PKD3 floxed mice (PKD3f/f) (20) and loxP-STOP-loxP-(3xFlag)PKD3ca mice, in which the two serines in the PKD3 kinase domain are mutated to glutamic acid (S731E/S735E) (generated by Cyagen with the PiggyBac transgenic method), were mated with mice expressing transgenic Cre recombinase under the control of the albumin promoter/enhancer (the Jackson laboratory) (21) to generate homozygous liver-specific PKD3-deficient mice (PKD3liverΔ/Δ) and heterozygous liver-specific transgenic PKD3ca mice (TgPKD3caliver), respectively. All animal studies were approved by the local animal welfare authorities (Regierung von Unterfranken) with the animal protocol nos. AK55.2-2531.01-124/13 and AK55.2.2-2532-2-741-13. The mice were housed in cages from Tecniplast in a green line IVC rack system with ad libitum supply of water and normal chow diet (ssniff Spezialdiaeten). In that case of HFD (Research Diets, D12331i), mice received the high-calorie diet (58% kcal from fat) from the age of 3 weeks for a duration of 24 weeks, and BW measurements were performed weekly. For dissection, mice were sacrificed by cervical dislocation, and organs [liver, gonadal white adipose tissue (gWAT), subcutaneous white adipose tissue (sWAT), brown adipose tissue (BAT), and skeletal muscle (SKM)] were weighted and snap frozen in liquid nitrogen. For fasting/refeeding experiments, mice were fasted overnight for 16 hours with free access to water, and food was restored for 4 hours before livers were removed as described before. For insulin injections, mice received an intraperitoneal dose of 8 U/kg BW of insulin (Sigma), and livers were collected after 15 min according to standard procedures. For in vivo experiments using CRT0066101, C57BL/6JRj mice (Janvier Labs) were fed an HFD for 8 weeks and were randomly assigned to two groups that either received an intraperitoneal injection 10 mg/kg BW of CRT0066101 in 5% dimethyl sulfoxide (DMSO) or vehicle for five consecutive days.

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