**2. Vitamin K2 and its mechanism of action – beyond xenobiotic metabolism**

The steroid and xenobiotic receptor (SXR), which is synonymous with the pregnane X receptor = PXR, is characterized as a nuclear hormone receptor that is stimulated by a plethora of hormones, dietary steroids, pharmaceutically active agents, as well as xenobiotic compounds. SXR exhibits a binding domain, which diverges across mammalian species. SXR is construed as a xenobiotic sensor, facilitating xenobiotic clearance in the liver and intestine. However, newly published experiments unravel novel roles for SXR in modulating phenomena like inflammation, bone turnover, metabolism of vitamin D, lipid, and energy homeostasis, as well as cancer. The characterization of SXR as conveyer of hormone-like signals has now been recognized as a key instrument for the study of novel mechanisms, through which diet may ultimately affect health and disease. The discovery and pharmacological development of new PXR modulators, like vitamin K2, might represent an interesting and innovative therapeutic approach to combat various ailments and diseases.

Without elaborating on the details of how different natural products modulate the activity SXR to affect gene expression, it should be noted that St. John's wort (hypericum perforatum), vitamin E (tocopherols and tocotrienols), as well as sulforaphane and *Coleus forskohlii*, the latter producing forskolin which is able to stimulate adenylate cyclase activity, suffice to say that K2 emerges as an interesting "player" on the scene featuring major metabolic pathways accounting for a plethora of actions to be reckoned with [16]. One good example is the effect of SXR activation on the metabolism of cholesterol and lipid turnover, a second one is its impact on the Fox transcription factors (i.e., FoxO1 and FoxA2), and their influence on energy homeostasis [16]. FoxO1 and FoxA2 are both members of the "forkhead" family tree of transcription factors, serving critical roles in both lipid metabolism, as well as gluconeogenesis in the liver [17]. FoxO1 stimulates hepatic gluconeogenesis during fasting through the activation of gluconeogenic genes, such as PEPCK1 (phosphoenolpyruvate carboxykinase 1), G6P (glucose-6-phosphatase), as well as and insulin-like growth factor-binding protein 1.

FoxA2, and the ensuing interaction will halt the "coupling" between FoxA2 and its response in DNA elements. The communication between SXR and FoxO1/FoxA2 signifies that SXR, apart from being a modulator of drug metabolism in the liver, is also serving as an important regulator in hepatic in glucose and energy homeostasis. Hence, since vitamin K2 binds to and enhances SXR-mediated transcription, it may serve as the natural "drug" to ingest, when one is aiming to treat insulin resistance and type II diabetes mellitus. The impact of vitamin K2 via SXR on the metabolic functions in general, is described in more detail in the forthcoming

The Impact of Vitamin K2 on Energy Metabolism

http://dx.doi.org/10.5772/67152

89

In **Figure 3**, we show our own experiments indicating that the adipocyte phenotype, characterized by the expression of the transcription factor PPARγ, is heavily dependent on the presence of FoxO1 and FoxA3, respectively, since siRNA against either of them completely obliterate the stimulatory effect of vitamin K2 obtained through its binding to SXR. However, the literature in general advocates a plethora of PIK3/Akt-stimulated transcription factors of the FoxA and FoxO families in the cascade of insulin/IGF-1 mediated signaling in general. However, the present data show, for the first time that vitamin K2 (i.e., the MK-7 variant) directly stimulates FoxO1 and FoxA3, short-cutting the insulin/IGF-1 activation cascade. The assertion to be drawn is simply: Vitamin K2 may directly fortify the action of insulin, thus ensuring a better glucose homeostasis, as well as protection from a detrimental turnover of

**Figure 3.** The impact of vitamin K2 (in the form of MK-7) on the signaling mechanism of insulin, via the PIK3/Akt/ FoxO/FoxA cascade. *Left panel*: MK-7, bound to the transcription factor SXR activates FoxO1 and FoxA3, with ensuing stimulation of Sirt1. *Right panel*: Expression (mRNA analyses) of the transcription factor PPARγ in adipocytes generated from stem cells, either stimulated by vitamin K2 (MK-7) or inhibited in the presence of siRNA against FoxO1 or FoxA3. Reference to the figures: Left – DN Gross et al. (Oncogene, 2320–2336, 2008); Michael P. Czech (PNAS, 11198–11200,

paragraphs ("SXR in glucose handling" and "SXR in lipid turnover") [22].

lipids and protein structures of the body.

September 30, 2003).

FoxA2, on the other hand serves as a key switch, representing one of the regulatory factors of the breakdown of hepatic fatty-acids during calorie-restriction (i.e., fasting). Via mammalian cell-based two-hybrid screening, it was feasible to identify FoxO1 as a coactivator of both CAR (constitutive androstane receptor)-mediated, as well as SXR-stimulated transcription [18]. FoxO1 may directly associate with CAR and SXR as a hormone-ligand-receptor complex and stimulate their transcriptional ability. And, both CAR and SXR function as corepressors of FoxO1, suppressing the FoxO1-mediated transcription by counteracting its association with its response elements within the susceptible genes. As well as obliterating the FoxO1 activity, drug-stimulated SXR and CAR species were also shown to downregulate HNF4α transcriptional power via suppression of PGC1α, thus suppressing the transcription of both PEPCK1 and G6P [19]. This indicates that metabolic turnover of both drugs and glucose, the two major functions of the liver which are regulated independently, happens to be reciprocally coregulated via communication between xenobiotic sensors on one hand, and transcription factors in the liver, on the other. When blood sugar concentrations are rendered low due to fasting or subsequent to periods of exercise, the liver funnels energy rich molecules to extra-hepatic tissues and peripheral organs via either β-oxidation or the production of ketone bodies [20]. FoxA2 is stimulating both ketogenesis and β-oxidation via enhancement of the transcriptional activity of a plethora of genes, such as mitochondrial 3-hydroxy-3 methylglutarate-CoA synthase 2 (HMGCS2) and carnitine palmitoyltransferase 1A (CPT1A) in energy-depleted conditions (fasting), or subsequent to periods where extensive exercise prevails [21].

FoxA2 is phosphorylated and thus inactivated by the Akt pathway, and serves to decrease lipid turnover in response to insulin. Treatment with other drugs such as barbiturates has been shown to suppress lipid metabolism in an insulin-independent fashion [22]. Furthermore, it was reported that SXR may crosstalk with FoxA2 in order to induce the repression of lipid turnover in livers of fasting mice. By applying wild-type and SXR-/- animals, it was demonstrated that treatment with PCN (pregnenolone-16d-carbonitrile) diminished the steady-state mRNA levels of HMGCS2 and CPT1A in control animals, but not in SXR-deficient mice. When conducting biochemical and cell-based analyses, it was shown that SXR markedly downregulates the ability of FoxA2 to bring about activation of the HMGCS2 and CPT1A genes. SXR that serves as the ligand binding domain will associate directly with the DNA-binding area of FoxA2, and the ensuing interaction will halt the "coupling" between FoxA2 and its response in DNA elements. The communication between SXR and FoxO1/FoxA2 signifies that SXR, apart from being a modulator of drug metabolism in the liver, is also serving as an important regulator in hepatic in glucose and energy homeostasis. Hence, since vitamin K2 binds to and enhances SXR-mediated transcription, it may serve as the natural "drug" to ingest, when one is aiming to treat insulin resistance and type II diabetes mellitus. The impact of vitamin K2 via SXR on the metabolic functions in general, is described in more detail in the forthcoming paragraphs ("SXR in glucose handling" and "SXR in lipid turnover") [22].

Without elaborating on the details of how different natural products modulate the activity SXR to affect gene expression, it should be noted that St. John's wort (hypericum perforatum), vitamin E (tocopherols and tocotrienols), as well as sulforaphane and *Coleus forskohlii*, the latter producing forskolin which is able to stimulate adenylate cyclase activity, suffice to say that K2 emerges as an interesting "player" on the scene featuring major metabolic pathways accounting for a plethora of actions to be reckoned with [16]. One good example is the effect of SXR activation on the metabolism of cholesterol and lipid turnover, a second one is its impact on the Fox transcription factors (i.e., FoxO1 and FoxA2), and their influence on energy homeostasis [16]. FoxO1 and FoxA2 are both members of the "forkhead" family tree of transcription factors, serving critical roles in both lipid metabolism, as well as gluconeogenesis in the liver [17]. FoxO1 stimulates hepatic gluconeogenesis during fasting through the activation of gluconeogenic genes, such as PEPCK1 (phosphoenolpyruvate carboxykinase 1), G6P (glucose-

FoxA2, on the other hand serves as a key switch, representing one of the regulatory factors of the breakdown of hepatic fatty-acids during calorie-restriction (i.e., fasting). Via mammalian cell-based two-hybrid screening, it was feasible to identify FoxO1 as a coactivator of both CAR (constitutive androstane receptor)-mediated, as well as SXR-stimulated transcription [18]. FoxO1 may directly associate with CAR and SXR as a hormone-ligand-receptor complex and stimulate their transcriptional ability. And, both CAR and SXR function as corepressors of FoxO1, suppressing the FoxO1-mediated transcription by counteracting its association with its response elements within the susceptible genes. As well as obliterating the FoxO1 activity, drug-stimulated SXR and CAR species were also shown to downregulate HNF4α transcriptional power via suppression of PGC1α, thus suppressing the transcription of both PEPCK1 and G6P [19]. This indicates that metabolic turnover of both drugs and glucose, the two major functions of the liver which are regulated independently, happens to be reciprocally coregulated via communication between xenobiotic sensors on one hand, and transcription factors in the liver, on the other. When blood sugar concentrations are rendered low due to fasting or subsequent to periods of exercise, the liver funnels energy rich molecules to extra-hepatic tissues and peripheral organs via either β-oxidation or the production of ketone bodies [20]. FoxA2 is stimulating both ketogenesis and β-oxidation via enhancement of the transcriptional activity of a plethora of genes, such as mitochondrial 3-hydroxy-3 methylglutarate-CoA synthase 2 (HMGCS2) and carnitine palmitoyltransferase 1A (CPT1A) in energy-depleted condi-

6-phosphatase), as well as and insulin-like growth factor-binding protein 1.

88 Vitamin K2 - Vital for Health and Wellbeing

tions (fasting), or subsequent to periods where extensive exercise prevails [21].

FoxA2 is phosphorylated and thus inactivated by the Akt pathway, and serves to decrease lipid turnover in response to insulin. Treatment with other drugs such as barbiturates has been shown to suppress lipid metabolism in an insulin-independent fashion [22]. Furthermore, it was reported that SXR may crosstalk with FoxA2 in order to induce the repression of lipid turnover in livers of fasting mice. By applying wild-type and SXR-/- animals, it was demonstrated that treatment with PCN (pregnenolone-16d-carbonitrile) diminished the steady-state mRNA levels of HMGCS2 and CPT1A in control animals, but not in SXR-deficient mice. When conducting biochemical and cell-based analyses, it was shown that SXR markedly downregulates the ability of FoxA2 to bring about activation of the HMGCS2 and CPT1A genes. SXR that serves as the ligand binding domain will associate directly with the DNA-binding area of In **Figure 3**, we show our own experiments indicating that the adipocyte phenotype, characterized by the expression of the transcription factor PPARγ, is heavily dependent on the presence of FoxO1 and FoxA3, respectively, since siRNA against either of them completely obliterate the stimulatory effect of vitamin K2 obtained through its binding to SXR. However, the literature in general advocates a plethora of PIK3/Akt-stimulated transcription factors of the FoxA and FoxO families in the cascade of insulin/IGF-1 mediated signaling in general. However, the present data show, for the first time that vitamin K2 (i.e., the MK-7 variant) directly stimulates FoxO1 and FoxA3, short-cutting the insulin/IGF-1 activation cascade. The assertion to be drawn is simply: Vitamin K2 may directly fortify the action of insulin, thus ensuring a better glucose homeostasis, as well as protection from a detrimental turnover of lipids and protein structures of the body.

**Figure 3.** The impact of vitamin K2 (in the form of MK-7) on the signaling mechanism of insulin, via the PIK3/Akt/ FoxO/FoxA cascade. *Left panel*: MK-7, bound to the transcription factor SXR activates FoxO1 and FoxA3, with ensuing stimulation of Sirt1. *Right panel*: Expression (mRNA analyses) of the transcription factor PPARγ in adipocytes generated from stem cells, either stimulated by vitamin K2 (MK-7) or inhibited in the presence of siRNA against FoxO1 or FoxA3. Reference to the figures: Left – DN Gross et al. (Oncogene, 2320–2336, 2008); Michael P. Czech (PNAS, 11198–11200, September 30, 2003).
