**2. Liver X Receptors (LXR)**

#### **2.1. LXR structure and function**

LXR(NR1H3) and LXR (NR1H2) are ligand-activated transcription factors belonging to the nuclear receptor (NR) superfamily (Lehmann JM (Lehmann et al., 1997; Willy et al., 1995; Janowski et al., 1996). LXR is primarily expressed in metabolically active tissues, such as liver, intestine, adipose tissue, kidney and macrophages, whereas LXR is ubiquitously expressed (Apfel et al., 1994; Teboul et al., 1995; Teboul et al., 1995). LXRs are intracellular sensors of cholesterol and oxidized cholesterol derivatives (oxysterols) have been identified as their endogenous ligands (Janowski et al., 1996; Lehmann et al., 1997). The two isotypes originates from two different genes on separate chromosomes, but share the same modular structure, which is characteristic of most NRs (Fig. 1).

**Figure 1.** Structure of the LXRs

The DNA-binding domain (DBD) and the ligand binding domain (LBD) are highly structured domains. LXRα and LXRβ share 78 % amino acid sequence identity in these regions, while the N-terminal domain (NTD) and the hinge domain are far more disordered and less conserved. DNA binding requires dimerization with RXR. Transactivation by the LXRs is mediated through the ligand independent activation function (AF1) in NTD and the ligand dependent activation function 2 (AF2) in the LBD. Binding of a ligand to the hydrophobic ligand binding pocket leads to a conformational change that releases corepressors (CR) and exposes binding sites for coactivators (CA), recruiting the general transcription machinery and RNA polymerase II (RNA Pol II) (Fig. 2). This leads to changes in LXR dependent gene expression. The interactions with coregulators can also occur independently of ligand to AF1, however this is far less characterized. Upon activation, LXRs regulate a number of genes involved in lipid, cholesterol and glucose metabolism by binding to LXR response elements (LXREs) in their promoter region. These consist of a direct repeat of the nucleotide hexamer AGGTCA spaced by four nucleotides. Insights into LXR function in metabolism was provided by the generation of LXR mutant mice. These mice accumulate hepatic cholesterol, ultimately causing liver dysfunction (Peet et al., 1998; Ulven et al., 2005). It was found that LXRcontrols cholesterol metabolism by conversion of cholesterol to bile acid by induction of the cholesterol 7 alpha-hydroxylase (Cyp7A1) gene, biliary cholesterol excretion and cholesterol efflux via induction of ABCG5/8 and ABCA1/ABCG1, respectively (Lehmann et al., 1997; Chiang et al., 2001; Yu et al., 2003; Repa et al., 2002; Graf et al., 2002; Costet et al., 2000; Sabol et al., 2005; Venkateswaran et al., 2000; Venkateswaran et al., 2000). LXRs are strongly implicated in the development of metabolic disorders and associated pathologies, notably, hyperlipidemia and atherosclerosis (Peet et al., 1998; Calkin & Tontonoz, 2010). Thus, LXRs are key players in maintaining metabolic homeostasis in health and disease by regulating inflammation and lipid/carbohydrate metabolism.

**Figure 2.** Activation of LXR by coregulator switching

62 Lipid Metabolism

insulin resistant conditions will be discussed.

structure, which is characteristic of most NRs (Fig. 1).

**2. Liver X Receptors (LXR)** 

**Figure 1.** Structure of the LXRs

**2.1. LXR structure and function** 

translocation to the nucleus (Havula & Hietakangas, 2012). Interestingly, both LXR and ChREBP were recently shown to be post-translationally modified by O-linked -Nacetylglucosamine (O-GlcNAc) in response to glucose potentiating their lipogenic capacity (Anthonisen et al., 2010; Guinez et al., 2011). Glucose flux through the hexosamine signaling pathway generates UDP-N-acetyl-glucosamine (UDP-GlcNAc), a substrate for O-GlcNAc modification of nucleocytoplasmic proteins by the enzyme O-GlcNAc transferase (OGT). We have shown that O-GlcNAcylation of LXR is increased in mouse livers in response to feeding and in livers from hyperglycemic diabetic mice potentiating SREBP1c expression (Anthonisen et al., 2010). Furthermore, preliminary studies in our laboratory indicate that LXR potentiate ChREBP activity under hyperglycemic conditions establishing a link between glucose metabolism, LXR and ChREBP. These observations suggest that LXR, SREBP1c and ChREBP contribute to converting carbohydrates into fat in a cooperative manner in response to high circulating glucose levels and that O-GlcNAc signaling plays a role in this process. As O-GlcNAc cycling appear to be essential for proper insulin signaling and the sensitivity of OGT to glucose increases with decreasing insulin signaling (Mondoux et al., 2011; Hanover et al., 2010) the relative roles of LXR, SREBP1c and ChREBP in regulating *de novo* lipogenesis in response to feeding and modification by O-GlcNAc signaling under insulin sensitive and

LXR(NR1H3) and LXR (NR1H2) are ligand-activated transcription factors belonging to the nuclear receptor (NR) superfamily (Lehmann JM (Lehmann et al., 1997; Willy et al., 1995; Janowski et al., 1996). LXR is primarily expressed in metabolically active tissues, such as liver, intestine, adipose tissue, kidney and macrophages, whereas LXR is ubiquitously expressed (Apfel et al., 1994; Teboul et al., 1995; Teboul et al., 1995). LXRs are intracellular sensors of cholesterol and oxidized cholesterol derivatives (oxysterols) have been identified as their endogenous ligands (Janowski et al., 1996; Lehmann et al., 1997). The two isotypes originates from two different genes on separate chromosomes, but share the same modular

#### **2.2. Modulation of LXR activity by coregulators and PTMs**

The transcriptional activity of LXRs is highly dependent on the presence of coregulators which has been linked to several metabolic processes (Jakobsson et al., 2009; Kim et al., 2003; Huuskonen et al., 2004; Kim et al., 2008; Oberkofler et al., 2003). Coregulators constitutes large multisubunit protein complexes containing chromatin-remodelling and/or –modifying enzymes with intrinsic histone acetylase (HAT)/ deacetylase (HDAC) and histone methylase (HMT)/demethylase (HDM) activities, depending on whether they act as activators or repressors, respectively (Kato et al., 2011). It has been assumed that that the unliganded LXRs are localized in the nucleus and interact with CRs, including nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid receptor (NcoR/SMRT) (Wagner et al., 2003). However, recent chromatin immunoprecipitation (ChIP) studies, including ChIPsequencing (ChIP-Seq), have challenged this classical model. These studies put forward a more complex view, that ligands, pioneer factors, coregulators and posttranslational modifications (PTMs) play different roles in determining the LXR binding sites and actions *in vivo* (Boergesen et al., 2012; Heinz et al., 2010; Pehkonen et al., 2012). Furthermore, some coregulators have been shown to act as dual function activators/repressors, such as the coregulator protein receptor interacting protein 140 (RIP140). RIP140 has been shown to serve as a CA for LXR in lipogenesis but as a CR in gluconeogenesis independent of ligand activation (Herzog et al., 2007). General mechanisms of coregulator actions are assumed to be conserved between LXRs, but based on the low amino acid sequence identity in the NTD (32%) and the hinge domain (25%) it is possible that they contain novel isotype specific interaction surfaces. Also, the specific coregulator requirement to lipogenic LXR target genes in response to different feeding regiments under normal and diabetic conditions remain largely unexplored. In addition to ligand binding, LXRs can be posttranslationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively (Li et al., 2007; Ghisletti et al., 2007; Chen et al., 2006; Yamamoto et al., 2007). We have recently shown that LXR can be modified by O-GlcNAcylation in response to glucose (see section 4.3), increasing its transactivation of the SREBP1c promoter (Anthonisen et al., 2010). PTMs may alter the structural conformation of LXR thereby modifying the affinity of coregulators that determines whether a target gene is induced or suppressed. Modulation by PTMs can occur both in the absence and presence of natural ligand tuning LXR activities in a cell- and gene-specific manner (Rosenfeld et al., 2006) depending on the nutritional stimuli.

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 65

**Figure 3.** Regulation of hepatic lipogenesis by LXR, SREBP1c and ChREBP.

in hepatic lipogenesis. SREBP1c null mice treated with an LXR agonist results in induction of a subset of lipogenic genes and a modest increase in fatty acid synthesis (Liang et al., 2002), which implies that LXR can act independently of SREBP1c. In particular, the SCD1 gene is directly regulated by LXR in response to synthetic ligands, also in the absence of SREBP1c (Chu et al., 2006). SCD1 is central in desaturation of saturated fatty acyl-CoAs important for formation of cholesterol esters (CEs) and TGs. Thus, specific LXR-mediated regulation of SCD1 can be explained by the essential role of LXR in limiting toxic free

#### **3. LXR in hepatic** *de novo* **lipogenesis**

#### **3.1. LXR lipogenic target genes**

In addition to being central regulators of cholesterol metabolism, the LXRs are involved in induction of fatty acid and triglyceride (TG) biosynthesis in response to feeding. *De novo* lipogenesis ensures that excess acetyl-CoA, which is an intermediate product of glucose metabolism, is converted into fats and subsequent TGs. LXRs are involved in hepatic lipogenesis through direct regulation of SREBP1c and ChREBP expression (Repa et al., 2000; Cha & Repa, 2007; Shimano, 2001). SREBP1c is a well described transcriptional regulator of hepatic lipogenesis (Shimano, 2001), and together with LXR and glucose-regulated ChREBP (see section 4.1), it controls expression of essential enzymes in lipogenesis, lipid storage and secretion (Fig. 3). SREBP1c deficiency does not fully abolish the expression of genes involved 64 Lipid Metabolism

depending on the nutritional stimuli.

**3.1. LXR lipogenic target genes** 

**3. LXR in hepatic** *de novo* **lipogenesis** 

enzymes with intrinsic histone acetylase (HAT)/ deacetylase (HDAC) and histone methylase (HMT)/demethylase (HDM) activities, depending on whether they act as activators or repressors, respectively (Kato et al., 2011). It has been assumed that that the unliganded LXRs are localized in the nucleus and interact with CRs, including nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid receptor (NcoR/SMRT) (Wagner et al., 2003). However, recent chromatin immunoprecipitation (ChIP) studies, including ChIPsequencing (ChIP-Seq), have challenged this classical model. These studies put forward a more complex view, that ligands, pioneer factors, coregulators and posttranslational modifications (PTMs) play different roles in determining the LXR binding sites and actions *in vivo* (Boergesen et al., 2012; Heinz et al., 2010; Pehkonen et al., 2012). Furthermore, some coregulators have been shown to act as dual function activators/repressors, such as the coregulator protein receptor interacting protein 140 (RIP140). RIP140 has been shown to serve as a CA for LXR in lipogenesis but as a CR in gluconeogenesis independent of ligand activation (Herzog et al., 2007). General mechanisms of coregulator actions are assumed to be conserved between LXRs, but based on the low amino acid sequence identity in the NTD (32%) and the hinge domain (25%) it is possible that they contain novel isotype specific interaction surfaces. Also, the specific coregulator requirement to lipogenic LXR target genes in response to different feeding regiments under normal and diabetic conditions remain largely unexplored. In addition to ligand binding, LXRs can be posttranslationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively (Li et al., 2007; Ghisletti et al., 2007; Chen et al., 2006; Yamamoto et al., 2007). We have recently shown that LXR can be modified by O-GlcNAcylation in response to glucose (see section 4.3), increasing its transactivation of the SREBP1c promoter (Anthonisen et al., 2010). PTMs may alter the structural conformation of LXR thereby modifying the affinity of coregulators that determines whether a target gene is induced or suppressed. Modulation by PTMs can occur both in the absence and presence of natural ligand tuning LXR activities in a cell- and gene-specific manner (Rosenfeld et al., 2006)

In addition to being central regulators of cholesterol metabolism, the LXRs are involved in induction of fatty acid and triglyceride (TG) biosynthesis in response to feeding. *De novo* lipogenesis ensures that excess acetyl-CoA, which is an intermediate product of glucose metabolism, is converted into fats and subsequent TGs. LXRs are involved in hepatic lipogenesis through direct regulation of SREBP1c and ChREBP expression (Repa et al., 2000; Cha & Repa, 2007; Shimano, 2001). SREBP1c is a well described transcriptional regulator of hepatic lipogenesis (Shimano, 2001), and together with LXR and glucose-regulated ChREBP (see section 4.1), it controls expression of essential enzymes in lipogenesis, lipid storage and secretion (Fig. 3). SREBP1c deficiency does not fully abolish the expression of genes involved

**Figure 3.** Regulation of hepatic lipogenesis by LXR, SREBP1c and ChREBP.

in hepatic lipogenesis. SREBP1c null mice treated with an LXR agonist results in induction of a subset of lipogenic genes and a modest increase in fatty acid synthesis (Liang et al., 2002), which implies that LXR can act independently of SREBP1c. In particular, the SCD1 gene is directly regulated by LXR in response to synthetic ligands, also in the absence of SREBP1c (Chu et al., 2006). SCD1 is central in desaturation of saturated fatty acyl-CoAs important for formation of cholesterol esters (CEs) and TGs. Thus, specific LXR-mediated regulation of SCD1 can be explained by the essential role of LXR in limiting toxic free

#### 66 Lipid Metabolism

cholesterol in response to diets rich in cholesterol and saturated fat. The expression of LXR in liver is rapidly upregulated by insulin *in vivo*, increasing mRNA expression of SREBP1c, malic enzyme (ME), ACC and FAS. Furthermore, expression of these lipogenic genes was abolished in insulin-injected LXR/ double knock out mice (Tobin et al., 2002), indicating an essential role for LXR in insulin-mediated regulation of hepatic lipogenesis. The mechanisms by which insulin activate LXR-mediated gene expression is not clearly understood, but may involve production of endogenous ligand for LXRα/β (Chen et al., 2004) and/or by signal transduction mechanisms downstream of the IR affecting CA recruitment to LXRs and/or PTMs of LXRs. This will be discussed in more detail below. Of note, PKA-induced phosphorylation of LXR has been shown to inhibit the expression of SREBP1c in liver from mice via reduced DNA binding and CA recruitment (Yamamoto et al., 2007). Since glucagon/cAMP/PKA signaling may, at least in part, explain downregulation of SREBP1c expression in response to fasting, it is likely that PKA-mediated phosphorylation of LXR contributes to the fasting signal on SREBP1c.

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 67

protein kinase (PDK1), the serine/threonine kinase Akt/protein kinase B and possibly also mammalian target of rapamycin complex 2 (mTORC2). PDK and mTORC2 are both necessary for full activation of Akt downstream of the insulin receptor via PDK1-mediated phosphorylation of Akt on threonine 308 and mTORC2-mediated phosphorylation on serine 472 (Saltiel & Kahn, 2001; White, 2003; Jacinto et al., 2006). All these events occur transiently in specific cholesterol rich plasma membrane microdomains called caveolae, generating a specific signaling unit for proper downstream insulin signaling where Akt plays a central

One of the targets of Akt is mTORC1 (Zoncu et al., 2011). Recent evidence suggests that mTORC1 is involved in LXR-mediated lipogenic gene transcription including induction of SREBP1c, FAS and ACC in liver from mice subjected to a high fat diet (Hwahng et al., 2009). The authors show that the mechanism by which mTORC1 activates LXR is via p70 S6 kinase (S6K)-mediated phosphorylation of LXR. Conversely, in the fasted state, LXR was shown to be inhibited by AMPK-mediated phosphorylation. In agreement with these observations, Li et al (Li et al., 2010) showed that insulin-activated hepatic transcription of SREBP1c, FAS and SCD1 is mediated by mTORC1, however independent of S6K. As both LXR and SREBP1c induce lipogenic promoters in response to insulin, this might suggest that activation of LXR in response to insulin/nutrients is mediated, at least in part, by mTORC1 and S6K, whereas insulin-signaling to SREBP1c requires mTORC2 independently of S6K, possibly via Akt-mediated inhibition of glycogen synthase kinase-3 (GSK3) (Hagiwara et al., 2012). In this way, GSK3-mediated phosphorylation and degradation of SREBP1c is prevented by insulin signaling to mTORC2 and Akt. Of note, insulin has primarily been shown to act on the SREBP1c promoter by activating LXRs and not SREBP1c (Chen et al., 2004) and the effect of insulin on SREBP1c is mainly at the posttranslational level. In a recent publication, mTORC1 was shown to phosphorylate a phosphatidic acid phosphatase, Lipin 1, preventing its nuclear entry and subsequent inhibition of SREBP1c-mediated activation of the FAS promoter (Peterson et al., 2011). Furthermore, Yecies JL et al (Yecies et al., 2011) showed that Akt2 independently of mTORC1 downregulate the mRNA expression of insulin induced gene 2 (Insig2a), an inhibitor of SREBP1c. This finding has been debated by Wan M et al (Wan et al., 2011), who could not observe any downregulation of Insig2a by Akt2. They postulate that Akt2 acts independently of mTORC1 and SREBP1c, possibly via posttranslational mechanisms, and that nutrients have a direct role in the liver to promote lipogenesis by a process dependent on both mTORC1 and other insulin-dependent signaling pathways. In light of the above mentioned studies, both mTORC1 and mTORC2 (Soukas et al., 2009; Guertin et al., 2006; Lamming et al., 2012; Hagiwara et al., 2012) appear to play important roles in lipid synthesis and storage in hepatocytes. Further studies will reveal the relative roles of Akt1, Akt2, mTORC1/C2 and S6kinase on activation of LXR and SREBP1c in this regulation under insulin sensitive and insulin resistant conditions and cross-talk with

role.

*3.2.2. Regulation by mTOR* 

glucose metabolism and signaling (Fig.4).

### **3.2. Putative mechanisms regulating LXR-mediated** *de novo* **lipogenesis in response to insulin**

Insulin is the most important anabolic hormone in the body, regulating many processes important for cellular growth and energy storage such as glucose uptake and metabolism, glycogen and lipid synthesis, gene transcription and translation. A classic action of insulin is to mediate a metabolic switch from fatty acid oxidation to synthesis and suppress hepatic glycogenolysis and gluconeogenesis in response to carbohydrate excess, a process that is largely regulated at the transcriptional level. In this way, hepatic insulin signaling maintains whole body energy homeostasis. In the insulin-resistant state, only the ability of insulin to suppress hepatic gluconeogenesis is lost, while its ability to activate lipogenesis is retained (Shimomura et al., 2000; Matsumoto et al., 2006; Brown & Goldstein, 2008). This bifurcated insulin resistance can be explained by failure of insulin to inhibit the gluconeogenic transcription factor Forkhead box protein O1 (FoxO1), but maintaining signaling to lipogenic transcriptional regulators including LXR and SREBP1c.

#### *3.2.1. The insulin signaling cascade*

The insulin signaling cascade is initiated by the binding of insulin to the extracellular subunits of the dimerized IR followed by autophosphorylation on several intracellular tyrosine residues on the IR. Insulin receptor substrate (IRS) is an essential protein docking onto the phosphorylated IR which in turn is phosphorylated itself on multiple tyrosine residues. This creates docking sites for src homology 2 (SH2) domain containing proteins. The best studied SH2 protein that binds to tyrosine phosphorylated IRS proteins is the regulatory subunit of the phosphoinositide 3-kinase (PI3K). PI3K catalyzes the formation of the lipid second messenger phosphatidylinositol (3,4,5) trisphosphate (PIP3), which is necessary to recruit downstream kinases. PIP3 generates a binding site for proteins containing Pleckstrin homology (PH) domains, such as 3'-phosphoinositide-dependent protein kinase (PDK1), the serine/threonine kinase Akt/protein kinase B and possibly also mammalian target of rapamycin complex 2 (mTORC2). PDK and mTORC2 are both necessary for full activation of Akt downstream of the insulin receptor via PDK1-mediated phosphorylation of Akt on threonine 308 and mTORC2-mediated phosphorylation on serine 472 (Saltiel & Kahn, 2001; White, 2003; Jacinto et al., 2006). All these events occur transiently in specific cholesterol rich plasma membrane microdomains called caveolae, generating a specific signaling unit for proper downstream insulin signaling where Akt plays a central role.

#### *3.2.2. Regulation by mTOR*

66 Lipid Metabolism

**response to insulin** 

cholesterol in response to diets rich in cholesterol and saturated fat. The expression of LXR in liver is rapidly upregulated by insulin *in vivo*, increasing mRNA expression of SREBP1c, malic enzyme (ME), ACC and FAS. Furthermore, expression of these lipogenic genes was abolished in insulin-injected LXR/ double knock out mice (Tobin et al., 2002), indicating an essential role for LXR in insulin-mediated regulation of hepatic lipogenesis. The mechanisms by which insulin activate LXR-mediated gene expression is not clearly understood, but may involve production of endogenous ligand for LXRα/β (Chen et al., 2004) and/or by signal transduction mechanisms downstream of the IR affecting CA recruitment to LXRs and/or PTMs of LXRs. This will be discussed in more detail below. Of note, PKA-induced phosphorylation of LXR has been shown to inhibit the expression of SREBP1c in liver from mice via reduced DNA binding and CA recruitment (Yamamoto et al., 2007). Since glucagon/cAMP/PKA signaling may, at least in part, explain downregulation of SREBP1c expression in response to fasting, it is likely that PKA-mediated

phosphorylation of LXR contributes to the fasting signal on SREBP1c.

lipogenic transcriptional regulators including LXR and SREBP1c.

*3.2.1. The insulin signaling cascade* 

**3.2. Putative mechanisms regulating LXR-mediated** *de novo* **lipogenesis in** 

Insulin is the most important anabolic hormone in the body, regulating many processes important for cellular growth and energy storage such as glucose uptake and metabolism, glycogen and lipid synthesis, gene transcription and translation. A classic action of insulin is to mediate a metabolic switch from fatty acid oxidation to synthesis and suppress hepatic glycogenolysis and gluconeogenesis in response to carbohydrate excess, a process that is largely regulated at the transcriptional level. In this way, hepatic insulin signaling maintains whole body energy homeostasis. In the insulin-resistant state, only the ability of insulin to suppress hepatic gluconeogenesis is lost, while its ability to activate lipogenesis is retained (Shimomura et al., 2000; Matsumoto et al., 2006; Brown & Goldstein, 2008). This bifurcated insulin resistance can be explained by failure of insulin to inhibit the gluconeogenic transcription factor Forkhead box protein O1 (FoxO1), but maintaining signaling to

The insulin signaling cascade is initiated by the binding of insulin to the extracellular subunits of the dimerized IR followed by autophosphorylation on several intracellular tyrosine residues on the IR. Insulin receptor substrate (IRS) is an essential protein docking onto the phosphorylated IR which in turn is phosphorylated itself on multiple tyrosine residues. This creates docking sites for src homology 2 (SH2) domain containing proteins. The best studied SH2 protein that binds to tyrosine phosphorylated IRS proteins is the regulatory subunit of the phosphoinositide 3-kinase (PI3K). PI3K catalyzes the formation of the lipid second messenger phosphatidylinositol (3,4,5) trisphosphate (PIP3), which is necessary to recruit downstream kinases. PIP3 generates a binding site for proteins containing Pleckstrin homology (PH) domains, such as 3'-phosphoinositide-dependent One of the targets of Akt is mTORC1 (Zoncu et al., 2011). Recent evidence suggests that mTORC1 is involved in LXR-mediated lipogenic gene transcription including induction of SREBP1c, FAS and ACC in liver from mice subjected to a high fat diet (Hwahng et al., 2009). The authors show that the mechanism by which mTORC1 activates LXR is via p70 S6 kinase (S6K)-mediated phosphorylation of LXR. Conversely, in the fasted state, LXR was shown to be inhibited by AMPK-mediated phosphorylation. In agreement with these observations, Li et al (Li et al., 2010) showed that insulin-activated hepatic transcription of SREBP1c, FAS and SCD1 is mediated by mTORC1, however independent of S6K. As both LXR and SREBP1c induce lipogenic promoters in response to insulin, this might suggest that activation of LXR in response to insulin/nutrients is mediated, at least in part, by mTORC1 and S6K, whereas insulin-signaling to SREBP1c requires mTORC2 independently of S6K, possibly via Akt-mediated inhibition of glycogen synthase kinase-3 (GSK3) (Hagiwara et al., 2012). In this way, GSK3-mediated phosphorylation and degradation of SREBP1c is prevented by insulin signaling to mTORC2 and Akt. Of note, insulin has primarily been shown to act on the SREBP1c promoter by activating LXRs and not SREBP1c (Chen et al., 2004) and the effect of insulin on SREBP1c is mainly at the posttranslational level. In a recent publication, mTORC1 was shown to phosphorylate a phosphatidic acid phosphatase, Lipin 1, preventing its nuclear entry and subsequent inhibition of SREBP1c-mediated activation of the FAS promoter (Peterson et al., 2011). Furthermore, Yecies JL et al (Yecies et al., 2011) showed that Akt2 independently of mTORC1 downregulate the mRNA expression of insulin induced gene 2 (Insig2a), an inhibitor of SREBP1c. This finding has been debated by Wan M et al (Wan et al., 2011), who could not observe any downregulation of Insig2a by Akt2. They postulate that Akt2 acts independently of mTORC1 and SREBP1c, possibly via posttranslational mechanisms, and that nutrients have a direct role in the liver to promote lipogenesis by a process dependent on both mTORC1 and other insulin-dependent signaling pathways. In light of the above mentioned studies, both mTORC1 and mTORC2 (Soukas et al., 2009; Guertin et al., 2006; Lamming et al., 2012; Hagiwara et al., 2012) appear to play important roles in lipid synthesis and storage in hepatocytes. Further studies will reveal the relative roles of Akt1, Akt2, mTORC1/C2 and S6kinase on activation of LXR and SREBP1c in this regulation under insulin sensitive and insulin resistant conditions and cross-talk with glucose metabolism and signaling (Fig.4).

#### *3.2.3. Regulation by FoxO1*

Another mechanism by which insulin may promote LXR-mediated SREBP1c transcription is through the transcription factor FoxO1. FoxO1, generally known as an activator of gluconeogenic genes during fasting, can repress the transactivating ability of LXR and cooperating transcription factors SREBP1c and Specificity protein 1 (Sp1) to activate SREBP1c transcription during fasting (Liu et al., 2010; Deng et al., 2012). FoxO1 does not seem to bind directly to the SREBP1c promoter, but appears to act as a repressor through protein-protein interactions, possibly by recruiting CR proteins (Deng et al., 2012). Upon feeding, FoxO1 is inhibited by insulin via PI3-kinase activation and phosphorylation by Akt, which excludes phosphorylated FoxO1 from the nucleus via association with the 14-3-3 protein (reviewed in (Tzivion et al., 2011)). In this way, at least under insulin sensitive conditions, inhibition mediated by FoxO1 and associating CRs is relieved, enabling LXR, Sp1 and SREBP1c to activate the SREBP1c promoter in a cooperative fashion. Of note, an important role for the E-box transcription factor Upstream Stimulatory Factor (USF) in mediating insulin activation of the SREBP1c promoter has also been reported (Wong & Sul, 2010). The relative roles of LXR, SREBP1c and cooperating transcription factors in regulation of the SREBP1c promoter after high-carbohydrate feeding under normal and insulin resistant conditions and the role of FoxO1 in this process in insulin resistance is currently not known. Recently, the role of Akt as a central regulator of both gluconeogenesis, through inhibition of FoxO1, and lipogenesis, through activation of mTORC1/2 in hepatic insulin signaling, was debated as the insulin resistant phenotype of mice lacking hepatic Akt1/2 were normalized in mice with concomitant liver-specific deletion of FoxO1 (Lu et al., 2012). This work suggests that a major role for Akt as a metabolic regulator in response to insulin is largely to restrain FoxO1 activity, at least for suppression of liver glucose output.

The Role of Liver X Receptor in Hepatic *de novo* Lipogenesis and Cross-Talk with Insulin and Glucose Signaling 69

complete summary of putative mechanisms of insulin-mediated signaling to LXR, SREBP1c

and lipogenesis is depicted in Fig. 4.

**Figure 4.** Insulin-mediated regulation of hepatic lipogenesis

#### *3.2.4. Regulation by insulin-mediated oxysterol production*

Considering the bifurcated nature of insulin resistance and the postulated central role of Akt in this process, a very recent work by Wu and Williams (Wu & Williams, 2012), put forward an interesting theory. They suggest that disturbance of a single molecule, NAD(P)H oxidase 4 (NOX4), is sufficient to induce the key harmful features of insulin resistance. NOX4 is activated upon IR activation, generating a transient burst of superoxide (O2- ) and its byproduct H2O2. This enhances signal transduction by disabling enzymes in the proteintyrosine phosphatase gene family. In this way, essential inhibiting enzymes in the insulin signaling cascade is blocked, notably the PI3K inhibitor PTEN and protein-tyrosine phosphatase-1B (PTP1B) (Wu & Williams, 2012). Intriguingly, NOX4 may also be the link between insulin signaling and production of oxysterol ligand for LXR, as NOX4 through its superoxide producing activity may mediate the production of oxygenated cholesterol. The evidence for this is that pharmacological inhibition of NOX4 blocked insulin-induction of SREBP1c mRNA in rat primary hepatocytes, even though phosphorylations upstream and downstream of mTORC1 remained responsive (Wu & Williams, 2012). Furthermore, NOX4 is transiently localized to caveolae (Han et al., 2012), possibly via recruitment to the IR, placing the enzyme in close proximity to cholesterol-rich areas of the plasma membrane. A complete summary of putative mechanisms of insulin-mediated signaling to LXR, SREBP1c and lipogenesis is depicted in Fig. 4.

68 Lipid Metabolism

*3.2.3. Regulation by FoxO1* 

Another mechanism by which insulin may promote LXR-mediated SREBP1c transcription is through the transcription factor FoxO1. FoxO1, generally known as an activator of gluconeogenic genes during fasting, can repress the transactivating ability of LXR and cooperating transcription factors SREBP1c and Specificity protein 1 (Sp1) to activate SREBP1c transcription during fasting (Liu et al., 2010; Deng et al., 2012). FoxO1 does not seem to bind directly to the SREBP1c promoter, but appears to act as a repressor through protein-protein interactions, possibly by recruiting CR proteins (Deng et al., 2012). Upon feeding, FoxO1 is inhibited by insulin via PI3-kinase activation and phosphorylation by Akt, which excludes phosphorylated FoxO1 from the nucleus via association with the 14-3-3 protein (reviewed in (Tzivion et al., 2011)). In this way, at least under insulin sensitive conditions, inhibition mediated by FoxO1 and associating CRs is relieved, enabling LXR, Sp1 and SREBP1c to activate the SREBP1c promoter in a cooperative fashion. Of note, an important role for the E-box transcription factor Upstream Stimulatory Factor (USF) in mediating insulin activation of the SREBP1c promoter has also been reported (Wong & Sul, 2010). The relative roles of LXR, SREBP1c and cooperating transcription factors in regulation of the SREBP1c promoter after high-carbohydrate feeding under normal and insulin resistant conditions and the role of FoxO1 in this process in insulin resistance is currently not known. Recently, the role of Akt as a central regulator of both gluconeogenesis, through inhibition of FoxO1, and lipogenesis, through activation of mTORC1/2 in hepatic insulin signaling, was debated as the insulin resistant phenotype of mice lacking hepatic Akt1/2 were normalized in mice with concomitant liver-specific deletion of FoxO1 (Lu et al., 2012). This work suggests that a major role for Akt as a metabolic regulator in response to insulin

is largely to restrain FoxO1 activity, at least for suppression of liver glucose output.

activated upon IR activation, generating a transient burst of superoxide (O2-

Considering the bifurcated nature of insulin resistance and the postulated central role of Akt in this process, a very recent work by Wu and Williams (Wu & Williams, 2012), put forward an interesting theory. They suggest that disturbance of a single molecule, NAD(P)H oxidase 4 (NOX4), is sufficient to induce the key harmful features of insulin resistance. NOX4 is

byproduct H2O2. This enhances signal transduction by disabling enzymes in the proteintyrosine phosphatase gene family. In this way, essential inhibiting enzymes in the insulin signaling cascade is blocked, notably the PI3K inhibitor PTEN and protein-tyrosine phosphatase-1B (PTP1B) (Wu & Williams, 2012). Intriguingly, NOX4 may also be the link between insulin signaling and production of oxysterol ligand for LXR, as NOX4 through its superoxide producing activity may mediate the production of oxygenated cholesterol. The evidence for this is that pharmacological inhibition of NOX4 blocked insulin-induction of SREBP1c mRNA in rat primary hepatocytes, even though phosphorylations upstream and downstream of mTORC1 remained responsive (Wu & Williams, 2012). Furthermore, NOX4 is transiently localized to caveolae (Han et al., 2012), possibly via recruitment to the IR, placing the enzyme in close proximity to cholesterol-rich areas of the plasma membrane. A

) and its

*3.2.4. Regulation by insulin-mediated oxysterol production* 

**Figure 4.** Insulin-mediated regulation of hepatic lipogenesis
