FABP1: A Novel Hepatic Endocannabinoid and Cannabinoid Binding Protein

Abstract

Endocannabinoids (ECs) and cannabinoids are very lipophilic molecules requiring the presence of cytosolic binding proteins that chaperone these molecules to intracellular targets. While three different fatty acid binding proteins (FABP3, -5, and -7) serve this function in brain, relatively little is known about how such hydrophobic ECs and cannabinoids are transported within the liver. The most prominent hepatic FABP, liver fatty acid binding protein (FABP1 or L-FABP), has high affinity for arachidonic acid (ARA) and ARA-CoA, suggesting that FABP1 may also bind ARA-derived ECs (AEA and 2-AG). Indeed, FABP1 bound ECs with high affinity as shown by displacement of FABP1-bound fluorescent ligands and by quenching of FABP1 intrinsic tyrosine fluorescence. FABP1 also had high affinity for most non-ARA-containing ECs, FABP1 inhibitors, EC uptake/hydrolysis inhibitors, and phytocannabinoids and less so for synthetic cannabinoid receptor (CBR) agonists and antagonists. The physiological impact was examined with liver from wild-type (WT) versus FABP1 gene-ablated (LKO) male mice. As shown by liquid chromatography and mass spectrometry, FABP1 gene ablation significantly increased hepatic levels of AEA, 2-AG, and 2-OG. These increases were not due to increased protein levels of EC synthetic enzymes (NAPEPLD and DAGL) or a decreased level of EC degradative enzyme (FAAH) but correlated with complete loss of FABP1, a decreased level of SCP2 (8-fold less prevalent than FABP1, but also binds ECs), and a decreased level of degradative enzymes (NAAA and MAGL). These data indicated that FABP1 not only is the most prominent endocannabinoid and cannabinoid binding protein but also impacts hepatic endocannabinoid levels.

Fig. 1. FABP1 and SCP2 directly bind native endocannabinoids (ECs)

In Panels A–C, direct binding of ECs to FABP1 and SCP2 was determined by impact on FABP1 and SCP2 aromatic amino acid fluorescence emission as described in Methods. (A) FABP1 (500nM) was incubated with (dashed line) or without (solid line) AEA (3μM) and fluorescence emission spectra of FABP1 Tyr determined over the range 295–450 nm, Ex 280 nm. (B) SCP2 (500nM) was incubated with (dashed line) or without (solid line) OEA (1.4 μM) and fluorescence emission spectra of SCP2 Trp determined over the range 295–420 nm, Ex 275 nm. (C) SCP2 (500nM) was titrated with increasing concentration of OEA (0–3 μM) and fluorescence emission maximum of SCP2 Trp monitored with Ex 275nm/Em 330nm. In Panel D, direct binding of inhibitors to SCP2 was determined by displacement of SCP2-bound NBD-stearic acid as described in Methods. SCP2 (500nM) was incubated with NBD-stearate (500nM) and then titrated with increasing concentration of SCP2 inhibitor: SCPI1 (closed black circles), SCPI3 (open circles), SCPI4 (closed black triangles), and FABP inhibitor BMS309403 (open triangles). With increasing amount of inhibitor, NBD-stearate emission decreased (Ex = 490 nm, Em max = 528nm). K_is were calculated from K_d = 0.22 ± 0.03 μM, which was determined by reverse and forward titrations of SCP2 and NBD-stearate, and the EC50 for displacement of NBD-stearate by the respective ligands as described in Methods. In Panels E–F, direct binding of EC to ACBP was determined by impact on ACBP Tyr/Trp fluorescence emission: (E,F) ACBP (500nM) was incubated with AEA (closed black circles), OEA (open circles), or Oleoyl CoA (solid black triangles) and fluorescence emission spectra of ACBP Tyr/Trp determined over the range 290–450 nm, Ex 274 nm.

Fig. 2. FABP1 binds N-acylethanolamides (NAEs): displacement of FABP1-bound fluorescent cis-parinaroyl-CoA

Displacement assays were performed as in Methods by equilibrating FABP1 (500nM) with cis-parinaroyl-CoA (500nM) and then titrating with increasing amount of: (A) AEA (0–8μM); (B) OEA (0–1.5 μM, solid black circles), PEA (0–2μM, open circles); (C). EPEA (0–14μM, solid black circles), DHEA (0–10μM, open circles). As N-acylethanolamide concentration increased, cis-parinaroyl-CoA emission (Ex = 304nm, Em max = 420nm) decreased.

Fig. 3. FABP1 binds 2-monoacylglycerols (2-MGs): displacement of FABP1-bound fluorescent cis-parinaroyl-CoA

Displacement assays were performed as in Methods by equilibrating FABP1 (500nM) with cis-parinaroyl-CoA (500nM) and then titrating with increasing amount of: 2-AG (0–2μM, solid black circles); 2-OG (0–1.2μM, open circles); or 2-PG (0–2.6μM, solid black triangle). As 2-MG concentration increased, emission of cis-parinaroyl-CoA (Ex = 304nm, Em max = 420) decreased.

Fig. 4. FABP1 binds inhibitors of N-arachidonoylethanolamide (AEA) uptake, FABPs and SCP2: displacement of FABP1-bound fluorescent cis-parinaroyl-CoA and NBD-stearate, or ANS

FABP1-bound cis-PnCoA, NBD-stearate, and ANS fluorescence displacement assays were performed as in Methods. Briefly, FABP1 (500nM) was equilibrated with cis-parinaroyl-CoA (500nM), NBD-stearate (500nM), or ANS (35μM), and then titrated with increasing amount of AEA uptake inhibitors: (A) AM404 (0–1.6μM, solid black circles), VDM11 (0–1.6μM, open circles), OMDM1 (0–1.2μM, solid black triangles), OMDM2 (0–1.2μM, open triangles); (B) general FABP inhibitor BMS309403 (0–2.4μM, solid black circles); (C,D) SCPI1 (solid black circles); (C) SCPI3 (open circles); and (C,D) SCPI4 (solid black triangles. As inhibitor concentration increased, fluorescence emission of FABP1-bound cis-parinaroyl-CoA (Ex = 304nm, Em max = 420), NBD-stearate (Ex = 490nm, Em max = 548nm), and ANS (Ex = 380nm, Em max = 480nm) decreased.

Fig. 5. FABP1 binds phytocannabinoids and synthetic cannabinoids: displacement of FABP1-bound fluorescent cis-parinaroyl-CoA and NBD-arachidonoylethanolamide (NBD-AEA)

FABP1-bound cis-PnCoA and NBD-AEA fluorescence displacement assays were performed as in Methods. FABP1 (500nM) was equilibrated with cis-parinaroyl-CoA (500nM) or NBD-AEA (1μM) and then titrated with increasing amount (0–5μM) of phytocannabinoids: (A) tetrahydrocannabinol (THC) (solid circle, cis-PnCoA displacement; open circle, NBD-AEA displacement); (B) Cannabidiol cis-PnCoA displacement; or synthetic cannabinoids (C) JWH018 cis-PnCoA displacement (solid circle), CP55940 cis-PnCoA displacement (open circles); (D) HU-210 cis-PnCoA displacement (solid circle), Rimonabant cis-PnCoA displacement (open circles); (E) JWH-133 (cis-PnCoA displacement, solid circles; NBD-AEA displacement, open circles); SR-144528 (cis-PnCoA displacement, solid triangles; NBD-AEA displacement, open triangles). As cannabinoid concentration increased, fluorescence emission of cis-parinaroyl-CoA emission (Ex = 304nm, Em max = 420) or NBD-AEA (Ex = 490nm, Em = 540nm) were recorded.

Fig. 6. Impact of FABP1 ablation on liver N-acylethanolamide and 2-monoacylglycerol levels

C57BL/6N male and female WT and FABP1 gene ablated mice (8 wk old) were fed phytol-free, phytoestrogen-free control chow for 3 months, overnight fasted, and livers removed/flash frozen and stored at −80°C. LC-MS analysis to quantitate N-acylethanolamides and 2-monoacylglycerols using deuterated internal standards (Cayman Chemical) was performed as described in Methods to quantitate: (A) arachidonoylethanolamide (AEA); (B) oleoylethanolamide (OEA); (C) palmitoylethanolamide (PEA); (D) docosahexaenoylethanolamide (DHEA), (E) eicosapentaenoylethanolamide (EPEA); (F) 2-arachidonoylmonoglycerol (2-AG); (G) 2-oleoylmonoglycerol (2-OG); H. 2-palmitoylmonoglycerol (2-PG). Values are the mean ± SEM, n=8. *p<0.05 for LKO vs WT; ^#p<0.05 for female vs male of same genotype.

Fig. 7. Effect of FABP1 ablation on protein levels of liver proteins involved in endocannabinoid synthesis, degradation, and action

C57BL/6N male and female WT and FABP1 gene ablate mice (8 wk old) were fed phytol-free, phytoestrogen-free control chow for 3 months, overnight fasted, and livers removed/flash frozen and stored at −80°C. Aliquots of liver homogenate proteins were examined by SDS-PAGE and subsequent western blot analysis as we described (5,76) to determine protein levels of: NAPE-PLD (A), DAGL-α (B), FAAH (C), NAAA (D), MAGL (E), and CB1 (F). Insets show representative western blots of the respective protein (upper blot) and the gel-loading control protein (GAPDH, lower blot). Relative protein was normalized to internal control and WT was set to 1. Values are the mean ± SEM, n=8. *p<0.05 for LKO vs WT; ^#p<0.05 for female vs male of same genotype.

Fig. 8. FABP1 ablation impacts protein levels of liver membrane proteins and cytosolic ‘chaperone’ proteins involved in fatty acid uptake

All conditions were as in legend to Fig. 7 except that western blot analysis was performed to determine levels of: FATP5 (A), FATP2 (B), and FATP4 (C). Insets show representative western blots of the respective protein (upper blot) and the gel-loading control protein (GAPDH, lower blot). Relative protein was normalized to internal control and WT was set to 1. Mean ± SEM (n=8 different subjects). Quantitative Western blotting to determine protein levels (ng/μg total protein) of FABP1, SCP2, ACBP, and HSP70 was performed by comparison to standard curves of the respective pure recombinant proteins similarly as described for FABP1(–80). (D) FABP1, (E) SCP2, (F) ACBP, (G) HSP70. Values are the mean ± SEM, n=8. *p<0.05 for LKO vs WT; ^#p<0.05 for female vs male of same genotype.