Animal Studies and Cold Exposure
C57BL/6J mice were purchased from the Jackson laboratory (Bar Harbor, ME, USA), bred and kept in a barrier animal facility in the Dasman Diabetes Institute (DDI) under constant temperature (22℃±1℃) and humidity in a 12 h controlled dark/light cycle (lights on: 7:00 am to 7:00 pm). All mice were kept with ad libitum access to a normal chew diet containing 6.2% calories as total fat (EURodent Diet 14%, 5LF2, LabDiet, St. Louis, MO, USA) and drinking water.
A total number of 45 male mice, age 12 weeks old were used in this study. Mice were individually kept in cages with minimum required bedding with free access to the food and water. The mice were randomly allocated in two groups, namely cold exposure group (n= 40) and room temperature control group (n=5). At the end of study, the levels of weight gain and food intake were measured, and the mice were sacrificed. Liver biopsies and fat depots including subcutaneous adipose tissue (SAT)and brown adipose tissue (BAT) were collected for further analysis. All the procedures were approved by the DDI Animal Care and Ethics Committee according to the international regulations and laws on animal research.
For cold exposure, 40 male mice were singly housed in prechilled cages with minimum bedding on a 12 -hour dark/light cycle with ad libitum access to chow diet and water in a cold controlled environment at 4°C. At days 1, 3, 5, and 10 post-cold induction, mice (n=10 in each group) were sacrificed and the required samples were harvested for further analysis.
In a preliminary experiment, 10 mice were kept under cold conditions at the mentioned time points and their physical activity was observed and rectal temperature was monitored using a microprobe (50-7221F, Harvard Apparatus, Holliston, MA, USA) to rule out stress and hypothermia incidences.
RNA isolation and quantitative real-time PCR
Dissected tissues were immediately placed in RNAlater RNA Stabilization Reagent (Qiagen Inc.) and stored at −800C for subsequent RNA extraction. Total RNA was extracted and isolated from the tissues using TRIzol reagent (Cat. No. 15596026, Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. For analyzing mRNA expression, the extracted samples were first quantified to assess their quality and concentration using the Epoch microplate spectrophotometer (Biotek Instruments, Inc., Winooski, VT, USA). cDNA was synthesized from 1 μg RNA using the High-Capacity cDNA Reverse Transcription kit (Cat. No. 4368814, Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed in duplicates using SYBR Green primers and on an Applied Biosystems 7500 Real-Time PCR System. Relative gene expression (normalized to 18S) was calculated using the comparative CT method formula 2-ΔΔCT and reported as mean fold change in gene expression. The following primers were used for amplification: ANGPTL3 (FW: TGCACCTTCAGAGCCAAAAT, RV: CATTGGTTCGAAGTGATAGGTCA), ANGPTL4 (FW: ACAGTGACTTTGGTTGTGGC, RV: CTCGAGCCCATGTTTTCTGG), ANGPTL8 (FW: CTCTCTGCCTCCTGTGGAC, RV: GCTCTGTACACGCCATTGAG), UCP1 (FW: CTTTGCCTCACTCAGGATTGG, RV: ACTGCC ACACCTCCAGTCATT) and 18S (FW: CTGAGAAACGGCTACCACATC, RV: GGCCTCGAAAGAGTCCTGTAT).
Immunohistochemistry and Confocal imaging
Formalin fixed and paraffin embedded sections (of 8µm thickness) from SAT of each time point were used. These sections were de-paraffinized, rehydrated and antigen retrieved at pH6 using DAKO reagents (Dako, Glostrup, Denmark). Quenching of endogenous peroxidase activity was done using 3% hydrogen peroxide solution (1 hr at room temperature). The sections were then blocked, first with 5% fat free milk followed by 1% BSA. They were then incubated with the primary antibody ANGPTL 8 (MAB8548, R&D systems, USA) at 4˚C, overnight. Alexa fluor 488 conjugated secondary antibody was used at RT for 1 hr (A-11008, Invitrogen, USA, 1:100 dilution). Nuclear staining was done using DAPI at 0.05%. Zeiss LSM 710 confocal laser scanning microscope (Zeiss, Germany) was used to acquire the fluorescence images. The images were taken at 40X magnification for each group. Zen software (Zeiss, Germany) was utilized for quantification of the image intensities.
The minimum sample size of 4-6 mice per group was estimated to obtain statistical significance, considering the error = 0.05, power = 0.80, percentage change in means (PC) = 20%, and co-efficient of variation (CV) = 10 ~ 15% (varies between the experiments). Together with our prior experience, we used a total of 5 mice as a room temperature control group, and n=10 mice in each group for cold exposure to ensure adequate power. Values are expressed as means of duplicate experiments ± SEM of each harvested sample. All data was analyzed, and figures were prepared using GraphPad Prism 6 (GraphPad Software). Statistical differences between two groups were assessed by unpaired, 2-tailed Student’s t test and between multiple groups using nonparametric Kruskal–Wallis test for multiple comparisons. A P value of less than 0.05 was considered statistically significant.