Dietary cholesterol facilitates
green tea-induced
hepatotoxicity
Prof. Oren Tirosh
The Institute of Biochemistry, Food Science
and Nutrition.
NAFLD/NASH
• Nonalcoholic steatohepatitis or NASH is a
common, often “silent” liver disease.
• The major feature in NASH is fat in the liver,
along with inflammation and damage.
• NASH affects 2 to 5 percent of Americans. An
additional 10 to 20 percent of Americans have
fat in their liver, but no inflammation or liver
damage.
NASH and CKD
• Nonalcoholic fatty liver disease (NAFLD) and
chronic kidney disease (CKD) share common
features.
• Chronic kidney disease was present in 24
(14%) of 174 NAFLD patients. The prevalence
of CKD was significantly higher in NASH
patients (19 of 92; 21%) than non-NASH
patients (5 of 82; 6%).
Metabolism. 2011 May;60(5):735-9. Nonalcoholic steatohepatitis
and increased risk of chronic kidney disease.
Yasui K1, et al.
Despite studies that show the
benefits of green tea, there have
been several recent reports that
demonstrated hepatotoxicity
following the consumption of
concentrated green tea extract.
[McKay and Blumberg, 2002].
Objective
To evaluate the effect of GTE in
high cholesterol diet-induced
NASH.
Experimental Design:
32 C57BL/6J mice 7 weeks old, were randomly divided into four
groups (n= 8) and fed the respective experimental diets for 6 weeks.
Group A - Normal Diet Control (ND): AIN-93G
Group B - Normal Diet (ND)+1% Polyphenols from Green tea
extract (GTE-purchased from Sigma) –ND+GTE
Group C - High Cholesterol+ Cholate Diet (HCD), 1% cholesterol+
0.5% Cholate
Group D - High Cholesterol Diet, 1% cholesterol+ 0.5% Cholate+ 1
% Green tea extract-HCD+GTE
** The mice were capped under controlled environment (22–24°C), with 12 hours light/dark
cycles and free access to food and water. All procedures were performed in accordance with
the institutions guidelines of animal care committee of the Hebrew University in Jerusalem.
6
HCD - induced liver damage is augmented
by GTE supplementation Bile acid level in
the serum was
higher compared
to other groups
Triglycerides & Cholesterol levels are increased in
the liver after HCD in the presence or absence of
GTE
Cholesterol accumulation in liver increases Triglycerides levels in liver are
as a result of HCD, compared to ND
significantly higher in HCD+GTE
groups. GTE significantly lower
group compared to ND+GTE group.
cholesterol levels.
Oxidative stress induced by GTE and cholesterol
iNOS levels is elevated in HCD+GTE only.
This finding confirms increased inflammation
caused by consumption of dietary cholesterol
combined with GTE. Suggesting potential liver
damage caused by GTE.
4-HNE expression, was significantly
higher in HCD+GTE . Supporting
the evidence of liver damage caused
by HCD+GTE
Inflammatory gene expression
Serum amyloids are
markers for inflammation
development
SAA1&SAA2 level significantly elevated in HCD+GTE. This finding confirms increased inflammation
caused by consumption of dietary cholesterol combined with GTE. Suggesting potential liver damage
Bile acid synthesis: Activation of the acidic pathway
and suppression of the classical pathway by GTE
FXR is the chief sensor of
intracellular levels of bile
acids, controlling their
synthesis and transport.
catalyzes the first and
rate-limiting step in the bile
acid synthesis pathway
SHP plays a key role in maintaining
cholesterol and bile acid homeostasis
by inhibiting cholesterol conversion to
bile acids.
sterol 27-hydroxylase
breaks down cholesterol to
form chenodeoxycholic
acid
Cholesterol
+ Cholic acid
intake
FXR
activation
CYP27a1
GTE
intake
SHP
?
Bile acids
synthesis
CYP7a1
CONCLUSION
In light of our results, we may suggest
that subjects diagnosed with fatty liver
should be taken into account of
avoiding the consumption of GTE
chronically.
Evaluation of the effect of GTE
supplements on NASH and CKD is
needed.
Acknowledgment
• Dr. Nina Hirsch
• Anya Konstantinov
• Prof. Zecharia Madar
• Prof. Zion Hagay
• Funding: Orion Foundation
The serum amyloid A (SAA) protein is an acute phase apolipoprotein reactant
produced mainly by hepatocytes and under regulation of inflammatory cytokines. The
SAA cleavage product, designated amyloid protein A (AA), is deposited systemically as
amyloid in vital organs including the liver, spleen, and kidneys in patients with chronic
inflammatory diseases
Serum amyloids are markers for inflammation development
15
Sterol 27-hydroxylase (CYP27A1) breaks down cholesterol to form a bile acid called
chenodeoxycholic acid. The formation of bile acids from cholesterol is the body's main
pathway for cholesterol removal. Sterol 27-hydroxylase plays a key role in maintaining
normal cholesterol levels in the body.
16
Figure 1
17
FIGURE 1. Bile acid biosynthetic pathways. Two major bile acid biosynthetic pathways
are shown. The classic pathway is the major bile acid synthetic pathway in the liver. In
this pathway, cholesterol is converted to 7a-hydroxycholesterol (7a-HOC) by the ratelimiting enzyme cholesterol 7a-hydroxylase (CYP7A1), which is located in the
endoplasmic reticulum. The sterol 12ahydroxylase (CYP8B1) converts the
intermediate 7a-hydroxy-4 cholesten-3-one (C4) to 7a,12a-dihydroxy-4-cholesten-3one,leading to the synthesis of cholic acid (CA). Without 12a hydroxylation by
CYP8B1, C4 is eventually converted to chenodeoxycholic acid (CDCA). The
mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes the steroid side-chain
oxidation in both CA and CDCA synthesis. In the alternative pathway, CYP27A1
converts cholesterol to 27-hydroxycholesterol (27-HOC), which eventually is converted
to CDCA. In mouse liver, most CDCA is converted to a and b muricholic acid
(MCA).MCA is only found in trace amount in humans. In the large intestine, bacterial
7-dehydroxylase removes a hydroxyl group from C-7 and converts CA to deoxycholic
acid (DCA) and converts CDCA to lithocholic acid (LCA). CYP3A1 and epimerases
also convert CDCA to the secondary bile acids, including hyocholic acid (HCA),
murideoxycholic acid (MDCA), v-muricholic acid (v-MCA), hyodeoxycholic acid
(HDCA) and ursodeoxycholic acid (UDCA). Most LCA and v-MCA are excreted into
feces.
18
19
Mechanisms of FXR regulation of enterohepatic circulation of bile acid. Bile acids
synthesized in the liver are excreted into bile via BSEP and stored in the gallbladder. After
each meal, bile acids are excreted into the intestinal tract. In the ileum, bile acids are
reabsorbed by ASBT in the brush border membrane. Bile acids activate FXR to induce IBABP
in enterocytes. OSTα/β transporter in the basolateral membrane effluxes bile acids to portal
circulation to hepatocytes where they are taken up by NTCP. In the liver, bile acids activate
FXR, which induces SHP expression. SHP then inhibits LRH-1 (or human FTF) and HNF4α
transactivation of CYP7A1 (FXR/SHP pathway 1). In the endocrine pathway, intestinal bile
acids activate FXR, which induces FGF19 expression. FGF19 may be transported to the liver
to activate a liver-specific receptor tyrosine kinase FGFR4 (FXR/FGF19/FGFR4 pathway 2). In
the autocrine pathway (pathway 3), cholestatic bile acids may activate FXR and
FGF19/FGFR4 signaling, which activates the MAPK/ERK1/2 pathway to inhibit CYP7A1
transcription. It is not clear how the FGF19/ERK1/2 pathway downregulates CYP7A1
transcription. The endocrine pathway may be a physiological pathway for bile acid inhibition
of bile acid synthesis, while the autocrine pathway may be an adaptive response to protect
liver from cholestatic injury. BARE-II contains 18 bp sequence of overlapping HNF4α and FTF
(α-fetoprotein transcription factor, a human homolog of mouse LRH-1) binding site, which is
completely conserved in all species.
20
Bile acids are nutrient signaling hormones
Steroids, Volume 86, 2014, 62 - 68
Fig. 3 Interrelationship between sphingosine 1-phosphate receptor 2 and the insulin signaling pathway in regulating hepatic nutrient
metabolism. S1PR2, sphingosine 1-phosphate receptor 2; Src, Src Kinase; EGFR, epidermal growth factor receptor; PPARα, pero...
Cholesterol excretion in feces.
Serum lipids
Green tea extract lowers triglycerides
level in mice that fed with normal diet.
TG level in mice that had received a diet
rich in cholesterol decreased
dramatically. Green tea extract led to a
similar effect as HCD alone
Cholesterol levels are obviously
significantly higher in groups
consuming HCD compared to ND.
GTE has no effect on cholesterol
levels in serum
Histopathological evidence of liver Injury
ND
ND+GTE
HCD
HCD+GTE
Insulin levels were significantly lower
in HCD and/or GTE supplementation
compared to ND
No significant effect
GTE led to decrease the insulin level with no effect on fasting
glucose level
HCD (high cholesterol diet) and GTE intake led to body
weight decrease compared to normal diet
Green Tea extract led to reduce the body weight of mice consuming Normal Diet.
Both HCD and HCD+GTE also decrease the body but with higher extent with green
tea indicating the effects of GTE on weight reduction.
SUMMARY
Dietary Cholesterol
Recent reports suggest that dietary
cholesterol could be a critical factor in
the development of steatohepatitis
[Subramanian et al, 2011].
Tea (Camellia sinensis) is a widely
consumed beverage worldwide.
Green tea has received considerable attention
as a strong antioxidant due to its high level of
polyphenols.