**3. SXR cross talks with biological signals in biological responses: implications in health and disease**

#### **3.1. The molecular mechanisms of SXR-mediated gene repression**

Currently, SXR has been described as a repressor of gluconeogenic gene expression, some of which are glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase 1 (PEPCK1), thus implicating vitamin K2 in metabolic (energy-related) reactions taking place in the liver [23–25]. The SXR-vitamin K2 complex may therefore interfere directly with transcription factors and thus be rendered responsive to both insulin and glucagon. Consequently, one would observe a release of transcription factors with their coactivators from target genes, which would lead to a general subactivation of gene transcription.

**3.4. SXR in glucose handling**

the G6Pase and PEPCK1.

loses its activity [41, 42].

levels within "healthy" limits.

**3.5. SXR in lipid turnover**

The pancreatic hormones (insulin and glucagon) reciprocally regulate the blood glucose level via transcriptional processes, where rate-limiting enzymes like G6Pase and PEPCK1 in the glucose metabolism play a decisive role [34–36]. The glucose-6-phosphatase dephosphorylates glucose-6-phospate (G6P), constituting the endpoint of both the glucose-forming and glucose-utilizing reactions, while PEPCK1 converts oxaloacetate to phosphoenolpyruvate (PEP) in the gluconeogenic reaction. These enzymes are instrumental in controlling blood glucose levels. During fasting and/or prolonged exercise, glucagon dominates glucose metabolism by activating the cAMP/PKA signaling pathway [35, 37, 38]. When phosphorylated by PKA, the cAMP-response element-binding protein (CREB) stimulates both

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However, insulin acts in an opposite manner, and in response to high blood glucose levels, insulin is secreted and activates the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway [39]. Thereafter, Akt phosphorylates and inactivates the transcription factor forkhead box O1 (FoxO1). FoxO1, being a key regulator of glucose turnover, subsequently stimulates the insulin response sequence (IRS)-bearing genes G6Pase and PEPCK1 [40]. When phosphorylation by Akt, FoxO1 cannot longer translocate to the nucleus, it is rapidly acetylated and therefore

But, when using SXR-KO and SXR-humanized animals, it has been demonstrated that SXR is an important regulator of xenobiotic-dependent glucose turnover in the liver. Treatment of mice with potent SXR activators consistently leads to lowered blood glucose levels in laboratory animals [32]. Furthermore, it was shown that there existed so-called "cross-talk" between SXR and FoxO1 as a molecular mechanism underlying the downregulation of glucose metabolism [18]. It was reported by Kodama and Negishi that liganded SXR directly interacts with phosphorylated CREB in primary hepatocytes [25], and that SXR disturbs the binding of CREB to CRE with an ensuing repression of the CREB-mediated transcription of G6Pase and PEPCK1 genes. Looking at hitherto available information, it can be asserted that SXR, by "targeting" a plethora of factors modulated by insulin and glucagon, leads to an activation of many genes being functional in the intrinsic regulatory machinery maintaining serum glucose

The liver provides lipid-derived energy-rich compounds to different parts of the body. Hepatic lipid metabolism is controlled by the net influence of the "reciprocal" hormones insulin and glucagon, as well as by nutritional conditions. Some 10 years ago, Kodama and Negishi, along with Nakamura [25, 32], published that treatment with PCN (an activator of SXR) decreased the mRNA levels of carnitine palmitoyltransferase 1a (CPT1a) and 3-hydroxy-3-methylglutarate-CoA synthase 2 (Hmgcs2) in livers of starved wild-type mice, but not in SXR-KO mice [32]. CPT1a is instrumental in the overall mitochondrial β-oxidation

Furthermore, it has been asserted that SXR represses the CYP-genes through interference with the vitamin D receptor, VDR [26]. Hence, in tissues short of vitamin D3, liganded SXR would associate directly to vitamin D response elements (VDREs) and modulate transcription. Therefore, it may be asserted that vitamin K2 and vitamin D, as well as vitamin A (via VDR and RXA, respectively), and many other transcription factor like molecules (e.g., PPARs, FXR, LXRα, LRH-1 = NR5A2, RXR), when associated with their ligands, may act synergistically on gene transcription in general [16, 27–29]. Hence, it is not straightforward to predict the net results of a certain combination of liganded transcription factors on biological processes. However, there are several excellent reports on the impact of vitamin K2, in association with the nuclear factor SXR, on cellular metabolism.

#### **3.2. Involvement of SXR in metabolic functions**

Xenobiotics are able to enhance SXR-mediated expression of xenobiotic-metabolizing enzymes in both the liver and intestines. Even though such a modulation normally serves to detoxify the xenobiotics in question, these "alien molecules" enhance the production of intermediates, which confer harmful attack of tissues on the body [30, 31]. Additionally, SXR affects the balance of endobiotics (e.g., steroid hormones, cholesterols, and bile acids) aided by the same biochemical pathways. Hence, an SXR activation will consequently stimulate a plethora of physiological responses, i.e., in the liver, which plays an important role in the processing of, among many substances, glucose and lipids. Disruption of their metabolic fate may result in diseases, of which type II diabetes (T2DM) and obesity are most frequently encountered. Newly published studies of SXR-KO and SXR-humanized animals clearly shed light on the metabolic functions of SXR in man.

#### **3.3. SXR and its role in energy metabolism of the liver**

The liver provides energy sources to the rest of the body, i.e., carbohydrates and lipids are catabolized in order to fuel both central and peripheral tissues and organs. The effect of SXR on hepatic energy turnover was discovered with the aid of SXR-KO mice [25, 32, 33].

#### **3.4. SXR in glucose handling**

**3. SXR cross talks with biological signals in biological responses:** 

Currently, SXR has been described as a repressor of gluconeogenic gene expression, some of which are glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase 1 (PEPCK1), thus implicating vitamin K2 in metabolic (energy-related) reactions taking place in the liver [23–25]. The SXR-vitamin K2 complex may therefore interfere directly with transcription factors and thus be rendered responsive to both insulin and glucagon. Consequently, one would observe a release of transcription factors with their coactivators from target genes,

Furthermore, it has been asserted that SXR represses the CYP-genes through interference with the vitamin D receptor, VDR [26]. Hence, in tissues short of vitamin D3, liganded SXR would associate directly to vitamin D response elements (VDREs) and modulate transcription. Therefore, it may be asserted that vitamin K2 and vitamin D, as well as vitamin A (via VDR and RXA, respectively), and many other transcription factor like molecules (e.g., PPARs, FXR, LXRα, LRH-1 = NR5A2, RXR), when associated with their ligands, may act synergistically on gene transcription in general [16, 27–29]. Hence, it is not straightforward to predict the net results of a certain combination of liganded transcription factors on biological processes. However, there are several excellent reports on the impact of vitamin K2, in association with

Xenobiotics are able to enhance SXR-mediated expression of xenobiotic-metabolizing enzymes in both the liver and intestines. Even though such a modulation normally serves to detoxify the xenobiotics in question, these "alien molecules" enhance the production of intermediates, which confer harmful attack of tissues on the body [30, 31]. Additionally, SXR affects the balance of endobiotics (e.g., steroid hormones, cholesterols, and bile acids) aided by the same biochemical pathways. Hence, an SXR activation will consequently stimulate a plethora of physiological responses, i.e., in the liver, which plays an important role in the processing of, among many substances, glucose and lipids. Disruption of their metabolic fate may result in diseases, of which type II diabetes (T2DM) and obesity are most frequently encountered. Newly published studies of SXR-KO and SXR-humanized animals clearly shed light on the

The liver provides energy sources to the rest of the body, i.e., carbohydrates and lipids are catabolized in order to fuel both central and peripheral tissues and organs. The effect of SXR

on hepatic energy turnover was discovered with the aid of SXR-KO mice [25, 32, 33].

**3.1. The molecular mechanisms of SXR-mediated gene repression**

which would lead to a general subactivation of gene transcription.

**implications in health and disease**

90 Vitamin K2 - Vital for Health and Wellbeing

the nuclear factor SXR, on cellular metabolism.

**3.2. Involvement of SXR in metabolic functions**

metabolic functions of SXR in man.

**3.3. SXR and its role in energy metabolism of the liver**

The pancreatic hormones (insulin and glucagon) reciprocally regulate the blood glucose level via transcriptional processes, where rate-limiting enzymes like G6Pase and PEPCK1 in the glucose metabolism play a decisive role [34–36]. The glucose-6-phosphatase dephosphorylates glucose-6-phospate (G6P), constituting the endpoint of both the glucose-forming and glucose-utilizing reactions, while PEPCK1 converts oxaloacetate to phosphoenolpyruvate (PEP) in the gluconeogenic reaction. These enzymes are instrumental in controlling blood glucose levels. During fasting and/or prolonged exercise, glucagon dominates glucose metabolism by activating the cAMP/PKA signaling pathway [35, 37, 38]. When phosphorylated by PKA, the cAMP-response element-binding protein (CREB) stimulates both the G6Pase and PEPCK1.

However, insulin acts in an opposite manner, and in response to high blood glucose levels, insulin is secreted and activates the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway [39]. Thereafter, Akt phosphorylates and inactivates the transcription factor forkhead box O1 (FoxO1). FoxO1, being a key regulator of glucose turnover, subsequently stimulates the insulin response sequence (IRS)-bearing genes G6Pase and PEPCK1 [40]. When phosphorylation by Akt, FoxO1 cannot longer translocate to the nucleus, it is rapidly acetylated and therefore loses its activity [41, 42].

But, when using SXR-KO and SXR-humanized animals, it has been demonstrated that SXR is an important regulator of xenobiotic-dependent glucose turnover in the liver. Treatment of mice with potent SXR activators consistently leads to lowered blood glucose levels in laboratory animals [32]. Furthermore, it was shown that there existed so-called "cross-talk" between SXR and FoxO1 as a molecular mechanism underlying the downregulation of glucose metabolism [18]. It was reported by Kodama and Negishi that liganded SXR directly interacts with phosphorylated CREB in primary hepatocytes [25], and that SXR disturbs the binding of CREB to CRE with an ensuing repression of the CREB-mediated transcription of G6Pase and PEPCK1 genes. Looking at hitherto available information, it can be asserted that SXR, by "targeting" a plethora of factors modulated by insulin and glucagon, leads to an activation of many genes being functional in the intrinsic regulatory machinery maintaining serum glucose levels within "healthy" limits.

#### **3.5. SXR in lipid turnover**

The liver provides lipid-derived energy-rich compounds to different parts of the body. Hepatic lipid metabolism is controlled by the net influence of the "reciprocal" hormones insulin and glucagon, as well as by nutritional conditions. Some 10 years ago, Kodama and Negishi, along with Nakamura [25, 32], published that treatment with PCN (an activator of SXR) decreased the mRNA levels of carnitine palmitoyltransferase 1a (CPT1a) and 3-hydroxy-3-methylglutarate-CoA synthase 2 (Hmgcs2) in livers of starved wild-type mice, but not in SXR-KO mice [32]. CPT1a is instrumental in the overall mitochondrial β-oxidation by funneling long-chain fatty acids into mitochondria [43], and the mitochondrial enzyme HMGCS2 facilitates the initial reaction of ketogenesis [44].

**4. The effect of vitamin K2 on other genes related to metabolic processes** 

In 2009, Slatter [49] and coworkers published a paper, featuring oligonucleotide microarrays with the intention to reveal the heterogeneity of drug metabolism associated gene expression in liver tissue from healthy humans. Their intention was to define clusters of so-called "absorption, distribution, metabolism, and excretion" = ADME genes to define subgroups of coregulated genes. When analyzing the gene sets, they discovered distinct patterns of "parallel" gene expressions featuring gene "clusters", which proved to be modulated by the nuclear receptor SXR. So called "fold range metrics and frequency distributions" were applied in order to reveal the variability of solitary PKDM genes. The most variable gene entities chiefly correlated to: (1) drug metabolism, (2) intermediary metabolism, (3) inflammation, and (4) cell cycle control. Unique expression patterns of these genes allowed for a further correlation with a parallel expression of a plethora of other genes. Of major interest was the identification of SXR responsive genes.

A comprehensive list of these genes can be found in the article, however, quite a few of which are related to metabolic processes in the cell. The genes are the following (in alphabetical order): CLOCK, DUSP7, GCDH, IGFBP2, MAP2K2, NUCB2, OGT, PFKB1, PTPN11, and SLC16A2. By "looking up" current descriptions of the genes in "Gene-Cards", the following features of these SXR-sensitive genes were obtained (of which parts of their description is cited as presented):

CLOCK Clock circadian regulator (The protein encodes a transcription factor, and serves as DNA-binding

insulin and growth factors, ensuring energy homeostasis within optimal limits. GCDH The protein encoded by this gene belongs to the acyl-CoA dehydrogenase family. It catalyzes the

DUSP7 Dual-specific phosphatase (DUSPs) constitutes a large subgroup of cysteine-base protein-

oxidative carboxylation of glutaryl-CoA to crotonyl-CoA and CO<sup>2</sup>

stability of mitochondria, and hence energy metabolism in general. IGFBP2 Insulin-like growth factor binding protein, type 2. This protein inhibits IGF-mediate growth.

MAP2K2 This MAP-kinase catalyzes the concomitant phosphorylation of a threonine and a tyrosine

metabolism via MEK1/2 and the FoxO- and FoxA-family of transcription factors. NUCB2 Anorexigenic peptide; seems to play an important role in hypothalamic pathways regulating

regulator fortifying the effect of leptin, but independent of the size of fat depots.

histone acetyl transferase). *Interpretation:* Polymorphisms in this gene may be associated with obesity and metabolic syndrome. CLOCK normally regulates gene products (proteins) in an optimal fashion, adapted to diurnal demands on the body related to food ingestion, physical

tyrosine phosphatases characterized by their ability to dephosphorylate both tyrosine and serine/ threonine residues. *Interpretation:* DUSP7 may function as a modulator of cellular exposure to

L-lysine, L-hydroxylysine, and L-tryptophan metabolism. *Interpretation:* GCDH is involved in the

residue in a Thr-Glu-Tyr sequence located in MAP-kinases. It activates the ERK1 and ERK2 MAPkinases (by similarity). *Interpretation:* This kinase may block tumorigenesis and normalize energy

food intake and energy homeostasis, acting in a leptin-dependent manner. *Interpretation:* Appetite

*Interpretation:* A reduction in IGFBP2 may be responsible for organ hyperplasia and the

in the degradative pathway of

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http://www.genecards.org/cgi-bin/carddisp.pl?gene=NR1I2

**in the cell**

Name of gene Description of cellular function(s)

activity and recreation/sleep.

development of neoplasia (cancer).

Additionally, the stimulation of SXR by PCN has been demonstrated to enhance the mRNA steady state level of stearoyl-CoA desaturase 1 (Scd1) in hepatic tissue of starved wild-type experimental animals. SCD1, which serves as a key enzyme in hepatic lipogenesis, facilitates the rate-limiting step in the synthesis of unsaturated fatty acids [45]. The plasma concentrations of 3-OH-butylate were decreased, while the hepatic level of triglyceride (TG) was increased by the PCN treatment in wild-type mice during assay conditions. However, neither TG nor cholesterol levels in the blood were altered in those animals, despite the fact that there was a significant rise in TG accumulated in their liver. Hence, as a means of survival during fasting, SXR is thought to slow down hepatic lipid turnover by repressing β-oxidation and ketogenesis, while stimulating the transcription of lipogenic enzymes, in much the same way as induced by insulin.

The Akt-regulated forkhead transcription factor FoxA2 which serves as a facilitator of insulin-dependent modulation of β-oxidation and ketogenesis, enhances expression of both the CPT1a and HMGCS2 gene, respectively [21, 46]. It is well known that Insulin activates the PI3K/Akt signaling pathway to phosphorylate FoxA2, in order to translocate it from nucleus to cytosol, thereby downregulating both the genes. And, it has been asserted that a direct interaction between SXR and FoxA2 serves as the mechanism, by which SXR represses the transcription of CPT1a and HMGCS2 in the liver [32].

A plethora of transcription factors and coregulators have been asserted to serve as modulators of hepatic lipid metabolism, e.g., the peroxisome proliferator-activated receptors (PPARs), the liver X receptor α (LXRα), as well as the sterol regulatory element-binding proteins (SREBPs) [47]. The expression of SREBP1c, which is construed as the dominant regulator of hepatic lipogenesis, is under the control of LXRα, and mediates the insulin- and fatty acids-dependent responses of lipogenic genes such as fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), stearoyl-CoA-desaturase-1 (SCD1), and fatty acid elongase (FAE). SXR is believed to upregulate lipogenesis in the liver, independently of SREBP1c action, and it is not deemed to be associated with the steady state expression levels of both the Fas and Acc1 genes. Among the cluster of lipogenic genes, Cd36 (cluster of differentiation 36) is deemed to be serve as a direct target of SXR in the liver. And, upon stimulation by ligands, the receptor is believed to become recruited to a DR3-type SXR response element within its promoter region of the liver of experimental animals [48]. Furthermore, SXR has been asserted to serve as a link, facilitating the upregulation of the Pparγ-gene, which functions as a strong regulator of lipid-synthesizing enzymes [48]. Such a cross-talk involving nuclear receptors should confer a significant impact on the body's lipid homeostasis. Our data are in line with the published literature, however, it should be asserted that SXR probably affects a larger spectrum of FoxO and FoxA species than those presented in this review. In this way, one might speculate that SXR is able to recruit a "moving" representation of these transcription factors simultaneously, and that the net effect on various cell phenotypes depends on: (1) the distribution of FoxOs and FoxAs at any time within the cell or tissue, as well as (2) the epigenetic machinery or "make up" at any time within the same cells or tissues.
