Fig 2: Leptin improves the body fat composition and weight of piglets by up-regulating the expression of Plin5. (A) The leptin kit was used to detect the level of leptin protein in various tissues of piglets. (B) The expression of leptin receptors in various tissues of piglets. The effect of leptin treatment on: (C) the average daily gain, (D) the intramuscular fat content, (E) abdominal fat rate and back fat rate and (F) blood lipid level etc. (G) The change of piglet’s body temperature for 15 days after leptin treatment. (H,I) The mRNA and protein expression levels of the Plin family were analyzed by real-time quantitative PCR and western blot. (J) Immunohistochemical detection of piglet adipose tissue. (K) Western blot analysis of lipolytic protein expression. n = 4 in each group, values are means ± SD. vs. control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Leptin inhibits the m6A methylation of Plin5 through FTO. (A) Porcine adipocytes in vitro were treated with 50 nmol/µL leptin for 24 h to detect the mRNA expression of methylation-related genes. (B) The protein expression of FTO under the same treatment as (A). (C) Dot Blot detected the level of total RNA m6A in 50 nmol/µL leptin-treated porcine adipocytes in vitro. (D) The colorimetric assay was used to quantify the relative m6A levels of 50 nmol/µL leptin-treated porcine adipocytes in vitro. (E) m6A-IP was used to detect Plin5 M6A methylation in 50 nmol/µL leptin-treated porcine adipocytes in vitro. (F) Real-time quantitative PCR was used to detect FTO overexpression and interference efficiency in vitro. (G) m6A-IP detected the m6A level of Plin5 after overexpression or interference with FTO in vitro. (H) The relative M6A level of 50 nmol/µL leptin treated porcine adipocytes in vitro after overexpression or interference with FTO. (I) Plin5 M6A levels in 50 nmol/µL leptin-treated porcine adipocytes in vitro after overexpression or interference with FTO. n = 6 for each group of cell samples, values are means ± SD. vs. control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: Leptin promotes lipolysis by up-regulating the expression of Plin5 in pig adipocytes. (A,B) Real-time quantitative PCR was used to detect the mRNA expression of Plin5, lipolytic genes and mitochondrial complex-related genes in piglets subcutaneous adipose tissue. n = 4 in each group. (C) Oil red O staining was used to observe the number and size of LD in the subcutaneous fat tissue of piglets. (D) Real-time quantitative PCR was used to detect Plin5 and lipolytic gene expression in porcine adipocytes in vitro treated with 50 nmol/µL leptin for 24 h. The effect of 50 nmol/µL leptin treatment of porcine adipocytes in vitro on: (E) the expression of Plin5 and lipolytic protein and (F) the mRNA expression of mitochondrial complex-related genes. (G,H) Porcine adipocytes in vitro were treated with 50 nmol/µL leptin for 24 h to detect Plin5 immunofluorescence or BODIPY staining analysis. n = 3 for each group of cell samples, values are means ± SD. vs. control group, * p < 0.05, ** p < 0.01.

Fig 5: FTO up-regulates Plin5 protein expression by inhibiting the m6A methylation of Plin5. The effect of FTO interference or overexpression in vitro on: (A) Plin family mRNA expression and (B) Plin family protein levels. (C) The relative M6A levels of pig adipocytes in vitro treated with CL and Bet after overexpression or interference with FTO. (D) The total M6A level of CL-treated pig adipocytes in vitro after overexpression or interference with FTO. (E) Plin family mRNA expression in CL-treated pig adipocytes in vitro after overexpression or interference with FTO. (F) The total M6A level of Bet-treated pig adipocytes after overexpression or interference with FTO. (G) Plin family mRNA expression in Bet-treated pig adipocytes in vitro after overexpression FTO. (H) Plin5 protein expression in pig adipocytes in vitro treated with CL and Bet after overexpression FTO. (I,J) Plin5 3'UTR end M6A site mutation vector construction. n = 4 for each group of cell samples, values are means ± SD. vs. control group, * p < 0.05, ** p < 0.01.