To investigate protective effects and molecular mechanisms of green tea polyphenols (GTP) on non-alcoholic fatty liver disease (NAFLD) in Zucker fatty (ZF) rats.

Male ZF rats were fed a high-fat diet (HFD) for 2 wk then treated with GTP (200 mg/kg) or saline (5 mL/kg) for 8 wk, with Zucker lean rat as their control. At the end of experiment, serum and liver tissue were collected for measurement of metabolic parameters, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), inflammatory cytokines and hepatic triglyceride and liver histology. Immunoblotting was used to detect phosphorylation of AMP-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC), and sterol regulatory element-binding protein 1c (SREBP1c).

Genetically obese ZF rats on a HFD presented with metabolic features of hepatic pathological changes comparable to human with NAFLD. GTP intervention decreased weight gain (10.1%, P = 0.052) and significantly lowered visceral fat (31.0%, P < 0.01). Compared with ZF-controls, GTP treatment significantly reduced fasting serum insulin, glucose and lipids levels. Reduction in serum ALT and AST levels (both P < 0.01) were observed in GTP-treated ZF rats. GTP treatment also attenuated the elevated TNFα and IL-6 in the circulation. The increased hepatic TG accumulation and cytoplasmic lipid droplet were attenuated by GTP treatment, associated with significantly increased expression of AMPK-Thr172 (P < 0.05) and phosphorylated ACC and SREBP1c (both P < 0.05), indicating diminished hepatic lipogenesis and triglycerides out flux from liver in GTP treated rats.

The protective effects of GTP against HFD-induced NAFLD in genetically obese ZF rats are positively correlated to reduction in hepatic lipogenesis through upregulating the AMPK pathway.

Non-alcoholic fatty liver disease (NAFLD), characterised by excess accumulation of fat in the liver, has a dramatically rising incidence parallel with the increasing population of metabolic syndrome worldwide[1,2]. NAFLD is emerging as a potential health burden in society because hepatic lipid accumulation can trigger hepatocyte damage, inflammation and fibrogenesis leading to more serious liver disorders such as nonalcoholic steatohepatitis (NASH), cirrhosis and hepatic cellular carcinoma (HCC)[3]. In addition, increased liver lipids [triglyceride (TG), diglyceride, and ceramides] exacerbate hepatic insulin resistance subsequently leading to type 2 diabetes and cardiovascular complications[4,5]. Therefore, identifying strategies to reduce excessive fat accumulation in the liver, especially to decrease hepatic TG, is critical for treatment of NAFLD and prevention of its potential consequences such as cardiometabolic disease.

Currently, no pharmacological therapy is approved for NAFLD. Several drug treatments for the management of NAFLD have been proposed, but none have shown considerable efficacy on the whole spectrum of liver damage[6]. Lifestyle interventions involving exercise and weight loss are the only accepted treatments for this disease but are often difficult to maintain for NAFLD sufferers. Thus, there is an urgent need to discover agents targeted at elevated hepatic lipids and safe for long-term administration.

Green tea (Camellia sinensis) contains natural catechins that is a heterogeneous class of polyphenolic compounds, mainly (-)-epigallocatechin-3-gallate (EGCG), with beneficial effects related to their antioxidant and anti-inflammatory properties[7]. Recently, green tea polyphenols (GTP) has received considerable attention for its metabolic effects against metabolic syndrome and type 2 diabetes[8]. Several lines of studies suggest that the anti-diabetic effects of GTP are probably due to suppressing appetite, modifying dietary fat emulsification in the gastrointestinal tract, inhibiting gastrointestinal lipolysis and reducing nutrient absorption[9-11]. Our previous studies have demonstrated that GTP has direct effects on glucose and lipid metabolism through enhancing glycogen synthesis and inhibiting lipogenesis in hepatocytes[12]. Based on the results of this in vitro study, we hypothesis that GTP can reduce hepatic lipid accumulation against fatty liver in vivo.

NAFLD is a complex metabolic disease. Many epidemiological studies have demonstrated that overnutrition or inappropriate diet leads to insulin resistance, dyslipidaemia and chronic inflammation, which significantly contribute to the development of NAFLD[13,14], in particular for those who have tendency for obesity genetically[15]. To evaluate effects of GTP on a rodent model that mimics most pathophysiological features of NAFLD in humans, Zucker fatty (ZF) rat, a genetic model of metabolic syndrome with obesity[16] was used in the study and fed with a high fat diet (HFD). There is increasing evidence that increased liver TG content is highly correlated to increased de novo lipogenesis[17]. AMP-activated protein kinase (AMPK) activity plays a vital role in mediating hepatic lipogenesis[18]. Recent studies have shown that AMPK activity is reduced by the factors implicated in the development of NAFLD, such as inflammation and obesity[19,20]. Therefore, inhibition of hepatic lipogenesis by AMPK activation is envisaged as a viable therapeutic strategy to prevent the initiation and progression of NAFLD[21]. Whether GTP improves both genetic and dietary factors implicated in NAFLD through activation of AMPK pathway, however, has not previously been addressed. In the current study, we tested the hypothesis that GTP would confer an effective therapy against obese- and HFD-induced NAFLD in rats through upregulating the AMPK pathway. Thus, protein molecules involved in AMPK-mediated hepatic lipogenesis were detected followed by a metabolic study in HFD-ZF rats with or without GTP treatment.

Hepatic steatosis is the first stage of NAFLD, which is mostly associated with increased de novo lipogenesis and obesity, however, it can progress to a more advanced form of the disease, NASH, hepatic necrosis, fibrosis and cirrhosis and hepatocellular carcinoma. Therefore, it is important to control NAFLD at the initial stage. Here, we report for the first time that GTP can protect against HFD-induced NAFLD in genetically obese rats. HFD-fed Zucker fatty rats recapitulated the key metabolic features seen in humans with NAFLD, including central obesity, hyperlipidemia, and hyperinsulinemia and elevated fasting glucose levels. Hepatic manifestations of NAFLD in the rats were evidenced by the elevated ALT and AST levels, increased lipids droplets and TG content in the liver. The parameters associated with obesity and metabolisms shown in Table 1 indicate that HFD-fed ZF rat is an optimal model of NAFLD to test preventive and therapeutic agents against NAFLD.

The key finding of this study is that GTP, as a natural agent, protected liver damage by HFD feeding and genetic obese, evidenced by improved hepatic enzymes (AST and ALT) levels and significantly lowered TG content in the liver of HFD-ZF rats. Results attained from histological analysis showed that GTP also markedly reduced lipid droplets in the liver of HFD-ZF rats. Elevated serum ALT and AST levels are the primary abnormality seen in patients with NAFLD and other chronic liver disease in human[23]. These finding indicate that GTP can protect hepatocytes against HFD- and obesity-induced hepatic steatosis. Traditionally, beneficial effects of GTP on the liver have been attributed to their antioxidant properties[8]. Accumulating evidence has revealed that NAFLD is strongly related to inflammation. For example, IL-6, as a proinflammatory cytokine, has been demonstrated as a potential mediator leading to NAFLD. TNFα has been implicated in liver fibrosis and advanced stages of NAFLD in human[24,25]. In this study, HFD fed obese rats presented systemic inflammation by markedly raised circulating TNFα and IL-6 levels. Although no change was found in anti-inflammatory cytokine IL-10, raised TNFα and IL-6 were attenuated by GTP treatment. This funding is consistent with a recent study in which Xiao et al[26] reported EGCG, a major active compound in GTP, against HFD-induced liver fibrosis associated with inflammation in rats through deregulation of TGF/SMAD, PI3 K/Akt/FoxO1, and NF-κB pathways. Taken together, intervention with GTP and EGCG can effectively prevent and reverse inflammation-mediated liver damage and fibrosis in rats, suggesting that GTP is beneficial in humans with NAFLD-induced by dietary factors and genetic obesity.

In addition to anti-inflammatory and antioxidant action, protective effects of GTP on NAFLD may be related to improved hyperinsulinemia and hyperlipidaemia. In NAFLD, there is excess of (free fatty acids) FFAs/NEFAs delivered to the liver due to increased adipose lipolysis under insulin resistant status, which lead to hepatic steatosis by over conversion of FFAs into TG and cause hepatocytes injury-induced by lipotoxicity[27]. In this study, serum NEFAs level was significantly higher in HFD-ZF rats than the lean control, whereas, GTP treatment significantly attenuated the elevated NEFAs levels, which in turn, could reduce substrate influx to the liver for hepatic TG synthesis and also may diminish unoxidized fatty acids accumulation, therefore, prevent lipoperoxidative stress-induced hepatic injury[27,28]. Furthermore, reduction of hepatic lipids in GTP treated rats may be also attributed for improved hyperglycemia. Consequently, glucose influx to the liver was reduced and ultimately decreased de novo lipogenesis (DNL) in the liver.

Hepatic lipogenesis is the metabolic pathway by which acetyl-CoA carboxylase (ACC) converts acetyl-CoA derived from glucose to fats, which involves sub-processes of fatty acid synthesis and subsequent triglyceride synthesis utilizing carbohydrate in the liver[17]. In healthy individuals, DNL contributes < 5% to liver TG content, whereas in individuals with NAFLD, DNL contributes > 26% of liver TG[17]. The significant increase (approximately five fold) in DNL suggests that this pathway may play an important role in the development of NAFLD. Thus, controlling hepatic DNL highlights a potential therapeutic utility for alleviating NAFLD and preventing its potential consequences (NASH and HCC). DNL is regulated by complex enzyme cascades/pathways. Among these, AMPK-mediated phosphorylation of target substrates plays a critical role in the regulation of hepatic lipogenesis[21]. The activity of AMPK is stimulated by insulin through promoting its phosphorylation at Thr172[29], subsequently, AMPK activation leads to phosphorylation of ACC1 at Ser79 and ACC2 at Ser221, causing a reduction in ACC activity that lowers malonyl-CoA from acetyl-CoA, which leads to decreased hepatic TG synthesis[30,31].

We have previously reported that GTP inhibits lipogenesis associated with enhanced expression of phosphorylated AMPKα and ACC proteins in hepatocytes[12]. To elucidate molecular mechanisms by which GTP improves hepatic steatosis in vivo, AMPK-ACC pathway was examined by immunoblotting analysis. We found that obese rats exposed to HFD expressed less AMPK Thr172 associated with reduced phosphorated ACC-Ser79 protein in the liver than the lean control rats. GTP intervention significantly enhanced protein expression of AMPK Thr172 and ACC Ser79 compared to saline-treated ZF rats, indicating that decreased hepatic TG and reversed liver histopathology are likely through diminishing hepatic lipogenesis in GTP-ZF rats. In addition to suppression of de novo lipogenesis, increased AMPK activity associated with inactivation of ACC by GTP may prevent hepatic injury by increasing fatty acid oxidation. Several lines of studies have demonstrated that reduction in malonyl-CoA secondary to AMPKα1-mediated ACC inactivation enhance carnitine palmitoyltransferase I (CPT I) activity, which in turn, increases in mitochondrial fatty acid oxidation by increasing CPT I flux[29,32]. Thus, by enhancing AMPK activation, reduced hepatic TG in GTP treated rats is due, at least in part, to suppression of hepatic lipogenesis and fatty acid oxidation in the liver through upregulating AMPK activation.

SREBP-1c is a key transcription factor regulating the gene expression of key enzymes implicated in hepatic lipogenesis and triglyceride-rich lipoprotein secretion[33]. In this study, SREBP1c levels were lower in HFD-ZF rats and GTP enhanced expression SREBP-1c, which is likely to reduce hepatic triglyceride biosynthesis and out flux. This finding indicates that GTP inhibited lipogenesis also through inactivation of SREBP-1c. This finding explained the molecular mechanism of decreased serum cholesterol and TG levels in GTP-treated ZF rats.

Significant reduction of serum ALT and AST levels and hepatic TG content in GTP-treated HFD ZF rats strongly supports that GTP can protect HFD-induced liver damage. Mechanisms are likely to include anti-inflammation and suppression de novo lipogenesis through enhancing AMPK activation and promoting fatty-acid oxidation thereby reducing hepatic lipotoxicity. In summary, we provide the first evidence that consistent administration of GTP protect genetically obese rodent against HFD induced hepatic steatosis. This protection was associated with increased activation of AMPK pathway to control hepatic de novo lipogenesis and hepatic TG secretion. Our results encourage further study into the effects and safety for GTP conferring protection against NAFLD in human.

We are grateful to Zuyi Lushen Kangyuan Co (Meitan, Guizhou, China) for supporting this study by providing GTP. The authors also would like to thank Dr Yali Sun’s advice for chemical analysis of GTP.