Understanding activity of butyrate at a cellular level

Butyrate is a short-chain fatty acid of four carbons in length that is a by-product produced by the microbial fermentation of dietary fiber and undigested carbohydrates within the colon. Over the years, butyrate has attracted significant attention due to its diverse roles within cells. These roles include that it serves as an energy source, regulates histone deacetylation, impacts cell development, influences various cellular processes such as apoptosis and autophagy, exerts immunomodulatory functions, and maintains intestinal barrier integrity. Recent studies indicate that butyrate also plays a major role in brain function by strengthening the integrity of blood–brain barrier (BBB), can cross the BBB to exert beneficial effects including modulation of neuroinflammation and neurotransmitter secretion, while acting as a key signal carrier molecule to improve bi-directional communication between the gut and the brain via the gut–brain axis. This perspective aims to summarise butyrate’s intricate cellular mechanisms and their effects on overall health, including neurological well-being. One of the most well-known functions of butyrate is to serve as an energy source for the cells lining the colon, also known as colonocytes. In fact, butyrate has been reported to provide approximately 70% of the energy requirements of colonocytes and 5%–15% of total human caloric requirements (reviewed in Liu et al., 2018). This energy production occurs through butyrate entering mitochondria, undergoing rapid oxidation to acetyl-CoA, which enters the Krebs cycle, resulting in NADH production, which enters the electron transport chain producing ATP and carbon dioxide (Donohoe et al., 2011). Evidence of the importance of this activity has been demonstrated by Donohoe et al. (2011) who found that marked decreases in NADH/NAD+ and ATP levels are present in the colon and colonocytes of germ-free mice compared to controls (Donohoe et al., 2011). They also found that these colonocytes had decreased mitochondrial respiration. They then colonized germ-free mice with a butyrate-producing bacterial strain called Butyrivibrio fibrisolvens and found that this rescued the decreased colonocyte levels of NADH/NAD+, ATP and mitochondrial respiration to that of control animals (Donohoe et al., 2011). Butyrate is a ligand at G-protein coupled receptors (GPR). These receptors include GPR41 and GPR43, with GPR43 expressed in a range of tissues, particularly immune cells, and GPR41 expressed even more broadly (reviewed in Siddiqui and Cresci, 2021). Butyrate preferentially binds to GPR41 over GPR43, which has higher affinities for acetate and propionate. Butyrate is also a ligand at GPR109A receptors, which are involved in regulating immune responses and inflammation. The interaction between butyrate and GPR receptors present in immune cells (neutrophils, mast cells, macrophages, dendritic cells, polymorphonuclear cells) helps to activate a cascade of anti-inflammatory signaling pathways (reviewed in Siddiqui and Cresci, 2021; Liu et al., 2023). At a cellular level, butyrate exerts beneficial effects through the inhibition of histone deacetylase (HDAC) enzymes, leading to increased acetylation of histones and more relaxed chromatin structure, which in turn improves gene accessibility to aid transcription (reviewed in Siddiqui and Cresci, 2021). This action of butyrate as an HDAC inhibitor is critical for several cellular processes, such as cell growth, differentiation, and apoptosis. Butyrate has been found to modulate the phosphoinositide 3-kinase/Akt pathway, mitogen-activated phosphate kinase pathway, and nuclear factor-κB pathway, all of which play crucial roles in regulating cell survival and apoptosis (Chen et al., 2019). Treatment with butyrate on human cancer lines has been demonstrated to induce death-associated protein kinase mediated apoptosis, promoting expression of B-cell lymphoma 2 (Bcl-2) while suppressing the action of Bcl-2-associated X protein (BAX) (Chen et al., 2019). Treatment with sodium butyrate also induces apoptosis in human colorectal cancer lines via a p53 independent pathway (Chen et al., 2019). Moreover, butyrate can also maintain cell homeostasis by inducing autophagy (Wen et al., 2024). Through histone deacetylase activity, butyrate alters the expression of autophagy-related genes, crucial to exerting anti-cancer effects. A study treating a carbon monoxide-induced brain injury rat model with sodium butyrate reported increased expressions of autophagy related genes such as LC3-II and beclin 1, which in turn induced autophagy (Wen et al., 2024). Additionally, our team has demonstrated that treating a transgenic zebrafish model of the neurodegenerative disease Machado Joseph disease with sodium butyrate results in the induction of autophagy, together with improved swimming of the zebrafish, suggesting disease amelioration (Watchon et al., 2024). Our study investigated the potential mechanism of the induction of autophagy by sodium butyrate and found that it likely involves increases in protein kinase A and adenosine monophosphate kinase activity (Watchon et al., 2024). Furthermore, butyrate’s inhibition of HDAC enzymes is at the center of its anti-inflammatory activity (reviewed in Liu et al., 2018). Through its HDAC inhibitor activity, butyrate suppresses the production of pro-inflammatory cytokines such as interferon-γ, tumor necrosis factor-alpha, interleukin-6, and interleukin-8 and promotes the production of the anti-inflammatory factors interleukin-10. These actions have been demonstrated to occur through the HDAC inhibitor effect of modulating the activity of transcription factors such as nuclear factor-κB and signal transducer and activator of transcription 3 (Liu et al., 2018). Consequently, this promotes the expression of anti-inflammatory genes, upregulating expression of peroxisome proliferator-activated receptor gamma, and differentiation of regulatory T cells. Examples of the anti-inflammatory effect of butyrate are that it has been reported to downregulate proinflammatory factors in lamina propria macrophages of patients with ulcerative colitis and regulate cytokine expression of T cells (Liu et al., 2018). Research has also suggested that butyrate plays a crucial role in maintaining gut barrier function by promoting the production of tight junction proteins including occludin and claudins (Peng et al., 2009). These proteins are essential for maintaining the integrity of the gut epithelium (Figure 1). Butyrate stimulates the expression of these tight junction proteins, which helps to strengthen and regulate the permeability of the intestinal barrier, ultimately contributing to overall gastrointestinal health. A study where butyrate was administered to a Caco-2 cell monolayer model demonstrated that butyrate affects intestinal barrier function by regulating the assembly of tight junction proteins via activation of adenosine monophosphate kinase, important for phosphorylation of tight junction proteins, affecting the stability and assembly (Peng et al., 2009). Further, a study using E12 mucus-producing epithelial cells reported that a dose of 1–10 mM of butyrate was able to improve intestinal epithelial integrity (Siddiqui and Cresci, 2021).Figure 1: Effects of butyrate at a cellular level.The short-chain fatty acid, butyrate, plays a role in regulating various physiological processes at the cellular level, including within the gastrointestinal tract where it is released, as well as through effects on the gut–brain axis. Butyrate has effects on epigenetic modifications through HDAC inhibition, influencing gene expression. Butyrate also regulates appetite and promotes satiety and glucose homeostasis, contributing to energy balance and metabolic health. Together, butyrate aids gut epithelium homeostasis, supporting the integrity of the gut barrier, and fostering a healthy gut environment. Created with BioRender.com. ATP: Adenosine triphosphate; Ca2+: calcium ion; GLP-1: glucagon-like peptide 1; HDAC: histone deacetylase; IL-6: interleukin-6; IL-8: interleukin-8; IL-10: intereukin-10; TGF-β; tumor growth factor-beta; TNF-α: tumor necrosis factor-alpha.Similarly, studies have reported that butyrate plays a crucial role in maintaining the integrity of the BBB, which is essential for protecting the brain from harmful substances. For example, sodium butyrate administration in the PD mouse model upregulated occludin and zonulin-1 protein expression, required to restore the integrity of the BBB (Liu et al., 2017). Butyrate has been identified to have vital impacts on the brain and gut health via the gut-brain axis. The gut–brain axis is a bidirectional communication pathway involving signaling contributions from the enteric nervous system, vagus nerve activity, tryptophan metabolism, and microbial metabolites (Holzer and Farzi, 2014). Butyrate acts within this pathway as a signal carrier molecule to exchange information between the brain and gut. For example, within the distal ileum and colon, butyrate stimulates enteroendocrine L cells via activation of GPRs such as GPR41 resulting in the secretion of hormones, such as satiety hormones glucagon-like peptide-1 and peptide YY (Holzer and Farzi, 2014; Figure 1). These hormones reach the brain through vagal afferents and the circulation, where they act on receptors within the hypothalamus to regulate appetite, food intake, and glucose homeostasis. Evidence of this comes from findings that whilst colonization of the mouse colon with microbial communities can increase plasma levels of peptide YY, this effect is lost by knockout of GPR41 (reviewed in Holzer and Farzi, 2014). GPR41 deficiency has also been found to result in decreased peptide YY levels, increased intestinal transit times, and an attenuation of energy harvest. Further, supplementation of sodium butyrate to the db/db mouse model of Type II diabetes and obesity has been found to elevate levels of serum glucagon-like peptide-1, confirming that butyrate can modulate the secretion of this hormone (Yang et al., 2020). As described above, butyrate has been examined as a treatment for several ailments including inflammatory bowel syndrome, ulcerative colitis, cancer, and type 2 diabetes mellitus. Recently, butyrate has also been explored as a viable therapeutic agent for neurological conditions such as Parkinson’s disease and Alzheimer’s disease. For example, treatment with sodium butyrate was found to improve motor deficits and elevate levels of dopamine within the substantia nigra of a mouse model of Parkinson’s disease (Zhang et al., 2023). This treatment was found to have reduced oxidative stress and neuroinflammation within the brains of the mice. Whilst butyrate has been found to have beneficial effects for a range of conditions, the effect of butyrate greatly varies among cell types, route of drug delivery, and type of condition. Despite a range of positive reports, it is also unlikely that butyrate is a panacea, having beneficial effects for all conditions. Therefore, more research is required to understand the cellular mechanisms at play when butyrate treatment is applied to these various conditions. Together, these findings may help us to unlock the full potential of butyrate as a treatment. This work was supported by an NHMRC Project Grant GNT2012895 (to ASL). C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y