Teklad global 18 protein rodent diet 2918

IRAKM knockout mice were generated by Dr. Richard Flavell (Yale School of Medicine, New Haven) as described^{51 (link)}. IRAKM flox/flox mice were generated by our lab as described^{52 (link)}. Adipocyte-specific deletion of IRAKM (IRAKM^AKO) was obtained by breeding IRAKM flox/flox mice with Adiponectin-Cre transgenic mice (Jackson Laboratory, 028020). IRAKM kinase-dead(K205A) knock-in mice were generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences). Six- to eight-week-old male mice were maintained on a high-fat diet composed of 60% kcal derived from fat (TD06414, Envigo) for 12 weeks, or on high carbohydrate (62% Sucrose)/zero-fat diet (TD03314, Envigo) for 8 weeks while normal mice maintained on either standard rodent chow diet (2918 Teklad Global 18% Protein Rodent Diet, Envigo). Animals were housed in a specific pathogen-free facility at a temperature of 21 °C, relative humidity of 50–70%, and under a constant 12-h light/dark cycle and given free access to food and water. All procedures using animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Cleveland Clinic (protocol number: 2020–2316). Ethical compliance with IACUC protocols and institute standards was maintained.

Female C57/BL6 mice (#000664) were purchased from Jackson Laboratory. Car^−/− mice were obtained from Dr. David Moore's laboratory as previously described [19 (link)]. The mice were housed in flow cages at 24 °C on a 12/12-hour-light/dark cycle, with lights on starting at 6 Am CST, corresponding to zeitgeber time (ZT) 0. Female 8- to 10-week-old mice were used for all experiments. Mice were allowed ad libitum access to a standard chow diet (Teklad Global 18% Protein Rodent Diet, 2918, ENVIGO) and water. WT and Car^−/− mice were randomly divided into a control or treatment group. With oral pipetting, control mice were dosed with vehicle (honey, water, and DMSO mixture, 4:1:1 ratio). Treatment group mice were dosed twice daily (at 7 Am and 7 Pm) for 21 days with 50 mg/kg imidacloprid (PESTANAl, analytical standard, Millipore Sigma) solubilized in a honey-DMSO solvent. Mice were monitored for lethargy, shaking, diarrhea, and body weights were measured once daily over the 21-day treatment period. All mice were sacrificed at ZT4-6. Blood serum and tissues (liver, ileum, colon, kidney, and spleen) were collected for analysis. Tissue was flash-frozen for RNA analysis or fixed in 10% formalin for histological analysis. Genotype was confirmed by PCR (n = 5-8 mice per genotype) as described [19 (link)].