To assess the role of the PI3K/Akt pathway in sitagliptin-induced NGF suppression, infarcted rat hearts were treated with the PI3K specific inhibitor, wortmannin (WM), together with sitagliptin. MI was induced by coronary ligation, and 4 weeks later the infarcted rat hearts were isolated and subjected to no treatment (vehicle), sitagliptin (5 μM), sitagliptin + WM (100 nM, Sigma–Aldrich) or sitagliptin + resistin (10 nM). The drugs were perfused for 60 min. The doses of WM [27 ] and resistin [27 ] used in that study have been used previously. All of the hearts (n=5 each group) were used for western blot analysis for NGF at the remote zone.
Sitagliptin
Manufactured by Merck Group
Sourced in United States, China, United Kingdom, Israel
Sitagliptin is a pharmaceutical product developed by Merck Group. It is a dipeptidyl peptidase-4 (DPP-4) inhibitor, which functions by inhibiting the enzyme DPP-4 to regulate blood glucose levels.
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Lab products found in correlation
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Metformin, by Merck Group (3 mentions)
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Aprotinin, by Merck Group (3 mentions)
Sdf 1α, by ProSpec (3 mentions)
Chitosan, by Merck Group (2 mentions)
Acarbose, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Gly pro p nitroanilide, by Merck Group (2 mentions)
Streptozotocin (stz), by Merck Group (2 mentions)
Dpp 4 inhibitor screening assay kit, by Cayman Chemical (2 mentions)
Dpp 4, by Merck Group (2 mentions)
C57bl 6 cd45 2 thy1, by Charles River Laboratories (2 mentions)
Balb c mice, by Charles River Laboratories (2 mentions)
Januvia tablets, by Merck Group (2 mentions)
Amd3100, by R&D Systems (2 mentions)
Pdgf bb, by Merck Group (2 mentions)
Thioglycolic acid, by Merck Group (1 mentions)
Sodium taurodeoxycholate hydrate, by Merck Group (1 mentions)
68 protocols using sitagliptin
1
Sitagliptin's Effect on NGF via PI3K/Akt
2
DPP4 Activity and Total Levels Assay
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Total circulating DPP4 was assessed by a specific enzyme-linked immunosorbent assay (ELISA) (R&D Systems, DuoSet, lot P185646), using a 1:1 dilution (1 part serum to 1 part saline) as sample and performed according to the manufacturer's instructions.
DPP4 serum enzymatic activity was determined as the rate of cleavage of 7-amino-4-methylcoumarin (AMC) from the fluorogenic substrate Glycyl-Prolyl-AMC (Gly-Pro-AMC; Sigma, USA) in a 96-well microplate in SpectraMax® M5 ROM v3.0.22 Molecular Devices spectrofluorometer (USA) at 360 nm excitation and 460 nm emission for 15 min and in duplicate. The rate of liberated fluorescence was calculated per minute (U/min). All enzymatic reactions were performed with 10 µL of serum sample in activity buffer (25 mM HEPES, pH 7.5) in the presence of 20 µM of the substrate in a final volume of 100 µL (30 (link)). To confirm the specificity of DPP4 activity in these assays, a highly selective DPP4 inhibitor (Sitagliptin; Sigma) was incubated with some serum samples for 15 min before proceeding to hydrolysis of the substrate, in triplicate, with Sitagliptin at a final concentration of 1 µM.
DPP4 serum enzymatic activity was determined as the rate of cleavage of 7-amino-4-methylcoumarin (AMC) from the fluorogenic substrate Glycyl-Prolyl-AMC (Gly-Pro-AMC; Sigma, USA) in a 96-well microplate in SpectraMax® M5 ROM v3.0.22 Molecular Devices spectrofluorometer (USA) at 360 nm excitation and 460 nm emission for 15 min and in duplicate. The rate of liberated fluorescence was calculated per minute (U/min). All enzymatic reactions were performed with 10 µL of serum sample in activity buffer (25 mM HEPES, pH 7.5) in the presence of 20 µM of the substrate in a final volume of 100 µL (30 (link)). To confirm the specificity of DPP4 activity in these assays, a highly selective DPP4 inhibitor (Sitagliptin; Sigma) was incubated with some serum samples for 15 min before proceeding to hydrolysis of the substrate, in triplicate, with Sitagliptin at a final concentration of 1 µM.
3
Sitagliptin Protects Against Kainic Acid-Induced Epilepsy
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Mice were randomly grouped (each group contained at least 6 mice) and were treated as follows: mice in kainic acid + sitagliptin (KA + Sita) group received sitagliptin (30 mg/kg/d i.p., Sigma, USA) for 7 days before KA injection and mice in control (Ctrl) group and kainic acid (KA) group received an equal volume of saline for 7 days.
The animal model of epilepsy was established according to a previous method [27 (link)–29 (link)]. On the last day of the sitagliptin or saline treatment, a lateral ventricle injection operation was performed. All mice were anaesthetized under 2–3% isoflurane. The right lateral brain ventricle (AP = – 0.20 mm, ML = 1.00 mm, DV = – 2.40 mm, the anterior fontanelle as the origin) was localized with a stereotactic instrument. The mice were treated as follows: Ctrl group received saline (5 μl) and KA and KA + Sita groups received kainic acid (10 μg in 5 μl i.c.v., MCE, USA). After the operation, skin was sutured, and the mice were kept under a warming place until they woke up.
The animal model of epilepsy was established according to a previous method [27 (link)–29 (link)]. On the last day of the sitagliptin or saline treatment, a lateral ventricle injection operation was performed. All mice were anaesthetized under 2–3% isoflurane. The right lateral brain ventricle (AP = – 0.20 mm, ML = 1.00 mm, DV = – 2.40 mm, the anterior fontanelle as the origin) was localized with a stereotactic instrument. The mice were treated as follows: Ctrl group received saline (5 μl) and KA and KA + Sita groups received kainic acid (10 μg in 5 μl i.c.v., MCE, USA). After the operation, skin was sutured, and the mice were kept under a warming place until they woke up.
4
Sitagliptin-Chitosan-Thioglycolic Acid Protocol
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Sitagliptin, chitosan (low molecular weight, 100,000 Da and 75–85% deacetylated) and thioglycolic acid were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Other chemicals were of analytical grade. Double-distilled deionized water was used for the experiments.
5
Comprehensive Enzyme Inhibition Assays
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Folin and Ciocalteu’s phenol reagent, 2-Aminoethyl diphenylborinate 97%, ABTS (2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), DPPH: 2,2-Diphenyl-1-picrylhydrazyl, Trolox®: (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, MOPS: 3-(N-Morpholino) propanesulfonic acid, 4-Morpholinepropanesulfonic acid (≥99.5%), Orlistat: tetrahydrolipstatin (≥98%), DMSO: Dimethyl sulfoxide, α-Amylase from porcine pancreas (EC3.2.1.1), α-Glucosidase from Saccharomyces cerevisiae (EC3.2.1.20), lipase from porcine pancreas (EC3.1.1.3), Dipeptidyl Peptidase IV human (EC 3.4.14.5), quercetin-3-d -galactoside (≥98%), delphinidin 3-O-glucoside (≥98%), malvidin 3-O-glucoside (≥98%) and cyanidin 3-O-glucoside (≥98%), acarbose (≥95%), 3,5-Dinitrosalicylic acid (≥98%), p-Nitrophenyl-α-d -glucopyranoside (≥99%), catechin (99%), rutin (94%), gallic acid (97.5%), vanillin (99%), sodium nitroprusside dihyrate (≥99%), sitagliptin, tributyrin, sodium taurodeoxycholate hydrate, 4-Nitrophenol, tert-butanol and Griess reagent, were purchased from Sigma Aldrich (St. Louis, MO, USA). DPP-IV GLO® protease assay kit G8351 was purchased from Promega (Madison, WI, USA). All chemicals were analytical grade.
6
Sitagliptin Modulates Peptide Cellular Uptake
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Dulbecco’s modified Eagle medium (DMEM) was obtained from GIBCO (Thermo Fisher Scientific, Waltham, MA USA). Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT, USA). Stable L-glutamine, 1% non-essential amino acids, penicillin/streptomycin, and PBS were from Euroclone (Milan, Italy). Sitagliptin and Gly-Pro-amido-4-methylcoumarin hydrobromide (Gly-Pro-AMC) were from Sigma-Aldrich (St. Louis, MO, USA). Polycarbonate filters, 12 mm in diameter, 0.4 m in pore diameter, were from Transwell Corning Inc. (Lowell, MA, USA). Peptides Lup1 (LTFPGSAED) and Soy1 (IAVPTGVA) were synthetized by the company PRIMM (Milan, Italy) at >95% purity. Human serum was freshly collected from a healthy female volunteer.
7
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).
8
Caspase Activation and Pyroptosis Assays
9
Compound Acquisition for Experimentation
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Cyclosporine (CAS No.: 59865-13-3), Sitagliptin (CAS No.: 486460-32-6), and Hesperidin (CAS No.: 520-26-3) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals used for the investigation were of analytical grade.
10
Inhibition of DPP4 in Mice
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A selective and specific inhibitor of DPP4, sitagliptin (provided by Sigma-Aldrich, PHR1857.14) was administered once a day by intraperitoneal injections at a dose of 15 mg/kg (i.e., 0.3 mg/day for a 20-g mouse) for a total of 6 weeks of treatment before animal sacrifices. To evaluate cerebral DPP4 activity, sitagliptin was administrated daily for 8 days and then DPP4 activity was measured as described above.
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