Our study discovered that a sodium salt of gut microbial metabolite valeric acid reduces binge-like alcohol consumption in mice. This effect is associated with an increase of GABA levels in the periphery and brain, modulation of brain epigenetics and transcriptomics, and an impact on the gut microbiome composition.
Previous research has demonstrated the involvement of gut microbiota in alcohol consumption, [8–10] and the disruption of gut microbiota through antibiotics has been shown to increase ethanol consumption in mice [33]. In agreement with this study, we found that Abx treatment significantly increased voluntary ethanol consumption levels in a binge-like ethanol drinking paradigm in mice. However, the opposite effect was reported in wistar-derived high-drinker UChB rats [34], it's worth noting that it utilized an ad libitum ethanol access paradigm leading to lower BEC. In addition, a different antibiotics regimen, and animals were also used in their study. Regardless, our work reveals an important relationship between the gut microbiota and ethanol consumption behavior and supports the use of microbial-targeted approaches to study gut-brain interactions in alcohol drinking behavior.
In our mouse model, intestinal SCFA production was significantly suppressed by the Abx treatment. This finding is consistent with previous reports. Additionally, in our study where we provided various SCFAs to mice as supplements, we observed no statistical changes in alcohol intake when sodium acetate and butyrate were supplemented. Prior research suggests that acetate might encourage heavy drinking, providing a reward in the form of added energy from calories or by influencing adenosinergic adaptation mechanisms [22]. Studies have shown that sodium butyrate does not influence alcohol self-administration in non-dependent rodents but may reduce drinking in alcohol-dependent or antibiotic treated rodents. Interestingly, when we supplemented valeric acid, we observed a significant reduction in ethanol consumption. Valeric acid can also be found in plants such as Valeriana wallichii and Valeriana officinalis. One study has shown that Valeriana wallichii extract reduces chronic ethanol intake in animal models. However, Valeriana wallichii extract contains a variety of active constituents. The exact compound that is responsible for reduced ethanol intake has not been studied. In our study, sodium valerate supplementation did not affect body weight, food intake, or fluid drinking. This suggests that the observed reduction in alcohol consumption was not due to changes in fluid or weight regulation. A recent study reported a decrease in fecal isovalerate (an isomer of valeric acid) linked to increased alcohol drinking in humans, which further reiterates the potential of valeric acid in regulating ethanol consumption.
The molecular mechanisms that underlie alcohol drinking behaviors are intricate and multifaceted [35]. Anxiety can promote alcohol drinking behaviors in both humans and animals, and excessive drinking increase anxiety-like behavior [36–38]. Our study suggests that sodium valerate supplementation have potential anxiolytic effects in mice. Interestingly, Valeriana wallichii and Valeriana officinalis, plant reservoirs of valeric acid and other compounds, have been used as supplements to address insomnia and anxiety due to their sedative attributes [39–41]. GABA may plays a role in reducing depression and anxiety linked to alcohol dependence, as lower GABA levels are associated with these conditions [42, 43]. In our study, sodium valerate supplement led to increased GABA levels in stool and the amygdala. Valproic acid, a structural analog of valeric acid, is a popular antiepileptic drug with GABAergic activity. Some research suggests that valproic acid may raise GABA levels in the brain by inactivating α-ketoglutarate dehydrogenase involved in the breakdown of GABA [44]. Whether valeric acid acts in a similar fashion warrants further investigation.
Increased levels of GABA detected in stool samples from mice supplemented with sodium valerate suggest that the gut microbiome may be involved in GABA regulation. Previous studies have identified a variety of GABA-producers and degraders in the gut microbiome. Indeed, our gut-brain module analysis revealed a decrease in GABA degradation by the gut microbiome in sodium valerate supplemented mice. It will be interesting to examine the ability of GABA modulation by Ileibacterium and Dubosiella, two bacterial genera that are significantly increased during sodium valerate supplementation. However, it is also possible that changes in Ileibacterium and Dubosiella abundance are responses to administered sodium valerate or increased GABA. A report indicated that the metabolic disorder induced by chronic alcohol consumption caused a decrease in the relative abundance of Ileibacterium. Under physiological conditions, it has been widely believed that GABA does not cross blood brain barrier [45, 46]. The impact of gut-derived GABA on brain function and drinking behavior, therefore, warrants further investigation. It is also possible that valeric acid can directly cross blood brain barrier and regulates GABA levels in the brain or acts indirectly through gut-brain axis.
There is consistent evidence that acute and chronic alcohol exposure modulate histone acetylation in the amygdaloid circuitry, leading to alcohol tolerance and dependence. Our study revealed increased acetylation of histone H4 in the amygdala of sodium valerate-supplemented mice. Previous findings confirm that intermittent alcohol exposure decreased histone acetylation in the amygdala, which may be related to the ethanol-induced increase in histone deacetylase (HDAC) [47]. Similarly, administration of HDAC inhibitors like sodium butyrate increase histone acetylation and suppress anxiety or depression-like behaviors in mice. Our results suggest that HDAC inhibitors such as sodium valerate may be able to reverse the effects of ethanol via HDAC-induced epigenetic changes in the amygdala.
Increased histone acetylation leads to a more open structure of chromosomes, thus promoting gene transcription. Our data suggests that another potential mechanism by which valeric acid attenuates alcohol drinking is through its effects on transcriptional regulation in the brain. A downregulation of inflammatory molecules such as Ptgs2 and MAPK was identified in valerate-treated mice. The immune modulatory effect of valerate has been shown in experimental mouse models of colitis and multiple sclerosis, mediated by suppressing Th17 cells and the enhancement of IL-10 production [48]. Numerous studies have demonstrated that SCFAs modulate transcription of a wide range of genes associated with behaviors [19]. SCFAs are known to regulate GPCRs such as GPR41, GPR43, and GPR109A, all of which are critical in regulating neuroinflammation, depression, and anxiety-like behaviors [49]. Our bulk RNAseq analysis showed upregulation of GPR56 and downregulation of GPR158 in the amygdala region of the brain in valerate treated mice. GPR158 is a novel regulator of stress-responsive behaviors and is highly upregulated in people with major depression disorder [50]. By contrast, GPR 56 activation has an antidepressant effect [51]. Valerate acid may regulate two GPRs of opposing effects to control anxiety or depression behavior, thus indirectly influencing moderating drinking behavior.
Our study has some limitations. All experiments in the current study were performed in male mice, further investigation is necessary to ascertain if there also exists an association between sodium valerate and alcohol intake in females. The study was tested on alcohol-independent mice. Future studies will be conducted to assess treatment effects of sodium valerate supplementation on alcohol-dependent animals. Further research is needed to fully understand the underlying mechanisms of action of valerate acid on voluntary alcohol drinking.