Vildagliptin

6-week-old male C57BL/6J mice were randomly grouped into 8, 12, 16 and 24 weeks in a cross-sectional study using randomizer.com. In a longitudinal study, 6-8-week-old male Pak2^fl/fl and Pak2^cKO mice were randomly allocated and maintained on high fat high sucrose diet (HFHSD, 45% fat and 20% sucrose) (SDS, 824018) or standard chow (SDS, 801960) for 16 weeks.
Glycyrrhizic acid/Glycyrrhizin (a HMGB1 inhibitor, Sigma-Aldrich, G2137), and Vildagliptin (a DPP4 inhibitor, Sigma-Aldrich, SML2302) were administered to C57BL/6J mice and Pak2^cKO mice ad libitum in drinking water, at an estimated dose of 150 mg/kg/day and 3 mg/kg/day, respectively, along with HFHSD according to individual experimental design (see Results). The water pouch was changed every 3 days. For cardiac specific over-expression of PAK2, adeno-associated virus (AAV9-Pak2) was generated previously (Binder et al., 2019 (link)) using human Pak2 cDNA (Source Bioscience, NCBI Accession # BC069613). The recombinant virus (0.5 × 10¹¹ genomic particles) was administered via tail vein after 8 weeks of HFHSD feeding.

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).

Indicated chemicals were obtained from companies (in parentheses): brefeldin A (Sigma-Aldrich), golgicide A (Sigma-Aldrich), AG1478/tyrphostin (Sigma-Aldrich), Erastin (MedChem Express), ferrostatin-1 (Sigma-Aldrich), liproxstatin-1 (Sigma-Aldrich), NOX1/4 inhibitor (GKT137831, Cayman Chemical), Sulfasalazine (Sigma-Aldrich), Sorafenib (Sigma-Aldrich), RSL3 (MedChem Express), Glutathione (Sigma-Aldrich), N-acetyl-cysteine (Sigma-Aldrich), Ciclopirox olamine (CPX, Sigma-Aldrich), LOXi (PD-146176, Santa Cruz Biotechnology), Trolox (Sigma-Aldrich), Prankulast (Biomol), tunicamycin (Sigma-Aldrich), Erastin (MedChemExpress), BSO (Santa Cruz Biotechnology), cisplatin (Santa Cruz Biotechnology), doxorubicin (Sigma-Aldrich), 2-ME (Sigma-Aldrich), PPG (Sigma-Aldrich),
Nutlin-3 (Sigma-Aldrich), vildagliptin (Sigma-Aldrich), Q-VD-OPh hydrate (Sigma-Aldrich), bafilomycin A (Sigma-Aldrich), nocodazole (Sigma-Aldrich), panobinostat (Sigma-Aldrich), doxorubicin (Sigma-Aldrich). AMF-26 was synthesized by Merck KGaA^{23 (link)}.