MicroRNAs (miRNAs) are small ∼22-nt regulatory RNAs that regulate the stability and translation of cognate mRNAs. MiRNAs participate in the regulation of adipogenesis, and identification of the full repertoire of miRNAs expressed in adipose tissue is likely to significantly increase our understanding of adipose tissue growth and development. Here, we adopted a deep sequencing approach to determine the identity and abundance of miRNAs in developing swine adipose tissue. Via this approach, we identified the sequences and relative expression levels of 227 conserved miRNAs (of which 59 were novel) and 66 potential porcine miRNAs. The expression levels displayed a large range, as reflected by the number of sequence reads, which varied from several counts for rare miRNAs to several million reads for the most abundant miRNAs. The abundant miRNAs principally belonged to 32 miRNA gene families, including miR-143, miR-103, let-7, and miR-148. Of the conserved miRNAs, 93 miRNAs were up-regulated and 33 miRNAs were down-regulated in the adult pig adipose tissue. Moreover, we observed sequence variants and seed edits of the miRNAs. KEGG pathway analysis and GO term enrichment suggested that highly expressed miRNAs are involved in adipose tissue development, signal transduction, cell-cell and cell-extracellular matrix communication, neural development and function, and lipid metabolism including carboxylic acid, oxoacid, fatty acid, steroid, glycerolipid, alcohol and phospholipid metabolism. Our results expand the number of known porcine miRNAs and provide a thorough account of the miRNA transcriptome in porcine adipose tissue.

MicroRNAs are small non-coding RNAs that partially bind to the 3' untranslated (3'UTR) regions of target genes in animals and regulate protein production of the target transcripts. MiR-103 has been confirmed to play a critical role in lipid metabolism, however, the target genes and signaling pathway regulated by miR-103 is still unclear. In our experiment, we observed a positive function of miR-103 on the adipogenic differentiation of 3T3-L1 pre-adipocyte. Furthermore, we proved that this function of miR-103 worked through activating AKT/mTOR signal pathway and impairing target gene MEF2D. By inhibiting and over-expressing the MEF2D gene, we found that MEF2D had a negative role in regulating adipocyte key genes, and this function of MEF2D could be impaired by miR-103. In conclusion, we found that miR-103 can promote 3T3-L1 cells differentiation by targeting MEF2D and activating AKT/mTOR signal pathway. These results will shed a light on further study of microRNAs.

Alpha melanocyte stimulating hormone (αMSH) abates inflammation in multiple tissues, while Forkhead box proteins O (FoxOs) stimulate inflammatory cascade. However, the relationship between αMSH and FoxOs in adipose inflammation remains unclear. In this study, we used LPS-induced inflammation model, attempted to interpret the function of αMSH in inflammation and the interactions with FoxOs. Results indicated that upon inflammatory situation, the secretion of αMSH and the expression of its receptor MC5R were greatly decreased, but FoxOs expressions were elevated. After the treatment with αMSH, LPS-induced adipose inflammation together with FoxOs expressions was significantly reduced. Conversely, when Foxo1, Foxo3a or Foxo4 overexpressed in αMSH treated inflammatory mouse model, all the anti-inflammatory impacts of αMSH were found disappeared. We further studied the mechanisms by which αMSH exerts its anti-inflammatory impacts and how FoxOs reverse αMSH's function. Foxo4 was found as a negative regulator for MC5R transcription in αMSH inhibited inflammation. Moreover, a negative role was found of αMSH in regulating both Akt and JNK signal pathways by observing the enhanced the anti-inflammatory impacts of pathway-specific inhibitors with αMSH treatment. Our findings demonstrate αMSH plays a key role in the prevention of adipose inflammation and inflammatory diseases by down-regulating Akt/JNK signal pathway and negatively interacting with FoxOs, which brings up αMSH as a novel candidate factor in the adipose anti-inflammation process in obesity.

Extracellular matrix (ECM), as an essential component of adipose tissue, not only provides mechanical support for adipocyte growth, but also participates in ECM-adipocyte communication via various secreted proteins, including highly enriched collagens. Collagen XV (ColXV) is a secreted non-fibrillar collagen within ECM Basement Membrane (BM) zones and well recognized as a tumor suppressor. However, the role of ColXV in adipose tissue is still unknown. In this study, high fat diet (HFD) fed mice were used as obese model, in which we deeply investigated the interaction between ColXV and adipocyte differentiation or adipose metabolism. We found great elevated ColXV expression and positive effect of ColXV on lipid deposition during adipocyte differentiation or obesity both in vitro and in vivo. cAMP response element binding protein (CREB) is a cellular transcription factor that can inhibit adipogenesis and promote lipolysis. Here we proposed ColXV as a newly discovered downstream gene of CREB. We further proved that CREB can repress adipocyte differentiation and enhance lipolysis by negatively regulating ColXV transcription. Mechanistic studies showed ColXV enhanced adipocyte differentiation and lipid deposition through reducing its DNA methylation and repressing the cAMP/PKA signaling pathway. Collectively, our study identified ColXV as a novel downstream gene for CREB and could promote adipocyte differentiation, inhibit lipolysis through repressing cAMP/PKA signaling pathway and positively regulating adipogenic markers expressions by repressing the activity of maintenance methyltransferase Dnmt1. Our data discovered a novel role of ColXV in adipocyte differentiation and provide insight into obesity and related metabolic diseases.

Alpha-melanocyte stimulating hormone (αMSH) is an important adenohypophysis polypeptide hormone that regulates body metabolic status. To date, it is well known that the disorder of hypothalamic αMSH secretion is related to many metabolic diseases, such as obesity and type II diabetes. However, the underlying mechanisms are poorly understood. In our study, we focused on the reactive oxygen species (ROS)-induced adipocyte apoptosis and tried to unveil the role of αMSH in this process and the signal pathway which αMSH acts through. Kunming white mice were used and induced to oxidative stress status by hydrogen peroxide (H2O2) injection and a significant reduction of αMSH were found in mice serum, while elevated ROS level and mRNA level of pro-apoptotic genes were observed in mice adipose tissue. What is more, when detect the function of αMSH in ROS-induced apoptosis, similar inhibitory trend was found with the oxidative stress inhibitor N-acetyl-L-cysteine (NAC) in ROS-induced adipocyte apoptosis and this trend is αMSH receptor melanocortin 5 receptor (MC5R) depended, while an opposite trend was found between αMSH and Foxo1, which is a known positive regulator of adipocyte apoptosis. Further, we found that the repress effect of αMSH in adipocytes apoptosis is acting through Foxo1/mTORC2 pathway. These findings indicate that, αMSH has a strong inhibitory effect on ROS-induced adipocyte apoptosis and underlying mechanism is interacting with key factors in mTOR signal pathway. Our study demonstrated a great role of αMSH in adipocyte apoptosis and brings a new therapeutic mean to the treatment of obesity and diabetes.