Unconventional Secretion of Adipocyte Fatty Acid Binding Protein 4 Is Mediated By Autophagic Proteins in a Sirtuin-1–Dependent Manner
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Figures
- Figure 1
FABP4 secretion in response to lipolytic stimuli. A (top): Western analysis of FABP4 secretion from differentiated 3T3-L1 adipocytes in response to a 4-h treatment with VEH, buffer, 20 μmol/L FSK, 10 μmol/L isoproterenol (ISO), 1 mmol/L 8-Br-cAMP, or 500 nmol/L insulin (INS). The intracellular levels of β-actin and FABP4 were determined immunochemically. Because these samples were analyzed on different gels, the indicated break in the image denotes the two separate gels used. A (bottom): Secreted and intracellular FABP4 in differentiated OP9 adipocytes in response to VEH or 20 μmol/L FSK. B: Levels of FABP4 (quantified from A) and fatty acids (FFA) secreted (as measured by the colorometric assay described in research design and methods) by 3T3-L1 adipocytes treated with VEH or 20 μmol/L FSK. C: Tissue explants from eWAT, pWAT, and iWAT were isolated and treated with 20 μmol/L FSK or 500 nmol/L INS for 4 h, and the secretion of FABP4 was evaluated immunochemically. D: Quantitation of FABP4 (from C) and FFA secretion by eWAT and pWAT explants. E: Quantitation of FABP4 and FFA secretion by iWAT explants from male or female C57BL/6J mice as shown in C. F: FABP4 secretion from primary adipocytes derived from eWAT and iWAT of high-fat–fed C57BL/6J mice in response to 2-h treatment with either 20 μmol/L FSK or 500 nmol/L INS. G: Explant tissue from interscapular BAT of male C57BL/6J mice was isolated as described and treated with 20 μmol/L FSK or 500 nmol/L INS for 4 h. The secretion of FABP4 was evaluated immunochemically. H: Quantitation of FABP4 and FFA secretion by BAT explants from male C57BL/6J mice as shown in G. *P < 0.05; **P < 0.01; ***P < 0.001. A.U., arbitrary units; F, female; hr, hours; M, male.
- Figure 2
FABP4 secretion in response to chemical regulators of autophagy. A: Differentiated 3T3-L1 adipocytes were pretreated for 2 h with or without 5 mmol/L 3MA, washed, and then incubated in secretion media with VEH or 20 μmol/L FSK in the presence of 3MA for 4 h. Secreted FABP4 and cellular actin, FABP4, and LC3BI and -II levels were determined immunochemically. B: Quantification secreted FFA and FABP4 from A. C: Differentiated 3T3-L1 adipocytes were pretreated for 2 h with or without 30 μmol/L NAS, washed, and then incubated in secretion media with VEH or 20 μmol/L FSK in the presence of NAS for 4 h. Secreted FABP4 and cellular β-actin, FABP4, and P62 levels were determined immunochemically. D: Levels of secreted FFA and FABP4 from C. E: Differentiated 3T3-L1 adipocytes were pretreated for 2 h with or without 7.5 μmol/L of the PI3K inhibitor PIK-III, washed, and then incubated in secretion media with VEH or 20 μmol/L FSK in the presence of PIK-III for 4 h. Secreted FABP4 and cellular β-actin, FABP4, and LC3BI and -II levels were determined immunochemically. F: Quantitation of FFA and FABP4 secretion from E. *P < 0.05; **P < 0.01. hr, hours; kd, knockdown.
- Figure 3
FABP4 secretion in response to molecular regulators of early autophagic proteins. A: Lentiviral-infected control (Ctrl) or Atg5-silenced 3T3-L1 adipocytes were treated with VEH or 20 μmol/L FSK for 4 h, and the secretion of FABP4 and RBP4, as well as the cellular levels of ATG5, β-actin, FABP4, and histone H4, was evaluated immunochemically. B: Quantitative analysis of the levels of secreted FFA and FABP4 from A. C: MEFs from a wild-type C57BL/6J or beclin-1 null mouse were differentiated as described in research design and methods, and secreted FABP4 and RBP4 and cellular levels of beclin-1, β-actin, FABP4, and P62 were determined immunochemically. D (top): Lentiviral-infected control or Fip200-silenced 3T3-L1 adipocytes were treated with VEH or 20 μmol/L FSK for 4 h, and the secretion of FABP4, FABP5, eNAMPT, and RBP4 as well as cellular FABP4 was determined immunochemically. D (bottom): Cellular levels of FIP200 and β-actin in control or Fip200-silenced 3T3-L1 adipocytes. E: Quantitative analysis of FABP4 (from D) and FFAs secreted by control or Fip200-silenced 3T3-L1 adipocytes. F: MEFs from wild-type C57BL/6J or ULK1/2 null mice were differentiated and the expression of secreted FABP4 and RBP4 and cellular levels of ULK1/2, β-actin, FABP4, and LC3B were determined immunochemically. The arrow indicates specific RBP4 band. G: Differentiated 3T3-L1 adipocytes were treated with or without 2 μmol/L of the ULK1/2 inhibitor MRT68921 for 2 h followed by addition of either VEH or 20 μmol/L FSK for 4 h. The levels of secreted FABP4 and cellular β-actin and FABP4 were evaluated immunochemically. Additionally, the cellular levels of phosphorylated ATG14 (pATG14), ATG14, and β-actin were evaluated immunochemically. H: Quantitation of FABP4 and FFA secreted by 3T3-L1 adipocytes in response to VEH or 20 μmol/L FSK stimulation in the presence and absence of the ULK1/2 inhibitor MRT68921. *P < 0.05; **P < 0.01; ***P < 0.001. hr, hours; kd, knockdown.
- Figure 4
FABP4 secretion in response to regulators of SIRT1. A: Differentiated 3T3-L1 adipocytes were treated with 10 μmol/L EX527 or 10 μmol/L resveratrol for 16 h, and the secretion of FABP4, FABP5, eNAMPT, and RBP4 was determined immunochemically. In parallel, the cellular extract was probed for acetylated lysine using the polyspecific anti-Kac antibody. The lysine acetylation results are from a single gel but are shown as different exposures to allow a better representation of the resulting signal across the entire blot. B: Differentiated Sirt1-deficient and control MEFs were treated with VEH or 20 μmol/L FSK for 4 h and secreted FABP4 and RBP4 and cellular SIRT1, β-actin, and FABP4 determined immunochemically. C: Quantitative analysis of the levels of FABP4 (from B) and FFA secreted by control and SIRT1-deficient adipocytes. D: Levels of Sirt1 mRNA relative to TFIIE measured in control and Sirt1 silenced 3T3-L1 adipocytes. E: Control and Sirt1-silenced 3T3-L1 adipocytes were preincubated for 18 h with 400 μmol/L potassium oleate complexed to 100 μmol/L BSA. The basal levels of FABP4, FABP5, and RBP4 secretion were determined immunochemically. F: Differentiated 3T3-L1 adipocytes were incubated with 50 μmol/L of the pan-FABP inhibitor HTS01037 (HTS) or 20 μmol/L FSK, and the levels of secreted FABP4 were determined immunochemically. G: Serum FABP4 levels in chow-fed wild-type (WT) and Sirt1 null C57BL/6J mice as evaluated by an ELISA (Lifespan Biosciences Inc.) conducted per the manufacturer’s protocol. *P < 0.05. hr, hour; kd, knockdown; ND, not detected.
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