Complete ultra tablet

NIH 3T3 mouse fibroblast cells (ATCCRCRL-1658) were maintained in Dulbecco’s modification of Eagle’s medium (DMEM, with 4.5 g/l glucose, L-glutamine & sodium pyruvate, Corning) with fetal bovine serum (FBS, 10% v/v, Biowest) and Penicillin/Streptomycin (100 U/mL, Roche Diagnostics GmbH). Cells were cultured on 10 cm sterile plates in 37°C, in humidified a chamber with 5% CO_2. Lipofectamine 2000 (Invitrogen) was used to transfect cells (at 70% confluence, one day after seeding) according to manufacturer’s protocol. 10 μg of plasmid DNA (4 variants of shRNA carrying plasmids, OriGene TL501619) DNA was used to transfect one plate. The medium was replaced 6 hours after transfection with a standard one. 3 days after transfection cells were washed twice with cold PBS (137 mM NaCl, 7.97 mM Na₂HPO₄ × 12 H₂O, 2.68 mM KCl, 1.47 mM KH₂PO₄), detached in PBS and moved to a 1.5 ml tube. After centrifugation the cell pellet was lysed in 1 ml of cold RIPA buffer (50 mM Tris-HCl pH7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF) with freshly added protease (cOmplete ULTRA tablets, EDTA-free, Roche Diagnostics GmbH) and phosphatase inhibitors (PMSF 1mM, Na₃VO₄ 0.2mM). The protein isolate was separated from cell debris by centrifugation (15 min, 14000g, 4°C) and stored at -20°C.

The Helicobacter hepaticus strain Hh-2 (ATCC 51448)^{59 (link)} was purchased from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures (DSM No.22909) and cultivated at the Max von Pettenkofer-Institute, LMU Munich. Bacteria from cryo stock were resuspended in Brain Heart Infusion (BHI) medium and put on blood agar plates (Columbia agar with 5% sheep blood, BD, Cat: 4354005). Plates were incubated in a chamber with anaerobic conditions (83% N₂, 10% CO₂, 7% H₂) for 4 days at 37°C. A subculture was cultivated further on in BHI medium with 3% sheep serum in a culture flask in the chamber with anaerobic conditions for additional 4 days at 37°C.
For Hh lysate (HhL) preparation, bacterial cells were harvested and washed 2–3 times with PBS. Cell pellets were resuspended in PBS and lyzed by sonification with the Sonifier 150 Cell Disruptor (Branson) 6 times for 3 min at level 3 on ice. Lyzed cells were centrifuged at 20,000 x g for 30 min at 4°C and the supernatant was mixed with a protease inhibitor (cOmplete ULTRA Tablets, Roche, Sigma-Aldrich, Cat: 05892953001). Protein concentration was determined using the Qubit Protein Assay Kit and Fluorometer (Invitrogen), according to the manufacturer’s instructions and the lysate was stored at −20°C until use for immunoblot or ELISA.

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).

Florets were detached from the spadices and used for the preparation of mitochondria. Purification was performed as previously reported23 (link), with the exception that protease inhibitors (cOmplete ULTRA tablets (Roche), or 10 μM E-64 and 0.5 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) were added upon initiation of the grinding of the florets.
Oxygen consumption rates were determined using freshly prepared mitochondria as described previously20 21 (link). Mitochondrial respiration with citrate as a substrate was measured in an assay buffer21 (link) containing 10 mM citrate, 0.5 mM ADP, 10 mM malonate and 0.1 mM rotenone at 8°C, 15 °C, 23 °C and 30 °C. For respiration assays with NADH as a substrate, 1 mM NADH, 0.5 mM ADP and 0.1 mM rotenone were added to the assay buffer21 (link) at 15 °C, 23 °C, 30 °C and 37 °C. Capacities for AOX-mediated pathway were determined as KCN-insensitive (0.5 mM) and n-propyl gallate-sensitive (0.1 mM) respiration. Pyruvate (10 mM) was added where indicated. COX capacities were also determined as KCN-sensitive (0.5 mM) and n-propyl gallate-insensitive (0.1 mM) respiration. Protein concentrations of purified mitochondria were determined as in our previous report24 (link). Mitochondria were stored at −80 °C and used for enzyme assays.

Samples were lysed in RIPA buffer (RIPA Lysis and Extraction Buffer, cat. 89900, Thermo Fisher Scientific) (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (cOmplete™ ULTRA Tablets, Roche). Proteins were separated by SDS-PAGE (Tris-glycine gels with Tris/glycine/SDS buffer) and transferred onto nitrocellulose membranes (Whatman, Dassel, Germany), using the Mini Trans-Blot^® Cell (Bio-Rad). Membranes were probed over night with antibodies directed against P2Y10 (Life Technologies GmbH, #PA570914, 1:1000) or GAPDH (Cell Signalling Technology, #2118, 1:1000). After washing, membranes were probed with horseradish peroxidase-conjugated antibodies directed against rabbit or mouse IgG (1:3000, Cell Signalling Technology Europe). The target proteins were visualized by enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) and a ChemiDoc MP Imaging System (Bio-Rad).