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Dipeptidyl Peptidase IV (DPIV/CD26) Degradation of Glucagon

CHARACTERIZATION OF GLUCAGON DEGRADATION PRODUCTS AND DPIV-RESISTANT ANALOGS*
Open AccessPublished:February 11, 2000DOI:https://doi.org/10.1074/jbc.275.6.3827
      Over the past decade, numerous studies have been targeted at defining structure-activity relationships of glucagon. Recently, we have found that glucagon1–29 is hydrolyzed by dipeptidyl peptidase IV (DPIV) to produce glucagon3–29 and glucagon5–29; in human serum, [pyroglutamyl (pGlu)3]glucagon3–29 is formed from glucagon3–29, and this prevents further hydrolysis of glucagon by DPIV (H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation). In the current study, the biological activity of these peptides was examined in vitro. The amino-terminally truncated peptides all behaved as partial agonists in cyclic AMP stimulation assays, with Chinese hamster ovary K1 cells overexpressing the human glucagon receptor (potency: glucagon1–29 > [pGlu3]glu- cagon3–29 > glucagon3–29 > glucagon5–29 > [Glu9]glu- cagon2–29). In competition binding experiments, [pGlu3]glucagon3–29 and glucagon5–29 both demonstrated 5-fold lower affinity for the receptor than glucagon1–29, whereas glucagon3–29 exhibited 18-fold lower affinity. Of the peptides tested, only glucagon5–29 showed antagonist activity, and this was weak compared with the classical glucagon antagonist, [Glu9]glucagon2–29. Hence, DPIV hydrolysis of glucagon yields low affinity agonists of the glucagon receptor. As a corollary to evidence indicating that DPIV degrades glucagon (Demuth, et al., manuscript in preparation), DPIV-resistant analogs were synthesized. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to assess DPIV resistance, and it allowed kinetic analysis of degradation. Of several analogs generated, only [d-Ser2] and [Gly2]glucagon retained high affinity binding and biological potency, similar to native glucagon in vitro. [d-Ser2]Glucagon exhibited enhanced hyperglycemic activity in a bioassay, whereas [Gly2]glucagon was not completely resistant to DPIV degradation.
      DPIV
      dipeptidyl peptidase IV
      pGlu
      pyroglutamyl
      Fmoc
      N-9-fluorenylmethyloxycarbonyl
      HPLC
      high pressure liquid chromatography
      MALDI-TOF
      matrix-assisted laser desorption/ionization-time of flight
      EC50
      half-maximal effective concentration
      (I/A)50
      ratio of inhibitor to agonist resulting in reduction of agonist alone 2-fold
      CHO
      Chinese hamster ovary
      hGlucR cell
      cell stably expressing the human glucagon receptor
      Glucagon is a 29-amino acid peptide hormone that is released from pancreatic α-cells and acts to raise blood glucose in the fasted state by increasing hepatic glycogenolysis and gluconeogenesis (
      • Holst J.
      • Ørskov C.
      ,
      • Chiasson J.
      • Cherrington A.
      ). The circulating half-life of immunoreactive glucagon is estimated to be between 5 and 6 min in dogs and humans (
      • Jaspan J.
      • Polonsky K.
      • Lewis M.
      • Pensler J.
      • Pugh W.
      • Moossa A.
      • Rubenstein A.
      ,
      • Alford F.
      • Bloom S.
      • Nabarro J.
      ). The tissues responsible for the clearance of glucagon from the circulation are somewhat controversial; however, it is generally accepted that the kidneys play the dominant role (reviewed in Refs.
      • Holst J.
      and
      • Lefebvre P.
      • Luyckx A.
      ). Interestingly, there have been reports that glucagon degradation in blood or plasma is negligible (
      • Hendriks T.
      • Benraad T.
      ,
      • Märki F.
      ). Recently, evidence has been presented suggesting that dipeptidyl peptidase IV (DPIV)1 is responsible in part for the inactivation of glucagon (
      • Demuth H.-U.
      • Glund K.
      • Heiser U.
      • Hinke S.
      • Hoffman T.
      • Pospisilik J.
      • Rosche F.
      • Schlenzig D.
      • Wermann M.
      • McIntosh C.
      • Pederson R.
      ).
      H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation.
      2H.-U. Demuth, K. Glund, U. Heiser, J. Pospisilik, S. Hinke, T. Hoffmann, F. Rosche, D. Schlenzig, M. Wermann, C. McIntosh, and R. Pederson, manuscript in preparation.
      This finding is consistent with renal clearance of glucagon but conflicts with the absence of plasma degradation previously reported, as DPIV is found on the surface of lymphocytes and as a freely circulating enzyme in addition to the apical surface of the renal proximal tubules (
      • Yaron A.
      • Naider F.
      ).
      The glucagon receptor is a class B serpentine G-protein coupled receptor, belonging to the same family of hormone receptors as those for secretin, vasoactive intestinal peptide, glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide, glucagon-like peptide-1, calcitonin, parathyroid hormone, and pituitary adenylyl cyclase activating polypeptide (
      • Horn F.
      • Weare J.
      • Beukers M.
      • Hörsch S.
      • Bairoch A.
      • Chen W.
      • Edvardsen Ø.
      • Campagne F.
      • Vriend G.
      ). The ligand specificity of the glucagon receptor is primarily conferred by its extracellular amino terminus (
      • Graziano M.
      • Hey P.
      • Strader C.
      ,
      • Buggy J.
      • Livingston J.
      • Rabin D.
      • Yoo-Warren H.
      ); activation of the receptor results in activation of both the adenylyl cyclase/cyclic AMP and the phospholipase C/inositol trisphosphate intracellular cascades (
      • Li J.
      • Larocca J.
      • Rodriguez-Gabin A.
      • Charron M.
      ).
      To date, a plethora of structure-activity studies on glucagon have been performed (the most recent comprehensive review in Ref.
      • Hruby V.
      ). These have generally consisted of rational and systematic investigations of ligand-receptor agonism and antagonism, resulting in an increased understanding of the charge-charge interactions between the hormone and receptor resulting in its ability to bind and activate the receptor (
      • Unson C.
      • Wu C.-R.
      • Cheung C.
      • Merrifield R.
      ). Other key findings include the importance of the amino terminus of glucagon in receptor activation (
      • Smith R.
      • Sisk R.
      • Lockhart P.
      • Mathewes S.
      • Gilbert T.
      • Walker K.
      • Piggot J.
      ), as well as important residues within the primary sequence of glucagon (
      • Unson C.
      • Wu C.-R.
      • Cheung C.
      • Merrifield R.
      ,
      • Azizeh B.
      • Van Tine B.
      • Trivedi D.
      • Hruby V.
      ).
      Recently, it was discovered that purified pork kidney DPIV is capable of hydrolyzing glucagon1–29 to glucagon3–29and glucagon5–29 in vitro and that, in human serum, it is converted first to glucagon3–29, and subsequently its amino terminus is cyclized by a serum enzyme (possibly to pyroglutamyl-glucagon3–29([pGlu3]glucagon3–29)), thus preventing further DPIV degradation, as it does not fulfil the substrate requirements of the enzyme (i.e. it lacks a protonable amino terminus).2 The specificity of DPIV was characterized to preferentially release dipeptides from the amino terminus of polypeptides with proline or alanine in the penultimate position (
      • Yaron A.
      • Naider F.
      ). However, amino-terminal degradation of nontypical substrates has been reported, including sequential cleavage of amino-terminal dipeptides (
      • Proost P.
      • Struyf S.
      • Schols D.
      • Opdenakker G.
      • Sozzani S.
      • Allavena P.
      • Mantovani A.
      • Augustyns K.
      • Bal G.
      • Haemers A.
      • Lambeir A.-M.
      • Scharpé S.
      • Van Damme J.
      • De Meester I.
      ,
      • Demuth H.
      • Heins J.
      ).
      In the current study, the effects of the potentially physiologically relevant amino-terminally truncated glucagon fragments on the human glucagon receptor were examined. The glucagon fragments glucagon3–29, glucagon5–29, and [pGlu3]glucagon3–29 were characterized on Chinese hamster ovary K1 (CHO-K1) cells transfected with the human glucagon receptor, with respect to agonist and antagonist activity as well as binding affinity, and compared with glucagon1–29and [Glu9]glucagon2–29. Further studies using DPIV-resistant glucagon analogs were performed to support existing evidence for DPIV degradation of glucagon using in vitro methods and a bioassay.

      DISCUSSION

      Several lines of evidence have resulted in the necessity for reassessment of glucagon degradation in vivo. Controversy in the past regarding glucagon degradation, with respect to specific enzymes and organs involved, needs to be clarified. Evidence presented here indicates that dipeptidyl peptidase IV is a prime candidate for enzymatic inactivation of glucagon. The recent finding demonstrating DPIV in the secretory granules of the pancreatic islet α-cell compels one to question how much of the pancreatic glucagon enters the circulation intact (
      • Grondin G.
      • Hooper N.
      • LeBel D.
      ). Grondin et al. (
      • Grondin G.
      • Hooper N.
      • LeBel D.
      ) further argue that the low pH of the secretory granule would not permit activity of DPIV, and thus DPIV would not be active until granule contents are secreted. The discovery that glucagon is successively hydrolyzed by dipeptidyl peptidase IV into amino-terminally truncated peptides2 raises a number of questions. The first question is the role of the hydrolyzed peptides: are they simply degradation products, or do they have a physiological role? The emergence of the “mini-glucagon” story in the pancreas suggests the hypothesis of a local action of amino-terminally truncated glucagon. Processing of glucagon by miniglucagon-generating endopeptidase to glucagon19–29, results in a peptide having differential effects on cardiac myocytes (
      • Pavoine C.
      • Brechler V.
      • Kervran A.
      • Blache P.
      • Le-Nguyen D.
      • Laurent S.
      • Bataille D.
      • Pecker F.
      ) and having the ability to inhibit insulin release in the picomolar range (
      • Dalle S.
      • Smith P.
      • Blache P.
      • Le-Nguyen D.
      • Le Brigand L.
      • Bergeron F.
      • Ashcroft F.
      • Bataille D.
      ). This report forms the foundation for further work on glucagon degradation products and their possible function in vivo.
      Fragments similar to those described here have been tested for agonism and antagonism in other biological systems. Glucagon5–29was found to have <0.001% of the potency of native glucagon in the rat hepatocyte membrane adenylyl cyclase activity assay (
      • Frandsen E.
      • Grønvald F.
      • Heding L.
      • Johnsen N.
      • Lundt B.
      • Moody A.
      • Markussen J.
      • Volund A.
      ), and it was found that this fragment also acted as an antagonist in this tissue ((I/A)50 ≅ 71). In the current study, using cells overexpressing the human glucagon receptor, it was found that glucagon5–29 has 28.5% of the potency of glucagon1–29 (Fig. 1 and Table I), and indeed, it does act as a weak antagonist on these cells (Fig. 2). Similar glucagon analogs to those tested here, [Glu9]glucagon3–29 and [Glu9]glucagon5–29, have also been previously characterized (
      • Unson C.
      • Gurzenda E.
      • Iwasa K.
      • Merrifield R.
      ). The binding affinities reported on the Glu9 substituted analogs (
      • Unson C.
      • Gurzenda E.
      • Iwasa K.
      • Merrifield R.
      ) are consistent with the trend observed with native fragments on transfected cells (Fig. 3); however, the amino acid substitution at position 9 alone ([Glu9]glucagon1–29) was also shown to have dramatic effects on binding affinity (
      • Unson C.
      • Gurzenda E.
      • Iwasa K.
      • Merrifield R.
      ). Similarly, [Glu9]glucagon1–29 had significantly reduced potency, and amino-terminally truncated (desHis1) peptides showed negligible adenylyl cyclase stimulating activity (
      • Unson C.
      • Gurzenda E.
      • Iwasa K.
      • Merrifield R.
      ). In light of the finding that only glucagon5–29 showed antagonism on hGlucR cells, it is likely that the Glu9 substitution was responsible for the antagonism observed for [Glu9]glucagon3–29, and resulted in (I/A)50 ratios similar to [Glu9]glucagon2–29, as was also the case for [Glu9]glucagon5–29 (
      • Unson C.
      • Gurzenda E.
      • Iwasa K.
      • Merrifield R.
      ). Native glucagon5–29 showed only weak antagonism compared with [Glu9]glucagon2–29 (Fig. 2). The (I/A)50 value for [Glu9]glucagon2–29 obtained in the current study was higher than that reported previously (
      • Unson C.
      • Gurzenda E.
      • Iwasa K.
      • Merrifield R.
      ); however, it is likely that this is simply due to overexpression of glucagon receptors in the system used for the current study. The activities of the fragments tested support the importance of the amino terminus in glucagon signal transduction. Cyclization of the side chain of Gln3 to form [pGlu3]glucagon3–29increased both binding affinity and potency as compared with glucagon3–29 (Fig. 1 and 3). Glucagon5–29also retained high affinity binding (greater than glucagon3–29) but showed lower potency when compared with either [pGlu3]glucagon3–29 or glucagon3–29.
      Surprisingly, the use of heterologous expression systems for the testing of glucagon antagonists have not been reported in the literature, and the hepatocyte adenylyl cyclase assay is the most widely used assay system. The only exception was Hjorth et al. (
      • Hjorth S.
      • Ørskov C.
      • Schwartz T.
      ), who examined the possible inverse agonism of [Glu9]glucagon2–29 using constitutively active glucagon receptor mutants [H178R] transfected into COS-7 cells. Recently, potent phosphodiesterase inhibitors have been used to characterize “pure” glucagon antagonists (
      • Azizeh B.
      • Van Tine B.
      • Trivedi D.
      • Hruby V.
      ). The overexpression of the glucagon receptor in CHO-K1 cells has also proven to be highly sensitive to partial agonism and thus may also serve the same purpose.
      Characterization of DPIV-resistant, amino-terminally modified glucagon analogs is consistent with published literature. The general conclusion from random molecular mutagenesis screening was that modification of the amino terminus of glucagon reduces biological activity, implicating it as an important domain necessary for receptor activation (
      • Smith R.
      • Sisk R.
      • Lockhart P.
      • Mathewes S.
      • Gilbert T.
      • Walker K.
      • Piggot J.
      ). Robberecht et al. (
      • Robberecht P.
      • Damien C.
      • Moroder L.
      • Coy D.
      • Wunsch E.
      • Cristophe J.
      ) found that altering the chirality at positions 2 and 3 of glucagon has minor effects on potency in the hepatocyte adenylyl cyclase assay; [d-Ser2]glucagon was equivalent to native glucagon in terms of cAMP formation and binding affinity, whereas reversing the chirality of position 3 had significant effects on both parameters. Unson and Merrifield (
      • Unson C.
      • Merrifield R.
      ) also substituted thed-isomer of serine in position 2; however, they found that it dramatically reduced affinity and potency of this analog in vitro. Our work using cells transfected with the human glucagon receptor is consistent with the earlier studies using hepatocyte membranes (
      • Robberecht P.
      • Damien C.
      • Moroder L.
      • Coy D.
      • Wunsch E.
      • Cristophe J.
      ). The [d-Ser2] substitution was better tolerated than [d-Gln3], when looking at in vitro cAMP stimulatory activity and receptor binding affinity (Fig. 6 and Table II).
      Due to the difficulty in obtaining second order rate constants from substrates that are hydrolyzed so slowly, and because some of the peptide analogs undergo different degradation fates, the more general parameter of half-life (t 12) was chosen as a general means of comparison for degradation of glucagon and amino-terminally modified peptides (Table III). Using this method, a measure of degradation in human serum could be obtained; however, as only 20% serum was used, values are underestimates of the true degradation by serum. Furthermore, serum DPIV activity represents only a fraction of the DPIV activity in vivo, as DPIV is found on endothelial cells and the surface of lymphocytes, among other tissues (
      • Yaron A.
      • Naider F.
      ). Using surface plasmon resonance measurements, binding constants (K d) of amino-terminally modified glucagon analogs and purified DPIV could be obtained, and all showed a significant reduction in affinity (6–23-fold; Table II). However, despite their binding kinetics, analogs exhibited variable resistance to purified DPIV (Table III). [d-Ser2]Glucagon had the greatest affinity for DPIV of the modified peptides but was completely resistant to DPIV degradation; in contrast, [Gly2]glucagon had a K d similar to that of [d-Ser2]glucagon but was degraded at a rate only slightly slower than native glucagon by purified DPIV. Hence, it appears that a moderate reduction in substrate-enzyme affinity by amino-terminal modification does not necessarily affect the rate-limiting step of DPIV-mediated hydrolysis to a great degree.
      Previous studies on [d-Ser2] and [d-Gln3] were limited to in vitrostructure-function studies. With the objective of generating DPIV-resistant glucagon analogs, to support the hypothesis of DPIV degradation of glucagon, an in vivo assay system was necessary. The [d-Ser2] substitution was the only analog that possessed enhanced ability to increase circulating glucose levels relative to native glucagon. The greater potencyin vivo can be attributed to the lack of degradation by DPIV, as the in vitro potency was found to be moderately reduced (Fig. 6). However, this substitution rendered the peptide more susceptible to degradation by trypsin-like enzymes (Fig. 5). Other amino-terminally modified glucagon analogs were not suitable to demonstrate the contribution of DPIV to the degradation of glucagon, as they possessed reduced biological activity in vitro, andin vivo, or were susceptible to DPIV degradation.
      The question remains as to the physiological relevance of DPIV-mediated glucagon hydrolysis. In general, K d,K m, or K i values of peptide substrate/inhibitor-enzyme binding are mainly of theoretical interest, as they are usually 1–5 orders of magnitude greater than circulating levels of peptide. However, if the second-order rate constant of a protease-catalyzed reaction can be determined (e.g. K cat/K m), values obtained from different peptides can be helpful in interpreting biological significance. The second order rate constant for DPIV-catalyzed glucagon hydrolysis was 2.0 × 105m−1 s−1, which is comparable to the rate constants for the incretins glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide and glucagon-like peptide-1 and other known DPIV substrates (data not shown)2 (
      • Demuth H.
      • Heins J.
      ,
      • Pauly R.
      • Rosche F.
      • Wermann M.
      • McIntosh C.
      • Pederson R.
      • Demuth H.-U.
      ,
      • Rosche F.
      • Schmidt J.
      • Hoffmann T.
      • Pauly R.
      • McIntosh C.
      • Pederson R.
      • Demuth H.-U.
      ,
      • Mentlein R.
      • Gallwitz B.
      • Schmidt W.
      ). Notably, the incretins were found to be DPIV substrates in vitro (
      • Mentlein R.
      • Gallwitz B.
      • Schmidt W.
      ), prior to demonstration of in vivo relevance (
      • Kieffer T.
      • McIntosh C.
      • Pederson R.
      ). Pauly et al. (
      • Pauly R.
      • Rosche F.
      • Wermann M.
      • McIntosh C.
      • Pederson R.
      • Demuth H.-U.
      ) hypothesized that in vivo inhibition of DPIV would enhance the incretin effect, a hypothesis that was later shown to be correct (
      • Pederson R.
      • White H.
      • Schlenzig D.
      • Pauly R.
      • McIntosh C.
      • Demuth H.-U.
      ,
      • Pauly R.
      • Demuth H.-U.
      • Rosche F.
      • Schmidt J.
      • White H.
      • Lynn F.
      • McIntosh C.
      • Pederson R.
      ,
      • Deacon C.
      • Hughes T.
      • Holst J.
      ). Similar studies investigating the physiological importance of DPIV-mediated glucagon degradation will undoubtedly be forthcoming.
      In summary, several structure-activity relationships of glucagon have been assessed in vitro and in vivo, with specific reference to degradation of glucagon by dipeptidyl peptidase IV. Amino-terminally truncated glucagon fragments were all weak partial agonists of the human glucagon receptor and showed no glycemic effectin vivo. The role of DPIV degradation in glucagon metabolism was also studied using amino-terminally modified glucagon analogs. Of these peptides, [d-Ser2] and [Gly2]glucagon were the best tolerated modifications, as assessed by cAMP production and competitive binding studies on hGlucR cells. [d-Ser2] was the only peptide suitable for in vivo studies, as [Gly2] was not DPIV-resistant; [d-Ser2] exhibited enhanced biological activity relative to native glucagon in a bioassay. We have thus provided further evidence that DPIV is likely a primary enzyme involved in glucagon degradation. With the foundation of research set, the physiological roles of potentially biologically relevant amino-terminally truncated glucagon peptides remain to be elucidated.

      Acknowledgments

      We thank Dr. R. W. Gelling for development of the hGlucR cells. We are grateful for the technical assistance of Cuilan Nian and Irene Bremsak.

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