To elucidate the key miRNAs and the signalling pathways that are involved in porcine oocyte maturation, we performed a deep sequencing analysis of the miRNAs of pig germinal vesicle (GV) oocytes and metaphase II (MII) oocytes. Seven differentially expressed (DE) miRNAs were identified and the expression levels of miR-21 and miR-27b-3p were further confirmed by QPCR analysis. The target genes of 7 DE miRNAs were predicted and subjected to pathway analysis. Interestingly, fatty acid metabolism and fatty acid biosynthesis were the top two significantly enriched molecular functions during oocyte maturation. Heat map, which was built with 7 DE miRNAs and the enriched the molecular functions, revealed that miR-21, miR-27b-3p, miR-10a-5p and miR-10b-5p were involved in fatty acid metabolism. In particular, the regulatory role of miR-27b-3p on peroxisome proliferator-activated receptor-γ (PPARγ) was confirmed by their inversed expression patterns in GV and MII oocytes and luciferase report assays. In addition, we observed that PPARγ agonist (rosiglitazone) treatment significantly enhanced porcine oocyte maturation rate and early embryo developmental competent. Taken together, our results demonstrated that miR-27b and its target, PPARγ, play the vital roles in pig oocyte maturation through regulating the fatty acid metabolism. These data increased our understanding of the regulatory gene networks in porcine oocyte maturation and development.

RNF20, an E3 ligase critical for monoubiquitination of histone H2B at lysine 120 (H2Bub), has been implicated in the regulation of various cellar processes; however, its physiological roles in adipocytes remain poorly characterized. Here, we report that the adipocyte-specific knockout of Rnf20 (ASKO) in mice led to progressive fat loss, organomegaly and hyperinsulinemia. Despite signs of hyperinsulinemia, normal insulin sensitivity and improved glucose tolerance were observed in the young and aged CD-fed ASKO mice. In addition, high-fat diet-fed ASKO mice developed severe liver steatosis. Moreover, we observed that the ASKO mice were extremely sensitive to a cold environment due to decreased expression levels of brown adipose tissue (BAT) selective genes, including uncoupling protein 1 (Ucp1), and impaired mitochondrial functions. Significantly decreased levels of peroxisome proliferator-activated receptor gamma (Pparγ) were observed in the gonadal white adipose tissues (gWAT) from the ASKO mice, suggesting that Rnf20 regulates adipogenesis, at least in part, through Pparγ. Rosiglitazone-treated ASKO mice exhibited increased fat mass compared to that of the non-treated ASKO mice. Collectively, our results illustrate the critical role of RNF20 in control of white and brown adipose tissue development and physiological function.

Fat deposition, which influences pork production, meat quality and growth efficiency, is an economically important trait in pigs. Numerous studies have demonstrated that stearoyl-CoA desaturase (SCD), a key enzyme that catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, is associated with fatty acid composition in pigs. As SCD was observed to be significantly induced in 3T3-L1 preadipocytes differentiation, we hypothesized that it plays a role in porcine adipocyte differentiation and fat deposition. In this study, we revealed that SCD is highly expressed in adipose tissues from seven-day-old piglets, compared to its expression in tissues from four-month-old adult pigs. Moreover, we found that SCD and lipogenesis-related genes were induced significantly in differentiated porcine adipocytes. Using CRISPR/Cas9 technology, we generated SCD-/- porcine embryonic fibroblasts (PEFs) and found that the loss of SCD led to dramatically decreased transdifferentiation efficiency, as evidenced by the decreased expression of known lipid synthesis-related genes, lower levels of oil red O staining and significantly lower levels of triglyceride content. Our study demonstrates the critical role of SCD expression in porcine adipocyte differentiation and paves the way for identifying it as the promising candidate gene for less fat deposition in pigs.

RNF20 is recognized as a main E3 ligase for monoubiquitination of histone H2B at lysine 120 (H2Bub). The critical role of RNF20 and H2Bub in various molecular events, such as DNA replication, RNA transcription, and DNA damage response, has been widely investigated and documented. However, its role in porcine adipogenesis remains unknown. In this study, we aimed to clarify the effect of RNF20 on porcine preadipocyte differentiation.

Beige adipose tissue has been considered to have potential applications in combating obesity and its related metabolic diseases. However, the mechanisms of acute cold-stimulated beige formation still remain largely unknown. Here, transcriptional analysis of acute cold-stimulated (4 °C for 4 h) subcutaneous white adipose tissue (sWAT) was conducted to determine the molecular signatures that might be involved in beige formation. Histological analysis confirmed the appearance of beige adipocytes in acute cold-treated sWAT. The RNA-sequencing data revealed that 714 genes were differentially expressed (p-value < 0.05 and fold change > 2), in which 221 genes were upregulated and 493 genes were downregulated. Gene Ontology (GO) analyses showed that the upregulated genes were enriched in the GO terms related to lipid metabolic process, fatty acid metabolic process, lipid oxidation, fatty acid oxidation, etc. In contrast, downregulated genes were assigned the GO terms of regulation of immune response, regulation of response to stimulus, defense response, etc. The expressions of some browning candidate genes were validated in cold-treated sWAT and 3T3-L1 cell browning differentiation. In summary, our results illustrated the transcriptional response of sWAT to acute cold exposure and identified the genes, including Acad11, Cyp2e1, Plin5, and Pdk2, involved in beige adipocyte formation in mice.

Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.