No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Novel DPP-IV-resistant analogs of GLP-1: The N-terminal extension of GLP-1 by a single amino acid
... Many approaches have been designed in order to increase the short in vivo half-life of the molecule. The modification of amino acids at the cleavage site of GLP-1 showed resistance to the activity by DPP-IV (8,9). In spite of this, the clinical limitation of these GLP-1 analogs remains due to the short duration of the peptides in the body. ...
... In recent studies, few research groups focused on the addition of an amino acid to the N-terminus of GLP-1R agonists (8,9) and the fusion to HSA, IgG, or transferrin (8,14,15). In a previous study, we demonstrated that a modified GLP-1 peptide with additional alanine (AGLP-1) at the N-terminus was resistant to DPP-IV. ...
... In a previous study, we demonstrated that a modified GLP-1 peptide with additional alanine (AGLP-1) at the N-terminus was resistant to DPP-IV. AGLP-1 fusion protein with IgG-Fc was also prevented from rapidly degrading by the enzyme, and had biological activity which stimulated the expression levels of IRS-2 protein in vitro (9). Furthermore, the effect of AGLP-1/IgG-Fc fusion protein which suppressed the glucose levels was prolonged for up to 24 h in vivo (19). ...
Glucagon like peptide-1 (GLP-1) regulates glucose mediated-insulin secretion, nutrient accumulation, and β-cell growth. Despite the potential therapeutic usage for type 2 diabetes (T2D), GLP-1 has a short half-life in vivo (t1/2 <2 min). In an attempt to prolong half-life, GLP-1 fusion proteins were genetically engineered: GLP-1 human serum albumin fusion (GLP-1/HSA), AGLP-1/HSA which has an additional alanine at the N-terminus of GLP-1, and AGLP-1-L/HSA, in which a peptide linker is inserted between AGLP-1 and HSA. Recombinant fusion proteins secreted from the Chinese Hamster Ovary-K1 (CHO-K1) cell line were purified with high purity (>96%). AGLP-1 fusion protein was resistant against the dipeptidyl peptidase-IV (DPP-IV). The fusion proteins activated cAMP-mediated signaling in rat insulinoma INS-1 cells. Furthermore, a C57BL/6N mice pharmacodynamics study exhibited that AGLP-1-L/HSA effectively reduced blood glucose level compared to AGLP-1/HSA.
... Human serum albumin (HSA), immunoglobulin gamma heavy chain (IgG-Fc), or polyethylene glycol (PEG) has commonly been tethered to the C-terminus of GLP-1 to increase the half-life of GLP-1 in vivo, and the fusion proteins showed increased efficacy of glucose-dependent insulin secretion789. In our previous study, we have constructed fusion proteins of GLP-1 and IgG-Fc in which the former had an additional Ala or Gly on the N-terminus of GLP-1 (A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc) along with the wild-type GLP-1/IgG-Fc fusion protein [10]. Here, we examined whether the A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc fusion protein is resistant to DPP-IV, and also performed pharmacokinetic and pharmacodynamic studies using Sprague–Dawley rats and db/db mice. ...
... Since DPP-IV cleaves the N-terminal dipeptide of substrates, especially after Pro and Ala [10], we determined the N-terminal sequence of Ala-or Gly-extended GLP-1/IgG-Fc fusion proteins after incubation with DPP-IV. The N-terminal sequence analysis revealed that both A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc fusion proteins maintained their intact sequences whereas the dipeptide, His-Ala, was cleaved off from the wild-type GLP-1/IgG-Fc fusion protein, suggesting the additional N-terminal location of Ala or Gly conferred resistance to DPP-IV action (seeTable 1). ...
... The N-terminal sequence analysis revealed that both A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc fusion proteins maintained their intact sequences whereas the dipeptide, His-Ala, was cleaved off from the wild-type GLP-1/IgG-Fc fusion protein, suggesting the additional N-terminal location of Ala or Gly conferred resistance to DPP-IV action (seeTable 1). These results are consistent with our previous finding [10]. ...
The aim of this study was to develop novel long-acting glucagon-like peptide 1 (GLP-1) analogs resistant to dipeptidyl peptidase-IV (DPP-IV). We constructed three fusion proteins comprising GLP-1 and the human immunoglobulin gamma heavy chain (IgG-Fc); wild-type GLP-1 and IgG-Fc (GLP-1/IgG-Fc) and two N-terminal-extended fusion proteins in which an additional Ala (A) or Gly (G) was located on the N-terminus of GLP-1 (A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc). The fusion proteins expressed in CHO-K1 cells were secreted into medium and purified by Protein A affinity chromatography. Here, we show that the Ala or Gly-extended GLP-1/IgG-Fc fusion protein is resistant to DPP-IV and has increased half-life in vivo. To our surprise, the A-GLP-1/IgG-Fc fusion protein was more effective than wildtype GLP-1/IgG-Fc fusion protein in reducing blood glucose levels in db/db mice. Our findings suggest that the A-GLP-1/IgG-Fc fusion protein could be a potential long-acting GLP-1 receptor agonist for the treatment of insulin-resistant type 2 diabetes.
The incretins glucose-dependent insulinotropic polypeptide (GIP1-42) and glucagon-like peptide-1-(7-36)-amide (GLP-17-36), hormones that potentiate glucose-induced insulin secretion from the endocrine pancreas, are substrates of the circulating
exopeptidase dipeptidyl peptidase IV and are rendered biologically inactive upon cleavage of their N-terminal dipeptides.
This study was designed to determine if matrix-assisted laser desorption/ionization-time of flight mass spectrometry is a
useful analytical tool to study the hydrolysis of these hormones by dipeptidyl peptidase IV, including kinetic analysis. Spectra
indicated that serum-incubated peptides were cleaved by this enzyme with only minor secondary degradation due to other serum
protease activity. Quantification of the mass spectrometric signals allowed kinetic constants for both porcine kidney- and
human serum dipeptidyl peptidase IV-catalyzed incretin hydrolysis to be calculated. The binding constants (Km) of these incretins to purified porcine kidney-derived enzyme were 1.8 ± 0.3 and 3.8 ± 0.3 μ, whereas the binding constants
observed in human serum were 39 ± 29 and 13 ± 9 μ for glucose-dependent-insulinotropic polypeptide and glucagon-like peptide-1-(7-36)-amide
respectively. The large range of Km values found in human serum suggests a heterogeneous pool of enzyme. The close correlation between the reported kinetic constants
and those previously described validates this novel approach to kinetic analysis.
Glucagon-like peptide 1 (GLP-1) has great potential in diabetes therapy due to its glucose-dependent stimulation of insulin secretion, but this is limited by its rapid degradation, primarily by dipeptidyl peptidase IV. Four analogues, N-terminally substituted with threonine, glycine, serine or alpha-aminoisobutyric acid, were synthesised and tested for metabolic stability. All were more resistant to dipeptidyl peptidase IV in porcine plasma in vitro, ranging from a t1/2 of 159 min (Gly8 analogue) to undetectable degradation after 6 h (Aib8 analogue; t1/2 for GLP-1 (7-36) amide, 28 min). During i. v. infusion in anaesthetised pigs, over 50% of each analogue remained undegraded compared to 22.7 % for GLP-1 (7-36) amide. In vivo, analogues had longer N-terminal t1/2 (intact peptides: means, 3.3-3.9 min) than GLP-1 (7-36) amide (0.9 min; p < 0.01), but these did not exceed the C-terminal t1/2 (intact plus metabolite: analogues, 3.5-4.4 min; GLP-1 (7-36) amide, 4.1 min). Analogues were assessed for receptor binding using a cell line expressing the cloned receptor, and for ability to stimulate insulin or inhibit glucagon secretion from the isolated perfused porcine pancreas. All bound to the receptor, but only the Aib8 and Gly8 analogues had similar affinities to GLP-1 (7-36) amide (IC50; Aib8=0.45 nmol/l; Gly8=2.8 nmol/l; GLP-1 (7-36) amide=0.78 nmol/l). All analogues were active in the isolated pancreas, with the potency order reflecting receptor affinities (Aib8 > Gly8 > Ser8 > Thr8). N-terminal modification of GLP-1 confers resistance to dipeptidyl peptidase IV degradation. Such analogues are biologically active and have prolonged metabolic stability in vivo, which, if associated with greater potency and duration of action, may help to realise the potential of GLP-1 in diabetes therapy.
Two fragments of the receptor for glucagon-like peptide-1 (GLP-1), each containing the N-terminal domain, were expressed and characterized in either bacterial or mammalian cells. The first fragment, rNT-TM1, included the N-terminal domain and first transmembrane helix and was stably expressed in the membrane of human embryonic kidney 293 cells. The second, 6H-rNT, consisted of only the N-terminal domain of the receptor fused with a polyhistidine tag at its N terminus. The latter fragment was expressed in Escherichia coli in the form of inclusion bodies from which the protein was subsequently purified and refolded in vitro. Although both receptor fragments displayed negligible (125)I-labeled GLP-1(7-36)amide-specific binding, they both displayed high affinity for the radiolabeled peptide antagonist (125)I-exendin-4(9-39). Competition binding studies demonstrated that the N-terminal domain of the GLP-1 receptor maintains high affinity for the agonist exendin-4 as well as the antagonists exendin-4(3-39) and exendin-4(9-39) whereas, in contrast, GLP-1 affinity was greatly reduced. This study shows that although the exendin antagonists are not dependent upon the extracellular loops and transmembrane helices for maintaining their normal high affinity binding, the endogenous agonist GLP-1 requires regions outside of the N-terminal domain. Hence, distinct structural features in exendin-4, between residues 9 and 39, provide additional affinity for the N-terminal domain of the receptor. These data are consistent with a model for the binding of peptide ligands to the GLP-1 receptor in which the central and C-terminal regions of the peptides bind to the N terminus of the receptor, whereas the N-terminal residues of peptide agonists interact with the extracellular loops and transmembrane helices.
Glucagon-like peptide-1(7-36)amide (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. Rapid removal of the N-terminal dipeptide, His7-Ala8, by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) curtails the biological activity of GLP-1. Chemical modifications or substitutions of GLP-1 at His7 or Ala8 improve resistance to DPP-IV action, but this often reduces potency. Little attention has focused on the metabolic stability and functional activity of GLP-1 analogues with amino acid substitution at Glu9, adjacent to the DPP IV cleavage site. We generated three novel Glu9-substituted GLP-1 analogues, (Pro9)GLP-1, (Phe9)GLP-1 and (Tyr9)GLP-1 and show for the first time that Glu9 of GLP-1 is important in DPP IV degradation, since replacing this amino acid, particularly with proline, substantially reduced susceptibility to degradation. All three novel GLP-1 analogues showed similar or slightly enhanced insulinotropic activity compared with native GLP-1 despite a moderate 4-10-fold reduction in receptor binding and cAMP generation. In addition, (Pro9)GLP-1 showed significant ability to moderate the plasma glucose excursion and increase circulating insulin concentrations in severely insulin resistant obese diabetic (ob/ob) mice. These observations indicate the importance of Glu9 for the biological activity of GLP-1 and susceptibility to DPP IV-mediated degradation.
Peptide hormones exert unique actions via specific G protein-coupled receptors; however, the therapeutic potential of regulatory peptides is frequently compromised by rapid enzymatic inactivation and clearance from the circulation. In contrast, recombinant or covalent coupling of smaller peptides to serum albumin represents an emerging strategy for extending the circulating t(1/2) of the target peptide. However, whether larger peptide-albumin derivatives will exhibit the full spectrum of biological activities encompassed by the native peptide remains to be demonstrated. We report that Albugon, a human glucagon-like peptide (GLP)-1-albumin recombinant protein, activates GLP-1 receptor (GLP-1R)-dependent cAMP formation in BHK-GLP-1R cells, albeit with a reduced half-maximal concentration (EC(50)) (0.2 vs. 20 nmol/l) relative to the GLP-1R agonist exendin-4. Albugon decreased glycemic excursion and stimulated insulin secretion in wild-type but not GLP-1R(-/-) mice and reduced food intake after both intracerebroventricular and intraperitoneal administration. Moreover, intraperitoneal injection of Albugon inhibited gastric emptying and activated c-FOS expression in the area postrema, the nucleus of the solitary tract, the central nucleus of the amygdala, the parabrachial, and the paraventricular nuclei. These findings illustrate that peripheral administration of a larger peptide-albumin recombinant protein mimics GLP-1R-dependent activation of central and peripheral pathways regulating energy intake and glucose homeostasis in vivo.
Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid inactivation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His(7)-modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1(9-36)amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC(50) values 32.9 and 6.7 nM, respectively) compared with native GLP-1 (IC(50) 0.37 nM). Similarly, both analogues stimulated cAMP production with EC(50) values of 16.3 and 27 nM respectively compared with GLP-1 (EC(50) 4.7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5.6 mM glucose (P<0.05 to P<0.001) similar to native GLP-1. Both analogues (25 nM/kg body weight) lowered plasma glucose and increased plasma insulin levels when administered in conjunction with glucose (18 nM/kg body weight) to adult obese diabetic (ob/ob) mice. N-pyroglutamyl-GLP-1 was substantially better at lowering plasma glucose compared with the native peptide, while N-acetyl-GLP-1 was significantly more potent at stimulating insulin secretion. These studies indicate that N-terminal modification of GLP-1 results in DPP IV-resistant and biologically potent forms of GLP-1. The particularly powerful antihyperglycaemic action of N-pyroglutamyl-GLP-1 shows potential for the treatment of type 2 diabetes.
The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.
Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide)
are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated
by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification
or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic
GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16–0.52 nm) and IC50s (4.3–8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly
right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and
thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank
potency: [Ser(P)2]incretins ≥ [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least
in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.
Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala2, its clinical application is limited. Hence, understanding the structure−activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [d-Ala2]GLP-1 and [Gly2]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [d-Ala2]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His1-GLP-1, glucagon(1-5)-, glucagon(1-10)-, PACAP(1-5)-, VIP(1-5)-, and secretin(1-10)-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala8/11/12/16] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4−12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the α-amino group of His1 and replacement of Ala2 result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.
Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biological half-time limits the therapeutic use of GLP-1. DPP IV requires an intact α-amino-group of the N-terminal histidine of GLP-1 in order to perform its enzymatic activity. Therefore, the following GLP-1 analogues with alterations in the N-terminal position 1 were synthesized: N-methylated- (N-me-GLP-1), α-methylated (α-me-GLP-1), desamidated- (desamino-GLP-1) and imidazole–lactic-acid substituted GLP-1 (imi-GLP-1). All GLP-1 analogues except α-me-GLP-1 were hardly degraded by DPP IV in vitro. The GLP-1 analogues showed receptor affinity and in vitro biological activity comparable to native GLP-1 in RINm5F cells. GLP-1 receptor affinity was highest for imi-GLP-1, followed by α-me-GLP-1 and N-me-GLP-1. Only desamino-GLP-1 showed a 15-fold loss of receptor affinity compared to native GLP-1. All analogues stimulated intracellular cAMP production in RINm5F cells in concentrations comparable to GLP-1. N-terminal modifications might therefore be useful in the development of long-acting GLP-1 analogues for type 2 diabetes therapy.
The design and development of specific substrates for proteolytic enzymes is reviewed. Particular attention is given to substrates containing the leaving groups 4-methoxy-2-naphthylamide (MNA) and 7-amino-4-trifluoromethylcoumarin (AFC). The MNA substrates are used for histochemical and cytochemical purposes, and they yield a coloured final reaction product when azo-coupled with a diazonium salt, an osmiophilic product for electron microscopy when coupled with hexazotized Pararosaniline, or a fluorescent final reaction product when coupled with 5-nitrosalicylaldehyde. AFC substrates are considerably more sensitive, and they yield the fluorescent product AFC after enzymatic cleavage of the substrate. AFC is not sufficiently water-insoluble to allow (intra)cellular localization, but AFC substrates are successfully used for incubations in microwells (Immu-Probe technique) and for the demonstration of banding patterns after gel electrophoresis (enzyme-directed overlay membrane technique). The methods are discussed with the example of the elucidation of the role of dipeptidylpeptidase IV in autoimmune diseases.
The metabolism of glucagon-like peptide-1 (GLP-1) has not been studied in detail, but it is known to be rapidly cleared from the circulation. Measurement by RIA is hampered by the fact that most antisera are side-viewing or C-terminally directed, and recognize both intact GLP-1 and biologically inactive. N-terminally truncated fragments. Using high pressure liquid chromatography in combination with RIAs, methodology allowing specific determination of both intact GLP-1 and its metabolites was developed. Human plasma was shown to degrade GLP-1-(7-36)amide, forming an N-terminally truncated peptide with a t1/2 of 20.4 +/- 1.4 min at 37 C (n = 6). This was unaffected by EDTA or aprotinin. Inhibitors of dipeptidyl peptidase-IV or low temperature (4 C) completely prevented formation of the metabolite, which was confirmed to be GLP-1-(9-36)amide by mass spectrometry and sequence analysis. High pressure liquid chromatography revealed the concentration of GLP-1-(9-36)amide to be 53.5 +/- 13.7% of the concentration of endogenous intact GLP-1 in the fasted state, which increased to 130.8 +/- 10.0% (P < 0.01; n = 6) 1 h postprandially. Metabolism at the C-terminus was not observed. This study suggests that dipeptidyl peptidase-IV is the primary mechanism for GLP-1 degradation in human plasma in vitro and may have a role in inactivating the peptide in vivo.
Glucagon-like peptide-1(7-37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 +/- 0.14 and 1.39 +/- 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 +/- 0.05 and 0.36 +/- 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.
A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined.
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 beta-cell-associated gene expression, including insulin and glucokinase. Further investigation of the effects of stable GLP-1 analogues on beta-cell differentiation is required to assess their potential in diabetic therapy.
The proper regulation of blood glucose homeostasis in mammals requires an adequate relation between the capacity to produce insulin and metabolic demand. Insulin receptor substrate proteins (IRS) are signalling intermediates that are required to keep this balance because they are needed for insulin action in target tissues but also for insulin production in pancreatic beta-cells. The total functional beta-cell mass in an individual sets the limit of how much insulin can be produced at a given time. It can change adaptively to meet demand and studies in vivo indicate that the regulation of beta-cell mass involves IRS2, while IRS1 is only required for proper insulin production in beta-cells. Overexpression studies in isolated islets have shown that IRS2, but not IRS1 or Shc, is sufficient to induce proliferation of beta-cells and to protect against d-glucose-induced apoptosis. In light of the finding that many growth factors can regulate Irs2 in islets, this signalling intermediate could balance capacity for insulin production with demand. This review summarizes observations in mouse models and in primary beta-cells and proposes a new hypothetical model of how IRS2 might control beta-cell mass.
Glucagon-like peptide 1 (GLP-1) is a hormone that is encoded in the proglucagon gene. It is mainly produced in enteroendocrine L cells of the gut and is secreted into the blood stream when food containing fat, protein hydrolysate, and/or glucose enters the duodenum. Its particular effects on insulin and glucagon secretion have generated a flurry of research activity over the past 20 years culminating in a naturally occurring GLP-1 receptor (GLP-1R) agonist, exendin 4 (Ex-4), now being used to treat type 2 diabetes mellitus (T2DM). GLP-1 engages a specific guanine nucleotide-binding protein (G-protein) coupled receptor (GPCR) that is present in tissues other than the pancreas (brain, kidney, lung, heart, and major blood vessels). The most widely studied cell activated by GLP-1 is the insulin-secreting beta cell where its defining action is augmentation of glucose-induced insulin secretion. Upon GLP-1R activation, adenylyl cyclase (AC) is activated and cAMP is generated, leading, in turn, to cAMP-dependent activation of second messenger pathways, such as the protein kinase A (PKA) and Epac pathways. As well as short-term effects of enhancing glucose-induced insulin secretion, continuous GLP-1R activation also increases insulin synthesis, beta cell proliferation, and neogenesis. Although these latter effects cannot be currently monitored in humans, there are substantial improvements in glucose tolerance and increases in both first phase and plateau phase insulin secretory responses in T2DM patients treated with Ex-4. This review will focus on the effects resulting from GLP-1R activation in the pancreas.
- R P Pauly
- F Rosche
- M Wermann
- C H Mcintosh
- R A Pederson
- H U Demuth
Pauly, R. P.; Rosche, F.; Wermann, M.; McIntosh, C. H.;
Pederson, R. A.; Demuth, H. U. J. Biol. Chem. 1996, 271, 23222.
- Q Xiao
- J Giguere
- M Parisien
- W Jeng
- S A St-Pierre
- P L Brubaker
- M B Wheeler
Xiao, Q.; Giguere, J.; Parisien, M.; Jeng, W.; St-Pierre, S. A.;
Brubaker, P. L.; Wheeler, M. B. Biochemistry 2001, 40, 2860.
- H K Liu
- B D Green
- V A Gault
- J T Mccluskey
- N H Mcclenaghan
- F P O 'harte
- Flatt
Liu, H. K.; Green, B. D.; Gault, V. A.; McCluskey, J. T.; McClenaghan, N. H.; O'Harte, F. P.; Flatt, P. R. Cell Biol. Int. 2004, 28,
69.
- D J Leahy
- C E Dann
- P Longo
- B Perman
- K X Ramyar
Leahy, D. J.; Dann, C. E., 3rd; Longo, P.; Perman, B.; Ramyar,
K. X. Protein Expr. Purif. 2000, 20, 500.
- B D Green
- V A Gault
- N Irwin
- M H Mooney
- C J Bailey
- P Harriott
- B Greer
- P R Flatt
- F O 'harte
Green, B. D.; Gault, V. A.; Irwin, N.; Mooney, M. H.; Bailey, C.
J.; Harriott, P.; Greer, B.; Flatt, P. R.; O'Harte, F. P. Biol. Chem.
2003, 384, 1543.
- R Burcelin
- W Dolci
Burcelin, R.; Dolci, W.; Thorens, B. Metabolism 1999, 48, 252.
- M E Doyle
- J M Egan
Doyle, M. E.; Egan, J. M. Pharmacol. Ther. 2007, 113, 546.
- B D Green
- M H Mooney
- V A Gault
- N Irwin
- C J Bailey
- P Harriott
- B Greer
- F P O 'harte
- P R Flatt
Green, B. D.; Mooney, M. H.; Gault, V. A.; Irwin, N.; Bailey, C.
J.; Harriott, P.; Greer, B.; O'Harte, F. P.; Flatt, P. R. J. Endocrinol.
2004, 180, 379.
- B Gallwitz
- T Ropeter
- C Morys-Wortmann
- R Mentlein
- E G Siegel
- W E Schmidt
Gallwitz, B.; Ropeter, T.; Morys-Wortmann, C.; Mentlein, R.;
Siegel, E. G.; Schmidt, W. E. Regul. Pept. 2000, 86, 103.
- S A Hinke
- S Manhart
- K Kuhn-Wache
- C Nian
- H U Demuth
- R A Pederson
- C H Mcintosh
Hinke, S. A.; Manhart, S.; Kuhn-Wache, K.; Nian, C.; Demuth,
H. U.; Pederson, R. A.; McIntosh, C. H. J. Biol. Chem. 2004,
279, 3998.
- D Parkes
- C Jodka
- P Smith
- S Nayak
- L Rinehart
- R Gingerich
- K Chen
- A Young
Parkes, D.; Jodka, C.; Smith, P.; Nayak, S.; Rinehart, L.; Gingerich,
R.; Chen, K.; Young, A. Drug. Dev. Res. 2001, 54, 260.
- R Lopez De Maturana
- A Willshaw
- A Kuntzsch
- R Rudolph
- D Donnelly
Lopez de Maturana, R.; Willshaw, A.; Kuntzsch, A.; Rudolph,
R.; Donnelly, D. J. Biol. Chem. 2003, 278, 10195.
- L L Baggio
- Q Huang
- T J Brown
- D Drucker
Baggio, L. L.; Huang, Q.; Brown, T. J.; Drucker, D. J. Diabetes
2004, 53, 2492.
- J Eng
- W A Kleinman
- L Singh
- G Singh
- J P Raufman
Eng, J.; Kleinman, W. A.; Singh, L.; Singh, G.; Raufman, J. P. J.
Biol. Chem. 1992, 267, 7402.
- S Runge
- H Thogersen
- K Madsen
- J Lau
- R Rudolph
Runge, S.; Thogersen, H.; Madsen, K.; Lau, J.; Rudolph, R. J.
Biol. Chem. 2008, 283, 11340.
- C F Deacon
- A H Johnsen
- J J Holst
Deacon, C. F.; Johnsen, A. H.; Holst, J. J. J. Clin. Endocrinol.
Metab. 1995, 80, 952.
- M Niessen
Niessen, M. Arch. Physiol. Biochem. 2006, 112, 65.