The Jinhua pig has become one of the most important local breeds in China due to its excellent meat quality and it exhibits the characteristics of early sexual maturity, low prolificacy, and a high body fat content .The purpose of this study was to explore the evolutionary relationship of ANGPTL family genes in Jinhua Pig, a model animal of lipid metabolism, and the important roles of ANGPTL3, ANGPTL4 and angptl8 in lipid metabolism. In the present study, amino acid sequence analysis, phylogenetic analysis, chromosome adjacent gene analysis, and RT-qPCR analysis showed that Jinhua pig ANGPTL family members had common characteristics with homologous human genes, including the high similarity of amino acid sequence and chromosome flanking genes. Consistent with earlier studies [51, 52], pig ANGPTL3-4 had typical angiopoietin family structures. The structures of pig ANGPTL3-4 was similar to those of humans and mice, and both had FReD, which affects angiogenic activity, and CCD, which regulated fat metabolism by inhibiting LPL. The eight ANGPTL family member genes in Jinhua pigs were 1100–2000 bp in length and consisted of 4–9 exons. The functional helical domain and fibrinogen-like domain in the ANGPTL1-8 amino acid sequence were strongly conserved. Through the identification and expression analysis of the bovine ANGPTL1-7 genes, all of the deduced amino acid sequences had an N-terminal crimped domain and a C-terminal fibrinogen-like domain, and both had the characteristics of angiogenin. The bovine ANGPTL1-7 amino acid sequences are similar to those of humans, and the corresponding members had 95%, 97%, 82%, 85%, 87%, 85%, and 87% homology . Related studies reported the cloning, chromosomal location, and expression analysis of pig ANGPTL3 and ANGPTL4. The deduced amino acid sequence of pig ANGPTL3 had 83% and 73.7% homology to humans and mice, respectively. While pig ANGPTL4 had 79.4% and 79.1% homology with humans and mice . This research showed that pig ANGPTL1-8 amino acid sequence had 95.93%, 97.57%, 84.13%, 79.37%, 91.75%, 88.35%, and 89.28% homology with corresponding human members, respectively.
In vertebrates, the ANGPTL family of genes evolved by gene duplication, gene deletion, and gene mutation. They share the same evolutionary origin with angiogenin and share similarities in sequence and structure. ANGPTL family members expanded in early vertebrate genome doubling and genome segment replication before vertebrate radiation. Five major ANGPTL vertebrate protein clusters, ANGPTL1-2-6, ANGPTL3-4, ANGPTL5, ANGPTL7, ANGPTL8, derived from the replication of ancestor ANGPTL [53, 54]. The ANGPTL8 cluster was isolated from the other four protein clusters since ANGPTL8 has a different structure from other ANGPTL family members . Clustering in phylogenetic trees indicates that pig sequences are not significantly different from those of other vertebrates.
Recent studies have shown that ANGPTL3 , ANGPTL4 , and ANGPTL8  are directly related to lipid metabolism and fat deposition in the body. In this study, tissue expression profiles showed that ANGPTL3 and ANGPTL8 were mainly expressed in the liver of pigs, with similar results in humans and mice [58, 59]. However, ANGPTL4 in pigs is widely expressed in many tissues, including the gastrointestinal tract, subcutaneous fat, liver, muscles, heart, and lungs. In humans and mice, ANGPTL4 is also expressed mainly in white adipose tissue and the liver as well as in the intestine and heart tissue [18, 60]. By analyzing the expression levels of ANGPTL3 and ANGPTL8 in the liver and the relative expression levels of ANGPTL4 in the liver, subcutaneous fat, and ileum of Jinhua pigs with distinct fatness, we found that the expression of ANGPTL3 in the liver of Jinhua pigs in the H group was significantly lower than that of Jinhua pigs in the L group (P = 0.005). Moreover, the expression level of ANGPTL4 in the liver (P = 0.008), subcutaneous fat (P = 0.025), and ileum (P = 0.043) of the H group pigs was significantly lower than that of the L group pigs. This might be due to the inhibitory effect of ANGPTL3 and ANGPTL4 on LPL. With the decrease in the expressions of ANGPTL3 and ANGPTL4, the activity of LPL would increase, which in turn would decrease the serum TG level and increase the lipid absorption. Early research showed that in sterile mice with routine feeding, the microbiota promoted the storage of triglycerides in fat cells by inhibiting the intestinal expression of ANGPTL4, illustrating the decrease of ANGPTL4 expression is beneficial to fat storage . Recent studies have indicated that the level of circulating ANGPTL4 in obese children was lower than that of normal weight children. Waist circumference, body weight, and other major obesity indicators were negatively correlated with the level of circulating ANGPTL4 . Meanwhile, mice with ANGPTL3 and ANGPTL4 deficiency had severe hypertriglyceridemia while mice with ANGPTL3 overexpression had hyperlipidemia [62, 63]. Furthermore, compared with other breeds of pigs, the fat percentage of Large White pigs was lower with a higher expression of ANGPTL4 mRNA . These studies are consistent with the results of the present study.
Although there was no significant difference in ANGPTL8 expression between the liver of Jinhua pigs in the H and L groups, it might be resulted from the function of ANGPTL8, which promotes the cleavage of ANGPTL3 and binds to the N-terminal of ANGPTL3 . The interaction of ANGPTL8 with ANGPTL3 would form a complex with the N-terminal of ANGPTL3, synergistically inhibiting the LPL activity and modulating the plasma triglyceride levels . Additionally, the overexpression of ANGPTL8 in the liver of ANPTL3 gene knockout mice had no effect on the triglyceride metabolism .
Fat metabolism and faat storage are closely related to the meat quality and carcass quality. Relevant research showed that by comparing the carcass composition and development patterns of Jinhua and Landrace pigs at 35 ~ 125 days of age, the carcass fat content of Jinhua pigs was significantly higher than that of Landrace pigs (P < 0.05), and the lean meat rate of the carcass was significantly lower than that of Landrace pigs (P < 0.01) . Similar studies showed that Jinhua pigs had a higher tendency to deposit fat compared with Landrace pigs. These are consistent with the results of this experiment. Therefore, Jinhua Pigs with high body fat percentage and good meat quality play an important role in the study of lipid metabolism and carcass quality.