Resistin and adenylyl cyclase-associated protein 1 (CAP1) regulate the expression of genes related to insulin resistance in BNL CL.2 mouse liver cells

Resistin is an adipokine produced in white adipose tissue that is thought to modulate insulin sensitivity in peripheral tissues (such as liver, skeletal muscle or adipose tissue). Human and murine resistin molecules share only about 60% sequence homology. [1] Contrary to humans, in which resistin is secreted mostly by macrophages, Park and Ahima 2013 resistin in rodents is produced primarily by the mature adipocytes of the white adipose tissue. Although resistin can bind to toll-like receptor 4 (TLF4) activating proinflammatory responses in human and rodents, [3], [4], [5], [6], [7], [8] the inflammatory actions of resistin in human monocytes were found to be mediated by resistin binding to adenylyl cyclase-associated protein 1 (CAP1). [9] In this study, we aimed to investigate the in vitro effects of resistin on the expression of various genes related to insulin resistance in mouse liver cells. Using BNL CL.2 cells, we investigated the effect of resistin in untransfected or CAP1 siRNA-transfected cells on the expression of 84 key genes involved in insulin resistance.


Optimization of CAP1 siRNA transfections
Our optimization experiments (data not shown) demonstrated that there was no statistically significant difference in CAP1 mRNA expression levels between untransfected and siRNA negative control-transfected cells. There was a significant decrease (86%) in CAP1 siRNA levels in CAP1 siRNAtransfected cells, compared to untransfected controls. There was no statistically significant difference in the cell viability between all of the treatment conditions.

Effect of resistin on the expression of genes related to insulin resistance in BNL CL.2 cells
Using quantitative RT-PCR array, we measured the expression levels of 84 key genes involved in the mechanisms behind type 2 diabetes mellitus (T2DM) in adipose tissue (the full list of genes is presented in Table 1, and list of genes grouped by function is provided in Table 2) in BNL CL.2 cells in the presence or absence of resistin (25 ng/ml for 24 hours) (Fig. 1).

Reagents
Mouse recombinant resistin (Sigma-Aldrich, Cat. # SRP4560) was resuspended in water to a stock concentration of 100 mg/ml and further diluted to 25 mg/ml before cell treatment.

Experiment design
BNL CL.2 cells were seeded in 6-well tissue culture plates with 2 ml tissue culture medium, in a density of 0.5 Â 10 [6] cells and grown for one day (to approximately 60% confluency). Resistin treatment was performed by adding 2 ml (25 mg/ml) resistin to the appropriate wells.  CAP1 Silencer Select siRNA (Life Technologies, Cat. # 4390771, siRNA ID# s63297) following manufacturer's protocol. Transfection was performed for 6 hours; the cell culture medium was then replaced with complete medium for overnight cell growth.

RNA extraction and reverse transcription
After completing the experiments, the cells were washed one time with ice-cold PBS and RNA was extracted using TRIzol Reagent (Ambion, Cat. # 15596018), chloroform, and iso-propanol. Total RNA concentration was quantified using NanoDrop One spectrophotometer (Thermo Scientific). All of the  samples were normalized to 1 mg/ml of total RNA. Reverse transcriptase (RT) reaction was performed using qScript cDNA SuperMix kit (QuantaBio, Cat. # 95048). RT reaction was performed using the following conditions: 25 C/5 min, 42 C/30 min, 85 C/5 min, 4 C/∞. After RT reaction, the cDNA samples were diluted 10 times with water.

Data analysis
Results were obtained from 4 separate experiments. Data analysis was performed using QIAGEN web-based software (https://dataanalysis.qiagen.com). Fold change values were calculated using DDCt method (2^(-DDCt)). p-values were calculated based on Student's t-test of the replicate 2^(-DCt) values for each gene in the control group and the treatment groups.