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CHAPTER (3)
ROLE OF VITAMIN D IN NAFLD
As mentioned previously vitamin D is an important steroid
hormone with pleiotropic effects. Recently there is increasing recognition
that vitamin D has immunomodulatory, anti-inflammatory, anti-fibrotic
properties and plays an important role in the regulation of cell
proliferation and differentiation. These extraskeletal effects are relevant
in the pathogenesis and treatment of many causes of chronic liver disease.
(Lazo et al., 2011).
Vitamin D mediates its intracellular signals via its receptor VDR
which is constitutively expressed in the liver (Han et al., 2010). It has
been estimated that VDR regulates over 200 genes involved in glucose
and lipid metabolism (Zeitz et al., 2003), inflammation (Chun et al.,
2014), cellular proliferation and differentiation and apoptosis (Jiang et
al., 2013). In NAFLD, four single nucleotide polymorphisms (SNPs)
were found to have significant association with NAFLD. Among these
four SNPs GCgene was included which is predominately expressed in
hepatocytes and codes for vitamin D binding protein, the main carrier
protein for vitamin D (Adams et al., 2013).
In NAFLD, liver VDR expression is inversely correlated with
severity of NAFLD on histopathology.
Vitamin D deficient individuals are more likely to develop
alterations in glucose metabolism, such as impaired glucose tolerance, the
MS and T2DM (Hypponen et al., 2008).Based on these evidences, it can
be hypothesized that vitamin D deficiency should not be considered an
exclusive feature of patients with osteo mineral disorders. Vitamin D is
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capable to reduce free FFA-induced IR both in peripheral tissues and in
hepatocytes. Therefore, low serum vitamin D may predispose to
intrahepatic lipid accumulation leading to NAFLD (Zhou et al., 2004).
Vitamin D may prevent liver pathology and the development of
NAFLD through the suppression of related and potentially interacting
pathways that involve hepatocyte apoptosis, liver inflammation and
fibrosis, oxidative stress, the expression of protective adipokines, and
changes to the composition of the gut microbiome.
Potential mechanisms and evidence that show the benefit effect
of vit D on NAFLD:

Improvement in insulin secretion and insulin resistance:
Due to the presence of VDR in pancreatic beta cells (Maestro et
al., 2003).And expression of 1-α-hydroxylase enzyme in pancreatic beta
cells (Bland et al., 2004), Vitamin D deficiency impairs glucosemediated insulin secretion from rat pancreatic beta cells in vitro and in
vivo (Cade et al., 1986).
Vitamin D enhances insulin responsiveness for glucose transport in
muscle cells (Alkharfy et al., 2013). Also vitamin D up-regulates glucose
transporter 4 (GLUT4) translocation and glucose utilization in adipocytes
(Manna and Jain, 2012).

Improvement in hepatic inflammation:
Vitamin D deficiency causes toll like receptor (TLR ) activation
and exacerbates hepatic inflammation (Roth et al., 2012).
TLRs are a class of proteins that play a key role in the innate
immune system. They are single, membrane-spanning, receptors usually
expressed in sentinel cells such as macrophages and dendritic cells, that
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recognize structurally conserved molecules derived from microbes. Once
these microbes reach physical barriers such as the skin or intestinal tract
mucosa, they are recognized by TLRs, which activate immune cell
responses (Mahla, 2013).
Artificial sunlight therapy in rats reduced liver inflammation and
apoptosis (Nakano et al., 2011). VDR expression is inversely correlated
with steatosis severity and nonalcoholic fatty liver disease score in NASH
patients (Barchetta et al., 2012).

Vitamin D Inhibits Hepatocyte Apoptosis:
Vitamin D act by inhibiting hepatocyte apoptosis, as suggested by
its ability to regulate the expression of apoptosis-associated proteins in
the liver, increasing anti-apoptotic Bcl-2 and Bcl-xL proteins, and
decreasing pro-apoptotic Bax and Caspase-3 proteins. Calcitriol also
inhibit expression of FasL, a protein important for the ability of cytotoxic
T cells to target foreign cells (Zhang et al.,2007).

Vitamin D Modulate Adipokine Expression:
Adiponectin is an adipokine (secreted from adipose tissue), which
regulates glucose and fatty acid oxidation, but may play a protective role
in metabolic processes. Adiponectin may suppress liver fibrosis by
inhibiting secretion of TNF-α by HSCs (Giby and Ajith, 2014). Some
studies suggest a positive relationship between serum levels of
adiponectin and vit D (Husemoen et al., 2014), increased serum
adiponectin reduce the hepatic expression of inflammatory genes TNF-α
and TGF-β as well as a-smooth muscle actin (a-SMA)known to be a
marker of HSCS activation (Nakano et al., 2011),
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
Vitamin D Alters the Gut Microbiome and Production of Bile
Acids:
Pathogen-associated molecular patterns (PAMPS) derived from the
gut may contribute towards liver inflammation (Roth et al., 2012).
PAMPS are molecules associated with groups of pathogens that by
TLR and other pattern recognition receptors (PRRS).PAMPS acitvate
immune response protect the host from infection (Maverakis et al.,
2015).
In absence of vitamin D the gut microbiome may change, resulting
in enhanced endotoxin exposure and TLR activation (Roth et al., 2012).
Vitamin D may induce antimicrobials and promote immune tolerance to
change the gut microbiome (Lucas et al., 2014). Other important
pathways regulated by vitamin D include the metabolism of bile acids by
gut microflora and hepatocytes. Bile acids are produced by hepatocytes
and stored in the gallbladder before release into the gut for fat digestion
(Lade et al., 2014). Bile acids share structural similarity with vitamin D
and regulate energy metabolism by interacting with bile acid receptors.
Loss in the expression of these receptors has been linked with
development of NAFLD (Lade et al., 2014). Vitamin D may directly
suppress the synthesis of bile acids by hepatocytes (Han et al., 2013). In
addition, changes to the gut microbiome driven by vitamin D deficiency
may alter the levels or type of bile acids present in the gut. Indeed, the
hepatotoxic bile acid, lithochloric acid, interacts with the vitamin D
receptor (VDR), which acts as bile acid receptor and targets the acid for
degradation .Activation of the VDR by other bile acids or active
1,25(OH)2D may enable the degradation of toxic bile acids in the gut and
protect from subsequent liver inflammation (Bouillon et al., 2014).
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
Vitamin D Suppresses Proinflammatory Cytokines and Mediators
of Oxidative Stress:
Increased lobular inflammation and expression of mRNAs
encoding the pro-inflammatory cytokines IL-1β, IL-4 and IL-6 was
detected in the livers of vitamin D-deficiency (Roth et al., 2012).
Oxidative stress pathways in the liver may also be regulated by vitamin
D, vitamin D administration increase the gene expression of antioxidant
defense molecules such as glutathione peroxidase and superoxide
dismutase in the livers (Roth et al., 2012). Furthermore, vitamin D
significantly reduced free radical liver peroxidation substances and
increased hepatic glucose uptake. These findings suggest that vitamin D
deficiency may exacerbate NAFLD at least in part via an inflammatorymediated pathway (George et al., 2012).

Vitamin D reduces liver fibrosis:
There is VDR in HSCs (Gascon-Barré et al., 2003). Vitamin D
treatment suppresses HSCs proliferation (Abramovitch et al., 2011). Also
it
down
regulates
pro-fibrotic
marker
tissue
inhibitors
of
metallopeptidase-1 (TIMP-1) and collagen type Ⅰproduction in HSCs
(Potter et al., 2013). VDR knockout mice develop spontaneous liver
injury with fibrosis (Ding et al., 2013).
HSC that obtained from patients with biopsy-proven NAFLD, liver
fibrosis was associated with increased fragmentation of the VDR protein.
Vitamin D suppressed the pro-fibrogenic activity of TGFβ from hepatic
stellate cells, modifying the expression of the SMAD2 protein (Beilfuss et
al., 2015). Other studies suggest that vitamin D may slow the
proliferation of hepatic stellate cells. Together, these observations
indicate that vitamin D may affect liver fibrosis controlled by HSC
through multiple mechanisms (Kwok et al., 2013).
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Transforming growth factor β1:
Transforming growth factor beta (TGF-β) is a multifunctional
cytokine, it has multiple profibrogenic but also anti-inflammatory and
immunosuppressive effects. The balance of these actions is required for
maintaining tissue homeostasis and an aberrant expression of TGF- β is
involved in a number of disease processes in the liver (Bissell et al.,
2001).
There are three major isoforms of TGF-β (TGF-β1, TGF-β2, and
TGF-β3), and TGF-β1 is the principal isoform implicated in liver fibrosis
(Inagaki and Okazaki, 2007). TGF-β1 plays a central role in liver
fibrogenesis. TGF-β1 is one of TGF-β isoforms, which arrests the cell
cycle in the G1 phase and elicits inhibition of cell proliferation and
triggering apoptosis (Pasche, 2001). Although it is a growth inhibitor, the
overexpression of hepatic TGF-β1 was found in HCC tissues and shown
to be correlated with carcinogenesis, progression and prognosis of HCC
(Okumoto et al., 2004). TGF-β1 promotes the transcription, synthesis and
secretion of numerous proteins of the extracellular matrix (ECM)
including collagens, fibronectin and proteoglycans. In addition to its
fibrogenic action leading
to
transdifferentiation of HSCS
into
myofibroblasts (Friedman, 2003).
HSCS activation is a key event in the natural process of wound
healing as well as in fibrosis development in liver. HSC are the most
important source of liver-derived TGF- β1 which exerts its function
through binding to specific receptors, including TGF-receptors I (TGFRI), TGF-RII and endoglin, TGF-β is also an important negative
regulator of proliferation and an inducer of apoptosis (Marja et al., 2005).
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As mentioned previously the mechanism of cell injury in NAFLD
involve excess fatty acids in the liver induces formation of free radicals,
which cause lipid peroxidation and induce proinflammatory cytokines
(Tolman et al., 2007). TGF-β1 is one of the cytokines secreted as a
response to cell injury and plays the dominant role in mediating fibrosis
(Friedman, 1999).
TGF-β1 stimulates extracellular matrix production not only by
HSCs but also by sinusoidal endothelial cells, however, its effect varies
from one condition to another. In the context of hepatic regeneration,
TGF-β1 is antiproliferative rather than pro-fibrogenic (Bissell, 2001).
Different reports have linked TGF-B1 to different pathological hepatic
states like cirrhosis and tumors (Hayashi and Sakai, 2012).
Mechanism of action of TGF-β1in NASH:
Following liver injury,TGF-β1, derived from both paracrine and
autocrine sources, binds to type I and type II serine/threonine receptor
kinases on the cell surface of HSCs (Inagaki and Okazaki, 2007).
Subsequently, its downstream effectors SMAD2 and SMAD3 are
phosphorylated and released into the cytosol, where they form a complex
with SMAD4. This SMAD complex can then translocate into the nucleus,
recognize SMAD-binding elements (SBE) on the genome, and directly
regulate target genes (Feng and Derynck, 2005). Thus, the TGFβ/SMAD transcriptional network in HSCs is responsible for anti fibrotic
strategies.
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Figure (4): Vitamin D may limit the progression of non-alcoholic fatty liver
disease through multiple, potentially interacting mechanisms
(Roth et al., 2012).
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