Amastatin

Buffalo worm (A. diaperinus larvae powder) was provided by Protifarm NV (Ermelo, Netherlands). Tenebrio molitor flour of subadult insects was obtained from Iberinsect, S.L. (Tarragona, Spain), and processed by FoodIE Research Group, University Rovira i Virgili, Spain. The nutritional composition of these samples is shown in Supporting Information Table S1.
Chemicals, porcine digestive enzymes (α‐amylase, pepsin, and pancreatin), bile salts, bovine serum albumin (fatty acid free), d‐glucose, d‐mannitol, amino acids, aprotinin, protease inhibitor cocktail (cOmplete™ ULTRA Tablets; Roche), and foetal bovine serum were purchased from Sigma‐Aldrich (Madrid, Spain). Amastatin was from Enzo Life Sciences (Madrid, Spain). Glutamine, penicillin, streptomycin, and Matrigel from Lonza (O Porriño, Spain).
The enzyme‐linked immunosorbent assay kits for total ghrelin (catalogue no. EZGRT‐91K) and total GLP‐1 (catalogue no. EZGLPT1‐36K) were purchased from Millipore (Billerica, MA, USA). Plasmatic parameters were measured with commercial kits according to manufacturer's instructions: insulin with an insulin enzyme‐linked immunosorbent assay kit (catalogue no. EZRMI‐13K) from Millipore (Madrid, Spain), and glucose triglycerides and cholesterol from QCA (Amposta, Spain). The Pierce BCA Protein Assay kit was from ThermoFisher (Waltham, MA, USA).

After the intestinal tube was washed with cold PBS buffer, the outer muscular layer was removed from the serosa layer with a scalpel. For each segment, the intestinal tube was then cut longitudinally and circular slices of tissue 5 mm in diameter were taken using a biopsy punch. The samples were kept at low temperature in an ice-cold Krebs–Ringer bicarbonate (KRB) buffer bath (Hepes 11.5 mM, CaCl₂ 2.6 mM, MgCl₂ 1.2 mM, KCl 5.5 mM, NaCl 138 mM, NaHCO₃ 4.2 mM, NaH₂PO₄ 1.2 mM) supplemented with 10 mM D mannitol for the whole procedure. The tissue segments were placed in a prewarmed (37 °C) KRB-D-mannitol buffer for 15 min to stabilize the tissue. The medium was then replaced for the treatments: KRB-D- glucose buffer as a control and peptone from bovine meat, enzymatically digested (Cat. no: 70175, Sigma-Aldrich, Madrid, Spain), to stimulate enterohormone secretion. The tissue segments were incubated for 30 min in a humidified incubator at 37 °C, 95% O₂ and 5% CO₂. After this period, the whole volume was aliquoted and stored at −80 °C for further measurements. KRB containing 10 mM D-glucose was supplemented with protease inhibitors amastatin 10 µM (Enzo Life Sciences, Madrid, Spain), aprotinin 100 KIU (Sigma, Barcelona, Spain) and 0.1% fatty acid free-bovine serum albumin (BSA).

GSPE was obtained from Les Dérivés Résiniques et Terpéniques (Dax, France). The same batch (#124029) was used in all studies. According to the manufacturer, the extract contains monomers of flavan-3-ols (21.3%), and dimeric (17.4%), trimeric (16.3%), tetrameric (13.3%), and oligomeric (5–13 U; 31.7%) proanthocyanidins. Catechin, (-)-Epicatechin, gallic acid (GA), 3-hydroxyphenyl acetic acid (3-HPAA), and protocatechuic acid (PCA) were obtained from Sigma (St. Louis, MO, USA), while procyanidin dimer B2 (B2) was obtained from Extrasynthese (Genay, France). For all studies, stocks were prepared in dimethyl sulfoxide (DMSO) and further diluted in the specific buffer required for each experiment. To transport and treat the intestinal samples, we used Krebs–Ringer bicarbonate (KRB) buffer (Hepes 11.5 mM, CaCl₂ 2.6 mM, MgCl₂ 1.2 mM, KCl 5.5 mM, NaCl 138 mM, NaHCO₃ 4.2 mM, NaH₂PO₄ 1.2 mM) pH 7.4, prepared with either 10 mM D-Glucose (KRB-D-Glucose buffer) or 10 mM D-Mannitol (KRB-D-Mannitol buffer). For the enterohormone secretion studies, KRB-D-Glucose was supplemented with protease inhibitors: 10 µM amastatin (Enzo Life Sciences, Madrid, Spain), 500 KIU aprotinin (Sigma, Madrid, Spain), and 0.1% fatty acid free-bovine serum albumin.