Genetic and pharmacological manipulation of glyoxalase 1 regulates voluntary ethanol consumption in mice

Abstract

Previous studies have identified an association between the gene glyoxalase 1 (Glo1) and anxiety-like behavior in mice and have shown that the substrate of GLO1, methylglyoxal, is a competitive partial agonist at GABA_A receptors. Given the well-established role of GABA_A receptors in the behavioral effects of ethanol (EtOH), we investigated the role of Glo1 in voluntary EtOH consumption in mice using the drinking in the dark (DID) paradigm. Transgenic mice overexpressing Glo1 on both FVB/NJ (FVB) or C57BL/6J (B6) backgrounds showed increased voluntary EtOH consumption compared to their wild-type littermates in DID. Furthermore, transgenic Glo1 knockdown mice on a B6 background showed decreased voluntary EtOH consumption in DID. These genetic manipulations of Glo1 had no effect on sucrose, saccharin or water consumption. Finally, we found that a small molecule GLO1 inhibitor (S-bromobenzylglutathione cyclopentyl diester (pBBG; 6.25, 12.5 mg/kg)) reduced EtOH consumption compared to vehicle treated B6 mice without altering saccharin or water consumption. Sucrose consumption was only reduced by the higher (12.5 mg/kg) dose of pBBG. We did not observe differences in the loss of righting reflex (LORR) or EtOH-induced foot slips on the balance beam in response to acute EtOH administration (LORR: 4 g/kg, Balance Beam: 1.25 g/kg) in B6 or FVB mice overexpressing Glo1, nor in B6 mice treated with pBBG. These data are the first to implicate Glo1 in EtOH-related behaviors and suggest that GLO1 inhibitors may have therapeutic potential for the treatment of alcohol use disorders.

Keywords: Alcohol use disorder; GLO1; drinking in the dark; ethanol consumption; methylglyoxal.

Figure 1. Glo1 expression regulates EtOH consumption

Mice overexpressing Glo1 (TG) on a (A) FVB background (n=20 WT, 16 TG) and (B) B6 background showed increased EtOH consumption over a 4 hr period in DID (n=21 per genotype). (C) Glo1 knockdown (KD; B6 background) mice show reduced EtOH consumption over a 4 hr period in DID (n=21 WT, 26 KD). *p<0.05 by Two-Way ANOVA.

Figure 2. The GLO1 inhibitor pBBG reduces EtOH consumption

Acute IP injection (2hrs before testing) with the indicated doses of pBBG reduces (A) EtOH consumption at multiple doses, but has no effect on (B) water or (C) 0.2% saccharin consumption. (D) Sucrose consumption was reduced only at the 12.5 mg/kg dose. n=14-15 per group for each test *p<0.05, ** p<0.01 by Holm-Sidak (comparisons to VEH).

Figure 3. EtOH induced LORR and balance beam foot slips are not altered in Glo1 TG overexpressing mice or in mice treated with MG or pBBG

Mice overexpressing Glo1 (TG) on an (A) FVB background (n=9 WT, 13 TG), or (B) B6 background (n=15 WT, 11 TG) show no differences in duration of LORR following a 4g/kg EtOH injection. (C) WT male B6 mice treated with 0, 6.25 or 12.5 mg/kg pBBG 2 hours before EtOH injections showed no differences in duration of LORR (n=19-20 per group). On the balance beam, mice overexpressing Glo1 (TG) showed no differences in foot slips at baseline or following 1.25g/kg EtOH injections on either an (D) FVB background (n=8 WT, 9 TG), or (E) B6 background (n=8 WT, 9 TG). (F) In WT B6 mice (n=11-12 per group) EtOH treatment significantly increased foot slips (p<0.001 Two-way ANOVA), but there was no interaction between drug (VEH, 50mg/kg MG or 6.25mg/kg pBBG) and EtOH treatment (G) Using a higher dose of pBBG (50mg/kg) in WT B6 mice (n=8 per group), EtOH treatment significantly increased foot slips (p<0.001 by Two-way ANOVA), but again, there was no interaction between drug and EtOH treatment.