DNA Microarray Study On The Molecular Mechanism Of Adipose Reduction By Clenbuterol-HCL Administration In Pigs

China is a country with a large production of porcine. However, pigs of Chinese local breed have a low lean rate, which is undesirable for porcine industry. Clenbuterol-HCL (CLB-HCL), as a kind of beta-agonist, can intermediate the reduction of fat deposition dramatically when fed to animals. It has been banned because it is possible to poison people who eat the porcine containing CLB-HCL. To understand the mechanism of CLB-HCL reducing fat accumulation on molecular level, we analyzed fat tissue gene expression profile of pig xiang (Totally 8 pigs were employed for 4 biological repetitions. In each group, the test pig was fed CLB-HCL, while the control pig was fed without CLB-HCL.) with cDNA microarray which was developed by our laboratory. We also conducted the gene differential expression profile of two groups' liver tissues again with Affymetrix Porcine Genome Genechips. The lean rates of the test pigs in these two groups were improved more than 10%. Furthermore, we used Affymetrix Genechip of Rat Expression Set 230A to analyze the gene expression profile of rat adipocyte co-cultured with CLB-HCL in vitro.In the fat tissue gene differential expression profile, we found the transcription of two genes up regulated more than 2 fold. They are the gene of cAMP dependent protein kinase type I regulatory (GenBank access: X05942) and the gene of small GTP-binding protein rab30 (GenBank access:U57092). Both of them are involved in the G-protein coupled signal pathway. We suppose that these two molecules transfer the signal when CLB-HCL combined beta-androgenic receptor. We also found that the transcription of gene Apolipprotein D and Apolipprotein R was up regulated 2.20 and 2.24 fold respectively, while the transcription of gene stearoyl-CoA desaturase (SCD) was down regulated 0.41 fold. All of these three genes involve in lipid metabolism. Besides, we found 9 clone standing for 3 genes of collagen protein [ α 1 (COL1 Al) and α 2(COL1 A2) of Collagen I and α 1 of collagen III ] up regulated more than 2 fold. This may indicate that the differentiation rate of preadipocyte co-cultured with CLB-HCL became slower than control cells. We also found 9 expression sequence tag (EST) which do not have homologous sequence in National Center of Biotechnology Information(NCBI) nucleotide database (Oct., 2005). We suppose they are new genes which may involve in lipid metabolism. Apolipoprotein D, stearoyl-CoA desaturase and EST rpfat 15972 were confirmed by real-time quantitive PCR.In liver gene differential expression profile, we found 276 genes changed 2 fold or more (158 up regulated and 118 down regulated). According to molecular function classification method of Geneontology provided by European Bioinformationtic Institute, 276 genes were divided into six groups, binding (11), catalytic activity (27), defense immunity activity (8), signal transducer activity (4) transportor activity (9) and unclassification (212). In 27 gene of catalytic activity, the EST, which is moderately similar to carbonyl reductase 1, was upregulated 3.94 fold. Carbonyl reductase 1 (GenBank access:NP₀01748.1) involves in metabolism of prostaglandin that can promote lipid hydrolysis. Also in catalytic activity group, type I iodothyronine deiodinase (GenBank access: NM 001001627.1), which belongs to iodothyronine deiodinase family, was upregulated 2.81 fold. Itencodes a selenoprotein that deiodinates prohormone thyroxine (T4, 3,5,3',5'-tetraiodothyronine) to bioactive T3 (3,3',5-triiodothyronine). One effect of Thyroxin is to reduce fat accumulation and improve muscle growth. The 4 differential genes of signal transducer activity group includes 3 genes related with G-protein signal pathway, which are the gene of regulator of G-protein signalling 1 (GenBank access: AF139837.1) and two EST moderately similar to regulator of G-protein signalling 18. We chose 6 genes for real-time PCR to confirm our results. 100% of them agree with the genechip data.In the experiment of adipocyte co-cultured with CLB-HCL in vitro, we found 508 genes differential expressed 2 fold or more, including 271 up regulated and 237 down. In the differential expressed genes, biological function of 68 up regulated and 72 down regulated were known. There were 31 genes involve in lipid metabolism in biological function known genes. The gene expression of glycerol 3-phosphate acyltransferase (GenBank access: AF021348.1) was suppressed 0.27 fold, which catalyzes the initial and committing step in glycerolipid biosynthesis. This genes is predicted to play a pivotal role in the regulation of cellular triacylglycerol and phospholipid levels. While the gene expression of adipocyte lipid-binding protein (GenBank access: NM₀53365.1) was induced 4.0 fold, which take part in lipid hydrolysis. Besides, we found 5 members of Apolipprotein family differential expressed more than 2 fold.In addition, frozen tissue section data of subcutaneous adipose tissues indicate that adipocyte size became smaller when pig fed with CLB-HCL.In summary, we conclude that the molecules involved in G-protein coupled signal pathway were stirred up (such as small GTP-binding protein rab30, regulator of G-protein signalling 1 and cAMP dependent protein kinase type I regulatory) when CLB-HCL combines to beta- androgenic receptor. Then the genes, which take part in lipids biological synthesis, were down regulated, such as 3-phosphate acyltransferase. While the genes involving in lipids hydrolysis were upregulated, such as adipocyte lipid-binding protein (FABP). Some Apolipprotein members expression were changed to alter the transportation and distribution of lipids. All of these change lead to reduction of lipid droplets in adipocytes, then adipocytes won't grow as large as usual. Finally, the fat deposition was reduced.There are many research works concerning either pig fat accumulation or CLB-HCL effects. We first combine these two things together. We explore pig fat accumulation mechanism on molecular level with animal model treated with CLB-HCL. This research lays a good foundation for pig breeding. According to our results, we can modify the genes which transcriptions have been changed in our experiment to obtain new pig breed with high lean rate and high meat quality in a short time.