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Cell Biology International (2004) 28, 69–73 (Printed in Great Britain)
N-acetyl-GLP-1: a DPP IV-resistant analogue of glucagon-like peptide-1 (GLP-1) with improved effects on pancreatic β-cell-associated gene expression
Hui‑Kang Liuab, Brian D. Greena*, Victor A. Gaulta, Jane T. McCluskeya, Neville H. McClenaghana, Finbarr P.M. O'Hartea and Peter R. Flatta
aSchool of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland, UK
bSchool of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK


Abstract

Glucagon-like peptide-1(7-36)amide (GLP-1) is a key insulinotropic hormone with the reported potential to differentiate non-insulin secreting cells into insulin-secreting cells. The short biological half-life of GLP-1 after cleavage by dipeptidylpeptidase IV (DPP IV) to GLP-1(9-36)amide is a major therapeutic drawback. Several GLP-1 analogues have been developed with improved stability and insulinotropic action. In this study, the N-terminally modified GLP-1 analogue, N-acetyl-GLP-1, was shown to be completely resistant to DPP IV, unlike native GLP-1, which was rapidly degraded. Furthermore, culture of pancreatic ductal ARIP cells for 72 h with N-acetyl-GLP-1 indicated a greater ability to induce pancreatic β-cell-associated gene expression, including insulin and glucokinase. Further investigation of the effects of stable GLP-1 analogues on β-cell differentiation is required to assess their potential in diabetic therapy.


Keywords: Glucagon-like peptide-1 (GLP-1), GLP-1 analogue, Cell differentiation.

*Corresponding author. Tel: +44-28-70324313; fax: +44-28-70324965


1 Introduction

Glucagon-like peptide-1(7-36)amide (GLP-1) is widely considered to have therapeutic potential in diabetes because of its ability to regulate blood glucose. This is primarily achieved through glucose-dependent stimulation of insulin secretion (Drucker, 2002). Other potentially important actions relevant to the treatment of diabetes include stimulation of insulin gene transcription, islet cell growth and neogenesis (Buteau et al., 1999; Stoffers et al., 2000; Xu et al., 1999). The effects of GLP-1 on insulin gene transcription and β-cell proliferation are mediated by upregulation of the transcription factor, pancreatic duodenal homeobox-1 (PDX-1)(Buteau et al., 1999).

PDX-1 translocation to the nucleus of RIN 1046-38 cells was shown to be mediated through the cAMP/protein kinase A (PKA) pathway, the predominant pathway associated with GLP-1-induced insulin secretion (Wang et al., 2001). Binding of PDX-1 to DNA leads to the upregulation of insulin, glucose transporter 2 (GLUT2) and glucokinase genes (McKinnon and Docherty, 2001). A second pathway, phosphatidylinositol 3-kinase (PI3-K), is also involved in the proliferation effects caused by GLP-1 (Buteau et al., 2001). In addition to these actions, GLP-1 has been shown to promote cellular differentiation, which increases islet and β-cell mass (Hui et al., 2001; Stoffers et al., 2000; Xu et al., 1999; Zhou et al., 1999).

GLP-1 has been shown to induce the differentiation of rat ARIP, pancreatic exocrine AR4J, and human islet progenitor cells, by a process involving the activation of PDX-1 (Hui et al., 2001; Zhou et al., 1999). In vivo studies in diabetic animal models have shown that prolonged GLP-1 administration causes enhanced β-cell neogenesis and islet cell mass (Stoffers et al., 2000; Xu et al., 1999). Such observations encourage the development of GLP-1 as a diabetic therapy, but the short biological half-life of GLP-1, due to rapid degradation by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), is a major obstacle (Drucker, 2002). The N-terminal modification of GLP-1 by glycation has been shown to improve resistance to DPP IV whilst retaining insulinotropic action on β-cells (O'Harte et al., 2000). The current study aimed to assess the stability of the GLP-1 analogue, N-acetyl-GLP-1, and to compare its biological effects with native GLP-1 on the differentiation of pancreatic ductal cells into pancreatic β-cell phenotype.

2 Materials and methods

2.1 Degradation of GLP-1 peptides by DPP IV and serum

GLP-1 and N-acetyl-GLP-1 (final peptide concentration 2 mM) were incubated with either DPP IV (1.25 mU) or serum (7.5 μl) (37 °C; 50 mM triethanolamine–HCl buffer; pH 7.8). Reactions were terminated by the addition of TFA/H2O (15 μl, 10% (v/v)). The reaction products were then applied to a Vydac C-18 analytical column (4.6×250 mm) and the major degradation fragment GLP-1(9-36)amide was separated from intact GLP-1 or N-acetyl-GLP-1 using linear gradients of acetonitrile in water (O'Harte et al., 2000). Absorbance was monitored at 206 nm using a SpectraSystem UV 2000 detector (Thermoquest Limited, Manchester, UK) and peaks were collected manually prior to electrospray ionization–mass spectrometry analysis. Using peak areas, degradation was expressed as a percentage of peptide remaining relative to the major degradation fragment GLP-1(9-36)amide.

2.2 Cells and cell culture

Rat ARIP pancreatic epithelial carcinoma cells purchased from American Type Culture Collection (ATCC, Maryland, USA) and BRIN BD11 cells (McClenaghan et al., 1996) were grown in RPMI 1640 medium containing 10% foetal bovine serum (v/v), 1% antibiotics (v/v) (100 U/ml penicillin and 0.1 mg/ml streptomycin). ARIP cells for cultivation with GLP-1 or N-acetyl-GLP-1 were seeded (1×106cells) into 75 cm3tissue culture flasks. After overnight attachment, the culture medium was replaced with culture medium supplemented with either GLP-1 (10 nM) or N-acetyl-GLP-1 (10 nM) and cultured for a further 3 days.

2.3 Total RNA extraction

At the end of the 3rd day of culture, cells were directly lysed by TRI-reagent. Lysate was aliquoted into Eppendorf tubes (1.5 ml) and total RNA was extracted by standard RNA extraction techniques. Briefly, lysate was centrifuged (12,000×g; 10 min; 4 °C) and the supernatant transferred to another Eppendorf tube. Following addition of chloroform (300 μl) and centrifugation, the upper aqueous phase (containing RNA) was transferred into a fresh Eppendorf tube and isopropanol added (750 μl). Centrifugation was again carried out and the pellet washed with 75% ethanol (2 ml) and centrifuged again. The remaining pellet was dried under vacuum for 5 min (Eppendorf concentrator 5301). RNA was dissolved in DEPC-treated distilled water (100 μl; 60 °C) and quantified. DNase I was used to remove genomic DNA contamination during total RNA preparation and the quality of de-contamination was verified by agarose gel electrophoresis.

2.4 Reverse transcription polymerase chain reaction (RT–PCR)

Gene expression of cultured cells was determined by RT–PCR with total RNA (100 ng) used as a template. Genes were amplified using specific primers for β-actin (forward: 5′-CGT AAA GAC CTC TAT GCC AA and reverse: 5′-AGC CAT GCC AAA TGT GTC AT), PDX-1 (forward: 5′-CTC GCT GGG AAC GCT GGA ACA and reverse: 5′-GCT TTG GTG GAT TTC ATC CAC GG), GLUT2 (forward: 5′-CAT TGC TGG AAG AAG CGT ATC AG and reverse: 5′-GAG ACC TTC TGC TCA GTC GAC), insulin (forward: 5′-TGC CCA GGC TTT TGT CAA ACA GCA CCT T and reverse: 5′-CTC CAG TGC CAA GGT CTG AA), glucokinase (forward: 5′-AAG GGA ACT ACA TCG TAG GA and reverse: 5′-CAT TGG CGG TCT TCA TAG TA) or GLP-1R (forward: 5′-GGC TGT CTT GTA CTG CTT TGT C and reverse: 5′-ATG CCT GTT TGA TAG GTT TGA G), following individual optimization of the PCR.

For reverse transcription: 50 °C for 30 min was used followed by 94 °C for 2 min. For PCR: 35 cycles were carried out with the primer specific annealing temperature for 30 s, followed by 72 °C for 45 s, and then 94 °C for 30 s. Once the reaction was completed, RT–PCR products (10 μl) were mixed with loading dye (2 μl) and loaded on to a 2% agarose gel containing ethidium bromide (500 ng/ml). Bands were visualized under UV light and recorded using a digital camera following electrophoresis. The intensity of the bands (volume) was analyzed by Phoretix 1D software (Nonlinear Dynamics, Newcastle upon Tyne, UK). The level of gene expression was expressed as a percentage of β-actin ((intensity of specific gene)/(intensity of β-actin)×100).

3 Results and discussion

The current investigation demonstrated that N-acetyl-GLP-1 was completely resistant to enzymatic degradation by DPP IV and serum, with no metabolite being observed even after 12 h (half-life >12 h). In contrast, native GLP-1 was progressively degraded by DPP IV and serum (Fig. 1A and B) with 77–82% converted to GLP-1(9-36)amide by 12 h. The corresponding half-lives were 4.4 h and 4.7 h, as indicated in Fig. 1. These observations reinforce N-acetyl-GLP-1 as a potentially useful GLP-1 analogue (Siegel et al., 1999).


Fig. 1

Degradation profiles of native GLP-1 (●) and N-acetyl-GLP-1 (□) after incubation with either (A) purified DPP IV or (B) serum. Data are expressed as a percentage of intact peptide remaining relative to the major degradation fragment GLP-1(9-36)amide. Values are mean±SEM of two separate experiments.


ARIP cells were employed as a model to compare the ability of GLP-1 and N-acetyl-GLP-1 to stimulate β-cell associated gene expression, based on the fact that pancreatic ductal epithelium contains pancreatic stem cells that can differentiate into insulin secreting β-cells (Bouwens et al., 1994). The effectiveness of GLP-1 and N-acetyl-GLP-1 to induce a pancreatic β-cell associated gene profile, as modelled by insulin-secreting BRIN-BD11 cells (McClenaghan et al., 1996), is shown in Table 1. Under control conditions, the rat ductal cell line, ARIP, expressed PDX-1 and GLUT2 genes, but none of the other β-cell or incretin hormone receptor genes. Treatment with native GLP-1 elevated mRNA levels of PDX-1 and had minor effects on expression of GLUT2. Additional markers, insulin, glucokinase and GLP-1R, were not detected in the cells. In contrast, treatment of ARIP cells with N-acetyl-GLP-1 did not change PDX-1 or GLUT2, but did enhance expression of insulin, glucokinase, and GLP-1R genes.


Table 1. Gene expression profiles of BRIN BD11 cells and ARIP cells after 3 days of culture with GLP-1 or N-acetyl-GLP-1. Gene expression was detected using RT–PCR

Image

Values are means for three observations±SEM and are normalized relative to the expression of β-actin, set at 100%: (intensity of specific gene)/(intensity of β-actin)×100.

Previous studies involving the culture of ARIP cells with native GLP-1 reported PDX-1-mediated differentiation into insulin secreting cells ([Hui et al., 2001]). This was not the case with ARIP cells cultured in the present study. Differences in the molecular phenotype, especially PDX-1 expression, of ARIP cells have been noted previously, despite common origin from the ATCC ( [Hui et al., 2001]; [Silver and Yao, 2001]). Our observations suggest that rapid enzymatic degradation of native GLP-1 by DPP IV in serum containing media (measured at 1.9 nmol/ml/min, unpublished observation) curtails the action of the peptide. Thus, N-acetyl-GLP-1 exhibited profound resistance to DPP IV, giving rise to a much superior gene expression profile in ARIP cells compared with culture using native peptide. Thus, genes for insulin, glucokinase and GLP-1R, not normally present in ARIP cells, were expressed, but a lack of improvement in PDX-1 expression was puzzling in relation to observations by [Hui et al., 2001].

Prevention of DPP IV degradation may not only improve the duration of GLP-1's insulinotropic action ([Drucker, 2002]), but also enhance its ability to stimulate insulin gene transcription, islet cell growth and neogenesis ( [Buteau et al., 1999]; [Stoffers et al., 2000]; [Xu et al., 1999]). These actions, together with other effects, such as stimulation of glucose-dependent insulin release, inhibition of glucagon secretion and retardation of gastric emptying, support the growing interest in stable GLP-1 analogues as potential therapeutic agents for diabetes mellitus.

Acknowledgements

These studies were funded by University of Ulster Strategy Funding, the Research and Development Office of the Department of Health and Personal Social Services for Northern Ireland and an Overseas Research Studentship to Hui-Kang Liu from the Committee of Vice Chancellor and Principals.

References

Bouwens et al., 1994. L. Bouwens, R.N. Wang, E. De-Blay, D.G. Pipeleers and G. Kloppel, Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas. Diabetes 43 (1994), pp. 1279–1283. View Record in Scopus | Cited By in Scopus (131)

Buteau et al., 1999. J. Buteau, R. Roduit, S. Susini and M. Prentki, Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells. Diabetologia 42 (1999), pp. 856–864. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (209)

Buteau et al., 2001. J. Buteau, S. Foisy, C.J. Rhodes, L. Carpenter, T.J. Biden and M. Prentki, Protein kinase C activation mediates glucagon-like peptide-1-induced pancreatic β-cell differentiation. Diabetes 50 (2001), pp. 2237–2243. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (109)

Drucker, 2002. D.J. Drucker, Biological actions and therapeutic potential of the glucagon-like peptides. Gastroenterology 122 (2002), pp. 531–544. Abstract | PDF (2875 K) | View Record in Scopus | Cited By in Scopus (226)

Hui et al., 2001. H. Hui, C. Wright and R. Perfetti, Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells. Diabetes 50 (2001), pp. 785–796. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (137)

McClenaghan et al., 1996. N.H. McClenaghan, C.R. Barnett, E. Ah Sing, Y.H.A. Abdel Wahab, F.P.M. O'Harte, T.W. Yoon et al., Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. Diabetes 45 (1996), pp. 1132–1140. View Record in Scopus | Cited By in Scopus (151)

McKinnon and Docherty, 2001. C.M. McKinnon and K. Docherty, Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function. Diabetologia 44 (2001), pp. 1203–1214. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (108)

O'Harte et al., 2000. F.P.M. O'Harte, M.H. Mooney, A. Lawlor and P.R. Flatt, N-terminally modified glucagon-like peptide-1(7-36) amide exhibits resistance to enzymatic degradation while maintaining its antihyperglycaemic activity in vivo. Biochim Biophys Acta 1474 (2000), pp. 13–22. View Record in Scopus | Cited By in Scopus (27)

Siegel et al., 1999. E.G. Siegel, B. Gallwitz, G. Scharf, R. Mentlein, C. Morys-Wortmann, U.R. Folsch et al., Biological activity of GLP-1-analogues with N-terminal modifications. Regul Pept 79 (1999), pp. 93–102. Abstract | Article | PDF (825 K) | View Record in Scopus | Cited By in Scopus (52)

Silver and Yao, 2001. K. Silver and F. Yao, ARIP cells as a model for pancreatic beta cell growth and development. Pancreas 22 (2001), pp. 141–147. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (6)

Stoffers et al., 2000. D.A. Stoffers, T.J. Kieffer, M.A. Hussain, D.J. Drucker, J.M. Egan, S. Bonner-Weir et al., Insulinotropic glucagon-like peptide-1 agonists stimulate expression of homeodomain protein IDX-1 and increase β-cell mass in mouse pancreas. Diabetes 49 (2000), pp. 741–748. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (288)

Wang et al., 2001. X. Wang, J. Zhou, M.E. Doyle and J.M. Egan, Glucagon-like peptide-1 causes pancreatic duodenal homeobox-1 protein translocation from the cytoplasm to the nucleus of pancreatic β-cells by a cyclic adenosine monophosphate/protein kinase A-dependent mechanism. Endocrinology 142 (2001), pp. 1820–1827. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (66)

Xu et al., 1999. G. Xu, D.A. Stoffers, J.F. Habener and S. Bonner-Weir, Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48 (1999), pp. 2270–2276. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (499)

Zhou et al., 1999. J. Zhou, X. Wang, M.A. Pineyro and J.M. Egan, Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 48 (1999), pp. 2358–2366. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (198)

Corresponding Author Contact InformationCorresponding author. Tel: +44-28-70324313; fax: +44-28-70324965


Cell Biology International
Volume 28, Issue 1, January 2004, Pages 69-73
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a P<0.01 compared with β-actin control.
b P<0.001 compared with β-actin control.
c P<0.01 compared with ARIP control.
d P<0.001 compared with ARIP control.

Previous studies involving the culture of ARIP cells with native GLP-1 reported PDX-1-mediated differentiation into insulin secreting cells (Hui et al., 2001). This was not the case with ARIP cells cultured in the present study. Differences in the molecular phenotype, especially PDX-1 expression, of ARIP cells have been noted previously, despite common origin from the ATCC (Hui et al., 2001; Silver and Yao, 2001). Our observations suggest that rapid enzymatic degradation of native GLP-1 by DPP IV in serum containing media (measured at 1.9 nmol/ml/min, unpublished observation) curtails the action of the peptide. Thus, N-acetyl-GLP-1 exhibited profound resistance to DPP IV, giving rise to a much superior gene expression profile in ARIP cells compared with culture using native peptide. Thus, genes for insulin, glucokinase and GLP-1R, not normally present in ARIP cells, were expressed, but a lack of improvement in PDX-1 expression was puzzling in relation to observations by Hui et al. (2001).

Prevention of DPP IV degradation may not only improve the duration of GLP-1's insulinotropic action (Drucker, 2002), but also enhance its ability to stimulate insulin gene transcription, islet cell growth and neogenesis (Buteau et al., 1999; Stoffers et al., 2000; Xuet al., 1999). These actions, together with other effects, such as stimulation of glucose-dependent insulin release, inhibition of glucagon secretion and retardation of gastric emptying, support the growing interest in stable GLP-1 analogues as potential therapeutic agents for diabetes mellitus.

Acknowledgements

These studies were funded by University of Ulster Strategy Funding, the Research and Development Office of the Department of Health and Personal Social Services for Northern Ireland and an Overseas Research Studentship to Hui-Kang Liu from the Committee of Vice Chancellor and Principals.

References

Bouwens L, Wang, RN, De-Blay, E, Pipeleers, DG, Kloppel, G. Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neonatal rat pancreas. Diabetes 1994:43:1279-83
Crossref   Medline   

Buteau J, Roduit, R, Susini, S, Prentki, M. Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells. Diabetologia 1999:42:856-64
Crossref   Medline   

Buteau J, Foisy, S, Rhodes, CJ, Carpenter, L, Biden, TJ, Prentki, M. Protein kinase C activation mediates glucagon-like peptide-1-induced pancreatic β-cell differentiation. Diabetes 2001:50:2237-43
Crossref   Medline   

Drucker DJ. Biological actions and therapeutic potential of the glucagon-like peptides. Gastroenterology 2002:122:531-44
Crossref   Medline   

Hui H, Wright, C, Perfetti, R. Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells. Diabetes 2001:50:785-96
Crossref   Medline   

McClenaghan NH, Barnett, CR, Ah Sing, E, Abdel Wahab, YHA, O'Harte, FPM, Yoon, TW. Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion. Diabetes 1996:45:1132-40
Crossref   Medline   

McKinnon CM, Docherty, K. Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function. Diabetologia 2001:44:1203-14
Crossref   Medline   

O'Harte FPM, Mooney, MH, Lawlor, A, Flatt, PR. N-terminally modified glucagon-like peptide-1(7-36) amide exhibits resistance to enzymatic degradation while maintaining its antihyperglycaemic activity in vivo. Biochim Biophys Acta 2000:1474:13-22
Medline   

Siegel EG, Gallwitz, B, Scharf, G, Mentlein, R, Morys-Wortmann, C, Folsch, UR. Biological activity of GLP-1-analogues with N-terminal modifications. Regul Pept 1999:79:93-102
Crossref   Medline   

Silver K, Yao, F. ARIP cells as a model for pancreatic beta cell growth and development. Pancreas 2001:22:141-7
Crossref   Medline   

Stoffers DA, Kieffer, TJ, Hussain, MA, Drucker, DJ, Egan, JM, Bonner-Weir, S. Insulinotropic glucagon-like peptide-1 agonists stimulate expression of homeodomain protein IDX-1 and increase β-cell mass in mouse pancreas. Diabetes 2000:49:741-8
Crossref   Medline   

Wang X, Zhou, J, Doyle, ME, Egan, JM. Glucagon-like peptide-1 causes pancreatic duodenal homeobox-1 protein translocation from the cytoplasm to the nucleus of pancreatic β-cells by a cyclic adenosine monophosphate/protein kinase A-dependent mechanism. Endocrinology 2001:142:1820-7
Crossref   Medline   

Xu G, Stoffers, DA, Habener, JF, Bonner-Weir, S. Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 1999:48:2270-6
Crossref   Medline   

Zhou J, Wang, X, Pineyro, MA, Egan, JM. Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 1999:48:2358-66
Crossref   Medline   


Received 8 July 2003/26 October 2003; accepted 31 October 2003

doi:10.1016/j.cellbi.2003.10.004


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)