Metformin effects on dipeptidylpeptidase iv degradation of glucagon-like peptide-

Biochemical and Biophysical Research Communications 291, 1302–1308 (2002)
doi:10.1006/bbrc.2002.6607, available online at http://www.idealibrary.com on Metformin Effects on Dipeptidylpeptidase IV Degradationof Glucagon-like Peptide-1 Simon A. Hinke,* Kerstin Ku ¨ hn-Wache,† Torsten Hoffmann,† Raymond A. Pederson,* Christopher H. S. McIntosh,* and Hans-Ulrich Demuth†,1†Probiodrug Research, Biocenter, Weinbergweg 22, D-06120 Halle (Saale), Germany; and*Department of Physiology, University of British Columbia, Vancouver, Canada V6T 1Z3 Received February 14, 2002 States of America, and similar projections are made There is current interest in the use of inhibitors of
worldwide (1). Of those diagnosed with diabetes melli- dipeptidyl peptidase IV (DP IV) as therapeutic agents
tus, it is thought that type 2 diabetes (T2D), defined to normalize glycemic excursions in type 2 diabetic
primarily by peripheral insulin resistance with concur- patients. Data indicating that metformin increases the
rent hyperglycemia, accounts for ninety to ninety-five circulating amount of active glucagon-like peptide-1
percent of diagnosed diabetic patients (1). Therapies (GLP-1) in obese nondiabetic subjects have recently
for T2D include insulin injection and various oral phar- been presented, and it was proposed that metformin
maceuticals (sulfonylureas, metformin, acarbose, and might act as a DP IV inhibitor. This possibility has
certain glitazones), however, resistance to monothera- been investigated directly using a number of in vitro
pies as the disease progresses usually results in the methods. Studies were performed on DP IV enzyme
necessity of combinatorial treatment in order to im- from three sources: 20% human serum, purified por-
prove blood glucose levels (2). As such, there is added cine kidney DP IV, and recombinant human DP IV.
pressure on the pharmaceutical industry to develop Inhibition of DP IV hydrolysis of the substrate Gly-
more potent forms of existing therapies and new oral Pro-pNA by metformin was examined spectrophoto-
agents with novel cellular targets that can be used as metrically. Effects of metformin on GLP-1
monotherapies or in combination with other antidia- radation were assessed by mass spectrometry. In ad-
dition, surface plasmon resonance was used to estab-
betic drugs (3).
lish whether or not metformin had any effect on GLP-
One such novel molecular target with potential an- interaction with immobilized
tihyperglycemic effects is the ubiquitous proteolytic porcine or human DP IV. Metformin failed to alter the
enzyme, dipeptidyl peptidase IV (DP IV, or known as kinetics of Gly-Pro-pNA hydrolysis or GLP-1 degrada-
CD26 to immunologists; EC3.4.14.5). The unique prop- tion tested according to established methods. Surface
erty of DP IV with respect to diabetes mellitus is that plasmon resonance recordings indicated that both
it is the primary enzyme responsible for degradation of and GLP-1
show micromolar affinity
the incretins in vivo (4). Incretins are the hormonal (K ) for DP IV, but neither interaction was influenced
arm of the enteroinsular axis, the link between the gut by metformin. The results conclusively indicate that
and the endocrine pancreas (5). Glucose-dependent in- metformin does not act directly on DP IV, therefore
sulinotropic polypeptide (GIP) and amino-terminally alternative explanations for the purported effect of
truncated glucagon-like peptide-1 (GLP-1 metformin on circulating active GLP-1 concentrations
the only hormones which have been proven to fulfill the must be considered.
2002 Elsevier Science (USA)
requirements to be defined as an incretin: they are Key Words: incretin; entero-insular axis; CD 26; DPP
released into the blood stream in response to luminal IV; MALDI–TOF mass spectrometry; BIAcore; surface
nutrients, and act to augment nutrient-induced insulin release in a glucose-dependent fashion (6). Mentlein etal. (7) first showed that GIP and GLP-1 were sub-strates for DP IV in vitro, and shortly thereafter, in Derangement of glucose homeostasis affects approx- vivo degradation was also demonstrated (4). It was imately six percent of the inhabitants of the United Pauly and colleagues who first postulated the link be-tween the possible benefits of DP IV inhibition and glycemic control due to enhancement of the incretin To whom correspondence should be addressed. Fax: ⫹49-345- effect (8). The hypothesis that DP IV inhibition would 0006-291X/02 $35.00 2002 Elsevier Science (USA)All rights reserved.
Vol. 291, No. 5, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS improve glucose tolerance was later shown to be cor- (purified porcine) and 32.4 units/mg (recombinant human; rhuman).
rect in both Wistar rats and diabetic fatty Zucker rats One unit of DP IV activity is defined as the release of 1.0 ␮mol/lnitroaniline (yellow product) per minute measured spectrophoto- (9, 10). These findings have been corroborated by sim- metrically at 390 nm under standard conditions (defined below).
ilar studies in mouse, rat and pig (11–13).
Human serum was obtained from healthy donors, pooled and stored Metformin is a derivative of the antidiabetic bigua- at ⫺20°C until use, described previously (19).
nide alkaloids found in French lilac (Galeg officinalis), Effect of metformin on DP IV hydrolysis of GP-pNA. a medieval treatment for diabetes (14). It has been were carried out under standard conditions: 30°C in pH 7.6 40 commercially available since the 1950s, and is com- mmol/l HEPES (Sigma-Aldrich) buffer containing 0.4 mmol/l H-Gly- monly used worldwide as an initial monotherapy for Pro-4-nitroaniline, and 2.5 mU of DP IV (porcine or rhuman) or 20% newly diagnosed T2D patients, as it is equally effective human serum. Metformin (1,1-dimethylbiguanide; Sigma-Aldrich)was added over the concentration range of 0 to 100 ␮mol/l. Nitro- as sulfonylurea treatment (2, 14). However, metformin aniline production was monitored using a HTS 7000⫹ microplate was not available for clinical use in the United States reader (PerkinElmer, U ¨ berlingen, Germany).
until 1995 (15). The specific molecular target of it is Effect of metformin on DP IV hydrolysis of GLP-1[7-36NH2] using still unknown, although biguanides generally act to MALDI-TOF MS. Similar to spectrophotometric studies, matrix- sensitize peripheral tissues to insulin action (in partic- assisted laser-desorption ionization time of flight mass spectrometry ular skeletal muscle) and inhibit hepatic gluconeogen- (MALDI-TOF MS) experiments were carried out at 30°C at pH 7.6, esis and glycogenolysis (2, 3, 14). Notably, unlike the but in 0.1 mol/l Tris/HCl (Sigma-Aldrich) buffer with 12 ␮mol/lGLP-1 incretins, metformin does not improve glucose toler- The degradation fate of GLP-1 was measured by monitoring the signal intensity of the pseudomolecular ion peaks of ance via an increase in circulating insulin levels, im- GLP-1[7-36NH2] ([M ⫹ H]⫹ ⫽ 3299.7) and GLP-1[9-36NH2] ([M ⫹ H]⫹ ⫽ 3090.4) plicating other modes of action.
versus time when incubated with 2.5 mU DP IV (porcine or rhuman) or Recently, data were presented demonstrating the 20% human serum, with or without metformin (0 –1 mmol/l). The mass effect of metformin on plasma active (amino-terminally spectrometer employed was a Hewlett-Packard G2025 model with alinear time of flight analyzer; samples (4 ␮l) were mixed 1:1 v/v with intact) GLP-1 concentrations in obese non-diabetic matrix (44 mg diammonium-hydrogen-citrate and 30 mg 2⬘,6⬘-dihy- male patients (16) (first appearing in abstract form droxyacetophenone in 1 ml aqueous solution containing 50% acetoni- (17)). In this study, administration of metformin (2550 trile and 0.05% trifluoroacetic acid; Sigma-Aldrich), transferred to a mg/day) over a two week period appeared to signifi- probe tip and immediately evaporated using the Hewlett-Packard G2024A cantly increase active GLP-1 levels relative to the con- sample preparation vacuum chamber. 250 single laser-shot spectrawere accumulated. This method of monitoring degradation has been trol group after an oral glucose load with a euglycemic validated in several prior publications (8, 19, 20), and allows the general hyperinsulinemic clamp protocol, but did not affect comparison of half-degradation times (t1/2) under various conditions.
basal active GLP-1 concentration. Furthermore, dur- Effect of metformin on substrate binding to DP IV using surface ing incubation of GLP-1 in human serum or Surface plasmon resonance is a highly sensi- buffer containing porcine DP IV in vitro, metformin tive technique which measures biomolecular interactions by detect- concentrations that would be expected in vivo appeared ing the change in refractive properties at the surface of a sensor chip.
to dose-dependently preserve intact GLP-1 (as mea- Purified pork DP IV and recombinant human DP IV were immobi- sured using an N-terminally specific ELISA) (16). The lized on the surface of a CM5 chip (BIAcore AB, Uppsala, Sweden)using amine coupling chemistry, precisely as previously described purpose of the current study was to reinvestigate these (19). Baseline values for porcine DP IV and recombinant human DP findings using alternative biochemical methods. Ex- IV were 5000 and 3500 resonance units (RU), respectively. Baseline periments were designed such that the effect of met- values affect the maximal possible change in RU upon analyte bind- formin on DP IV activity in human serum, purified ing (proportional to the ratio of molecular masses of the analyte to porcine DP IV, and purified recombinant human DP the immobilized ligand multiplied by the baseline value), however itdoes not in theory alter the outcome of kinetic analyses. Experiments IV could be determined. Gly-Pro-para-nitroaniline was were carried out using a flow rate of 20 ␮l/min, in HBS-EP buffer (10 used as a DP IV substrate for spectrophotometric stud- mmol/l HEPES, 150 mmol/l NaCl, 3 mmol/l EDTA, 0.005% v/v Sur- ies, and synthetic GLP-1 was employed during factant P-20, pH 7.4; BIAcore AB). Apparent K D values were mea- kinetic studies with matrix-assisted laser-desorption sured at 4°C and 25°C for both GLP-1[7-36NH2] and GLP-1[9-36NH2], over a ionization-time of flight mass spectrometry (MALDI- concentration range of 1.56 ␮mol/l to 100 ␮mol/l, and measured fromnon-linear regression curves on plots of R TOF MS). Surface plasmon resonance is able to detect Eq (steady-state equilibrium difference in resonance units) versus peptide concentration. To es- real-time interactions between proteins, and thus this tablish if metformin had an effect on GLP-1[7-36NH2] or GLP-1[9-36NH2] technique was applied to establish if metformin af- interaction with DP IV, 20 ␮mol/l of peptide was co-injected with binding to DP IV.
metformin in the concentration range 0 – 0.3 mmol/l.
GLP-1[7-36NH2] and GLP-1[9-36NH2] were synthesized MATERIALS AND METHODS in house, using the automated Symphony peptide synthesizer (RaininInstrument Co., Woburn, MA). Peptides were purified to ⬎95% purityby HPLC (Merck-Hitachi, Darmstadt, Germany) and MALDI-TOF MS Purified pork kidney dipeptidyl peptidase was used to confirm identity and purity of the products.
IV was prepared by the method of Wolf et al. (18). Recombinantsoluble human DP IV was kindly provided by J. Ba¨r (Probio- Data analysis. Data points represent compiled data from at least drug, Germany). Using the chromogenic substrate, H-Gly-Pro-4- three independent measurements, given as the mean ⫾ standard nitroaniline (GP-pNA; Probiodrug, Germany), the specific activity of error of the mean (SEM). Data were analysed using the Prism 3.0 DP IV used in the current study was measured to be 31.2 units/mg (GraphPad, San Diego), BIAevaluation 3.0.1 (BIAcore AB) or Excel Vol. 291, No. 5, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Metformin fails to alter interaction between GLP- Spectrophotometric Studies Using Gly-Pro-pNA and DP IV, as measured with surface plasmon resonance. Initial studies using surface plasmon resonance examined the apparent DP IV activity (%) binding affinity of GLP-1 Note. Hydrolysis was monitored under standard conditions as de- scribed under Materials and Methods.
a Purified porcine DP IV.
b Purified recombinant human DP IV.
97 (Microsoft) software packages for PC. MALDI-TOF MS degrada-tion curves were fitted to first-order exponential decay equations,whereas BIAcore binding curves were fitted to first-order exponen-tial association equations, both using appropriate non-linear regres-sion software. Significance of difference was ascertained using anal-ysis of variance (ANOVA) or a student's t test, where appropriate,with P ⬍ 0.05 considered significant.
Hydrolysis of Gly-Pro-pNA is not altered by met- A broad concentration range of metformin was used to monitor any influence it had on the stan-dard colorimetric determination of DP IV activity. Ta-ble 1 shows the effect of metformin on Gly-Pro-pNAhydrolysis by purified pig kidney DP IV, recombinanthuman DP IV and by human serum. No significanteffects were observed at any concentration tested. Theconcentration range of metformin used includes clini-cally relevant concentrations, as well as those higherthan found in vivo (normally less than 18 ␮mol/l; (21)).
With either competitive or non-competitive enzyme in-hibition, one would expect dose dependent effects.
Measurement of GLP-1 by MALDI-TOF mass spectrometry shows no effect ofmetformin. Matrix-assisted laser-desorption time of flight mass spectrometry was used to monitor the hy-drolysis of intact GLP-1 and purified DP IV homologs from pig and human.
MALDI-TOF MS has been used to measure classicalenzyme kinetic constants (8, 20), however, more rou-tinely performed is the comparison of half degradationtime (t ) in the presence or absence of an inhibitor (19, 20). Figure 1 depicts representative spectra obtained inthe presence and absence of 10 mmol/l metformin at 0min and 60 min after incubation with porcine, rhumanDP IV or 20% human serum at 30°C, pH 7.6. Qualita-tively, metformin appears not to prevent GLP-1 Representative MALDI-TOF mass spectra of GLP-1 deg- hydrolysis by DP IV or serum. Comparison of exponen- radation by (A) purified pork DP IV, (B) recombinant human DP IV,or (C) 20% human serum, with or without 1 mol/l metformin. The tial decay curves quantitatively verifies this conclu- abscissa is relative peak intensity, and the ordinate is mass to charge values with or without metformin did not ratio (m/z). See text for detailed methods. Quantitative kinetic pa- significantly differ (Table 2).
rameters can be found in Table 2.
Vol. 291, No. 5, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Degradation Kinetics of GLP-1 by MALDI-TOF Mass Spectrometry Half-time of degradation (t1/2; min) Note. See text for detailed methods.
a Purified porcine DP IV.
b Purified recombinant human DP IV.
c ND, not determined.
dextran immobilized DP IV (purified porcine and re-combinant human), as previously described for gluca-gon analogs (19). Apparent K values were obtained by plotting change in R from baseline versus peptide concentration (Fig. 2). As immobilized DP IV retainsenzymatic activity, experiments first measured at 25°Cwere also performed at 4°C, to obtain more accurate KDvalues. This was hypothesized to be more important forthe measurement of GLP-1 binding affinity at 25°C, where the measured K would also be influenced by the hydrolysis to GLP-1 at the sensor chip Binding kinetics of GLP-1 for dextran-immobilised DP IV surface. In fact, the measured K for GLP-1 measured by surface plasmon resonance. (A) A representative sen- peared to be only moderately reduced (i.e. higher affin- sorgram showing binding of GLP-1[7-36NH2] to DP IV immobilised onthe surface of the sensor chip versus time (flow rate ⫽ 20 ␮l/min, ity) at 4°C relative to 25°C (Table 3). Furthermore, the 25°C, pH 7.4). Baseline measurements were taken at 60 s and REq net effect of reduction of temperature was to decrease was measured at 360 s, at the end of the 5-min peptide injection. At , reduced by 35.6% and 58.2%, GLP- least 10 min of wash out was allowed in between peptide injections , reduced by 48.9% and 61.9%, for porcine and to allow return to baseline. (B) Saturation binding curves of GLP- rhuman DP IV isoforms, respectively. Metformin (0 – [7-36NH2] or GLP-1[9-36NH2] and DP IV at 25°C using equilibrium surface plasmon resonance deflections plotted versus peptide concentration.
0.3 mmol/l), had no effect on either GLP-1 See text and Table 3 for complete quantitative comparisons.
(20 ␮mol/l) binding to immobilized DP IV (Fig. 3). Metformin concentrations above this rangeinteracted non-specifically with the dextran matrix inhibitor, thus explaining the anorectic effect of met- (the reference chamber) in the absence of peptide, pre- formin and the concurrent improvement in glucose tol- venting the testing of higher doses (although 0.3 erance. By their own admission, in the Mannucci re- mmol/l metformin is already a suprapharmacological port only preliminary findings are included, and concentration). Results also indicated that constant 30 ␮mol/l metformin did not produce consistent effects onother concentrations of GLP-1 over the concentration range 0 –100 ␮mol/l (n ⫽ 2, Binding Constants (K ) of GLP-1 data not shown).
Dextran-Immobilised Porcine and Recombinant Human DPIV, Measured Using Surface Plasmon Resonance The current manuscript addresses whether or not metformin acts directly on dipeptidyl peptidase IV (DPIV) in order to retard degradation of GLP-1 inactive N-terminally truncated form, GLP-1 The recent manuscript by Mannucci et al. (16) sug- gested that because GLP-1 levels were elevated in metformin-treated non-diabetic obese males relative a Purified porcine DP IV.
to non-treated subjects, metformin may act as a DP IV b Purified recombinant human DP IV.
Vol. 291, No. 5, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Effect of graded metformin concentrations on interactions of GLP-1[7-36NH2] or GLP-1[9-36NH2] with porcine or recombinant human DP IV. See text for specific methods.
similar experiments have not yet been carried out in glucose sensitivity of the pancreatic alpha cell and healthy or diabetic subjects, animal models, or in vitro. enteroendocrine L-cell, or the secretory rate of these We have addressed the latter deficiency, and per- cells, resulting in greater hormone release with met- formed several direct in vitro enzymological experi- formin treatment. The work of Lugari et al. is sup- ments to determine if metformin inhibits DP IV or ported by studies published previously (23), which alters the substrate-enzyme interaction. In contrast to found that metformin significantly increased release of findings by Mannucci and co-workers, we have been pancreatic and gut glucagon (glicentin and oxynto- unable to show that metformin has any effect on DP IV, modulin intestinal products of proglucagon processing and thus we offer alternative explanations for their released in equal amounts to GLP-1 from enteroendo- crine L-cells in response to luminal nutrients (24)).
Mannucci et al. (16) continued the work of Lugari et Mannucci and colleagues tested obese non-diabetic al. (22), with respect to the effect of metformin on subjects using a euglycemic hyperinsulinemic clamp GLP-1 levels in obese or T2D patients. The latter test protocol, as opposed to a test meal, in order to manuscript examined the effect of metformin (1 week, avoid glycemia induced alterations in GLP-1 release, 500 mg three times per day) on plasma glucagon and rather than direct effects of metformin (16). Under total GLP-1 (GLP-1 these conditions, it was found that GLP-1 diabetic subjects after a test meal (550 kcal). Met- significantly greater in the metformin treated group, formin significantly increased both glucagon and total using a commercially available assay specific for GLP-1 levels after one week; glucagon release was not N-terminally intact GLP-1 (16). Unfortunately, total altered by the test meal, but was significantly greater GLP-1 levels were not measured. An increase in than paired data obtained prior to metformin treat- N-terminally intact GLP-1 was interpreted as indicat- ment (22). While plasma GLP-1 increased postprandi- ing protection from degradation by DP IV, and the ally in both control and metformin treated subjects, in possibility of an increase in total GLP-1 levels, yielding the metformin treated group GLP-1 levels were signif- a proportional rise in intact GLP-1 concentrations, was icantly greater than the control group at several time not considered. This possibility is consistent with prior points (22). Perhaps the most simplistic interpretation studies examining glucagon and GLP-1 levels after of these findings is that metformin either increases the metformin treatment (22, 23). Experiments were con- Vol. 291, No. 5, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS tinued in vitro using human plasma from healthy do- within the catalytic site of DP IV contributes little to nor subjects and purified pig kidney DP IV, with or the overall affinity of the interaction between enzyme without graded metformin concentrations (16). After a and substrate.
30 min incubation, detection of GLP-1 In summary, we have attempted to determine N-terminally specific ELISA was reduced by 24% in whether metformin has direct effects on DP IV- human serum and 84% in purified DP IV in the ab- mediated GLP-1 degradation in vitro, and additionally sence of metformin, while addition of 0.5 ␮g/ml met- have enhanced our understanding of GLP-1/DP IV in- formin (approx. 3 ␮mol/l) appeared to moderately re- teractions. We have been unable to support the claim verse the loss in detection to 12% and 55% respectively.
that metformin inhibits DP IV activity by a number of These findings compelled us to perform in vitro exper- different experimental approaches. The most likely ex- iments using alternative enzymological methods to re- planation for the findings of Mannucci et al. (16) with assess the role of metformin on DP IV.
respect to preservation of N-terminally intact GLP-1 In contrast to the in vitro findings of Mannucci et al. with metformin treatment is that metformin increases (16), we were unable to detect any significant effect of the secretion of total GLP-1, and thus a proportional metformin on Gly-Pro-pNA hydrolysis, the prototypical increase in intact GLP-1 would be expected. It is diffi- DP IV substrate, in healthy human serum, purified pig cult to explain the disparate findings in vitro between kidney DP IV or recombinant human DP IV. Further the current report and that published previously, how- experiments using MALDI-TOF mass spectrometry ever, the primary experimental omission of measuring which can concurrently detect disappearance and ap- only intact GLP-1 , and not total GLP-1 under- pearance of the molecular species corresponding to mines the earlier data, as sample recovery cannot be , incubated in the same assessed. In contrast, MALDI-TOF mass spectrometry enzyme preparations, yielded a t allowed direct detection of both intact and inactive exponential decay curves which can be compared under GLP-1, and hence is more convincing. Surface plasmon different experimental conditions. Consistent with resonance allowed measurement of affinity of interac- Gly-Pro-pNA enzymological experiments, metformin tion between enzyme and substrate. This was not sig- did not alter the degradation kinetics of GLP-1 nificantly altered by metformin or the presence of an over a wide range of concentrations in any of the en- intact N-terminus.
zyme preparations.
In conclusion, it appears that metformin may in- Surface plasmon resonance (SPR) was used to exam- crease hormone secretion from both the pancreatic al- ine the interaction between purified DP IV homologs pha cell and intestinal L-cell, resulting in greater glu- and GLP-1, irrespective of catalytic activity. The cagon and total GLP-1 levels in metformin treated amine-coupling reaction does not affect enzyme activ- individuals. The latter effect may be one of the mech- ity (19), and SPR allows measurement of intermolecu- anisms by which metformin improves glucose toler- lar interactions not necessarily confined to the cata- ance. With the emergence of potent specific DP IV lytic site. From these studies, apparent K values were inhibitors for the treatment of type 2 diabetes mellitus, measured for GLP-1 an even greater potential may lie in combinatorial formin failed to alter the binding interaction between treatment with both metformin and DP IV inhibitors to and DP IV. Remarkably, maximize the incretin effect.
the apparent affinity of GLP-1 is very similar to , even at reduced temperature.
Binding constants for N-terminally truncated glucagonfragments and purified DP IV were not previously This work was funded in part by Department of Science and tested, however, studies indicated that substitution or Technology of Sachsen Anhalt (HUD Grant 9704/00116) and by the modification of the penultimate amino acid of glucagon Medical Research Council of Canada (CHSM and RAP Grant590007) and the Canadian Diabetes Association. Simon Hinke is reduced the apparent K by approximately 10-fold, and grateful for the support of the Killam Trusts, the Medical Research that altering the chirality of Gln3 produced more pro- Council of Canada, and the Deutscher Akademischer Austausch- nounced effects on glucagon/DP IV interactions (19).
dienst (DAAD). The authors thank Madeleine Speck, Joachim Studies comparing DP IV binding constants for GIP Ba¨r, Michael Wermann, and Dr. Susanne Manhart for technical ⫽ 1.7 ␮mol/l) and DP IV hydrolysis product, assistance.
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Source: http://www.cyclicamp.ca/pdfs/PDF%20Articles/Hinke2002BBRC.pdf