Bile acids as global regulators of hepatic nutrient metabolism

Abstract

Bile acids (BA) are synthesized from cholesterol in the liver. They are essential for promotion of the absorption of lipids, cholesterol, and lipid-soluble vitamins from the intestines. BAs are hormones that regulate nutrient metabolism by activating nuclear receptors (farnesoid X receptor (FXR), pregnane X receptor, vitamin D) and G protein-coupled receptors (e.g., TGR5, sphingosine-1-phosphate receptor 2 (S1PR2)) in the liver and intestines. In the liver, S1PR2 activation by conjugated BAs activates the extracellular signal-regulated kinase 1/2 and AKT signaling pathways, and nuclear sphingosine kinase 2. The latter produces sphingosine-1-phosphate (S1P), an inhibitor of histone deacetylases 1/2, which allows for the differential up-regulation of expression of genes involved in the metabolism of sterols and lipids. We discuss here the emerging concepts of the interactions of BAs, FXR, insulin, S1P signaling and nutrient metabolism.

1. History of research into bile acids

Historically, bile salts were thought to be (i) “detergent molecules” required for cholesterol solubilization in the gallbladder and (ii) stimulation for the absorption of lipids, cholesterol and fat-soluble vitamins from the intestines upon formation of mixed micelles [1]. However, investigations into the role of bile acids were awakened in 1999 when three research teams reported that bile acids were natural ligands for the nuclear receptor (NR) farnesoid X receptor (FXR-α) (see Glossary) [2–4]. FXR-α is highly expressed in the liver and gastrointestinal tract. Subsequent studies showed that FXR-α is a key regulator of the homeostasis of bile acids, glucose and lipids in the liver and intestines [5]. In addition, FXR-α may play a crucial part in the pathophysiology of fatty liver, non-alcoholic steatohepatitis (NASH) and liver cancers [6]. In 2003, the G protein-coupled receptor (GPCR) TGR5 was discovered and reported to be activated by bile acids in the order lithocholic acid (LCA) > deoxycholic acid (DCA) > chenodeoxycholic acid (CDCA) > cholic acid (CA) > ursodeoxycholic acid (UDCA) [7,8]. TGR5 is a Gα_s-linked GPCR that activates the activity of adenylate cyclase, increases intracellular levels of cyclic adenosine monophosphate, and activates expression of protein kinase A. In the liver, TGR5 is expressed in macrophages, endothelial cells and cholangiocytes, but not in hepatocytes [9]. In 2012, it was reported that conjugated bile acids (CBAs) activate another Gα_i-linked GPCR, sphingosine-1-phosphate receptor 2 (S1PR2), in primary rodent hepatocytes and in vivo [10]. S1PR2 is highly expressed in liver hepatocytes and cholangiocytes. CBAs are natural ligands of S1PR2, which rapidly activates the extracellular signal-regulated kinase (ERK)1/2 and AKT signaling pathways as well as nuclear sphingosine kinase 2 (SphK2) in hepatocytes [10,11]. The physiologic and pathophysiologic importance of activation of specific NRs and GPCRs by different bile acids continues to be elucidated.

2. Enterohepatic circulation of bile acids during feeding

The primary bile acids in humans, CA and CDCA, are synthesized from cholesterol by two biosynthetic pathways in liver hepatocytes. The “classic” or “neutral” pathway is the major pathway for the synthesis of bile acids in humans. This pathway is initiated by the rate-limiting enzyme cholesterol 7α-hydroxylase to synthesize the two primary bile acids CA and CDCA [1,5]. The sterol 27α-hydroxylase is needed for the synthesis of CA in the classic pathway. The “acidic” or “alternative” pathway is initiated by mitochondrial sterol 27-hydroxylase, the activity of which is limited by the rate of transport of free cholesterol into the inner mitochondrial membrane [12]. The acidic pathway generates mostly CDCA along with the oxysterols 25- and 27-hydroxycholesterol. Oxysterols are agonists for the liver X receptor, which is a key NR regulating the homeostasis of cholesterol within the cell [13]. Once formed, bile acids are conjugated to glycine or taurine and secreted actively across the canalicular membrane of hepatocytes into bile by adenosine triphosphate-binding cassette (ABC) transporters (mainly ABCB11) along with phosphatidylcholine (by ABCB4) and free cholesterol (by ABCG5/ABCG8) [14]. These three major hepatic lipids form mixed micelles that are stored in the gallbladder.

Feeding stimulates gallbladder contraction, emptying its contents into the upper part of the small intestine, where CBAs activate pancreatic lipase to release monoglycerides and free fatty acids from triglycerides. Mixed micelles with monoglycerides, fatty acids, cholesterol, and fat-soluble vitamins (A, D, K and E) are formed and their absorption from the small bowel is promoted [1]. During their passage through the small intestine, a variable portion of CBAs is deconjugated by gut bacteria to release free bile acids and glycine or taurine. Unconjugated dihydroxy bile acids and some glycine-conjugated dihydroxy bile acids are absorbed via passive diffusion from the small intestine. In the ileum, bile acids are reabsorbed efficiently (>95%) by the apical sodium-dependent bile acid transporter. Bile acids are transported into portal blood on the basolateral side of ileocytes by organic solute transporter-α/β, which is a facilitated diffusion transporter [15]. Each day, several hundred milligrams of bile acids are not absorbed by the small intestine or ileum and enter the colon, where CA and CDCA are 7α-dehydroxylated by specific anaerobic gut bacteria to form the secondary bile acids DCA and LCA, respectively [16]. DCA and, to a much smaller extent, LCA, is absorbed passively from the colon and enters portal blood. Bile acids returning from the intestines comprise a mixture of CBAs as well as unconjugated primary and secondary bile acids. DCA can accumulate in the bile-acid pool (>50%) in some humans because DCA is not converted back to CA [17]. Bile acids are transported actively from the blood into hepatocytes primarily by the sodium taurocholate co-transporting polypeptide SLC10A1 [18]. CBAs are again secreted from hepatocytes to stimulate bile flow. Bile acids undergo the enterohepatic circulation several times each day (Fig. 1). The enterohepatic circulation of bile acids is increased by Western-type diets high in red meat, fructose and saturated fats and low in complex carbohydrates. Each day, there is a loss of 200–600 mg of bile acids into the feces, which is replaced by increased synthesis of bile acids in the liver.

Figure 1. Enterohepatic circulation of bile acids.

Bile acids are synthesized in liver hepatocytes, stored in the gallbladder, and released into the upper part of the small intestine after consumption of a meal. Conjugated bile acids promote the solubilization and absorption of cholesterol, lipids (long-chain fatty acids, monoglycerides) and lipid-soluble vitamins by forming mixed micelles. Bile acids are transported actively from the ileum (C) by apical sodium-dependent bile acid transporter (ASBT) or via passive diffusion of hydrophobic bile acids from the colon (D) and returned to the liver via the portal circulation. A–D indicate the duodenum, jejunum, ileum and colon, respectively.

3. Interplay between bile acids, insulin and FXR in the regulation of hepatic metabolism

Since 2002 it has been known that bile acids activate different signaling pathways (ERK1/2, AKT, c-Jun N-terminal kinase (JNK)1/2, protein kinase C (PKC)) in the liver and that CBAs and free BAs activate these pathways by different mechanisms [19–21]. In 2005, it was reported that taurocholic acid (TCA), but not DCA, activates the ERK1/2 and AKT signaling pathways in primary hepatocytes via a pertussis toxin (PTX)-sensitive mechanism, which suggests a role for a Gα_i-linked GPCR in this signaling pathway [19]. Nevertheless, activation of the AKT signaling pathway by TCA or DCA in primary rodent hepatocytes was shown to activate the activity of glycogen synthase significantly to the same extent as that observed with insulin alone. Interestingly, the addition of DCA or TCA plus insulin resulted in an additive effect on AKT activation and the activity of glycogen synthase in primary hepatocytes in vivo [22]. Bile acids also down-regulated expression of mRNA of the gluconeogenic genes PEPCK and G-6-Pase in primary rat hepatocytes in vivo in a manner similar to that observed with insulin [23]. The down-regulation of expression of gluconeogenic genes by TCA was blocked by PTX in primary rat hepatocytes (PRHs) [23]. Finally, in PRHs, TCA plus insulin showed stronger inhibition of the secretion/synthesis of glucose than TCA alone or insulin alone. In total, these results suggest that bile acids may “collaborate” with insulin to regulate glucose metabolism in the liver after consumption of a meal. This is a reasonable hypothesis because bile acids and co-absorbed nutrients returning from the intestines along with insulin secretion appear to be regulated in a coordinated manner.

Activation of the AKT signaling pathway appears to be important for FXR activation by bile acids. In 2010, it was reported that wortmannin (an inhibitor of the phosphoinositide 3-kinase (PI3K) signaling pathway) significantly inhibited the induction of expression of small heterodimeric partner (SHP) mRNA by TCA in PRHs [23]. SHP is a key FXR target gene that is induced by most bile acids. Further studies showed that chemical inhibition or knockdown of PKCζ mRNA significantly inhibited the ability of TCA to induce SHP [23]. PKCζ is activated by phosphoinositide-dependent protein kinase-1, which is activated by PI3K (a kinase in the insulin signaling pathway). Moreover, it has been reported that PKCζ can phosphorylate FXR, possibly allowing this NR to be translocated to the nucleus and to induce the expression of genes involved in the metabolism of bile acids, glucose and lipids [24]. Moreover, other PKC isoforms activated by bile acids have been reported to phosphorylate FXR [25]. These studies indicate a physiologic link between cell-signaling pathways (AKT, PKC) and a NR (FXR) in coordination with nutrient metabolism in the liver.

4. Discovery of S1PR2 as a CBA sensor in the liver

Early studies reported that activation of the ERK1/2 and AKT signaling pathways by CBAs was PTX-sensitive, suggesting a role for a Gα_i-linked GPCR [19]. To identify which GPCR activates the ERK1/2 and AKT signaling pathways, the cDNAs encoding more than a dozen lipid-activated GPCRs were expressed individually in HEK293 cells and screened for activation of these cell signaling pathways by TCA [10]. It was discovered that, if TCA was added to HEK293 cells over-expressing S1PR2, the ERK1/2 and AKT signaling pathways were activated significantly in a concentration-dependent manner. These results led to discovery of a link between CBAs and sphingosine-1-phosphate (S1P) signaling. S1P is synthesized from sphingosine by sphingosine kinase (SphK)1 or SphK2. SphK1 is located primarily in the cytoplasm of mammalian cells, whereas SphK2 is located in the nucleus and mitochondria [26]. S1P is secreted from mammalian cells by the transporters ABCC1 and ABCG2 as well as a facilitated diffusion transporter (spinster homolog 2) probably in a regulated manner [27]. Secreted S1P can act in an autocrine or paracrine manner to activate five GPCRs (Fig. 2), but the expression pattern of S1PRs is specific according to cell type. For example, hepatocytes express only S1PRs 1, 2 and 3, but immune cells express all S1PRs.

Figure 2. “Inside-out signaling by sphingosine-1-phosphate (S1P)”.

S1P is synthesized intracellularly from sphingosine by sphingosine kinase 1 (SphK1) or SphK2. Then, S1P is transported actively out of the cell by ABC transporters (ABCC1 and ABCG2) or via the facilitated diffusion transporter spinster homolog 2 (Spn2). Then, S1P can activate, in an autocrine or paracrine manner, five G-protein coupled receptors (S1PR1-5) to initiate a physiologic response. Intracellular S1P serves as a regulatory molecule by inhibiting specific histone deacetylases 1 and 2.

Additional studies showed that S1PR2 was the most highly expressed S1PR in primary rodent hepatocytes and human primary hepatocytes. Activation of the ERK1/2 and AKT signaling pathways by TCA was blocked significantly by an S1PR2 antagonist (JTE-013) or knockdown of S1PR2 mRNA by specific shRNA in primary hepatocytes [10]. Moreover, JTE-013 was shown to inhibit AKT activation significantly and to decrease the induction of expression of SHP mRNA by TCA using a model of chronic bile fistulae in rats. This study demonstrated a link between the insulin-signaling pathway and FXR activation in vivo. Finally, molecular modeling of the five known S1PRs showed that only S1PR2 could accommodate TCA binding in its putative active site [10].

What is the physiologic significance of S1PR2 activation in the liver? It was observed that feeding of a high-fat diet (HFD) to mice up-regulated expression of SphK2 [11]. Moreover, intestinal infusion of TCA into chronic bile fistulae in rats or over-expression of the gene encoding S1PR2 up-regulated hepatic expression of SphK2 significantly, but did not increase the levels of mRNA, protein or activity of SphK1 [11]. In contrast, primary hepatocytes isolated from S1PR2^−/− mice had significantly lower levels of nuclear SphK2 protein and activity. Measurement of S1P in nuclei isolated from the hepatocytes of wild-type and S1PR2^−/− mice showed lower levels of S1P in the nuclei from S1PR2^−/− mice. In total, these results showed an important link between CBAs, S1PR2, as well as up-regulation of nuclear levels of SphK2 and S1P.

It has been reported that S1P strongly inhibits expression of histone deacetylases 1/2 (HDAC1/2) in cancer cells [28]. Histone acetylation is a key regulator of gene expression in mammalian cells. When the levels of histone acetylation (H3K9, H4K5, and H2BK12) were measured in the nuclei of hepatocytes isolated from S1PR2^−/−or SphK2^−/− mice, acetylation was decreased markedly [11]. Moreover, there was a differential down-regulation of expression of genes encoding the enzymes involved in nutrient metabolism in hepatocytes isolated from S1PR2^−/− and SphK2^−/− mice [11]. Over-expression of the gene encoding S1 PR2 in hepatocytes from wild-type mice up-regulated expression of SphK2 markedly but not of SphK1. Expression of several genes involved in the metabolism of sterols and lipids was also up-regulated significantly, especially genes encoding the enzymes involved in fatty-acid oxidation and cholesterol transport (Table 1). As a control, the cDNA encoding mouse S1PR2 was over-expressed in mouse hepatocytes isolated from SphK2^−/− mice. The results showed no significant increase in expression of genes encoding the enzymes involved in lipid metabolism, suggesting that SphK2 is required for the up-regulation of expression of these genes [11].

Table 1.

Effect of S1PR2 over-expression on hepatic gene expression

	Expression levels (Mean ± SEM, n=3)
Gene symbol	Wild type	S1 PR2 OV (n=3)	P value
Cholesterol synthesis and transport
Cyp26a1	2423±127	3426±133*	2.15E-03
Cyp26b1	150±1	274±43*	2.87E-04
Cyp26c1	33±2	37±1	5.34E-01
Hmgcr	17249±486	12840±741*	4.33E-04
Hmgcs1	115279±9054	132452±11628	2.11E-01
Hmgcs2	1063±147	8795±628*	5.29E-71
Nceh1	9761±503	14949±416	1.95E-07
Abca1	19387±780	36536±1996*	8.32E-18
Abca2	686±16	746±14	6.58E-01
Abca3	10981±423	14474±805*	1.91E-03
Abca6	45906±2361	79400±1848*	7.64E-16
Abcg5	96±6	170±6*	5.86E-05
Abcg8	99±5	156±8*	3.36E-03
Ldlr	17713±1249	12291±271*	2.05E-05
Bile acid synthesis and transport
Cyp27a1	6780±227	7134.79±301	7.14E-01
Cyp7a1	7.0±0.1	8.6±0.56	1.11E-02
Cyp7b1	443±10	858±54*	4.04E-07
Cyp8b1	14±0.7	15±1	3.11E-01
Abcb11	1560±87	1106±88*	9.70E-03
Fatty acid synthesis, oxidation, transport
fasn	10277±724	8669±676	1.46E-01
Abcd2	1666±152	4303±130*	1.38E-18
Abcd3	34724±921	72041±1866*	5.31E-33
Dgat2	8246±160	9531±165	1.13E-01
Acaa2	7396±322	12121±432*	3.72E-09
Acaca	16834±265	22915±825*	2.88E-05
Acad11	7451±403	17331±361*	4.69E-27
Acat1	7646±257	13647±116*	4.27E-14
Acat2	10854±783	12834±478	1.18E-01
Acat3	8370±316	16691±380*	5.56E-20
Cpt1a	30659±836	46219±288l*	4.14E-08
Phospholipid transport
Apob100	3257097±111106	4333542±448539*	1.27E-03
Abcb4	10895±515	34076±945*	1.18E-56
Regulatory proteins
Nr0b2	22±0.5	31±3.5	N/A
Nr1h4	8047±74	19569.30±263*	7.99E-37
Srebf1	3981±270	3343±186	1.69E-01
Srebf2	2292±238	1844±112	1.39E-01
Sphk1	32±2	29±1	6.40E-01
Sphk2	524±36	489±68	7.97E-01

All samples were sequenced on the Illumina Hi-Seq RAPID SE100 sequencer. ~20 million 100 bp single-end reads per sample were obtained. Sequencing adapters were removed using Trimmomatic v.0.33. Quality control at each processing step will be performed using the FastQC tool v0.11.2 (quality base calls, CG content distribution, duplicate levels, complexity level). Reads were aligned to the latest assembly of the mouse genome (GRCm38/mm10) using the splice-aware aligner TopHat v2.0.9. Read counts per gene were summarized using the htseq-count tool v0.6.1p1. Differentially expressed genes were determined using the DESeq2 v1.12.4 R packages. P-values for differentially expressed genes will be corrected using a False Discovery Rate (FDR) multiple testing correction method. The biological interpretation of differentially expressed genes was performed using “gold standard” bioinformatics tools, such as DAVID 2016 version, GSEA (Gene Set Enrichment Analysis). All statistical calculations were performed within R/Bioconductor environment v3.3.1.

5. Epigenetic regulation of genes involved in hepatic lipid metabolism by S1P and SHP

The signaling cascade of CBA > S1PR2 > SphK2 > S1P > HDAC1/2 > increased hepatic gene expression can differentially up-regulate blocks of genes involved in the hepatic metabolism of sterols and lipids (Fig. 3, Table 1) [11]. Therefore, how are bile acid-induced genes repressed after feeding? There are four main mechanisms that down-regulate expression of hepatic genes induced by bile acids. The first mechanism is an increase in the serum levels of fibroblast growth factor-15/19 that is induced in the intestines by bile acids via an FXR-dependent mechanism. Fibroblast growth factor-15/19 is known to down-regulate expression of cholesterol 7α-hydroxylase and bile-acid synthesis by activating specific cell signaling pathways in the liver [29]. The second mechanism is CBA sequestration in the gallbladder that quenches bile-acid signaling in the intestines and liver. The third mechanism is reduction of insulin secretion as glucose is metabolized. The fourth mechanism is epigenetic repression by SHP-dependent mechanisms [30,31]. In this regard, SHP is a NR without a DNA binding domain, and is synthesized in response to bile-acid activation of FXR. SHP interacts with known transcription factors (hepatocyte nuclear factor 4α, sterol regulatory element-binding protein 2, liver receptor homolog-1 and others), allowing repression of expression of numerous hepatic genes involved in nutrient metabolism [32,33]. Moreover, it has been reported that SHP is activated post-translationally by phosphorylation (Thr-55) by PKCζ and then methylation (Arg-57) by protein arginine methyltransferase 5, which allows SHP to interact with the proteins that regulate gene repression [31]. SHP activation has been demonstrated to interact with HDAC1/2, G9A histone lysine methyltransferase, and chromatin-remodeling enzymes in the nucleus that promote gene repression [30]. Therefore, nuclear S1 P and activated SHP may act together as a “biologic rheostat” to regulate expression of the hepatic genes involved in nutrient metabolism during the feed/fast cycle (Fig. 3). The activation of cell signaling pathways and SphK2 by TCA occurs very rapidly (minutes) [11,23]. In contrast, induction of SHP mRNA by TCA in chronic bile fistulae in rats occurs ≤3 h after TCA infusion [23]. Therefore, SHP induction may be timed to repress gene expression after hepatic nutrient metabolism in the feed/fast cycle.

Figure 3. Cell signaling cascade activated by sphingosine-1-phosphate receptor 2 in hepatocytes.

Conjugated bile acids (CBA) or S1P activate S1PR2, which then activates the epidermal growth factor receptor (EGFR) followed by ERK1/2 and AKT activation (phosphorylation). Then, ERK1/2 phosphorylates nuclear SphK2 to stimulate S1P synthesis and inhibition of histone deacetylase 1 and 2 (HDAC1/2) to stimulate gene expression. Activation of the AKT signaling pathway results in the phosphorylation and activation of PKCζ by phosphoinositide-dependent protein kinase-1 (PDK-1). PKCζ phosphorylates FXR-α, thereby stimulating SHP induction. SHP is also phosphorylated by PKCζ, which allows interaction with nuclear HDAC1/2, G9A histone lysine methyltransferase (G9a) and chromatin-remodeling enzymes (not shown) allow the deacetylation and methylation of histones as well as chromatin remodeling to elicit repression of gene expression. In this manner, S1P and SHP may act as “biologic rheostats” to regulate expression of hepatic genes by epigenetic mechanisms during the feed/fast cycle.

6. Bile acids as cell-signaling molecules, SphK2 and fatty liver disease

A HFD is associated with obesity and nonalcoholic fatty liver disease (NAFLD), which is a precursor of NASH and liver cancer. Cell signaling by bile acids and SphK2 may play important parts in NAFLD. In this regard, feeding S1PR2^−/− or SphK2^−/− mice a HFD for 2 weeks resulted in rapid accumulation of lipids in the liver [11]. Recent studies by Lee et al. [34] reported that SphK2 is induced by activation of the unfolded protein response (UPR) in mouse livers. These investigators demonstrated that ATF4 (a transcription factor induced by the UPR) is responsible for the up-regulation of expression of SphK2 mRNA. A HFD can induce endoplasmic reticulum stress and activate the UPR. These investigators over-expressed SphK2 using a recombinant adenovirus in mice fed a HFD. They found that, in animals over-expressing SphK2, there was a marked induction of genes encoding enzymes involved in β-oxidation of fatty acids and a significant decrease in hepatic levels of triglycerides and cholesterol. Moreover, over-expression of SphK2 increased AKT^S473 phosphorylation significantly and improved tolerance to glucose and insulin without altering insulin secretion. CBAs may help regulate the metabolism of lipids, sterols and glucose by up-regulation of SphK2 expression (Fig. 4). It is not known if insulin up-regulates hepatic expression of SphK2.

Figure 4. Role of sphingosine-1-phosphate receptor 2 and conjugated bile acids in regulation of the hepatic metabolism of sterols and lipids.

S1PR2 activation by conjugated bile acids activates the ERK 1/2 and AKT signaling pathways and expression of sphingosine kinase 2 (SphK2) in the nuclei of liver hepatocytes. SphK2 produces S1P to inhibit histone deacetylases 1 and 2 with up-regulation of expression of the genes encoding enzymes involved in the metabolism of sterols and lipids. SphK2 activation also activates AKT in a non-insulin-dependent manner. Activation of the insulin signaling pathway is required for optimal activation of FXR and SHP induction, which represses expression of the genes involved in the metabolism of sterols and lipids.

The signaling cascade of CBAs > S1PR2 > SphK2 > S1P > hepatic gene expression may be important in the prevention of accumulation of sterols and fats in the livers of animals on a HFD. However, pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6) may quench bile acid-activated cell signaling pathways and dysregulate the hepatic metabolism of glucose, sterols and lipids. For example, pro-inflammatory cytokines that activate the JNK1/2 signaling pathway are known to increase phosphorylation of insulin receptor substrate 2 and decrease the strength of insulin signaling [35,36]. The activation of the insulin signaling pathway is important for FXR activation and SHP induction, which are key regulators of the hepatic metabolism of sterols and lipids [23]. Hence, inflammation may be a key factor in the disruption of cell signaling pathways in the liver, and lead to fatty liver and an increased risk of NASH, cirrhosis and liver cancer.

NAFLD and fatty liver is the accumulation of fat and cholesterol in hepatocytes. NAFLD is one of the most common liver diseases in the USA and worldwide [37,38]. NAFLD is associated with obesity, inflammation and insulin resistance. Fat accumulation in the liver is due to the uptake and synthesis (intake) of fats being greater than β-oxidation and secretion via very low density lipoprotein (export). A small percentage of patients with NAFLD will go on to develop NASH, which increases the risk of cirrhosis and liver cancer [39]. Diet is a major factor in NAFLD development. A Western-type diet high in saturated fats and fructose, which stimulates lipogenesis, appears to overload the ability of the liver to oxidize and secrete fats [40]. Saturated fats and fructose are associated with an increase in expression of inflammatory markers, which can disrupt the normal bile acid- and insulin-activated cell signaling pathways that regulate the metabolism of glucose, sterols and fats in the liver. CBA-activated nuclear SphK2 appears to be a key mechanism by which liver hepatocytes increase the oxidation and secretion of fats (Fig. 4, Table 1). Moreover, up-regulation of SphK2 expression appears to increase AKT^S473 phosphorylation and enhance hepatic glucose metabolism [34]. It is not known if inflammation suppresses hepatic signaling from CBA > S1PR2 > SphK2 > S1P synthesis and gene regulation.

7. Future directions, S1PR2, and liver cancers

Future approaches for the prevention and treatment of NAFLD might include ways to up-regulate hepatic expression of nuclear SphK2 by developing specific agonists for S1PR2, which would up-regulate expression of nuclear SphK2 and increase β-oxidation of fatty acids and secretion of lipids and cholesterol via very-low-density lipoprotein. However, long-term use of such agonists may lead to the proliferation of cholangiocytes and other intestinal epithelial cells, and to an increased risk of cancers of the liver and gastrointestinal tract. In this regard, it has been reported that CBAs stimulate the proliferation of rodent and human cholangiocarcinoma cell lines. Moreover, S1PR2 and SphK2 are highly expressed in human cholangiocarcinoma cancers, and inhibition of S1PR2 by a chemical antagonist (JTE-013) or knockdown of S1PR2 by shRNA significantly inhibits the proliferation of cholangiocarcinoma cell lines [41,42]. Finally, the S1PR2/SphK2 cell signaling pathway illustrates the physiologic link between nutrient metabolism and cell proliferation. Efforts should be made to ascertain if other cell signaling pathways are activated via S1PR2.