Adipocyte fatty acid-binding protein (AFABP) is an adipokine, which induces insulin resistance. However, AFABP does not possess any secretion-directed signals and the mechanisms for AFABP release have not been thoroughly assessed so far. In the current study, mechanisms for AFABP secretion were elucidated in 3T3-L1 adipocytes in vitro in the presence or absence of hormonal stimulation, calcium ionophore and secretion inhibitors by cell fractionation experiments, immunoblotting and ELISAs. We demonstrate that AFABP secretion is upregulated during adipocyte differentiation. AFABP secretion is not influenced by treatment with protein secretion inhibitors that block vesicular traffic at the endoplasmic reticulum and the Golgi apparatus. AFABP is secreted partially by adipocyte-derived microvesicles (ADMs), an established mechanism for unconventional secretion from adipocytes. Both total and ADM-secreted AFABP are downregulated by insulin and upregulated by the calcium ionophore ionomycin. Furthermore, murine RAW 264.7 macrophages secrete AFABP and AFABP release from these cells is upregulated by lipopolysaccharide treatment. Taken together, these results suggest that AFABP is actively released by unconventional mechanisms and by ADMs from 3T3-L1 adipocytes. Furthermore, AFABP secretion from fat cells is regulated by insulin and intracellular calcium.
Adipocyte fatty acid-binding protein (AFABP), also known as fatty acid-binding protein-4 and aP2, has recently been suggested as a novel adipokine, which is preferentially expressed in adipocytes.1 Animal studies indicate that AFABP is not only a marker for adipocyte differentiation,2 but also an intracellular protein directly promoting metabolic and cardiovascular disease. Thus, AFABP knockout mice are protected from obesity-associated insulin resistance and diabetes mellitus.3 Concerning cardiovascular disease, AFABP-/ApoE-double knockout animals show significantly reduced atherosclerotic lesions in the aorta as compared with ApoE-deficient controls.4 In accordance with these findings, the oral AFABP inhibitor BMS309403 protects mice from atherosclerosis and type 2 diabetes mellitus.5
In 2006, it was demonstrated for the first time that AFABP is also a circulating protein, serum levels of which are significantly associated with body weight, a finding that has been confirmed by various studies.1 Most interestingly, Cao et al. very recently demonstrated convincingly for the first time that circulating AFABP directly induces insulin resistance in mice because of increased liver glucose production.6
The precise intracellular compartmentalization and trafficking pathways leading to secretion of the protein have not been comprehensively assessed so far. There is no secretory signal sequence in the primary and three-dimensional structure of AFABP.1 Therefore, AFABP might cross the plasma membrane by unconventional secretion mechanisms. Furthermore, murine adipocytes release various proteins lacking a secretory signal sequence by adipocyte-derived microvesicles (ADMs)7 and this might also be a way for AFABP secretion. In the current study, we elucidated for the first time mechanisms by which AFABP is secreted from 3T3-L1 adipocytes.
Material and methods
Culture of 3T3-L1 and RAW 264.7 cells
3T3-L1 adipocytes (American Type Culture Collection, Rockville, MD, USA) were cultured and differentiated as described previously.8 The RAW 264.7 monocyte/macrophage cell line was purchased from American Type Culture Collection and cultured according to the manufacturer’s instructions. All stimulations were carried out in DMEM-H without any additions.
Preparation of ADMs
ADMs were prepared from 3T3-L1 adipocyte supernatants as previously described.7
Antibodies and ELISAs
Rabbit anti-AFABP and anti-caveolin-1 polyclonal antibodies were supplied by Abcam (Cambridge, UK), secondary antibodies by Cell Signaling Technologies (Boston, MA, USA). ELISAs were from Biovendor (AFABP, Modrice, Czech Republic) and Mediagnost (adiponectin, Reutlingen, Germany).
Immunoblotting was performed as previously described.8
Results are shown as mean±s.e. Differences between various treatments were analyzed by unpaired Student’s t-test with P values <0.01 considered highly significant and <0.05 considered significant.
AFABP release from 3T3-L1 adipocytes is induced during differentiation
AFABP secretion significantly increased in a differentiation-dependent manner (Figure 1a) similar to the control adipokine adiponectin (Figure 1b). Interestingly, maximal differentiation-dependent AFABP content in supernatants was >10-fold higher as compared with adiponectin (Figures 1a and b). Subcellular fractionation experiments showed that AFABP was localized in the cytoplasmic and the membrane fractions but not in the nuclear fraction (data not shown).
AFABP is not secreted by conventional endoplasmic reticulum (ER)-Golgi mechanisms
3T3-L1 adipocytes were incubated with different concentrations of the ER inhibitor brefeldin A. As expected, adiponectin secretion was significantly inhibited by brefeldin A (Figure 1c). In contrast, brefeldin A did not exhibit a significant effect on AFABP secretion (Figure 1c). Similar findings were obtained with the Golgi apparatus inhibitor monensin (data not shown).
AFABP release is regulated by insulin and calcium
Insulin significantly downregulated AFABP secretion into culture medium as compared with controls (Figure 1d). Furthermore, the calcium-raising ionophore ionomycin significantly elevated AFABP secretion as compared with untreated cells (Figure 1d). Interestingly, neither insulin nor ionomycin significantly affected adiponectin secretion from 3T3-L1 adipocytes (data not shown). AFABP content in supernatants from RAW 264.7 cells, an established model for murine macrophages, was comparable to the secretion from undifferentiated 3T3-L1 cells (Figure 1e). Interestingly, lipopolysaccharide treatment significantly induced AFABP secretion by RAW 264.7 cells as compared with untreated macrophages (Figure 1e).
AFABP is significantly released by ADMs
AFABP was readily detectable in ADMs besides the ADM marker protein caveolin-1 (Figure 2a). AFABP secretion by ADMs was induced during adipocyte differentiation (Figure 2b). As expected, treatment of differentiated adipocytes with inhibitors of the conventional ER-Golgi pathway, that is, brefeldin A and monensin, did not affect AFABP secretion by ADMs (Figure 2c). Similarly, inhibitors of tubulin polymerization including nocodazole, tubulozole and cytochalasin D (Figure 2d), as well as ATP-binding cassette transporter inhibitors including glyburide and probenecide (data not shown), did not influence AFABP amounts in ADMs.
AFABP release by ADMs is regulated by insulin and calcium
Treatment of differentiated 3T3-L1 adipocytes with insulin significantly decreased AFABP amounts in ADMs (Figure 2e). Furthermore, AFABP protein content was significantly elevated in ADMs after ionomycin stimulation (Figure 2f). In contrast, incubation with interleukin-1β, dexamethasone, growth hormone and troglitazone evoked no changes in AFABP in ADMs as compared with untreated cells (data not shown).
In the current study, we demonstrate that AFABP is secreted by adipocytes via non-classical mechanisms. These findings are in accordance with recent results from Cao et al.6 In contrast, adiponectin secretion is mediated by a classical pathway in agreement with findings from other groups.9, 10, 11
In the present study, we demonstrate that AFABP is released from cells partially by ADMs, an established mechanism for unconventional secretion from adipocytes. As expected, ADM-mediated AFABP secretion is not mediated by conventional secretion, that is, it is not influenced by treatment with brefeldin A and monensin. Recent reports demonstrate that unconventional secretion is also mediated by tubulin polymerization12 and ATP-binding cassette transporters.13 As inhibitors for both mechanisms do not alter ADM-secreted AFABP, it is unlikely that they contribute to unconventional AFABP release by ADMs. Moreover, Lamounier-Zepter et al. show convincingly that ADMs isolated from human adipocytes contain up to 8% of the total amount of secreted AFABP, which is accompanied by a low AFABP-dependent cardio-depressant effect on rat cardiomyocytes.14 However, in their system with mature human adipocytes, secreted AFABP is mainly found in the microvesicle-free fraction.14
Various reports have determined the role of ADMs in adipocyte physiology in more detail. ADMs contain several angiogenic and antiangiogenic proteins including leptin, tumor necrosis factor-α, acidic fibroblast growth factor, interferon-γ, matrix metalloprotease-2 and matrix metalloprotease-9.15 ADMs induce cell migration and tube formation of human umbilical vein endothelial cells, which is partially suppressed by neutralizing antibodies to leptin, tumor necrosis factor-α and acidic fibroblast growth factor.15 ADMs also promote cell invasion of human umbilical vein endothelial cells, which is suppressed by matrix metalloprotease inhibitors.15 Most interestingly, ADMs induce angiogenesis in C57BL/6 mice in vivo.15 ADMs mediate transport of adiponectin and resistin gene transcripts into macrophages.16 Thus, ADMs might have a role as novel intercellular communication tools by transporting proteins and RNA in paracrine and endocrine manners.16 Taking these results and the insulin resistance-inducing properties of AFABP6 into consideration, AFABP secreted by ADMs might well contribute to adverse metabolic and vascular effects in obesity. Clearly, more work is needed to further evaluate this hypothesis.
In the current study, we demonstrate that insulin consistently downregulates both total and ADM-secreted AFABP. It is interesting to note in this context that Cao et al. show significantly decreased AFABP levels in supernatants of cultured adipocytes after insulin treatment similar to our findings.6 Most interestingly, circulating AFABP levels in mice fasted for 24 h are higher as compared with the fed state and ad libitum feeding.6 Taking these results into consideration, it appears plausible that insulin is a major mediator suppressing AFABP release under fed conditions. Furthermore, it is tempting to speculate that impaired insulin signaling found in insulin resistance contributes to increased circulating AFABP concentrations in obese/diabetic humans. Moreover, we demonstrate for the first time in the current study that the calcium ionophore ionomycin significantly upregulates both total and ADM-secreted AFABP. Our data suggest that calcium-mediated signal transduction contributes to unconventional release of total and ADM-secreted AFABP from 3T3-L1 adipocytes. It is interesting to note in this context that release of another adipokine, that is, leptin, is also calcium dependent.17
Interestingly, in their study, Schlottmann et al.18 introduce similar findings in human adipocytes. Briefly, in undifferentiated preadipocytes AFABP expression is barely detectable but increases continuously during differentiation. Furthermore, in differentiated human adipocytes AFABP secretion is not influenced by treatment with protein secretion inhibitors that block vesicular traffic at the ER and the Golgi apparatus. Moreover, AFABP secretion into supernatants is enhanced by intracellular calcium levels in a concentration-dependent manner. In addition, Schlottmann et al. and our group demonstrate that AFABP is also secreted by human THP-1 macrophages and murine RAW 264.7 cells, respectively.
Taken together, we demonstrate that AFABP is secreted via unconventional mechanisms from adipocytes. Further studies are needed to better define the physiological significance of total and ADM-secreted AFABP in the pathophysiology of obesity and related disorders.
This study was supported by grants to MF from the Deutsche Forschungsgemeinschaft (DFG, SFB 1052/1, C06), the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1001 (IFB Adiposity Diseases, project K7-3 and K7-58), and the Deutsche Hochdruckliga e.V. Furthermore, TE was supported by a junior research grant by the Medical Faculty, University of Leipzig, and the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1001 (IFB Adiposity Diseases, MetaRot program). We thank Stephan Lorenz for scientific insights and discussion.
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Utility of curcumin for the treatment of diabetes mellitus: Evidence from preclinical and clinical studies
Journal of Nutrition & Intermediary Metabolism (2018)