Study On The Cloning And Expression Of Angptls In Pig

Angptls are newly discovered family of secrete proteins which are not only related toangiogenesis but also involved into the metabolism of fat and glucose as well as thesensitivity to insulin. They belong to the fibrinogen protein superfamily with similarstructure of angiopoietins, which contain a signal peptide for secretion, an extendedhelical domain predicted to form dimeric or trimeric coiled-coils in the N-termimalprotion and a fibrinogen-like domain to regulate ligand activity in the C-terminal portion.However, they are not really angiepoietin for not binding to the Tie2 receptor, so they arecalled angiopoietin-related protein (ARP) or angiepoietin-like protein (Angptl).Using Meishan pigs, this study reported the cloning, chromosomal location andtissue expression pattern analysis of porcine Angptls. The expression difference in mRNAlevels of these genes was also compared between obese pigs (Tongcheng) and lean pigs(Landrace). Finally, the expression regulation of these genes by some factors wasconducted using HepG2 cells. The main results are as follows:1. Five Angptls were cloned using the techniques of RT-PCR and RACE. Sequenceanalysis showed that Angptll contained an open reading frame of 1,476bp, whichencoding 491 amino acids, Angptl2 contained a coding region of 1,482bp, whichencoding 493 amino acids, Angptl3 contained a coding region of 1,389bp, whichencoding 462 amino acids, Angptl4 contained a coding region of 1,239bp, whichencoding 412 amino acids, and Angptl6 contained a coding region of 1,362bp (30bp or soshorten in 5'), which encoding 453 amino acids.2. Five intron sequences were amplified using primers designed according to thesequences of cDNA from porcine and the sequence of mRNA and genome DNA fromhuman. Then primers were designed according to the sequences of these introns to mapthese 5 genes using radiation hybrid (RH) technique. The 5 genes were mapped on 4chromosomes. Among them, Angptl4 and Angptl6 were mapped on a same chromosomalregion. The positions of these genes are corresponding to that in human.3. Tissue expression patterns were analyzed by the method of RT-PCR in 10 porcine(Meishan) tissues including white adipose, muscle, heart, stomach, liver, lung, spleen, gut, brain and kidney. The results showed that porcine Angptl1, Angptl2 and Angptl4 wereubiquitously expressed in many tissues. Among them, Angptl1 was highly expressed inwhite adipose, stomach and spleen, Angptl2 was highly expressed in white adipose andgut, and Angptl4 was most abundant in white adipose. However, porcine Angptl3 andAngptl6 were exclusively expressed in liver except for trace expression in white adipose,stomach, gut and brain for Angptl6.4. Expression analysis of porcine Angptls was conducted in tissues of obese pigs(Tongcheng) and lean pigs (Landrace). RT-PCR analysis of Angptls expression indicatedthat Angptl1 and Angptl2 mRNA level in liver and heart were significantly higher inobsess pigs than in their lean counterparts, however, it was revise in white adipose. TheAngptl3 mRNA level in liver was significantly higher in obese pigs than in their leancounterparts. The expression of Angptl4 in liver and white adipose were higher in obesepigs than in lean pigs. However, Angptl6 mRNA level in liver was lower in obese pigsthan in their lean counterparts.5. Porcine Angptls expression in HepG2 cells was analyzed after treated by differentconcentration of glucose, insulin, clenbuterol and dexamethasone. In general, under themedia with higher concentration of glucose (25mmol/L, 50 mmol/L and 100mmol/L), themRNA level of Angptl4 was significantly increased, whereas the expression of Angptl6was decreased. The expression of Angptl3, Angptl4 and Angptl6 were significantlydecreased under different concentration of insulin. When treated with same concentrationof insulin (100nmol/L) and different concentration of glucose simultaneously, theexpression of Angptls were similar as treated by different concentration of glucose.Treated with clenbuterol, the mRNA level of Angptl3 and ChREBP were increased. Thetreatment of dexamethasone significantly increased the expression of Angptl4, butinhibited the expression of Angptl6 and ChREBP.