Interleukin 2 production by human T lymphocytes identified by antibodies to dipeptidyl peptidase IV

T-Cell subsets identified by polyclonal and monoclonal antibodies to dipeptidyl peptidase IV (DP IV) were investigated. Analysis in a cytofluorograf revealed 63 +/- 7% positive scatter-gated T lymphocytes. DP IV-positive cells were found to be T11+, 74-81% OKT4+, and 12-19% OKT8+. DP IV-negative cells were T11+ and comprise 16-40% OKT8+, and 10-30% OKT4+ T cells. Treatment of T lymphocytes with rabbit anti-DP IV and complement as well as the presence of rabbit anti-DP IV during culture resulted in a reduction of interleukin 2 (IL-2) production. This reduction was not observed with the mouse monoclonal anti-DP IV antibody II-19-4-7. Positive enrichment of DP IV-positive lymphocytes by cell sorting revealed excellent IL-2 production of DP IV-positive cells and very poor IL-2 activity in supernatants obtained from DP IV-negative lymphocytes. Thus, DP IV may serve as cell surface marker for IL-2-producing T lymphocytes.     

Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase playing a significant role in the process of activation and proliferation of human thymus-derived lymphocytes. This conclusion is drawn from (1) the induction of this enzyme on mitogen-activated T lymphocytes (cf. Sch?n, E. & Ansorge, S. (1990) Biol. Chem. Hoppe-Seyler 371, 699-705) and (2) the impairment of different functions of activated T cells in the presence of specific inhibitors and antibodies against DP IV (Sch?n, E. & al. (1987) Eur. J. Immunol 17, 1821-1826). This paper is aimed at testing new active site-specific peptide inhibitors for their efficiency as inhibitors of lymphocyte DP IV and DNA synthesis of mitogen-stimulated lymphocytes. These inhibitors comprise (i) diacylhydroxylamine derivatives of Xaa-Pro or Xaa-Ala peptides, (ii) different oligopeptides with N-terminal Xaa-Pro-sequences, and (iii) amino-acid amides of the pyrrolidide and the thiazolidide type. The thiazolidides of epsilon-(4-nitrobenzyloxycarbonyl)-L-lysine and of L-isoleucine as well as Ala-Pro-nitrobenzoylhydroxylamine are the most effective inhibitors in both test systems, yielding half-maximal inhibitory concentrations in the micromolar range. Cell viability was not impaired in this effective concentration range. Other inhibitors of DP IV are one to two orders of magnitude less efficient in the suppression of lymphocyte proliferation.     

Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation.

CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26-specific mAb anti-Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF-HSB-2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co-immobilized mAb AC7 and mAb OKT3 (anti-CD3). Cultures of T cells growing in the presence of IL-2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells.     

Down-regulation of T cell activation following inhibition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1-9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1-9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp(2)-Tat(1-9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1-9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1-9) induces the production of the immunosuppressive cytokine transforming growth factor-beta1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.     

Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites.

Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus-1 (HIV-1) Tat protein and the N-terminal peptide Tat(1-9) inhibit DP IV activity and T cell proliferation. Therefore, the N-terminal amino acid sequence of HIV-1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2-R(1-9), bearing the N-terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., St?ckel-Maschek, A., Lorey, S., K?hne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180-22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1-9) can result in a change of the inhibition type. Certain Tat(1-9)-related peptides are found to be competitive, and others linear mixed-type or parabolic mixed-type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed-type mechanism, attributed to both non-mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide-based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.     

The role of dipeptidyl peptidase IV (DP IV) enzymatic activity in T cell activation and autoimmunity

Activated T lymphocytes express high levels of dipeptidyl peptidase IV (DP IV)/CD26. Recent studies support the notion that DP IV may play an important role in the regulation of differentiation and growth of T lymphocytes. This article gives a short overview on DP IV/CD26 expression and effects on immune cells in vitro and in vivo. A major focus of this review are clinical aspects of the function of CD26 on hematopoietic cells and the potential usage of synthetic DP IV inhibitors as therapeutics in inflammatory disorders.     

Characterization of T lymphocyte defects in patients with Hodgkin's disease using enzyme chemical markers]

Patients suffering from lymphogranulomatosis were studied with respect to cellular immune deficiencies. For this purpose, mononuclear cells from venous blood were separated and subjected to analysis of lymphocyte markers. T-lymphocytes were enumerated by means of the sheep erythrocyte (SE) rosette test. T cell subpopulations were determined using enzyme cytochemical staining for dipeptidyl peptidase IV (DP IV) and unspecific acid alphanaphthylacetate esterase (ANAE). In 18 patients with M. Hodgkin a significant reduction in the T lymphocyte count in peripheral blood was found. This T cell defect is due to a selective decrease in the TM-subpopulation as identified by enzyme cytochemical markers DP IV and ANAE (focal reaction). From these results it is concluded that patients with lymphogranulomatosis have characteristic abnormalities in the immune system in the sense of a disturbed equilibrium of immune regulatory cells.     

Dipeptidylpeptidase IV--inactivation with N-peptidyl-O-aroyl hydroxylamines

Eleven N-peptidyl-O-aroyl hydroxylamines have been synthesized and their hydrolytic stability, acidity and properties during reaction with dipeptidyl peptidase IV (E.C. 3.4.14.5) investigated. N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines act as irreversible inhibitors of serine proteases. The serine enzyme, dipeptidyl peptidase IV (DP IV), is inactivated by substrate analog derivatives of this class by a suicide inactivation mechanism. During the enzyme reaction of DP IV with the suicide substrates most molecules are hydrolyzed but some irreversibly inactivate the target enzyme. In contrast to porcine pancreatic elastase and thermitase, DP IV exhibits a high ratio for hydrolysis of the compounds versus inhibition during their interaction with the enzyme. Variation of the leaving aroyl residue lowers this ratio. Variation of the substrate analog peptide moieties of the DP IV-inhibitors increases their ability to inhibit the enzyme to a remarkable extent. Possible reaction pathways are discussed.     

Comparison of different markers on blood lymphocytes of chronic lymphocytic leukemia

Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.     

Targeting dipeptidyl peptidase IV (CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo

CD26 or dipeptidyl peptidase IV (DP IV) is expressed on various cell types, including T cells. Although T cells can receive activating signals via CD26, the physiological role of CD26/DP IV is largely unknown. We used the reversible DP IV inhibitor Lys[Z(NO(2))]-pyrrolidide (I40) to dissect the role of DP IV in experimental autoimmune encephalomyelitis (EAE) and to explore the therapeutic potential of DP IV inhibition for autoimmunity. I40 administration in vivo decreased and delayed clinical and neuropathological signs of adoptive transfer EAE. I40 blocked DP IV activity in vivo and increased the secretion of the immunosuppressive cytokine TGF-beta1 in spinal cord tissue and plasma during acute EAE. In vitro, while suppressing autoreactive T cell proliferation and TNF-alpha production, I40 consistently up-regulated TGF-beta1 secretion. A neutralizing anti-TGF-beta1 Ab blocked the inhibitory effect of I40 on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not generally immunosuppressive, neither eliminating encephalitogenic T cells nor inhibiting T cell priming. These data suggest that DP IV inhibition represents a novel and specific therapeutic approach protecting from autoimmune disease by a mechanism that includes an active TGF-beta1-mediated antiinflammatory effect at the site of pathology.     

Soluble CD26/dipeptidyl peptidase IV induces T cell proliferation through CD86 up-regulation on APCs.

CD26 is a T cell costimulatory molecule with dipeptidyl peptidase IV enzyme activity in its extracellular region. We have previously reported that the addition of soluble CD26 (sCD26) resulted in enhanced proliferation of peripheral blood T lymphocytes induced by the recall Ag, tetanus toxoid (TT). However, the mechanism involved in this immune enhancement has not yet been elucidated. In this paper, we demonstrate that the enhancing effect of sCD26 on TT-induced T cell proliferation occurred in the early stages of immune response. The cells directly affected by exogenously added sCD26 are the CD14-positive monocytes in the peripheral blood. Mannose-6 phosphate interfered with the uptake of sCD26 into monocytes, suggesting that mannose-6 phosphate/insulin-like growth factor II receptor plays a role in the transportation of sCD26 into monocytes. When sCD26 was added after Ag presentation had taken place, enhancement in TT-induced T cell proliferation was not observed. In addition, enhancement of TT-mediated T cell proliferation by sCD26 does not result from trimming of the MHC-bound peptide on the surface of monocytes. Importantly, we also showed that exogenously added sCD26 up-regulated the expression of the costimulatory molecule CD86 on monocytes through its dipeptidyl peptidase IV activity, and that this increased expression of CD86 was observed at both protein and mRNA level. Therefore, our findings suggest that sCD26 enhances T cell immune response to recall Ag via its direct effect on APCs.     

Structural requirements for catalysis,expression, and dimerization in the CD26/DPIV gene family

Dipeptidyl peptidase IV (DP-IV/CD26), fibroblast activation protein (FAP), DP-like 1 (DPL1), DP8, DP9, and DPL2 comprise the CD26 gene family. CD26/DP-IV has roles in liver disease, T cell costimulation, chemokine biology, type II diabetes, and tumor biology. DPIV substrates include the glucagonlike peptides, neuropeptide Y, and the chemokines CCL3, CCL5, CCL11, CCL22, and CXCL12. We have proposed that the extracellular region of CD26 is analogous to prolyl oligopeptidase in consisting of an alpha/beta hydrolase domain contributed by both N- and C-terminal portions of the polypeptide and a seven-blade beta-propeller domain. Replacing the C-terminal portion of the predicted alpha/beta hydrolase domain of CD26 (residues 501-766) with the homologous portion of DP8 or DP9 produced intact proteins. However, these chimeric proteins lacked dimerization and peptidase activity, suggesting that CD26 dimerization requires the C-terminal portion of the alpha/beta hydrolase domain. Deleting some N-terminal residues of the alpha/beta hydrolase domain of CD26 ablated peptidase activity and greatly diminished cell surface expression. Together with previous data that CD26 peptidase activity requires the C-terminal 20 residues, this suggests that peptidase activity requires the entire alpha/beta hydrolase domain. The catalytic triad of DP8 was shown to be Ser(739)-Asp (817)-His(849). Glu(259) of DP8, a residue distant from the catalytic triad yet greatly conserved in the CD26 gene family, was shown to be required for peptidase activity. These data concord with our predicted CD26 structure, indicate that biosynthesis of a functional fragment of CD26 is difficult, and confirm the functional homology of DP8 with CD26.     

Dipeptidyl peptidase IV as a new surface marker for a subpopulation of human T-lymphocytes

Plasma membranes were isolated from normal human lymphocytes as well as from cells of chronic lymphocytic leukemia of the T type. In both cases the bulk of the dipeptidyl peptidase IV activity paralleled the distribution of 5′-nucleosidase and, therefore, was localized in the plasmalemma. Immunofluorescence experiments with normal human lymphocytes and with antibodies against dipeptidyl peptidase IV revealed that this peptidase was accessible on the surface of viable cells. Further, it was demonstrated that the relative number of dipeptidyl peptidase IV-positive cells is much higher in lymphocytes reacting with the OKT4 antibody than in OKT8-positive cells. On the other hand, it has been reported that this peptidase is absent in B lymphocytes and is predominantly found in T cells bearing the Fc receptor for IgM (Tμ lymphocytes). Thus, it is concluded that dipeptidyl peptidase IV represents an easily demonstrable surface marker of this lymphocyte subset.     

Alteration of the antigenicity of human lymphocytes by plant lectins and short-term tissue culture.

Human lymphocytes studied after being placed in culture for 1–6 wk progressively lost stimulating ability, i.e., lymphocyte defined antigens, when tested in one way mixed lymphocyte culture (MLC) but retained several other identifiable membrane components as well as the capacity to respond to mitogenic stimuli. Lymphocytes placed in culture with motogenic doses of PHA and Con-A after 1 and 2 wk strongly stimulated autologous responding fresh lymphocytes, but the MLC response of allogeneic fresh lymphocytes to stimulating lectin treated cells was even lower than the response to stimulating allogeneic cultured lymphocytes. The HL-A antigens on lectin treated cells or on lymphocytes through 6 wk in culture were clearly identifiable. Assays for T cell rosettes and B cell surface immunoglobulin showed both cell types to be present in numbers equal to fresh lymphocytes for up to 5 wk after culturing. However, the Fc receptor site on B cells was lost from cultured lymphocytes at the same time that MLC stimulation was lost. It is concluded that plant lectins can unmask new mitogenic sites on the cell surface as well as mask or delete existing sites, and that culturing lymphocytes for 1–6 wk will produce somewhat similar modulations. Modulation of surface membrane components by tissue culture or lectins may, therefore, have a profound effect in altering transplantation immunogenicity.     

Differential regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, synthase, and low density lipoprotein receptor genes.

The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.     

Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes

A panel of B cell-specific monoclonal antibodies that identify the CR2/EBV receptor were examined for their ability to mimic the T-independent mitogenic agent, EBV, and thus activate human peripheral blood B lymphocytes. Two of four different anti-CR2/EBV monoclonal antibodies, OKB7 and AB-1, produced a 50-fold to 200-fold dose-dependent stimulation of DNA synthesis of peripheral blood mononuclear cells. One of the other monoclonal antibodies, anti-B2, had slight activity, and the other, HB-5, was completely inactive. One of the mitogenic antibodies, OKB7, which directly inhibits binding and infection of B cells by EBV in the absence of a second anti-immunoglobulin antibody, was examined in further detail. Both the intact antibody in soluble form and its pepsin-derived F(ab')2 fragment stimulated DNA synthesis of unseparated B and T lymphocytes. Peak stimulation of DNA synthesis in peripheral blood mononuclear cells occurred between 4 to 6 days. B cells were responsible for incorporation of [3H]thymidine. However, T cells were required for activation of peripheral blood mononuclear cells by OKB7. OKB7, as well as the other mitogenic monoclonal anti-EBV/CR2 receptor antibody, also induced B cells to differentiate after 6 to 10 days of culture as indicated by polyclonal Ig secretion. IgM was the predominate immunoglobulin secreted. These studies thus indicate that certain epitopes on the EBV/CR2 receptor trigger B cells to divide and differentiate. This pathway of B cell activation, in contrast to that produced by EBV, is T cell dependent.     

Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lymphocytes.

Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.     

Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.     

Intestinal digestive resistance of immunodominant gliadin peptides

Two recently identified immunodominant epitopes from alpha-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogenous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food.     

Metformin effects on dipeptidylpeptidase IV degradation of glucagon-like peptide-1

There is current interest in the use of inhibitors of dipeptidyl peptidase IV (DP IV) as therapeutic agents to normalize glycemic excursions in type 2 diabetic patients. Data indicating that metformin increases the circulating amount of active glucagon-like peptide-1 (GLP-1) in obese nondiabetic subjects have recently been presented, and it was proposed that metformin might act as a DP IV inhibitor. This possibility has been investigated directly using a number of in vitro methods. Studies were performed on DP IV enzyme from three sources: 20% human serum, purified porcine kidney DP IV, and recombinant human DP IV. Inhibition of DP IV hydrolysis of the substrate Gly-Pro-pNA by metformin was examined spectrophotometrically. Effects of metformin on GLP-1([7-36NH2]) degradation were assessed by mass spectrometry. In addition, surface plasmon resonance was used to establish whether or not metformin had any effect on GLP-1([7-36NH2]) or GLP-1([9-36NH2]) interaction with immobilized porcine or human DP IV. Metformin failed to alter the kinetics of Gly-Pro-pNA hydrolysis or GLP-1 degradation tested according to established methods. Surface plasmon resonance recordings indicated that both GLP-1([7-36NH2]) and GLP-1([9-36NH2]) show micromolar affinity (K(D)) for DP IV, but neither interaction was influenced by metformin. The results conclusively indicate that metformin does not act directly on DP IV, therefore alternative explanations for the purported effect of metformin on circulating active GLP-1 concentrations must be considered.