Butyric acid is one of short-chain fatty acids (SCFAs) that are generated
physiologically by anaerobic microbial fermentation of dietary fibers in the
gastrointestinal tract (Bugaut and Bentéjac, 1993). Major SCFAs contain acetic acid,
propionic acid, and butyric acid. SCFAs offer an important energy source for animals,
providing ~10-25% of total energy requirement for monogastric animals, and ~60-70%
for ruminants (Bergman, 1990). Besides, SCFAs not only benefit the colonic mucosa
and intestinal epithelial cells, but also play an important role in colonic homeostasis
(den Besten et al., 2013). Colonocytes utilize SCFAs as their primary energy source,
and they tend to undergo apoptosis easily when there is no SCFA available (Hamer et
al., 2008). There are four carbons in the molecule of butyric acid (CH3CH2CH2COO-H),
and butyric acid becomes sodium butyrate (NaB, CH3CH2CH2COO-Na) after receiving
sodium. NaB is a sodium salt of butyrate used for research experiments because it has
the same physiologic function as butyric acid. Moreover, it is much stable than butyric
acid. NaB is a natural nutrient and dietary component existed in milk products. It is also
produced in a large quantity (from 40 to 100 mM) (den Besten et al., 2013), and
resistant starch, nonstarch polysaccharides, and other low-digestible saccharides (Pryde
et al., 2002).
In addition to serve as an energy resource, butyric acid or sodium butyrate is also
to mediate several physiological functions. Numerous studies suggested that butyrate
plays a role in rumen development, gastrointestinal motility, insulin and glucagon
secretion, gastrointestinal blood flow, epithelial cell proliferation, cell differentiation,
and apoptosis (Bergman, 1990). Recent studies also revealed that dietary
supplementation of sodium butyrate can prevent diet-induced obesity and decrease the
occurring rate of insulin resistance in mouse models of obesity (Gao et al., 2009).
Sodium butyrate has also been shown to alleviate metabolic impairments, with overtly
functions in anti-obesity and maintaining β-cell function in pregnant obese mouse (Li et
al., 2013). Butyrate is likely influence insulin sensitivity by stimulating the excretion of
gastric inhibitory polypeptide (GIP) and incretin, a glucagon-like peptide 1 (GLP-1),
and affect the intestinal barrier function by stimulating the release of GLP-2 (Brahe et
al., 2013).
In addition, the biofunction for butyrate in the development of colon cancer has
been supported by the downregulation of butyrate transporters (SLC5A8, a tumor
suppressor gene silenced by methylation) in human colon tumor tissues, which results
in a reduced intake and metabolism of butyrate in the colonocytes (Li et al., 2003).
Butyrate not only has an anti-cacinogenic property, but processes the anti-inflammatory
potential in intestinal mucosa (Hamer et al., 2008). Butyrate regulates different cells
during immune and inflammatory response. It presents multiple effects on migration of
leukocytes, production of inflammatory mediators and inducing apoptosis in
lymphocytes (Vinolo et al., 2011). NF-κB (nuclear factor kappa-light-chain-enhancer of
activated B cells), a transcription factor that has an important role in regulating immune
response to infection, and IκBα (nuclear factor of kappa light polypeptide gene
enhancer in B-cells inhibitor, alpha) is a cellular protein that functions to bind and
inhibit the NF-κB (Jacobs and Harrison, 1998). The potential anti-inflammatory
mechanism of butyrate is mediated by preventing the degradation of IκBα, and thereby
blocked the ability of NF-κB (Yin et al., 2001). Furthermore, suppression of NF-κB
activation, which may also result from the inhibition of histone deacetylase (HDAC), is
the most frequently reported anti-inflammatory function of butyrate (Andoh et al., 1999;
Yin et al., 2001).
Up to sixteen HDACs of human and yeasts were classified into three categories.
Class I HDACs are expressed ubiquitously, and with four variances, HDAC1, 2, 3, and
8. Class II HDACs are abundantly expressed in brain, heart, and skeletal muscle,
consisting with HDAC4, 5, 6, 7, 9, and 10. Class III HDACs belong to the sirtuin family
of HDACs. Besides, HDAC11, a new member of the HDAC family, has also been
identified (Gao et al., 2002; de Ruijter et al., 2003). Biochemically, butyrate can act as a
class I HDACs selective inhibitor, and is an necessary agent to inhibit
chromatin-remodeling activity by affecting gene expressions and arresting cell
proliferation (Davie, 2003). Considering the effect of butyrate on inhibiting HDAC
activity and diminishing condensation of the chromatin structure, it is surprising to learn
that only 2% of the mammalian gene expression is affected when HDAC activity is
inhibited (Davie, 2003). Nevertheless, butyrate is still an effective fatty acid in
repressing or stimulating the specific gene expression (Yang et al., 2001). Besides
butyrate, it has been reported that treatment with other HDAC inhibitors such as
trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) induced either cell
cycle arrest or cell death, resisted cellular invasion and inhibited angiogenesis leading to a reversed transformation of phenotypes of cancer cells (Xu et al., 2007). Many studies
have suggested that HDAC inhibitors, in addition to their modulation in gene expression, inflammatory responses, and anticancer effects, also offer an effect on differentiation of normal cells. Culture with HDAC inhibitors suppressed osteoclast-like cells formation (Rahman et al., 2003), induced to pancreatic and hepatic cell differentiation (Haumaitre et al., 2008; Henkens et al., 2007), accelerated intestine epithelial differentiation (Tou et al., 2004), and also regulated myogenesis (McKinsey et al., 2001). Additionally, HDAC inhibitors have been demonstrated to modulate adipogenesis, whereas the role of HDACs during adipocyte differentiation is somewhat contentious. For example, several teams demonstrated that HDAC inhibitors from nature sources like TSA or others synthetically developed inhibitors such as valproic
acid (VPA), MS-275, and suberoylanilide hydroxamic acid (SAHA) suppressed adipose
conversion and adipogenic gene expressions in human preadipocytes or mouse 3T3-L1 cells (Burton et al., 2004; Catalioto et al., 2009; Kim et al., 2009; Lagace and Nachtigal, 2004). In contrast, other groups showed that butyrate can activate transcription factor expressions related to adipogenesis and thus stimulate adipocyte differentiation by reducing HDAC activity in porcine preadipocytes or mouse 3T3-L1 cells (Li et al., 2014; Yoo et al., 2006). These notions revealed that different kinds of HDAC inhibitors
may influence adipogenesis by suppressing distinct HDACs, or alterative mechanisms
that butyrate may go through to regulated adipogenesis.
G protein-coupled receptors (GPRs), also called serpentine receptors, heptahelical
receptors, and G protein-linked receptors (GPLR), represents a largest protein family of
receptors (Trzaskowski et al., 2012). GPRs are also known as seven-transmembrane
(7TM) domains. These receptors can sense extremely diverse physiological ligands
outside the cell such as amines, amino acids, peptides, proteins, glycoproteins, fatty
acids, lipids, phospholipids, steroids, nucleotides hormones, pheromones, ions, odors,
and photons (Yonezawa et al., 2013). With these various ligands, GPRs transduce
extracellular signals across the cell membrane, activate heterotrimeric G proteins and
then trigger the second messenger cascades such as the cAMP-dependent protein kinase
A and C signaling pathways (Trzaskowski et al., 2012; Bindels et al., 2013). Lately, a
number of orphan GPRs were shown to combine extracellular free fatty acids including
SCFAs, middle-chain fatty acids (MCFAs) and long-chain fatty acids (LCFAs), and
thus they were renamed as free fatty acid receptor (FFAR) (Brown et al., 2003; Le Poul
et al., 2003; Talukdar et al., 2011; Wang et al., 2006). FFAR1 (GPR40) is predominantly expressed in the pancreatic β-cells responding to MCFAs and LCFAs
and plays a pivotal role in the enhancement of glucose stimulated insulin secretion
(Yonezawa et al., 2013). GPR120 (FFAR4) is also activated by MCFAs and LCFAs
and related with LCFAs-induced glucagon-like peptide-1 (GLP-1) secretion, increasing
insulin sensitivity as well as repression of macrophage-induced inflammation. FFAR1
and GPR120 preferentially bind to LCFAs as compared to MCFAs (Bindels et al., 2013;
Talukdar et al., 2011). Unlike FFAR1 and GPR120, GPR84 is selectively bound to
MCFAs only (Wang et al., 2006). GPR84 is mainly expressed in bone marrow and
several kinds of leukocytes and monocytes, suggesting a role to mediate the functions of
free fatty acids on immune system (Blad et al., 2012). In contrast to FFAR1, GPR120
and GPR84, FFA2 (GPR43) and FFA3 (GPR41) are activated by SCFAs (Ulven, 2012).
In 2003, two different groups simultaneously identified that formate, acetate,
propionate, butyrate, isobutyrate, and pentanoate are all ligand of FFAR3 and FFAR2
(Brown et al., 2003; Le Poul et al., 2003). They also reported that FFAR3 is related to
FFAR2 (52% similarity; 43% identity) and both of them are coupled with Gα i/o family
proteins. Action of FFAR3 and 2 with Gα i/o results in an increase of intracellular Ca2+
concentration and inhibition of the adenylate cyclase pathway. Whereas only FFAR2
couples to Gα q/11 family proteins, and thus activation of FFAR2 with Gα q/11 causes to
stimulation of the phospholipase C (PLC) pathway and raises of intracellular Ca2+ levels
(Milligan et al., 2009). Although FFAR3 and FFAR2 are activated by SCFAs, they
have different specificities for carbon chain length. SCFAs bind to FFAR3 in the
following order of potency: propionate = butyrate = pentanoate > acetate = formate,
while acetate and propionate are the most selective and efficacious ligands for FFAR2
(acetate = propionate > butyrate > pentanoate = formate) (Milligan et al., 2009). Current
studies, mainly in humans and rodents, have reported that FFAR2 and FFAR3 are
expressed in intestine, spleen, and immune tissues, and both receptors mediate the
function of SCFAs. For example, FFAR2 and FFAR3 promote inflammatory response
in mouse model (Frost et al., 2014; Tazoe et al., 2008; Kim et al., 2013; Li et al., 2014;
Trompette et al., 2014). Additionally, FFAR2 and FFAR3 are also detected in
pancreatic tissues and adipose tissues, and thus impact on energy metabolites, but
FFAR3 mRNA is not detected in mouse adipose tissues (Blad et al., 2012; Frost et al.,
2014; Hong et al., 2005). Acetate can stimulate mouse leptin secretion from epididymal
adipocytes, and the mechanism is mediated through FFAR2 pathway (Zaibi et al., 2010).
However, another group demonstrated that SCFAs augment leptin expression in both
mouse primary adipose cells and hasmer CHO-K1 cells by activating FFAR3 (Xiong et
al., 2004). It is also reported that acetate and propionate increased lipid accumulation
and adipocyte differentiation in mouse 3T3-L1 adipocytes by activating FFAR2, but not
through FFAR3 (Hong et al., 2005). Besides, acetate and propionate also can activate
FFAR2 and result in inhibition of lipolysis in mouse models (Ge et al., 2008). As for
the effect of FFAR2 on obesity and metabolic abnormalities, it still remains unclear.
One group suggested that FFAR2-deficient mice are fatter than wild type mice, whereas
mice overexpressing FFAR2 specifically in adipose tissue maintained normal body
weight and had suppressed adipose accumulation by blocking insulin signaling in
adipocytes (Kimura et al., 2013). However, the other groups hold a different view, they
showed that Ffar2-KO mice changed body composition with increased lean body mass
and lower body fat mass, and were able to escape from HFD-induced obesity, leading to
better insulin secretion as well as an improvement of glucose tolerance (Bjursell et al.,
2011; Tang et al., 2015). Therefore, whether the role of FFAR2 in obesity needs further
stidies.
Adipogenesis is a complex process of cell differentiation by which undifferentiated
precursor cells become mature adipocytes (Symonds, 2012). In vitro studies using
mouse 3T3-L1 and 3T3-F442A cell line and primary human and rodent
stromal-vascular cells (SVC) isolated from various fat regions have defined the morphological, biochemical and functional changes between the period of adipocyte differentiation (Ntambi and Kim, 2000). When the adipogenic program executes, the
transcription factors such as CCAAT/enhancer-binding proteins (CEBPs), peroxisome
proliferator-activated receptor gamma (PPARγ), and adipocyte determination and
differentiation-dependent factor 1/sterol response element-binding factor 1c
(ADD1/SREBF1c) are involved in the cell morphology. The transcriptional cascades
also regulate other adipogenic genes giving rise to cell phenotype conversion and accumulation of lipid droplets (Spiegelman et al., 1997; Darlington et al., 1998; Payne et al., 2010). On the other hand, adipose tissue has main role in storing energy in the
form of lipid and a major influence on the development of obesity and related
complications. Dysregulation of adipose tissue is thought to occupy an important
position of many obesity metabolic syndromes (Wang et al., 2008). Adipogenetic
capability of preadipocytes affects the insulin resistance indeed. Limiting quantities of
mature adipocytes in a hypercaloric condition ameliorates the metabolic disease of
many organs (Unger et al., 2010). For these reasons, a greater understanding of
adipogenesis is significant for the healthy metabolism. We previously mentioned that
acetate and propionate stimulate adipogenesis via FFAR2 (Hong et al., 2005), and
butyrate augments adipocyte differentiation by down regulating HDAC in mouse
3T3-L1 cells (Haberland et al., 2010; Yoo et al., 2006), there are still some conflicting
results. Sodium butyrate inhibits mouse mesenchymal stem cells differentiating into
adipose cells or fails to influence adipocyte differentiation of primary human
preadipocytes (Chatterjee et al., 2011; Chen et al., 2007). Moreover, there is no study to
discuss whether butyrate influences the adipogenesis through FFAR2.
In this study, we observed distinct effects of butyrate on adipocyte differentiation
of mouse and porcine SVCs and mouse 3T3-L1 cells. We also found a potential
signaling pathway that butyrate may go through to affect adipogenesis in mouse SVC.