Plasma butyrylcholinesterase regulates ghrelin to control aggression

Abstract

Ongoing mouse studies of a proposed therapy for cocaine abuse based on viral gene transfer of butyrylcholinesterase (BChE) mutated for accelerated cocaine hydrolysis have yielded surprising effects on aggression. Further investigation has linked these effects to a reduction in circulating ghrelin, driven by BChE at levels ∼ 100-fold above normal. Tests with human BChE showed ready ghrelin hydrolysis at physiologic concentrations, and multiple low-mass molecular dynamics simulations revealed that ghrelin's first five residues fit sterically and electrostatically into BChE's active site. Consistent with in vitro results, male BALB/c mice with high plasma BChE after gene transfer exhibited sharply reduced plasma ghrelin. Unexpectedly, such animals fought less, both spontaneously and in a resident/intruder provocation model. One mutant BChE was found to be deficient in ghrelin hydrolysis. BALB/c mice transduced with this variant retained normal plasma ghrelin levels and did not differ from untreated controls in the aggression model. In contrast, C57BL/6 mice with BChE gene deletion exhibited increased ghrelin and fought more readily than wild-type animals. Collectively, these findings indicate that BChE-catalyzed ghrelin hydrolysis influences mouse aggression and social stress, with potential implications for humans.

Keywords: BChE; aggression; ghrelin; mice; viral vector.

Fig. 1.

In vitro enzymatic deacylation of human ghrelin by human BChE (125 nM). (A) Reciprocal changes in ghrelin and desacyl-ghrelin (0.5 nM initial substrate). (B) Generation of desacyl-ghrelin from ghrelin, 0.5 nM (Left) or 25 µM (Right). (C) Inhibitor sensitivity. BChE was incubated 10 min with iso-OMPA or proteinase inhibitors (1 µM aprotinin, 20 µM leupeptin, 15 mM pepstatin). Ghrelin was then added (0.5 nM). Hydrolysis activities compared with no-inhibitor controls were determined 1 h later by Ellman assay and desacyl-ghrelin immunoassay.

Fig. 2.

Ghrelin-bound BChE model. (A) Acylated ghrelin bound in the active site of human BChE at a reversible complex state before hydrolysis obtained from 100 2.0-ns low-mass molecular dynamics simulations. Ghrelin is in ball-and-stick model; BChE is in cartoon model with key amino acids indicated. (B) Close-up of ghrelin’s acyl group in relation to catalytic triad of human BChE.

Fig. 3.

BChE and ghrelin levels after gene transfer in C57BL/6 mice. (A) Plasma BChE activity with butyrylthiocholine in mice given 10¹³ particles of AAV-luciferase or AAV-mBChE mutant vector. Mean values ± SEM (n = 8 per group). (B) Ghrelin levels after AAV-luciferase or AAV-mBChE mutant vector treatment as percentage of untreated control. (C and D) BChE activity and ghrelin levels under fasting conditions, 6 h after treatment with iso-OMPA, 50 mg/kg, i.p., or saline. Values are means ± SEM (n = 4); ***P < 0.001 compared with saline group.

Fig. 4.

Bite scores in confrontations between a resident male mouse and a male intruder. (A) Three-month-old BALB/c mice with AAV-luciferase vector (n = 18) vs. mBChE mutant vector-treated mice (n = 7) and AAV-CocH-6 ΔT-treated mice (n = 14). (B) Untreated 3-mo-old C57BL/6 wild-type mice (n = 18) vs. same-age BChE knockouts (n = 9) and vector-treated BChE knockouts (n = 9). (C) AAV-luciferase–treated 3-mo-old C57BL/6 wild-type (n = 9) vs. same-age C57BL/6 treated simultaneously with AAV vectors encoding cDNA for ghrelin and GOAT (n = 6) and same-age mice treated triply with vectors for ghrelin, GOAT, and mBChE mutant (n = 9). (D) C57BL/6 mice given AAV-luciferase (n = 9) or mBChE mutant vector (n = 9) at 6 wk and tested for aggression at 12 mo. Data were analyzed by two-way ANOVA with a Holm–Sidak multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.