Leptin Reduces Plin5 m6A Methylation through FTO to Regulate Lipolysis in Piglets

Perilipin5 (Plin5) is a scaffold protein that plays an important role in lipid droplets (LD) formation, but the regulatory effect of leptin on it is unclear. Our study aimed to explore the underlying mechanisms by which leptin reduces the N6-methyladenosine (m6A) methylation of Plin5 through fat mass and obesity associated genes (FTO) and regulates the lipolysis. To this end, 24 Landrace male piglets (7.73 ± 0.38 kg) were randomly sorted into two groups, either a control group (Control, n = 12) or a 1 mg/kg leptin recombinant protein treatment group (Leptin, n = 12). After 4 weeks of treatment, the results showed that leptin treatment group had lower body weight, body fat percentage and blood lipid levels, but the levels of Plin5 mRNA and protein increased significantly in adipose tissue (p < 0.05). Leptin promotes the up-regulation of FTO expression level in vitro, which in turn leads to the decrease of Plin5 M6A methylation (p < 0.05). In in vitro porcine adipocytes, overexpression of FTO aggravated the decrease of M6A methylation and increased the expression of Plin5 protein, while the interference fragment of FTO reversed the decrease of m6A methylation (p < 0.05). Finally, the overexpression in vitro of Plin5 significantly reduces the size of LD, promotes the metabolism of triglycerides and the operation of the mitochondrial respiratory chain, and increases thermogenesis. This study clarified that leptin can regulate Plin5 M6A methylation by promoting FTO to affect the lipid metabolism and energy consumption, providing a theoretical basis for treating diseases related to obesity.

PSIX-35 Quebracho Tannin Influences Lipolytic Activity in Mature Porcine Adipocytes

Fat deposition in pork enhances flavor of meat; however, too much fat is an undesirable commodity in a health-conscious society. Therefore, manipulating the nutritional components of a swine diet to aid in the deliberate deposition of fat for the purpose of flavor while avoiding overconditioning is an aim in production. Nutrient additives, such as condensed polyphenolic tannins, inhibit pre-adipocyte maturation, but the role on lipid metabolism in mature adipocytes (MA) remains unclear. Therefore, it is hypothesized that quebracho tannin will alter lipid metabolism in porcine MA. Subcutaneous adipose tissue was collected from 5 ± 0 month old (n = 3) barrows weighing 37.7 ± 1.84kg. Tissue was enzymatically dispersed (collagenase type II) to isolate lipid filled adipocytes. After enzymatic separation the cells were rinsed and divided into 2 groups for separate incubation periods plus tannin treatment: 1) 2 hr incubation time with/without tannin (Quebracho Schinopsis lorentzii; 0M, 0.1mg, 0.5mg, and 1mg) or 2) 24 hr incubation time with/without tannin (0M, 0.1mg, 0.5mg, and 1mg). Approximately 4x105 cells/well were cultured in triplicate/treatment dose at 37 °C with 5% CO2 in atmosphere. Upon termination of the culture period, media was processed for analysis of glycerol content to determine lipolytic activity using an enzymatic colorimetric assay. The MIXED procedure of SAS for factorial treatment design was utilized to determine the effect of time and tannin treatment on lipolytic activity in cultured MA. Glycerol content was significantly higher (P£0.001) in tannin treated cultures. Time tended (P = 0.1) to influence the magnitude of lipolytic activity. Hence, quebracho tannin appears to augment lipolytic activity in cultured porcine MA. Determining the effect of tannin on lipolytic regulators will support the supposition that tannins influence MA lipid metabolism.

Upregulated microRNA-106a Promotes Porcine Preadipocyte Proliferation and Differentiation by Targeting Different Genes

Adipose tissue is one of the main organs for the energy storage and supply of organisms. Adipose deposition and metabolism are controlled by a cascade of transcription factors and epigenetic regulatory mechanisms. Previous studies have also shown that miR-106a plays a considerable role in the development of organisms. The regulatory mechanism of miR-106a on porcine preadipocytes is still not clear. In this study, preadipocytes were isolated from the neck subcutaneous deposits of 3–5-day old Chinese native Guanzhong black pigs using 5-ethynyl-20-deoxyuridine (EdU) staining and a CCK-8 assay to detect the number of proliferous cells and real-time qPCR (RT-qPCR) and western blot analysis to detect gene expression, as well as Oil Red O and BODIPY staining dye lipid droplets and flow cytometry (FCM) to detect cell cycles. We also used the double luciferase method to detect the relative luciferase activities. Upregulated miR-106a increased the number of proliferous cells and enhanced the expression of cell proliferation-related genes in porcine adipocytes. The double luciferase reporter vector confirmed that p21 was a target gene of miR-106a in the cell proliferation phase. miR-106a upregulation increased the number of lipid droplets and the expression of lipogenic genes and directly targeted BMP and activin membrane-bound inhibitor (BAMBI) in the process of differentiation. Our results indicated that miR-106a promotes porcine preadipocyte proliferation and differentiation by targeting p21 and BAMBI.