*Mechanism, Role and Functions of SCFAs*

SCFAs are versatile molecules involved in cell signalling in a wide range of physiological and pathological conditions [40]. Numerous pieces of evidence report that the disruption of SCFAs generated by the gut microbiota is associated with diseases, including inflammatory bowel disease, obesity, diabetes mellitus type 1 and 2, autism, major depression, colon cancer, and renal diseases [41,42]. The functions of SCFAs are mainly related to their activation of free fatty acid receptors (FFARs) belonging to the family of orphan G-protein-coupled receptors (GPCRs) and olfactory receptors (Olfr) or as histone deacetylase inhibitors (HDACs). FFARs are G-protein-coupled transmembrane receptors that bind fatty acids with carbon chains of different lengths. In particular, SCFAs are ligands of the GPR41 and GPR109A receptors coupled to the Gi/0 protein and the GPR43 receptor coupled to both Gi/0 and Gq proteins. GPR109A responds predominantly to butyrate, which also has a high affinity for the GPR43 receptor, while propionate is a potent agonist for both GPR41 and GPR43; acetate is more selective for the GPR43 receptor. Furthermore, both acetate, propionate, and butyrate also bind to the Olfr78 receptor [39,43]. In addition to their metabolic and structural roles, SCFAs possess several metabolic and signalling properties. Indeed, after intestinal microbial fermentation, they can act locally (Table 1; [44–46]). In the intestine, butyrate is used more as an energy source for colonocytes and can be converted into glucose by intestinal gluconeogenesis, leading to satiety and a decrease in hepatic glucose production. Butyrate binds to the GPR109A receptor, which is mainly expressed intestinally on the apical membrane of colonic and small intestinal epithelial cells, and whose activation is responsible for both the activation of the NLRP3 inflammasome, which is essential for intestinal homeostasis, and anti-inflammatory effects through increased IL (Interleukin)-18. Butyrate also binds to the GPR109A expressed on dendritic cells (DCs) and induces the differentiation of naive T cells into Th1 and Th17 cells and the increase in IL-10 in T-Reg cells, providing an anti-inflammatory function and enhancing the intestinal immune response. Moreover, in the intestine, but to a lesser extent, acetate and propionate bind to the GPR43 receptor, whose signalling functions are mediated by the Gq protein to induce the release of pancreatic peptide YY (PYY) and Glucagon-like peptide 1 (GLP-1) and influence satiety and intestinal transit. The binding of propionate and acetate to the intestinal receptor GPR43 also induces the activation of the transcription factor forkhead box P3 (FOXP3) by regulatory T lymphocytes, resulting in cell expansion and differentiation into mast cells, neutrophils, and eosinophils, respectively, implementing intestinal immunity. Then, SCFAs bind to the receptors on the immune cells of the lamina propria and enteric nervous system cells, where propionate activates the GPR41 receptor and stimulates motility and secretory activity in the colon and intestine [24,47–50]. In addition to acting locally, SCFAs from the gut can also be absorbed into the bloodstream either through anion exchange between SCFAs and HCO3 or through the membrane by a diffusive process promoted by the pH gradient during the diffusion of protonated SCFAs [51]. Systemically, however, acetate concentrations are higher than propionate and butyrate, and the functions of SCFAs mainly depend on the binding to the GPR43 and GPR41 receptors, inducing beneficial effects throughout the body. The GPR43 receptor has a potential role in inflammation and is most highly expressed in immune cells, adipose tissue and the subset of large renal vessels, renal afferent arteriole and juxtaglomerular apparatus, where it is involved in the regulation of renin secretion. The GPR41 receptor is expressed in the peripheral nervous system and adipose tissue and at low levels in the spleen, lymph nodes, bone marrow, peripheral blood mononuclear cells, and blood vessel endothelial cells. In the bone marrow, the activation of GPR41 induces the hematopoiesis of DCs, and in the brain, it reduces the permeability of the blood-brain barrier, increases neurogenesis, and stimulates microglia activity. In the peripheral nervous system, it induces sympathetic activation through the release of norepinephrine, leading to an increase in heart rate, energy expenditure, and satiety. However, these events are also associated with a number of hepatic events, such as increased hepatic insulin sensitivity and the activation of AMPK-dependent signalling, reduced gluconeogenesis, and reduced lipid accumulation. GPR43, GPR41, and GPR109A

promote anti-lipolytic activity through increased glucose and lipid metabolism. SCFAs are also inhibitors of intracellular HDAC [48,52,53]. SCFAs enter the cell by diffusion and/or the transport mediated by sodium channel-coupled transporter protein SLC5A8 and, through HDAC inhibition, act on epigenetic modulation. In particular, butyrate and propionate as HDAC inhibitors in the intestine and colon protect against colorectal cancer and inflammation. Systemically, however, HDAC inhibition influences gene expression to exhibit anti-tumour, anti-fibrotic, and anti-inflammatory activities. In the lungs, for example, acetate reduces asthma symptoms and increases T-reg cells through HDAC9 inhibition [54,55].

**Table 1.** Acetate, propionate, and butyrate are formed in the human colon in an estimated ratio of approximately 3:1:1. Different bacteria are involved in SCFAs production, and once produced, SCFAs are able to bind to different receptors. In the table are indicated the receptors for which each SCFA has a major affinity and their intestinal and non-intestinal expression.

