Tnf α

Islets isolated from wild-type and Oxtr^−/− mice were isolated and cultured in RPMI medium supplemented with 5% FBS overnight. Subsequently, only size-matched and well-shaped islets were transferred to a 12-well plate (20 islets/well), with each well represents one replicate. The islets were treated with 100 pM OXT (Sigma), 1 nM AVP (Sigma), 2 ng/mL Tunicamycin (Sigmga-Aldrich), or a cytokine mixture [IL1-β (50 U/mL) •TNF-α (1 × 10³ U/mL) •INF-α (1 × 10³ U/mL), Wako] for 24 hours. Cell death was measured by the Cell Death Detection ELISA Assay Kit (Roche) according to the manufacturer’s protocol. The cell death level was measured as the absorbance at 405 nm with respect to a substrate solution blank. The experiment was repeated for three times. The n number represents the total number of replicates from these independent experiments.

To prepare soft or stiff endothelial cells, HUVECs were treated with 10 μM of blebbistatin (Merck Millipore, Darmstadt, Germany) or 1 μM of carbenoxolone (Sigma). They were then stimulated by tumor necrosis factor-α (TNF-α) (10 units/mL, FUJIFILM Wako, Osaka, Japan) for 4 h. THP-1 cells were stained with calcein-AM and co-cultured with HUVECs for 1 h. After incubation, to remove non-adhesive cells, HUVECs were washed twice with PBS. THP-1 cells bound to HUVECs were counted using an Olympus IX71 fluorescence microscope.
To assess the effect of rsTM-mediated cellular softening on monocyte adhesion, HUVECs were treated with rsTM (0, 1, 3, 10 μg/mL) in the presence or absence of LPS (1 μg/mL) for 4 h. HUVECs were washed twice with PBS and then cocultured with HUVECs for 1 h. After incubation, to remove non-adhesive cells, HUVECs were washed twice with PBS. THP-1 cells bound to HUVECs were observed under an Olympus IX71 fluorescence microscope. Quantification of the THP-1 cells that had adhered to HUVECs was performed using ImageJ 1.53a software.

Serum was obtained by centrifugation at 1500×g for 15 min at 4 °C. The serum levels of total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed using a Hitachi 7060 Automatic Analyzer (Hitachi, Tokyo, Japan) with commercial kits (Fujifilm Wako Pure Chemical Co., Osaka, Japan). The serum insulin (Morinaga Institute of Biological Science, Yokohama, Japan), adiponectin (Otsuka Pharmaceutical Co. Ltd. Tokyo, Japan), tumor necrosis factor-α (TNF-α; Fujifilm Wako Pure Chemical Co., Osaka, Japan), and monocyte chemoattractant protein-1 (MCP-1; Proteintech, Rosemont, IL, USA) levels were also determined using a commercial ELISA kit. Serum malondialdehyde (MDA) levels were determined using a commercial kit (Japan Institute for the Control of Aging Co. Ltd., Shizuoka, Japan). The homeostasis model assessment insulin resistance (HOMA-IR), an insulin resistance index, was calculated using the following equation: HOMA-IR = fasting glucose (mg/dL) × fasting insulin (ng/mL)/22.5.

MIN6 cells or islets were incubated with a cytokine cocktail (DMEM containing 5 ng/mL IL-1β (Fujifilm Wako Pure Chemical Co.), 10 ng/mL TNF-α (Fujifilm Wako Pure Chemical Co.) and 50 ng/mL IFN-γ (Fujifilm Wako Pure Chemical Co.) for 4 h to induce cytokine production within islets (defined as “CC”). The groups incubated with MCC950 (10 µM) or not treated were defined as “CC + MCC950” and “control”, respectively. After treatments, MIN6 cells or islets were lysed with TRIzol Reagent (Thermo Fisher Scientific) and RNA was extracted from lysates using the PureLink RNA Mini Kit (Thermo Fisher Scientific). RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time PCR for cDNA was performed using the LightCycler96 System (Roche) with TB Green Premix Ex Ta II (Takara Bio Inc., Shiga, Japan) for amplification and detection. The expressions of Il1b and Nlrp3 were normalized to the expression of Rplp0. The primers were as follows: 5′-TCCAGGATGAGGACATGAGCAC-3′ (forward) and 5′-GAACGTCACACACCAGCAGGTTA-3′ (reverse) for Il1b, 5′-AATGCCCTTGGAGACACAGGA-3′ (forward) and 5′-TGAGGTGAGGCTGCAGTTGTCTA-3′ (reverse) for Nlrp3, 5′-GGCAGCATTTATAACCCTGAAGTG-3′ (forward) and 5′-TGTACCCATTGATGATGGAGTGTG-3′ (reverse) for Rplp0.

Cells were pretreated with 10 μM Z-VAD-FMK (Peptide Institute) for 1 h and then stimulated with 10 ng/ml TNF-α (Wako Chemicals) and 20 μg/ml CHX (Calbiochem) for the indicated times. Cells were then lysed with lysis buffer (30 mM Tris-HCl [pH 7.5], 120 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM PMSF, 2 mM EDTA, 2 mM imidazole, and 10 mM NaF), followed by centrifugation at 20,000 × g for 20 min at 4°C. The cleared lysates were immunoprecipitated with an anti-caspase-8 antibody and Dynabeads protein A. Immunoprecipitants were analyzed by immunoblotting.

Cells were transfected with the luciferase reporter plasmid pGL4.32[luc2P/NF-κB-RE/Hygro] (Promega, Madison, WI, USA), and stably expressing cells were selected with hygromycin. Before the luciferase assay, cells were plated on 96-well plates at a density of 1.5 × 10⁴ cells/well and cultured in DMEM for 24 h. The cells were then treated with 20 ng/mL of TNF-α (Wako, Osaka, Japan) for 5 h. Luciferase activity was measured by using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega). Each experiment was independently conducted three times, with five replicates for each sample.

HUVECs (C2519A) were purchased from Lonza Japan (Tokyo, Japan). Cells were cultured in EGM-2 Endothelial Cell Growth Medium-2 BulletKit (CC-3162, Lonza Japan). The cells were used between passages 4 and 6. All experiments were carried out with the same batch of HUVECs, which were from pooled donors. HUVECs were incubated with TNFα (10 ng/mL) (203–15263, Wako Pure Chemical Industries, Ltd., Osaka, Japan), Irisin (5 nM) (067–29, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA), and BAIBA (10 µM) (A0324, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) for 24 hours. After incubation, total RNA was extracted using 200 μL RNA iso plus. Reverse transcription and qPCR protocols are described above.