ChREBP-Mediated Regulation of Lipid Metabolism: Involvement of the Gut Microbiota, Liver, and Adipose Tissue

Carbohydrate response element-binding protein (ChREBP) plays an important role in the development of type 2 diabetes, dyslipidemia, and non-alcoholic fatty liver disease, as well as tumorigenesis. ChREBP is highly expressed in lipogenic organs, such as liver, intestine, and adipose tissue, in which it regulates the production of acetyl CoA from glucose by inducing Pklr and Acyl expression. It has recently been demonstrated that ChREBP plays a role in the conversion of gut microbiota-derived acetate to acetyl CoA by activating its target gene, Acss2, in the liver. ChREBP regulates fatty acid synthesis, elongation, and desaturation by inducing Acc1 and Fasn, elongation of long-chain fatty acids family member 6 (encoded by Elovl6), and Scd1 expression, respectively. ChREBP also regulates the formation of very low-density lipoprotein by inducing the expression of Mtp. Furthermore, it plays a crucial role in peripheral lipid metabolism by inducing Fgf21 expression, as well as that of Angptl3 and Angptl8, which are known to reduce peripheral lipoprotein lipase activity. In addition, ChREBP is involved in the production of palmitic-acid-5-hydroxystearic-acid, which increases insulin sensitivity in adipose tissue. Curiously, ChREBP is indirectly involved in fatty acid β-oxidation and subsequent ketogenesis. Thus, ChREBP regulates whole-body lipid metabolism by controlling the transcription of lipogenic enzymes and liver-derived cytokines.


INTRODUCTION
Excess carbohydrate intake causes hepatic triglyceride accumulation through the activation of carbohydrate response element binding protein (ChREBP) and de novo lipogenesis. Dietary carbohydrate is metabolized to acetyl CoA, which is a key intermediate in lipid metabolism (1). Acetyl CoA is produced by the oxidation of pyruvate, the end product of glycolysis, through the action of pyruvate dehydrogenase (PDH) and fatty acid b-oxidation, for use as a substrate in triglyceride and cholesterol synthesis, as well as ketogenesis and protein acetylation (1). In the fed state, the resulting high nucleocytosolic acetyl CoA is readily utilized for lipid synthesis and histone acetylation (1,2). In contrast, in the fasted state or under more extreme conditions, acetyl CoA is preferentially directed to the mitochondria to permit greater synthesis of adenosine triphosphate (ATP) and ketone bodies (1,2). Acetyl CoA is produced in the liver from glucose and fructose, which are rapidly converted to glyceraldehyde-3 phosphate (GAP) by glycolysis and fructolysis ( Figure 1). GAP is then converted to pyruvate by several glycolytic enzymes, including liver-type pyruvate kinase (encoded by Pklr), which is activated by ChREBP and converts phosphoenolpyruvate to pyruvate (1). Pyruvate is then converted to acetyl CoA by the PDH complex in the mitochondria. When acetyl CoA enters the tricarboxylic acid cycle, there is an increase in the production of citrate, which is exported from the mitochondria and converted to acetyl CoA by ATP citrate lyase (encoded by Acly) (1,2). Cytosolic acetyl CoA is converted into long-chain fatty acyl CoA by lipogenic enzymes such as acetyl CoA carboxylase 1 (encoded by Acc1) and fatty acid synthase (encoded by Fasn).
Carbohydrate response element-binding protein (ChREBP) plays a pivotal role in the pathogenesis of metabolic diseases and tumors (3)(4)(5)(6). ChREBP was initially identified as a glucoseactivated transcription factor that binds to the carbohydrate response element (ChoRE) in the promoter of Pklr (6). ChoREs are also found in the promoters of lipogenic genes, including Acc1 and Fasn (7,8), and ChREBP, together with its heterodimerization partner Max-like factor X, controls de novo lipogenesis in the liver FIGURE 1 | High-fructose/high-sucrose diet-feeding causes an increase in de novo lipogenesis through the regulation of lipogenic gene expression and gut microbiota-derived acetate utilization. Fructose is normally absorbed and converted to glucose and lactate. Glucose, lactate, and a small amount of fructose enter the portal vein. In the liver, glucose and fructose activate ChREBP transcriptional activity through increases in the concentrations of glucose and fructose-derived metabolites, such as xylulose-5-phosphate and glucose-6-phosphate, and this results in greater expression of lipogenic genes, such as Acc1, Fasn, Elovl6, and Scd1. This higher expression causes the metabolism of glucose and fructose to generate acetyl CoA and fatty acyl CoA in the liver. Fructose that is not absorbed in the small intestine is absorbed in the colon and enters the portal vein. In the liver, acetate is converted to acetyl CoA by acyl-coenzyme A synthetase short-chain family member 2 (encoded by Acss2), and this is used for fatty acyl CoA synthesis, sterol synthesis, and histone acetylation. ChREBP transcription activity is also regulated by acetyl CoA and uridine diphosphate-N-acetylglucosamine, through acetylation and O-GlcNAcylation, respectively. These substances are involved in epigenic regulation, such as histone acetylation and histone methylation. GLUT5, glucose transporter 5; KHK, ketohexokinase; ALDOB, aldolase B; GLUT2, glucose transporter 2; PKLR, liver-type pyruvate kinase; ACSS2, acyl-coenzyme A synthetase short-chain family member 2; G6P, glucose 6-phosphate; Xu5P, xylulose 5phosphate; ACLY, ATP citrate lyase; ACC1, acetyl CoA carboxylase; FASN, fatty acid synthase; ELOVL6, fatty acid elongase 6; SCD1, stearyl CoA desaturase; BCKDK, branched-chain ketoacid dehydrogenase kinase; CBP, CREB binding protein; OGT, O-linked N-acetylglucosamine (GlcNAc) transferase; HCF-1, host cell factor-1; PHF2, plant homeodomain finger 2; H3K4me3, trimethylated H3K4; H3K9me2, dimethylated H3K9. by upregulating the expression of lipogenic genes (9)(10)(11)(12). Studies that used DNA microarray, chromosome immunoprecipitationsequencing (ChIP-seq) and Chrebp knockout mice have confirmed a critical role for ChREBP in hepatic glucose and lipid metabolism (13)(14)(15)(16). Interestingly, ChREBP also modulates lipolysis in adipose tissue by directly regulating the expression of Fgf21 and Angptl genes (14,(17)(18)(19)(20)(21)(22)(23). Furthermore, it regulates the hepatic and intestinal metabolism of fructose, which is tightly linked to lipogenesis and the pathogenesis of the metabolic syndrome (24)(25)(26)(27)(28). In addition, ChREBP plays a critical role in the conversion of gut microbiota-derived acetate, the production of which is increased by excess dietary fructose intake, to acetyl coenzyme A (CoA) by activating its target, Acss2, in the liver, which contributes to hepatic triglyceride accumulation (29). ChREBP also modulates hepatic b-oxidation and ketogenesis (30)(31)(32). This review focuses on recent advances in knowledge of the ChREBP-mediated regulation of lipid metabolism in the liver, adipose tissue, and gut.

CARBOHYDRATE RESPONSE ELEMENT-BINDING PROTEIN REGULATES HEPATIC DE NOVO LIPOGENESIS
The Conventional Pathway for the Production of Acetyl Coenzyme A As described in the Introduction, ACLY is an important mediator of the supply of acetyl CoA from glycolysis and fatty acid oxidation. Because the ACLY-mediated conversion of citrate to acetyl CoA is critical for the synthesis of triglycerides and sterols, efforts have been made to develop ACLY inhibitors for the treatment of hypertriglyceridemia and hypercholesterolemia (2,3). Acly is expressed mainly in the liver and white adipose tissue and its expression is upregulated at the transcriptional level by glucose-activated ChREBP and the insulin-induced transcription factor sterol response element-binding protein-1c (SREBP-1c) (13,14,33,34). The enzymatic activity of ACLY is post-translationally regulated by the phosphatidylinositol 3kinase (PI3K)/Akt pathway and acetylation (35,36). In addition, recent studies have demonstrated that ACLY is activated by branched-chain ketoacid dehydrogenase kinase (BDK) and mitochondrial protein phosphatase 1K (PPM1K), which are respectively positively and negatively regulated by ChREBP (37-39) ( Figure 1). Furthermore, the branched-chain a-ketoacid dehydrogenase complex (BCKDH) is an enzyme that catalyzes the commitment step of branched-chain amino acid (BCAA) catabolism and is negatively and positively regulated by BDK and PPM1K, respectively. Thus, ChREBP regulates the use of BCAAs in triglyceride synthesis by regulating BDK/PPM1K (39). Therefore, ChREBP might contribute to the development of BCAA-induced insulin resistance (39).

A Novel Pathway for the Production of Acetyl Coenzyme A
Recent studies have revealed that gut microbiota-derived acetate also represents an important source of acetyl CoA for hepatic lipogenesis (29). Dietary fructose is absorbed and metabolized to glucose in the intestine, whereas unabsorbed fructose is fermented by the gut microbiota to produce acetate (26,27,29,(40)(41)(42). Glucose induces the expression of both ChREBP-b and SREBP-1c, whereas fructose only induces the expression of SREBP-1c in the liver (28). Our group and others have recently reported that ChREBP regulates intestinal fructose absorption by inducing the expression of Glut5, Khk, and Aldob in the intestine and that Chrebp knockout mice consuming a sucrose-based diet show an irritable bowel syndrome-like phenotype, which develops because of fructose malabsorption and an increase in the numbers of acetateproducing bacteria in the intestine (26,27). These findings suggest that the inhibition of intestinal ChREBP increases microbial acetate production by increasing the entry of unabsorbed fructose into the colon. Gut microbiota-derived acetate is absorbed and reaches the liver via the portal vein. Acetate is then converted to acetyl CoA by acyl-CoA synthetase short-chain family member, which is encoded by Acss2, and used as a substrate for lipogenesis (43,44). Acss2 is highly expressed in the kidney and liver and is present in both the cytosol and nucleus (45,46). Importantly, depletion of the gut microbiota using antibiotics suppresses acetate production in the colon, and subsequent acetyl CoA synthesis and de novo lipogenesis in the liver; and the knock-down of Acss2 mRNA using siRNA also reduces the hepatic production of acetyl CoA (29). In addition, it has been shown that SREBP-1c, a critical regulator of hepatic lipogenesis that is transcriptionally activated by insulin, induces Acss2 mRNA expression (47)(48)(49). ChREBP also induces Acss2 expression ( Figure 1) (14,29,33). Thus, inhibition of hepatic ChREBP is likely to prevent fructose-induced triglyceride accumulation by suppressing lipogenic gene expression and hepatic acetyl CoA production from gut microbial acetate, while the inhibition of intestinal ChREBP may increase acetate production in the intestine (27).
During weaning, Fasn mRNA expression is increased by greater binding of ChREBP and SREBP-1c to Fasn promoter and greater histone H3 and H4 acetylation (70). Moreover, ChREBP induces Fasn mRNA expression and triglyceride synthesis by facilitating H3 and H4 acetylation, but these effects are prevented by the inhibition of histone acetylation using garcinol, a histone acetyltransferase (HAT) inhibitor (71). The role of the histone deacetylase activity of CREB-binding protein (CBP)/p300 in the mechanism of ChREBP-induced histone acetylation is well known (72). In human hepatocytes, the binding of farnesoid X receptor (FXR) to the ChREBP-Hepatocyte Nuclear Factor 4a (HNF4a) complex triggers the release of ChREBP from CBP/p300, leading to the recruitment of the histone deacetylase, silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), to the Lpk promoter, where it acts as a co-repressor of ChREBP transcriptional activity (73). Interestingly, CBP/p300 HAT acetylates ChREBP, which promotes transactivation by this molecule (72). Therefore, CBP/ p300 regulates ChREBP transcriptional activity through the acetylation of substrates such as histones and ChREBP (Figure 1).

THE REGULATION OF LIPOPROTEIN METABOLISM BY CARBOHYDRATE RESPONSE ELEMENT BINDING PROTEIN
It has been suggested that ChREBP may regulate lipoprotein metabolism, because the plasma concentrations of cholesterol, triglyceride, and very low density lipoprotein (VLDL)triglyceride, as well as the number of VLDL particles, are lower in Chrebp knockout mice (13,31,58,76). Our detailed analysis demonstrated that the mRNA and protein expression of hepatic microsomal triglyceride transfer protein (MTP) is lower in these mice (58,76). MTP catalyzes the rate-limiting step in the production of apoB-containing VLDL, and therefore plays an important role in VLDL secretion (77). Furthermore, the consumption of a high fat/high sucrose diet is associated with lower VLDL secretion in Chrebp knockout mice than in WT mice, which is consistent with the effect of the genetic manipulation on hepatic MTTP expression (76). Thus, the lower MTP expression, together with the suppression of de novo lipogenesis, in the livers of Chrebp knockout mice may contribute to the lower hepatic VLDL secretion and plasma lipid concentrations (58,76). Interestingly, hepatic Chrebp overexpression is also associated with lower plasma triglyceride concentrations, but a different mechanism is involved (32). Hepatic Chrebp overexpression is associated with higher hepatic triglyceride content but lower plasma triglyceride concentrations (32). The hepatic Mtp mRNA expression of mice with hepatic Chrebp overexpression is similar to that of control mice (32). Because hepatic Chrebp overexpression reduces the concentrations of VLDL-triglycerides and lowdensity lipoprotein-triglycerides (32), it has been suggested that lipoprotein lipase (LPL) might be activated by hepatic Chrebp overexpression. Consistent with this, we found that hepatic Chrebp overexpression increased hepatic Fgf21 mRNA expression and the plasma FGF21 concentration (20,32,57). We also found that hepatic Chrebp overexpression was associated with lower hepatic mRNA and protein expression of ANGPTL3 (32). Because LPL is activated by FGF21 and suppressed by the ANGPTL3/ANGPTL8 complex, it is conceivable that high FGF21 and low ANGPTL3 might activate peripheral lipoprotein metabolism by increasing LPL activity, and thereby reducing plasma triglyceride concentration (32). Thus, ChREBP controls lipoprotein metabolism through the regulation of de novo lipogenesis, VLDL secretion, and the secretion of hepatokines that influence peripheral lipoprotein metabolism (Figure 3).

CARBOHYDRATE RESPONSE ELEMENT BINDING PROTEIN AND FATTY ACID ESTERS OF HYDROXYFATTY ACIDS
Fatty acid esters of hydroxyfatty acids (FAHFAs) are a recently discovered class of endogenous lipids that have anti-diabetic and anti-inflammatory properties (78)(79)(80)(81)(82)(83). FAHFAs, and especially palmitic-acid-hydroxy-stearic-acid (PAHSA), have been found to be present in much higher concentrations in mice that overexpress GLUT4 in adipose tissue, which is a major site of FAHFA synthesis (78,79). PAHSA is hydrolyzed by carboxyl ester lipase, mutations of which are known to cause maturity-onset diabetes of the young type 8 (80). In humans, FAHFAs can be found in serum, breast milk, meconium, and adipose tissue; and the serum PHASA concentration correlates with insulin sensitivity (81). It has been shown that PAHSA activates G-protein coupled receptor 120 (GPR120) to increase insulin-stimulated glucose uptake by adipocytes and glucagon-like-peptide-1 (GLP-1) secretion in the intestine (82). In addition, PAHSA activates G-protein coupled receptor 40 (GPR40) to increase glucose-induced insulin secretion (82) (Figure 4). PAHSA reduces adipose tissue inflammation and potentiates the insulin-induced suppression of hepatic glucose production (78,79,83). Whole-body Chrebp knockout mice have lower PAHSA concentrations in their adipose depots (78). Recently, adipose tissue-specific Chrebp knockout mice were also shown to have lower PAHSA concentrations in their serum and adipose tissue (83). Although the relationship between carboxyl ester lipase and ChREBP remain unclear, ChREBP regulates de novo lipogenesis in adipose tissue. Therefore, ChREBP may regulate PAHSA concentration via effects on biosynthetic and/or degradative pathways in adipose tissue. because a reduction in adipose Chrebp expression causes insulin resistance, the administration of PAHSA might be a means of ameliorating this defect, but further investigation is needed to clarify the mechanism involved.

CARBOHYDRATE RESPONSE ELEMENT BINDING PROTEIN REGULATES B-OXIDATION AND KETOGENESIS
b-oxidation and ketogenesis are upregulated in the fasting state to produce ketone bodies that are used as an energy source in peripheral tissues, including brain, heart, and muscle. Through b-oxidation, fatty acyl CoA is converted to acetyl CoA; and the resulting acetyl CoA is then converted to 3-hydroxybutyric acid (OHBA) or acetoacetyl CoA through ketogenesis. Although ChREBP is activated by feeding, it is also involved in boxidation and ketogenesis in the fasting state (30,32,76), which was suggested by the observation that hepatic Chrebp overexpression reduces plasma OHBA concentration and increases free fatty acid concentrations (32). Hepatic Chrebp overexpression causes an increase in the expression of Acc2, which catalyzes the conversion of acetyl CoA to malonyl CoA. Malonyl CoA reduces the entry of acyl CoA into mitochondria by inhibiting carnitine palmitoyl transferase-1 (84). Hepatic Chrebp overexpression also reduces the expression of acyl CoA oxidase (58). Therefore, it has been suggested that ChREBP activation suppresses b-oxidation and ketogenesis. However, Chrebp knockout mice have paradoxically low plasma OHBA concentrations, compared to their FFA concentrations, which suggests that ChREBP inhibition also suppresses b-oxidation and ketogenesis (30,31,58,76). Recently, it was reported that peroxisome proliferator-activated receptor-a (PPARa) is required for the ChREBP-mediated transcriptional activation of the Fgf21 gene (85). Although ChREBP and PPARa mRNA expression are reciprocally regulated in other tissues such brown adipose tissues (86), these results suggest that ChREBP may act in concert with PPARa to regulate gene transcription, even in the fasting state, to fine-tune b-oxidation and ketogenesis.

CONCLUSIONS AND FURTHER PERSPECTIVES
The last two decades of research have revealed critical roles of ChREBP in lipid metabolism. ChREBP regulates hepatic lipogenesis via acetyl-CoA produced by the conventional pathway (glycolysis and fructolysis) and a novel pathway (conversion of gut microbiota-derived acetate). It also regulates lipoprotein metabolism in the liver and PAHSA production in adipose tissue. Furthermore, ChREBP regulates b-oxidation and ketogenesis in concert with PPARa. However, despite remarkable progress in understanding of the roles of ChREBP in these processes, a number of questions remain regarding its relationship with lipid metabolism. First, to what extent does gut microbiota-derived acetate induce hepatic triglyceride accumulation? Although ChREBP regulates Acss2 mRNA, does acetate itself affect ChREBP transcriptional activity? Second, the role of ChREBP in cholesterol metabolism remains to be determined. Chrebp knockout mice have lower plasma concentrations of cholesterol, despite normal cholesterol synthesis in their livers, and the low plasma cholesterol concentrations may affect physiological processes, such as steroidogenesis in the adrenal gland. Third, the physiological and pathophysiological roles of ChREBP in the fasting state require more in-depth investigation. Because ChREBP is normally activated by glucose, it is conceivable that it regulates post-prandial lipid metabolism. However, it remains unclear why the ability of ChREBP to regulate b-oxidation and ketogenesis evolved. Finally, it remains unclear whether ChREBP suppression modifies the microbiota and lipid metabolism in humans. As discussed above, Chrebp knockout mice have an altered gut microbiota due to impaired fructose absorption from the gut (25). Consistent with this, it has been demonstrated that metformin treatment modifies the gut microbiota (87). AMP-activated protein kinase (AMPK) is known to be a major target of metformin, and it also regulates the activity of several transcription factors, such as SREBP-1c, PGC-1, FOXO1, and CREB. In addition, both AMPK and metformin reduce the transcriptional activity of ChREBP. Therefore, the effects of metformin on the microbiota might be mediated partly through ChREBP (88)(89)(90). Genome-wide scanning has also identified a human ChREBP homologue, MLXIPL, genetic variants of which are associated with plasma triglyceride concentration (91). Thus, it would be interesting to determine the effects of certain dietary habits (e.g., high fructose consumption) on the gut microbiota of individuals with MLXIPL variants (92). Further investigation is necessary to clarify the various physiological and pathophysiological roles of ChREBP in lipid metabolism.

AUTHOR CONTRIBUTIONS
All the authors (KI, KT, and DY) have made a substantial, direct intellectual contribution to the work, and approved it for publication.