Elsevier

Nutrition

Volumes 55–56, November 2018, Pages 63-70
Nutrition

Basic nutritional investigation
Caffeic acid ameliorates hepatic steatosis and reduces ER stress in high fat diet–induced obese mice by regulating autophagy

https://doi.org/10.1016/j.nut.2018.03.010Get rights and content

Highlights

Abstract

Objective

Non-alcoholic fatty liver disease is characterized by high hepatic triacylglycerol contents, which is associated with endoplasmic reticulum (ER) stress and insulin resistance. Caffeic acid (CA) has antioxidant, immunomodulatory, and antiinflammatory effects. We investigated the effects of CA on hepatic steatosis and its mechanism of action.

Methods

We treated CA (50 µM) with AML12 cells. We categorized mice into three groups as follows: low-fat diet mice (LFD, n = 10), high-fat diet-induced obese mice (HFD, n = 10), and HFD fed with CA (50 mg/kg/d, n = 10) for 10 wk.

Results

CA did not cause any cytotoxic effect on AML12 cell line within the range of concentrations tested (0–200 µM). We found that CA (50 µM) treatment in palmitate-treated AML12 hepatocytes reduced lipid accumulation and lipogenesis markers, decreased ER stress, and increased autophagy markers. However, there was no significant difference in lipid droplets of palmitate-treated AML12 hepatocytes and CA-treated autophagy-related protein 7 deficiency AML12 hepatocytes with palmitate. Similarly, CA significantly lowered body and liver weights. Lipid accumulation in the liver decreased in the HFD + CA group compared with the HFD group. Glucose intolerance and insulin sensitivity also were markedly improved in the HFD + CA group. Moreover, the levels of ER stress markers were decreased in the livers of the HFD + CA group.

Conclusion

Autophagy markers were increased in the livers of the HFD + CA group. These results suggest that caffeic acid may ameliorate hepatic steatosis and decrease ER stress by increasing autophagy.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disorders [1], and it is related to insulin resistance, obesity, and metabolic syndrome [2]. Recent increases in the prevalence of obesity and diabetes have contributed to rapid increase in NAFLD globally. NAFLD has become a major public health problem because of its associated hepatic and extrahepatic morbidity and mortality [3]. Thus the control of fat accumulation in the liver is an important way to prevent or treat NAFLD. However, the number of effective therapeutic options for treating NAFLD is limited. Hepatic steatosis refers to the overaccumulation of triacylglycerol in hepatocytes. Triacylglycerol accumulation results from increased lipid delivery to the liver, increased de novo lipogenesis, reduced fatty acid oxidation, reduced lipolysis, and decreased lipid secretion [4]. A growing body of evidence indicates that endoplasmic reticulum (ER) stress and autophagy are associated with the development of hepatic steatosis [5], [6].

Several studies have reported that autophagy plays an important role in hepatic fatty acid metabolism [7], [8] and accumulated lipid droplets (LDs) decreased the expression of autophagy [7], [9]. Lipophagy is an autophagy that breaks down accumulated LDs and decomposes accumulated fat; the degraded fat is used as intracellular energy [7], [10]. A recent study found that autophagy/lipophagy degrade LDs. Also, autophagy is an important intracellular catabolic process that allows cell components such as damaged organelles and unfolded proteins to be degraded by lysosomes [11]. Because autophagy eliminates misfolded and unfolded proteins, its impairment could result in the induction of ER stress and aggravate hepatic steatosis [12].

ER stress disturbs hepatic lipid metabolism by regulating lipogenic gene expression and apolipoprotein secretion and by promoting insulin resistance [13]. ER stress also activates nuclear factor (NF) erythroid 2–related factor 2, c-Jun N-terminal kinase (JNK), and NF-κB pathways, which play important roles in inflammatory process. Therefore, targeting the regulation of autophagy-mediated ER stress might be a good therapeutic option for patients with NAFLD. A major cause of hepatic insulin resistance is ER stress in the liver [14]; in addition, ER stress induces hepatic fibrosis and inflammation [15]. It is mediated by a signaling pathway of several markers (such as pancreatic ER kinase, inositol-requiring enzyme 1 [IRE1], and activating transcription factor 6) in the ER membrane. First, ER stress activates IRE1 and JNK, which leads to apoptosis and inflammation. Phosphorylation of JNK induces insulin receptor substrate 1 (IRS-1) phosphorylation at serine 307 and reduces IRS-1 phosphorylation at tyrosine [16].

Caffeic acid (CA) is an organic compound classified as hydroxycinnamic acid, which is biosynthesized by the hydroxylation of coumaroyl ester of quinic acid. CA is an antioxidant in vitro and in vivo [17] and has immunomodulatory and antiinflammatory activities [18]. Recently, several studies have found that caffeine intake in humans and animals is inversely correlated with the severity of NAFLD and type 2 diabetes [19], [20], [21]. Although CA has been reported to improve inflammation and oxidative stress in chronic metabolic disease, the effects of CA on hepatocytes through the mechanism regarding ER stress and autophagy have not been investigated. The purpose of this study was to examine whether CA ingestion affects hepatic steatosis via regulating ER stress and autophagy in high-fat diet (HFD)–fed mice.

Section snippets

Cell culture

AML12 hepatocytes (ATCC, USA) were grown at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium/F12 (Gibco BRL, Grand Island, NY, USA) containing 10% fetal calf serum, 10 mL/L penicillin streptomycin (Invitrogen, Carlsbad, CA, USA), and ×100 insulin-transferrin-selenium (Gibco BRL).

CRISPR/Cas9 knockout plasmid transfection

The CRISPR/Cas9 system can be used to generate knockout cells by coexpressing a guide RNA specific to the target gene and the endonuclease Cas9. Autophagy-related protein 7.

(ATG7) CRISPR/Cas9 KO plasmid containing

Caffeic acid prevents palmitate-induced hepatic steatosis in AML12 hepatocytes

Hepatocytes (AML12) were treated with palmitate (250 µM) for 24 h. The cell viability assay indicated that caffeic acid had no significant difference the growth of AML12 cells in a dose-dependent manner (Fig. 1A). However, 50 µM caffeic acid reduced the lipid accumulation by 60% compared with palmitate (Fig. 1B). We examined the expression of fatty acid synthesis and transport genes and found that they were significantly reduced in CA-treated AML12 hepatocytes (Fig. 1C and D). This finding

Discussion

In the present study we found that CA protected against HFD-induced hepatic steatosis by regulating ER stress and autophagy. Furthermore, we found that CA-induced autophagy could reduce liver inflammation and regulate hepatic lipid metabolism in mice fed with HFD. Caffeic acid have been reported in metabolic syndrome and inflammatory disease [22], [23]. Caffeic acid and CA analog have antioxidant, antihypertensive, antiinflammatory, anticancer, and antibacterial activities. CA methyl ester

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2016 R1 A6 A3 A11933581).

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