Basal Receptor Activation by Locally Produced Glucagon-Like Peptide-1 Contributes to Maintaining ß-Cell Function
Kai Masur,
Elmi C. Tibaduiza,
Ci Chen,
Brooke Ligon and
Martin Beinborn
Molecular Pharmacology Research Center (K.M., E.C.T., C.C., M.B.), Molecular Cardiology Research Institute, Tufts-New England Medical Center, and Department of Neuroscience (B.L.), Tufts University, Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: Martin Beinborn, Tufts-New England Medical Center, Mailbox 7703, 750 Washington Street, Boston, Massachusetts 02111. E-mail: MBeinborn{at}Tufts-NEMC.org.
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ABSTRACT
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Glucagon-like peptide 1 (GLP-1) is a physiological stimulus of pancreatic ß-cell function. This enteroendocrine hormone is produced by intestinal L cells, and is delivered via the bloodstream to GLP-1 receptors (GLP-1Rs) on pancreatic ß-cells. In addition, there is evidence that ß-cell GLP-1Rs maintain sustained basal activity even in the absence of intestinal peptide, an observation that has raised the question whether these receptors have some degree of ligand-independent function. Here, we provide an alternative explanation for basal receptor activity based on our finding that biologically relevant amounts of fully processed GLP-1 are locally generated by insulinoma cell lines, as well as by
-cells of isolated rat islets in primary culture. Presence of GLP-1 was established by immunocytochemistry, as well as by selective ELISAs and bioassays of cell supernatants. A GLP-1R antagonist significantly reduced insulin secretion/production in ß-TC-6 insulinoma cells and isolated rat islets, suggesting a functionally important loop between locally produced GLP-1 and its cognate receptor. Treatment with this antagonist also inhibited the growth of ß-TC-6 cells. These observations provide novel insight into the function of insulin-producing cell lines and native ß-cells during in vitro culture, and they support the idea that locally produced GLP-1 may play a role in intraislet regulation.
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INTRODUCTION
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GLUCAGON-LIKE PEPTIDE-1 (GLP-1) is an enteroendocrine hormone that plays an important physiological role in maintaining normal blood glucose homeostasis (1). This peptide acts through a G protein-coupled receptor, which primarily triggers cAMP production and also activates multiple other signal transduction mechanisms (2, 3). The GLP-1 receptor (GLP-1R)-mediated reduction of postprandial blood glucose excursions is attributed to a combination of effects in multiple target tissues, including amplification of glucose-stimulated insulin secretion from pancreatic ß-cells, reduced glucagon secretion from adjacent
-cells, delayed gastric emptying, and induction of satiety (1, 4). Recent evidence further suggests that, in addition to these acute functions, GLP-1 also contributes to the long-term maintenance of the pancreatic ß-cell mass by mechanisms that rely on enhanced proliferation and differentiation, as well as a reduced apoptosis rate of these endocrine cells (5, 6, 7). Based on this broad range of functions, which converge on the maintenance of normal blood glucose homeostasis, drugs that mimic the function of endogenous GLP-1 (8, 9, 10) or extend the short biological half-life of this peptide (11, 12) are currently evaluated as a therapeutic option for the treatment of diabetes.
The majority of endogenous GLP-1 is synthesized in enteroendocrine L cells of the intestinal mucosa and is secreted into the bloodstream in response to food ingestion (4, 13). A critical step in the biosynthesis of GLP-1 is the cleavage of this peptide from an intracellular precursor, proglucagon, through the action of prohormone convertase 1/3 (PC1/3) (14). In particular, PC1/3 enables the formation of bioactive, amino-terminally truncated GLP-1, i.e. GLP-1(736) amide (unless otherwise noted, "GLP-1" in the current report refers to this fully processed peptide). Other forms of GLP-1, which either lack the amino-terminal truncation [e.g. GLP-1(137)] or are further cleaved during the process of rapid enzymatic degradation in the bloodstream [GLP-1(936) amide], have markedly reduced affinity/efficacy at the GLP-1R and, as a consequence, do not have biologically relevant agonist activity (13, 15). In addition to intestinal L cells, the proglucagon precursor is also expressed in the
-cell population of pancreatic islets (16). However, in the latter location proglucagon is processed to glucagon, a structurally related peptide that acts on its own cognate receptors (distinct from those for GLP-1) (3). The alternative processing of proglucagon in pancreatic
- vs. intestinal L cells is, at least in part, explained by expression of prohormone convertase 2 instead of PC1/3 in the former cell type (17). In the pancreas, PC1/3 is considered a selective marker of the insulin-producing ß-cell population. In ß-cells (which lack proglucagon expression), PC1/3 plays an important role in the processing of insulin (18, 19).
Studies with recombinant GLP-1Rs, which were expressed in nonendocrine fibroblast cell lines, have shown that this receptor is basally silent unless stimulated with an agonist (20, 21). In contrast, these receptors display enhanced basal activity when expressed in their native microenvironment in pancreatic ß-cells, even if no GLP-1 is added. Thus, it has been reported that the production of cAMP in islets that were isolated from wild-type mice was higher during in vitro culture compared with the level observed in corresponding islets from genetically engineered GLP-1R-deficient (GLP1R/) animals (22). Furthermore, in an immortalized ß-cell-derived cell line that was developed from wild-type mice, the basal level of cAMP production was reduced, in a concentration-dependent manner, by the GLP-1R antagonist exendin(939), whereas such inhibition was not apparent in a parallel line derived from GLP1R/ animals (23). Given that no indication for the presence of endogenous GLP-1 was detected in these in vitro experiments, it was concluded 1) that the enhanced basal level of GLP-1R activity reflects constitutive (i.e. ligand-independent) receptor function that may occur in a ß-cell microenvironment even without the supply of intestinal GLP-1 through the circulation, and 2) that exendin(939) may function as an inverse agonist rather than a true antagonist (22, 23). By definition, inverse agonists are distinguished from antagonists by their ability to inhibit constitutive, in addition to ligand-induced, receptor function (24).
In the current study, we have further investigated the molecular basis of tonic GLP-1R activity in an insulinoma cell line, ß-TC-6, as well as in isolated primary rat islets. Although our findings confirm enhanced basal GLP-1R function in both of these systems, the data suggest that this observation does not reflect constitutive receptor activity. Instead, in the models we studied, basal GLP-1R function was plausibly explained by local production of fully processed endogenous GLP-1. This peptide acted in an autocrine or paracrine fashion, respectively, on ß-TC-6 cells or on native ß-cells in isolated islets (the latter being stimulated by GLP-1 from adjacent
-cells). Suggesting a biological significance of this mechanism in preserving normal ß-cell function, we also found that pharmacological disruption of basal GLP-1R stimulation significantly diminished the basal level of insulin secretion and production. These in vitro observations, together with recent indications that developing or regenerating pancreatic islets can express the machinery for producing GLP-1 in vivo (25, 26), suggest a possible mechanism whereby locally generated GLP-1 may contribute to the physiological maintenance of ß-cell function and differentiation.
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RESULTS
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ß-TC-6 cells are responsive to GLP-1 stimulation (27) and are thus predicted to express cognate receptors for this peptide. Consistent with this expectation, analysis of ß-TC-6 cells by competition binding experiments vs. the GLP-1R-specific radioligand [125I]exendin(939) (Fig. 1A
) revealed a peptide affinity rank order that matched the well-established profile of GLP-1Rs (i.e. GLP-1 > exendin(939) >>GLP-2, glucagon) (15, 28, 29, 30). In view of the functional studies discussed below, note that the binding affinity of GLP-1 (IC50 = 170 pM) was approximately 4 orders of magnitude higher than that of glucagon (IC50 > 1 µM).

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Fig. 1. Expression and Function of GLP-1Rs in ß-TC-6 Cells
A, Competition studies vs. [125I]exendin(939), a GLP-1R-specific radioligand. Data were normalized to control binding in the absence of competitors (=100%). IC50 values (pIC50± SEM; n = 3) were as follows: GLP-1, 0.17 nM (9.76 ± 0.04); exendin(939), 1.0 nM (9.00 ± 0.06); GLP-2, 1.2 µM (5.92 ± 0.03); glucagon, 1.3 µM (5.89 ± 0.03). B, Glucose-induced insulin secretion by ß-TC-6 cells is enhanced by a GLP-1R agonist (exendin-4, 100 nM) or a phosphodiesterase inhibitor (IBMX, 1 mM) and is abolished by a GLP-1R antagonist [exendin(939), 100 nM]. Inhibition by the antagonist is reversed in the presence of an equimolar concentration of agonist (exendin-4). Means ± SEM; n = 3.
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Data shown in Fig. 1B
demonstrate a role of GLP-1Rs in amplifying glucose-induced insulin secretion by ß-TC-6 cells. In control experiments (no GLP-1R ligands added), a glucose-dependent increase in insulin secretion (2.8-fold) was detected with a half-maximal effect between 2.5 mM and 5 mM glucose. The GLP-1R agonist, exendin-4 (100 nM), further enhanced glucose-induced insulin secretion, with a half-maximal effect observed at a slightly lower (2.5 mM) glucose concentration. The effects of exendin-4 (i.e. an increased maximal effect with enhanced glucose sensitivity) were mimicked, to an even stronger extent, by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (1 mM), consistent with the concept that GLP-1R modulation of insulin secretion is mediated by an increased cAMP production in ß-cells (3, 31). Unexpectedly, we noted that the ability of glucose to enhance insulin secretion in this cell line was virtually abolished in the presence of exendin(939), a well-established GLP-1R-specific antagonist (28, 32, 33). If the observed loss of function reflected a receptor-mediated effect, inhibition of glucose-induced insulin secretion by exendin(939) should be prevented by coincubation with an equimolar concentration of exendin-4, a GLP-1R agonist with slightly higher receptor affinity than that of the antagonist (34). In fact, exendin-4 more than compensated for the inhibitory effect of exendin(939) on insulin secretion. Together, these findings suggest that ß-TC-6 function was largely dependent on basal GLP-1R activity (a function that occurred when no exogenous GLP-1R agonists were added).
It has been demonstrated that native rat and human ß-cells express glucagon receptors in addition to GLP-1Rs and that stimulation of either receptor type can enhance insulin secretion (35, 36). Given that ß-TC-6 cells can produce small, yet detectable, amounts of glucagon (37), we considered the possibility that auto-/paracrine stimulation of glucagon receptors contributes to the basal function of these cells. We found, to the contrary, that glucose-induced insulin secretion was only inhibited by exendin(939) but not by Des-His1, Asp9Glu glucagon 129 amide (DHG, Fig. 2A
). These compounds are selective antagonists at the GLP-1R (38) or glucagon receptor (39), respectively. Consistent with the differential effects of these antagonists on insulin secretion, we also found that only exendin(939), but not DHG, blocked basal cAMP production in ß-TC-6 cells (Fig. 2B
). Similar to what was observed for insulin secretion (see above), inhibition of basal cAMP production by exendin(939) was slightly overcompensated by coincubation with the GLP-1R agonist, exendin-4. Again, the latter finding supports the view that exendin(939)-induced inhibition of basal cAMP production was specifically mediated via GLP-1Rs.

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Fig. 2. Glucose Competence and Basal cAMP Production of ß-TC-6 Cells Result from Basal Activity of GLP-1 But Not Glucagon Receptors
A, Glucose (2.5 mM)-induced insulin secretion is inhibited by the GLP-1R antagonist, exendin(939) (100 nM), whereas the glucagon receptor antagonist DHG (1 µM) has no effect. GLP-1R agonists (exendin-4 or GLP-1; 100 nM each) further enhance glucose-induced insulin secretion. B, Basal cAMP production by ß-TC-6 cells is inhibited by the GLP-1R antagonist exendin(939) (100 nM) but not by the glucagon receptor antagonist DHG (1 µM). In contrast, the GLP-1R agonist exendin-4 (100 nM) stimulates cAMP production above basal and reverses inhibition by the antagonist. Data are expressed as a percentage of forskolin (100 µM)-induced cAMP production (5.4 ± 0.7 pmol/well). Means ± SEM, n = 4 (panel A), or n > 12 (panel B); **, P < 0.01 vs. basal.
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Further suggesting that GLP-1Rs in ß-TC-6 cells are tonically active, the time-dependent growth of these cells was considerably decelerated when exendin(939) was added to the medium. A significant inhibition of proliferation was detected as early as 2 d after exendin(939) treatment had been initiated (Fig. 3A
). The exendin(939)-induced growth inhibition was prevented by coincubation with an equimolar concentration of the GLP-1R agonist, exendin-4 (Fig. 3B
). ß-TC-6 cells thus show parallel patterns of GLP-1R-modulated cAMP production and proliferation, consistent with prior evidence that cAMP is an important messenger in the pathway of GLP-1-induced ß-cell growth and survival (5, 7, 40, 41). In further support of this concept, we noted that the proliferation of ß-TC-6 cells was enhanced by forskolin, a direct stimulus of adenylyl cyclase activity and cAMP production (42) (see supplemental Fig. 1
published as supplemental data on The Endocrine Societys Journals Online web site at http://mend.endojournals.org).

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Fig. 3. The Growth of ß-TC-6 Cells Is Decelerated by a GLP-1R Antagonist
ß-TC-6 cells (50,000) were seeded in 10-cm dishes and grown with daily medium changes, in the presence of exendins or in the absence of these peptides (control). Cell counts were increased or decreased, respectively, by a GLP-1R agonist (exendin-4, 100 nM) or antagonist [exendin(939), 100 nM]. Means ± SEM, n = 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control.
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Tonic GLP-1R function may result either from constitutive activity (i.e. ligand-independent induction of a conformationally active receptor state) (43) or from local production of endogenous agonist (e.g. GLP-1). In favor of the latter scenario, immunohistochemical analysis revealed that, in clusters of ß-TC-6 cells, virtually each individual cell stained positive with antibodies directed against GLP-1 (Fig. 4A
), a peptide that is typically found in intestinal L cells. In contrast, nonendocrine COS-7 cells that were seeded around the insulinoma clusters as negative controls did not show GLP-1 staining. The antibody that was used for immunocytochemistry is directed against the carboxy terminus of GLP-1, a domain that is distinct from the corresponding region in processed glucagon but is also present in proglucagon, the common intracellular precursor that is shared between these two peptides (13). Thus, it is difficult to establish by GLP-1 staining per se that the fully processed, bioactive form of this peptide is present. In our experiments, production of processed GLP-1 is suggested by the additional observation that virtually all ß-TC-6 cells are also immunoreactive for PC1/3 (Fig. 4B
), the key enzyme that effects processing of bioactive, amino terminally truncated GLP-1(736)amide from the proglucagon precursor (14). In addition to immunostaining, expression of both proglucagon and PC1/3 in these cells was confirmed by detection of respective mRNAs by RT-PCR analysis (see supplemental Fig. 2
published as supplemental data on The Endocrine Societys Journals Online web site at http://mend.endojournals.org). At the same time, consistent with the origin of this cell line from immortalized ß-cells (44), ß-TC-6 cell clusters also showed relatively homogeneous staining for insulin (Fig. 4C
). Together, these findings suggest that ß-TC-6 cells, although maintaining key aspects of the ß-cell phenotype (i.e. presence of GLP-1Rs, PC1/3, and insulin), have also the ability to produce fully processed GLP-1 (which may, in turn, trigger autocrine stimulation of GLP-1Rs).

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Fig. 4. GLP-1 Production Is Found in the Entire Population of ß-TC-6 cells but Only in a Distinct Population of Islet Cells
Top row, ß-TC-6 cells grow in clusters where immunostaining (brown) reveals expression of GLP-1 (panel A), PC1/3 (panel B), and insulin (panel C) in virtually every cell. Nonendocrine COS-7 cells that were seeded as negative controls (surrounding monolayer) show only minimal nonspecific staining. Bottom row, Fluorescence immunostaining is positive for GLP-1 in a distinct subset of cells at the islet periphery (panel D) whereas PC1/3 is found in virtually all cells (panel E) including GLP-1-positive putative -cells (merged pictures D and E, panel F).
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Immortalized pancreatic cell lines, although providing useful surrogate models of native ß-cells, typically approach, but do not fully recapitulate, the differentiated phenotype of the latter (44, 45). This holds also true for ß-TC-6 cells, which show a lower insulin content than native ß-cells and aberrantly produce trace amounts of glucagon as well as somatostatin (37). To address this caveat, we conducted further studies with isolated rat islets in parallel with pancreatic ß-cell lines. Immunostaining of isolated rat islets revealed a limited population of GLP-1-positive cells at the islet periphery (Fig. 4D
). These cells also stained positive for glucagon (a marker for pancreatic
-cells) but were clearly distinct from centrally located insulin-positive cells that accounted for most of the islet mass (data not shown). The latter, putative ß-cells also expressed PC1/3 (Fig. 4E
), consistent with the established role of this enzyme in insulin processing (18, 19). However, it is of note that expression of PC1/3 was also found in GLP-1 (and glucagon)-positive putative
-cells at the islet periphery (Fig. 4F
), contrary to prior findings that this enzyme was not detectable in pancreatic
-cells when examined in situ (17). As discussed for ß-TC-6 cells (see above), colocalization of PC1/3 with GLP-1/proglucagon immunoreactivity suggests the capacity of pancreatic
-cells to produce bioactive GLP-1. Different from the insulinoma line (where an autocrine mechanism of action is likely), it is conceivable that locally produced GLP-1 in native
-cells acts as a paracrine stimulus on adjacent ß-cell GLP-1Rs.
The local production of bioactive GLP-1 (as predicted by immunocytochemical colocalization of proglucagon and PC1/3) was further assessed by determining the identity of secreted proglucagon-derived peptides in cell culture supernatants. Analysis by specific RIA revealed that supernatants from both ß-TC-6 cells and isolated rat islets contained glucagon (Fig. 5A
). The RIA we used selectively detects carboxy-terminal portions of glucagon and showed virtually no cross-reactivity even when 100 nM of exogenous GLP-1(736) amide was added to the culture medium (Fig. 5A
). At the same time, ß-TC-6 cells and isolated islets also released lower, yet clearly detectable, amounts of amino-terminally processed GLP-1 (Fig. 5B
). This was demonstrated using a specific ELISA assay in which positive readouts depend on antibody detection of both the carboxy terminus and the correctly processed amino terminus of bioactive GLP-1. We found that bioactive GLP-1, in addition to ß-TC-6 cells, was also produced by the MIN6 ß-cell line (46) but not by AR42J cells, considered to resemble primordial epithelia in the pancreatic duct (47). Compared with bioactive GLP-1(736) amide, inactive forms of the peptide with an altered amino terminus showed little [GLP-1(137); < 0.5%] or no detectable cross-reactivity [GLP-1(936)] when added to the medium at high concentrations to assess ELISA specificity (Fig. 5C
).

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Fig. 5. ß-TC-6 Cells and Rat Pancreatic Islets Produce Both Glucagon and Fully Processed GLP-1
Cell supernatants were analyzed with either a glucagon-specific RIA (panel A) or an ELISA for amino-terminally processed bioactive GLP-1 (panel B). Panel C, The ELISA specifically detects exogenously added GLP-1(736)amide (added at 1 nM to the samples) vs. other control peptides (added at 100 nM). Means ± SEM; n = 3.
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Observed glucagon levels (
100 pM; see Fig. 5A
) were markedly lower than those required for half-maximal occupancy of GLP-1Rs (>1 µM; see Fig. 1A
). In contrast, the levels of secreted GLP-1 (
60 pM; Fig. 5B
) came close to the binding affinity of GLP-1 at its cognate receptor (170 nM; Fig. 1A
) and should thus enable at least partial receptor stimulation. Consistent with this expectation, supernatants from either ß-TC-6 cell or islet cultures led to a significant increase in cAMP production when added to GLP-1R-expressing COS-7 cells that served as functional sensors for the presence of bioactive peptide (Fig. 6
). Two controls confirmed that the observed increase in cAMP production was due to GLP-1-induced receptor stimulation: 1) absence of this response when assessed with COS-7 cells that lack GLP-1R expression; and 2) inhibition by the GLP-1R-specific antagonist, exendin(939).

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Fig. 6. ß-TC-6 and Rat Islet Cell Supernatants Induce cAMP Production When Applied to GLP-1R-Expressing COS-7 Cells
No increase in cAMP production occurs in COS-7 cells that lack GLP-1R expression (transfected with empty expression vector; negative control). Means ± SEM, n 7. *, P < 0.05; ***, P < 0.001; paired t test, absence vs. presence of antagonist [exendin(939), 100 nM].
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Reminiscent of our observations with the ß-TC-6 cell line, it appears that locally produced GLP-1 also plays a role in preserving the ability of isolated rat islets to produce insulin during in vitro culture (Fig. 7
). When the islets were kept in tissue culture, a constant rate of daily insulin release into the medium was maintained over 7 d; however, this rate was reduced in the presence of 100 nM exendin(939), the GLP-1R antagonist (data not shown). Consistent with this observation, total insulin content in islets after 7 d in culture was significantly lower in exendin(939)-treated vs. control cells (Fig. 7
, left panel). Maintenance of islets in the presence of a GLP-1R agonist (exendin-4, 100 nM) did not further increase insulin content vs. controls; however, exendin-4, when coadministered at equimolar concentrations with exendin(939), abolished the inhibitory effect of this antagonist. Together, these observations suggest that a sufficient level of endogenously produced GLP-1 was present in islet cultures to effect optimal GLP-1R stimulation for preserving insulin production. In contrast to the effect on insulin release, culture with exendin(939) did not significantly alter the total amount of islet protein (Fig. 7
, right panel).

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Fig. 7. Insulin Production by Rat Islets in Primary Culture Is, in Part, Dependent on Basal GLP-1R Activity
Isolated rat islets were maintained for 7 d in the presence of exendins or without these peptides (untreated controls), and insulin/protein contents were subsequently measured. A, Treatment with the GLP-1R antagonist exendin(939) (100 nM) reduces islet insulin content, an effect that is reversed by coincubation with an equimolar concentration of the GLP-1R agonist, exendin-4. Data are normalized to the insulin content in untreated controls (4.7 ± 0.5 mU/well). B, Exendin treatment (agonist or antagonist) for 7 d does not significantly alter the amount of total islet protein. Data are normalized to the protein content in untreated controls (21 ± 4 µg/well). Means ± SEM, n 4; *, P < 0.05 (Wilcoxon signed rank test vs. untreated control).
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DISCUSSION
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The present study suggests that locally produced GLP-1 maintains a significant level of basal GLP-1R activity in the ß-TC-6 insulinoma cell line as well as in isolated rat pancreatic islets in primary culture. The observation that this autostimulatory loop (autocrine in insulinoma cells vs. paracrine in islets) is a prerequisite for maintaining glucose-induced insulin secretion and production in either of these models has important implications for in vitro studies of ß-cell function. Furthermore, this finding may also shed new light on the understanding of physiological intraislet regulation.
To our knowledge, the existence of an endogenous GLP-1/GLP-1R loop in insulinoma cell lines has not been previously reported. This mechanism may result from a phenotypic drift of ß-TC-6 cells (a concept discussed in Ref.48) that is possibly driven by growth advantages due to a GLP-1R-mediated increase in glucose sensitivity (Figs. 1B
and 2A
) and in cell proliferation (Fig. 3
). Our findings raise the question whether a similar autocrine GLP-1/GLP-1R loop may also occur in other insulinoma cell lines, which would be relevant given the common use of such lines for studying ß-cell functions, e.g. insulin secretion. There is emerging evidence that among well-established insulinoma/ß-cell lines (44, 45) several have the capacity of producing bioactive GLP-1. In addition to the ß-TC-6 line, we have found this property with MIN6 cells (Fig. 5
), whereas others have reported GLP-1 production by INS-1 cells (49). With INS-1 cells, an impact of endogenously produced GLP-1 on basal activity was not readily appreciable unless these cells were transfected with recombinant GLP-1R cDNA (49). The latter observation illustrates that biologically relevant autostimulation, although potentially applicable to many ß-cell lines, is dependent on sufficient endogenous expression of both GLP-1 and its cognate receptors. Even for a given cell line, either of these two parameters may be altered by multiple factors, including culture conditions, as has been demonstrated for endogenous GLP-1R expression by MIN6 cells (50).
Stimulation of GLP-1Rs by locally produced agonist is conceptually different from the constitutive (i.e. putatively ligand-independent) activity of GLP-1Rs that has been previously postulated for two other insulinoma cell lines, RIN104638 (51) and ß-TC-Tet (23). In ß-TC-Tet cells during tetracycline-induced growth arrest, no preglucagon mRNA expression was detected by Northern blot analysis, leading the authors to attribute basal GLP-1R function to constitutive activity. It is possible that this difference vs. our findings with ß-TC-6 cells reflects truly divergent biological properties between the two insulinoma cell lines. On the other hand, it should be considered that even picomolar concentrations of GLP-1 are sufficient for partial receptor stimulation, and that such small quantities of the peptide may only be detectable with a sensitive bioassay as has been applied in the current study (see Fig. 6
). Consistent with the possibility that ß-TC-Tet cells may have the capacity of producing small, yet potentially relevant, amounts of GLP-1, trace levels of preproglucagon mRNA were observed in these cells when tetracycline induction was omitted (23).
The question of whether tonic GLP-1R activity in insulinoma cell lines is due to autocrine stimulation vs. constitutive activity is relevant for the distinction of putative antagonists vs. inverse agonists. Different from conventional antagonists that, by definition, only inhibit agonist-induced signaling, inverse agonists have the additional ability of inhibiting ligand-independent signaling of G protein-coupled receptors (24). In both ß-TC-Tet (23) and ß-TC-6 cells (current study), basal GLP-1R function was inhibited by exendin(939). Whereas the traditional view of exendin(939) as an antagonist (28, 32, 33, 52, 53) is sufficient for explaining its effects on the basal function of ß-TC-6 cells, an ability of this peptide to attenuate constitutive receptor activity, as has been postulated for ß-TC-Tet cells, would require the reclassification of this compound as an inverse agonist. Supporting the view of exendin(939) as a traditional antagonist, we have found that this peptide did not inhibit basal function of a genetically engineered constitutively active GLP-1R mutant when recombinantly expressed in nonendocrine fibroblasts that lack the potential for GLP-1 production (54).
Comparison of findings with ß-TC-6 cells and native pancreatic islets in primary culture revealed production of GLP-1 in both systems. However, whereas in the ß-TC-6 line GLP-1 and insulin production occurred within the same cells, these two hormones are produced separately (i.e. in
- and ß-cells, respectively) in pancreatic islets (Fig. 4
). Thus, locally produced GLP-1 likely acts as an autocrine stimulus in the insulinoma cell line but is a candidate paracrine mediator in pancreatic islets. Our conclusion that detectable amounts of fully processed and biologically active GLP-1 are produced in the
-cells of pancreatic islets is consistent with a prior immunohistochemical analysis of rat pancreatic tissue sections (55) and is also supported by HPLC/RIA analysis of rat pancreatic extracts in which small amounts of fully processed GLP-1 were detected (26, 56). Furthermore, the idea that GLP-1Rs in pancreatic islets are tonically active has been previously raised, based on functional comparison of mouse islets isolated from either wild-type or GLP-1R-deficient mice (22). This study showed that the level of basal cAMP production was lower in islets from GLP-1R-deficient mice compared with islets from wild-type animals, and that glucose-induced insulin secretion from the latter was attenuated in the presence of exendin(939). However, the authors did not consider the possibility that sustained GLP-1R function may be due to locally produced GLP-1 but alternatively proposed that these receptors may be constitutively active and/or partially stimulated by high concentrations of locally accumulated glucagon. Linking these prior histochemical (55) and biochemical (26, 56) with functional (22) observations, our findings suggest that basal GLP-1R activity in ß-cells is attributable to local GLP-1 production by pancreatic
-cells.
Although a physiological role of locally produced GLP-1 in islet function under in vivo conditions remains to be established, there is emerging evidence that
-cells in situ have the potential to generate fully processed, biologically active GLP-1. Wilson and colleagues (25) reported that the earliest endocrine cells in the developing mouse pancreas not only produce glucagon, as is well established, but also express PC1/3 and thus have the potential to release bioactive GLP-1. The authors raised the hypothesis that locally produced GLP-1 may be an important trigger of islet development. Recently, it was observed that
-cells in the adult pancreas can express PC1/3 in mice that have been engineered to lack expression of the related prohormone convertase, PC2 (a key player in normal glucagon production by this cell type) (57). Taken together with our finding that GLP-1 can also be produced by
-cells from normal rat islets during in vitro culture, it is possible that this peptide is up-regulated under certain conditions in adult life, e.g. when required during the process of islet neogenesis, regeneration, or repair.
In support of this idea, experiments with adult rats have shown that experimentally induced ß-cell injury and hyperglycemia trigger an increase in PC1/3 expression in pancreatic
-cells (26). It was concluded from these observations that hyperglycemia may induce GLP-1 production in
-cells, which then enhances the function of adjacent surviving ß-cells. In view of these in vivo findings, generation of GLP-1 by isolated rat islets during in vitro culture (as demonstrated in the current study) may present a parallel survival/repair mechanism, one that is possibly triggered by challenges such as enzymatic cell isolation and maintenance outside the physiological tissue environment.
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MATERIALS AND METHODS
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Materials
Tissue culture medium and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA) and from Intergen Co. (Purchase, NY), respectively. Bolton-Hunter labeled [125I]exendin(939) (2200 µCi/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA). Nonradioactive exendin-4, exendin(939), glucagon, GLP-1 (bioactive and -inactive forms), and glucagon-like peptide-2 (GLP-2) were purchased from American Peptide Co. (Sunnyvale, CA), and DHG from Bachem Bioscience, Inc. (King of Prussia, PA). Primary antibodies were purchased from the following suppliers: goat anti-GLP-1, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); guinea pig antiinsulin, DAKO Corp. (Carpinteria, CA); rabbit anti-PC1/3, Chemicon (Temecula, CA). Horseradish peroxidase-conjugated secondary antibodies were either from Santa Cruz Biotechnology (antigoat or antirabbit) or from Zymed Laboratories, Inc. (South San Francisco, CA) (antirabbit). Fluorescein isothiocyanate (FITC) or cyanine3 (Cy3)-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Methods
Cell Culture
Cell lines or isolated islets were kept at 37 C, in a humidified atmosphere with 5% CO2. Indicated media were supplemented with heat-inactivated FBS at the specified concentration and with penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively). Pancreatic cell lines were maintained between passages 18 and 35. ß-TC-6 cells (37) (kindly provided by Dr. R. P. Robertson) were grown in RPMI medium/10% FBS; MIN6 cells (46) (kindly provided by Drs. J. Miyazaki and D. F. Steiner) were grown in DMEM/10% FBS, and AR42J cells (47) (purchased from the American Type Culture Collection, Manassas, VA) were grown in F12K medium/20% FBS. Islets of Langerhans were prepared from male Sprague Dawley rats (200225 g), as previously described (58). The islets were maintained in DMEM/10% FBS on Matrigel extracellular matrix (BD Biosciences, Bedford, MA).
Radioligand-Binding Assay
ß-TC-6 cells were seeded into 24-well plates at an initial density of 50,000/well and grown for 2 d. Receptor-binding assays were performed using 15 pM [125I]exendin(939) as the radioligand and increasing concentrations of unlabeled peptides as competitors (20).
Measurement of Insulin, Glucagon, and GLP-1
ß-TC-6 cells (50,000/well) were seeded into 24-well plates and grown for 2 d. The cells were then starved for 30 min in Krebs/Ringer/HEPES solution and subsequently incubated for another 30 min in fresh Krebs/Ringer/HEPES containing indicated concentrations of glucose, GLP-1 related peptides, and/or IBMX (Sigma-Aldrich, St. Louis, MO). Secreted insulin in the cell supernatant was determined using a RIA kit (MP Biomedicals, Irvine, CA).
Additional experiments were carried out to determine the insulin content in primary rat islet cultures. Freshly isolated islets (516 islets per Matrigel-coated well in a 24-well plate) were maintained for 7 d with daily medium changes. The islets were then extracted with M-Per mammalian protein extraction buffer (Pierce Chemical Co., Rockford, IL), and the insulin content in each well was determined by RIA. Also, the protein content in each well was determined using the bicinchoninic acid method (Pierce) (59).
The concentrations of glucagon and of fully processed GLP-1 in 24-h insulinoma cell or islet culture supernatants were determined by RIA and ELISA, respectively (Linco Research, Inc., St. Charles, MO). Parallel samples were supplemented with 1 mM IBMX and applied to COS-7 fibroblasts as a reporter system for the presence of bioactive GLP-1. Two days before the assay, the COS-7 cells had been transiently transfected with human GLP-1R cDNA (20) and thus responded with receptor-mediated cAMP production if bioactive GLP-1 was present.
Measurement of cAMP Formation
ß-TC-6 (50,000/well) or GLP-1R-expressing COS-7 cells (100,000/well) were seeded in 24-well plates and grown for 3 d or 1 d, respectively. The production of cAMP during a 1-h incubation in the presence of 1 mM IBMX, with or without receptor agonists/antagonists, was determined by FlashPlate proximity scintillation assays (PerkinElmer Life Sciences), as previously described (20).
Immunohistochemical Staining/Light and Confocal Fluorescence Microscopy
To detect individual antigens by peroxidase staining, 50,000 ß-TC-6 cells were seeded on coverslips that had been placed in six-well culture plates. After maintenance for 5 d, when the majority of ß-TC-6 cells had formed large clusters, COS-7 cell fibroblasts (500,000/well) were added as negative controls that lack expression of endocrine hormones. After an additional 24 h, the cells were fixed with 3.7% formaldehyde solution in PBS and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). The samples were incubated with PBS/10% FBS for 20 min to block nonspecific binding, followed by two sequential overnight incubations at 4°C with primary and secondary antibodies, respectively. The following primary antibodies were used at the indicated dilutions: guinea pig antiinsulin (1:250); rabbit anti-PC1/3 (1:500), or goat anti-GLP-1 (1:500). Corresponding secondary antibodies were horseradish peroxidase conjugated, and each used at a 1:1600 dilution. Cell-bound antibodies were visualized by incubation with the peroxidase substrate, diaminobenzidine (Sigma-Aldrich), for 30 min. The coverslips were mounted on object slides, and images were taken with an Olympus BH-2 microscope (Olympus America Inc., Melville, NY).
To assess colocalization of GLP-1 and PC1/3, isolated rat islets were plated on Matrigel-coated coverslips and maintained for 2 d. After fixation and permeabilization, an overnight incubation was performed with primary antibodies (see above). Another overnight incubation was then performed with fluorescence-labeled secondary antibodies (each diluted at 1:2000), mouse-Cy3 (for GLP-1 staining) and mouse-fluorescein isothiocyanate (for PC1/3 staining). The samples were mounted on object slides, and images were taken using a Leica TCS SP2 confocal laser scan microscope (Leica Microsystems Inc., Exton, PA).
Data Analysis
GraphPad Prism software version 3.0 (GraphPad, San Diego, CA) was used to calculate radioligand competition binding and agonist/antagonist concentration-response curves. Unless otherwise specified, statistical comparisons were made by ANOVA and Tukey-Kramer multiple comparison posttests.
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ACKNOWLEDGMENTS
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We thank Drs. R. P. Robertson, D. F. Steiner, and J. Miyazaki for generously providing ß-TC-6 and MIN6 cells. The excellent technical assistance of Sophia Morin and Christine I. Worrall is greatly appreciated.
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FOOTNOTES
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This work was supported by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK56674, T32 DK07471, and P30 DK34928 (Center for Gastroenterology Research on Absorptive and Secretory Processes); and by a research grant from the American Diabetes Association.
Current address for K.M.: Institut für Immunologie, Universität Witten-Herdecke, D-58448 Witten, Germany.
First Published Online January 27, 2005
Abbreviations: DHG, Des-His1, Asp9Glu glucagon 129 amide; FBS, Fetal bovine serum; GLP-1, glucagon-like peptide 1; GLP-1R, GLP-1 receptor; IBMX, isobutylmethylxanthine; PC1/3, prohormone convertase 1/3.
Received for publication September 3, 2004.
Accepted for publication January 18, 2005.
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