Mice and treatments
Male LATY136F mutant C57BL/6J mice and CD3ε knockout C57BL/6J mice were obtained from Centre d’Immunologie de Marseille-Luminy in France. Mice were maintained under SPF conditions. All animal experiments in this study were approved by the Welfare and Ethical Committee for Experimental Animal Care of Xinxiang Medical University. These mice under standard laboratory conditions with controlled temperature (19–22 °C) and 12-h light/dark cycle. Purified Th2 cells were obtained from mesenteric lymph nodes of LATY136F mutant C57BL/6J mice with CD5 MicroBeads (130-049-301 Miltenyi) following the standard protocol. 2 x106 purified Th2 cells in 200 μl PBS or 200 μl PBS only were injected into CD3ε knockout C57BL/6J mice via tail vein respectively. After that, the two group mice were subjected to high-fat diet (D12492i, Research Diets, 60 kcal%) for 16 weeks.
Metabolic and physical activity measurements
Animals were placed individually in chambers for 5 consecutive days at ambient temperature with 12 h light/dark cycles. Animals had free access to food and water. At the end of 16 weeks, the mice were given 2 days of acclimation in metabolic chambers before the trial and then continuously recorded for 96 h with the following measurements being taken every 30 min: food intake, water intake, ambulatory activity (in X and Z axes), and gas exchange (O2 and CO2). All measurements were taken automatically through the use of the SABLE SYSTEMS INERNATIONAL (USA).
Glucose and insulin tolerance test
The glucose tolerance test (GTT), and insulin tolerance test (ITT) were conducted in mice after 6-h fasting. The mice were intraperitoneally injected with 2 g/kg glucose for GTT, and 0.75 U/kg insulin for ITT. In the studies, the blood glucose was tested at six time points (0, 15, 30, 60, 90 and 120 minutes).
RNA isolation and real time RT-PCR
Total RNA was isolated from animal tissues with TRIzol (Invitrogen, Carlsbad, CA). RNA concentration was measured by a spectrophotometer (Thermo Scientific, Ltd, Waltadult mice, MA). The first strand of cDNA was obtained using the PrimeaScript™ II 1st Strand cDNA synthesis kit (Takara, Ltd, Japan) according to the manufacturer’s instruction. Subsequently, SYBR green-based qPCR was performed using a power SYBR greenmaster mix (Applied Biosystems, Foster City, CA) and 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). The expression was normalized to mouse ribosome 18S rRNA.
EAT and liver were collected from mice and snap frozen in liquid nitrogen and splitted in RIPA buffer of protease inhibitors. Homogenates were cleared by centrifugation (10000 rpm for 30 min at 4°C). Protein lysates (30 μg) were separated on 10% SDS-PAGE and transferred on polyvinyldifluoride membranes (Millipore, Bedford, USA). Membrane were blocked in tris-buffered saline (TBS, pH 7.4) containing 5% nonfat milk at room temperature for 1 h and incubated with the primary antibodies overnight at 4 °C. After washing, the membranes were incubated with the HRP-conjugated secondary antibodies at room temperature for 1 h. Detection and analysis was performed with Chemidoc XRS Image system and Image Lab 5.0 software (Biorad).
Cell culture and induction of adipocyte differentiation
3T3-L1 cells were maintained in DMEM supplemented with 10% calf serum and 5% CO2. For adipocyte differentiation, the cells were differentiated with 10 μg/mL insulin, 1 μM dexamethasone and 0.5 mM 1-methyl-3-isobutylxanthine (IBMX) in DMEM containing 10% (v/v) fetal bovine serum (FBS) and 1% penicillin-streptomycin for 2 days. The medium was then replaced by a medium containing only insulin. After 2 days, this maintenance medium was discarded. Differentiated adipocytes were cultured in DMEM containing 10% FBS.
Mice were killed at indicated time points and adipose tissue and liver were carefully harvested. For hematoxylin and eosin (H&E) staining, tissues of mice were fixed in 4% paraformaldehyde (PFA) overnight at 4℃, followed by dehydration in 70% ethanol. After the dehydration procedure, tissues were embedded in paraffin, sectioned at a thickness of 5mm, and stained with H&E following the standard protocol. Images were acquired using a microscope (LEICA 500).
Co-culture of Th2 Cells and 3T3-L1 Preadipocytes
3T3-L1 preadipocyte cells were plated in 24 wells at a density of 1×105 cells/well and maintained in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS. To evaluate the effect of Th2 cells on 3T3-L1 induction and differentiation, 2-days-postconfluent cells (day 0) were treated with different number of purified Th2 cells (5×104 and 1×105 Th2 cells) and induction medium Ⅰ (DMEM contains10% FBS, 1.73μM insulin, 1μM dexamethasone, 0.5mM 3 -isobutyl-1-methylzanthine, 0.2μM indomethaein and 1μM pioglitazone) every 2 days until day4, and control group was treated without Th2 cells. The medium was replaced with induction medium Ⅱ (DMEM contains10% FBS and 1.73μM insulin) every 2 days until day 8.
All results were expressed as mean ± SEM. Statistical significance between two groups was evaluated using the Student's t-test, while comparisons of multiple groups were assessed by two-way analysis of variance (ANOVA), followed by Student-Newman-Keul's test. p<0.05 was considered significant. For morphometric analyses, quantification of micrographs was independently reviewed by two observers and the average of their scoring was used for each micrograph. Quantification was performed in images after appropriate thresholding using the Image J software (NIH Image).