ACSL4 Directs Intramuscular Adipogenesis and Fatty Acid Composition in Pigs

The intramuscular fat is a major quality trait of meat, affecting sensory attributes such as flavor and texture. Several previous GWAS studies identified Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) gene as the candidate gene to regulate intramuscular fat content in different pig populations, but the underlying molecular function of ACSL4 in adipogenesis within pig skeletal muscle is not fully investigated. In this study, we isolated porcine endogenous intramuscular adipocyte progenitors and performed ACSL4 loss- and gain-of-function experiments during adipogenic differentiation. Our data showed that ACSL4 is a positive regulator of adipogenesis in intramuscular fat cells isolated from pigs. More interestingly, the enhanced expression of ACSL4 in pig intramuscular adipocytes could increase the cellular content of monounsaturated and polyunsaturated fatty acids, such as gamma-L eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA). The above results not only confirmed the function of ACSL4 in pig intramuscular adipogenesis and meat quality attributes, but also provided new clues for the improvement of the nutritional value of pork for human health.

KLF4 Inhibits the Differentiation of Goat Intramuscular Preadipocytes Through Targeting C/EBPβ Directly

Intramuscular fat (IMF) deposition is a complicated process, and most of the underlying regulators of this biological process are unknown. Here, we cloned the intact CDS of KLF4 gene, investigated the role of KLF4 by gaining or losing function in vitro and further explored the pathways of KLF4 regulating differentiation of intramuscular preadipocytes in goat. Our results show that goat KLF4 gene consists of 1,536 bp encoding a protein of 486 amino acids. The expression of KLF4 is higher in the lung while lower in the heart and muscle in goat. Knockdown of KLF4 mediated by siRNA technique significantly promotes intramuscular preadipocyte lipid accumulation and upregulates mRNA expression of adipogenic related genes including C/EBPα, C/EBPβ, and PPARγ in vivo cultured cells. Consistently, overexpression of KLF4 inhibits intramuscular adipocyte lipid accumulation and significantly downregulation gene expression of C/EBPβ, PPARγ, aP2, and Pref-1. Further, we found that other members of KLFs were upregulated or downregulated after interference or overexpression of KLF4, including KLF2 and KLF5–7. We also found that C/EBPβ was a potential target of KLF4, because it had an opposite expression pattern with KLF4 during the differentiation of intramuscular preadipocytes and had putative binding sites of KLF4. The dual-luciferase reporter assay indicated that overexpression of KLF4 inhibited the transcriptional activity of C/EBPβ. These results demonstrate that KLF4 inhibits the differentiation of intramuscular preadipocytes in goat by targeting C/EBPβ.

The Effects of Age at Weaning and Length of Lipid Supplementation on Growth, Metabolites, and Marbling of Young Steers

The objective of this study was to determine how weaning age, days on supplements, and lipid supplementation affected the growth and marbling deposition of steers. Steers from a single sire were early weaned (n = 24) at 150 ± 11 days of age or traditionally weaned (n = 24) at 210 ± 11 days of age. Steers were assigned to control (n = 12/weaning group) or an isocaloric, isonitrogenous rumen by-pass lipid (RBL, n = 12/weaning group) for either 45 (n = 6/treatment) or 90 (n=6/treatment) days then harvested. Steer body weight (BW) was recorded on days −14 and −7, then BW and blood samples were collected on days 0, 22, 45, 66, and 90. The right rib section of each animal was collected for proximate analysis. Longissimus dorsi from RBL steers had increased lipids compared with control steers (3.6 ± 0.2 vs. 2.4 ± 0.2% on a wet basis; p < 0.0001). Steers fed for 90 days had greater (p = 0.02) concentrations of Longissimus dorsi lipid (3.3 ± 0.2%) than those fed for 45 days (2.7 ± 0.2%). There was a weaning age by treatment by days on feed interaction for intramuscular adipocyte diameter (p = 0.02) in which early weaned RBL fed for 90 days steers had an increased adipocyte diameter compared to the early weaned control fed for 90 and early weaned fed for 45 days steers with all other treatment groups as intermediates. Supplementation of RBL increased concentrations of C18:2, C20:4, and total fatty acids on days 45 and 90 (p ≤ 0.05). Data show that RBL supplementation increased the marbling content of the Longissimus dorsi. Furthermore, a longer period of supplementation resulted in increased adipose diameter.

LncRNA IMFlnc1 Promotes Porcine Intramuscular Adipocyte Adipogenesis by Sponging Mir-199a-5p to Up-regulate CAV-1

Background: Local Chinese local pig breeds have thinner muscle fiber and higher intramuscular-fat (IMF) content. But its regulation mechanism has not been discussed in-depth. Studies indicated that long non coding RNAs (lncRNAs) play important role in muscle and fat development.Results: The lncRNAs expressional differences in the longissimus dorsi (LD) muscle were identified between Huainan pigs (local Chinese pigs, fat-type, HN) and Large White pigs (lean-type, LW) at 38, 58, and 78 days post conception (dpc). In total, 2131 novel lncRNAs were identified in 18 samples, and 291, 305, and 683 differentially expressed lncRNAs (DELs) were found between these two breeds at three stages, respectively. GO and KEGG analysis of the DEL co expressed mRNAs showed that muscle development and energy metabolism were more active at 58 dpc in HN, but at 78 dpc in LW pigs. Muscle cell differentiation and myofibril assembly might associated with earlier myogenesis and primary-muscle-fiber assembly in HN, and cell proliferation, insulin, and the MAPK pathway might contribute to longer proliferation in LW pigs. The PI3K/Akt and cAMP pathways were associated with higher IMF deposition in HN. IMFlnc1 was selected for functional verification, and results indicated that it regulated the expressional level of caveolin-1 (CAV-1) by acting as competing endogenous RNA (ceRNA) to sponge miR-199a-5p.Conclusions: Our data contributed to understanding lncRNAs in porcine-muscle development and IMF deposition, and provided valuable information for improving pig-meat quality.

A Newly Identified LncRNA LncIMF4 Controls Adipogenesis of Porcine Intramuscular Preadipocyte through Attenuating Autophagy to Inhibit Lipolysis

Intramuscular fat (IMF) is implicated in juiciness, tenderness, and flavor of pork. Meat quality of Chinese fat-type pig is much better than that of lean-type pig because of its higher IMF content. LncRNA is a vital regulator that contributes to adipogenesis. However, it is unknown about the regulation of lncRNA on IMF content. Here, by RNA sequence analysis of intramuscular adipocyte from Bamei pig (fat-type) and Yorkshire pig (lean-type), we found that a novel lncRNA, lncIMF4, was associated with adipogenesis. LncIMF4, abundant in adipose, differently expressed along with intramuscular preadipocyte proliferation and differentiation. Meanwhile, it is located both in cytoplasm and nucleus. Besides, lncIMF4 knockdown promoted proliferation and differentiation of porcine intramuscular preadipocytes, whereas inhibited autophagy. Moreover, lncIMF4 knockdown facilitated intramuscular adipogenesis through attenuating autophagy to repress the lipolysis. Our findings will contribute to understand better the mechanism of lncRNA controlling intramuscular adipogenesis for promoting pork quality.