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Endocrinology Vol. 143, No. 9 3529-3539
Copyright © 2002 by The Endocrine Society

ARTICLE

Transfection of Pancreatic-Derived ß-Cells with a Minigene Encoding for Human Glucagon-Like Peptide-1 Regulates Glucose-Dependent Insulin Synthesis and Secretion

Hongxiang Hui, Run Yu, Corinne Bousquet and Riccardo Perfetti

Division of Diabetes, Endocrinology, and Metabolism (H.H., R.Y., C.B., R.P.), Cedars-Sinai Medical Center and University of California Los Angeles (R.P.), Los Angeles, California 90048

Address all correspondence and requests for reprints to: Riccardo Perfetti, M.D., Ph.D., Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, 8723 Alden Drive, SSB 290, Los Angeles, California 90048. E-mail: perfettir{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from the proglucagon gene, capable of regulating the transcription of the three major genes that determine the pancreatic ß-cell-specific phenotype: insulin, GLUT-2, and glucokinase. The aim of this study was to investigate the potential role of GLP-1 for the gene therapy of glucose-insensitive pancreatic ß-cells. We transfected mouse insulinoma cells with a DNA fragment of the human proglucagon gene containing the nucleotide sequence encoding for human GLP-1 but lacking the coding region for glucagon. Two constructs were generated: In one, the expression of GLP-1 was under the control of the cytomegalovirus (CMV) promoter (CMV/GLP-1), and the second was regulated by the rat insulin II promoter (RIP)/GLP-1). Northern blot, HPLC, and RIA analyses confirmed that the minigene was transcribed and the protein appropriately translated, processed, and secreted in the extracellular environment. Gene expression studies revealed that although CMV/GLP-1 cells did not gain a greater glucose sensitivity as a result of the transfection with GLP-1, compared with cells transfected with the plasmid alone, RIP/GLP-1 was capable of regulating the gene expression of insulin and GLP-1 based on the concentration of glucose in the culture medium. Detection of the counterpart proteins (insulin and GLP-1) in the culture medium paralleled the observation derived from the Northern blot analysis. GLP-1 action was mediated by an IDX-1 (islet/duodenum homeobox-1) dependent transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis. This was further suggested by a significant increase of the glucose-dependent binding of IDX-1 to the insulin promoter in RIP/GLP-1 cells but not in CMV/GLP-1 cells or control cells. Finally, we observed that although the GLP-1-dependent secretion of insulin was mediated by an increase in cAMP levels, the transcription of the insulin gene, in response to GLP-1, was in large part cAMP independent. The present study lays the research foundation to investigate the potential use of GLP-1 for the gene or cell therapy of diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC ISLET ß-CELLS regulate the synthesis and secretion of insulin in response to glucose and other stimuli by constantly resetting the amount of insulin secreted to maintain the excursion of plasma glucose levels within a very tight range. The inability of compensating for abnormally elevated glucose levels in the setting of insulin resistance and/or impaired insulin secretion is not adequately addressed by current modality of treatment of type 2 diabetes (1, 2). The current repertoire of pharmacological agents for the treatment of diabetes, although based on drugs with diverse mechanisms of action, does not include factors capable of promoting a glucose-dependent secretion of insulin.

The inability of restoring the physiological coupling between glucose and insulin represents a fatal limitation of the current modalities of treatment for hyperglycemia. This results in having subjects with diabetes that are frequently undertreated for fear of reactive hypoglycemia, leading to a greater incidence of diabetic complications because of the toxic effect of elevated plasma glucose.

Studies from various laboratories have demonstrated that an intestinal hormone, termed glucagon-like peptide-1 (GLP-1), induces insulin secretion in response to glucose, leading to propose that its use may be beneficial for the treatment of type 2 diabetes (3, 4). Interestingly, the capability of GLP-1 to enhance glucose-dependent insulin secretion is preserved in subjects with diabetes, even after many years from the onset of the disease and/or when they are no longer responding to oral hypoglycemic sulfonylureas (5). A major limitation in extending these biological observations to the design of new strategies for the treatment of diabetes resides in the very short half-life of GLP-1 in vivo (3, 4) and in its method of administration, which would have to be via an injectable route because of its peptide structure.

In the present study, we demonstrated that gene transfection with a minigene encoding for human GLP-1 is capable of restoring a normal synchronization of insulin synthesis in response to glucose.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Mouse insulinoma (MIN-6) cells were a gift from Dr. K. Silver (University of Maryland, Baltimore, MD). Cells were cultured in DMEM medium (Life Technologies, Inc., Gaithersburg, MD) containing 100 µg/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum (FBS) (Gemini Bio-Products, Inc., Woodland, CA) at 37 C under a humidified condition of 95% air and 5% CO2. Gene and protein expression experiments were carried out using cells grown to 80% of confluence after washing the cell layer with serum-free medium and a washout incubation with fresh medium (according to experiment-specific protocols described in the figure legends). To determine the response to various stimuli, cells were cultured in serum-free, glucose-free medium and then exposed to medium containing increasing concentration of glucose (as described in the figure legend of individual experiments). At the completion of the experiments, media and cells were collected separately and assayed for the experiments described hereafter.

Isolation of GLP-1 minigene construct
Human proglucagon was a kind gift from Dr. Daniel J. Drucker (University of Toronto, Toronto, Canada). Proglucagon cDNA was used to generate by PCR a DNA sequence spanning from nucleotide 289 to nucleotide 516 (Fig. 1Go, top panel). This PCR-generated sequence included the coding region for human GLP-1, a partial fragment of the intervening peptide sequence at the 5'-end, and a fragment of the GLP-2 sequence at the 3' end. The identity of the PCR product was confirmed by DNA sequencing.



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  		Figure 1. Top panel, Plasmid construct schematic representation of the two plasmid constructs (A, CMV/GLP-1; B, RIP/GLP-1) generated with the human GLP-1 minigene. C, Area of the human proglucagon gene used to generate the GLP-1 insert. Bottom panel, Northern blot analysis of transfected cell. Parental MIN-6 cells and MIN-6 cells transfected with the vector alone or with a vector containing a DNA sequence encoding for human GLP-1 were cultured in 10% FBS in the presence of 12 mM glucose. Cells were subjected to RNA extraction and Northern blot analysis for insulin, GLP-1, and ß-actin mRNA levels. Left, Cells transfected with a plasmid containing human GLP-1 driven by the CMV promoter (lane 1); parental MIN-6 cells (lane 2); and MIN-6 cells transfected with the vector alone (lane 3). Right, Cells transfected with a plasmid containing human GLP-1 driven by the rat insulin promoter (lane 4) and parental MIN-6 cells (lane 5). Each experiment was repeated twice, using RNA samples from independent cultures.

 
Plasmid construction and cell transfection with human GLP-1 minigene
MIN-6 cells were transfected with a pSecTag2 A plasmid (Invitrogen, Carlsbad, CA) harboring the GLP-1 fragment of the human proglucagon gene using LipoTAXI (mammalian transfection kit, Stratagene, La Jolla, CA). Two GLP-1-expressing vectors were generated (Fig. 1BGo): in the first one, the DNA fragment encoding for GLP-1 was inserted at the HindIII and PstI sites of the pSecTag2 A plasmid, downstream of the cytomegalovirus (CMV) promoter. For the second expression vector, the CMV sequence was deleted by restriction enzyme digestion using the enzymes BglII and SacI and replaced with a DNA fragment encoding for the rat insulin II promoter (RIP) (a gift from Dr. Theodore Friedman, King Drew Medical Center, Los Angeles, CA). The fragment of the rat insulin promoter that was used corresponds to the previously characterized RIP-7 promoter (6) and was inserted after digestion with BamH1 and HindIII restriction enzymes. Vector DNA and promoter sequence were blunted by incubation with the Klenow fragment before the ligation. The correct orientation of the sequence was verified by digesting the plasmid with EcoR1.

Both constructs contained a signal peptide sequence on the 5'-end of the insert (Fig. 1Go, top panel). Control cells were transfected with the vector alone or with a plasmid encoding solely the RIP-7 sequence. The selection of positive (i.e. transfected) cells was carried out by culturing the cells in the presence of 400 µg/ml G418 sulfate (Omega, Tarzana, CA).

Two independently transfected cultures (for each of the constructs used) were tested by Northern blot analysis in the presence of different concentration of glucose.

RNA isolation and Northern blot analysis
Cellular RNA was extracted as routinely described. Northern blots were hybridized with full-length rat insulin II cDNA probe, human GLP-1 cDNA, and rat ß-actin cDNA probe. The human GLP-1 cDNA probe was generated by PCR of the proglucagon plasmid used for cell transfection. The PCR product was generated using the following primers: 5'-ACAAGCTTGGTTGATGAACACCAAGAGG-3 and 5'-CGCTGCAGAGAGAAGGATCCATCAGCAT-3'. All cDNA probes were labeled with [32P]dCTP (Amersham Life Science, Arlington Heights, IL) by the random priming procedure using the enzyme sequence (United States Biochemical Corp., Cleveland, OH). Hybridization and washing conditions were carried out as routinely described. The mRNA level for individual transcripts was evaluated by densitometric analysis and normalized for the relative abundance of ß-actin mRNA (Fig. 1Go, bottom panel).

HPLC and RIA for GLP-1
To determine the identity of GLP-1 immunoreactivity, culture medium from MIN-6 RIP/GLP-1 cells and parental MIN-6 cells was separated by reversed-phase HPLC using a liquid chromatography system and a C18 µBondapak column (Waters Corp., Milford, MA) as described previously (7). Extracts were separated using a 30-min linear gradient of 45–68% solvent B (solvent A: 0.1% phosphoric acid, 0.3% triethylamine, buffered with NaOH to pH 7.0; solvent B: 60% acetonitrile, 40% solvent A) at a flow rate of 1.0 ml/min. This gradient was followed by a 10-min isocratic run at 99% solvent B. HPLC fractions were lyophilized before RIA. GLP-1 was measured in HPLC fractions using a GLP-1 (7–36) amide antiserum (Linco Research, Inc., St. Charles, MA). Commercially available human recombinant GLP-1 (Bachem, King of Prussia, PA) was used as a standard for HPLC analysis of medium samples.

Immunofluorescence microscopy
Cells were cultured on monocoated chamber slides (Nalge Nunc International, Naperville, IL) in the presence of different concentrations (0, 6, and 12 mM) of glucose (Sigma, St. Louis, MO) for 8 h. Cells were washed and fixed with 2% paraformaldehyde for 4 h at room temperature in PBS (Life Technologies, Inc.), solubilized with 0.1% (vol/vol) Triton X-100 (Sigma) in PBS for 5 min. Cells were then washed with 0.01 M PBS three times for 3–5 min, and nonspecific binding was inhibited by using 5% chick serum (Life Technologies, Inc.) in 0.01 M PBS at room temperature for 60 min in a humid chamber. A rabbit IDX-1 (islet/duodenum homeobox-1) antibody (a kind gift from Dr. Chris Wright, Vanderbilt University, Nashville, TN) directed against the N terminus of the frog homolog of the IDX-1 gene was used as the primary antibody (1:500 diluted with 0.1% Triton X-100, 1% BSA in 0.01 M PBS), and slides were incubate at 4 C overnight in a humid chamber. After washing, cells were incubated with a fluorescein- conjugated goat antirabbit IgG antibody (Molecular Probes, Inc., Eugene, OR) and incubated at room temperature for 1 h in a humid chamber.

Immunofluoresce experiments were repeated at least three times using independent cell cultures.

Measurement of insulin and GLP-1 secretion
MIN-6 cells (parental, CMV/GLP-1, RIP/GLP-1 and transfected with the pSecTag2 A plasmid alone) were plated at density of 106 cells/well in 6-well plates. Once the cells reached 80% of confluence, they were washed and exposed to fresh serum-free medium for 8 h in the presence of various concentrations of glucose (0, 0.1, 1, 3, 6, 10, 20 mM). The level of insulin and GLP-1 in the culture medium was measured by RIA (Linco Research, Inc.). Insulin and GLP-1 levels were then normalized for total cellular protein content per each individual culture.

To validate the specificity of GLP-dependent secretion of insulin, MIN-6 RIP/GLP-1 cells were cultured in the presence of the receptor antagonist Exendin-9 (American Peptide Co., Sunnyvale, CA). After an overnight washout period (in medium deprived of glucose and FBS), cells were cultured in serum-free medium containing 10 mM glucose for various lengths of time (10 min, 30 min, 1 h, 24 h), in the presence or absence of 10-6 M of Exendin-9. Conditioned media were then collected for RNA analysis for insulin level.

Protein assay
Total cellular protein content was measured by using the Bradford method (Bio-Rad Laboratories, Inc., Richmond, CA). The amount of proteins measured was used as a correction factor for the detection of the relative amount of insulin or GLP-1 in the culture medium, as assayed by RIA.

Protein extraction and Western blotting
To compare GLP-1 receptor level among the various cell lines studied, parental, CMV/GLP-1-transfected, and RIP/GLP-1-transfected cells were cultured in the presence of 6 mM glucose; to investigate the potential effect of glucose on receptor expression MIN-6 RIP/GLP-1 cells were grown in the presence of 0, 3, 6, or 15 mM glucose. For both experiments cells were cultured for 12 h and then washed and the pellet collected and homogenized in ice-cold TE buffer (40 mM Tris, pH 7.4; 1 mM EDTA; 1 mM dithiothreitol) (Sigma) in the presence of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 8.3 µM aprotinin, 50 µM leupeptin, 30 mM sodium orthovanadate) (Sigma) and spun at 90,000 x g for 30 min. Cell pellets were resuspended in TE buffer, supplemented with 0.3% wt/vol deoxycholate and 1% wt/vol digitonin (Sigma), rotated at 4 C, and spun at 90,000 x g for 30 min. Supernatants were collected. Cell homogenates were separated by SDS-PAGE on an 8% polyacrylamide gel (Novex, San Diego, CA) and electroblotted onto polyvinyl difluoride membrane (Millipore Corp., Bedford, MA). The blot was then blocked in wash solution (PBS containing 3% milk and 0.1% Tween-20), probed with 1 µg/ml of a mouse antihuman GLP-1 receptor (GLP-1R) antibody (kindly donated by Dr. Daniel J. Drucker) for 90 min at 25 C, washed twice with wash solution, incubated with antimouse secondary antibody for 1 h at 25 C, and washed three times with wash solution. The protein band corresponding to the GLP-1R was visualized by the enhanced chemiluminescence method (Amersham, Piscataway, NJ).

Gel shift analysis
For gel shift analysis, an oligonucleotide corresponding to rat insulin-II A1 element (-89 to -69, AGAGCCCTTAATGGCCAAA) was annealed and end labeled using T4-polynucleotide kinase (Life Technologies, Inc.) and [{gamma}-32P]ATP (Amersham Life Science). Nuclear extracts from parental MIN-6, MIN-6 transfected solely the RIP promoter, MIN-6 CMV/GLP-1, and MIN-6 RIP/GLP-1 cells were cultured in the presence of various concentrations of glucose (0, 3, 10, 15 mM) for 8 h were prepared as described previously (8). Gel shift reactions with nuclear proteins were carried out in 1 µg poly-DI-DC, 25 mM HEPES, 1.5 mM EDTA, 5% glycerol, 1.0 mM dithiothreitol, and 150 mM KCl in a final volume of 25 µl. The mixture of 10 µg of nuclear extracts and [32P]-labeled DNA was incubated for 20 min at 25 C. For competition assays, an excess of specific or nonspecific competitor oligonucleotide was added (100-fold) 5 min before the addition of [32P]-labeled DNA. Reaction mixtures were loaded onto a 4% polyacrylamide gel and subjected to electrophoresis at 90 V in 0.5x TBE [0.9 M Tris, 0.9 M borate, 2 mM EDTA (pH 8.0)]. Gels were dried and protein-DNA complexes were visualized by exposure to x-ray film for 4–12 h. For supershift assays, nuclear extracts derived from MIN-6 RIP/GLP-1 cultured in the presence of 15 mM glucose were incubated with 2 µg IDX-1 antibody (a gift from Dr. Chris Wright) for 30 min on ice. Extracts were then subjected to electrophoresis and detected as described above.

Inhibition of cAMP-dependent GLP-1 signaling
To investigate whether the ability of GLP-1-transfected cells to release insulin in a glucose-dependent fashion was dependent on cAMP- dependent GLP-1 signaling, we tested the effect of the cAMP inhibitor Rp-cAMP (cAMP, Rp-isomer, triethylammonium salt), which blocks the activation of protein kinase A, resulting from an elevation of cAMP, on insulin secretion and insulin mRNA levels. MIN-6 RIP/GLP-1 cells routinely cultured in the presence of 10% FBS and 12 mM glucose, were subjected to an overnight washout period with medium deprived of glucose and FBS. They were then cultured with serum-free medium containing either 3 mM or 10 mM glucose for various lengths of time (10 min, 30 min, 1 h, 24 h), in the presence or absence of 10-6 M Rp-cAMP (Biosciences Inc., La Jolla, CA). Cells and conditioned media were collected for RNA analysis and RIA of insulin and GLP-1 levels. Culture conditions, RNA extraction, RIA, and protein assay were performed as described elsewhere. Determination of cAMP levels was performed with a cAMP detection kit (Amersham Pharmacia Biotech, Piscataway, NJ), according to the manufacturer.

Statistical analysis
The data were expressed as mean ± SE. Significance of the data was evaluated by unpaired t test. One-way ANOVA was used to evaluate statistical significance when more than two data points were analyzed. Statistical analyses by unpaired t test or ANOVA are explicitly identified in the text or figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HPLC and RIA of cell culture medium
To determine whether GLP-1 was both processed correctly and secreted in the extracellular environment, HPLC and RIA were used. The major GLP-1 immunoreactive HPLC fraction detected in medium extracts from MIN-6 RIP/GLP-1 cells eluted at the same position of synthetic GLP-1 (Fig. 2Go). HPLC and RIA of medium collected from parental MIN-6 did not reveal any trace of GLP-1, but its level in the culture medium collected from MIN-6 RIP/GLP-1 cells was greatly enhanced by the presence of glucose in the culture medium (Fig. 2Go).



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  		Figure 2. HPLC analysis of transfected cells. A, HPLC of human recombinant GLP-1. B, HPLC of medium collected from MIN-6 RIP/GLP-1 cells cultured for 12 h in the presence of 15 mM glucose. C, RIA for GLP-1 of HPLC fractions collected from the culture medium of MIN-6 RIP/GLP-1 cells cultured for 12 h in the presence of 15 mM glucose. D, RIA for GLP-1 of HPLC fractions collected from the culture medium of MIN-6 RIP/GLP-1 and parental MIN-6 cells cultured for 12 h in the presence of 0 or15 mM glucose. GLP-1 levels were normalized for protein content in the cell pellet extract for each individual culture. The graph represents the average ± SE of three independent experiments. Statistical significance of the data was evaluated by unpaired t test.

 
Northern blot analysis for insulin and GLP-1 mRNAs
CMV/GLP-1, RIP/GLP-1 cells and cells transfected with the pSecTag2A plasmid alone or with a plasmid encoding solely the RIP-7 sequence were cultured in the presence of increasing concentrations of glucose and subjected to Northern blot analysis for detection of insulin, GLP-1, and ß-actin mRNA levels. Northern blot analysis of MIN-6 cells transfected solely with the pSecTag2A as well as cells transfected with RIP-7 showed that the insulin gene was constitutively transcribed and that the insulin mRNA level was not affected by varying the concentration of glucose in the culture medium (Fig. 3Go, A and C). Cellular transfection of MIN-6 cells with a plasmid harboring the CMV/GLP-1 construct did not promote a glucose-dependent transcription of the insulin gene characteristic of normal islet ß-cells (Fig. 3BGo). Transfection with RIP/GLP-1 construct induced a profoundly different profile in the glucose-dependent accumulation of insulin mRNA (Fig. 3DGo). There was a sharp increase in the expression of insulin mRNA that paralleled the increase in the concentration of glucose in the culture medium (P < 0.001). Detection of GLP-1 mRNA in the various cell lines exposed to increasing concentrations of glucose paralleled the response observed with insulin. GLP-1 mRNA was constitutively expressed by CMV/GLP-1 MIN-6 cells, and its expression was not controlled by glucose (Fig. 3BGo). On the other hand, when the expression of GLP-1 was under the control of the rat insulin promoter (RIP/GLP-1), a significant glucose-dependent expression of both insulin and GLP-1 genes was observed (Fig. 3DGo).



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  		Figure 3. Glucose-dependent regulation of insulin and GLP-1 mRNA levels. Cells routinely grown in the presence of 10% FBS and 12 mM glucose were subjected to a 2-h washout period with medium deprived of glucose and FBS. They were then cultured in serum-free medium for 8 h in the presence of various concentrations of glucose (0, 0.1, 1, 3, 6, 10, 20 mM). After RNA extraction, the membranes were hybridized with cDNA probes for insulin, GLP-1, and ß-actin. Autoradiograph represents one individual experiment; graphs are the average of four independent Northern blot analyses. Two independently transfected cultures (for each of the constructs used) were tested. A, Parental MIN-6 cells; B, MIN-6 RIP-transfected cells; C, MIN-6 CMV/GLP-1 transfected cells; D, MIN-6 RIP/GLP-1 transfected cells. Insulin and GLP-1 mRNA levels were normalized by ß-actin mRNA levels for each individual blot. Statistical significance of the data was evaluated by ANOVA.

 
Glucose-dependent secretion of insulin
CMV/GLP-1, RIP/GLP-1 cells, parental MIN-6 cells, and cells transfected with the pSecTag2A plasmid alone were cultured in the presence of increasing concentration of glucose as described for Northern blot analysis. Culture medium from various culture conditions was then collected and subjected to RIA for insulin and for protein assay to detect the total protein content in each medium sample.

MIN-6 cells transfected solely with the pSecTag2A plasmid as well as parental cells transfected with the GLP-1 construct under the CMV promoter all showed a very similar glucose-dependent pattern of insulin secretion (Fig. 4Go). When the responsiveness to glucose at 3 and 20 mM was compared, the insulin levels in the culture medium were increased by 1.6-, 1.5-, 1.7-fold for parental cells, cells transfected with plasmid alone, and CMV/GLP-1 MIN-6 cells, respectively.



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  		Figure 4. Glucose-dependent insulin secretion in the culture medium. Insulin accumulation in the culture medium was determined after a 2-h washout period carried out with serum-free and glucose-free medium. Parental MIN-6, MIN-6 transfected with the vector alone, MIN-6 cells transfected with CMV/GLP-1, and MIN-6 cells transfected with RIP/GLP-1 were incubated in the presence of various concentrations of glucose for 8 h. Each experiment was repeated at least four times, and the data plotted on the graph represent the mean ± SD. Insulin levels were normalized for total proteins from cell extracts. Statistical significance of the data was evaluated by ANOVA.

 
Cellular transfection with the RIP/GLP-1 construct promoted a profoundly different pattern of insulin secretion. In these cells, insulin secretion appeared to be strongly regulated by the concentration of glucose in the culture medium (Fig. 4Go). Insulin levels in the culture medium obtained from cells grown in the absence of glucose was significantly lower, compared with the other cell lines tested [parental, plasmid-transfected, and CMV/GLP-1 transfected (P < 0.05)]. Increasing the concentration of glucose in the culture medium induced an increase in the accumulation of insulin into the culture medium (P < 0.001), such that in the presence of 20 mM glucose, insulin levels were approximately 10-fold greater than those observed in the presence of 3 mM glucose (P < 0.001).

RIP/GLP MIN-6 cultured in the presence of the GLP-1 receptor antagonist Exendin-9 showed a significant reduction of the glucose-dependent secretion of insulin, compared with cells transfected with the plasmid alone (Fig. 5Go). Although there was a 160% increase of insulin content into the culture medium within the first 30 min of culture in control cells grown in the presence of 10 mM glucose, at the same time point the presence of Exendin-9 prevented the time dependent accumulation of insulin (P < 0.01).



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  		Figure 5. Inhibition of insulin secretion by the GLP-1R antagonist Exendin-9. MIN-6 RIP/GLP-1 cells routinely cultured in the presence of 10% FBS and 12 mM glucose were subjected to an overnight washout period with medium deprived of glucose and FBS. They were then cultured in serum-free medium in the presence of 10 mM glucose in the presence of Exendin-9 (10-6 M) for increasing lengths of time. Insulin levels were normalized for total proteins from cell extracts. The data plotted represent the average ± SD of three independent experiments. Statistical significance of the data was evaluated by unpaired t test.

 
Regulation of GLP-1 secretion
Parental MIN-6 cells and cells transfected with the plasmid alone, cultured in the presence of various concentrations of glucose, did not secrete any detectable amount of GLP-1 (Fig. 6Go). Cellular transfection with human GLP-1 was associated with the secretion of the counterpart protein into the culture medium. However, a major difference between the two GLP-1 transfected cell lines was observed. Although the presence of the insulin promoter placed upstream to the GLP-1 coding sequence induced an increase in the GLP-1 accumulation into the culture medium in response to glucose (P < 0.001), cells transfected with the GLP-1 gene driven by the CMV promoter exhibited a constitutive release of GLP-1, and this did not vary in response to ambient concentrations of glucose (Fig. 6Go).



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  		Figure 6. Glucose-dependent GLP-1 secretion in the culture medium. GLP-1 accumulation in the culture medium was determined after a 2-h washout period carried out with serum-free and glucose-free medium. Parental MIN-6, MIN-6 transfected with the vector alone, MIN-6 cells transfected with CMV/GLP-1, and MIN-6 cells transfected with RIP/GLP-1 were incubated in the presence of various concentrations of glucose for 8 h. Each experiment was repeated at least four times, and the data plotted on the graph represent the mean ± SD. GLP-1 levels were normalized for total proteins from cell extracts. Statistical significance of the data was evaluated by ANOVA.

 
Immunofluorohistochemistry for IDX-1
MIN-6 (parental, CMV/GLP-1, RIP/GLP-1) cells were cultured for 12 h in serum-free medium containing 0, 6, or 12 mM glucose. Using anti-IDX-1 antibody, a positive immunoreactivity for IDX-1 was detected in all culture conditions (Fig. 7Go). Transfected cells (with either one of the two GLP-1 constructs) showed a greater abundance of IDX-1 cytoplasmic protein levels, compared with parental MIN-6 cells. Exposure to increasing concentrations of glucose appeared to further increase the expression level of IDX-1 in cells that were transfected with the CMV/GLP-1 construct (Fig. 7Go, D–F). A much greater glucose-dependent increase of IDX-1 was observed in cells transfected with the RIP/GLP-1 construct when exposed to different concentrations of glucose (Fig. 6Go, G–I), compared with either control or CMV/GLP-1 MIN-6 cells. Although a different degree of positivity for IDX-1 was present in all cell lines tested, the percentage of IDX-1-positive cells and the intensity of the staining for IDX-1 varied significantly among the various cell and treatment groups. RIP/GLP-1 MIN-6 cells appeared to be the best responder to glucose. In those cultures, the number of cells positive for IDX-1 immunostaining increased from 10% ± 7% in the absence of glucose to 65% ± 17% when exposed to 6 mM glucose, reaching 90% ± 8% in the presence of 12 mM glucose (vs. 10% ± 4%, 18% ± 12%, 35% ± 15% in parental cells culture in the presence of 0, 6, and 12 mM glucose, respectively). CMV/GLP-1 cells showed an intermediate level of response to glucose between the values observed for parental cells and cell transfected with the RIP/GLP-1 construct. In the absence of glucose, 17% ± 5% of cells were IDX-1 positive, increasing to 40% ± 8% and 55% ± 11% in the presence of 6 and 12 mM glucose, respectively.



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  		Figure 7. Immunofluorocytochemistry for IDX-1 in cells exposed to various concentrations of glucose. CMV/GLP-1, RIP/GLP-1 cells, and parental MIN-6 cells were cultured in the presence of 6 mM glucose with 10% FBS. After a 2-h washout incubation with glucose-free, serum-free medium, they were incubated with 0, 6, or 12 mM glucose for 12 h and subjected to immunostaining with an anti-IDX-1 antibody. A–C represent parental MIN-6 cells cultured in glucose-free medium (A), 6 mM glucose (B), or 12 mM glucose (C). D–F represent CMV/GLP-1 MIN-6 cells cultured in glucose-free medium (D), 6 mM glucose (E), or 12 mM glucose (F). G–I represent RIP/GLP-1 MIN-6 cells cultured in glucose-free medium (G), 6 mM glucose (H), or 12 mM glucose (I).

 
Western blotting analysis for GLP-1R
GLP-1R immunodetection was performed for MIN-6 cells transfected with the RIP/GLP-1 construct after culturing cells in the presence of various concentrations of glucose (0, 3, 6, and 15 mM) for 12 h. Western blot analysis was carried out with a monoclonal antibody that recognizes an epitope of the native protein located on the extracellular surface of human cells. A 62-kDa protein was detected in all culture conditions studied. No significant changes in GLP-1R protein levels were observed as a result of treatment with different concentration of glucose (Fig. 8Go). This suggested that the glucose-dependent increase in GLP-1 levels (Figs. 2Go and 4Go) was not associated with a down-regulation of the protein levels of GLP-1R receptor (Fig. 8Go). Similarly, that was not a significant changes in GLP-1R level between MIN-6 cells transfected with the RIP/GLP-1 construct and those with the CMV/GLP-1 plasmid, compared with parental cells (Fig. 8Go).



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  		Figure 8. GLP-1R expression in MIN-6 cells transfected with RIP/GLP-1. Parental, CVM/GLP-1-transfected, and RIP/GLP-1 transfected MIN-6 cells were cultured for 12 h in the presence of 6 mM glucose. After removal of the culture medium, the cells were collected and the protein extract subjected to Western blot analysis with a polyclonal antibody directed against human GLP-1R. A, An individual Western blot analysis. B, A graph representing the average of three independent experiments, with the GLP-1R levels normalized by the total protein content of each individual cell extract. For glucose response study, MIN-6 cells transfected with RIP/GLP-1 were cultured in serum-free medium in the presence of various concentrations of glucose (0, 3, 6, 15 mM) for 12 h. C, An individual Western blot analysis. D, A graph representing the average of three independent experiments, with the GLP-1R levels normalized by the total protein content of each individual cell extract.

 
Gel shift analysis
The presence of increasing concentrations of glucose in the culture medium of MIN-6 RIP/GLP-1 cells induced an in the binding of IDX-1 to the A1 element of the insulin promoter (Fig. 9Go). This glucose-dependent activation of the insulin gene mirrored the glucose-dependent secretion of GLP-1 (Fig. 6Go). Control nuclear extracts from parental MIN-6 cells, as well as cells transfected solely with the RIP-7 promoter, showed no increase in IDX-1 binding to the insulin promoter in response to glucose (Fig. 9Go, A and C). To confirm the specificity of IDX-1 binding, we performed a supershift analysis with an anti-IDX-1 antibody in addition to a competition assay with unlabeled A1 oligonucleotide. The presence of the anti-IDX-1 antibody in the nuclear extracts induced a shift in the size of the detected band (Fig. 9EGo), but the presence of unlabeled A1 oligonucleotide abolished the nuclear protein binding (Fig. 9Go). On the other hand, gel shift analysis of MIN-6 CMV/GLP-1 cells revealed a modest IDX-1 binding to insulin promoter, and this did not appear to be significantly regulated by the concentration of glucose in the culture medium (Fig. 9BGo).



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  		Figure 9. Glucose- and GLP-1-dependent binding of IDX-1 to the rat insulin promoter Al element. Nuclear extract from cells cultured in the presence of different concentrations of glucose was analyzed by EMSA for binding to the Al element of the insulin promoter {gamma}-32P-labeled probe. Lane 1, Incubation of the radiolabeled A1 oligonucleotide sequence in the absence of nuclear extracts; lane 2, nuclear extracts incubated in the presence of 100x nonlabeled A1 oligonucleotide sequence, and labeled element (cells were cultured in the presence of 10 mM glucose); lanes 3–7, nuclear extracts of cells cultured in the presence of 0, 3, 6, 10, and 15 mM glucose. A–D, Binding of nuclear proteins to the A1 element of the insulin promoter from cells cultured in the presence of different concentrations of glucose. The bar graph represents the average of three independent experiments and is expressed in arbitrary units with the binding to cells cultured in the absence of glucose considered equal to 1. A, Parental MIN-6 cells. B, MIN-6 CMV/GLP-1-transfected cells. C, MIN-6 RIP transfected cells. D, MIN-6 RIP/GLP-1 cells. E, Supershift analysis for MIN 6 RIP/GLP-1 cells cultured in 15 mM glucose before incubation of the nuclear extracts with or without an anti IDX-1 antibody. Statistical significance of the data was evaluated by t test.

 
Inhibition of cAMP-dependent signaling pathway
To investigate whether the observed glucose-dependent insulin synthesis and secretion of RIP/GLP-1 MIN-6 cells was mediated by a cAMP-dependent signaling pathway, cells were grown in the presence of Rp-cAMP. Cells and culture media were collected for Northern blot analysis and RIAs. Determination of cAMP levels demonstrated that Rp-cAMP was capable of inhibiting cAMP accumulation (Fig. 10AGo). Accumulation of insulin in the culture medium was also inhibited by Rp-cAMP, suggesting that insulin secretion was entirely under the cAMP-dependent signaling pathway (Fig. 10BGo). Detection of insulin mRNA levels showed a very different pattern, compared with insulin secretion, suggesting that there was a dissociation between the mechanism(s) by which GLP-1 controls the secretion of insulin and its action at the transcription level of the insulin gene (Fig. 10CGo).



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  		Figure 10. Effect of cAMP inhibition on glucose and GLP-1-dependent secretion of insulin and insulin mRNA levels. MIN-6 RIP/GLP-1 cells routinely cultured in the presence of 10% FBS and 12 mM glucose were subjected to an overnight washout period with medium deprived of glucose and FBS. They were then cultured in serum-free medium in the presence of either 3 mM or 10 mM glucose in the presence of Rp-cAMP (10-6 M). A, The cAMP levels normalized for protein content. B, Insulin levels normalized for protein content. C, The mRNA levels for insulin, normalized for ß-actin mRNA content. The blot in C indicates one individual experiment; repetition of the experiment using RNA extracts from independent cultures produced very similar results. Statistical significance of the data for mRNA and protein levels was evaluated by ANOVA. P values indicated in the figure were derived by comparing the two curves obtained from cells treated with Rp-cAMP vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The hypoglycemic action of GLP-1 results from various acute mechanisms of action, which include an enhancement of glucose-dependent insulin secretion, an inhibition of glucagon release, a slowdown of gastric motility, and a prosatiety effect (9, 10, 11, 12, 13). The latter is mostly dependent on the synthesis of GLP-1 at the level of the central nervous system, an additional site for the transcription of the proglucagon gene. In addition to those well-characterized mechanisms for the acute lowering postprandial glucose elevation, GLP-1 has been shown to have longer lasting effects on islet ß-cells. These include a positive effect on insulin biosynthesis and proinsulin gene expression as well as the regulation of the transcription of the glucose transporter GLUT-2 and the glucose phosphorylating enzyme glucokinase, two major regulators in the utilization of glucose by islet ß-cells. To date, no known insulin-secreting agent, other than glucose and GLP-1, has also been shown to regulate insulin synthesis and insulin gene expression in addition to promoting the secretion of insulin. Glucose-dependent insulinotropic peptide, another incretin hormone capable of such biological activity has been shown to be ineffective in controlling the production of insulin in subjects with diabetes (14, 15, 16). The unique broad ability of GLP-1 to improve the function of insulin secretion is limited by its very short half-life in vivo (3, 4) so that before considering its potential use for the treatment of diabetes, the issue of its very rapid degradation will have to be addressed. Alternatively, mechanisms to provide a constant delivery of GLP-1 may have to be designed.

In the present study, we demonstrated that cellular transfection of an islet-derived ß-cell line (MIN-6) with a minigene encoding solely for the GLP-1 fragment of the proglucagon gene (when driven by a glucose responsive promoter) was capable of regulating the synthesis and secretion of insulin in a glucose-dependent manner. HPLC and RIA for GLP-1 demonstrated that the transgene was translated and secreted correctly. Inhibition of GLP-1-dependent secretion of insulin using the GLP-1R antagonist Exendin-9 abolished the capability of secreting insulin in response to glucose. This suggested that the gain in glucose-sensing activity of RIP/GLP-1 cells was not simply the result of a clonal selection, but it was specifically dependent on the cellular release of GLP-1 in a glucose-dependent fashion. To further demonstrate that GLP-1 was the factor responsible for the restored link between ambient glucose concentration and insulin production in our cells (MIN-6 RIP/GLP-1), we also studied an additional cell line transfected solely with RIP promoter. The rationale for generating this cell line was to investigate whether the mere presence of additional copies of a glucose responsive promoter in the cell genome was proving an alternatively binding site for transcription factors regulating the expression of the endogenous insulin gene. Indeed, it has been proposed that the nonregulated (constitutive) synthesis and secretion of insulin by insulinoma cell lines results from a constant saturation of the regulatory regions of the insulin gene. The aim of this experiment was to test whether the presence of the transfected glucose responsive promoter (alone and not linked to the GLP-1 encoding sequence) was responsible for a mop-out effect capable of restoring a greater glucose-dependent secretion of insulin. Our data demonstrate that the presence of RIP was not sufficient per se to ameliorate the capability of cells to sense glucose (as showed by Northern blot and gel shift analyses).

Increasing concentrations of glucose in the culture medium were also responsible for increasing IDX-1 levels. In turn, in RIP/GLP-1 cells, binding of IDX-1 to the insulin promoter was associated with a glucose-dependent accumulation of insulin mRNA into the cellular cytoplasm, which resulted in a greater accumulation of the counterpart protein and a more significant amount of insulin secreted into the culture medium.

Interestingly, it was not the mere presence of GLP-1 in the medium that restored a more physiological regulation of insulin synthesis. This was demonstrated by the observation that cells with a constitutive transcription of the GLP-1 gene (by means of having the minigene under the control of the CMV promoter) were not capable of increasing their insulin secretory capability in response to glucose, even when the concentration of glucose in the culture medium reached 20 mM. These observations are supported by studies of others (17, 18, 19, 20) leading us to propose that GLP-1 may regulate the transcription and the secretion of insulin via partially independent mechanism(s) of action. In the present study, we were able to selectively block the GLP-1-dependent secretion of insulin with nearly no effect on the regulation of insulin mRNA levels. Indeed, incubation of RIP/GLP-1-transfected cells with the cAMP inhibitor Rp-cAMP produced the expected inhibitory effect on cAMP levels and insulin secretion, but it had no effect on the regulation of insulin mRNA levels.

The present study shows that transfection of insulin- producing cells with a gene fragment encoding for human GLP-1 is capable of various biological activity characteristically observed when the corresponding recombinant protein is administered in vitro or in vivo, and it aims at laying the experimental foundations for a GLP-1-based gene therapy of poorly glucose-sensitive insulin-secreting cells.


   Acknowledgments
 
We would like to thank Alexander Y. Kariagin for his technical support and Olga Garcia for her assistant in the writing of the manuscript.


   Footnotes
 
This work was supported, in part, by the Foundation for Diabetes Research.

Abbreviations: CMV, Cytomegalovirus; FBS, fetal bovine serum; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; IDX-1, islet/duodenum homeobox-1; MIN-6, mouse insulinoma cell; RIP, rat insulin II promoter; Rp-cAMP, Rp-isomer, triethylammonium salt, cAMP.

Received August 23, 2001.

Accepted for publication May 23, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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