Unconventional secretion of FABP4 by endosomes and secretory lysosomes

Abstract

An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.

Figure 1.

FABP4 secretion is induced by lipolytic agonists in adipocytes. (A and B) Left: Adipocytes were incubated with increasing concentrations of FSK (A) and IBMX (B) and at the indicated time, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Right: Quantification of FABP4 secretion. Results are shown as the mean ± SD of three independent experiments. (C) Top: Adipocytes were incubated with or without 20 µM FSK and at indicated times, media fractions were collected and cells lysed. For each time point, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Bottom: Quantification of FABP4 secretion. Results are shown as the mean ± SD of three independent experiments. (D) Top: Adipocytes were incubated with 20 µM FSK or 20 µM 1,9-ddFSK and at the indicated time, medium was collected and cells lysed. For each condition, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Bottom: Quantification of FABP4 secretion. Results are shown as the mean ± SD of three independent experiments. (A–D) FABP4 secretion was calculated as a percentage of the signal detected in the medium compared with the total amount (the sum of FABP4 in both medium and lysate).

Figure 2.

FABP4 secretion is independent of conventional secretory pathway, autophagy processes, and MVBs, and is not secreted in exosomes. (A) Adipocytes were incubated in the presence or absence of 20 µM FSK, 10 µg/ml BFA, or 5 µM monensin for 2 h. The medium fractions were collected and cells lysed, and for each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4, anti-ACDC, and anti–α-tubulin antibodies. (B) Top: Adipocytes were incubated with 10 µM FSK for 4 h, and the medium fraction was harvested and subjected to differential centrifugation. An aliquot (10%) of each membrane pellet fraction and 0.1% of the soluble fraction were analyzed by immunoblotting with anti-CD63, anti-ACDC, and anti-FABP4 antibodies. Bottom: Quantification of the percentage of the indicated protein in each fraction compared with the total expression. Results are representative of two independent experiments. (C and D) Wild-type adipocytes and adipocytes depleted for Hrs (C) and TSG101 (D) using the CRISPR/Cas9 system were incubated with 20 µM FSK, and at indicated times, medium fractions were collected and cells lysed. For each condition, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Gene disruption efficiencies were validated by immunoblotting with anti-Hrs (C) and anti-TSG101 (D) antibodies. (E, F, and H) Adipocytes were incubated with 20 µM FSK in the presence or absence of 20 nM wortmannin (Wort; E), 5 µM PI3K inhibitor 3-methyladenin (3MA; F) or 100 µM H89 (H), and at indicated times, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium fractions was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. (G) Wild-type and knockout (KO) ATG5 MEF cells were transfected to express Flag-FABP4 and incubated in EBSS. At the indicated time, cells were lysed and medium fractions were concentrated using TCA precipitation. For each condition, 3% of the total cell lysate and 18% of the medium were analyzed by immunoblotting with anti-Flag, anti–α-tubulin and anti-LC3 antibodies.

Figure 3.

FABP4 secretion is mediated by secretory lysosomes. (A and B) Left: Adipocytes were incubated with 20 µM FSK in the presence or absence of 50 µM CQ (A) or 50 mM ammonium chloride (NH₄Cl; B), and at indicated times, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4, anti–α-tubulin, anti-LC3, and anti-P62 antibodies. Quantification of P62 expression in cell lysates is indicated. Right: Quantification of FABP4 secretion. For each condition, FABP4 secretion was calculated as a percentage of the signal detected in the medium compared with the total amount (the sum of FABP4 in both medium and lysate). Results are shown as the mean ± SD of three independent experiments. *, P < 0.05. (C and D) Adipocytes were incubated with or without 20 µM FSK for 2 h, and LAMP1 expression at the cell surface was analyzed by immunofluorescence microscopy (C) and flow cytometry (D). For immunofluorescence microscopy analysis, three inserts are shown for each condition. Bar, 10 µm. For flow cytometry analysis, results are representative of three independent experiments. (E and F) Wild-type adipocytes and adipocytes depleted for VAMP7 (E) and SytVII (F) using the CRISPR/Cas9 system were incubated with 20 µM FSK, and at the indicated time, medium was collected and cells lysed. For each condition, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Knockout efficiencies were validated by immunoblotting with anti-VAMP7 (E) and anti-SytVII (F) antibodies. Quantification of FABP4 secretion was performed as described in A and B. Results are representative of three independent experiments. Ctr, control.

Figure 4.

FABP4 accumulates in membrane compartments when FSK-induced FABP4 secretion is inhibited with CQ. (A) Left: Adipocytes were incubated in the presence or absence of 20 µM FSK with or without 50 µM CQ for 1 h. Adipocytes were homogenized, and the lysates were centrifuged to separate cytosol from the total membrane fraction. The total PC content of each membrane fraction was measured to sample the same amount of membranes for each condition. Cytosol and membrane fractions were analyzed by immunoblotting with anti-FABP4, anti-ERGIC53, anti-Sec22, and anti-LAMP1 antibodies. Right: Quantification of FABP4 expression. FABP4 expression was calculated as a percentage of the signal detected in each fraction compared with the signal detected in the cytosolic fraction in the control condition. Results are shown as the mean ± SD of three independent experiments. *, P < 0.05. (B) Adipocytes were incubated in the presence of 20 µM FSK with 50 µM CQ for 1 h. Adipocytes were homogenized, and the lysate was centrifuged to separate cytosol from the total membrane fraction. The total membrane fraction was subjected to Nycodenz density gradient centrifugation, after which 10 fractions were collected from the top and the indicated markers were detected by immunoblotting. (C) Adipocytes were incubated in the presence of 20 µM FSK with 50 µM CQ for 1 h. Medium was collected, adipocytes were homogenized, and the lysate was centrifuged to separate cytosol from the total membrane fraction. Medium and total membrane fractions were subjected to proteinase K treatment (see Materials and methods section) in the presence or absence of detergent, and specific marker proteins were analyzed by immunoblotting. (D) Membrane fractionation scheme. Adipocytes were homogenized, and the lysate was subjected to differential centrifugations. A first centrifugation at 1,000 g (1K) removed unbroken cells and nuclei. The first supernatant fraction was subjected successively to centrifugation at 3,000 g (3K), 25,000 g (25K), and 100,000 g (100K). The 25K pellet fraction, which had the highest level of FABP4, was selected, and a sucrose gradient ultracentrifugation was performed to separate the 25K pellet into L (light) and P (pellet) fractions. The L fraction, which contained the majority of FABP4, was further resolved on an OptiPrep gradient, after which 10 fractions were collected from the top. (E) Top: Adipocytes were incubated in the presence or absence of 20 µM FSK with or without 50 µM CQ for 1 h. Adipocytes were homogenized, and a differential centrifugation experiment was performed as depicted in D. The total PC content of each membrane fraction was measured to sample the same amount of membranes for each condition. Expression of FABP4 and the indicated membrane markers was analyzed in the cytosol and membrane fractions by immunoblotting. Bottom: Quantification of FABP4 expression. FABP4 expression was calculated as a percentage of the signal detected in each fraction compared with the signal detected in the cytosolic fraction in the control condition. Results are shown as the mean ± SD of three independent experiments. *, P < 0.05. (F) A sucrose step gradient ultracentrifugation to further separate the 25K pellet fraction was performed as depicted in D from adipocytes incubated in the presence of 20 µM FSK with 50 µM CQ for 1 h. The total PC content of each membrane fraction was measured to sample the same amount of membranes, and FABP4 and LAMP1 were detected by immunoblotting. (G) Top: OptiPrep gradient ultracentrifugation was used to resolve membranes in the L fraction as depicted in D from adipocytes incubated in the presence or absence of 20 µM FSK with or without 50 µM CQ for 1 h. 10 fractions were collected, and the total PC content of each membrane fraction was measured to sample the same amount of membranes, and FABP4 and specific membrane markers were analyzed by immunoblotting. Bottom: Heat maps showing the relative expression of FABP4, ERGIC, Transferrin receptor, and LAMP1. For each condition and each marker, the fraction with the highest value was defined as 1. Results are representative of three independent experiments.

Figure 5.

FABP4 localizes in the endosomal/lysosomal compartment when FSK-induced FABP4 secretion is inhibited with CQ. (A and B) Adipocytes were incubated in the presence or absence of 20 µM FSK with or without 50 µM CQ for 30 min. The localization of FABP4 was monitored by immunofluorescence microscopy in cells permeabilized or not with saponin before fixation. Bar, 10 µm. (C–F) Left: Adipocytes were incubated in the presence of 20 µM FSK with 50 µM CQ for 30 min, and cells were permeabilized with saponin before fixation. The colocalization of FABP4 with the indicated membrane markers was monitored by immunofluorescence microscopy. Bars: (C and D) 5 µm; (E and F) 10 µm. (D) Colocalization analysis using SIM. The close proximity between FABP4 and LAMP1 labeling is indicated by arrows and by an arrowhead identifying FABP4 labeling surrounded by LAMP1 labeling. Pearson correlation coefficient (r) of colocalization, FABP4/LAMP1 r: 0.317 (C); FABP4/LAMP1 r: 0.182 (D); FABP4/M6PR r: 0.530 (E); FABP4/RAB7 r: 0.456 (F); n = 10 for each colocalization analysis. Right: Intensity profile graphs of FABP4 colocalization with the indicated membrane markers.

Figure 6.

Transport through the endocytic pathway is required for FABP4 secretion. (A and B) Left: Adipocytes were incubated with 20 µM FSK in the presence or absence of 20 µM dynasore (A) or 60 µM dynasore (B), and at indicated times, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Right: Quantification of FABP4 secretion. For each condition, FABP4 secretion was calculated as a percentage of the signal detected in the medium compared with the total amount (the sum of FABP4 in both medium and lysate). Results are shown as the mean ± SD of three independent experiments. *, P < 0.05. (C–H) Left: Adipocytes were incubated in the presence of 20 µM FSK with 60 µM dynasore for 30 min, and cells were permeabilized with saponin before fixation. The colocalization of FABP4 with the indicated membrane markers was monitored by immunofluorescence microscopy. Bar, 10 µm. Pearson correlation coefficient (r) of colocalization, FABP4/M6PR r: 0.068 (C); FABP4/LAMP1 r: 0.079 (D); FABP4/EEA1 r: 0.427 (E); FABP4/CD63 r: 0.152 (F); FABP4/GRASP65 r: 0.029 (G); FABP4/GM130 r: 0.010 (H); n = 10 for each colocalization analysis. Right: Intensity profile graphs of FABP4 colocalization with the indicated membrane markers.

Figure 7.

CQ treatment inhibits FABP4 secretion in isoproterenol-treated mice. (A) Left: Adipocytes were incubated with increasing concentrations of isoproterenol, and at indicated times, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Right: Quantification of FABP4 secretion. Results are shown as the mean ± SD of three independent experiments. (B) Left: Adipocytes were incubated with 1 µM isoproterenol in the presence or absence of 50 µM CQ, and at indicated times, medium fractions were collected and cells lysed. For each condition, performed in duplicate, 1% of total cell lysate and medium was analyzed by immunoblotting with anti-FABP4 and anti–α-tubulin antibodies. Right: Quantification of FABP4 secretion. Results are shown as the mean ± SD of three independent experiments. *, P < 0.05. (A and B) FABP4 secretion was calculated as a percentage of the signal detected in the medium compared with the total amount (the sum of FABP4 in both medium and lysate). (C) Plasma FABP4 levels in mice injected with saline (Ctr) or isoproterenol (Iso, 1 mg/kg). (D) Plasma FABP4 levels at T20 in mice preinjected with saline or CQ (50 mg/kg) 24 h and 1 h before isoproterenol injection (Iso, 1 mg/kg). Results in C and D are shown as the mean ± SEM. Twelve mice were used in each group. *, P < 0.05.

Figure 8.

Unconventional secretion of FABP4. Schematic representation of the signaling and trafficking pathways required for FABP4 secretion in adipocytes. Upon lipolytic agonist stimulation (1), triglyceride hydrolysis (2) leads to FABP4 packaging in the endosomal/lysosomal compartment (3–5), followed by lysosome exocytosis and release of FABP4 to the extracellular space (6). Once circulating in the blood stream, FABP4 targets different cell types regulating essential biological functions such as the control of glucose production by hepatocytes, insulin secretion by pancreatic β-cells, or the control of cardiomyocyte contraction among others (Lamounier-Zepter et al., 2009; Cao et al., 2013; Girona et al., 2013; Wu et al., 2014; Hotamisligil and Bernlohr, 2015). AC, adenylate cyclase; ATGL, adipocyte triglyceride lipase; B-AR, β-adrenergic receptor; FFA, free fatty acid; HSL, hormone-sensitive lipase; TG, triglyceride.