**2. Genetic factors and muscle/bone phenotypes**

Genes such as myostatin, α-actinin-3, proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and myocyte enhancer factor 2C (MEF-2C) are included in GWAS (genomewide association study) as believed to be involved in a concurrent loss of muscle and bone tissue [3]. Myostatin, on the other hand, has been shown to be a negative regulator of muscle mass. Alpha-actinin-3 has been demonstrated to be abundantly expressed in fast-twitch skeletal muscle fibres and may also affect their differentiation towards fast-twitch fibres. Finally, it was shown [4] that a lack of α-actinin-3 may lead to a reduction in bone mineral density (BMD) in both humans and rodents.

of HDACs, transcription factors (TFs), as well as microRNAs and the impact of vitamin K2 (MK-7) on mineralizing cells (osteoblasts) and striated muscle cells exposed to either normal growth conditions or mediators of inflammation (i.e. Th-cells, macrophages, or interleukins), indicating that it is possible to engineer cells displaying an adapted phenotype where: (a) towards mineralization is reinforced, (b) untoward mineral deposition is halted and finally (c)

With the aid of various algorithms, one may reveal regulatory loops involving both TFs and microRNAs. The subjects TFs and microRNA species appeared to be part of an intricate hierarchical structure encompassing several classes of HDACs, including the Sirtuins, known to

vitamin K2 (MK-7, via binding to the transcription factor SXR) interfered with a plethora of signalling pathways (such as the FoxA and FoxO families of transcription factors), the downstream of the signalling mechanisms represented by the PI3-kinase system (i.e. Akt/PKB and SGK, respectively), thus potentiating the cross-talk signals or suppressing the mineralizing character. It was concluded that vitamin K2 plays a pivotal role by optimizing the endocrine interaction between osteoblasts and striated muscle cells, facilitating a 'win-win' situation. Furthermore, we have shown that vitamin K2 may confer the ability for cross-talk between striated muscle cells and bones to include cells, such as insulin-producing β-cells, thyroid follicular cells, PTH-producing parathyroid cells and hepatocytes, in the absence or presence of

Sarcopenia (reduced muscle mass and/or function) and osteoporosis (bone brittleness) have generally been known for their relations to the locomotive syndrome and are linked to old age. Contrastingly, an increased muscle mass correlates with an enhanced bone mass and thus with a reduced fracture incidence. Genetic, as well as endocrine and mechanical factors, inflammation and nutritional states concurrently impinge on muscle tissue and bone metabolism.

Furthermore, a plethora of genes like myostatin and α-actinin-3 associate with both conditions. Factors such as vitamin D, growth hormones (like GH and IGF-1) and testosterone and pathological conditions with excess cortisol, as well as type I diabetes (T1DM), affect both muscle and bone tissues. It was shown that the genes Tmem119, osteoglycin and FAM5C may be critical for the commitment of myoprogenitor cells to the osteoblast lineage. Furthermore, osteoglycin and FAM5C might serve as muscle-derived humoral osteogenic factors. Others, encompassing myostatin, osteonectin, as well as IGF-1, irisin and osteocalcin, may also be

Genes such as myostatin, α-actinin-3, proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and myocyte enhancer factor 2C (MEF-2C) are included in GWAS (genomewide association study) as believed to be involved in a concurrent loss of muscle and bone tissue [3]. Myostatin, on the other hand, has been shown to be a negative regulator of muscle mass. Alpha-actinin-3 has been demonstrated to be abundantly expressed in fast-twitch skeletal muscle fibres and may also affect their differentiation towards fast-twitch fibres. Finally,

associated with reciprocal metabolic interactions between muscle and bone [2].

**2. Genetic factors and muscle/bone phenotypes**

ratios). Finally, it was demonstrated that

mutual musculoskeletal interactions are 'reinforced'.

200 Vitamin K2 - Vital for Health and Wellbeing

respond to cellular energy status (i.e. NADH/NAD+

inflammatory cells or their secreted cytokines/interleukins ± TNFα.

PGC-1α seems to be instrumental in the modulation of mitochondrial biogenesis [5], and a further study established that PGC-1α elicited by physical activity seems to be crucial for oxidative metabolism in skeletal muscle fibres [6]. Furthermore, it was demonstrated that mitochondrial biogenesis induced by an enhancement in PGC-1α levels facilitates Wnt-mediated induction of osteoblastic differentiation of mesenchymal C3H10T1/2 cells [7]. These findings indicate that PGC-1α serves as a 'commitment' factor or inductor of stem cells to produce osteoblastic cells. Another essential factor is MEF-2C, which interacts with other myogenic regulatory factors, like Myf5 and MyoD, which in a synergistic fashion activates specific muscle-phenotypic genes. Animals devoid of MEF-2C in osteocytes make less sclerostin, a humoral factor acting as an inhibitor of the Wnt family of signalling molecules involved in osteoblast differentiation and bone formation. Thus, the MEF-2C-sclerostin signalling inhibits the formation of excessive bone mass and a 'healthy' turnover, which normally ensures minimal bone brittleness.

Qiu et al. [8] demonstrates that NF-κb-mediated signalling modulates myostatin transcription in myoblasts during cirrhosis-induced hyperammonaemia. This suggests that NF-κB antagonists are useful to reverse cirrhosis induced by sarcopenia. This observation also indicates that vitamin K2-induced modulation of NF-κB may determine the levels of humoral factors, which reciprocally regulate muscle and bone physiology. We have found [Gordeladze et al., 2015, unpublished] that preadipocytes, with mutated and superactive Gsα-induced adenylate cyclase activity, in the presence of vitamin K2 (MK-7), produce more beige-like adipocytes (see **Figure 1**) than large white adipocytes (ref), with an enhancement in PGC-1α levels (ref). Hence, it may be asserted that vitamin K2 facilitates Wnt-mediated induction of osteoblastic differentiation by

**Figure 1.** Putative working model showing how vitamin K2 may affect the hormonal signalling systems and transcription factors responsible for the transition of «white» adipocytes to «beige» adipocytes, thus blocking fat deposition and enhancing the production of heat from fatty acids.

enhancing the β-adrenoceptor and PKA-mediated signalling through PGC-1α of mesenchymal cells/stem cells, in order to fortify metabolic mechanisms 'ruled' by c/EBPβ, PPARα/γ and DiO<sup>2</sup> .

Since most of our cells throughout the body express the transcription factor PXR/SXR, binding vitamin K2, it may be asserted that vitamin K2 not only affects the phenotype of muscle and bone cells, shown here to interact in a reciprocal endocrine fashion but also (a) plays a major role in the determination and stabilization of the phenotype of a plethora of specialized cells in our body and (b) plays a pivotal role in the reciprocal interaction of various organ systems in our body to ensure optimal organ functions ('inter-organ cross-talk'). An interesting and elegant article written by Lara Pizzorno (see reference on last page) underscores the different effects of vitamin K2 in a comprehensive manner, supporting the notion that vitamin K2 is an essential biological factor supporting disease-free old age, which may be construed as

Longevity requires, of course, optimized and 'healthy' organ functioning throughout the body. Therefore, it is mandatory for the different organs of the body to communicate with each other and together form a 'cyn-organic' lattice where each organ communicates with the most part or all the others. Vitamin K2 may be one factor contributing to this inter-organ 'cross-talk', and there are several ways this little molecule exerts its integrative power. In this respect, the present book's chapter entitled 'Vitamin K2—small molecule with a large biological impact' featuring the molecular mechanisms, by which K2 exerts its actions, describes in detail how DIO1,2,3 impacts the regulation of cell proliferation, lipogenesis, lipolysis, cholesterol metabolism, carbohydrate metabolism, muscle contraction, thermogenesis, cell communication, exocytosis, cell cycle regulation and growth regulation. Of particular interest is

cells, the liver as well as white and brown adipose tissues, converts T4 to its active form, T3, ensuring an integrated metabolic and hormonal homeostasis and 'steady state' or endocrine equilibrium between the different organ systems of the body. In this context, we have shown (ref) that vitamin K2 is able to sustain the cell phenotypes of different organ systems, such as bones, striated muscles and others in the presence of sub-chronical inflammation, as induced by the presence of either Th-1 cells, Th-17 cells, as well as a mixture of TNFα or inflammatory

Others have more directly proven that vitamin K2, via binding to PXR, affects both triglyceride turnover and gluconeogenesis in the liver (ref). The authors of this chapter describe how the MK-PXR complex via CD36, CPTA1 and SCD1 stimulates ketogenesis and hampers triglyceride production. Furthermore, they also show how the MK-PXR complex, via a cluster of transcription factors (FoxO1, CREB, PGC-1α and HNF4), stimulates the enzymes PEPCK1 and G6Pase in order to facilitate the metabolic conversion of lactate and amino acids to glucose (i.e. gluconeogenesis). This mechanism is remarkably like the one sketched in **Figure 1**,

only that there are other members of the Fox family of transcription factors involved!

A summary of a literature survey, extracting articles from PubMed, featuring new research on the biological impact of vitamin K2 published in 2015 and 2016, gave the following results:

**3. Novel findings related to the biology of vitamin K2**

), which, via higher brain centres, the pancreas, striated muscle

Vitamin K2 Facilitating Inter-Organ Cross-Talk

http://dx.doi.org/10.5772/67153

203

if vitamin K2 is one important alimentary ingredient ensuring 'longevity'.

the type 2 deiodinase,(DIO2

interleukins (e.g. IL-1 and IL-17).

However, the interaction between striated muscle cells and bones is illustrated in a better way in **Figure 2**. Here, it is shown that striated muscle cells communicate with 'the environment', consisting of other organs, such as white adipose tissue, liver, pancreas and bones through a multitude of endocrine/hormonal factors (see **Figure 2**(left)). However, if we just look closer at the reciprocal interactions between striated muscle cells and bone cells (osteoblasts, osteocytes and osteoclasts), there are still a large number of humoral factors, such as IGF-1, myostatin, osteoglycin, FAM5C, Irisin, Osteonectin, FGF2, IL-6, IL-7, IL-15, MMP-2, Sclerostin, Osteocalcin, MGP, VEGF and HGF (see **Figure 2**(right)), which 'capture' the two organs in a reciprocal regulatory 'looping system'. We have recently shown, for most part, the 'cross-talk' exchanged between these organs, particularly within the muscle-bone axis.

Vitamin K2 serves as a 'coupling agent', fine-tuning muscle-bone interactions, while concomitantly preserving its precision and strengthening it in the presence of inflammatory interleukins and INFα, and/or Th-1 and Th-17 cells.

**Figure 2.** Endocrine communication between striated muscle cells and other organ systems, such as white adipose tissue, liver, bone and pancreas. Pay special attention to the plethora of hormones/cytokines being exchanged between the muscle and the bone. Ref.: Left: Benatti, F. B. & Pedersen, B. K. Nat. Rev. Rheumatol. 11, 86–97 (2015); published online 25 November 2014; doi:10.1038/nrrheum.2014.193. Right: Kawao N, Kaji H. J Cell Biochem. 2015 May;116(5):687–95. doi: 10.1002/jcb.25040. PMID: 25521430.

Since most of our cells throughout the body express the transcription factor PXR/SXR, binding vitamin K2, it may be asserted that vitamin K2 not only affects the phenotype of muscle and bone cells, shown here to interact in a reciprocal endocrine fashion but also (a) plays a major role in the determination and stabilization of the phenotype of a plethora of specialized cells in our body and (b) plays a pivotal role in the reciprocal interaction of various organ systems in our body to ensure optimal organ functions ('inter-organ cross-talk'). An interesting and elegant article written by Lara Pizzorno (see reference on last page) underscores the different effects of vitamin K2 in a comprehensive manner, supporting the notion that vitamin K2 is an essential biological factor supporting disease-free old age, which may be construed as if vitamin K2 is one important alimentary ingredient ensuring 'longevity'.

enhancing the β-adrenoceptor and PKA-mediated signalling through PGC-1α of mesenchymal cells/stem cells, in order to fortify metabolic mechanisms 'ruled' by c/EBPβ, PPARα/γ and DiO<sup>2</sup>

However, the interaction between striated muscle cells and bones is illustrated in a better way in **Figure 2**. Here, it is shown that striated muscle cells communicate with 'the environment', consisting of other organs, such as white adipose tissue, liver, pancreas and bones through a multitude of endocrine/hormonal factors (see **Figure 2**(left)). However, if we just look closer at the reciprocal interactions between striated muscle cells and bone cells (osteoblasts, osteocytes and osteoclasts), there are still a large number of humoral factors, such as IGF-1, myostatin, osteoglycin, FAM5C, Irisin, Osteonectin, FGF2, IL-6, IL-7, IL-15, MMP-2, Sclerostin, Osteocalcin, MGP, VEGF and HGF (see **Figure 2**(right)), which 'capture' the two organs in a reciprocal regulatory 'looping system'. We have recently shown, for most part, the 'cross-talk' exchanged between these organs, particularly within

Vitamin K2 serves as a 'coupling agent', fine-tuning muscle-bone interactions, while concomitantly preserving its precision and strengthening it in the presence of inflammatory interleu-

**Figure 2.** Endocrine communication between striated muscle cells and other organ systems, such as white adipose tissue, liver, bone and pancreas. Pay special attention to the plethora of hormones/cytokines being exchanged between the muscle and the bone. Ref.: Left: Benatti, F. B. & Pedersen, B. K. Nat. Rev. Rheumatol. 11, 86–97 (2015); published online 25 November 2014; doi:10.1038/nrrheum.2014.193. Right: Kawao N, Kaji H. J Cell Biochem. 2015 May;116(5):687–95. doi:

the muscle-bone axis.

202 Vitamin K2 - Vital for Health and Wellbeing

10.1002/jcb.25040. PMID: 25521430.

kins and INFα, and/or Th-1 and Th-17 cells.

.

Longevity requires, of course, optimized and 'healthy' organ functioning throughout the body. Therefore, it is mandatory for the different organs of the body to communicate with each other and together form a 'cyn-organic' lattice where each organ communicates with the most part or all the others. Vitamin K2 may be one factor contributing to this inter-organ 'cross-talk', and there are several ways this little molecule exerts its integrative power. In this respect, the present book's chapter entitled 'Vitamin K2—small molecule with a large biological impact' featuring the molecular mechanisms, by which K2 exerts its actions, describes in detail how DIO1,2,3 impacts the regulation of cell proliferation, lipogenesis, lipolysis, cholesterol metabolism, carbohydrate metabolism, muscle contraction, thermogenesis, cell communication, exocytosis, cell cycle regulation and growth regulation. Of particular interest is the type 2 deiodinase,(DIO2 ), which, via higher brain centres, the pancreas, striated muscle cells, the liver as well as white and brown adipose tissues, converts T4 to its active form, T3, ensuring an integrated metabolic and hormonal homeostasis and 'steady state' or endocrine equilibrium between the different organ systems of the body. In this context, we have shown (ref) that vitamin K2 is able to sustain the cell phenotypes of different organ systems, such as bones, striated muscles and others in the presence of sub-chronical inflammation, as induced by the presence of either Th-1 cells, Th-17 cells, as well as a mixture of TNFα or inflammatory interleukins (e.g. IL-1 and IL-17).

Others have more directly proven that vitamin K2, via binding to PXR, affects both triglyceride turnover and gluconeogenesis in the liver (ref). The authors of this chapter describe how the MK-PXR complex via CD36, CPTA1 and SCD1 stimulates ketogenesis and hampers triglyceride production. Furthermore, they also show how the MK-PXR complex, via a cluster of transcription factors (FoxO1, CREB, PGC-1α and HNF4), stimulates the enzymes PEPCK1 and G6Pase in order to facilitate the metabolic conversion of lactate and amino acids to glucose (i.e. gluconeogenesis). This mechanism is remarkably like the one sketched in **Figure 1**, only that there are other members of the Fox family of transcription factors involved!
