Fig 2: Effect of GST-irisin on lipolysis in 3T3-L1 mature adipocytes.A: Effect of GST-irisin on the release of glycerol in 3T3-L1 mature adipocytes. The adipocytes were cultured with various GST-irisin concentrations (0, 50, 100, and 200 nM) for 2 (a), 4 (b), 6 (c) and 8 (d) days. B: The mRNA expression of FABP4, ATGL, and HSL was determined by qPCR. C: The protein levels of FABP4, ATGL, FNDC5, and ß-actin were determined by western blotting. The adipocytes were cultured with various GST-irisin concentrations, and the concentration was 0 (a), 50 (b), 100 (c), and 200 (d) nM. Values are expressed as the mean ± SD of three independent experiments. *P < 0.05 versus control; **P < 0.01 versus control.

Fig 3: The proposed molecular mechanism of irisin in adipocytes.AQP7: aquaporin 7, ATGL: adipose triglyceride lipase, DAG: diacylglycerol, ERK: extracellular signal-regulated kinase, FABP: fatty acid-binding protein, FFA: free fatty acid, FNDC5: Fibronectin Type III Domain Containing 5, HSL: hormone-sensitive lipase, MAGL: monoacylglycerol lipase, MAPK: mitogen-activated protein kinase, MG: monoacylglycerol, PGC1a: peroxisome proliferator-activated receptor gamma coactivator 1-alpha, TG: triglyceride, UCP1: uncoupling protein 1.

Fig 4: Insulin inhibits isoproterenol-stimulated lipolysis in 3T3-L1 adipocytes via the AKT/PKA/HSL pathway.We treated differentiated adipocytes with 100 nM insulin and 1 µM isoproterenoland examined the effects on lipolysis(in triplicate). (a)Western blot analyses ofAKT, PDE3B, PKA, HSL and ATGL protein levels in the insulin, isoproterenol and control groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (b)The phosphorylated protein/total protein ratios of AKT, PKA and HSL were calculated, and the control ratio was normalized to 1. (c)Western blot analyses of PDE3B protein levels in the insulin, isoproterenol and control groups. Differentiated 3T3-L1 adipocytes were treated with different doses of insulin (1, 10,100 nM) and 1 µM isoproterenol for 24 h(in triplicate). (d) Cellular triglycerides were stained with Oil red O. Bar, 50 µm. (e) The amount of lipids was quantified by the Oil red O staining technique. (f)The concentration of glycerol in the medium was detected in different groups(g, i)Western blot analyses of AKT, PDE3B, PKA, HSL and ATGL protein levels in different groups. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (h) The phosphorylated protein/total protein ratios were calculated, and the control ratio was normalized to 1.(j)cAMP levels in different groups. (k) The expression of PDE3Bwas determined in different groups by Western blot.

Fig 5: Effects of PID1 expression on lipolysis and the phosphorylation of AKT/PDE3B/PKA/HSL signaling molecules and ATGL.Preadipocytes were subjected to PID1knockout or upregulation and allowed to differentiate into 3T3-L1 adipocytes; these cells were treated with 1 µM isoproterenol and 100 nM insulin for 24 h(in triplicate). (a-b)Immunofluorescence analysis was performed to assess the expression of the PID1 gene in empty vector cells, PID1-overexpressing cells, and control cells.(c)RT-PCR analyses of the mRNA expression of PID1 in empty vector cells, PID1-overexpressing cells, and control cells. (d)Glycerol released into the medium after the upregulation of PID1.(e-f)Protein expression of AKT, PDE3B, PKA, HSL and ATGL in empty vector cells, PID1-overexpressing cells, and control cells. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control.(g)The phosphorylated protein/total protein ratios for AKT, PKA, HSL, and ATGL in 3T3-L1 adipocytes after transfection with the PID1 overexpression plasmid. (h)The expression of PDE3B was determined by Western blot after the PID1 overexpression plasmid. (i-j)Protein expression of AKT, PDE3B, PKA, HSL and ATGL after transfection with PID1 shRNA. Phosphorylated AKT (p-AKT), phosphorylated PKA (p-PKA), phosphorylated HSL (p-HSL) and phosphorylated ATGL (p-ATGL) expression was normalized to their total protein level as a loading control. (k)RT-PCR analyses of mRNA after transfection with PID1 shRNA. (l) Glycerol was released into the medium after knockdown of PID1. (m)Phosphorylated protein/total proteinratios for AKT, PKA, HSL, and ATGL after PID1 knockdown. (n)The expression of PDE3B was determined by Western blot after transfection with PID1 shRNA.