The Role of Dipeptidyl Peptidase IV in the Cleavage of Glucagon Family Peptides

IN VIVO METABOLISM OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE-(1–38)*

Lan Zhu {ddagger}, Constantin Tamvakopoulos §, Dan Xie {ddagger}, Jasminka Dragovic §, Xiaolan Shen ¶, Judith E. Fenyk-Melody ¶, Keith Schmidt ||, Ansuman Bagchi ||, Patrick R. Griffin § **, Nancy A. Thornberry {ddagger} and Ranabir Sinha Roy {ddagger} {ddagger}{ddagger}

From the {ddagger}Metabolic Disorders, §Medicinal Chemistry, Comparative Medicine, and ||Applied Computer Science and Mathematics, Merck Research Laboratories, Rahway, New Jersey 07065

Received for publication, December 4, 2002 , and in revised form, March 20, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Dipeptidyl peptidase IV (DP-IV) is a cell surface serine dipeptidase that is involved in the regulation of the incretin hormones, glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). There is accumulating evidence that other members of the glucagon family of peptides are also endogenous substrates for this enzyme. To identify candidate substrates for DP-IV, a mass spectrometry-based protease assay was developed that measures cleavage efficiencies (kcat/Km) of polypeptides in a mixture, using only a few picomoles of each substrate and physiological amounts of enzyme in a single kinetic experiment. Oxyntomodulin and the growth hormone-(1–43) fragment were identified as new candidate in vivo substrates. Pituitary adenylate cyclase-activating polypeptide-(1–38) (PACAP38), a critical mediator of lipid and carbohydrate metabolism, was also determined to be efficiently processed by DP-IV in vitro. The catabolism of exogenously administered PACAP38 in wild type and DP-IV-deficient C57Bl/6 mice was monitored by tandem mass spectrometry. Animals lacking DP-IV exhibited a significantly slower clearance of the circulating peptide with virtually complete suppression of the inactive DP-IV metabolite, PACAP-(3–38). These in vivo results suggest that DP-IV plays a major role in the degradation of circulating PACAP38.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Dipeptidyl peptidase IV (DP-IV)1 is a ubiquitous serine amino-terminal dipeptidase that has been implicated in the degradation of several peptides and hormones (1). The enzyme is identical to the T-cell surface antigen CD26 (2). Interest in DP-IV as a target for type 2 diabetes has arisen due to its role in the proteolytic inactivation of the incretin GLP-1-(7–36)-amide (3, 4), which has a clearly established role in glucose homeostasis in humans (5). Removal of the amino-terminal His-Ala dipeptide by DP-IV generates GLP-1-(9–36)-amide, which is unable to elicit glucose-dependent insulin secretion from the islets (6). Evidence that DP-IV inhibition may be therapeutically beneficial has been provided by mice engineered to be deficient in the cd26 gene (7), which exhibit elevated levels of endogenous GLP-1 and reduced glucose excursion in an oral glucose tolerance test. Indeed, DP-IV inhibitors increase endogenous levels of active GLP-1 and improve glucose tolerance in diabetic animals (812) and human subjects (13).

A DP-IV inhibitor improves glucose tolerance in GLP-1 receptor-deficient mice, but not in DP-IV-deficient mice (7), indicating that substrates beyond GLP-1 are important for efficacy in glucose control. One likely candidate is GIP, another incretin in the glucagon family of peptides, which is also regulated by DP-IV in vivo (3, 14). DP-IV has also been implicated in functions beyond glucose homeostasis; specifically, DP-IV-deficient Fischer rats gain less weight than wild type animals on a high fat diet (15), and DP-IV-deficient mice are also resistant to diet-induced obesity,2 suggesting that the enzyme may be involved in the regulation of additional bioactive peptides that control feeding behavior. In this regard, a number of peptides have been shown to be efficiently processed in vitro, including glucagon family peptides, neuropeptides, and chemokines.

To better understand the biological role of DP-IV and to determine the potential therapeutic benefit and safety issues that may be associated with DP-IV inhibition, a thorough understanding of endogenous substrate specificity is required. Numerous strategies have been devised to determine protease substrate specificity and provide insight into their biological roles. For serine- and cysteine-type proteases, "positional scanning" combinatorial libraries of peptide substrates are often effective in identifying residue specificities proximal to the scissile amide bond and revealing consensus amino acid sequences for substrate recognition (16, 17). These data can facilitate the identification of potential substrates from protein sequence databases, although rapid assays to quantitatively measure cleavage efficiencies have been lacking to date.

We have been evaluating mass spectrometry (MS) as the method of choice for developing sensitive and quantitative assays of protease activity. MS techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (18, 19) and electrospray ionization-liquid chromatography/mass spectrometry (ESI-LC/MS) (20) can facilitate quantitative measurements of polypeptide cleavage based upon the specific changes in m/z that accompany each proteolytic event (21). MS is of particular utility in assays of amino/carboxypeptidase or dipeptidase activity involving the cleavage of only one or two residues, where the quantitation of substrate and product peptides can be limited by the poor resolution and low sensitivity of conventional separation techniques such as high performance liquid chromatography and gel electrophoresis. Here, we describe a general MS-based approach for screening libraries of bioactive polypeptides against a target protease, measuring specificity constants of cleavage (kcat/Km) from a single kinetic run, and evaluating the effects of genetic ablation (or inhibition) of protease activity on the in vivo metabolism of exogenously administered candidate substrates in mice. These studies have led to the identification of oxyntomodulin and growth hormone-(1–43) as novel in vitro substrates for DP-IV and provide compelling evidence that PACAP38 is regulated by this enzyme in vivo.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
DP-IV Assay—The panel of 15 hormones and neuropeptides (purchased from Anaspec, Synpep, Bachem, and American Peptide Company) was assayed for DP-IV processing in groups of 5–6 peptides each. A validated physiological DP-IV substrate (GLP-1-(7–36)) was included in each assay as a positive control. Peptide mixtures were compiled so as to avoid any overlap in the mass spectra of prominent charge states for all species present (including potential products of DP-IV-mediated cleavage). A charge state conflict analysis algorithm was implemented using combinatorial optimization to automate the grouping of peptides in each assay while preventing overlaps in their mass spectra with respect to a minimally acceptable mass/charge separation (typically 6 m/z). Equimolar mixtures containing 250 nM of each peptide in assay buffer (100 mM HEPES, pH 7.5, 0.05 mg/ml bovine serum albumin) were incubated at 37 °C with soluble recombinant human DP-IV (500 pM, corresponding to 0.96 milliunits) in a volume of 2 ml (one unit of enzyme activity is defined as the amount of enzyme required to hydrolyze 1 µmol/min of 50 µM Gly-Pro-7-amido-4-methyl-coumarin in 100 mM HEPES buffer containing 0.1 mg/ml bovine serum albumin, pH 7.5, 37 °C). The kinetic parameters for substrate cleavage by the recombinant enzyme (comprising residues 29–766) were indistinguishable from those of wild type DP-IV. At appropriate intervals (0–120 min after the addition of enzyme), 150-µl aliquots were withdrawn and quenched with 2 µl of 6 N HCl. A 90-µl volume of each quenched aliquot was analyzed by ESI-LC/MS, and kinetic progress curves for each processed peptide were obtained as described below.

LC/MS Analysis of in Vitro Substrates—The MS instrumentation consisted of an Agilent 1100 HPLC and autosampler connected to a Thermo Finnigan LCQ ion trap mass spectrometer equipped with a standard electrospray ionization (ESI) source. Sample aliquots containing 20–80 pmol of each peptide analyte were injected on a 100 x 1 mm HAISIL 300 C18 column (Higgins Analytical) at a flow rate of 150 µl/min (solvent A = water, 0.03% trifluoroacetic acid, solvent B = acetonitrile, 0.03% trifluoroacetic acid, initial equilibration at 5% B, linear gradients of 3.75% B/min over 3–15 min and 2.1% B/min over 15–37 min after injection). The flow was diverted to waste for 5.2 min before the column eluate was introduced to the electrospray source. All samples were analyzed in positive ion mode with the source voltage set at 4500 V and the capillary voltage at 5 V. The heated capillary was maintained at 200 °C. Full scan data were collected as centroid spectra (400–1800 m/z). Extracted ion chromatograms (Fig. 1) were generated for each peptidyl species using prominent charge states that were empirically determined for each substrate and product species (typically [M+3H]+3 and/or [M+4H]+4, see Table I). MS acquisition, peak integration, and data analysis were performed using Xcalibur software version 1.2 (Thermo Finnigan).



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FIG. 1.
Base peak ESI-LC/MS chromatogram for a sample aliquot obtained at 30 min from a typical DP-IV assay mixture (top trace). Each time point in the chromatogram reflects the abundance of the most intense signal in the range 1000–1400 m/z of the corresponding mass spectrum. Extracted ion chromatograms for 6 of the 11 peptide species present in the assay (GHRH-(1–29), GLP-1-(7–36), PACAP-(1–38), and their corresponding des-dipeptidyl DP-IV metabolites) are also shown.

 

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TABLE I
MS parameters and kinetic data for the bioactive peptides investigated in this study

Equimolar peptide mixtures were incubated with soluble recombinant human DP-IV and assayed for cleavage by ESI/LC-MS. Kinetic progress curves were generated using peak areas of substrate and product ions that were normalized for differences in ionization efficiencies using previously determined response factors as described under "Experimental Procedures." Specificity constants were determined from first-order fits to the kinetic data using the integrated Michaelis Menten equation. Literature values, where available, are listed for comparison. ND, not detected; NA, not applicable.

 

Measurement of Response Factors—Quantitative measurements of substrate conversion were made by ratiometric analysis of substrate (S) and product (P) peak areas in the extracted ion chromatograms of each species. For an accurate analysis, instrument response factors were determined for every substrate-product pair. Peptide standards corresponding to the DP-IV cleavage products of each identified in vitro substrate were obtained by incubating the latter (1 µM in 528 µl of assay buffer) with excess enzyme (100 nM, 192 milliunits) at 37 °C for 3 h. The reactions were subsequently quenched with 10 µl of 0.2 N HCl. Under these conditions, even relatively inefficient substrates of DP-IV (kcat/Km < 104M–1s1) were cleaved to >95% completion. Peptide mixtures containing 20 pmol of each substrate and 20–80 pmol of the corresponding DP-IV product in 100 µl of assay buffer were prepared using a grouping of peptides identical to that of the in vitro screen. Aliquots of these mixtures (90 µl) were analyzed by ESI-LC/MS. The m/z of predominant charge states for each substrate and product peptide (see Table I) was used to generate extracted ion chromatograms from which integrated peak areas were measured. The ratio of the product:substrate peak areas was plotted against the known ratio of product:substrate in the mixtures (see Fig. 2). The slope of the resulting linear plot was used as a response factor to correct the measured peak area of product peptide in the kinetic assays (Pcorr = (Pobs)/(slope)).



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FIG. 2.
Measurement of instrument response factors for GHRH-(1–29), PACAP38, GLP-1-(7–36), and their DP-IV metabolites. The ratio of product:substrate (P/S) determined by mass spectrometry for each peptide is plotted against the actual ratio present in a series of standard mixtures, and the slope of the plot is used as a correction factor in the kinetic assays (see "Experimental Procedures").

 

Calculation of Specificity Constants—Progress curves for the cleavage of individual substrates (see Fig. 3) were generated from extracted ion chromatograms using peak areas of substrate and product, percent conversion = ((Pcorr)/(Pcorr + Sobs)) x 100. For data obtained from repeat assays, the standard error of this measurement was typically <15% of the mean. The specificity constant (kcat/Km) for cleavage of a particular substrate was directly calculated from a first order fit of the enzyme kinetics: kobs = (kcat)/(Km) · Eo, where kobs is the first order rate constant and Eo is the total enzyme concentration (22).



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FIG. 3.
Typical kinetic progress curves for DP-IV substrates, generated using the multiplexed MS-based assay. Equimolar mixtures of 5–6 glucagon family peptides were incubated with enzyme, and aliquots were sampled by LC/MS at appropriate intervals. The extent of cleavage for each peptide was calculated by ratiometric analysis of the corresponding product and substrate ions. The data followed first order kinetics, and kcat/Km was calculated from the observed rate constant as described under "Experimental Procedures."

 

In Vivo Metabolism of PACAP38 —Animal procedures were approved by the Merck-Rahway Institutional Animal Care and Use Committee and performed in accordance with the Guide for the Care and Use of Laboratory Animals (47). The generation of DP-IV-deficient (CD26–/–) C57Bl/6 mice has been reported previously (7). These animals were obtained from INSERM, Paris, France. A solution of PACAP38 was prepared in saline (0.4 mg/ml) for intravenous dosing. Two groups of naive 9-week-old male C57Bl/6mice (wild type and DP-IV-deficient, respectively) were administered an intravenous bolus of PACAP38 (2 mg/kg). Blood samples were collected by cardiocentesis after CO2 euthanasia from 4–6 animals at each time point (1–5 min after dose). Blood from individual animals was placed immediately into chilled EDTA-coated tubes containing a DP-IV inhibitor (2S,3S-isoleucyl thiazolidide hemi-fumarate salt, 100 µM, obtained from Heumann Pharma) and aprotinin (0.04 mg/ml, purchased from Sigma) to prevent further degradation of PACAP38 and its metabolites. The samples were centrifuged at 6000 rpm for 10 min at 4 °C. Plasma was transferred to a 96-deep-well plate and stored at –70 °C until extraction and analysis.

Plasma Extraction and Sample Preparation—PACAP38 and related peptides were extracted from mouse plasma using Oasis HLB 10 mg 96-well solid phase extraction plates (Waters). Plasma aliquots (100 µl) were diluted with 100 µl of water, acidified with 100 µl of 0.1 N acetic acid, and loaded on the extraction plate (preconditioned as per the manufacturer's instructions). Plasma samples of known peptide concentrations (for calibration curves) were prepared by spiking mouse plasma with 25 µl of a stock solution containing equimolar mixtures of PACAP-(1–38) and PACAP-(3–38) (synthesized by Invitrogen) in 10% acetonitrile, 0.1% trifluoroacetic acid. After loading all samples, the extraction plate was washed with 0.5 ml of 5% methanol. Peptides were eluted with 50% acetonitrile, 0.1% trifluoroacetic acid (2 x 0.25 ml), and the eluate was collected in a 96-well plate. The eluted samples were dried at 40 °C under a stream of nitrogen. Recoveries of PACAP-(1–38) and PACAP-(3–38) were typically 65 and 78%, respectively. Dried samples were reconstituted in 190 µl of an aqueous solution containing 5% acetonitrile, 5% 1-propanol, and 0.03% trifluoroacetic acid. The samples were transferred to 0.5-ml microcentrifuge tubes and centrifuged at 13,000 rpm for 5 min. Supernatants (180 µl) were transferred to plastic LC vials, and 45-µl aliquots were analyzed in triplicate by tandem mass spectrometry.

Quantitation of Peptides by Tandem Mass Spectrometry—Extracted plasma samples were injected on a Symmetry-300 C4 column (2.1 x 50 mm, Waters) at a flow rate of 200 µl/min (solvent A = water, 0.03% trifluoroacetic acid, solvent B = 1:1 acetonitrile:1-propanol, 0.03% trifluoroacetic acid, initial equilibration at 10% B, linear gradients of 2.2% B/min over 1–10 min and 10% B/min over 12–17 min after injection). The flow was diverted to waste for 4.5 min before introducing the column eluate to the ESI source. All samples were analyzed in positive ion mode (centroid spectra) with the source voltage at 4.5 kV, capillary voltage at 42 V, and capillary temperature at 200 °C. For the analysis of intact PACAP38, the parent ion at m/z 1134.6 (corresponding to [M+4H]+4) yielded three significant fragment ions at m/z 1045.5 , 1049.9 , and 1221.6 , with 30% relative collision energy (see Fig. 4). For the PACAP-(3–38) metabolite, the parent ion at m/z 1437.7 (corresponding to [M+3H]+3) yielded fragment ions at m/z 1126.4 and 1431.5 ([M-H2O+3H]+3) with 30% relative collision energy. The total integrated peak area for these major fragment ions was used to quantitate the corresponding peptides, using standard curves generated from plasma samples of known peptide concentrations (see Fig. 5). The standard error for repeat measurements was <15% of the mean.



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FIG. 4.
Product ion spectra for PACAP38 (PACAP-(1–38), precursor ion at m/z 1134.6), and the corresponding DP-IV metabolite PACAP-(3–38) (precursor ion at m/z 1437.7). Fragment ions that were used to quantitate each species are labeled.

 


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FIG. 5.
Calibration curves (plotted as mean + S.E., n = 3) for the quantitation of intact PACAP38 (PACAP-(1–38)) and the inactive DP-IV metabolite PACAP-(3–38) in mouse plasma. Plasma samples were spiked with increasing amounts of each peptide (0–6600 ng/ml) and extracted as described under "Experimental Procedures." The extracted peptides were quantified by LC/MS-MS using the total integrated area of major fragment ions obtained in the MS-MS spectrum of each species (Fig. 4).

 


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
A Multiplexed MS-based Kinetic Assay for Proteases—Endogenous levels of circulating bioactive peptides are generally 3–6 orders of magnitude lower than the micromolar Km values for substrates of typical proteases. At such low concentrations, the rate of proteolysis is determined by the specificity constant (kcat/Km), which represents the apparent second order rate constant for substrate cleavage. Measurement of this kinetic parameter allows substrates to be ranked in order of processing efficiency, with kcat/Km > 105 M–1 s1 being characteristic of efficient cleavage by a particular protease. The in vitro substrate specificity of DP-IV for glucagon family peptides was determined using a mass spectrometry assay that provided a direct measurement of the specificity constant for each substrate. This approach was used to rapidly identify candidate DP-IV substrates for subsequent in vivo analysis. Although MS-based assays of DP-IV activity have been reported previously (23, 24), our approach quantitatively measures kcat/Km for multiple substrates in a single kinetic experiment. For the glucagon family peptides described in this work, mixtures of 5–6 peptides were assayed simultaneously, allowing automated LC/MS analysis of all 16 peptides to be completed in 36 h. The increased throughput of this method as compared with standard HPLC-based assays is further illustrated by our subsequent screen of a library of ~150 neuropeptides, hormones, and chemokines, and measurement of kcat/Km for all identified in vitro DP-IV substrates, which was completed in less than a week.3

Measurement of Specificity Constants by MS—Since mass spectrometry measures analyze concentrations indirectly, appropriate precautions were taken to ensure that the measured ion signals reflected accurate estimates of substrate turnover. These steps included the measurement of instrument response factors for each substrate-product pair (Fig. 2 and Table I) and operation within the linear dynamic range of the assay (0–10,000 ng/ml). The high sensitivity of ESI-LC/MS allowed kinetic data to be collected at relatively low (nanomolar) concentrations of substrate as compared with the Michaelis constant (Km), which is typically in the high micromolar range for peptides that are cleaved by DP-IV in vitro (14, 2527). Each potential substrate could therefore effectively compete for the enzyme active site without a bias toward high affinity substrates. Under these conditions of low substrate concentrations ([S]< 0.1 Km), the specificity constant for cleavage could be directly calculated from a single progress curve for each substrate (Fig. 3) using the integrated Michaelis-Menten equation (22). Repeat analyses confirmed that kinetic measurements were unaffected by the composition of peptides in the assay mixture. Identical results were also obtained when substrates were incubated individually with the peptidase.

Efficient in Vitro Cleavage of P1-Pro/Ala-containing Peptides—Table I lists the bioactive peptides that were screened in this study, ranked in descending order of their measured specificity constants for cleavage by DP-IV. The efficient processing of GHRH-(1–44), GRP, GLP-1-(7–36), peptide histidine methionine, GLP-2, and GIP (kcat/Km ≥ 105 M–1 s1) was consistent with previous reports of their in vitro processing by DP-IV (23, 25, 27, 28), which typically cleaves the amide bond after a penultimate amino-terminal Pro or Ala residue (the P1 site, according to the nomenclature of Berger and Schechter (29)). For GRP, sequential processing of the Val-Pro-Leu-Pro amino terminus produced the intermediate GRP-(3–27), which was further cleaved to GRP-(5–27) as has been reported previously (25, 27). Rate constants for these cleavages were determined from the progress curves for each species using standard equations (30). In addition, oxyntomodulin, a glucagon family peptide that inhibits food intake when administered centrally in rats (31), and the pituitary growth hormone fragment GH-(1–43), which exhibits insulin potentiating activity in obese or diabetic rodents (32, 33), were established as novel in vitro substrates of DP-IV.

Differential Processing of P1-Ser-containing Peptides in Vitro—In general, where comparisons can be made, the specificity constants determined in this study are in good agreement with values reported previously in the literature. The exceptions in this regard are data for the P1-Ser-containing peptides glucagon, PACAP27, and PACAP38. The specificity constant determined for glucagon was 10-fold lower than that reported using MALDI-TOF mass spectrometry (34). Indeed, most P1-Ser peptides were either inefficient substrates in our assay (kcat/Km < 105 M–1 s1, PACAP27, secretin) or not detectably cleaved by DP-IV (VIP, VIP-(1–12)). Similar results have been reported for P1-Ser-containing analogs of GHRH. Substitution of Ala2 by Ser in Ala15-GHRH-(1–29) led to a 400-fold decrease in kcat/Km (26), and first order rate constants for DP-IV cleavage of Ser-containing GHRH analogs were typically 25-fold lower than that of the corresponding P1-Ala substrates (35). Unexpectedly, PACAP38 and oxyntomodulin, both of which contain basic carboxyl-terminal extensions relative to PACAP27 and glucagon, respectively, were processed 15-fold and 8-fold more efficiently than their shorter isoforms in each case. Such differential processing indicates that residues remote from the scissile bond can modulate the efficiency of DP-IV cleavage, as was further demonstrated by a modest (1.5-fold) increase in kcat/Km for GHRH-(1–44) cleavage relative to GHRH-(1–29).

Differential in vitro processing of the pancreatic neuropeptides PACAP38 and PACAP27 by DP-IV has been described previously (27). The reported specificity constants of these peptides, however, were an order of magnitude lower than the values obtained in this work. Although the reason for these discrepancies is unknown, the evidence linking PACAP to metabolic control (see Ref. 38) and the requirement of an intact amino terminus for PACAP bioactivity (36, 37) prompted us to investigate the potential role of DP-IV in the in vivo catabolism of PACAP38.

In Vivo Metabolism of PACAP Peptides Monitored by Tandem Mass Spectrometry—Increased steady-state levels of PACAP38 in DP-IV-deficient mice would provide conclusive evidence of a physiological role for DP-IV in the regulation of this neuropeptide. Unfortunately, measurements of endogenous PACAP38 in mice were not possible for several reasons. First, specific immunoassays that discriminate between intact PACAP-(1–38) and its DP-IV metabolite PACAP-(3–38) were not available. Second, since PACAP38 functions as a non-cholinergic sympathoadrenal neurotransmitter (38), local stabilization of the neuropeptide at nerve ganglia in DP-IV–/– mice would probably not be reflected in assays that measured circulating peptide. Finally, PACAP38 binds the plasma protein ceruloplasmin (39), which further complicates the quantitation of endogenous peptide, estimated to be ~25–100 pM (40). For a typical 100-µl mouse plasma sample, this low abundance of endogenous PACAP38 would require attomole sensitivity of detection, which is approximately 3 orders of magnitude lower than what can be typically achieved for extracted plasma peptides using conventional ion trap mass spectrometers. We therefore adopted an alternate approach that compared the metabolic fates of exogenously administered PACAP38 in wild type and DP-IV-deficient C57Bl/6 mice.

Measurement of intact PACAP-(1–38) and the inactive DP-IV metabolite PACAP-(3–38) in mouse plasma was accomplished using tandem mass spectrometry (MS-MS) following intravenous dosing of animals with supraphysiological levels of neuropeptide. Although the concentration of infused peptide was much higher than that of endogenous PACAP38, the metabolic fate of this neuropeptide when exposed to a milieu of competing peptidases in vivo (including DP-IV) would still be expected to provide valuable insight regarding the enzyme(s) responsible for its regulation. Indeed, similar infusion experiments using radioiodinated GLP-1 and GIP in DP-IV-positive and -negative rats provided among the first in vivo evidence for inactivation of these incretins by the enzyme (3).

The enhanced selectivity and improved sensitivity of tandem mass spectrometry is ideal for the quantitation of peptides in biological samples, where the complexity of the matrix otherwise results in unacceptable chemical noise. The MS-MS experiments for PACAP-(1–38) and PACAP-(3–38) monitored the production of characteristic fragment ions from a specific parent ion following collision-activated dissociation (Fig. 4). Despite the high abundance of ceruloplasmin in plasma (1.7 µM, (39)), an ESI-LC/MS-MS assay was developed that simultaneously measures PACAP-(1–38) and PACAP-(3–38) with a limit of quantitation of ~45 nM (200 ng/ml, Fig. 5). The sensitivity and dynamic range of this assay confirmed that the protocols developed for sample preparation were effective in separating PACAP38 from bound proteins and allowed the metabolism of infused PACAP38 in C57Bl/6 mice to be monitored over a period of several minutes.

PACAP38 but Not PACAP27 Is Processed in Vivo by DP-IV— As illustrated in Fig. 6, exogenous PACAP38 was rapidly degraded in wild type C57Bl/6 mice with concomitant formation of the inactive DP-IV metabolite PACAP-(3–38), consistent with our in vitro observations. In contrast, formation of PACAP-(3–38) was virtually absent in age- and gender-matched DP-IV–/– mice, and clearance of intact PACAP38 from the circulation was significantly slower in these animals (presumably by renal filtration, as has been reported for GLP-1-(41)). Significantly, the total concentration of PACAP38 and its DP-IV metabolite in wild type mice was similar to levels of intact PACAP38 in DP-IV–/– animals at each time point (Fig. 6), consistent with DP-IV playing a major role in the degradation of circulating PACAP38. A similar experiment monitoring the catabolism of exogenous PACAP27 in wild type C57Bl/6 mice found no evidence of the corresponding des-dipeptidyl PACAP-(3–27) metabolite (data not shown), consistent with the ~15-fold lower specificity constant of this peptide relative to PACAP38. Nevertheless, since PACAP38 constitutes ~90% of the endogenous PACAP pool, metabolic inactivation by DP-IV would be expected to regulate overall PACAP bioactivity in vivo.



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FIG. 6.
In vivo metabolism of exogenous PACAP38 in wild type (+/+) and DP-IV-deficient (–/–) C57Bl/6 mice. An intravenous bolus of neuropeptide (2 mg/kg) was administered at 0 min. Clearance of intact PACAP-(1–38) from the circulation was significantly slower in –/– mice (•) than in +/+ mice ({circ}) (*, p < 0.05; **, p < 0.01, unpaired t test). Formation of the inactive DP-IV metabolite PACAP-(3–38) was observed in +/+ animals ({square}) but was suppressed in –/– mice ({blacksquare}). At each time point, the total concentration of intact PACAP38 and degraded PACAP-(3–38) in +/+ mice ({triangleup}) was similar to the concentration of intact PACAP38 in –/– animals (•). All data are plotted as mean + S.E., n = 4–6.

 

The Metabolic Effects of DP-IV Inhibition May Involve Stabilization of Multiple Substrates—Improvements in metabolic control obtained upon inhibition of DP-IV in human subjects and diabetic animals have been classically attributed to stabilization of the incretins GLP-1 and GIP. Although other bioactive peptides are cleaved by DP-IV in vitro, most research on DP-IV-mediated peptide processing has been conducted without attention to turnover efficiency (kcat/Km) and biologically relevant enzyme levels. A critical evaluation of potential and purported substrates, including members of the glucagon/VIP/secretin superfamily, was therefore undertaken to measure specificity constants in vitro under physiologically relevant conditions and to identify new candidate substrates for in vivo validation. Oxyntomodulin and GH-(1–43) were identified as novel in vitro substrates of DP-IV, but the unexpectedly efficient in vitro processing of the incretin PACAP38, a critical regulator of lipid and carbohydrate metabolism, led us to focus on the metabolism of this neuropeptide in vivo. Infusion studies in mice recapitulated the in vitro cleavage pattern of PACAP38 by DP-IV (except in DP-IV-deficient animals), although the substrate was exposed to multiple proteases. Furthermore, a closely related but relatively inefficient substrate (PACAP27) was not demonstrably processed by DP-IV in vivo, although infused at supraphysiological levels. These results suggest that DP-IV may participate in the inactivation of endogenous PACAP38.

Although a number of functions have been attributed to PACAP, recent studies with PACAP receptor-deficient mice and PACAP-deficient mice suggest a role for this neuropeptide in glucose control, lipid metabolism, and adaptive thermogenesis. PACAP signals via three receptor subtypes (PAC1, VPAC1, and VPAC2) (42). The PAC1 receptor is specific for PACAP; VPAC1 and VPAC2 are shared with VIP. PAC1-deficient mice display impaired glucose tolerance and fed hyperinsulinemia (43). PACAP-deficient mice have an impaired response to insulin-induced hypoglycemia and a 2–3-fold increase in serum triglycerides and cholesterol (44). Loss of PACAP in these animals also results in inadequate heat production due to reduced norepinephrine stimulation of brown adipose tissue (45). Our data on the in vivo degradation of exogenous PACAP38 by DP-IV therefore suggest that inhibitors of this peptidase may have therapeutic utility extending beyond improvements in glucose control, specifically in the regulation of lipid metabolism and/or adaptive thermogenesis. In this regard, we have noted that DP-IV-deficient mice maintained on a high fat diet exhibit reduced adiposity. Furthermore, chronic administration of the DP-IV inhibitor FE999011 in male Zucker diabetic fatty rats reportedly prevents a rise in circulating free fatty acid, in addition to postponing the development of diabetes (11). It is possible that increased endogenous pools of intact PACAP38 may mediate some of these effects, although stabilization of oxyntomodulin, GH-(1–43), or of an as yet undiscovered DP-IV substrate may also contribute to the improved metabolic control. Final confirmation of the in vivo regulation of such substrates by DP-IV will require quantitation of the corresponding endogenous peptides in the setting of enzyme inhibition or ablation. Encouragingly, rapid advances in sensitive MS instrumentation increase the feasibility of measuring picomolar plasma peptides in the near future, which should facilitate development of the requisite assays. Clearly, additional studies of substrate specificity will also be required to obtain an intimate understanding of the overall biological role of DP-IV and the prospects and liabilities of enzyme inhibition.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Present address: ExSAR Corporation, 11 Deer Park Dr., Ste. 103, Monmouth Junction, NJ 08852. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Metabolic Disorders, Mail code RY50G-241, Merck Research Laboratories, Rahway, NJ 07065. Tel.: 732-594-3132; Fax: 732-594-3664; E-mail: ranabir{at}merck.com.

1 The abbreviations used are: DP-IV, dipeptidyl peptidase IV; ESI-LC/MS, electrospray ionization-liquid chromatography/mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MS-MS, tandem mass spectrometry; GH, growth hormone; GHRH, growth hormone releasing hormone; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucagon-like peptide; GRP, gastrin releasing peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; PHM, peptide histidine methionine; VIP, vasoactive intestinal peptide; S, substrate; P, product. Back

2 Bei Zhang, personal communication. Back

3 S. Jagpal, N. A. Thornberry, and R. Sinha Roy, manuscript in preparation. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Barbara Leiting for providing us with recombinant soluble DP-IV and Dr. Nathan Yates for assistance with the MS instrumentation.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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