Differential Gene Expression Profile in Pig Adipose Tissue Treated with/without Clenbuterol

Background Clenbuterol, a beta-agonist, can dramatically reduce pig adipose accumulation at high dosages. However, it has been banned in pig production because people who eat pig products treated with clenbuterol can be poisoned by the clenbuterol residues. To understand the molecular mechanism for this fat reduction, cDNA microarray, real-time PCR, two-dimensional electrophoresis and mass spectra were used to study the differential gene expression profiles of pig adipose tissues treated with/without clenbuterol. The objective of this research is to identify novel genes and physiological pathways that potentially facilitate clenbuterol induced reduction of adipose accumulation. Results Clenbuterol was found to improve the lean meat percentage about 10 percent (P< 0.5). The adipose cells became smaller and the muscle fibers became thicker with the administration of clenbuterol. The mRNA abundance of 82 genes (ESTs) were found to be statistically differentially expressed based on the Student t-test (P<0.5) in the microarray analyses which containing 3358 genes (ESTs).Theses 82 genes (ESTs) were divided into four groups according to their Gene Ontology Biological Process descriptions. 16 genes were cellular metabolism related genes (including five lipids metabolism related such as apolipoprotein D and apolipoprotein R), 10 were signal transduction related genes, 45 were expressed sequence tags (ESTs) and other 11 others were of various categories. Eleven of the 82 genes (ESTs) were chosen for real-time PCR analysis, with eight of them induced to express similar magnitudes as in the microarray analyses, two could not be detected and one expressed magnitudes that disagreed with the microarray results. Apolipoprotein R was also found to be up-regulated by the proteomic analysis. Conclusions Profound changes were observed in adipose cells after administration of clenbuterol in pigs by histological section and cDNA microarray techniques. The adipose cells became smaller with stimulation by clenbuterol. Changes in mRNA abundance indicated that DNA transcription and protein translation were enhanced in the adipose cells with clenbuterol stimulation. Five lipids metabolism related genes, such as apoD and apoR, were significantly up-regulated. ApoR was up-regulated in both the mRNA transcription and the protein synthesis. ApoR may be one of the critical molecules through which clenbuterol reduces fat accumulation. Keywords: dose response Eight cDNA microarray slides were hybridized for the eight pigs. Eight Chinese miniature pigs were used in the experiments. Four hogs and four sows, all 4 weeks old, were housed in the Nutrition and Metabolism Laboratory at the China Agriculture University. They were raised under exactly the same conditions and were fed the same diets until 8 weeks (average body weight 17 kg). They were randomly divided into 4 groups with each group having two pigs with the same gender and the same parents. For the following 4 weeks, one pig in each group was fed 25 mg/kg clenbuterol twice daily in their diets as the test pig, while the other was fed the same diet without clenbuterol as the control. Then one group of hogs and one group of sows were slaughtered for analysis. These two groups are referred to as the 3 month-old pigs. The other two groups were fed with/without 50 mg/kg clenbuterol twice daily in their diets for another 4 weeks and slaughtered for analysis. These two groups are referred to as the 4 month-old pigs. Approximately 1 g biopsies were taken from the back fat adipose tissues (at the fifth lumbar vertebra level) of each pig. The adipose tissue samples were washed in sterile water, snap frozen in liquid nitrogen and stored at -80℃. Total RNA of the adipose tissue was extracted with TRIZOL reagent (Invitrogen, Gaithersburg, MD, USA) and further purified with an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturers instructions. The porcine cDNA microarray was produced at the China Agricultural University. A total of 11520 spots representing 3358 genes (ESTs) and 1446 quality control spots were included on the microarray slide. 3358 genes (ESTs) were cloned from the procine adipose cDNA library and were printed in triplicate on each slide. (The cDNA microarray gene list and clone numbers are listed in additional file 3. More details on the specific genes and probe sequences are given by Guo et al [39]). A cDNA microarray hybridization analysis was performed with Pronto!TM Universal Microarray Kits (Corning, MA, USA) according to the manufacturers instructions. Briefly, reverse transcription was done with 10 µg total RNA as the template to synthesize cDNA incorporated with flourence dye Cy3-dCTP or Cy5-dCTP. Probes were purified on a Qiagen spin column (Qiagen, Valencia, CA, USA). Mix dye-labeled cDNA together and dry dye-labeled cDNA were used for hybridization (see Pronto Universal hybridization kit Quick Reference Guide). To avoid dye bias, the experiments were performed in duplicate by dye swap with Cy5-dCTP first used with the test pig and Cy3-dCTP used with the control pig and then Cy3-dCTP used with the test pig and Cy5-dCTP used with the control pig.