**4. Lipogenic gene expression in response to glucose metabolism**

Hepatic glucose metabolism activates the transcription of various genes encoding enzymes of glycolysis and lipogenesis independently of insulin. However, the initial modification of glucose into Glucose-6-phosphate (G6P) by the enzyme Glucokinase (GK; Hexokinase 4) required for transcriptional regulation by glucose is highly dependent on insulin (Bosco et al., 2000), possibly via SREBP1c (Foretz et al., 1999; Kim et al., 2004) in concert with LXR and Peroxisome Proliferator-Activated Receptor gamma (PPAR) (Kim et al., 2009). Thus the actions of glucose and insulin may be considered interdependent and that regulation of gene expression in response to glucose seems to require active LXR, SREBP1c and/or PPAR. The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 71

is involved in regulation GK- and PFK2-expression in response to insulin, this may suggest that ChREBP is dependent on insulin signaling via LXR for proper substrate availability.

Glucose metabolism from F6P can follow the alternative hexosamine biosynthetic pathway (HBP) where the enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) controls

**Figure 5.** Nutrient flux and O-GlcNAc modification of nucleocytoplasmatic proteins through the HBP

The end product of this pathway is Uridine diphosphate *N*-acetylglucosamine (UDP-GlcNAc), an essential building block for N-and O-linked glycosylation of proteins and lipids. Cytoplasmic and nuclear proteins can be dynamically modified by O-linked -Nacetylglucosamine (*O*-GlcNAc) on serine and threonine residues by the enzyme O-GlcNAc transferase (OGT) using UDP-GlcNAc as substrate. OGT is an essential enzyme as targeted deletion of this gene is lethal (Shafi et al., 2000). The enzyme O-GlcNAc transferase (OGA) hydrolyses the sugar analogous to protein dephosphorylation of phosphorylated proteins

**4.2. Glucose metabolism via the hexosamine biosynthetic pathway and O-**

**GlcNAc signaling** 

the first and rate limiting step (Fig. 5).

#### **4.1. Glucose regulation via ChREBP**

A majority of hepatic glucose-responsive genes is thought to be regulated by the transcription factor ChREBP (Yamashita et al., 2001; Ishii et al., 2004). ChREBP mediates transcriptional regulation of glycolytic and lipogenic enzymes and is particularly important for the induction of liver-pyruvate kinase (L-PK), one of the rate limiting enzymes of glycolysis, which is exclusively dependent on glucose (Matsuda et al., 1990; Dentin et al., 2004). Furthermore, ChREBP is involved in regulating ACC and FAS in concert with LXR and SREBP1c in response to glucose and insulin, respectively, suggesting its involvement of the conversion of carbohydrates into fat (Joseph et al., 2002; Talukdar & Hillgartner, 2006). Moreover, stimulation by a synthetic LXR ligand, induces hepatic expression and activity of ChREBP (Cha & Repa, 2007). However, ChREBP is apparently not dependent on LXR for its hepatic expression and activity in mice fed a high carbohydrate/high fat diet (Denechaud et al., 2008), suggesting that ChREBP activity is reinforced by upstream LXR under certain nutritional conditions. At low glucose concentrations, the ChREBP protein is retained as an inactive phosphoprotein in the cytoplasm (reviewed in (Havula & Hietakangas, 2012)). The mechanisms by which glucose activate ChREBP is not clear, but involves induction of the ChREBP mRNA, dephosphorylation of the protein, shuttling to the nucleus and binding to the ChREBP response element at the promoter of its target genes (Uyeda & Repa, 2006). Early studies pointed to xylose 5-phosphate (Xu5P), an intermediate of the pentose phosphate pathway (PPP), as an activating signal through its ability to activate protein phosphatase 2A (PP2A) and subsequent dephosphorylation of ChREBP (Havula & Hietakangas, 2012). Recently, ChREBP was shown to be activated by fructose 2,6 biphosphate (F2,6BP) in hepatocytes (Arden et al., 2012). The level of F2,6BP is regulated by the bifunctional enzyme 6-phosphofructokinase-2-kinase/fructose-2,6-biphosphatase (PFK2/FBP2). Thus, PFK2 catalyzes the synthesis and degradation of F2,6BP and as a result, the enzyme is involved in both glycolysis and gluconeogenesis. In the fed state, insulin and carbohydrates dephosphorylate PFK2 in the liver making the enzyme kinase dominant. Subsequently, F6P is converted to F2,6BP that activates PFK1, which in turn stimulates glycolysis (Fig. 6). Interestingly, LXR was recently shown to be a central regulator of hepatic PFK2 mRNA expression (Zhao et al., 2012). Activation of ChREBP in response to glucose appears to depend on multiple glucose metabolites, including G6P, X5P and F2,6BP. As LXR

is involved in regulation GK- and PFK2-expression in response to insulin, this may suggest that ChREBP is dependent on insulin signaling via LXR for proper substrate availability.

## **4.2. Glucose metabolism via the hexosamine biosynthetic pathway and O-GlcNAc signaling**

70 Lipid Metabolism

**4.1. Glucose regulation via ChREBP** 

**4. Lipogenic gene expression in response to glucose metabolism** 

Hepatic glucose metabolism activates the transcription of various genes encoding enzymes of glycolysis and lipogenesis independently of insulin. However, the initial modification of glucose into Glucose-6-phosphate (G6P) by the enzyme Glucokinase (GK; Hexokinase 4) required for transcriptional regulation by glucose is highly dependent on insulin (Bosco et al., 2000), possibly via SREBP1c (Foretz et al., 1999; Kim et al., 2004) in concert with LXR and Peroxisome Proliferator-Activated Receptor gamma (PPAR) (Kim et al., 2009). Thus the actions of glucose and insulin may be considered interdependent and that regulation of gene expression in response to glucose seems to require active LXR, SREBP1c and/or PPAR.

A majority of hepatic glucose-responsive genes is thought to be regulated by the transcription factor ChREBP (Yamashita et al., 2001; Ishii et al., 2004). ChREBP mediates transcriptional regulation of glycolytic and lipogenic enzymes and is particularly important for the induction of liver-pyruvate kinase (L-PK), one of the rate limiting enzymes of glycolysis, which is exclusively dependent on glucose (Matsuda et al., 1990; Dentin et al., 2004). Furthermore, ChREBP is involved in regulating ACC and FAS in concert with LXR and SREBP1c in response to glucose and insulin, respectively, suggesting its involvement of the conversion of carbohydrates into fat (Joseph et al., 2002; Talukdar & Hillgartner, 2006). Moreover, stimulation by a synthetic LXR ligand, induces hepatic expression and activity of ChREBP (Cha & Repa, 2007). However, ChREBP is apparently not dependent on LXR for its hepatic expression and activity in mice fed a high carbohydrate/high fat diet (Denechaud et al., 2008), suggesting that ChREBP activity is reinforced by upstream LXR under certain nutritional conditions. At low glucose concentrations, the ChREBP protein is retained as an inactive phosphoprotein in the cytoplasm (reviewed in (Havula & Hietakangas, 2012)). The mechanisms by which glucose activate ChREBP is not clear, but involves induction of the ChREBP mRNA, dephosphorylation of the protein, shuttling to the nucleus and binding to the ChREBP response element at the promoter of its target genes (Uyeda & Repa, 2006). Early studies pointed to xylose 5-phosphate (Xu5P), an intermediate of the pentose phosphate pathway (PPP), as an activating signal through its ability to activate protein phosphatase 2A (PP2A) and subsequent dephosphorylation of ChREBP (Havula & Hietakangas, 2012). Recently, ChREBP was shown to be activated by fructose 2,6 biphosphate (F2,6BP) in hepatocytes (Arden et al., 2012). The level of F2,6BP is regulated by the bifunctional enzyme 6-phosphofructokinase-2-kinase/fructose-2,6-biphosphatase (PFK2/FBP2). Thus, PFK2 catalyzes the synthesis and degradation of F2,6BP and as a result, the enzyme is involved in both glycolysis and gluconeogenesis. In the fed state, insulin and carbohydrates dephosphorylate PFK2 in the liver making the enzyme kinase dominant. Subsequently, F6P is converted to F2,6BP that activates PFK1, which in turn stimulates glycolysis (Fig. 6). Interestingly, LXR was recently shown to be a central regulator of hepatic PFK2 mRNA expression (Zhao et al., 2012). Activation of ChREBP in response to glucose appears to depend on multiple glucose metabolites, including G6P, X5P and F2,6BP. As LXR

Glucose metabolism from F6P can follow the alternative hexosamine biosynthetic pathway (HBP) where the enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) controls the first and rate limiting step (Fig. 5).

**Figure 5.** Nutrient flux and O-GlcNAc modification of nucleocytoplasmatic proteins through the HBP

The end product of this pathway is Uridine diphosphate *N*-acetylglucosamine (UDP-GlcNAc), an essential building block for N-and O-linked glycosylation of proteins and lipids. Cytoplasmic and nuclear proteins can be dynamically modified by O-linked -Nacetylglucosamine (*O*-GlcNAc) on serine and threonine residues by the enzyme O-GlcNAc transferase (OGT) using UDP-GlcNAc as substrate. OGT is an essential enzyme as targeted deletion of this gene is lethal (Shafi et al., 2000). The enzyme O-GlcNAc transferase (OGA) hydrolyses the sugar analogous to protein dephosphorylation of phosphorylated proteins

#### 72 Lipid Metabolism

by phosphatases (Hart et al., 2007; Love, 2005). Because *O*-GlcNAc levels on proteins appear to be sensitive to increasing flux through this pathway in response to nutrient excess, OGT can be considered as a general sensor of glucose availability that modifies proteins according to changes in UDP-GlcNAc levels. There is no identified consensus sequence for GlcNAcylation, and unlike the multiple genes encoding kinases, there is only a single Xlinked gene encoding the catalytic subunit of OGT in mammals (Shafi et al., 2000). For this reason, it has been hypothesized that OGT is the catalytic subunit in large transient enzyme complexes where interacting proteins are able to target OGT to its many substrates. Many transcription factors are modified by O-GlcNAc in the liver (Dentin et al., 2008; Housley et al., 2008; Kuo et al., 2008; Ozcan et al., 2010). Interestingly, FoxO1 has been shown to be a target for O-GlcNAcylation in hepatocytes in response to hyperglycemia in the insulin resistant state, resulting in elevated transactivating capacity for FoxO1 against its gluconeogenic targets phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) reinforcing hepatic glucose production (Housley et al., 2008; Kuo et al., 2008). Moreover, this activation was later shown to be dependent on targeting of OGT to FoxO1 via interaction with the coactivator PGC1α, which itself was shown to be modified by O-GlcNAc upon interaction with OGT (Housley et al., 2009). As PGC1 have been shown to significantly amplify LXR mediated activation of the SREBP1c promoter (Oberkofler et al., 2003; Kim et al., 2008), a possible recruitment of an OGT/PGC1-complex to LXR on lipogenic target genes under insulin resistant conditions remains to be explored. Recently, ChREBP was also shown to be a target for O-GlcNAcylation in response to hyperglycemia (Guinez et al., 2011). Adenoviral overexpression of OGT in liver increased ChREBP O-GlcNAc modification, protein stability and transactivating activity of L-PK, as well as potentiating expression of ACC, FAS and SCD1 mRNA expression in response to refeeding (Guinez et al., 2011). In contrast, hepatic overexpression of OGA reduced lipogenic protein content (ACC and FAS) and hepatic steatosis (excessive accumulation of TGs and CEs) in db/db mice, suggesting that enhanced OGT signaling to ChREBP and cooperating transcription factors/coregulators contributes to hepatic steatosis under insulin resistant conditions.

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 73

mRNA expression. Moreover, general protein O-GlcNAcylation was increased in STZtreated hyperglycemic mice compared to control mice. Our results suggest that LXR is regulated by O-GlcNAc modification, thereby increasing its lipogenic potential. Whether O-GlcNAc-LXR is able to transactivate other lipogenic genes in addition to SREBP1c, is currently under investigation in our laboratory. Our preliminary studies point to a role for O-GlcNAc-LXR in upregulating ChREBP, FAS, ACC and SCD1 expression (Bindesbøll et al, unpublished). Furthermore, preliminary reChIP experiments in our laboratory (LXR ChIP followed by O-GlcNAc ChIP), show a strong induction of O-GlcNAc-associated LXR binding to LXRE on the promoters of SREBP1c, ChREBP, FAS and SCD1 in response to feeding both in control mice and STZ treated mice. Our study is supported by the observation that the SREBP1c promoter activity and protein levels of SREBP1c are increased in response to elevated glucose concentration in the mouse hepatocyte cell line H2-35 (Hasty et al., 2000). Furthermore, treatment with azaserine, an inhibitor of GFAT, completely suppressed expression of both cytoplasmic and nuclear SREBP1c protein, suggesting that hexosaminedependent O-GlcNAc signaling indeed is involved in glucose-induced SREBP1c mRNA expression, possibly via activation of LXR and/or cooperating transcription factors/CAs.

In our *in vitro* studies, we observed only modest LXR/RXR transactivation of the SREBP1c promoter in high glucose/low insulin-treated cells. This might be explained by constitutive phosphorylation competing for the same site(s) as GlcNAc on LXR and/or inhibitory phosphorylation occurring on adjacent GlcNAc sites. Housley et al. (Housley et al., 2008) reported elevated *O*-GlcNAc on FoxO1 by high glucose and a subsequent reduction by insulin. They further showed that *O*-GlcNAc modification increased substantially on the insulininsensitive mutant FoxO1 lacking three AKT phosphorylation sites (T24A, S256A, S319A), resulting in increased FoxO1-dependent luciferase reporter activity. These observations imply overlapping and/or adjacent phosphorylation and GlcNAc sites on FoxO1. Indeed, the authors also identified several O-GlcNAc sites on FoxO1, one of which is adjacent to an Akt phosphorylation site (Thr317). In the case of LXR, which is activated by insulin, apparently in part via S6K-mediated phosphorylation (Hwahng et al., 2009), GlcNAcylation and phosphorylation might act synergistically on LXR in response to glucose and insulin. In fact, extensive cross-talk between O-GlcNAcylation and phosphorylation appear to contribute to the pathology of various diseases (Hart et al., 2011). In addition, GlcNAc and inhibiting phosphate (in response to fasting via PKA and/or AMPK) may compete for the same sites or are situated at different serines and/or threonines on LXR. Furthermore, GlcNAcylation and phosphorylation of LXR might be affected by ligand binding, which has been shown for SUMOylation and acetylation of LXR (Venteclef et al., 2010; Lee et al., 2009). A study by Torra et al*.* (Torra et al., 2008) reported that Ser198 phosphorylation of LXRα in RAW macrophages was induced by both synthetic and natural oxysterol LXR ligands and reduced by the RXR ligand 9-*cis*-retinoc acid. As such, we cannot exclude the possibility that LXR O-GlcNAcylation may be positively or negatively regulated by LXR and/or RXR ligands. From our *in vitro* GlcNAcylation results (Anthonisen et al., 2010) we believe that the major O-GlcNAc site(s) on LXRα and LXRβ resides in the N-terminal region containing the AF1 and DBD, indicating that O-GlcNAcylation occur independently of ligand. However, under hyperglycemic conditions,

#### **4.3. O-GlcNAc signaling activates LXR and hepatic lipogenesis**

In 2007, glucose was reported as an endogenous ligand for LXR (Mitro et al., 2007). This has, however, been debated considering the hydrophobic nature of the ligand binding pocket (Lazar & Willson, 2007). Instead, we asked the question whether glucose exert its effect via hexosamine signaling and posttranslational O-GlcNAc modification of LXR. In a recent publication, we show that LXR is O-GlcNAc modified in response to high glucose (25 mM) in absence of insulin (cells cultured in 2 % serum, approximately 1-2 pmol/l insulin) and synthetic LXR-ligand in Huh7 cells, a human hepatoma cell line (Anthonisen et al., 2010). By pharmacological inhibition we demonstrated that hexosamine signaling and O-GlcNAc cycling mediates LXR dependent activation of the SREBP1c promoter in response to glucose. Furthermore, we observed increased O-GlcNAc modification of LXR in livers from refed mice and streptozotosin (STZ) treated diabetic mice corresponding with increased SREBP1c 72 Lipid Metabolism

hepatic steatosis under insulin resistant conditions.

**4.3. O-GlcNAc signaling activates LXR and hepatic lipogenesis** 

In 2007, glucose was reported as an endogenous ligand for LXR (Mitro et al., 2007). This has, however, been debated considering the hydrophobic nature of the ligand binding pocket (Lazar & Willson, 2007). Instead, we asked the question whether glucose exert its effect via hexosamine signaling and posttranslational O-GlcNAc modification of LXR. In a recent publication, we show that LXR is O-GlcNAc modified in response to high glucose (25 mM) in absence of insulin (cells cultured in 2 % serum, approximately 1-2 pmol/l insulin) and synthetic LXR-ligand in Huh7 cells, a human hepatoma cell line (Anthonisen et al., 2010). By pharmacological inhibition we demonstrated that hexosamine signaling and O-GlcNAc cycling mediates LXR dependent activation of the SREBP1c promoter in response to glucose. Furthermore, we observed increased O-GlcNAc modification of LXR in livers from refed mice and streptozotosin (STZ) treated diabetic mice corresponding with increased SREBP1c

by phosphatases (Hart et al., 2007; Love, 2005). Because *O*-GlcNAc levels on proteins appear to be sensitive to increasing flux through this pathway in response to nutrient excess, OGT can be considered as a general sensor of glucose availability that modifies proteins according to changes in UDP-GlcNAc levels. There is no identified consensus sequence for GlcNAcylation, and unlike the multiple genes encoding kinases, there is only a single Xlinked gene encoding the catalytic subunit of OGT in mammals (Shafi et al., 2000). For this reason, it has been hypothesized that OGT is the catalytic subunit in large transient enzyme complexes where interacting proteins are able to target OGT to its many substrates. Many transcription factors are modified by O-GlcNAc in the liver (Dentin et al., 2008; Housley et al., 2008; Kuo et al., 2008; Ozcan et al., 2010). Interestingly, FoxO1 has been shown to be a target for O-GlcNAcylation in hepatocytes in response to hyperglycemia in the insulin resistant state, resulting in elevated transactivating capacity for FoxO1 against its gluconeogenic targets phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) reinforcing hepatic glucose production (Housley et al., 2008; Kuo et al., 2008). Moreover, this activation was later shown to be dependent on targeting of OGT to FoxO1 via interaction with the coactivator PGC1α, which itself was shown to be modified by O-GlcNAc upon interaction with OGT (Housley et al., 2009). As PGC1 have been shown to significantly amplify LXR mediated activation of the SREBP1c promoter (Oberkofler et al., 2003; Kim et al., 2008), a possible recruitment of an OGT/PGC1-complex to LXR on lipogenic target genes under insulin resistant conditions remains to be explored. Recently, ChREBP was also shown to be a target for O-GlcNAcylation in response to hyperglycemia (Guinez et al., 2011). Adenoviral overexpression of OGT in liver increased ChREBP O-GlcNAc modification, protein stability and transactivating activity of L-PK, as well as potentiating expression of ACC, FAS and SCD1 mRNA expression in response to refeeding (Guinez et al., 2011). In contrast, hepatic overexpression of OGA reduced lipogenic protein content (ACC and FAS) and hepatic steatosis (excessive accumulation of TGs and CEs) in db/db mice, suggesting that enhanced OGT signaling to ChREBP and cooperating transcription factors/coregulators contributes to mRNA expression. Moreover, general protein O-GlcNAcylation was increased in STZtreated hyperglycemic mice compared to control mice. Our results suggest that LXR is regulated by O-GlcNAc modification, thereby increasing its lipogenic potential. Whether O-GlcNAc-LXR is able to transactivate other lipogenic genes in addition to SREBP1c, is currently under investigation in our laboratory. Our preliminary studies point to a role for O-GlcNAc-LXR in upregulating ChREBP, FAS, ACC and SCD1 expression (Bindesbøll et al, unpublished). Furthermore, preliminary reChIP experiments in our laboratory (LXR ChIP followed by O-GlcNAc ChIP), show a strong induction of O-GlcNAc-associated LXR binding to LXRE on the promoters of SREBP1c, ChREBP, FAS and SCD1 in response to feeding both in control mice and STZ treated mice. Our study is supported by the observation that the SREBP1c promoter activity and protein levels of SREBP1c are increased in response to elevated glucose concentration in the mouse hepatocyte cell line H2-35 (Hasty et al., 2000). Furthermore, treatment with azaserine, an inhibitor of GFAT, completely suppressed expression of both cytoplasmic and nuclear SREBP1c protein, suggesting that hexosaminedependent O-GlcNAc signaling indeed is involved in glucose-induced SREBP1c mRNA expression, possibly via activation of LXR and/or cooperating transcription factors/CAs.

In our *in vitro* studies, we observed only modest LXR/RXR transactivation of the SREBP1c promoter in high glucose/low insulin-treated cells. This might be explained by constitutive phosphorylation competing for the same site(s) as GlcNAc on LXR and/or inhibitory phosphorylation occurring on adjacent GlcNAc sites. Housley et al. (Housley et al., 2008) reported elevated *O*-GlcNAc on FoxO1 by high glucose and a subsequent reduction by insulin. They further showed that *O*-GlcNAc modification increased substantially on the insulininsensitive mutant FoxO1 lacking three AKT phosphorylation sites (T24A, S256A, S319A), resulting in increased FoxO1-dependent luciferase reporter activity. These observations imply overlapping and/or adjacent phosphorylation and GlcNAc sites on FoxO1. Indeed, the authors also identified several O-GlcNAc sites on FoxO1, one of which is adjacent to an Akt phosphorylation site (Thr317). In the case of LXR, which is activated by insulin, apparently in part via S6K-mediated phosphorylation (Hwahng et al., 2009), GlcNAcylation and phosphorylation might act synergistically on LXR in response to glucose and insulin. In fact, extensive cross-talk between O-GlcNAcylation and phosphorylation appear to contribute to the pathology of various diseases (Hart et al., 2011). In addition, GlcNAc and inhibiting phosphate (in response to fasting via PKA and/or AMPK) may compete for the same sites or are situated at different serines and/or threonines on LXR. Furthermore, GlcNAcylation and phosphorylation of LXR might be affected by ligand binding, which has been shown for SUMOylation and acetylation of LXR (Venteclef et al., 2010; Lee et al., 2009). A study by Torra et al*.* (Torra et al., 2008) reported that Ser198 phosphorylation of LXRα in RAW macrophages was induced by both synthetic and natural oxysterol LXR ligands and reduced by the RXR ligand 9-*cis*-retinoc acid. As such, we cannot exclude the possibility that LXR O-GlcNAcylation may be positively or negatively regulated by LXR and/or RXR ligands. From our *in vitro* GlcNAcylation results (Anthonisen et al., 2010) we believe that the major O-GlcNAc site(s) on LXRα and LXRβ resides in the N-terminal region containing the AF1 and DBD, indicating that O-GlcNAcylation occur independently of ligand. However, under hyperglycemic conditions, ligand binding may recruit OGT to LXR via CAs, possibly PGC1 as reported for FoxO1 (Housley et al., 2009). A more detailed mapping of the GlcNAc sites on LXR and site-directed mutagenesis as well as identification of coregulators of LXR under hyperglycemic conditions, are under way in our laboratory to elucidate the biological role of O-GlcNAc on LXR. A complete summary of putative mechanisms of glucose-signaling to LXR, ChREBP and lipogenesis is depicted in Figure 6.

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 75

Studies in *C.elegans* demonstrate that O-GlcNAc cycling phenotypes are very sensitive to insulin as well as nutrient composition and that levels of insulin and nutrients influence the role of O-GlcNAc cycling and vice versa (Mondoux et al., 2011; Hanover et al., 2010; Hanover et al., 2010; Whelan et al., 2008). Intriguingly, O-GlcNAc-marked promoters in *C.elegans* are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism (Love et al., 2010a). Defects in O-GlcNAc cycling results in deregulation of genes necessary for carbohydrate and lipid metabolism in response to insulin (Forsythe et al., 2006; Hanover et al., 2010) suggesting that both O-GlcNAc cycling and insulin-signaling are required for a robust and adaptable response to hyperglycemia. Several studies have implicated O-GlcNAc cycling in the development of insulin resistance (reviewed in (Mondoux et al., 2011)). Mice overexpressing OGT in muscle or fat and mammalian cells overexpressing OGA develop insulin resistance (McClain, 2002; Arias et al., 2004; Vosseller et al., 2002). Later studies revealed that a subset of OGT was able to transiently translocate to the plasma membrane via association with PIP3 generated by insulin-activated PI3K (Yang et al., 2008). In response to increased glucose metabolism, PIP3-associated OGT can O-GlcNAcylate IR, IRS and Akt antagonizing insulin signaling (Yang et al., 2008; Whelan et al., 2010). Moreover, OGT may also interact with the mTOR pathway (Hanover et al., 2010). As mentioned in section 3.2.3, the downstream target for insulin signaling, FoxO1, is also modified by O-GlcNAc, apparently via OGT recruitment to PGC1, providing another mechanism for OGT to contribute to insulin resistance, at least for sustained hepatic glucose production in response to hyperglycemia (Housley et al., 2009). Directing OGT to transcriptional targets implies that PGC1α can integrate multiple nutrient signals to regulate gene expression. Whether OGT via PGC1 or other CAs is also recruited to ChREBP- and LXR-regulated promoters is currently not known. OGT is recruited to and O-GlcNAcylate several coregulators and histone modifying enzymes (acetylases/deacetylases, metylases/demetylases) and even histones themselves (Fujiki et al., 2009; Hanover et al., 2012; Fujiki et al., 2011; Sakabe et al., 2010). Depending on the nutritional stimuli, all components of the transciptional machinery from specific transcription factors to coregulators, histones and RNA polymerase II are subject to epigenetic regulation by acetylation, ubiquitinylation, SUMOylation, phosphorylation and/or O-GlcNAcylation (Rosenfeld et al., 2006; Venteclef et al., 2011; Love et al., 2010b; Kato et al., 2011). The finetuning of these modifications determines whether a gene is activated or repressed. Furthermore, as the substrate specificity of OGT is believed to be spatio-temporally regulated by transient interactions with large enzyme complexes, its binding to PIP3 may not occur solely at the plasma membrane, as PI3K is also active in the nucleus where it is involved in regulation of protein-chromatin interactions, transcription and mRNA export (Viiri et al., 2012; Kebede et al., 2012; Okada & Ye, 2009). As protein O-GlcNAcylation is rapidly increased at both the plasma membrane and the nucleus in response to serumstimulation (Carrillo et al., 2011), OGT-binding to nuclear PIP3 may also be instrumental in transcriptional regulation in response to feeding. Interestingly, nonalcoholic fatty liver

**5. Cross-talk between O-GlcNAc- and insulin signaling** 

**Figure 6.** Glucose-mediated regulation of hepatic lipogenesis

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 75

#### **5. Cross-talk between O-GlcNAc- and insulin signaling**

74 Lipid Metabolism

lipogenesis is depicted in Figure 6.

**Figure 6.** Glucose-mediated regulation of hepatic lipogenesis

ligand binding may recruit OGT to LXR via CAs, possibly PGC1 as reported for FoxO1 (Housley et al., 2009). A more detailed mapping of the GlcNAc sites on LXR and site-directed mutagenesis as well as identification of coregulators of LXR under hyperglycemic conditions, are under way in our laboratory to elucidate the biological role of O-GlcNAc on LXR. A complete summary of putative mechanisms of glucose-signaling to LXR, ChREBP and

Studies in *C.elegans* demonstrate that O-GlcNAc cycling phenotypes are very sensitive to insulin as well as nutrient composition and that levels of insulin and nutrients influence the role of O-GlcNAc cycling and vice versa (Mondoux et al., 2011; Hanover et al., 2010; Hanover et al., 2010; Whelan et al., 2008). Intriguingly, O-GlcNAc-marked promoters in *C.elegans* are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism (Love et al., 2010a). Defects in O-GlcNAc cycling results in deregulation of genes necessary for carbohydrate and lipid metabolism in response to insulin (Forsythe et al., 2006; Hanover et al., 2010) suggesting that both O-GlcNAc cycling and insulin-signaling are required for a robust and adaptable response to hyperglycemia. Several studies have implicated O-GlcNAc cycling in the development of insulin resistance (reviewed in (Mondoux et al., 2011)). Mice overexpressing OGT in muscle or fat and mammalian cells overexpressing OGA develop insulin resistance (McClain, 2002; Arias et al., 2004; Vosseller et al., 2002). Later studies revealed that a subset of OGT was able to transiently translocate to the plasma membrane via association with PIP3 generated by insulin-activated PI3K (Yang et al., 2008). In response to increased glucose metabolism, PIP3-associated OGT can O-GlcNAcylate IR, IRS and Akt antagonizing insulin signaling (Yang et al., 2008; Whelan et al., 2010). Moreover, OGT may also interact with the mTOR pathway (Hanover et al., 2010). As mentioned in section 3.2.3, the downstream target for insulin signaling, FoxO1, is also modified by O-GlcNAc, apparently via OGT recruitment to PGC1, providing another mechanism for OGT to contribute to insulin resistance, at least for sustained hepatic glucose production in response to hyperglycemia (Housley et al., 2009). Directing OGT to transcriptional targets implies that PGC1α can integrate multiple nutrient signals to regulate gene expression. Whether OGT via PGC1 or other CAs is also recruited to ChREBP- and LXR-regulated promoters is currently not known. OGT is recruited to and O-GlcNAcylate several coregulators and histone modifying enzymes (acetylases/deacetylases, metylases/demetylases) and even histones themselves (Fujiki et al., 2009; Hanover et al., 2012; Fujiki et al., 2011; Sakabe et al., 2010). Depending on the nutritional stimuli, all components of the transciptional machinery from specific transcription factors to coregulators, histones and RNA polymerase II are subject to epigenetic regulation by acetylation, ubiquitinylation, SUMOylation, phosphorylation and/or O-GlcNAcylation (Rosenfeld et al., 2006; Venteclef et al., 2011; Love et al., 2010b; Kato et al., 2011). The finetuning of these modifications determines whether a gene is activated or repressed. Furthermore, as the substrate specificity of OGT is believed to be spatio-temporally regulated by transient interactions with large enzyme complexes, its binding to PIP3 may not occur solely at the plasma membrane, as PI3K is also active in the nucleus where it is involved in regulation of protein-chromatin interactions, transcription and mRNA export (Viiri et al., 2012; Kebede et al., 2012; Okada & Ye, 2009). As protein O-GlcNAcylation is rapidly increased at both the plasma membrane and the nucleus in response to serumstimulation (Carrillo et al., 2011), OGT-binding to nuclear PIP3 may also be instrumental in transcriptional regulation in response to feeding. Interestingly, nonalcoholic fatty liver disease is often accompanied by hepatic insulin resistance, metabolic syndrome, and diabetes (reviewed in (Scorletti et al., 2011)) and the sensitivity of OGT to glucose increases with decreasing insulin signaling (Mondoux et al., 2011). These findings suggest that elevated O-GlcNAc cycling on key nuclear proteins contributes to the development of hepatic steatosis. This notion is also in line with the above mentioned observation by Guinez et al (Guinez et al., 2011), where overexpression of OGA reduced hepatic steatosis in db/db mice. A complete summary of a putative glucose-insulin cross-talk in regulation of hepatic *de novo* lipogenesis is depicted in Fig. 7.

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 77

In mice and humans, hepatic *de novo* lipogenesis is activated by a high intake of both glucose and fructose (Scorletti et al., 2011; Schwarz et al., 1995; Schwarz et al., 2003). Fructose increase hepatic hexosamine signaling (Hirahatake et al., 2011) and induce SREBP1c and ChREBP expression in hepatic cells (Matsuzaka et al., 2004; Haas et al., 2012; Koo et al., 2009), which may, in part be mediated by LXR. The response of LXR to glucose has been debated (Lazar & Willson, 2007), but a recent study support the notion of LXR as a glucose/fructose sensor as high sucrose fed mice exhibit elevated hepatic expression of SREBP1c and increased TG levels, which was not observed in LXR/ double knock out mice (Korach-Andre et al., 2011). LXR increases lipogenesis, in part by activating SREBP1c and ChREBP proteins. Thus, in response to feeding, they can cooperately activate most of the genes required for hepatic lipogenesis and TG secretion. Whether hepatic LXR drives the expression of SREBP1c and/or ChREBP to the same degree under different nutritional conditions is currently not known, as most studies have been performed using synthetic LXR agonists. We have preliminary results showing that hepatic expression of SREBP1c and ChREBP is upregulated in refed control mice and to a lesser extent in STZ-treated hyperglycemic mice, which is not observed in LXR/ double knock out mice (Bindesbøll et al, unpublished). O-GlcNAc modification of LXR is increased in STZ-treated mice (Anthonisen et al., 2010) and we postulate that O-GlcNAc modification of LXR in response to glucose activates LXR and drives the expression of ChREBP and SREBP1c and in particular the lipogenic genes, ACC and SCD1. Furthermore, RNA Pol II ChIP-Seq data show reduced binding of RNA Pol II to the L-PK promoter and no binding of RNA Pol II to the SCD1 promoter in LXR / double knock out mice compared to control mice. Moreover, a novel LXRE immediately downstream of SCD1 was found, to which LXR bound more strongly than the previously published upstream LXR binding site (Boergesen et al., 2012). This suggests an important role for LXR as an upstream activator of ChREBP-mediated transcription and argues for LXR acting independently on the SCD1 promoter, at least under certain nutritional conditions. Previous studies have demonstrated that LXR directly activates key lipogenic genes (Joseph et al., 2002), most notably SCD1 in the liver of SREBP1c knockout mice (Liang et al., 2002; Chu et al., 2006). Why there would be a need for LXR to activate lipogenic genes directly, may be explained by the nutritional conditions and redundancy in the system. Oxysterols bind the endoplasmic reticulum resident Insig protein and could inhibit the proteolytic maturation of SREBP1c (Radhakrishnan et al., 2007). This would limit transcription by SREBP1c, and direct activation by LXR would be required to stimulate lipogenesis. In the absence of active SREBP1c, however, LXR may act in concert with ChREBP in regulating lipogenic expression. A recent study show that hepatic overexpression of ChREBP induces SCD1 expression and hepatic steatosis, but not insulin resistance (Benhamed et al., 2012). Whether overexpression of ChREBP affected LXR protein expression and transactivation of the SCD1 promoter was not investigated in this study. In later studies, it would be interesting to investigate the SCD1 expression and activity in livers or hepatocytes with targeted deletion of ChREBP. Benhamed et al (Benhamed et al., 2012) also showed that ChREBP expression was increased in liver biopsies from patients with

**6. Concluding remarks** 

**Figure 7.** Glucose-insulin cross-talk in regulation of hepatic lipogenesis

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 77

#### **6. Concluding remarks**

76 Lipid Metabolism

disease is often accompanied by hepatic insulin resistance, metabolic syndrome, and diabetes (reviewed in (Scorletti et al., 2011)) and the sensitivity of OGT to glucose increases with decreasing insulin signaling (Mondoux et al., 2011). These findings suggest that elevated O-GlcNAc cycling on key nuclear proteins contributes to the development of hepatic steatosis. This notion is also in line with the above mentioned observation by Guinez et al (Guinez et al., 2011), where overexpression of OGA reduced hepatic steatosis in db/db mice. A complete summary of a putative glucose-insulin cross-talk in regulation

of hepatic *de novo* lipogenesis is depicted in Fig. 7.

**Figure 7.** Glucose-insulin cross-talk in regulation of hepatic lipogenesis

In mice and humans, hepatic *de novo* lipogenesis is activated by a high intake of both glucose and fructose (Scorletti et al., 2011; Schwarz et al., 1995; Schwarz et al., 2003). Fructose increase hepatic hexosamine signaling (Hirahatake et al., 2011) and induce SREBP1c and ChREBP expression in hepatic cells (Matsuzaka et al., 2004; Haas et al., 2012; Koo et al., 2009), which may, in part be mediated by LXR. The response of LXR to glucose has been debated (Lazar & Willson, 2007), but a recent study support the notion of LXR as a glucose/fructose sensor as high sucrose fed mice exhibit elevated hepatic expression of SREBP1c and increased TG levels, which was not observed in LXR/ double knock out mice (Korach-Andre et al., 2011). LXR increases lipogenesis, in part by activating SREBP1c and ChREBP proteins. Thus, in response to feeding, they can cooperately activate most of the genes required for hepatic lipogenesis and TG secretion. Whether hepatic LXR drives the expression of SREBP1c and/or ChREBP to the same degree under different nutritional conditions is currently not known, as most studies have been performed using synthetic LXR agonists. We have preliminary results showing that hepatic expression of SREBP1c and ChREBP is upregulated in refed control mice and to a lesser extent in STZ-treated hyperglycemic mice, which is not observed in LXR/ double knock out mice (Bindesbøll et al, unpublished). O-GlcNAc modification of LXR is increased in STZ-treated mice (Anthonisen et al., 2010) and we postulate that O-GlcNAc modification of LXR in response to glucose activates LXR and drives the expression of ChREBP and SREBP1c and in particular the lipogenic genes, ACC and SCD1. Furthermore, RNA Pol II ChIP-Seq data show reduced binding of RNA Pol II to the L-PK promoter and no binding of RNA Pol II to the SCD1 promoter in LXR / double knock out mice compared to control mice. Moreover, a novel LXRE immediately downstream of SCD1 was found, to which LXR bound more strongly than the previously published upstream LXR binding site (Boergesen et al., 2012). This suggests an important role for LXR as an upstream activator of ChREBP-mediated transcription and argues for LXR acting independently on the SCD1 promoter, at least under certain nutritional conditions. Previous studies have demonstrated that LXR directly activates key lipogenic genes (Joseph et al., 2002), most notably SCD1 in the liver of SREBP1c knockout mice (Liang et al., 2002; Chu et al., 2006). Why there would be a need for LXR to activate lipogenic genes directly, may be explained by the nutritional conditions and redundancy in the system. Oxysterols bind the endoplasmic reticulum resident Insig protein and could inhibit the proteolytic maturation of SREBP1c (Radhakrishnan et al., 2007). This would limit transcription by SREBP1c, and direct activation by LXR would be required to stimulate lipogenesis. In the absence of active SREBP1c, however, LXR may act in concert with ChREBP in regulating lipogenic expression. A recent study show that hepatic overexpression of ChREBP induces SCD1 expression and hepatic steatosis, but not insulin resistance (Benhamed et al., 2012). Whether overexpression of ChREBP affected LXR protein expression and transactivation of the SCD1 promoter was not investigated in this study. In later studies, it would be interesting to investigate the SCD1 expression and activity in livers or hepatocytes with targeted deletion of ChREBP. Benhamed et al (Benhamed et al., 2012) also showed that ChREBP expression was increased in liver biopsies from patients with

#### 78 Lipid Metabolism

steatosis and decreased in liver of patients with severe insulin resistance, suggesting that ChREBP, alone or in combination with LXR, drives SCD1 expression and steatosis independent of insulin resistance. This is in line with recent human studies showing no relationship between hepatic TG accumulation and insulin resistance (Cohen et al., 2011; Hooper et al., 2011). Thus, hepatic steatosis can either be the result or cause of hepatic insulin resistance. The mechanisms of hepatic insulin resistance is still not clear (Farese, Jr. et al., 2012), but may involve specific lipids, nutrition-induced metabolites and PTMs including O-GlcNAc. Hepatic TG synthesis may be a protective mechanism to limit accumulation of toxic free fatty acids, liver damage and fibrosis (Choi & Diehl, 2008) where particularly SCD1 seem to play a protective role (Li et al., 2009).

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 79

Apfel, R., Benbrook, D., Lernhardt, E., Ortiz, M.A., Salbert, G., & Pfahl, M. (1994). A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily. *Mol.Cell Biol.,* Vol. 14,

Arden, C., Tudhope, S.J., Petrie, J.L., Al-Oanzi, Z.H., Cullen, K.S., Lange, A.J., Towle, H.C., & Agius, L. (2012). Fructose 2,6-bisphosphate is essential for glucose-regulated gene transcription of glucose-6-phosphatase and other ChREBP target genes in hepatocytes.

Arias, E.B., Kim, J., & Cartee, G.D. (2004). Prolonged incubation in PUGNAc results in increased protein O-Linked glycosylation and insulin resistance in rat skeletal muscle.

Benhamed, F., Denechaud, P.D., Lemoine, M., Robichon, C., Moldes, M., Bertrand-Michel, J., Ratziu, V., Serfaty, L., Housset, C., Capeau, J., Girard, J., Guillou, H., & Postic, C. (2012). The lipogenic transcription factor ChREBP dissociates hepatic steatosis from insulin resistance in mice and humans. *J.Clin.Invest,* Vol. 122, No. 6, pp. 2176-2194, PM:22546860 Boergesen, M., Pedersen, T.A., Gross, B., van Heeringen, S.J., Hagenbeek, D., Bindesboll, C., Caron, S., Lalloyer, F., Steffensen, K.R., Nebb, H.I., Gustafsson, J.A., Stunnenberg, H.G., Staels, B., & Mandrup, S. (2012). Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor alpha in mouse liver reveals extensive sharing of binding sites. *Mol.Cell Biol.,* Vol. 32, No. 4, pp. 852-867,

Bosco, D., Meda, P., & Iynedjian, P.B. (2000). Glucokinase and glucokinase regulatory protein: mutual dependence for nuclear localization. *Biochem.J.,* Vol. 348 Pt 1, No.pp.

Brown, M.S., & Goldstein, J.L. (2008). Selective versus total insulin resistance: a pathogenic

Calkin, A.C., & Tontonoz, P. (2010). Liver x receptor signaling pathways and atherosclerosis.

Carrillo, L.D., Froemming, J.A., & Mahal, L.K. (2011). Targeted in vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction. *J.Biol.Chem.,* 

Cha, J.Y., & Repa, J.J. (2007). The liver X receptor (LXR) and hepatic lipogenesis. The carbohydrate-response element-binding protein is a target gene of LXR. *J.Biol.Chem.,* 

Chen, G., Liang, G., Ou, J., Goldstein, J.L., & Brown, M.S. (2004). Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver. *Proc.Natl.Acad.Sci.U.S.A,* Vol. 101, No. 31, pp. 11245-11250,

Chen, M., Bradley, M.N., Beaven, S.W., & Tontonoz, P. (2006). Phosphorylation of the liver X

receptors. *FEBS Lett.,* Vol. 580, No. 20, pp. 4835-4841, PM:16904112

*Arterioscler.Thromb.Vasc.Biol.,* Vol. 30, No. 8, pp. 1513-1518, PM:20631351

paradox. *Cell Metab,* Vol. 7, No. 2, pp. 95-96, PM:18249166

Vol. 286, No. 8, pp. 6650-6658, PM:21138847

Vol. 282, No. 1, pp. 743-751, PM:17107947

No. 10, pp. 7025-7035, PM:7935418

PM:22158963

PM:15266058

215-222, PM:10794734

*Biochem.J.,* Vol. 443, No. 1, pp. 111-123, PM:22214556

*Diabetes,* Vol. 53, No. 4, pp. 921-930, PM:15047606

As LXR is shown also to act anti-inflammatory in liver (Wouters et al., 2008; Venteclef et al., 2010), LXR activation may be an important compensative mechanism in response to excess nutrients to limit liver damage, inflammation and fibrosis. SUMOylation is an important ligand-activated transrepressional PTM of LXR on inflammatory genes (Venteclef et al., 2011) and future studies in our laboratory aim to elucidate a putative cross-talk between OGT and E3 ligases (SUMO conjugating enzymes) in liver in response to excess nutrients, especially high sugar levels (glucose and fructose). The relative roles of LXR, SREBP1c and ChREBP in driving lipogenesis is clearly dependent on both insulin and glucose signaling and cross-talk between these pathways. Both phosphorylation and GlcNAcylation appear instrumental in hepatic lipogenesis and future focus in our laboratory will be to elucidate a possible cross-talk between these PTMs, endogenous LXR ligands and interacting CAs in response to various feeding conditions (high glucose, fructose and/or fatty acids, cholesterol) and the impact on downstream ChREBP, SREBP1c and lipogenic enzyme expression and activity. ChIP and reChIP analysis in combination with loss of function studies have become powerful tools to analyze activation of specific genes by specific transcription factors in response to extracellular stimuli. By these methods, we anticipate that the signaling mechanisms and relative roles of LXR, ChREBP, SREBP1c and cooperating transcription factors in driving hepatic *de novo* lipogenesis will be revealed in the not too distant future.
