Asiatic Acid Ameliorates Hepatic Lipid Accumulation and Insulin

Article pubs.acs.org/JAFC

Asiatic Acid Ameliorates Hepatic Lipid Accumulation and Insulin Resistance in Mice Consuming a High-Fat Diet Sheng-Lei Yan,†,∥ Hui-Ting Yang,#,∥ Yi-Ju Lee,§ Chun-Che Lin,○ Ming-Hui Chang,○ and Mei-Chin Yin*,#,⊥ †

Division of Gastroenterology, Department of Internal Medicine, Chang Bing Show-Chwan Memorial Hospital, Changhua County, Taiwan # Department of Nutrition, China Medical University, Taichung City, Taiwan § Department of Pathology, Chung Shan Medical University Hospital, Taichung City, Taiwan ○ Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung City, Taiwan ⊥ Department of Health and Nutrition Biotechnology, Asia University, Taichung City, Taiwan ABSTRACT: Effects of asiatic acid (AA) at 10 or 20 mg/kg/day upon hepatic steatosis in mice consuming a high-fat diet (HFD) were examined. AA intake decreased body weight, water intake, feed intake, epididymal fat, and plasma and hepatic triglyceride levels in HFD-treated mice (P < 0.05). HFD enhanced 2.85-fold acetyl coenzyme A carboxylase (ACC1), 3.34-fold fatty acid synthase (FAS), 3.71-fold stearoyl CoA desaturase (SCD)-1, 3.62-fold 3-hydroxy-3-methylglutaryl coenzyme A reductase, 2.91-fold sterol regulatory element-binding protein (SREBP)-1c, and 2.75-fold SREBP-2 expression in liver (P < 0.05). Compared with HFD groups, AA intake at two doses reduced 18.9−45.7% ACC1, 25.1−49.8% FAS, 24.7−57.1% SCD-1, and 21.8−53.3% SREBP-1c protein expression (P < 0.05). Histological results indicated AA intake at two doses reduced hepatic lipid accumulation and inflammatory infiltrate. HFD increased hepatic production of reactive oxygen species, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α, as well as decreased hepatic glutathione content and glutathione peroxidase and catalase activities (P < 0.05). AA intake at two doses reversed these alterations (P < 0.05). AA intake suppressed 32.4−58.8% nuclear factor kappa (NF-κ)B p65 and 24.2−56.7% p-p38 expression (P < 0.05) and at high dose down-regulated 29.1% NF-κB p50 and 40.7% p-JNK expression in livers from HFD-treated mice. AA intake at two doses lowered plasma insulin secretion and HOMR-IR (P < 0.05). These results suggest that AA is a potent hepatic protective agent against HFD-induced hepatic injury. KEYWORDS: asiatic acid, high-fat diet, hepatic steatosis, insulin resistance

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INTRODUCTION Hepatic steatosis, hyperlipidemia, and obesity due to lipid accumulation are risk factors associated with the prevalence of cardiovascular diseases and metabolic disorders,1,2 in which both oxidative and inflammatory reactions are involved.3 Enhanced lipogenesis is a major contributor to lipid accumulation in the liver and other organs. Thus, any agent with antilipogenic, antioxidative, and anti-inflammatory effects may improve lipid metabolism disorders and alleviate hepatic steatosis and related injury. Asiatic acid (AA, Figure 1) is a pentacyclic triterpene naturally occurring in many vegetables and fruits such as basil

(Ocimum basilicum), brown mustard (Brassica juncea), and centella (Centella asiatica L.).4,5 It has been reported that this triterpene could provide antioxidative and anti-inflammatory protection for mouse liver against D-galactosamine or lipopolysaccharide-induced acute liver injury.6,7 Wei et al.8 observed that AA intragastrical administration protected liver against ethanol-induced hepatotoxicity through attenuating oxidative stress. Pakdeechote et al.9 indicated that 3 weeks of AA intake at 10 or 20 mg/kg/day improved high-carbohydrate and high-fat diet induced metabolic abnormalities including glucose intolerance, hypertension, and circulating oxidative and inflammatory stress in rats. However, it remains unknown whether AA could suppress high-fat diet caused hepatic lipogenesis or oxidative and inflammatory injury. Acetyl coenzyme A carboxylase (ACC), fatty acid synthase (FAS), stearoyl CoA desaturase (SCD)-1, and 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase are lipogenic enzymes and involved in the biosynthesis of triglyceride and cholesterol in the liver and adipose tissue.10,11 Received: Revised: Accepted: Published:

Figure 1. Structure of AA. © 2014 American Chemical Society

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March 8, 2014 April 24, 2014 April 29, 2014 April 29, 2014 dx.doi.org/10.1021/jf501165z | J. Agric. Food Chem. 2014, 62, 4625−4631

Journal of Agricultural and Food Chemistry

Article

Table 1. Body Weight, Water Intake, Feed Intake, Liver Weight, and Epididymal Fat in Mice Treated with Normal Diet (ND), High-Fat Diet (HFD), or HFD with AA-Low or AA-High for 7 Weeksa body wt (g/mouse) water intake (mL/mouse/day) feed intake (g/mouse/day) liver wt (g/mouse) epididymal fat (g/mouse) a

ND

HFD

HFD + AA-low

HFD + AA-high

24.6 ± 1.1a 2.0 ± 0.4a 1.5 ± 0.3a 1.28 ± 0.13a 0.24 ± 0.05a

36.4 ± 1.7d 5.8 ± 0.6c 5.3 ± 0.8d 2.16 ± 0.23c 1.41 ± 0.15d

32.8 ± 0.9c 4.7 ± 0.3b 4.5 ± 0.4c 2.01 ± 0.14c 1.12 ± 0.10c

28.5 ± 1.2b 4.5 ± 0.5b 3.8 ± 0.5b 1.74 ± 0.18b 0.72 ± 0.09b

Values are means ± SD, n = 10. Means in a row without a common letter differ, P < 0.05. determined by commercial kits (Randox Laboratories Ltd., Crumlin, UK). Plasma levels of glucose (mmol/L) and insulin (nmol/L) were measured by using a glucose HK kit (Sigma Chemical Co., St. Louis, MO, USA) and mouse ultrasensitive ELISA kit (DRG Instruments GmbH, Marburg, Germany), respectively. Insulin resistance, expressed as homeostasis model assessment-insulin resistance (HOMA-IR), was calculated via the formula [glucose (mmol/L) × insulin (mU/L)]/ 22.5. Plasma leptin and adiponectin levels were measured using a Mouse Leptin Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) and a Rat/mouse Adiponectin ELISA kit (Phoenix Europe GmbH, Karlsruhe, Germany), respectively. Plasma immunoreactive ghrelin concentration was measured using a radioimmunoassay kit (Phoenix Pharmaceuticals, Belmont, CA, USA). Hepatic TG and TC. Liver homogenate, 1 mL, was mixed with 2.5 mL of chloroform/methanol (2:1, v/v). The chloroform layer was collected and concentrated. After mixing with 10% Triton X-100 in isopropanol, the sample was assayed by using Wako Triglyceride ETest and Total Cholesterol E-Test kits (Wako Pure Chemical). Fecal Lipid Analysis. Feces, at 0.5 g, were mixed with 3.5 mL of deionized water. After sitting at 4 °C overnight, feces were homogenized by vortexing. The fecal lipid was extracted with methanol/chloroform (2:1, v/v) using a method described in Tsujita et al.18 The lipophilic layer was collected and dried under a nitrogen stream. Measurement of Oxidative and Inflammatory Factors in Liver. Liver tissue was homogenized with cold phosphate-buffered saline containing 0.05% Tween 20 and 1 mM EDTA. After centrifuging, the supernatants were used for measurements. Sample was mixed with 25 mM 2′,7′-dichlorofluorescein diacetate. After 30 min of incubation at room temperature, reactive oxygen species (ROS) level was determined by monitoring fluorescence change at an excitation wavelength of 488 nm and an emission wavelength of 515 nm. GSH content was measured by using a commercial kit (OxisResearch, Portland, OR, USA). The activities of GPX and catalase were assayed by GPX and catalase kits (Calbiochem, EMD Biosciences, Inc., San Diego, CA, USA). Levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were determined using ELISA kits (R&D Systems). Western Blot Analysis. Liver tissue was homogenized in buffer containing 0.5% Triton X-100 and protease-inhibitor cocktail (1:1000, Sigma-Aldrich Chemical Co.). This homogenate was further mixed with buffer (60 mM Tris-HCl, 2% SDS, and 2% β-mercaptoethanol, pH 7.2) and boiled for 5 min. Sample at 40 μg protein was applied to 10% SDS−polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) for 1 h. After blocking with a solution containing 5% nonfat milk for 1 h to prevent nonspecific binding of antibody, membrane was incubated with mouse anti-ACC1, anti-FAS, anti-SCD-1 (1:1000), anti-HMG-CoA reductase, anti-SREBP-1c, anti-SREBP-2 (1:2000), and anti-NF-κB and antiMAPK (1:1000) monoclonal antibodies (Boehringer-Mannheim, Indianapolis, IN, USA) at 4 °C overnight and followed by reaction with horseradish peroxidase-conjugated antibody for 3.5 h at room temperature. The detected bands were quantified by an image analyzer (ATTO, Tokyo, Japan), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The blot was quantified by densitometric analysis. Results were normalized to GAPDH and given as arbitrary units (AU).

Sterol regulatory element-binding protein (SREBP)-1c and SREBP-2 are important transcription factors responsible for the expression of genes encoded for fatty acid and cholesterol biosynthesis, respectively.12,13 In addition, hormones such as insulin, leptin, and adiponectin also affect lipid metabolism. Abnormal levels of these hormones in circulation due to a highfat diet enhance lipogenesis.14 So far, less information is available regarding the influence of AA upon these lipogenic enzymes, SREPBs, and hormones. It is reported that a high-fat diet promoted hepatic oxidative and inflammatory stress by activating nuclear factor kappa (NF-κ)B and mitogen-activated protein kinase (MAPK) pathways.15,16 Our previous study found that a high-fat diet decreased hepatic glutathione (GSH) content, lowered catalase and glutathione peroxidase (GPx) activities, and increased cytokine release.17 If AA could alleviate high-fat diet caused oxidative and inflammatory injury, it may protect the liver against the development of steatohepatitis. The major purpose of this study was to investigate the effects of AA at 10 or 20 mg/kg/day on lipid deposit and protein expression of lipogenic enzymes and SREBPs in livers from high-fat diet treated mice. The influence of this triterpene upon hepatic oxidative and inflammatory stress, as well as the variation of insulin, leptin, adiponectin, and ghrelin in circulation, was also evaluated.

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MATERIALS AND METHODS

Materials. Asiatic acid (AA, 98%) was purchased from SigmaAldrich Co. (St. Louis, MO, USA). High-fat diet (HFD) containing 60% of calories as fat was purchased from Research Diets Co. (New Brunswick, NJ, USA), in which saturated fat and monounsaturated fat were 55 and 35% of total fat, respectively. Animals and Diets. Male 3-week-old C57BL/6 mice were obtained from the National Laboratory Animal Center (National Science Council, Taipei City, Taiwan). Use of the mice was reviewed and approved by the China Medical University animal care committee. After 1 week of acclimation, mice were used for experiments. Experimental Design. Mice were divided into four groups: normal group (normal diet); HFD group (high-fat diet); AA-low group (high-fat diet plus AA at 10 mg/kg/day); AA-high group (highfat diet plus AA at 20 mg/kg/day). AA, suspended in 1.2% methyl cellulose (MC), was administered daily by oral gavage. After 7 weeks, mice were killed with carbon dioxide. Blood, liver, and epididymal fat from each mouse were collected and weighed. Protein concentration of tissue homogenate was determined by a commercial assay kit (Pierce Biotechnology Inc., Rockford, IL, USA) with bovine serum albumin as standard. Our preliminary study found that there was no significant difference in all measurements between mice consuming the normal diet and normal diet plus AA-high or between mice consuming the high-fat diet and the high-fat diet plus vehicle (1.2% MC). Thus, two groups, normal diet plus AA-high and high-fat diet plus vehicle were omitted in the present study. Blood Analysis. Triglyceride (TG) and total cholesterol (TC) levels in plasma were assayed by kits purchased from Wako Pure Chemical Co. (Osaka, Japan). Plasma activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was 4626

dx.doi.org/10.1021/jf501165z | J. Agric. Food Chem. 2014, 62, 4625−4631


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Table 2. Plasma Levels of ALT, AST, TG, and TC, Hepatic Levels of TG and TC, and Fecal Lipid Level in Mice Treated with Normal Diet (ND), High-Fat Diet (HFD), or HFD with AA-Low or AA-High for 7 Weeksa ND

HFD

HFD + AA-low

HFD + AA-high

ALT (U/L) AST (U/L) TG (g/L) TC (g/L)

33 ± 3a 36 ± 4a 2.24 ± 0.08a 1.28 ± 0.05a

118 ± 10d 104 ± 8d 5.76 ± 0.21d 3.90 ± 0.11c

91 ± 5c 82 ± 7c 5.01 ± 0.13c 3.56 ± 0.06c

60 ± 6b 51 ± 4b 3.98 ± 0.10b 2.94 ± 0.07b

TG (mg/g wet wt) TC (mg/g wet wt)

25.9 ± 1.2a 2.8 ± 0.2a

64.5 ± 2.5d 7.6 ± 0.4c

51.1 ± 1.6c 7.1 ± 0.3c

38.2 ± 0.9b 6.4 ± 0.2b

lipid (mg/g feces)

8.3 ± 0.5a

13.6 ± 0.4b

14.0 ± 0.6b

15.7 ± 0.5c

plasma

hepatic

fecal a

Values are means ± SD, n = 10. Means in a row without a common letter differ, P < 0.05.

Figure 2. Hepatic protein expression of ACC1, FAS, SCD-1, HMG-CoA reductase, SREBP-1c, and SREBP-2 in mice treated with normal diet (ND), high-fat diet (HFD), or HFD with AA-low or AA-high for 7 weeks. Values are means ± SD, n = 10. Means among bars without a common letter differ, P < 0.05.

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Histological Analysis. Partial liver tissue from each mouse was fixed in 10% phosphate-buffered formalin and embedded in paraffin. Paraffin section at 5 μm thickness was cut and stained with Oil Red O (ORO) stain and hematoxylin-eosin (H&E) stain, and followed by examination under a light microscope for histological analysis. Statistical Analysis. The effect of each treatment was analyzed from 10 mice (n = 10) in each group. All data were expressed as the mean ± standard deviation (SD). Statistical analysis was done using one-way analysis of variance, and post-hoc comparisons were carried out using Dunnet’s t test. Statistical significance was considered at P < 0.05.

RESULTS

Effects of AA on Body Weight. HFD increased mouse body weight, water intake, feed intake, liver weight, and epididymal fat (Table 1, P < 0.05). Compared with HFD groups, AA intake at low and high doses decreased body weight, water intake, feed intake, and epididymal fat in HFDtreated mice (P < 0.05). AA intake only at high dose lowered liver weight of mice treated with HFD (P < 0.05). 4627



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Figure 3. Effects of AA upon hepatic lipid accumulation (upper row), determined by ORO statin; hepatic inflammation (lower row), determined by H&E stain in mice treated with normal diet (ND), high-fat diet (HFD), or HFD with AA-low or AA-high for 7 weeks. Magnification: 200×.

Table 3. Plasma Level of Glucose, Insulin, HOMA-IR, Leptin, Adiponectin, and Ghrelin in Mice Treated with Normal Diet (ND), High Fat Diet (HFD), or HFD with AA-Low or AA-High for 7 Weeksa glucose (mmol/L) insulin (nmol/L) HOMA-IR leptin (ng/mL) adiponectin (μg/mL) ghrelin (fmol/mL) a

ND

HFD

HFD + AA-low

HFD + AA-high

7.2 ± 0.3a 14.2 ± 0.6a 4.3 ± 0.8a 1.21 ± 0.20a 8.3 ± 0.5b 152 ± 13b

7.8 ± 0.5a 25.8 ± 1.8d 33.4 ± 2.1d 2.19 ± 0.17b 4.7 ± 0.2a 96 ± 7a

7.6 ± 0.2a 22.5 ± 1.1c 26.1 ± 1.7c 2.10 ± 0.11b 4.8 ± 0.4a 101 ± 6a

7.5 ± 0.4a 17.8 ± 0.9b 15.0 ± 1.0b 1.98 ± 0.08b 5.1 ± 4a 105 ± 9a


Table 4. Hepatic Levels of ROS and GSH, Activities of Catalase and GPX, and Levels of IL-1β, IL-6, and TNF-α in Mice Treated with Normal Diet (ND), High-Fat Diet (HFD), or HFD with AA-Low or AA-High for 7 Weeksa

a

ND

HFD

HFD + AA-low

± ± ± ±

1.30 ± 0.12d 7.3 ± 0.4a 12.5 ± 0.3a 11.3 ± 0.4a

1.02 ± 0.10c 8.4 ± 0.5b 14.1 ± 0.4b 12.5 ± 0.6b

0.66 10.1 15.5 14.3

108 ± 10d 94 ± 7d 103 ± 11d

87 ± 8c 77 ± 5c 85 ± 9c

60 ± 5b 52 ± 4b 56 ± 5b

ROS (RFU/mg protein) GSH (nmol/mg protein) GPX (U/mg protein) catalase (U/mg protein)

0.26 12.4 18.6 17.1

0.04a 0.6d 0.5d 0.7d

IL-1β (pg/mg protein) IL-6 (pg/mg protein) TNF-α (pg/mg protein)

19 ± 3a 15 ± 2a 18 ± 4a

HFD + AA-high ± ± ± ±

0.08b 0.7c 0.5c 0.6c


Effects of AA on Lipid Levels. HFD raised plasma ALT, AST, TG, and TC levels; hepatic TG and TC levels; and fecal lipid content (Table 2, P < 0.05). Compared with HFD groups, AA intake at two doses decreased plasma ALT, 22.9−48.5%; AST, 21.2−50.1%; and TG levels, 12.4−30.4%; and lowered hepatic TG level, 20.8−41.3% (P < 0.05). Plasma TC level and hepatic TC level were reduced by AA intake at high dose (P < 0.05). AA intake only at high dose increased fecal lipid content (P < 0.05). Effects of AA on Hepatic Lipogenesis. As shown in Figure 2, HFD enhanced 2.85-fold ACC1, 3.34-fold FAS, 3.71fold SCD-1, 3.62-fold HMG-CoA reductase, 2.91-fold SREBP1c, and 2.75-fold SREBP-2 expression in liver (P < 0.05). Compared with HFD groups, AA intake at two doses reduced ACC1, 18.9−45.7%; FAS, 25.1−49.8%; SCD-1, 24.7−57.1%; and SREBP-1c, 21.8−53.3%, protein expression (P < 0.05). AA

intake did not affect hepatic protein expression of HMG-CoA reductase and SREBP-2 (P > 0.05). As shown in Figure 3, HFD led to a great amount of fat deposition in liver, determined by ORO stain (upper row), and caused obvious foci of inflammatory cell infiltration in liver, determined by H&E stain (lower row). AA intake at two doses decreased hepatic lipid droplets and inflammatory infiltration. HFD increased plasma levels of insulin, leptin, and HOMA-IR and decreased adiponectin and ghrelin levels (Table 3, P < 0.05). AA intake at two doses lowered plasma insulin secretion and HOMR-IR (P < 0.05) but did not affect plasma glucose, leptin, ghrelin, or adiponectin levels (P > 0.05). Effects of AA on Hepatic Oxidative and Inflammatory Stress. HFD increased hepatic production of ROS, IL-1β, IL-6, and TNF-α as well as decreased hepatic GSH content and GPX and catalase activities (Table 4, P < 0.05). Compared with HFD 4628



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Figure 4. Hepatic protein expression of NF-κB and MAPK in mice treated with normal diet (ND), high-fat diet (HFD), or HFD with AA-low or AA-high for 7 weeks. Values are means ± SD, n = 10. Means among bars without a common letter differ, P < 0.05.

that AA could protect liver via antilipogenic, antioxidative, and anti-inflammatory activities to improve high-fat diet caused injury. ACC1 mediates the initial step of fatty acid synthesis via converting acetyl-CoA to malonyl-CoA.11 FAS is responsible for the last step in fatty acid biosynthesis and plays a determinant role for hepatic fatty acid generation in de novo lipogenesis.10 SCD-1 catalyzes the rate-limiting step in the cellular biosynthesis of monounsaturated fatty acids, primarily oleate and palmitoleate, which could be incorporated into and stored as triglyceride in liver.19 SREBP-1c is the major upstream transcription factor involved in hepatic triglyceride biosynthesis and modulates the expression of downstream targets including FAS, ACC1, and SCD-1.20−22 Thus, the suppression on the expression of these enzymes and SREBP-1c could inhibit hepatic lipogenesis and lower fat accumulation in liver. Our present study found that AA treatments effectively downregulated hepatic expression of ACC1, FAS, SCD-1, and SREBP-1c, which subsequently decreased triglyceride biosynthesis and deposit in liver, adipose tissue, and circulation and finally attenuated hepatic steatosis and lowered body weight. These findings indicated that AA was an effective agent against triglyceride biosynthesis. Obviously, the antilipogenic activities of AA could be mainly ascribed to its suppression upon hepatic SREBP-1c, an upstream lipogenic factor, and the less available SREBP-1c subsequently declined the expression of downstream

groups, AA intake at two doses lowered hepatic ROS, 21.5− 49.2%; IL-1β, 19.4−44.3%; IL-6, 18.1−44.7%; and TNF-α, 17.5−45.6%, levels but retained 15.1−38.4% GSH content and maintained 12.8−24.1% GPX and 10.6−26.5% catalase activities (P < 0.05). As shown in Figure 4, HFD up-regulated hepatic NF-κB and MAPK expression (P < 0.05). AA intake dose-dependently suppressed NF-κB p65, 32.4−58.8%, and pp38, 24.2−56.7%, expression (P < 0.05) and at high dose down-regulated NF-κB p50, 29.1%, and p-JNK, 40.7%, expression. AA intake did not affect ERK1/2 and p-ERK1/2 expression (P > 0.05).

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DISCUSSION The high-fat diet we used enhanced hepatic protein expression of lipogenic enzymes and factors including ACC1, FAS, SCD-1, HMG-CoA reductase, SREBP-1c, and SREBP-2, which clearly explained the observed lipid accumulation in liver, circulation, and epididymal white adipose tissue. We further found that the intake of AA markedly attenuated high-fat diet caused hyperlipidemia, hepatic steatosis, and epididymal fat deposit. Our Western blot data revealed that AA intake effectively suppressed hepatic expression of ACC1, FAS, SCD-1, and SREBP-1c. Our histological data indicated that AA intake reduced fat deposits in hepatocytes. Furthermore, AA intake alleviated high-fat diet induced insulin resistance and hepatic oxidative and inflammatory stress. These findings supported 4629



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absorbed and exerted its hepatic protection through regulating hepatic NF-κB and MAPK pathways. AA is a triterpene naturally occurring in many plant foods and herbs. Ramachandran and Saravanan32 reported that AA intake at 10 or 20 mg/kg body weight altered hepatic enzyme activities of carbohydrate metabolism in diabetic rats. In our present study, AA intake also at 10 or 20 mg/kg body weight markedly protected THE liver against high-fat diet induced injury in mice. On the basis of its natural property and support from other studies and our data, the application of this compound seems safe. The doses used in our present study were approximately equal to 0.7 or 1.4 g/day for a 70 kg man. Our previous study found that gynura (Gynura bicolor DC), brown mustard (Brassica juncea), and daylily (Hemerocallis fulva L.) contained AA in the range of 55−102 mg/g dry weight.4 Thus, the consumption of these vegetables, rich in AA, might also benefit the prevention of diet-induced lipid disorders. On the other hand, we noted that AA intake lowered mouse feed intake. It seems that this agent, a tasteless compound, was able to affect appetite and contributed to decreased body weight. In conclusion, high-fat diet caused hyperlipidemia, hepatic steatosis, and insulin resistance. The intake of asiatic acid markedly lowered lipid accumulation in the circulation, liver, and adipose tissue via suppressing the protein expression of ACC1, FAS, SCD-1, and SREBP-1c. This triterpene also improved insulin resistance and attenuated hepatic oxidative and inflammatory injury. These results suggest that asiatic acid is a potent hepatic protective agent against high-fat diet induced steatohepatitis.

factors, ACC1, FAS, and SCD-1 in the liver. On the other hand, HMG-CoA reductase, regulated by SREBP-2, is responsible for cholesterol biosynthesis.23 Our present study found AA intake did not affect hepatic SREBP-2 and HMG-CoA reductase expression. SREBP-1c is produced from a single gene, SREBF-1, located on chromosome 17p11.2, but SREBP-2 is generated from another gene, SREBF-2, located on chromosome 22q13.24 It is speculated that AA had less affinity to chromosome 22q13 and/or less interference with gene SREBF-2, which caused failure in regulating SREBP-2 in HFD-treated mice liver. However, it is interesting to find that AA at high dose lowered hepatic and circulating cholesterol levels. It is likely that AA at high dose mildly mediated other factor(s) involved in cholesterol biosynthesis. These results implied that AA was a weak inhibitor against cholesterol biosynthesis. In addition, our data revealed AA intake at high dose increased fecal lipid content. Thus, the lipid-lowering effect of this triterpene was slightly due to an increase in lipid excretion. Other studies25,26 and our present study found that a high-fat diet altered circulating levels of several hormones. Our data indicated that AA intake substantially alleviated hyperinsulinemia. It is highly possible that this triterpene suppressed hepatic lipogenesis and reduced lipid accumulation in liver and adipose tissue, which in turn restored insulin sensitivity and lowered the requirement for insulin. The reduction of HOMAIR in AA-treated mice we observed also agreed with this agent improving high-fat diet induced insulin resistance. In addition, a high-fat diet stimulates the release of inflammatory cytokines such as IL-1β and TNF-α, which blocks adipocyte insulin action and contributes to the development of obesity-related insulin resistance.27,28 Thus, the improved insulin resistance in AA-treated mice could partially result from the diminished hepatic release of these inflammatory cytokines. AA treatments did not affect high-fat diet induced hyper-leptinemia, hypoadiponectinemia, and hypo-ghrelinemia in our present study. Obviously, AA was not able to mediate these hormones. It is reported that excessive energy from a high-fat diet promoted oxidative enzyme activities, disturbed tricarboxylic acid cycle electron transport chain, and impaired mitochondrial respiratory capability in liver and other organs such as the heart, which led to the overproduction of ROS and enhanced oxidative stress and consequently activated MAPK and NF-κB pathways.29,30 The activation of these two pathways further augmented hepatic oxidative and inflammatory stress and contributed to the progression of liver disorders including steatohepatitis.30,31 The results of our present study agreed with those previous studies because our molecular analyses data revealed that a high-fat diet enhanced hepatic expression of NFκB p50, NF-κB p65, p38, JNK, and ERK1/2, which in turn evoked the generation of ROS and inflammatory cytokines including IL-6 and TNF-α. Furthermore, we found that AA intake limited phosphorylation of NF-κB p50, NF-κB p65, p38, and JNK in liver, which subsequently decreased the activation of NF-κB and MAPK. Because both NF-κB and MAPK pathways have been suppressed, the lower formation of ROS and inflammatory cytokines in livers from AA-treated mice could be explained. Our histological results indicated that AA intake ameliorated lobular inflammation and diffuse ballooning degeneration in hepatocytes. The restored antioxidative defenses such as GPX and catalase activities and lowered plasma ALT and AST levels in AA-treated mice also indicated that hepatic oxidative and inflammatory stress had been mitigated. These findings showed that dietary AA could be

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AUTHOR INFORMATION

Corresponding Author

*(M.-C.Y.) Mail: Department of Nutrition, China Medical University, 91 Hsueh-shih Road, Taichung City, Taiwan. Phone: 886-4-22053366, ext. 7510. Fax: 886-4-22062891. Email: [email protected]. Author Contributions ∥

S.-L.Y. and H-T.Y. contributed equally to this study.

Notes

The authors declare no competing financial interest.

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REFERENCES

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Asiatic Acid Ameliorates Hepatic Lipid Accumulation and Insulin

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