determine the GK expression (upper lanes). Glyceraldehyde-3-phosphate dehydrogenase served as a control and its expression is shown in the bottom lanes. Lanes 1, 2: scrambled siRNA, lanes 3, 4: PREB siRNA. An identical experiment independently performed gave similar results.

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cAMP induces PREB expression in INS-l cells

A previous report indicated that cAMP is an important regulator of the GK gene expression in pancreatic β-cells [12]. Fliss et al. also reported that PREB mediates transcriptional activation by PKA in pituitary cells [15]. Therefore, we examined the effect of this metabolic regulator on PREB expression in the INS-1 cells. Western blots probed with a PREB specific antiserum showed that the relative abundance of the PREB protein increased in response to cAMP in a dose-dependent manner (Fig. 4). In contrast, the basal level of the transcriptional factor TFIID (TBP) was not affected by cAMP. Furthermore, the relative abundance of PREB mRNA also increased in response to cAMP treatment in a dose-dependent manner (Fig. 4B). These results clearly suggest that the cAMP stimulates the expression of PREB in INS-1 cells.

imageimage

Figure 4. Effects of cAMP on PREB protein and mRNA expression in INS-1 cells. (A) Nuclear extract was purified from the INS-1 cells treated with varying concentrations of cAMP for 24 hrs. Western blot analysis was performed to examine PREB expression. Expression of TFIID was assayed as a control and the results are shown in the bottom lanes. The ratio of PREB to TFIID at each cAMP concentration is plotted in the bottom figure. Results are presented as the mean ± S.E. of three independent experiments. The asterisk denotes a significant difference (P < 0.01). (B) Total RNA was extracted from the INS-1 cells treated with the indicated concentrations of cAMP for 24 hrs. Real-time PCR was performed to analyse the PREB mRNA expression. Plot shows the ratio of PREB/GAPDH mRNA. Results shown are mean ± S.E.M. of three experiments for each treatment group. The asterisk denotes a significant difference (P < 0.01).

PREB siRNA inhibits GK expression and attenuates cAMP-induced stimulation

Next, we tested whether PREB affects the cAMP-induced GK protein expression. To answer this question, INS-1 cells were treated with specific or scrambled PREB siRNA for 48 hrs and then exposed to a fixed amount (10−7 M) of cAMP. Our results showed that the GK protein levels increased in the cells treated with the scrambled siRNA and cAMP (Fig. 5, comparing columns 1 and 2) and that GK protein expression was markedly reduced in the cells treated with the PREB-specific siRNA (Fig. 5, comparing columns 3 and 4). These findings suggest that the cAMP-mediated induction of GK expression requires PREB.

image

Figure 5. Effect of knockdown of PREB on cAMP-induced GK expression. PREB siRNA (lanes 3, 4) or scrambled siRNA (lanes 1, 2) was transfected into INS-1 cells. At 48 hrs after transfection, the cells were treated with 10−7 M cAMP and then western blot analysis was performed to determine the GK protein expression (upper portion). Expression of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured as a control, and the results are shown in the bottom lanes. The ratio of GK to GAPDH was plotted in the bottom figure. Results were shown as the mean ± S.E. of three experiments. The asterisk denotes a significant difference (P < 0.01).

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PREB expression induces GK promoter activity in INS-l cells

The preceding data point to a likely role for PREB in the GK promoter activity, but whether PREB is directly involved in controlling the GK promoter remains unclear. Therefore, we cotransfected a plasmid construct (p-1000 WT LUC) comprising the full-length rat pancreatic GK promoter fused to the luciferase open reading frame and a PREB-expressing plasmid into INS-1 cells and measured the transcription activity. As shown in Fig. 6A, PREB expression induced a five-fold increase in GK promoter activity (comparing columns 1 and 3). The cAMP-induced GK promoter activity was used as a positive control (comparing columns 1 and 2). As expected, cotransfection with the PREB-specific siRNA, but not the scrambled PREB siRNA, blocked the stimulation by cAMP (Fig. 6B). These findings support the idea that the cAMP-mediated induction of GK promoter activity requires PREB.

imageimage

Figure 6. Effects of PREB on GK promoter activity. (A) INS-1 cells were transfected with p-1000 WT LUC and empty vector (control), empty vector plus cAMP-treatment (cAMP), PREB expression vector (PREB). Results were expressed as relative luciferase activity compared to the control cells arbitrarily set at 100. Each data point represents the mean ± S.E.M. of four separate transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01). (B) Effect of PREB-knockdown on cAMP-induced GK promoter activity. Cells treated with PREB siRNA were transfected with p-1000 WT LUC, empty vector (control), or empty vector. At 48 hrs after transfection, the cells were treated with 10−7 M cAMP for 24 hrs. Results were expressed as relative luciferase activity compared to control cells arbitrarily set at 100. Each data point represents the mean ± S.E.M. of four independent transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01).

PREB is involved in cAMP stimulated GK gene expression

Next, we searched for a DNA motif within the GK promoter that may bind PREB. Examination of the promoter sequence revealed a 7-nt motif (ATTGTCC) corresponding to the deduced PREB core-binding element (PCBE) of the prolactin gene [18]. A ChIP assay was used to determine whether PREB bound to the GK promoter. Figure 7A shows the PCR amplification product after the immunoprecipitation of the cross-linked chromatin with the PREB antibody (Fig. 7A, lane 5). No PCR amplified product was found following the immunoprecipitation of the cross-linked chromatin with purified rabbit IgG (Fig. 7A, lane 4). These data support the idea that PREB binds to the GK promoter, which spans the nucleotides from −445 to −245 in the GK promoter sequence.

imageimageimage

Figure 7. PREB mediated cAMP-induced GK promoter activity. (A) Recruitment of PREB to the PBCE of GK promoter region. ChIP assay was used to detect the binding of PREB to the PBCE on the GK promoter of the INS-1 cells. DNAs and proteins were cross-linked with formaldehyde for 10 min., and the DNA was sheared. The cross-linked protein-DNA complexes were immunoprecipitated with the anti-PREB antibody (lane 5), or with a purified rabbit IgG as negative control (lane 3). The protein-DNA cross-links were reversed and the purified DNAs were used for a semi-quantitative PCR analysis using a primer set for amplifying the PBCE in the GK promoter region (nt −445 to −245). PCR of the input (sample representing PCR amplification from a 1:25 dilution of total input chromatin from the ChIP experiment) is shown in lane 2. The PCR control represents the PCR amplification in the absence of DNA (lane 3). (B) and (C), Site-directed mutagenesis of the PREB-binding site abrogates the response to PREB (B) or cAMP (C). The binding site (−350 to −348) was disrupted by altering three base pairs (5′-ATT to 5′-CAC) in the PREB-binding site of the wild-type plasmid construct p-1000 WT LUC (GK wild-type promoter) to create the mutant plasmid construct p-1000 mt LUC (GK promoter mutant) as described in the ‘Materials and methods’ section. Each data point represents the mean ± S.E.M. of three independent transfections. The asterisk denotes a significant difference (P < 0.01). N.S.; no significant difference.

This finding led us to create a plasmid construct, p-1000 mt LUC, that contains a mutated putative PCBE (5′-ATT-3′ to 5′-GCA-3′). Transfection studies showed that PREB failed to induce any luciferase activity in cells transfected with the p-1000 mt LUC plasmid, but as shown above, it stimulated luciferase activity in cells transfected with the wild-type p-1000 WT LUC plasmid (Fig. 7B). Together, these findings suggest that the putative PCBE in the GK promoter is involved in the PREB-mediated induction of the GK promoter. In addition, a mutation in PCBE inhibited the ability of cAMP to stimulate the GK promoter activity (Fig. 7C). These results suggest that the cAMP-mediated induction of the GK promoter activity requires an intact PCBE motif.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

PREB cDNA was recently isolated from a rat pituitary cDNA library, and the protein product was shown to transactivate the prolactin promoter [15]. PREB mRNA transcripts are present not only in the pituitary but a strong signal is also present in the pancreas. Recently, we reported that PREB might participate in the regulation of insulin gene transcription and hormone secretion in response to glucose stimulation [18]. In this report, we have examined the regulation of GK expression in rat pancreatic islet and the rat pancreatic insulinoma cell line INS-1. GK plays a key role in the pancreatic β-cell’s ability to sense ambient glucose and thus regulate insulin secretion. The role of GK in this process makes it a key component of glucose homeostasis. Small changes in GK activity have pronounced effects on the regulation of insulin secretion by glucose [27–30]. Thus, a better understanding of how the GK expression is regulated by PREB may lead to the identification of novel targets that will augment insulin release. This knowledge will also be useful for developing new treatments for diabetes mellitus (DM).

Previously, we have reported that PREB is located mainly in the nuclei of the rat pancreatic β-cells and cultured INS-1 insulinoma cells. Co-expression of PREB and the insulin promoter induced the activity of the latter. Addition of glucose to the INS-1 cells increased PREB expression. Furthermore, cells with higher PREB expression or knocked down PREB exhibited increased or decreased insulin expression, respectively. These observations suggested that PREB may play the role of a pancreatic cell transcription factor [18]; this prompted us to examine the potential role of PREB in GK gene expression.

In this study, we observed that overexpression of PREB stimulated the GK protein expression in both rat pancreatic islets and INS-1 cells (Fig. 1). The cells overexpressing PREB also showed increased GK catalytic activity (Table 1). These findings are in keeping with the idea that glucose-induced GK expression requires PREB.

Analysis of the primary sequence of PREB reveals that it is a novel transcription factor that is distinct from Pit-1. The PREB protein has three motifs (WD I, WD II and WD III) with significant degrees of homology to the consensus WD repeat, suggesting that PREB is a member of the WD-repeat protein superfamily [17]. The highly conserved WD-repeats within PREB have sequence similarity to a subset of this family that comprises proteins that are gene regulators [17]. Unlike other WD-repeat proteins, PREB exerts transcriptional regulation as shown by its ability to stimulate gene expression by directly binding to DNA [15]. Our results show that PREB binds to the GK promoter and enhances GK promoter activity in INS-1 cells.

Several lines of evidence suggest that the isoenzymes of GK are differentially regulated in pancreatic β-cells and the liver [4, 31, 32]. These observations led to the idea that alternate promoters and the first exon confer differential isoenzyme expression. Consistent with this idea, GK expression in the islet cells and liver is different during development. GK is already present in the pancreatic β-cells of a foetus [33], while it appears in the liver at 2 weeks after birth [34, 35]. Furthermore, repression of GK gene transcription in response to glucagon via cAMP is an essential feature of hepatic GK regulation [36–38]. In contrast, cAMP increases pancreatic GK activity and expression [12]. Previous studies showed that cAMP activates the −1000 kb β-cell GK promoter, which provided further evidence that cAMP affects pancreatic GK gene transcription. However, a consensus cAMP response element sequence was not identified in the −1000 kb β-cell GK promoter [2, 12]. Our results showed that PREB bound to the GK promoter, while a mutant of the PREB binding site abrogated the effect of not only PREB but also that of cAMP. These results suggest that PREB may be involved in the cAMP-mediated stimulation of GK promoter activity.

Several studies have examined the mechanisms by which an increase in cAMP potentiates glucose-induced insulin secretion. Increased rates of phosphorylation of ion channels in the β-cell membrane via cAMP-dependent PKA may be one pathway that leads to increased β-cell sensitivity to primary stimuli [39–42]. In addition, cAMP promotes insulin release via an action that occurs downstream of the regulatory steps of the secretory machinery [42–44]. However, it appears that increases in GK gene expression may also contribute to the glucose-stimulated insulin secretion induced by cAMP. A previous report indicated that PREB could mediate the PKA stimulation of prolactin promoter activity [15], suggesting a role for this protein in cAMP-mediated transcriptional responses. Previously, we showed that INS-1 cells that stably express PREB exhibited increased insulin secretion as compared to mock-transfected cells [18]. A possible model for this potential role of PREB may involve the activation of PREB via PKA-mediated phosphorylation. Although it is not yet known whether PREB can serve as a PKA substrate in an in vitro or in vivo system, the predicted sequence of this protein contains a number of motifs resembling consensus PKA phosphorylation sites [45].

A mutation in GK can lead to maturity-onset DM in youth [9, 10]. What is the clinical relevance of the preceding findings in the context of human DM? A recent report showed that haploinsufficiency of β-cell-specific GK (GK+/−) caused impaired insulin secretion in response to glucose stimulation. However, the β-cell mass was normal in these animals. When fed a high-fat (HF) diet, wild-type mice showed marked β-cell hyperplasia. In contrast, GK+/− mice showed decreased β-cell replication and insufficient β-cell hyperplasia despite showing a similar degree of insulin resistance. These findings suggested that GK regulates the β-cell mass as well as the β-cell function [46]. Type-2 DM patients with decreased insulin secretion may also show a decrease in the β-cell mass [47]. Changes of GK induction are probably significant when the β-cell adapts to altered mean blood glucose levels during extended exposure as it occurs in diabetes or hypoglycemia. Enzyme induction may compensate, in part at least, for defective β-cell function in diabetes, and persistent hypoglycemia would probably down-regulate the glucose sensor. Such changes, although speculative at this point, may be of relevance for the new therapeutic approach proposing the use of GK activator drugs [48]. The activation of GK by increasing cAMP production by using derivatives of glucagons-like peptide 1 may offer a potential strategy for treating the decreased insulin secretion and decreased β-cell mass in type-2 DM patients. The rationale underlying this approach is that the transcription factor PREB, which regulates the GK gene expression in response to cAMP, may be one of the target genes for treating diseases such as type-2 DM.

In summary, our findings show that the PREB can function as a transcriptional regulator of the GK promoter. PREB binds to the GK promoter, and in cells expressing PREB, it increases both the GK protein expression and enzymatic activity. Further investigations will help us define a possible physiological role for PREB in pancreatic β-cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Miss Kazuko Yamaji and Kiyo Ueeda for their technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research 17590937 and 19591054 (K.M. and T.I.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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