FADD regulates adipose inflammation, adipogenesis and adipocyte survival

Animal welfare

The experimental mice used in this study were all of a pure C57BL/6N background and were bred and housed at the animal facility of CAM-SU (Suzhou, China). They had unlimited access to acidified water and regular rodent chow (irradiated and autoclaved). Mouse husbandry and experimental procedures were in accordance with the protocols approved by the CAM-SU Animal Care and Use Committee under protocol number ZJ-2021-1, which was approved on December 24, 2021. All animals were randomly selected according to genotyping.

Fashion Global knockout mice (Knockout First) were generated by injecting mutant mouse ES cells (containing cassettes with mouse En2-SA, LacZ, Neo, FRT and loxP sites inserted into introns) into mouse blastocysts, followed by implantation into mouse oviducts during phantom pregnancy. Chimeric mice were then sequenced and those with positive insertions were crossed with WT C57BL/6N mice to Fashion Tm1a mice (referred to as Fashion−/−).

Preadipocyte isolation and adipogenic differentiation in vitro

iWAT and BAT SVF were prepared from WT mice and digested with collagenase I, followed by density separation to isolate preadipocytes. Briefly, iWAT and brown adipose tissue (BAT) were minced and digested in 1.25 mg/ml collagenase I for 45 min at 37°C. Digestion was terminated by addition of DMEM with 20% FBS before centrifugation to remove undigested tissue. Cells were then centrifuged at 1700 rpm for 5 min at room temperature and SVF preadipocytes were obtained in the pellet. Freshly isolated SVF cells were seeded and cultured in growth medium containing DMEM, 20% FBS and 1% penicillin/streptomycin (P/S) at 37°C with 5% CO .₂ until 100% confluence was reached. For adipogenic differentiation, the growth medium was replaced with (IM, 10% FBS, 2.85 mM insulin, 0.3 mM dexamethasone, 1 mM rosiglitazone, and 0.63 mM 3-isobutylmethylxanthine in DMEM) for 4 days, followed by differentiation in differentiation medium (DM, 10% FBS, 200 nM insulin, and 10 nM T3 in DMEM).

Measuring body composition

Total body fat percentage and muscle mass in live animals were measured without anesthesia using a Minispec LF50 body composition analyzer located at CAM-SU’s animal facility. Animals were gently placed in a specially sized clear plastic holder without sedation or anesthesia. The holder was then inserted into a designated tubular space on the side of the Minispec LF50 system. To ensure accuracy, animals were kept still in the holder during the scanning process. Each scan lasted approximately 2 minutes.

Indirect calorimetry and body composition measurement

Oxygen consumption (VO₂) and carbon dioxide production (VCO₂) of WT and Fashion+/− Mice were assessed using an indirect calorimetry system (Oxymax, Columbus Instruments) in the CAM-SU animal facility. The system maintained stable environmental conditions, including 24°C temperature and humidity, with a cycle of 12 hours of light (8 a.m.–8 p.m.) and 12 hours of darkness (8 p.m.–8 a.m.). Mice were housed individually in each chamber of the indirect calorimetry system and had free access to food and water. Prior to experiments, mice were habituated to the chambers for 24 hours. Energy expenditure levels were reported as averages corrected for the body weight of the mice. Average energy expenditure values ​​during the daytime (8 a.m.–8 p.m.) and nighttime (8 p.m.–8 a.m.) periods were calculated as the mean of all points measured during the respective 12-hour periods.

Treadmill

Before the actual test, the mice underwent a 3-day adaptation period on a treadmill set to a constant electric shock of 0.7 mA and an incline of 15%. During this period, they habituated to a running speed of 10 m/min for 10 minutes. On the day of the experiment, the mice followed a program as follows: They ran at 5 m/min for 5 minutes, increasing the speed by 2.5 m/min every 2 minutes until it reached 25 m/min, which was maintained for 4 minutes. After 25 minutes, both the treadmill and indirect calorimetry programs were stopped and the mice removed. The treadmill was then cleaned with 75% alcohol.

Glucose tolerance test (GTT)

For the GTT, mice were fasted overnight for 14 hours before being injected with 200 mg/ml D-glucose diluted in saline (2 g/kg body weight in chow-fed mice). Tail blood glucose concentrations were measured 15, 30, 60, and 120 minutes after injection using a glucose meter (Accu-Check Active, Roche). During the test, mice were housed in blind cages in a random order.

Cold treatment

For cold exposure experiments, WT and Fashion+/− Mice were individually housed and exposed to cold temperatures (4 °C) in a climate chamber for 7 days according to established protocols (48). Littermate controls of both WT and Fashion+/− The mice were kept in the same room at room temperature.

Blood biochemistry

Blood biochemistry analysis was performed using a clinical chemistry analyzer (Hitachi 7100). Approximately 200 μL of plasma was collected from each mouse by centrifugation at 5000 rpm for 15 minutes at 4 °C. If plasma samples could not be analyzed immediately, they were stored at −80 °C until analysis. Plasma samples were used either undiluted or, if volume was insufficient, diluted 1:2 with deionized water.

Hematoxylin-eosin stain

iWAT and eWAT from WT and Fashion+/− Mice were fixed in 4% PFA for 48 hours at room temperature. Tissues were then dehydrated by a gradient ethanol process, then rehydrated and embedded in paraffin using a Leica EG1150H embedding machine. 6 mm thick sections were then cut using a Leica RM2235 manual rotary microtome. For H&E staining, sections were first deparaffinized, rehydrated in water and dried. Hematoxylin staining of nuclei was performed for 15 minutes followed by six washes with water. Sections were then stained with eosin for 1 minute, dehydrated and mounted. Images were captured using an OLYMPUS IX73 microscope.

Total RNA extraction and real-time PCR

Total RNA was extracted from cells or tissues using Trizol reagent according to the manufacturer’s instructions. The purity and concentration of extracted RNA were determined using a spectrophotometer (Nanodrop 3000, Thermo Fisher) at 260 and 280 nm. The absorbance ratios (260/280 nm) of all samples were confirmed using ~2–3 μg of RNA, which was then reverse-transcribed using random primers and M-MLV reverse transcriptase to generate cDNA. Real-time PCR was performed on a Roche Lightcycler 480 PCR system using SYBR Green Master Mix and gene-specific primers from PrimerBank. The 2^−ΔΔCT The method was used to analyze the relative changes in gene expression, normalized to mouse β-actin as an internal control.

Adenovirus production and infection

The empty adenovirus (pAd-Gfp) and adenovirus with Fadd insertion (pAd-Fadd-Gfp) were purchased from Genechem (Shanghai, China). Primary WAT SVF preadipocytes were either cultured with pAd-Gfp or pAd-Fadd-Gfp next to IM.

Protein extraction and Western blot analysis

Protein was extracted from homogenized liver samples with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) containing a protease inhibitor cocktail (Sigma) and the phosphatase inhibitors NaF and Na.₃VO₄. Protein concentrations were determined using Pierce BCA Protein Assay Reagent (Pierce Biotechnology). Equal amounts of proteins from each sample were loaded for electrophoresis (Bio-Rad). Proteins were separated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane (Millipore Corporation), incubated in blocking buffer (5% nonfat milk in TBS) for 1 hour at room temperature (RT), and then incubated with primary antibodies (anti-GFP, 50430-2-AP and anti-HSP90, 13171-1-AP from Proteintech; anti-FLAG, sab4301135 from SIGMA; anti-RIPK1, 3493, anti-RIPK3, 10188, anti-BCL2, 15071, and anti-cleaved-caspase-3, 9664 from Cell Signaling Technology) overnight at 4 °C in blocking buffer.

Statistical analysis

All analyses were performed using Student’s T-test (two-sided). Experimental data are expressed as mean ± SEM. Comparisons with P Values ​​< 0.05, < 0.01 or < 0.001 were considered statistically significant.