Glucose Dependence of the Regulated Secretory Pathway in TC1-6 Cells
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《内分泌学杂志》
Lawson Health Research Institute (R.M., C.E.E., S.D.), London, Ontario, Canada N6A 4V2
Departments of Medical Biophysics and Medicine (S.D.), University of Western Ontario, London, Ontario, Canada N6A 5B8
London Regional Genomics Centre (D.E.C.), London, Ontario, Canada N6A 5K8
Vascular Biology Group (J.D.A.), Robarts Research Institute, London, Ontario, Canada N6A 5K8
Loma Linda University (Y.N.), Loma Linda, California 92350
Charles R. Drew University (T.C.F.), Los Angeles, California 90059
Abstract
We have investigated the effects of chronically elevated glucose concentrations on the pancreatic -cell line TC1-6. We show that basal glucagon secretion and proglucagon gene expression were increased in response to high glucose levels. The extent of acute stimulated secretion of glucagon was also increased in response to high glucose, as was the transcription of the prohormone processing enzymes PC1/3 and PC2. The secretion of GLP-1, a proglucagon-derived peptide produced by cleavage of proglucagon by PC1/3, was also increased in response to high glucose. Gene expression profiling experiments showed that a number of components of the regulated secretory pathway were up-regulated at high glucose concentrations, including processing enzymes and exocytotic proteins. Immunoblot analysis showed that the expression of the exocytotic SNARE proteins, as well as that of PC1/3, chromogranin A, and 7B2, were all increased after chronic exposure to high glucose levels. Immunocytochemistry showed no changes in the expression of the mature -cell markers glucagon and brn-4 and no induction of the immature -cell marker pdx-1. We conclude that chronically elevated glucose concentrations up-regulate the regulated secretory response of the -cell.
Introduction
IN BOTH TYPE 1 and type 2 diabetes, -cell function becomes abnormal, and glucagon secretion is no longer suppressed by hyperglycemia (1). Several studies have documented the pathological increase in glucagon secretion in diabetic patients (2, 3), and a more recent study has documented an increase in -cell mass in individuals with type 2 diabetes (4). In patients with either type 1 or type 2 diabetes, a lack of glucagon suppression resulted in a worsening of glucose tolerance, due mainly to increased glycogenolysis (5). Therefore, in insulinopenic diabetes, hyperglucagonemia can lead to an increase in hepatic glucose output, thus exacerbating hyperglycemia. Suppression of glucagon secretion may therefore contribute toward more effective glycemic control in diabetes.
A few studies have examined the effects of sustained high glucose concentrations on the secretion and synthesis of glucagon. Rats made diabetic by streptozotocin have elevated levels of circulating glucagon concomitant with hyperglycemia (6, 7). The hyperglucagonemia of diabetes may likely be due to a loss in the secretion of insulin (6) or other cell factors (8). However, elevated glucose levels may have a direct effect on glucagon secretion in cell lines. Prolonged exposure to high glucose concentrations causes an increase in glucagon secretion and proglucagon gene transcription in the islet cell line InR1-G9, and these effects can be reversed by lowering the glucose concentration or by insulin treatment (9). However, the effects of high glucose on other proteins that may mediate glucagon secretion remain unknown.
The effects of chronic elevations in glucose concentrations on mediators of secretion have been determined in a number of cell lines and purified cell preparations. Gene expression profiling studies performed in a transformed cell line, MIN6, have shown that exposure to high glucose levels causes up-regulation of several genes involved in the secretory pathway, such as those involved in endoplasmic reticulum translocation, Golgi trafficking, and proinsulin processing (10). Proteins involved in stimulus-secretion coupling in cells, such as glucose and lipid metabolic enzymes, are up-regulated in response to long-term exposure to high glucose in isolated islets and INS-1 cells (11, 12), thus providing a model for the cell response to chronic hyperglycemia. We hypothesized that a similar approach could be used examine the molecular and cellular mechanisms of glucose-induced hyperglucagonemia in a mouse pancreatic -cell line, TC1-6. TC1-6 cells, a subclone of a glucagonoma-derived cell line originating in simian virus 40 transgenic mice (13), are a particularly good model with which to examine -cell gene expression because, unlike other glucagon-secreting cell lines, they are an homogenous cell population and do not express insulin or somatostatin (14). Proglucagon processing resembles that of normal -cells (15, 16), as does the glucagon secretory response to insulin (17) and nutrients (18).
We show here that exposure of TC1-6 cells to high glucose leads to increased basal glucagon secretion and proglucagon mRNA levels, results in an up-regulation of stimulated secretion of glucagon, and up-regulates some components of the regulated secretory pathway, including the exocytotic SNARE proteins and the processing enzyme PC1/3. We conclude that chronic exposure to high glucose levels may remodel the regulated secretory pathway of the -cell and therefore may account for the paradoxical increase in glucagon secretion under conditions of high glucose.
Materials and Methods
Cell culture and experimental protocol
Wild-type TC1-6 cells (a kind gift from C. Bruce Verchere, University of British Columbia, Vancouver, British Columbia, Canada) were maintained in DMEM containing 15% horse serum and 2.5% fetal bovine serum. Traditionally, TC1-6 cells have been maintained in media containing 25 mM glucose (13). To examine the effects of chronic high glucose on glucagon secretion, cells were incubated in DMEM containing high glucose (4.5 g/liter; 25 mM) or low glucose (1 g/liter; 5.5 mM) for 5 d. On d 6, cells were seeded into 24-well dishes, and, on d 7, cells were rinsed with Hanks’ buffered saline solution (HBSS) and incubated for 4 h in DMEM plus 0.5% fetal bovine serum. Cells and media were subsequently harvested and assessed for glucagon by RIA, as described below.
Assessment of cell number and proliferation
To measure cellular proliferation after high and low glucose treatments, a methyl thiazolyl tetrazolium (MTT) assay was performed. Cells were plated in replicates of six into 24-well plates at a density of 104 cells per well and incubated for 5 d in media containing high and low glucose levels as described above. Every 24 h time period, 20 μl of 2.5 mg/ml thiazolyl blue (MTT) was added to six wells of each treatment, and cells were incubated for 4 h at 37 C. Media were then removed, and the metabolized MTT was solubilized with 200 μl acidified isopropanol. Absorbance for duplicate 90 μl samples was read at 590 nm in a plate reader. Cell number was assessed after 24 h and 5 d of high and low glucose treatment by counting in a hemocytometer.
Stimulated secretion experiments
For stimulated secretion, cells were incubated for 24 h or 5 d in media containing high or low glucose as described above. Cells were subsequently seeded in 24-well plates in replicates of six at a density of 5 x 105 cells per well and allowed to settle overnight. The following day, cells were rinsed twice with warm HBSS and preincubated for 30 min in HBSS containing 25 mM glucose. For basal secretion, cells were then incubated for 1 h in HBSS containing 25 mM glucose. For stimulated secretion, parallel wells of cells were incubated for 1 h in HBSS containing 1 mM glucose and either 15 mM arginine (positive control) or 10 μM forskolin plus 10 μM 3-isobutyl-1-methylxanthine (IBMX).
We also assessed the direct response of TC1-6 cells to acute exposure to low glucose, as follows: cells were preincubated for 15 min in HBSS containing 1 mM glucose (basal), followed by 15 min in HBSS with 25 mM glucose, and finally for 15 min in HBSS containing 1 mM glucose, as described previously (19).
Glucagon and GLP-1 RIAs
After all secretion experiments, media were collected, and trifluoroacetic acid was added to 0.1%. Cells were rinsed twice in HBSS and scraped in 1 ml homogenization buffer [1 M HCl, 1 M formic acid, 1% (vol/vol) trifluoroacetic acid, and 1% (wt/vol) NaCl]. The cells were sonicated in one 12 s burst and centrifuged, the supernatant was collected, and both media and cell extracts were passed through a Sep-Pak C18 reverse-phase cartridge to elute glucagon and GLP-1 as described previously (20, 21). Glucagon and GLP-1 content were assessed by RIA using the glucagon and GLP-1 RIA kits from Linco (St. Charles, MO). The antibody provided with the glucagon RIA kit detects the C-terminal end of glucagon and does not cross-react with any other proglucagon-derived peptide. The antibody provided with the GLP-1 kit detects only the N terminus of active GLP-1; the antibody does not cross-react with GLP-1 (9–36NH2), GLP-1 (1–37/36NH2), or GLP-2.
RT-PCR
To examine effects on proglucagon gene expression, cells were seeded into 6-cm dishes and subject to treatment with media containing high and low glucose, as above. On d 6, cells were rinsed and harvested, and mRNA was extracted and purified using the RNeasy Mini Kit (Qiagen, Mississauga, Ontario, Canada). mRNA (1 μg) was using for first-strand cDNA synthesis with SuperScript II (Invitrogen, Burlington, Ontario, Canada). PCR was performed on half of the reaction using sequence-specific primers for rat proglucagon (forward, 5'-ATGAACGAGGACAAGCGC-3'; reverse, 5'-TTCACCAGCCAAGCAATG-3') and mouse -actin (forward, 5'-GCCCCTCTGAACCCTAAG-3'; reverse, 5'-CATCACAATGCCAGTGGTA-3'). PCR was performed over 25 cycles in a linear range. The primers amplified a 236-bp proglucagon fragment and a 138-bp -actin fragment. Both fragments were resolved on a 2% agarose gel, and bands were quantified using densitometry.
Microarray analysis and statistics
Total RNA was isolated and purified using RNeasy columns (Qiagen), and the purity of the RNA was checked by analysis on an Agilent Bioanalyser. For expression analysis, the Affymetrix (Santa Clara, CA) GeneChip Mouse Genome 430A 2.0 array (MOE430A), containing 22,600 probe sets representing transcripts and variants from 14,000 mouse genes, was used. All procedures, including cRNA synthesis and labeling and hybridization to Affymetrix MOE430A GeneChips, were performed as described in the Affymetrix Technical Analysis Manual. GeneChips were scanned with an Agilent Technologies (Palo Alto, CA) 2500A GeneArray Scanner.
Signal intensities for genes were generated using MAS 5.1 using default values for the statistical expression algorithm parameters and a target signal of 150 for all probe sets and a normalization value of 1. Normalization was performed using GeneSpring 6.1 (Silicon Genetics, Redwood, CA). Data were first transformed (measurements <0.01 set to 0.01) and then normalized per chip to the 50th percentile and per gene to "low glucose" expression on d 1 (for acute exposure experiments) and on d 5 (for chronic exposure experiments). Genes were considered changed by the following criteria: genes must be called "present" or "marginal" in at least one of eight arrays, thus excluding any genes that are called "absent" in all arrays, and through Welch’s t test and one-way ANOVA (variances not assumed equal) with a P value cutoff of 0.05.
Western blot analysis
To detect changes in the expression of secretory pathway proteins, TC1-6 cells were seeded into 10 cm dishes in replicates of three and grown for 24 h or 5 d in 25 or 5 mM glucose as described above. Cells were lysed on ice in 0.5 ml of 50 mM Tris-EDTA (pH 7.6) containing Complete Protease Inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and 5 μg/ml aprotinin. The lysate was sonicated and centrifuged at 10,000 rpm for 5 min to remove cellular debris and membranes, and the supernatant was removed for protein determination by the method of Bradford (22). Protein (10 μg) was resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with the following antibodies: syntaxin 1A and vesicle-associated membrane protein 2 (VAMP-2) (Sigma, St. Louis, MO); SNAP-25 (a kind gift from Dr. Herbert Gaisano, University of Toronto, Toronto, Ontario, Canada); carboxypeptidase E (CPE) and chromogranin A (CGA) (kind gifts from Dr. Y. Peng Loh, National Institutes of Health, Bethesda, MD); PC1/3, PC2, and 7B2 (kind gifts from Dr. Nabil G. Seidah, Institut de Recherches Cliniques de Montreal, Montreal, Quebec, Canada).
Promoter assays for PC1/3 and PC2
Cells were transferred to 6-mm dishes and, after overnight adherence, transfected using the calcium precipitation method (23). We used a human PC1/3-luciferase promoter construct containing the region of the human PC1/3 promoter (from –971 to –1 bp relative to the translation initiation codon) inserted in the pGL2-basic vector that contains a luciferase reporter (Promega, Madison, WI) (24). To study the involvement of the cAMP response elements (CREs) in the glucose regulation of PC1/3, the PC1/3 promoter construct (–971 to –1 bp relative to the translation start site) with the CRE motifs of both CRE-1 and CRE-2 mutated by changing the central core AC dinucleotides to TG (24) was used. For the human PC2-luciferase promoter construct, we used the PC2 promoter (–1102 to –177 bp relative to the translation initiation codon) inserted in the pGL2-basic vector (25). Cells were incubated with 3 μg DNA for 6 h, after which the medium was changed to glucose-free DMEM without serum for overnight incubation. Cells were treated with media containing 4.5 or 18 mM glucose and 5% charcoal-stripped fetal bovine serum for 6 h. All cells were washed with PBS and then lysed in 25 mM Tris phosphate [10 mM MgCl2, 0.1% BSA, 15% glycerol, 1% Triton X-100, and 1 mM EDTA (pH 7.8)]. After centrifugation, 180 μl of the cleared cell lysate was used for the luciferase assay. Luciferase activity was measured in a Berthold Lumat LB 9501 luminometer (Wallac, Gaithersburg, MD) in the presence of 0.8 mM ATP and 0.3 mM D-luciferin. Integrated light emission over 15 s was measured. All transfections were done in triplicate.
Immunocytochemistry
INS-1 832/13 and TC1-6 cells were prepared for immunocytochemistry by fixation in 2% paraformaldehyde (in PBS) for 30 min, followed by a 5 min incubation in 0.25% Triton-X-100. Cells were then incubated for 30 min in blocking buffer (1% BSA and 10% goat serum in PBS). INS-1 832/13 cells were incubated overnight with antibodies against insulin (1:500 dilution; Peninsula Laboratories, Belmont, CA) and pdx-1 (1:2000 dilution; a generous gift from C. V. E. Wright, Vanderbilt University, Nashville, TN). To visualize pdx-1 and insulin, fluorescent-conjugated secondary antibodies rabbit Alexa 594 (1:1000; Molecular Probes, Eugene, OR) and guinea pig Alexa 488 (1:1000) were used. TC1-6 cells were incubated overnight with antibodies against glucagon (1:1000; Peninsula Laboratories), insulin (1:500; Peninsula Laboratories), pdx-1 (1:2000), or brn-4 (1:20; Chemicon, Temecula, CA) along with anti-p115 (1:50; Transduction Laboratories, Lexington, KY) to visualize the Golgi. Secondary antibodies used with the TC1-6 cells were rabbit Alexa 488 (1:1000) and mouse Alexa 594 (1:1000) (Molecular Probes, Burlington, Ontario, Canada). After incubation with secondary antibodies, cells that were probed with anti-brn-4 were treated with 200 ng/ml Hoechst 33258 stain (Sigma) to visualize the nucleus.
Statistical analysis
To compare differences between control and treatment groups and between low and high glucose groups (except for the microarray experiments), statistical differences were determined using one-way ANOVA, followed by Student’s t test. Significance was set at P 0.05. Each experiment was repeated at least twice (indicated in the figure legends) and analyzed independently, with the indicated number of replicates, and one representative experiment is shown.
Results
Chronic changes in glucose concentration affects glucagon secretion, cell content, and proglucagon mRNA levels
We investigated the effects of high and low glucose concentrations on glucagon secretion, synthesis, and proglucagon mRNA levels in TC1-6 cells. Acute (24 h) exposure of cells to 25 mM glucose had no effect on glucagon secretion relative to cells incubated with low glucose (Fig 1A). However, after 5 d in media containing 25 mM glucose, glucagon secretion increased almost 2-fold (P < 0.05) relative to cells treated with 5 mM glucose (Fig. 1A). This increase in glucagon secretion was paralleled by an increase in proglucagon mRNA levels as measured by RT-PCR (Fig 1B); proglucagon mRNA levels increased by 60% (P < 0.05) after 5 d in 25 mM glucose. No difference in proglucagon mRNA levels was detected after 24 h (data not shown).
To ascertain that cell viability was not compromised by differences in glucose concentration, cells were counted after 24 h and 5 d of exposure to low or high glucose (Table 1), and cell proliferation was determined by MTT assay (Table 2). After incubation in 5 or 25 mM glucose for 5 d, there was no difference in cell number or in the rate of cell proliferation, indicating that changing the glucose concentration had no adverse effects on cell viability.
Chronic changes in glucose concentration affect the regulated secretory response of glucagon
We then investigated whether chronic exposure to high glucose affected the ability of TC1-6 cells to respond to acute stimulation by nutrients and secretagogues. Cells were incubated with 15 mM arginine, a potent stimulus of glucagon secretion, or with forskolin/IBMX, known to increase glucagon secretion through elevations in cAMP (26). Figure 2 shows that cells incubated in media containing 5 mM glucose had a small, but significant (P < 0.01), secretory response to arginine compared with basal secretion; however, there was no secretory response to stimulation by forskolin/IBMX when compared with basal secretion. In contrast, cells exposed to 25 mM glucose showed a 64% increase (P < 0.01) in secretion with arginine and a 25% increase (P < 0.05) in secretion with forskolin/IBMX treatment (Fig 2). These data indicate that the function of the regulated secretory pathway in TC1-6 cells is determined by the prevailing glucose concentration.
To determine the direct response of TC1-6 cells to glucose, we used a protocol to first suppress glucagon secretion with high glucose and then release glucagon by acute exposure to low glucose. Cells were incubated for 5 d in media containing 5 or 25 mM glucose, as described above. On the day of the experiment, cells from both treatment groups were rinsed and first incubated in media containing 25 mM glucose and then were incubated in media containing 1 mM glucose. TC1-6 cells incubated in media containing 25 mM glucose for 5 d showed a significant (P < 0.05) increase in glucagon secretion in response to acute stimulation by low glucose (Fig 3). However, cells exposed to 5 mM glucose failed to show a response to low glucose. These results suggest that the prevailing glucose concentrations determine the acute glucagon secretory response to low glucose in TC1-6 cells.
Gene transcription of both PC1/3 and PC2 is glucose dependent
Because the transcription of the proglucagon gene is glucose dependent in InR1-G9 cells (9), we examined the effects of increasing concentrations of glucose on the transcription of the proglucagon processing enzymes PC1/3 and PC2. We transiently transfected the PC1/3 promoter-luciferase and PC2 promoter-luciferase reporter constructs into TC1-6 cells and then exposed them to media containing either 4.5 or 18 mM glucose. As shown in Fig. 4, exposure to high glucose increased PC1/3 promoter activity by 45 ± 8% (P < 0.05), whereas PC2 promoter activity did not change.
The PC1/3 promoter, from –971 to –1 bp (relative to the translation initiation codon), contains two CREs (24). In TC1-6 cells transfected with the PC1/3 promoter construct with the CRE motifs of both CRE-1 and CRE-2 mutated (24), promoter activity was severely decreased (P < 0.001) compared with the wild-type promoter, and high glucose was without effect (Fig 4). This demonstrates that the glucose-mediated increase in PC1/3 activity may be mediated through a cAMP-dependent mechanism involving the CREs on the promoter region.
GLP-1 production is increased after chronic exposure to high glucose
The expression of PC1/3 and GLP-1 are coregulated by secretagogues in cell lines (27) and glucose in islets of intact rats (28). The glucose-dependent increase in the expression of PC1/3 (Fig. 4) led us to determine whether there were any corresponding increases in the production of GLP-1. Indeed, both GLP-1 cell content (P < 0.001) and GLP-1 secretion (P < 0.01) were increased in response to chronic exposure to high glucose (Fig. 5). It is noteworthy that the ratio of total active GLP-1 to total glucagon immunoreactivity was low: 0.038 ±.004 (n = 6) in cells exposed to low glucose and 0.035 ±.003 (n = 6) in cells exposed to high glucose.
Changes in glucose concentration alter the expression of genes encoding proteins in the regulated secretory pathway of -cells
We performed gene profiling experiments to identify those glucose-sensitive genes that encode proteins involved in the regulated secretory pathway. Microarray analyses using the Affymetrix murine 430A array were conducted on cells exposed to low and high glucose for 24 h and for 5 d. After 24 h, the expression of 194 genes and expressed sequence tags, of which 103 encoded specific biological function, were decreased and 114 were increased in response to high glucose. After 5 d, the expression of 186 genes with known biological functions was increased and 234 were decreased in response to high glucose. All genes that increased and decreased significantly (P < 0.05 with Welch’s t test and one-way ANOVA) were grouped according to function (Fig. 6, A and B).
By far, the largest group of genes was observed in genes that encoded proteins involved in "trafficking," which included cellular and molecular processes of translation, endoplasmic reticulum-Golgi transport, posttranslation modifications, processing, sorting, and secretion. Of all genes that changed in response to a 24-h exposure to high glucose, those involved in trafficking comprised 28% of the genes that increased and 24% of those that decreased. In response to a 5-d exposure to high glucose, genes encoding trafficking proteins comprised 29% of those that increased and 35% of those that decreased.
A list of selected genes encoding proteins involved in exocytosis, processing, and prohormones are presented in Table 3. Interestingly, genes encoding proteins involved in SNARE-dependent exocytosis, such as syntaxin 1A, VAMP, synaptophysin, synaptotagmin, all appeared to change in response to the prevailing glucose concentration (Table 3). We therefore examined glucose-induced changes in the expression of these SNARE proteins by Western blot analysis.
The expression of SNARE proteins increases in response to prolonged exposure to high glucose
After 24-h or 5-d incubation in media containing 5 or 25 mM glucose, the expression of SNARE proteins was examined in TC1-6 cells (Fig 7A). The expression of syntaxin 1A, VAMP-2, synaptophysin, and SNAP-25 were unchanged after 24 h. After 5-d incubation in media containing high glucose, there were significant increases in the expression of VAMP-2 (P < 0.05), SNAP-25 (P < 0.05), and syntaxin 1A (P < 0.001) relative to cells exposed to low glucose concentrations (Fig 7B). These results are consistent with the increase in regulated secretion seen in TC1-6 cells exposed to high glucose concentrations for 5 d.
Changes in secretory protein expression after prolonged exposure to high glucose
We examined the expression of the processing enzymes PC1/3, PC2, and CPE, as well as that of the granule-forming protein CGA and the PC2 chaperone 7B2 after a 24-h and 5-d exposure of TC1-6 cells to low or high glucose concentrations (Fig. 8A). The expression of the 66-kDa form of PC1/3 significantly increased (P < 0.05) after a 5-d exposure to high glucose, whereas PC2 and CPE expression did not change (Fig. 8B). As well, the expression of CGA (P < 0.001) and 7B2 (P < 0.05), proteins of the regulated secretory pathway, also increased in response to high glucose after 5 d. Expression of secretory proteins did not change significantly after 24 h.
Expression of -cell markers after exposure to high glucose
To examine the possibility that TC1-6 cells may have undergone a process of differentiation after exposure to high glucose, we conducted immunocytochemistry to determine the expression of mature and immature -cell markers. After exposure to high or low glucose for 5 d, cells were incubated with antibodies against glucagon (Fig. 9, A and B) and brn-4 (panels I and J), a known marker of mature -cells. Table 4 shows that the number of cells expressing glucagon and brn-4 approaches 100%, which is typical of mature -cells. Additionally, the number of cells expressing both glucagon and brn-4 is not altered after exposure to high or low glucose, indicating that differentiation after exposure to high glucose is not likely. Western blot analysis also showed that the expression of brn-4 was not altered after exposure to high glucose (data not shown).
To determine the effects of high glucose exposure on the de-differentiation of TC1-6 cells, we examined the expression of pdx-1, a transcription factor found in immature -cells. To examine the purity of the cell line, the number of cells expressing insulin was also determined. Figure 9 shows the expression of insulin in a cell line, INS-1 832/13 (panel E) but not in TC1-6 cells (panels C and D), regardless of the glucose concentration. Similarly, pdx-1, which is expressed in the nucleus of INS-1 cells (Fig 9H) and immature -cells, is not detected in the nucleus of TC1-6 cells (Fig. 9, F and G). Approximately 22% of TC1-6 cells showed some weak pdx-1 immunoreactivity in the cytoplasm (Table 4), regardless of the glucose concentration.
Discussion
In the present study, we have demonstrated that persistently high glucose concentrations elevate glucagon secretion and proglucagon mRNA levels in TC1-6 cells. The acute glucagon secretory response to nutrient or secretagogue stimuli was maintained in cells exposed to high glucose concentrations. Microarray analysis showed that some genes involved in the key steps of the -cell-regulated secretory pathway, such as proglucagon processing and exocytosis, are glucose sensitive. Western blot analysis showed increased expression of various proteins in the regulated secretory pathway, such as the processing enzyme PC1/3, the granule-forming protein CGA, the molecular chaperone 7B2, and the SNARE exocytotic proteins, after chronic exposure to high glucose levels. We show that increases in the promoter activity of PC1/3 occur in a glucose-dependent manner and correlate with the increase in proglucagon mRNA levels as well as GLP-1 secretion. Expression of the mature -cell marker brn-4 and of the cell marker pdx-1 is not altered, indicating that high glucose levels do not affect the differentiation state of these cells.
Our results are in agreement with those performed in InR1-G9 cells, a glucagon-producing cell line, which also showed an increase in glucagon secretion, proglucagon mRNA, and gene transcription after prolonged exposure to high glucose concentrations (9). That study also demonstrated that the effects of chronically high glucose concentrations may be mediated through the 138 bp region upstream of the transcriptional start site of the proglucagon promoter. Our study has now shown that the effects of high glucose may also be mediated by changes in the expression of other components of the regulated secretory pathway of TC1-6 cells. Arginine increased glucagon secretion approximately 2-fold after chronic exposure to high glucose. Although this is a somewhat muted response when compared with that of primary -cells, it is typical for this cell line (18, 29) and resembles the phenomenon of the augmented response to arginine in patients with type 2 diabetes with chronic hyperglycemia (30). Additionally, cAMP-stimulated secretion appeared to be dependent on exposure to high glucose, because cells cultured in low glucose failed to respond to such stimulation. Therefore, high glucose levels may prime the cAMP pathway to stimulate glucagon secretion through the regulated secretory pathway.
Our results suggest that the acute response to glucose is dependent on the ambient glucose concentration. Stimulation of glucagon secretion by acute exposure to low glucose shows that TC1-6 cells respond to low glucose in a manner similar to that seen in isolated islets and in vivo. Although incubation in 1 mM glucose to elicit a response is not a physiological stimulus, it has been used previously in this cell line (18) and isolated islets in culture (19) to maximally stimulate glucagon secretion and is routinely used in electrophysiological studies of glucagon secretion from isolated -cells (31, 32). Isolated islets and -cells in culture are in the artificial environment of a culture dish and may have different thresholds of glucose responsiveness than intact islets in vivo. The difference in glucose responsiveness when compared with primary -cells is a limitation in studying the regulation of glucagon secretion in TC1-6 cells.
We then used gene expression profiling to help us determine the nature of the changes in the -cell secretory response after prolonged exposure to high glucose. The TC1-6 cell line has been used previously for gene profiling studies (33, 34) and is preferable to primary islets, because TC1-6 cells are an homogenous cell population (13), and the amount of RNA recovered from primary -cells would be extremely limiting. Validation by Western blot showed for the first time that the expression of the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2 were regulated by glucose in TC1-6 cells. The increase in expression of these proteins after high glucose exposure is consistent with the extent of acute stimulated secretion of glucagon by nutrients and secretagogues. These proteins may therefore be implicated in the regulated secretion of glucagon, as well as insulin (35, 36).
The expression of SNARE proteins has been shown to be decreased in islets of the Zucker diabetic fatty (fa/fa) rat (37). These islets show defects in basal and glucose-stimulated insulin release, and the expression of both VAMP-2 and syntaxin 1A were decreased when measured by Western blot. These results, together with our findings, may provide an explanation for defects in both - and -cell secretion under conditions of prolonged hyperglycemia. It is conceivable that high glucose conditions down-regulate SNARE protein expression in cells but up-regulate SNARE protein expression in -cells. This would account for the decrease in the insulin secretory response seen in fa/fa islets and the increase in glucagon secretion seen in some models of type 2 diabetes (6, 38). In our study, we also found an increase in the granule-forming protein CGA. An increase in CGA expression would increase secretory granule formation (39) and therefore increase the expression of SNARE proteins associated with the secretory granule membranes, such as VAMP-2. The increase in the plasma membrane SNARE proteins syntaxin 1A and SNAP-25 may be mediated by increases in other components of the secretory pathway responsible for delivery of these proteins to the membrane.
Several studies have shown that the processing enzymes PC1/3 and PC2 are coregulated with their substrate prohormones. In particular, studies performed in isolated islets and cell lines have shown coordinate increases in the expression of proinsulin, PC1/3, and PC2 with high glucose levels (40, 41). In the intestinal GLUTag cell line, PC1/3 mRNA and protein levels are coregulated with GLP-1 secretion and proglucagon gene transcription in a cAMP-dependent manner (27). The mechanism is likely through phospho-cAMP response element binding protein binding to a CRE in the PC1/3 5'-flanking region (24, 25). In the present study, the increase in PC1/3 and GLP-1 after prolonged exposure to high glucose suggests that components of the cAMP pathway may be up-regulated by glucose.
Although we observed an increase in PC1/3 levels in response to high glucose by Western blot analysis, a similar increase was not seen with PC2, possibly due to the large amount of PC2 protein in the -cell and to the relative insensitivity of the Western blot-densitometry protocol to detect small changes in protein expression. We therefore examined the expression of both PC1/3 and PC2 by promoter assays. This is the first study to document an increase in PC1/3 gene transcription in response to high glucose in the -cell, in parallel to the increase in proglucagon mRNA levels and GLP-1 production. These results mirror those from in vivo experiments that showed an increase in PC1/3 expression and GLP-1 production in pancreatic -cells after treatment with streptozotocin (28). It is possible that an increase in PC1/3 expression could be an indication of de-differentiation of the -cell phenotype after prolonged exposure to high glucose levels, because immature -cells show an increase in PC1/3 expression (42). However, in our culture system, high glucose did not induce differentiation of TC1-6 cells, nor did de-differentiation to an immature phenotype occur, because there was no difference in the number of cells expressing brn-4 or pdx-1, markers for mature (43) and immature (44) -cells, respectively. Therefore, the changes observed in the regulated secretory pathway can be attributed to the direct effects of glucose and not to the state of differentiation.
In conclusion, we have shown that chronic exposure to high glucose concentrations resulted in an up-regulation of some components of the regulated secretory pathway in TC1-6 cells. However, the response of this cell line to glucose and other nutrients may differ from that of -cells in a perfused pancreatic preparation or within an intact islet in vivo. At present, there exists no satisfactory primary -cell preparation that can be used for studies in changes in global gene and protein expression in response to nutrients. Such an approach to examining changes in the regulated secretory pathway may provide clues to -cell dysfunction in diabetes.
Acknowledgments
We thank Dr. Chris Newgard (Duke University, Durham, NC) for providing the INS-1 832-13 cell line, Dr. Yves Bureau (Lawson Health Research Institute, London, Ontario, Canada) for assistance with statistical analysis, Dr. David J. Hill (Lawson Health Research Institute) for critical reading of this manuscript, and Anna Mugimba for excellent technical assistance.
Footnotes
This work was supported by operating grants from the Canadian Diabetes Association (in memory of the late Miklos Matyas) and the Lawson Health Research Institute (to S.D.), by National Institutes of Health Grant DA14659 (to T.C.F.), and Center of Clinical Research Excellence Grant U54 RR14616 (to Charles R. Drew University of Medicine and Sciences).
Abbreviations: CGA, Chromogranin A; CPE, carboxypeptidase E; CRE, cAMP response elements; HBSS, Hanks’ buffered saline solution; IBMX, 3-isobutyl-1-methylxanthine; MTT, methyl thiazolyl tetrazolium; VAMP, vesicle-associated membrane protein 2.
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Departments of Medical Biophysics and Medicine (S.D.), University of Western Ontario, London, Ontario, Canada N6A 5B8
London Regional Genomics Centre (D.E.C.), London, Ontario, Canada N6A 5K8
Vascular Biology Group (J.D.A.), Robarts Research Institute, London, Ontario, Canada N6A 5K8
Loma Linda University (Y.N.), Loma Linda, California 92350
Charles R. Drew University (T.C.F.), Los Angeles, California 90059
Abstract
We have investigated the effects of chronically elevated glucose concentrations on the pancreatic -cell line TC1-6. We show that basal glucagon secretion and proglucagon gene expression were increased in response to high glucose levels. The extent of acute stimulated secretion of glucagon was also increased in response to high glucose, as was the transcription of the prohormone processing enzymes PC1/3 and PC2. The secretion of GLP-1, a proglucagon-derived peptide produced by cleavage of proglucagon by PC1/3, was also increased in response to high glucose. Gene expression profiling experiments showed that a number of components of the regulated secretory pathway were up-regulated at high glucose concentrations, including processing enzymes and exocytotic proteins. Immunoblot analysis showed that the expression of the exocytotic SNARE proteins, as well as that of PC1/3, chromogranin A, and 7B2, were all increased after chronic exposure to high glucose levels. Immunocytochemistry showed no changes in the expression of the mature -cell markers glucagon and brn-4 and no induction of the immature -cell marker pdx-1. We conclude that chronically elevated glucose concentrations up-regulate the regulated secretory response of the -cell.
Introduction
IN BOTH TYPE 1 and type 2 diabetes, -cell function becomes abnormal, and glucagon secretion is no longer suppressed by hyperglycemia (1). Several studies have documented the pathological increase in glucagon secretion in diabetic patients (2, 3), and a more recent study has documented an increase in -cell mass in individuals with type 2 diabetes (4). In patients with either type 1 or type 2 diabetes, a lack of glucagon suppression resulted in a worsening of glucose tolerance, due mainly to increased glycogenolysis (5). Therefore, in insulinopenic diabetes, hyperglucagonemia can lead to an increase in hepatic glucose output, thus exacerbating hyperglycemia. Suppression of glucagon secretion may therefore contribute toward more effective glycemic control in diabetes.
A few studies have examined the effects of sustained high glucose concentrations on the secretion and synthesis of glucagon. Rats made diabetic by streptozotocin have elevated levels of circulating glucagon concomitant with hyperglycemia (6, 7). The hyperglucagonemia of diabetes may likely be due to a loss in the secretion of insulin (6) or other cell factors (8). However, elevated glucose levels may have a direct effect on glucagon secretion in cell lines. Prolonged exposure to high glucose concentrations causes an increase in glucagon secretion and proglucagon gene transcription in the islet cell line InR1-G9, and these effects can be reversed by lowering the glucose concentration or by insulin treatment (9). However, the effects of high glucose on other proteins that may mediate glucagon secretion remain unknown.
The effects of chronic elevations in glucose concentrations on mediators of secretion have been determined in a number of cell lines and purified cell preparations. Gene expression profiling studies performed in a transformed cell line, MIN6, have shown that exposure to high glucose levels causes up-regulation of several genes involved in the secretory pathway, such as those involved in endoplasmic reticulum translocation, Golgi trafficking, and proinsulin processing (10). Proteins involved in stimulus-secretion coupling in cells, such as glucose and lipid metabolic enzymes, are up-regulated in response to long-term exposure to high glucose in isolated islets and INS-1 cells (11, 12), thus providing a model for the cell response to chronic hyperglycemia. We hypothesized that a similar approach could be used examine the molecular and cellular mechanisms of glucose-induced hyperglucagonemia in a mouse pancreatic -cell line, TC1-6. TC1-6 cells, a subclone of a glucagonoma-derived cell line originating in simian virus 40 transgenic mice (13), are a particularly good model with which to examine -cell gene expression because, unlike other glucagon-secreting cell lines, they are an homogenous cell population and do not express insulin or somatostatin (14). Proglucagon processing resembles that of normal -cells (15, 16), as does the glucagon secretory response to insulin (17) and nutrients (18).
We show here that exposure of TC1-6 cells to high glucose leads to increased basal glucagon secretion and proglucagon mRNA levels, results in an up-regulation of stimulated secretion of glucagon, and up-regulates some components of the regulated secretory pathway, including the exocytotic SNARE proteins and the processing enzyme PC1/3. We conclude that chronic exposure to high glucose levels may remodel the regulated secretory pathway of the -cell and therefore may account for the paradoxical increase in glucagon secretion under conditions of high glucose.
Materials and Methods
Cell culture and experimental protocol
Wild-type TC1-6 cells (a kind gift from C. Bruce Verchere, University of British Columbia, Vancouver, British Columbia, Canada) were maintained in DMEM containing 15% horse serum and 2.5% fetal bovine serum. Traditionally, TC1-6 cells have been maintained in media containing 25 mM glucose (13). To examine the effects of chronic high glucose on glucagon secretion, cells were incubated in DMEM containing high glucose (4.5 g/liter; 25 mM) or low glucose (1 g/liter; 5.5 mM) for 5 d. On d 6, cells were seeded into 24-well dishes, and, on d 7, cells were rinsed with Hanks’ buffered saline solution (HBSS) and incubated for 4 h in DMEM plus 0.5% fetal bovine serum. Cells and media were subsequently harvested and assessed for glucagon by RIA, as described below.
Assessment of cell number and proliferation
To measure cellular proliferation after high and low glucose treatments, a methyl thiazolyl tetrazolium (MTT) assay was performed. Cells were plated in replicates of six into 24-well plates at a density of 104 cells per well and incubated for 5 d in media containing high and low glucose levels as described above. Every 24 h time period, 20 μl of 2.5 mg/ml thiazolyl blue (MTT) was added to six wells of each treatment, and cells were incubated for 4 h at 37 C. Media were then removed, and the metabolized MTT was solubilized with 200 μl acidified isopropanol. Absorbance for duplicate 90 μl samples was read at 590 nm in a plate reader. Cell number was assessed after 24 h and 5 d of high and low glucose treatment by counting in a hemocytometer.
Stimulated secretion experiments
For stimulated secretion, cells were incubated for 24 h or 5 d in media containing high or low glucose as described above. Cells were subsequently seeded in 24-well plates in replicates of six at a density of 5 x 105 cells per well and allowed to settle overnight. The following day, cells were rinsed twice with warm HBSS and preincubated for 30 min in HBSS containing 25 mM glucose. For basal secretion, cells were then incubated for 1 h in HBSS containing 25 mM glucose. For stimulated secretion, parallel wells of cells were incubated for 1 h in HBSS containing 1 mM glucose and either 15 mM arginine (positive control) or 10 μM forskolin plus 10 μM 3-isobutyl-1-methylxanthine (IBMX).
We also assessed the direct response of TC1-6 cells to acute exposure to low glucose, as follows: cells were preincubated for 15 min in HBSS containing 1 mM glucose (basal), followed by 15 min in HBSS with 25 mM glucose, and finally for 15 min in HBSS containing 1 mM glucose, as described previously (19).
Glucagon and GLP-1 RIAs
After all secretion experiments, media were collected, and trifluoroacetic acid was added to 0.1%. Cells were rinsed twice in HBSS and scraped in 1 ml homogenization buffer [1 M HCl, 1 M formic acid, 1% (vol/vol) trifluoroacetic acid, and 1% (wt/vol) NaCl]. The cells were sonicated in one 12 s burst and centrifuged, the supernatant was collected, and both media and cell extracts were passed through a Sep-Pak C18 reverse-phase cartridge to elute glucagon and GLP-1 as described previously (20, 21). Glucagon and GLP-1 content were assessed by RIA using the glucagon and GLP-1 RIA kits from Linco (St. Charles, MO). The antibody provided with the glucagon RIA kit detects the C-terminal end of glucagon and does not cross-react with any other proglucagon-derived peptide. The antibody provided with the GLP-1 kit detects only the N terminus of active GLP-1; the antibody does not cross-react with GLP-1 (9–36NH2), GLP-1 (1–37/36NH2), or GLP-2.
RT-PCR
To examine effects on proglucagon gene expression, cells were seeded into 6-cm dishes and subject to treatment with media containing high and low glucose, as above. On d 6, cells were rinsed and harvested, and mRNA was extracted and purified using the RNeasy Mini Kit (Qiagen, Mississauga, Ontario, Canada). mRNA (1 μg) was using for first-strand cDNA synthesis with SuperScript II (Invitrogen, Burlington, Ontario, Canada). PCR was performed on half of the reaction using sequence-specific primers for rat proglucagon (forward, 5'-ATGAACGAGGACAAGCGC-3'; reverse, 5'-TTCACCAGCCAAGCAATG-3') and mouse -actin (forward, 5'-GCCCCTCTGAACCCTAAG-3'; reverse, 5'-CATCACAATGCCAGTGGTA-3'). PCR was performed over 25 cycles in a linear range. The primers amplified a 236-bp proglucagon fragment and a 138-bp -actin fragment. Both fragments were resolved on a 2% agarose gel, and bands were quantified using densitometry.
Microarray analysis and statistics
Total RNA was isolated and purified using RNeasy columns (Qiagen), and the purity of the RNA was checked by analysis on an Agilent Bioanalyser. For expression analysis, the Affymetrix (Santa Clara, CA) GeneChip Mouse Genome 430A 2.0 array (MOE430A), containing 22,600 probe sets representing transcripts and variants from 14,000 mouse genes, was used. All procedures, including cRNA synthesis and labeling and hybridization to Affymetrix MOE430A GeneChips, were performed as described in the Affymetrix Technical Analysis Manual. GeneChips were scanned with an Agilent Technologies (Palo Alto, CA) 2500A GeneArray Scanner.
Signal intensities for genes were generated using MAS 5.1 using default values for the statistical expression algorithm parameters and a target signal of 150 for all probe sets and a normalization value of 1. Normalization was performed using GeneSpring 6.1 (Silicon Genetics, Redwood, CA). Data were first transformed (measurements <0.01 set to 0.01) and then normalized per chip to the 50th percentile and per gene to "low glucose" expression on d 1 (for acute exposure experiments) and on d 5 (for chronic exposure experiments). Genes were considered changed by the following criteria: genes must be called "present" or "marginal" in at least one of eight arrays, thus excluding any genes that are called "absent" in all arrays, and through Welch’s t test and one-way ANOVA (variances not assumed equal) with a P value cutoff of 0.05.
Western blot analysis
To detect changes in the expression of secretory pathway proteins, TC1-6 cells were seeded into 10 cm dishes in replicates of three and grown for 24 h or 5 d in 25 or 5 mM glucose as described above. Cells were lysed on ice in 0.5 ml of 50 mM Tris-EDTA (pH 7.6) containing Complete Protease Inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and 5 μg/ml aprotinin. The lysate was sonicated and centrifuged at 10,000 rpm for 5 min to remove cellular debris and membranes, and the supernatant was removed for protein determination by the method of Bradford (22). Protein (10 μg) was resolved on a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with the following antibodies: syntaxin 1A and vesicle-associated membrane protein 2 (VAMP-2) (Sigma, St. Louis, MO); SNAP-25 (a kind gift from Dr. Herbert Gaisano, University of Toronto, Toronto, Ontario, Canada); carboxypeptidase E (CPE) and chromogranin A (CGA) (kind gifts from Dr. Y. Peng Loh, National Institutes of Health, Bethesda, MD); PC1/3, PC2, and 7B2 (kind gifts from Dr. Nabil G. Seidah, Institut de Recherches Cliniques de Montreal, Montreal, Quebec, Canada).
Promoter assays for PC1/3 and PC2
Cells were transferred to 6-mm dishes and, after overnight adherence, transfected using the calcium precipitation method (23). We used a human PC1/3-luciferase promoter construct containing the region of the human PC1/3 promoter (from –971 to –1 bp relative to the translation initiation codon) inserted in the pGL2-basic vector that contains a luciferase reporter (Promega, Madison, WI) (24). To study the involvement of the cAMP response elements (CREs) in the glucose regulation of PC1/3, the PC1/3 promoter construct (–971 to –1 bp relative to the translation start site) with the CRE motifs of both CRE-1 and CRE-2 mutated by changing the central core AC dinucleotides to TG (24) was used. For the human PC2-luciferase promoter construct, we used the PC2 promoter (–1102 to –177 bp relative to the translation initiation codon) inserted in the pGL2-basic vector (25). Cells were incubated with 3 μg DNA for 6 h, after which the medium was changed to glucose-free DMEM without serum for overnight incubation. Cells were treated with media containing 4.5 or 18 mM glucose and 5% charcoal-stripped fetal bovine serum for 6 h. All cells were washed with PBS and then lysed in 25 mM Tris phosphate [10 mM MgCl2, 0.1% BSA, 15% glycerol, 1% Triton X-100, and 1 mM EDTA (pH 7.8)]. After centrifugation, 180 μl of the cleared cell lysate was used for the luciferase assay. Luciferase activity was measured in a Berthold Lumat LB 9501 luminometer (Wallac, Gaithersburg, MD) in the presence of 0.8 mM ATP and 0.3 mM D-luciferin. Integrated light emission over 15 s was measured. All transfections were done in triplicate.
Immunocytochemistry
INS-1 832/13 and TC1-6 cells were prepared for immunocytochemistry by fixation in 2% paraformaldehyde (in PBS) for 30 min, followed by a 5 min incubation in 0.25% Triton-X-100. Cells were then incubated for 30 min in blocking buffer (1% BSA and 10% goat serum in PBS). INS-1 832/13 cells were incubated overnight with antibodies against insulin (1:500 dilution; Peninsula Laboratories, Belmont, CA) and pdx-1 (1:2000 dilution; a generous gift from C. V. E. Wright, Vanderbilt University, Nashville, TN). To visualize pdx-1 and insulin, fluorescent-conjugated secondary antibodies rabbit Alexa 594 (1:1000; Molecular Probes, Eugene, OR) and guinea pig Alexa 488 (1:1000) were used. TC1-6 cells were incubated overnight with antibodies against glucagon (1:1000; Peninsula Laboratories), insulin (1:500; Peninsula Laboratories), pdx-1 (1:2000), or brn-4 (1:20; Chemicon, Temecula, CA) along with anti-p115 (1:50; Transduction Laboratories, Lexington, KY) to visualize the Golgi. Secondary antibodies used with the TC1-6 cells were rabbit Alexa 488 (1:1000) and mouse Alexa 594 (1:1000) (Molecular Probes, Burlington, Ontario, Canada). After incubation with secondary antibodies, cells that were probed with anti-brn-4 were treated with 200 ng/ml Hoechst 33258 stain (Sigma) to visualize the nucleus.
Statistical analysis
To compare differences between control and treatment groups and between low and high glucose groups (except for the microarray experiments), statistical differences were determined using one-way ANOVA, followed by Student’s t test. Significance was set at P 0.05. Each experiment was repeated at least twice (indicated in the figure legends) and analyzed independently, with the indicated number of replicates, and one representative experiment is shown.
Results
Chronic changes in glucose concentration affects glucagon secretion, cell content, and proglucagon mRNA levels
We investigated the effects of high and low glucose concentrations on glucagon secretion, synthesis, and proglucagon mRNA levels in TC1-6 cells. Acute (24 h) exposure of cells to 25 mM glucose had no effect on glucagon secretion relative to cells incubated with low glucose (Fig 1A). However, after 5 d in media containing 25 mM glucose, glucagon secretion increased almost 2-fold (P < 0.05) relative to cells treated with 5 mM glucose (Fig. 1A). This increase in glucagon secretion was paralleled by an increase in proglucagon mRNA levels as measured by RT-PCR (Fig 1B); proglucagon mRNA levels increased by 60% (P < 0.05) after 5 d in 25 mM glucose. No difference in proglucagon mRNA levels was detected after 24 h (data not shown).
To ascertain that cell viability was not compromised by differences in glucose concentration, cells were counted after 24 h and 5 d of exposure to low or high glucose (Table 1), and cell proliferation was determined by MTT assay (Table 2). After incubation in 5 or 25 mM glucose for 5 d, there was no difference in cell number or in the rate of cell proliferation, indicating that changing the glucose concentration had no adverse effects on cell viability.
Chronic changes in glucose concentration affect the regulated secretory response of glucagon
We then investigated whether chronic exposure to high glucose affected the ability of TC1-6 cells to respond to acute stimulation by nutrients and secretagogues. Cells were incubated with 15 mM arginine, a potent stimulus of glucagon secretion, or with forskolin/IBMX, known to increase glucagon secretion through elevations in cAMP (26). Figure 2 shows that cells incubated in media containing 5 mM glucose had a small, but significant (P < 0.01), secretory response to arginine compared with basal secretion; however, there was no secretory response to stimulation by forskolin/IBMX when compared with basal secretion. In contrast, cells exposed to 25 mM glucose showed a 64% increase (P < 0.01) in secretion with arginine and a 25% increase (P < 0.05) in secretion with forskolin/IBMX treatment (Fig 2). These data indicate that the function of the regulated secretory pathway in TC1-6 cells is determined by the prevailing glucose concentration.
To determine the direct response of TC1-6 cells to glucose, we used a protocol to first suppress glucagon secretion with high glucose and then release glucagon by acute exposure to low glucose. Cells were incubated for 5 d in media containing 5 or 25 mM glucose, as described above. On the day of the experiment, cells from both treatment groups were rinsed and first incubated in media containing 25 mM glucose and then were incubated in media containing 1 mM glucose. TC1-6 cells incubated in media containing 25 mM glucose for 5 d showed a significant (P < 0.05) increase in glucagon secretion in response to acute stimulation by low glucose (Fig 3). However, cells exposed to 5 mM glucose failed to show a response to low glucose. These results suggest that the prevailing glucose concentrations determine the acute glucagon secretory response to low glucose in TC1-6 cells.
Gene transcription of both PC1/3 and PC2 is glucose dependent
Because the transcription of the proglucagon gene is glucose dependent in InR1-G9 cells (9), we examined the effects of increasing concentrations of glucose on the transcription of the proglucagon processing enzymes PC1/3 and PC2. We transiently transfected the PC1/3 promoter-luciferase and PC2 promoter-luciferase reporter constructs into TC1-6 cells and then exposed them to media containing either 4.5 or 18 mM glucose. As shown in Fig. 4, exposure to high glucose increased PC1/3 promoter activity by 45 ± 8% (P < 0.05), whereas PC2 promoter activity did not change.
The PC1/3 promoter, from –971 to –1 bp (relative to the translation initiation codon), contains two CREs (24). In TC1-6 cells transfected with the PC1/3 promoter construct with the CRE motifs of both CRE-1 and CRE-2 mutated (24), promoter activity was severely decreased (P < 0.001) compared with the wild-type promoter, and high glucose was without effect (Fig 4). This demonstrates that the glucose-mediated increase in PC1/3 activity may be mediated through a cAMP-dependent mechanism involving the CREs on the promoter region.
GLP-1 production is increased after chronic exposure to high glucose
The expression of PC1/3 and GLP-1 are coregulated by secretagogues in cell lines (27) and glucose in islets of intact rats (28). The glucose-dependent increase in the expression of PC1/3 (Fig. 4) led us to determine whether there were any corresponding increases in the production of GLP-1. Indeed, both GLP-1 cell content (P < 0.001) and GLP-1 secretion (P < 0.01) were increased in response to chronic exposure to high glucose (Fig. 5). It is noteworthy that the ratio of total active GLP-1 to total glucagon immunoreactivity was low: 0.038 ±.004 (n = 6) in cells exposed to low glucose and 0.035 ±.003 (n = 6) in cells exposed to high glucose.
Changes in glucose concentration alter the expression of genes encoding proteins in the regulated secretory pathway of -cells
We performed gene profiling experiments to identify those glucose-sensitive genes that encode proteins involved in the regulated secretory pathway. Microarray analyses using the Affymetrix murine 430A array were conducted on cells exposed to low and high glucose for 24 h and for 5 d. After 24 h, the expression of 194 genes and expressed sequence tags, of which 103 encoded specific biological function, were decreased and 114 were increased in response to high glucose. After 5 d, the expression of 186 genes with known biological functions was increased and 234 were decreased in response to high glucose. All genes that increased and decreased significantly (P < 0.05 with Welch’s t test and one-way ANOVA) were grouped according to function (Fig. 6, A and B).
By far, the largest group of genes was observed in genes that encoded proteins involved in "trafficking," which included cellular and molecular processes of translation, endoplasmic reticulum-Golgi transport, posttranslation modifications, processing, sorting, and secretion. Of all genes that changed in response to a 24-h exposure to high glucose, those involved in trafficking comprised 28% of the genes that increased and 24% of those that decreased. In response to a 5-d exposure to high glucose, genes encoding trafficking proteins comprised 29% of those that increased and 35% of those that decreased.
A list of selected genes encoding proteins involved in exocytosis, processing, and prohormones are presented in Table 3. Interestingly, genes encoding proteins involved in SNARE-dependent exocytosis, such as syntaxin 1A, VAMP, synaptophysin, synaptotagmin, all appeared to change in response to the prevailing glucose concentration (Table 3). We therefore examined glucose-induced changes in the expression of these SNARE proteins by Western blot analysis.
The expression of SNARE proteins increases in response to prolonged exposure to high glucose
After 24-h or 5-d incubation in media containing 5 or 25 mM glucose, the expression of SNARE proteins was examined in TC1-6 cells (Fig 7A). The expression of syntaxin 1A, VAMP-2, synaptophysin, and SNAP-25 were unchanged after 24 h. After 5-d incubation in media containing high glucose, there were significant increases in the expression of VAMP-2 (P < 0.05), SNAP-25 (P < 0.05), and syntaxin 1A (P < 0.001) relative to cells exposed to low glucose concentrations (Fig 7B). These results are consistent with the increase in regulated secretion seen in TC1-6 cells exposed to high glucose concentrations for 5 d.
Changes in secretory protein expression after prolonged exposure to high glucose
We examined the expression of the processing enzymes PC1/3, PC2, and CPE, as well as that of the granule-forming protein CGA and the PC2 chaperone 7B2 after a 24-h and 5-d exposure of TC1-6 cells to low or high glucose concentrations (Fig. 8A). The expression of the 66-kDa form of PC1/3 significantly increased (P < 0.05) after a 5-d exposure to high glucose, whereas PC2 and CPE expression did not change (Fig. 8B). As well, the expression of CGA (P < 0.001) and 7B2 (P < 0.05), proteins of the regulated secretory pathway, also increased in response to high glucose after 5 d. Expression of secretory proteins did not change significantly after 24 h.
Expression of -cell markers after exposure to high glucose
To examine the possibility that TC1-6 cells may have undergone a process of differentiation after exposure to high glucose, we conducted immunocytochemistry to determine the expression of mature and immature -cell markers. After exposure to high or low glucose for 5 d, cells were incubated with antibodies against glucagon (Fig. 9, A and B) and brn-4 (panels I and J), a known marker of mature -cells. Table 4 shows that the number of cells expressing glucagon and brn-4 approaches 100%, which is typical of mature -cells. Additionally, the number of cells expressing both glucagon and brn-4 is not altered after exposure to high or low glucose, indicating that differentiation after exposure to high glucose is not likely. Western blot analysis also showed that the expression of brn-4 was not altered after exposure to high glucose (data not shown).
To determine the effects of high glucose exposure on the de-differentiation of TC1-6 cells, we examined the expression of pdx-1, a transcription factor found in immature -cells. To examine the purity of the cell line, the number of cells expressing insulin was also determined. Figure 9 shows the expression of insulin in a cell line, INS-1 832/13 (panel E) but not in TC1-6 cells (panels C and D), regardless of the glucose concentration. Similarly, pdx-1, which is expressed in the nucleus of INS-1 cells (Fig 9H) and immature -cells, is not detected in the nucleus of TC1-6 cells (Fig. 9, F and G). Approximately 22% of TC1-6 cells showed some weak pdx-1 immunoreactivity in the cytoplasm (Table 4), regardless of the glucose concentration.
Discussion
In the present study, we have demonstrated that persistently high glucose concentrations elevate glucagon secretion and proglucagon mRNA levels in TC1-6 cells. The acute glucagon secretory response to nutrient or secretagogue stimuli was maintained in cells exposed to high glucose concentrations. Microarray analysis showed that some genes involved in the key steps of the -cell-regulated secretory pathway, such as proglucagon processing and exocytosis, are glucose sensitive. Western blot analysis showed increased expression of various proteins in the regulated secretory pathway, such as the processing enzyme PC1/3, the granule-forming protein CGA, the molecular chaperone 7B2, and the SNARE exocytotic proteins, after chronic exposure to high glucose levels. We show that increases in the promoter activity of PC1/3 occur in a glucose-dependent manner and correlate with the increase in proglucagon mRNA levels as well as GLP-1 secretion. Expression of the mature -cell marker brn-4 and of the cell marker pdx-1 is not altered, indicating that high glucose levels do not affect the differentiation state of these cells.
Our results are in agreement with those performed in InR1-G9 cells, a glucagon-producing cell line, which also showed an increase in glucagon secretion, proglucagon mRNA, and gene transcription after prolonged exposure to high glucose concentrations (9). That study also demonstrated that the effects of chronically high glucose concentrations may be mediated through the 138 bp region upstream of the transcriptional start site of the proglucagon promoter. Our study has now shown that the effects of high glucose may also be mediated by changes in the expression of other components of the regulated secretory pathway of TC1-6 cells. Arginine increased glucagon secretion approximately 2-fold after chronic exposure to high glucose. Although this is a somewhat muted response when compared with that of primary -cells, it is typical for this cell line (18, 29) and resembles the phenomenon of the augmented response to arginine in patients with type 2 diabetes with chronic hyperglycemia (30). Additionally, cAMP-stimulated secretion appeared to be dependent on exposure to high glucose, because cells cultured in low glucose failed to respond to such stimulation. Therefore, high glucose levels may prime the cAMP pathway to stimulate glucagon secretion through the regulated secretory pathway.
Our results suggest that the acute response to glucose is dependent on the ambient glucose concentration. Stimulation of glucagon secretion by acute exposure to low glucose shows that TC1-6 cells respond to low glucose in a manner similar to that seen in isolated islets and in vivo. Although incubation in 1 mM glucose to elicit a response is not a physiological stimulus, it has been used previously in this cell line (18) and isolated islets in culture (19) to maximally stimulate glucagon secretion and is routinely used in electrophysiological studies of glucagon secretion from isolated -cells (31, 32). Isolated islets and -cells in culture are in the artificial environment of a culture dish and may have different thresholds of glucose responsiveness than intact islets in vivo. The difference in glucose responsiveness when compared with primary -cells is a limitation in studying the regulation of glucagon secretion in TC1-6 cells.
We then used gene expression profiling to help us determine the nature of the changes in the -cell secretory response after prolonged exposure to high glucose. The TC1-6 cell line has been used previously for gene profiling studies (33, 34) and is preferable to primary islets, because TC1-6 cells are an homogenous cell population (13), and the amount of RNA recovered from primary -cells would be extremely limiting. Validation by Western blot showed for the first time that the expression of the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2 were regulated by glucose in TC1-6 cells. The increase in expression of these proteins after high glucose exposure is consistent with the extent of acute stimulated secretion of glucagon by nutrients and secretagogues. These proteins may therefore be implicated in the regulated secretion of glucagon, as well as insulin (35, 36).
The expression of SNARE proteins has been shown to be decreased in islets of the Zucker diabetic fatty (fa/fa) rat (37). These islets show defects in basal and glucose-stimulated insulin release, and the expression of both VAMP-2 and syntaxin 1A were decreased when measured by Western blot. These results, together with our findings, may provide an explanation for defects in both - and -cell secretion under conditions of prolonged hyperglycemia. It is conceivable that high glucose conditions down-regulate SNARE protein expression in cells but up-regulate SNARE protein expression in -cells. This would account for the decrease in the insulin secretory response seen in fa/fa islets and the increase in glucagon secretion seen in some models of type 2 diabetes (6, 38). In our study, we also found an increase in the granule-forming protein CGA. An increase in CGA expression would increase secretory granule formation (39) and therefore increase the expression of SNARE proteins associated with the secretory granule membranes, such as VAMP-2. The increase in the plasma membrane SNARE proteins syntaxin 1A and SNAP-25 may be mediated by increases in other components of the secretory pathway responsible for delivery of these proteins to the membrane.
Several studies have shown that the processing enzymes PC1/3 and PC2 are coregulated with their substrate prohormones. In particular, studies performed in isolated islets and cell lines have shown coordinate increases in the expression of proinsulin, PC1/3, and PC2 with high glucose levels (40, 41). In the intestinal GLUTag cell line, PC1/3 mRNA and protein levels are coregulated with GLP-1 secretion and proglucagon gene transcription in a cAMP-dependent manner (27). The mechanism is likely through phospho-cAMP response element binding protein binding to a CRE in the PC1/3 5'-flanking region (24, 25). In the present study, the increase in PC1/3 and GLP-1 after prolonged exposure to high glucose suggests that components of the cAMP pathway may be up-regulated by glucose.
Although we observed an increase in PC1/3 levels in response to high glucose by Western blot analysis, a similar increase was not seen with PC2, possibly due to the large amount of PC2 protein in the -cell and to the relative insensitivity of the Western blot-densitometry protocol to detect small changes in protein expression. We therefore examined the expression of both PC1/3 and PC2 by promoter assays. This is the first study to document an increase in PC1/3 gene transcription in response to high glucose in the -cell, in parallel to the increase in proglucagon mRNA levels and GLP-1 production. These results mirror those from in vivo experiments that showed an increase in PC1/3 expression and GLP-1 production in pancreatic -cells after treatment with streptozotocin (28). It is possible that an increase in PC1/3 expression could be an indication of de-differentiation of the -cell phenotype after prolonged exposure to high glucose levels, because immature -cells show an increase in PC1/3 expression (42). However, in our culture system, high glucose did not induce differentiation of TC1-6 cells, nor did de-differentiation to an immature phenotype occur, because there was no difference in the number of cells expressing brn-4 or pdx-1, markers for mature (43) and immature (44) -cells, respectively. Therefore, the changes observed in the regulated secretory pathway can be attributed to the direct effects of glucose and not to the state of differentiation.
In conclusion, we have shown that chronic exposure to high glucose concentrations resulted in an up-regulation of some components of the regulated secretory pathway in TC1-6 cells. However, the response of this cell line to glucose and other nutrients may differ from that of -cells in a perfused pancreatic preparation or within an intact islet in vivo. At present, there exists no satisfactory primary -cell preparation that can be used for studies in changes in global gene and protein expression in response to nutrients. Such an approach to examining changes in the regulated secretory pathway may provide clues to -cell dysfunction in diabetes.
Acknowledgments
We thank Dr. Chris Newgard (Duke University, Durham, NC) for providing the INS-1 832-13 cell line, Dr. Yves Bureau (Lawson Health Research Institute, London, Ontario, Canada) for assistance with statistical analysis, Dr. David J. Hill (Lawson Health Research Institute) for critical reading of this manuscript, and Anna Mugimba for excellent technical assistance.
Footnotes
This work was supported by operating grants from the Canadian Diabetes Association (in memory of the late Miklos Matyas) and the Lawson Health Research Institute (to S.D.), by National Institutes of Health Grant DA14659 (to T.C.F.), and Center of Clinical Research Excellence Grant U54 RR14616 (to Charles R. Drew University of Medicine and Sciences).
Abbreviations: CGA, Chromogranin A; CPE, carboxypeptidase E; CRE, cAMP response elements; HBSS, Hanks’ buffered saline solution; IBMX, 3-isobutyl-1-methylxanthine; MTT, methyl thiazolyl tetrazolium; VAMP, vesicle-associated membrane protein 2.
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