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%).

steatohepatitis Nonalcoholic . 9-735):5(60May;2011 Metabolism.

and increased risk of chronic kidney disease.

et al. ,1K Yasui

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.

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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.

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

as a result of HCD, compared to ND

groups. GTE significantly lower

cholesterol levels.

Triglycerides levels in liver are

significantly higher in HCD+GTE

group compared to ND+GTE group.

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

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

caused by GTE.

Serum amyloids are markers for inflammation development

Bile acid synthesis: Activation of the acidic pathway

and suppression of the classical pathway by GTE

catalyzes the first and

rate-limiting step in the bile

acid synthesis pathway

FXR is the chief sensor of

intracellular levels of bile

acids, controlling their

synthesis and transport.

sterol 27-hydroxylase

breaks down cholesterol to

form chenodeoxycholic

acid

SHP plays a key role in maintaining cholesterol and bile acid homeostasis by inhibiting cholesterol conversion to bile acids.

Cholesterol + Cholic acid

intake

CYP27a1

GTE intake

FXR activation

SHP CYP7a1

Bile acids synthesis

?

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

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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

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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.

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Figure 1

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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 rate-

limiting 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-3-

one,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.

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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.

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...

Bile acids are nutrient signaling hormones

Steroids, Volume 86, 2014, 62 - 68

Cholesterol excretion in feces.

Serum lipids

Cholesterol levels are obviously

significantly higher in groups

consuming HCD compared to ND.

GTE has no effect on cholesterol

levels in serum

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

ND+GTE

HCD

HCD+GTE

Histopathological evidence of liver Injury

ND

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.