THE TWO GLUCAGON-LIKE peptides, GLP-1 and

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1 X/00/$03.00/0 Vol. 85, No. 8 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 2000 by The Endocrine Society In Vivo and in Vitro Degradation of Glucagon-Like Peptide-2 in Humans* BOLETTE HARTMANN, METTE B. HARR, PALLE B. JEPPESEN, MORTEN WOJDEMANN, CAROLYN F. DEACON, PER B. MORTENSEN, AND JENS J. HOLST Department of Medical Physiology, The Panum Institute, University of Copenhagen (B.H., M.B.H., C.F.D., J.J.H.); and Departments of Medicine CA-2121 (P.B.J., P.B.M.) and Surgery C (M.W.), Rigshospitalet, University Hospital of Copenhagen, DK-2200 Copenhagen, Denmark ABSTRACT Glucagon-like peptide-2 (GLP-2), an intestinal product of glucagon gene expression which induces intestinal growth in mice, has been proposed as a treatment for intestinal insufficiency. GLP-2 is metabolized extensively by dipeptidyl peptidase IV (DPP-IV) in rats, but less is known about its fate in humans. Therefore, GLP-2 metabolism was investigated in healthy volunteers after 1) a 500-Cal mixed meal (n 6), 2) iv infusion of synthetic human GLP-2 (0.8 pmol/kg min; n 8), 3) a sc bolus injection (400 g; n 9), and 4) in vitro incubation in plasma and blood (1000 pmol/l; n 4). GLP-2 concentrations were determined by N-terminal RIA measuring only intact GLP-2, sideviewing RIA measuring intact and degraded forms [e.g. GLP-2-(3 33) arising from DPP-IV degradation], and high performance liquid chromatography (HPLC). Meal ingestion elevated plasma GLP-2 (intact, 16 3to73 10 pmol/l at 90 min), and HPLC revealed two immunoreactive components: intact GLP-2 (57 2%) and GLP-2-(3 33). THE TWO GLUCAGON-LIKE peptides, GLP-1 and GLP-2, arise from tissue-specific posttranslational processing of the proglucagon precursor, proglucagon, within the intestinal mucosal L cells (1), and GLP-2 has recently been shown to induce intestinal growth in mice (2). Surprisingly, GLP-2 was much less effective in rats (3). Excessive degradation of GLP-2 by dipeptidyl peptidase IV (DPP-IV) in rats was suggested to explain this unexpected difference (3). Presently, the intestinotropic effect of GLP-2 is attracting considerable interest among clinical gastroenterologists, and the use of GLP-2 has been suggested for treatment of intestinal insufficiency, but little is known about the metabolism of GLP-2 in humans. Brubaker et al. (4), and Xiao et al. (5) showed that human, commercially available, DDP-IV was capable of cleaving human GLP-2, as suggested by Mentlein et al. in 1993 (6), and also provided HPLC profiles of immunoreactive GLP-2 in single human plasma samples obtained in the fasting and fed states that were consistent with the presence of both intact GLP-2-(1 33) and the DPP-IV metabolite GLP-2-(3 33). It is conceivable, therefore, that DPP- Received January 7, Revision received March 29, Rerevision received May 8, Accepted May 8, Address all correspondence and requests for reprints to: J. J. Holst, M.D., Department of Medical Physiology, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. holst@mfi.ku.dk. * This work was supported by the Danish Medical Research Council, the Løvens Research Foundation, and the Mimi and Victor Larsens Foundation. GLP-2 infusion increased plasma levels [intact, 9 4to pmol/l; total, 23 7to pmol/l; the differences represent GLP-2-(3 33)]. The elimination t 1/2 values were min (intact GLP-2) and min [GLP-2-(3 33)], and MCRs were and ml/kg min, respectively. Subcutaneous injection increased intact GLP-2 to maximally pmol/l at 45 min, whereas total GLP-2 increased to pmol/l at 90 min. At 60 min, plasma contained 69 1% intact GLP-2. In vitro the t 1/2 values were h (plasma) and h (blood). GLP-2-(3 33) was the only degradation product identified by HPLC, and a DPP-IV inhibitor abolished the degradation of GLP-2 in vitro. We conclude that GLP-2 is extensively degraded to GLP-2-(3 33) in humans, presumably by DPP-IV. Nevertheless, 69% remains intact 1 h after GLP-2 injection, supporting the possibility of sc use in patients with intestinal insufficiency. (J Clin Endocrinol Metab 85: , 2000) IV-mediated degradation could limit the effects of GLP-2 in humans. We, therefore, developed methodology to allow measurement of both intact GLP-2-(1 33) and possible metabolites, using a combination of high pressure liquid chromatography (HPLC) and specific RIAs to investigate the degradation of exogenous and endogenous human GLP-2 in vivo. Furthermore, in vitro studies were carried out to examine the effects of a specific DPP-IV inhibitor on the degradation of exogenous GLP-2 in human plasma. Materials and Methods Synthetic human GLP-2-(1 33) and GLP-2-(3 33) were purchased from PolyPeptides Laboratories (Wolfenbuttel, Germany), and human recombinant GLP-2-(1 33) was a gift from L. Thim, Novo Nordisk A/S (Bagsvaerd, Denmark). Purity and correctness of structure were confirmed by mass, sequence, and HPLC analysis (Dr. A. H. Johnsen, Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark). Valine pyrrolidide (VP) was a gift from R. D. Carr and L. B. Christiansen, Novo Nordisk A/S. RIAs GLP-2-(1 33) was measured with an N-terminal-specific antiserum (code no ) that measures only GLP-2 with an intact N-terminus, as described previously (7). For standards, we used recombinant human GLP-2-(1 33), and the tracer was bovine GLP-2 with a Thr 12 3Tyr 12 substitution, 125 I labeled using the standard stoichiometric chloramine-t method, as described previously (8). This assay shows a cross-reaction with synthetic human GLP-2-(3 33) of maximally %. Total GLP-2 was measured using a side-viewing antiserum (catalogue no. 2884

2 DEGRADATION OF HUMAN GLP RAS 7167, Peninsula Laboratories, Inc., Europe, St. Helens, UK; reacting with a midsequence of GLP-2), with rat GLP-2 with an Asp 33 3Tyr 33 substitution for iodination and human GLP-2-(1 33) for standards. In agreement with its specificity for a midregion of GLP-2, the antiserum does not bind the Tyr 12 -GLP-2 tracer. For both assays, the assay buffer was 80 mmol/l sodium phosphate buffer, ph 7.5, containing, in addition, 0.01 mmol/l VP, 0.1% (wt/vol) human serum albumin (HSA; ORHA 20/21, Behring, Marburg, Germany), 10 mmol/l ethylenediamine tetraacetic acid, and 0.6 mmol/l thimerosal (no. T-5125, Sigma, St. Louis, MO). Free and bound moieties were separated with plasmacoated charcoal (E. Merck, Darmstadt, Germany) (8). All plasma samples were extracted in a final concentration of 75% ethanol before GLP-2 measurements to remove unspecific cross-reacting substances. The recovery of synthetic GLP-2-(1 33) added to plasma before extraction and assay was 68%. For both assays, the experimental detection limit was less than 5 pmol/l, and the intraassay coefficient of variation was 5% at a concentration of 40 pmol/l. Endogenous GLP-2 in vivo Six normal healthy nonsmoking volunteers (four women and two men; age, yr; body mass index, kg/m 2 ), were studied after an overnight fast. The subjects received breakfast (500 Cal, with 58%, 33%, and 9% of the calories derived from carbohydrate, fat, and protein, respectively), and blood samples were drawn at regular intervals before and after the meal, collected into chilled tubes containing (in final concentrations) ethylenediamine tetraacetic acid (3.9 mmol/l) and VP (0.01 mmol/l), kept on ice, and centrifuged within 0.5 h. Plasma samples were stored at 20 C until assay. Metabolism of GLP-2 infused iv in vivo The study was approved by the regional ethical committee of Copenhagen (reference no. KF /95). Synthetic human GLP-2-(1 33) was dissolved in 0.9% NaCl containing 1% HSA (Novo Nordisk A/S), subjected to sterile filtration, checked for sterility and pyrogens, and kept at 20 C until use. Eight healthy fasting volunteers (five men and three women; age, yr; body mass index, kg/m 2 ) received infusions of synthetic human GLP-2-(1 33) at 0.8 pmol/kg min for 150 min. Blood samples were drawn (as described above) at regular intervals before, during, and after infusion. HPLC separation We used LKB equipment with an MN Nucleosil cartridge system (Macherey-Nagel, Duren, Germany) fitted with a Nucleosil mc 8 column at room temperature with on-line UV detection at 226 nm. The column was eluted at a flow rate of 2 ml/min, with stepwise linear gradients of acetonitrile (AcN; HPLC grade; Rathburn, Walkenburn, Scotland) in 0.1% trifluoroacetic acid (TFA; HPLC grade; Rathburn; 0% AcN for 5 min, followed by 0 33% over 3 min, 33 39% over 18 min, and 39 75% over 3 min). Fractions (500 L) were collected, lyophilized, and reconstituted in assay buffer. The HPLC system was capable of separating intact GLP-2-(1 33) from the putative metabolite, GLP-2-(3 33), used for calibration as well as intact GLP-2-(1 33). The recovery of endogenous GLP-2-(1 33) after HPLC (measured using the N-terminal assay) was %. Results Endogenous GLP-2 in vivo Mixed meal ingestion elevated plasma GLP-2-(1 33) from 16 3to73 10 pmol/l at 90 min (Fig. 1). HPLC analysis of plasma (2 ml) collected at 90 min (extracted by ethanol precipitation, lyophilized, and reconstituted in 0.1% TFA containing 0.2% HSA) followed by RIA with the side-viewing assay revealed two immunoreactive components. GLP- 2-(1 33) accounted for 57 2%, with the remainder being GLP-2-(3 33) (Fig. 2). The HPLC fractions were also analyzed with the N-terminal-specific RIA, which revealed a single immunoreactive component eluting at the position of the GLP-2-(1 33) marker (data not shown). The amount of immunoreactivity eluting at this position was similar for the two assays. Metabolism of GLP-2 infused iv in vivo The infusion increased plasma GLP-2-(1 33) from 9 4 pmol/l to a plateau of pmol/l (N-terminal RIA) and from 23 7to pmol/l (side-viewing RIA; Fig. Metabolism of GLP-2 injected sc in vivo The study was approved by the regional ethical committee of Copenhagen (reference no. KF /98). Synthetic human GLP-2-(1 33) was mixed with 0.9% NaCl, and 0.5% ammonia was added until the peptide was dissolved (ph 8 9), whereupon ph was neutralized using 0.1 mol/l acetic acid. The preparation was subjected to sterile filtration using a 22- m filter (Millex, Millipore Corp., Bedford, MA), dispensed into capped vials, and heat sterilized for 20 min at 100 C. Before use, the filter was saturated with 20% HSA (Statens Serum Institut, Copenhagen, Denmark) and washed with sterile water. The preparation was checked for sterility and kept at 20 C until use. The peptide content after sterile filtration was confirmed by amino acid analysis. Nine healthy fasting volunteers (three men and six women; age, yr; body mass index, kg/m 2 ) received a sc bolus injection of 400 g synthetic human GLP-2-(1 33). Again, blood samples were drawn as described above at regular intervals before, during, and after injection. For all studies in human volunteers, informed consent was obtained from all subjects before the studies. Degradation of GLP-2 in vitro Blood samples were collected from four healthy volunteers (staff members) into chilled heparinized tubes, and plasma was separated by centrifugation. Blood and plasma were incubated in a shaking water bath at 37 C with recombinant human GLP-2-(1 33) (1000 pmol/l, final concentration). At different time points (0, 1, 2, 4, and 6 h) aliquots were drawn, extracted by ethanol precipitation, and subjected to RIA and HPLC analysis. Similar incubation experiments were performed with VP (0.01 mmol/l, final concentration) added at 0 min. FIG. 1. Mean plasma concentrations of intact GLP-2-(1 33) in six healthy subjects after mixed meal ingestion. The plasma concentrations in picomoles per L ( SEM) are plotted against time.

3 2886 HARTMANN ET AL. JCE&M 2000 Vol 85 No 8 3). The elimination half-lives (t 1/2 ) after infusion were and min for GLP-2-(1 33) and GLP-2-(3 33), respectively, assuming that the concentration of the latter could be calculated as the difference between total GLP-2 and GLP-2-(1 33). The t 1/2 values were calculated after logarithmic transformation of the measured concentrations. This, in all cases, resulted in straight lines from which the slope and thereby the t 1/2 could be determined by regression analysis. The MCR was ml/kg min for GLP-2-(1 33) and ml/kg min for GLP-2-(3 33). The MCR for GLP- 2-(1 33) was calculated as MCR infusion rate (pmol/ FIG. 2. HPLC analysis of endogenous GLP-2. Human plasma (2 ml) was collected 90 min after mixed meal ingestion, extracted by ethanol precipitation, lyophilized, reconstituted in 0.1% TFA containing 0.2% HSA, and analyzed by reverse phase HPLC on a Nucleosil C 8 column, eluted with gradients of AcN in 0.1% TFA. All fractions were lyophilized, reconstituted in assay buffer, and analyzed for GLP-2 immunoreactivity using the side-viewing assay. The elution positions of the intact GLP-2-(1 33) and the metabolite GLP-2-(3 33) are indicated. The data shown are representative of six experiments. kg min)/plasma concentration increment (pmol/l). The MCR for GLP-2-(3 33) was calculated as MCR Vd app k, where k was derived from k ln2/t 1/2 [GLP-2-(3 33)], and Vd app, the apparent distribution space, was equal to that of GLP-2-(1 33), from the same equations, but solved for GLP- 2-(1 33). The Vd app was ml/kg. Metabolism of GLP-2 injected sc in vivo The sc bolus injection of 400 g GLP-2-(1 33) increased the plasma GLP-2-(1 33) concentration (measured with the N- terminal-specific RIA) to a maximum of pmol/l 45 min after injection, and the total GLP-2 concentration (measured with the side-viewing RIA) increased to a maximum of pmol/l at 90 min after injection (Fig. 4). HPLC analysis of plasma (700 L) collected 60 min after injection (n 6; extracted by ethanol precipitation, lyophilized, and reconstituted in 0.1% TFA containing 0.2% HSA) and analyzed using the side-viewing RIA showed 69 1% to be GLP-2-(1 33) (Fig. 5). Again, the HPLC fractions were also analyzed with the N-terminal-specific RIA, which revealed a single immunoreactive component eluting at the position of the GLP-2-(1 33) marker (data not shown). The amount of immunoreactivity eluting at this position was similar for the two assays. Degradation in vitro The elimination half-life for GLP-2-(1 33) after in vitro incubation was h in plasma and h in blood, measured with the N-terminal-specific RIA (Fig. 6). The concentrations of total GLP-2 measured using the side-viewing assay remained constant throughout the incubation periods (not shown). GLP-2-(3 33) was the only degradation product identified by HLPC. There was no degradation of GLP-2- FIG. 3. Mean plasma concentrations of intact human GLP-2 (f) and total human GLP-2 ( ) before, during, and after iv infusion of synthetic GLP-2-(1 33) at 0.8 pmol/kg min in eight healthy subjects. The plasma concentrations in picomoles per L ( SEM) are plotted against time. FIG. 4. Mean plasma concentrations of intact human GLP-2 (f) and total human GLP-2 ( ) after sc bolus injection of 400 g synthetic GLP-2-(1 33) in nine healthy fasting volunteers. The plasma concentrations in picomoles per L ( SEM) are plotted against time.

4 DEGRADATION OF HUMAN GLP FIG. 5. HPLC analysis of sc injected GLP-2. Plasma (700 L) was collected 60 min after injection, extracted by ethanol precipitation, lyophilized, reconstituted in 0.1% TFA containing 0.2% HSA, and analyzed by reverse phase HPLC on a Nucleosil C 8 column, eluted with gradients of AcN in 0.1% TFA. All fractions were lyophilized, reconstituted in assay buffer, and analyzed for GLP-2 immunoreactivity using the side-viewing assay. The elution positions of the intact GLP-2-(1 33) and the metabolite GLP-2-(3 33) are indicated. The data shown are representative of six experiments. (1 33) in plasma or blood when the incubation was performed in the presence of VP (Fig. 6). Discussion Recent research (9) has established that GLP-1, which is highly homologous to GLP-2, is extensively and rapidly metabolized by the ubiquitous enzyme, DPP-IV, whereby the peptide loses its two N-terminal amino acid residues as well as its biological activity. Thereby the elimination half-life of the intact peptide becomes as low as min, whereas its MCR exceeds cardiac output by a factor of 2 (10). The studies by Mentlein et al. (6) suggested that GLP-2 would also be a substrate for DPP-IV, and studies by Drucker et al. (3) indicated that a significant DPP-IV-mediated degradation of GLP-2 takes place in rats. As mentioned above, Brubaker et al. (4) showed that a commercial preparation of human DPP-IV could cleave off the two N-terminal amino acids of human GLP-2-(1 33), leading to the formation of GLP-2-(3 33). They also carried out HPLC analysis of two postprandial plasma samples, the single illustrated example of which was consistent with the presence in plasma of both GLP-2-(1 33) and GLP-2-(3 33). Subsequently, Xiao et al. showed single HPLC profiles of fasting and fat-stimulated GLP-2 in plasma, again consistent with the presence of both GLP-2-(1 33) and GLP-2-(3 33). In the present investigation the degradation rates of exogenous GLP-2-(1 33) was studied in humans, prompted by the recent demonstration of the intestinotropic effects of GLP-2 and the possibility that GLP-2 could be used to treat human intestinal diseases. We therefore studied the pharmacokinetics after both iv and sc administration. The N-terminal sequences of GLP-1 and GLP-2 are very similar, viz. His-Ala-Glu- for GLP-1 and His-Ala-Asp- for GLP-2. Because of this and in agreement with the known substrate specificity of DPP-IV (11), one would, therefore, expect the two peptides to be metabolized at about equal rates. However, our results show that they are metabolized at strikingly dissimilar rates. Thus, where GLP-1 is metabolized intravascularly and never reaches a true steady state FIG. 6.In vitro degradation (n 4) of GLP-2-(1 33) in plasma (A) and blood (B) (f) and in the presence of the DPP-IV inhibitor, VP ( ). GLP-2 immunoreactivity is expressed as a percentage ( SEM) ofthe initial concentration. (which, in fact, renders calculations of clearance rates and elimination constants according to single compartment, first order kinetics meaningless), GLP-2 was metabolized much more slowly. The intravascular degradation of GLP-1 is partly due to the presence of soluble DPP-IV in plasma, from which GLP-1 is eliminated with a half-life of 20 min (9), and partly due to the presence of DPP-IV proteolytic activity at the cell surface of endothelial and blood cells (10). Compared to this, the degradation of GLP-2 must be described as slow, 8 h in plasma and about 3 h in blood. The complete protection of GLP-2 by the specific DPP-IV inhibitor, VP, showed that the slow degradation was, in fact, due to the presence of DPP-IV proteolytic activity, both soluble in plasma and on the surface of blood cells. Also, the whole body metabolism of GLP-2 was remarkably slower than that of GLP-1, with half-lives of 7 min and a MCR of only about 7 ml/kg min, a rather slow elimination rate for this family of peptides (the glucagon-secretin family), where, for instance, glucagon is eliminated with reported half-lives of 5 6 min and clearances of 9 13 ml/kg min (12). In agreement with the observation that DPP-IV is indeed responsible for the slow degradation in blood, the process leads to the formation of a truncated form of GLP-2, GLP- 2-(3 33), from which the two N-terminal amino acid residues are cleaved off. The metabolite GLP-2-(3 33) apparently has no biological effect (3). In blood and plasma, no other degrading activity was observed, as also indicated by the constant concentration of total GLP-2 measured throughout the

5 2888 HARTMANN ET AL. JCE&M 2000 Vol 85 No 8 incubation period. The same metabolite was formed in vivo after iv or sc injection of GLP-2, indicating that DPP-IV is responsible for the initial metabolism of this peptide in the intact organism also. The metabolite, in turn, was eliminated with a half-life of 27 min and a clearance of about 2 ml/ kg min. Also these values are in great contrast to the corresponding values for the initial metabolite of GLP-1, generated by DPP-IV-mediated catalysis. The GLP-1 metabolite, which is generally designated GLP-1-(9 36)amide, is eliminated with a half-life of 4 5 min, mainly by renal clearance (10). Whereas renal fractional uptake of GLP-1 ranges from 50 60% (in vivo) up to more than 90% (in vitro) (10) and therefore indicates that renal clearance must include peritubular uptake in addition to glomerular filtration, the clearance of the GLP-2 metabolite is equivalent to what could be accounted for by filtration alone ( 2 ml/kg min). Again, this indicates major differences in the patterns of metabolism for the two peptides. Endogenous GLP-2 is apparently subjected to DPP-IV-mediated degradation. Thus, 43% of total GLP-2 at peak concentrations of meal-stimulated GLP-2 secretion consisted of the metabolite (the corresponding figure for GLP-1 was 60%) (9). This result is in agreement with data presented by Brubaker et al. (4), and Xiao et al. (5), reporting about 50 55% metabolite in postprandial plasma samples. GLP-1 was recently demonstrated to be degraded by DPP-IV activity in the capillaries of the intestinal mucosa before the hormone had left the gut with the venous drainage (13), an observation that supported the hypothesis that part of the biological role of GLP-1 is to act as a paracrine activator of intestinal sensory nerves. Whether GLP-2 is degraded by similar local mechanisms and therefore can be assumed to exert similar local actions remains to be seen. By both iv infusion and sc injection, a considerable fraction of GLP-2 was present as the degradation product rather than the intact hormone. Nevertheless, compared to GLP-1, a much larger fraction of GLP-2 survived intact, particularly after sc injection, whereas 70% was present as intact peptide at a time in between the peak concentrations of the intact peptide and the metabolite. For GLP-1 the corresponding fraction was only 5 10% (14), and because of this, the therapeutic potential of GLP-1 can only be exploited if the peptide is infused continuously (15) combined with inhibitors of DPP-IV (16) or if structural modifications are introduced rendering the peptide insensitive to DPP-IV (17). Similarly, for GLP-2, a structurally modified, DPP-IV-resistant peptide was shown to be effective in rats, where DPP-IV activity seems to be so strong that the peptide is prevented from generating its intestinotropic effect (3). However, in humans less than a third of the GLP-2 is apparently degraded after sc injection, and it therefore seems likely that the intestinotropic potential of the peptide can be realized by the use of single sc injections. In agreement with this, we recently showed that the intestinal absorptive capacity of patients with intestinal failure due to short bowel syndrome could be significantly improved by 5 weeks of treatment with sc injections of 400 g GLP-2 twice daily (our unpublished studies). Probably, a protracted formulation of GLP-2 could turn out to be useful, but our results suggest that the possible beneficial effects of GLP-2 under conditions of intestinal mucosal damage or insufficiency could be examined by simple, sc injections of the natural peptide. Acknowledgments We gratefully acknowledge the technical assistance of Lene Albæk and Rigmor Holck. References 1. Holst JJ Enteroglucagon. Annu Rev Physiol. 59: Drucker DJ, Erlich P, Asa SL, Brubaker PL Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci USA. 93: Drucker DJ, Shi Q, Crivici A, et al Regulation of the biological activity of glucagon-like peptide 2 in vivo by dipeptidyl peptidase IV. Nat Biotechnol. 15: Brubaker PL, Crivici A, Izzo A, Ehrlich P, Tsai CH, Drucker DJ Circulating and tissue forms of the intestinal growth factor, glucagon-like peptide-2. Endocrinology. 138: Xiao Q, Boushey RP, Drucker DJ, Brubaker PL Secretion of the intestinotropic hormone glucagon-like peptide 2 is differentially regulated by nutrients in humans. Gastroenterology. 117: Mentlein R, Gallwitz B, Schmidt WE Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7 36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur J Biochem. 214: Wojdemann M, Wettergren A, Hartmann B, Holst JJ Glucagon-like peptide-2 inhibits centrally induced antral motility in pigs. Scand J Gastroenterol. 33: Holst JJ, Bersani M Assays for peptide products of somatostatin gene expression. In: Conn PM, ed. Methods in Neuroscience. San Diego: Academic Press; Deacon CF, Johnsen AH, Holst JJ Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J Clin Endocrinol Metab. 80: Deacon CF, Pridal L, Klarskov L, Olesen M, Holst JJ Glucagon-like peptide 1 undergoes differential tissue-specific metabolism in the anesthetized pig. Am J Physiol. 271:E458 E Mentlein R Dipeptidyl-peptidase. IV. CD26: role in the inactivation of regulatory peptides. Regul Pept. 85: Holst JJ Degradation of Glucagons. In: Henriksen JH, ed. Degradation of bioactive substances. Physiology and pathophysiology. Boca Raton: CRC Press; Hansen L, Deacon CF, Orskov C, Holst JJ Glucagon-like peptide-1-(7 36)amide is transformed to glucagon-like peptide-1-(9 36)amide by dipeptidyl peptidase IV in the capillaries supplying the L cells of the porcine intestine. Endocrinology. 140: Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L, Willms B, Holst JJ Both subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the NH2-terminus in type II diabetic patients and in healthy subjects. Diabetes. 44: Toft-Nielsen MB, Madsbad S, Holst JJ Continuous subcutaneous infusion of glucagon-like peptide 1 lowers plasma glucose and reduces appetite in type 2 diabetic patients. Diabetes Care. 22: Holst JJ, Deacon CF Inhibition of the activity of dipeptidyl-peptidase IV as a treatment for type 2 diabetes. Diabetes. 47: Deacon CF, Knudsen LB, Madsen K, Wiberg FC, Jacobsen O, Holst JJ Dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1 which have extended metabolic stability and improved biological activity. Diabetologia. 41: