Diprotin a

Manufactured by Merck Group

Sourced in United States, France, United Kingdom, Germany

Diprotin A is a laboratory reagent produced by Merck Group. It is a naturally occurring peptide inhibitor that targets the enzyme dipeptidyl peptidase IV (DPP-IV). Diprotin A is used in research applications for the study of DPP-IV activity and function.

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34 protocols using diprotin a

Metabolic effects of high-fat diet and lactobacillus

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During the 5-week acclimatization period, mice were allowed ad libitum access to the same LFD and fresh water. Subsequent to acclimatization, mice were weight matched and provided with one of 3 diets: LFD, HFD, or HFD + Lac (N = 8). Weekly bodyweight and food intake measurements were recorded. To measure metabolic parameters, mice were housed individually for 72 h in TSE PhenoMaster system cages (TSE systems, Bad Homburg, DE) during weeks 9–11 before returning to their home cages. In week 13, immediately prior to termination, body composition was determined using a Bruker Minispec LF50H (Bruker Optics, Stillorgan, EI). Mice were fasted for 4–6 h, anesthetized (65 mg/kg ketamine and 13 mg/kg xylazine), and blood was collected into vacutainer EDTA tubes (Becton, Dickinson and Company, Dublin, EI). Upon collection, blood was mixed with 500,000 KUI/L Aprotinin (Sigma-Aldrich, Cambridge, UK) and 0.1 mM Diprotin A (Sigma-Aldrich, Cambridge, UK). Cervical dislocation was used to euthanize mice, and tissues were harvested, weighed, and snap frozen in dry ice (brain) or liquid nitrogen before being stored at −80 °C.

McManus B., Korpela R., O’Connor P., Schellekens H., Cryan J.F., Cotter P.D, & Nilaweera K.N. (2015). Compared to casein, bovine lactoferrin reduces plasma leptin and corticosterone and affects hypothalamic gene expression without altering weight gain or fat mass in high fat diet fed C57/BL6J mice. Nutrition & Metabolism, 12, 53.

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Plasma GLP-1 and GIP Quantification

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In Experiments 2 and 3, blood samples were collected at different time points (0, 15, 30, 60min) after infusion and placed in testing tubes containing chilled EDTA, aprotinin, and diprotin A (Sigma-Aldrich, St. Louis, MO, USA). Plasma GLP-1 and glucose-dependent insulinotropic peptide (GIP) were assessed using corresponding commercial ELISA kits (Sigma-Aldrich, St. Louis, MO, USA).

Dong Y., Zhang Y., Li S, & Chen J.D. (2021). Intestinal Electrical Stimulation Enhances Release of Postprandial Incretin Hormones via Cholinergic Mechanisms. Obesity surgery, 31(5), 1957-1966.

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Peptide-Functionalized AuNPs Synthesis

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Peptides with the sequences of Pro-Arg, Arg-Pro-Arg and Arg-Pro-Arg-Pro-Pro-Pro-Pro-Cys (≥98%) were synthesized and purified by Synpeptide Co., Ltd. (Shanghai, China). DPP-IV, diprotin A, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), trisodium citrate, 6-mercapto-1-hexanol (MCH), KH₂PO₄ and K₂HPO₄ were purchased from Sigma-Aldrich (Shanghai, China). Other reagents were analytical-grade and used without additional treatment. The peptide stock solution at the concentration of 2 mM was dissolved with deionized water and diluted with a phosphate-buffered saline solution (PBS buffer, 2 mM, pH 7.6) before use. The citrate-stabilized AuNPs with a size of 13 nm were prepared using a trisodium citrate reduction method [45 (link)]. The concentration of synthesized AuNPs was determined by Beer’s law with an extinction coefficient of 2.7 × 10⁸ M⁻¹ cm^–1 [18 (link)]. Unless otherwise noted, the reactions were conducted at room temperature.

Xia N., Wang X., Wang X, & Zhou B. (2016). Gold Nanoparticle-Based Colorimetric and Electrochemical Methods for Dipeptidyl Peptidase-IV Activity Assay and Inhibitor Screening. Materials, 9(10), 857.

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Oral Glucose Tolerance Test Protocol

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During standard oral glucose tolerance test (OGTT) and experiments, blood was collected to lithium heparin and EDTA tubes and was stored at −80°C until further analyses. Additional blood samples were collected in chilled EDTA tubes pretreated with 25 mg of DPP-IV inhibitor (Diprotin-A, Sigma-Aldrich); and 2000 KIE of trypsin inhibitor (Trasylol, Bayer AG) were added to chilled EDTA tubes before blood sampling. Determination of plasma glucose, insulin, C-peptide, FFA, HbA1c and cholesterol levels are described elsewhere (12 (link)). Total plasma GIP and active GLP-1 was measured using an enzyme-linked immunosorbent assay (ELISA) from Merck Millipore. GIP and GLP-1 assays were performed according to the instructions provided by the manufacturers.

Koffert J., Honka H., Teuho J., Kauhanen S., Hurme S., Parkkola R., Oikonen V., Mari A., Lindqvist A., Wierup N., Groop L, & Nuutila P. (2017). Effects of meal and incretins in the regulation of splanchnic blood flow. Endocrine Connections, 6(3), 179-187.

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Myocardial Infarction Induction and DPP4 Inhibitor Treatment

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Myocardial infarction (MI) was experimentally induced as described before [15 (link)]. The mice (n = 5–18 per group) were anesthetized with isoflurane (1.5–2.5%), intubated and ventilated, after which the left anterior descending (LAD) coronary artery was permanently ligated by placement of a suture. The mice were treated with the analgesic drug Temgesic, both pre-operative and 24 hrs post-operative to relieve pain. The mice were randomly allocated and treated i.p. with either 100 μl distilled water daily (Milli-Q ultrapure, sterile water = MQ treated or control group) or 100 μl DPP4 inhibitor (5 nMol, 55 μg/kg/day, Diprotin A, Sigma-Aldrich) for the first 5 or 14 days post-MI.

Dingenouts C.K., Bakker W., Lodder K., Wiesmeijer K.C., Moerkamp A.T., Maring J.A., Arthur H.M., Smits A.M, & Goumans M.J. (2017). Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction. PLoS ONE, 12(12), e0189805.

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GIP administration and blood collection

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WT mice were fasted for ∼4 hr and were administered 24 nmol/kg [D-Ala²]-GIP (Chi Scientific, Maynard, MA) or PBS (vehicle). Blood was collected via tail vein and mixed with 10% volume of TED (5000 kIU/ml Trasylol (Bayer), 32 mM EDTA, and 0.01 mM Diprotin A (Sigma)) at 0, 30 min, 1 hr, and 2 hr.

Beaudry J.L., Kaur K.D., Varin E.M., Baggio L.L., Cao X., Mulvihill E.E., Bates H.E., Campbell J.E, & Drucker D.J. (2019). Physiological roles of the GIP receptor in murine brown adipose tissue. Molecular Metabolism, 28, 14-25.

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Intrathecal Administration of Opioid Receptor Antagonists

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Diprotin A (isoleucin-prolin-isoleucin, IPI; Sigma-Aldrich, I9759) stock solution was made up in 25% (w/v) hydroxypropyl-beta-cyclodextrin (HPβCD, Sigma-Aldrich, H107) and dilutions were made with sterile saline and administered intrathecally (i.t.) in 30 nmol/rat dose. vildagliptin (VIL) was received from Prof. Ingrid De Meester of the Laboratory of Medical Biochemistry, University of Antwerp, Wilrijk, Belgium and was administered i.t. in 3 nmol/rat dose. Naltrexone hydrochloride (NTX) was a generous gift from DuPont Pharmaceuticals (Geneva, Switzerland), and was injected subcutaneously (s.c.) in 0.5 mg/g b.w. dissolved in saline.
The MOR antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP; Sigma-Aldrich, C6352; 200 pmol/rat), delta-opioid receptor (DOR) -antagonist H-Tyr-Tic(CH₂NH)-Phe-Phe-OH (TIPP[Ψ]; Sigma-Aldrich, T7075; 1 nmol/rat) and kappa-opioid receptor (KOR) -antagonist 5′-guanidinonaltrindole (gNTI; Sigma-Aldrich, G3416; 10 nmol/rat) were dissolved in distilled water.
I.t. injections were delivered in 5 μl volume by a 250 μl Hamilton syringe set into a Hamilton dispenser. The 23-Ga needle was introduced at the L5–6 intervertertebral space^{13 (link),21 (link)}.

Király K., Kozsurek M., Lukácsi E., Barta B., Alpár A., Balázsa T., Fekete C., Szabon J., Helyes Z., Bölcskei K., Tékus V., Tóth Z.E., Pap K., Gerber G, & Puskár Z. (2018). Glial cell type-specific changes in spinal dipeptidyl peptidase 4 expression and effects of its inhibitors in inflammatory and neuropatic pain. Scientific Reports, 8, 3490.

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Enzymatic Activity Assay of SgVnDPPIV

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The enzymatic activity of SgVnDPPIV was assayed with substrate Ala-Pro-p-nitroanilide (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China) following the method described by Tereshchenkova et al. [3 (link)]. The substrate was initially dissolved in dimethylformamide, and its final reaction concentration was 0.25 mM. SgVnDPPIV (1 µg) was combined with 2.5 µL substrate. The freshly prepared PBS (phosphate-buffered saline) (0.1 M, PH 8.0) was added to the final volume of 200 µL. The mixture was incubated at 40 °C for 30 min. Its absorbance was measured periodically using an Infinite F50 Plus and Infinite F50 Robotic microplate reader (TECAN, Männedorf, Switzerland) at 405 nm for 60 min. Venom of S. guani and gut extract from the T. molitor larvae were used as the positive controls. The effects of the inhibitors, including vildagliptin, sitagliptin, diprotin A, and diprotin B (Sigma-Aldrich (Shanghai) Trading Co., Ltd., Shanghai, China), on the enzymatic activity of SgVnDPPIV were tested. The inhibitor concentration was 0.1 mM in the incubate assay with the SgVnDPPIV before its reaction with the substrate, as described above. All assays were performed in three independent biological replicates. The enzymatic activity was expressed in units (U).

Wu C., Yang C., Wang Y., Wang J, & Zhu J. (2023). Molecular Characterization and Functional Analysis of the Dipeptidyl Peptidase IV from Venom of the Ectoparasitoid Scleroderma guani. Toxins, 15(5), 311.

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Pharmacological Modulators of Bile Acid Signaling

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The DPP-4 inhibitors diprotin A, FFA-free Bovine Serum Albumine (BSA), Chenodeoxycholic acid (CDCA), TβMCA, Exendin-4(9-39), Phloretin, DMSO and CMC were purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Sitagliptin was purchased from MSD. The synthetic FXR agonist GW4064 was purchased from Tocris (R&D Systems, Lille, France). For in vitro or ex vivo experiments, CDCA, TβMCA and GW4064 were dissolved in dimethylsulfoxide (DMSO) at 0.1% final. For in vivo experiments, GW4064 was dissolved in 1% Carboxymethylcellulose (CMC) and Exendin-4(9-39) was dissolved in NaCl 0.9%. Chow and high fat diets were purchased from UAR (A04, Villemoison/Orge, France). Colesevelam HCl was a kind gift of Daiichi Sankyo.

TRABELSI M.S., DAOUDI M., PRAWITT J., DUCASTEL S., TOUCHE V., SAYIN S.I., PERINO A., BRIGHTON C.A., SEBTI Y., KLUZA J., BRIAND O., DEHONDT H., VALLEZ E., DORCHIES E., BAUD G., SPINELLI V., HENNUYER N., CARON S., BANTUBUNGI K., CAIAZZO R., REIMANN F., MARCHETTI P., LEFEBVRE P., BÄCKHED F., GRIBBLE F.M., SCHOONJANS K., PATTOU F., TAILLEUX A., STAELS B, & LESTAVEL S. (2015). Farnesoid X Receptor Inhibits Glucagon-Like Peptide-1 Production by Enteroendocrine L-cells. Nature communications, 6, 7629.

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Blood Sampling for Metabolic Tests

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All blood samples (∼50–100 μl) during metabolic tests were collected from tail vein into lithium coated Microvette tubes (Sarstedt, Numbrecht, Germany) and mixed with a 10% volume of TED (5000 kIU/ml Trasylol (Bayer), 32 mM EDTA, and 0.01 mM Diprotin A (Sigma)). Samples were kept on ice and plasma was collected by centrifugation (>12, 000 rpm at 4 °C for 5 minutes) and stored at - 80 °C.

Beaudry J.L., Kaur K.D., Varin E.M., Baggio L.L., Cao X., Mulvihill E.E., Stern J.H., Campbell J.E., Scherer P.E, & Drucker D.J. (2019). The brown adipose tissue glucagon receptor is functional but not essential for control of energy homeostasis in mice. Molecular Metabolism, 22, 37-48.

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