asiatic acid ameliorates hepatic lipid accumulation and insulin resistance in mice consuming a...

Asiatic Acid Ameliorates Hepatic Lipid Accumulation and InsulinResistance in Mice Consuming a High-Fat DietSheng-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 hepatictriglyceride levels in HFD-treated mice (P < 0.05). HFD enhanced 2.85-fold acetyl coenzyme A carboxylase (ACC1), 3.34-foldfatty acid synthase (FAS), 3.71-fold stearoyl CoA desaturase (SCD)-1, 3.62-fold 3-hydroxy-3-methylglutaryl coenzyme Areductase, 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, and21.8−53.3% SREBP-1c protein expression (P < 0.05). Histological results indicated AA intake at two doses reduced hepatic lipidaccumulation 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-JNKexpression 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

■ INTRODUCTION

Hepatic steatosis, hyperlipidemia, and obesity due to lipidaccumulation are risk factors associated with the prevalence ofcardiovascular diseases and metabolic disorders,1,2 in whichboth oxidative and inflammatory reactions are involved.3

Enhanced lipogenesis is a major contributor to lipidaccumulation in the liver and other organs. Thus, any agentwith antilipogenic, antioxidative, and anti-inflammatory effectsmay improve lipid metabolism disorders and alleviate hepaticsteatosis 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), andcentella (Centella asiatica L.).4,5 It has been reported that thistriterpene could provide antioxidative and anti-inflammatoryprotection for mouse liver against D-galactosamine or lip-opolysaccharide-induced acute liver injury.6,7 Wei et al.8

observed that AA intragastrical administration protected liveragainst ethanol-induced hepatotoxicity through attenuatingoxidative stress. Pakdeechote et al.9 indicated that 3 weeks ofAA intake at 10 or 20 mg/kg/day improved high-carbohydrateand high-fat diet induced metabolic abnormalities includingglucose intolerance, hypertension, and circulating oxidative andinflammatory stress in rats. However, it remains unknownwhether AA could suppress high-fat diet caused hepaticlipogenesis or oxidative and inflammatory injury.Acetyl coenzyme A carboxylase (ACC), fatty acid synthase

(FAS), stearoyl CoA desaturase (SCD)-1, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase arelipogenic enzymes and involved in the biosynthesis oftriglyceride and cholesterol in the liver and adipose tissue.10,11

Received: March 8, 2014Revised: April 24, 2014Accepted: April 29, 2014Published: April 29, 2014

Figure 1. Structure of AA.

Article

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

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Sterol regulatory element-binding protein (SREBP)-1c andSREBP-2 are important transcription factors responsible for theexpression of genes encoded for fatty acid and cholesterolbiosynthesis, respectively.12,13 In addition, hormones such asinsulin, leptin, and adiponectin also affect lipid metabolism.Abnormal levels of these hormones in circulation due to a high-fat diet enhance lipogenesis.14 So far, less information isavailable regarding the influence of AA upon these lipogenicenzymes, SREPBs, and hormones. It is reported that a high-fatdiet promoted hepatic oxidative and inflammatory stress byactivating nuclear factor kappa (NF-κ)B and mitogen-activatedprotein kinase (MAPK) pathways.15,16 Our previous studyfound 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 alleviatehigh-fat diet caused oxidative and inflammatory injury, it mayprotect 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 proteinexpression of lipogenic enzymes and SREBPs in livers fromhigh-fat diet treated mice. The influence of this triterpene uponhepatic oxidative and inflammatory stress, as well as thevariation of insulin, leptin, adiponectin, and ghrelin incirculation, was also evaluated.

■ MATERIALS AND METHODSMaterials. Asiatic acid (AA, 98%) was purchased from Sigma-

Aldrich Co. (St. Louis, MO, USA). High-fat diet (HFD) containing60% of calories as fat was purchased from Research Diets Co. (NewBrunswick, NJ, USA), in which saturated fat and monounsaturated fatwere 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 (NationalScience Council, Taipei City, Taiwan). Use of the mice was reviewedand 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-lowgroup (high-fat diet plus AA at 10 mg/kg/day); AA-high group (high-fat diet plus AA at 20 mg/kg/day). AA, suspended in 1.2% methylcellulose (MC), was administered daily by oral gavage. After 7 weeks,mice were killed with carbon dioxide. Blood, liver, and epididymal fatfrom each mouse were collected and weighed. Protein concentrationof tissue homogenate was determined by a commercial assay kit(Pierce Biotechnology Inc., Rockford, IL, USA) with bovine serumalbumin as standard. Our preliminary study found that there was nosignificant difference in all measurements between mice consuming thenormal diet and normal diet plus AA-high or between mice consumingthe 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 vehiclewere omitted in the present study.Blood Analysis. Triglyceride (TG) and total cholesterol (TC)

levels in plasma were assayed by kits purchased from Wako PureChemical Co. (Osaka, Japan). Plasma activity of alanine amino-transferase (ALT) and aspartate aminotransferase (AST) was

determined by commercial kits (Randox Laboratories Ltd., Crumlin,UK). Plasma levels of glucose (mmol/L) and insulin (nmol/L) weremeasured by using a glucose HK kit (Sigma Chemical Co., St. Louis,MO, USA) and mouse ultrasensitive ELISA kit (DRG InstrumentsGmbH, Marburg, Germany), respectively. Insulin resistance, expressedas homeostasis model assessment-insulin resistance (HOMA-IR), wascalculated via the formula [glucose (mmol/L) × insulin (mU/L)]/22.5. Plasma leptin and adiponectin levels were measured using aMouse Leptin Quantikine ELISA kit (R&D Systems, Minneapolis,MN, USA) and a Rat/mouse Adiponectin ELISA kit (Phoenix EuropeGmbH, Karlsruhe, Germany), respectively. Plasma immunoreactiveghrelin concentration was measured using a radioimmunoassay kit(Phoenix Pharmaceuticals, Belmont, CA, USA).

Hepatic TG and TC. Liver homogenate, 1 mL, was mixed with 2.5mL of chloroform/methanol (2:1, v/v). The chloroform layer wascollected and concentrated. After mixing with 10% Triton X-100 inisopropanol, the sample was assayed by using Wako Triglyceride E-Test and Total Cholesterol E-Test kits (Wako Pure Chemical).

Fecal Lipid Analysis. Feces, at 0.5 g, were mixed with 3.5 mL ofdeionized water. After sitting at 4 °C overnight, feces werehomogenized by vortexing. The fecal lipid was extracted withmethanol/chloroform (2:1, v/v) using a method described in Tsujitaet al.18 The lipophilic layer was collected and dried under a nitrogenstream.

Measurement of Oxidative and Inflammatory Factors inLiver. Liver tissue was homogenized with cold phosphate-bufferedsaline containing 0.05% Tween 20 and 1 mM EDTA. Aftercentrifuging, the supernatants were used for measurements. Samplewas mixed with 25 mM 2′,7′-dichlorofluorescein diacetate. After 30min of incubation at room temperature, reactive oxygen species (ROS)level was determined by monitoring fluorescence change at anexcitation wavelength of 488 nm and an emission wavelength of 515nm. GSH content was measured by using a commercial kit(OxisResearch, Portland, OR, USA). The activities of GPX andcatalase were assayed by GPX and catalase kits (Calbiochem, EMDBiosciences, Inc., San Diego, CA, USA). Levels of interleukin (IL)-1β,IL-6, and tumor necrosis factor (TNF)-α were determined usingELISA kits (R&D Systems).

Western Blot Analysis. Liver tissue was homogenized in buffercontaining 0.5% Triton X-100 and protease-inhibitor cocktail (1:1000,Sigma-Aldrich Chemical Co.). This homogenate was further mixedwith 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 to10% SDS−polyacrylamide gel electrophoresis and transferred to anitrocellulose membrane (Millipore, Bedford, MA, USA) for 1 h. Afterblocking with a solution containing 5% nonfat milk for 1 h to preventnonspecific binding of antibody, membrane was incubated with mouseanti-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 anti-MAPK (1:1000) monoclonal antibodies (Boehringer-Mannheim,Indianapolis, IN, USA) at 4 °C overnight and followed by reactionwith horseradish peroxidase-conjugated antibody for 3.5 h at roomtemperature. The detected bands were quantified by an image analyzer(ATTO, Tokyo, Japan), and glyceraldehyde-3-phosphate dehydrogen-ase (GAPDH) was used as a loading control. The blot was quantifiedby densitometric analysis. Results were normalized to GAPDH andgiven as arbitrary units (AU).

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

ND HFD HFD + AA-low HFD + AA-high

body wt (g/mouse) 24.6 ± 1.1a 36.4 ± 1.7d 32.8 ± 0.9c 28.5 ± 1.2bwater intake (mL/mouse/day) 2.0 ± 0.4a 5.8 ± 0.6c 4.7 ± 0.3b 4.5 ± 0.5bfeed intake (g/mouse/day) 1.5 ± 0.3a 5.3 ± 0.8d 4.5 ± 0.4c 3.8 ± 0.5bliver wt (g/mouse) 1.28 ± 0.13a 2.16 ± 0.23c 2.01 ± 0.14c 1.74 ± 0.18bepididymal fat (g/mouse) 0.24 ± 0.05a 1.41 ± 0.15d 1.12 ± 0.10c 0.72 ± 0.09b

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

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Histological Analysis. Partial liver tissue from each mouse wasfixed 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 byexamination 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 themean ± standard deviation (SD). Statistical analysis was done usingone-way analysis of variance, and post-hoc comparisons were carriedout 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 HFD-

treated mice (P < 0.05). AA intake only at high dose lowered

liver weight of mice treated with HFD (P < 0.05).

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


plasmaALT (U/L) 33 ± 3a 118 ± 10d 91 ± 5c 60 ± 6bAST (U/L) 36 ± 4a 104 ± 8d 82 ± 7c 51 ± 4bTG (g/L) 2.24 ± 0.08a 5.76 ± 0.21d 5.01 ± 0.13c 3.98 ± 0.10bTC (g/L) 1.28 ± 0.05a 3.90 ± 0.11c 3.56 ± 0.06c 2.94 ± 0.07b

hepaticTG (mg/g wet wt) 25.9 ± 1.2a 64.5 ± 2.5d 51.1 ± 1.6c 38.2 ± 0.9bTC (mg/g wet wt) 2.8 ± 0.2a 7.6 ± 0.4c 7.1 ± 0.3c 6.4 ± 0.2b

fecallipid (mg/g feces) 8.3 ± 0.5a 13.6 ± 0.4b 14.0 ± 0.6b 15.7 ± 0.5c


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 letterdiffer, P < 0.05.



Effects of AA on Lipid Levels. HFD raised plasma ALT,AST, TG, and TC levels; hepatic TG and TC levels; and fecallipid 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 loweredhepatic TG level, 20.8−41.3% (P < 0.05). Plasma TC level andhepatic 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, 3.71-fold SCD-1, 3.62-fold HMG-CoA reductase, 2.91-fold SREBP-1c, and 2.75-fold SREBP-2 expression in liver (P < 0.05).Compared with HFD groups, AA intake at two doses reducedACC1, 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-CoAreductase and SREBP-2 (P > 0.05). As shown in Figure 3, HFDled to a great amount of fat deposition in liver, determined byORO stain (upper row), and caused obvious foci ofinflammatory cell infiltration in liver, determined by H&Estain (lower row). AA intake at two doses decreased hepaticlipid droplets and inflammatory infiltration. HFD increasedplasma levels of insulin, leptin, and HOMA-IR and decreasedadiponectin and ghrelin levels (Table 3, P < 0.05). AA intake attwo doses lowered plasma insulin secretion and HOMR-IR (P< 0.05) but did not affect plasma glucose, leptin, ghrelin, oradiponectin levels (P > 0.05).

Effects of AA on Hepatic Oxidative and InflammatoryStress. HFD increased hepatic production of ROS, IL-1β, IL-6,and TNF-α as well as decreased hepatic GSH content and GPXand catalase activities (Table 4, P < 0.05). Compared with HFD

Figure 3. Effects of AA upon hepatic lipid accumulation (upper row), determined by ORO statin; hepatic inflammation (lower row), determined byH&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) 7.2 ± 0.3a 7.8 ± 0.5a 7.6 ± 0.2a 7.5 ± 0.4ainsulin (nmol/L) 14.2 ± 0.6a 25.8 ± 1.8d 22.5 ± 1.1c 17.8 ± 0.9bHOMA-IR 4.3 ± 0.8a 33.4 ± 2.1d 26.1 ± 1.7c 15.0 ± 1.0bleptin (ng/mL) 1.21 ± 0.20a 2.19 ± 0.17b 2.10 ± 0.11b 1.98 ± 0.08badiponectin (μg/mL) 8.3 ± 0.5b 4.7 ± 0.2a 4.8 ± 0.4a 5.1 ± 4aghrelin (fmol/mL) 152 ± 13b 96 ± 7a 101 ± 6a 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 MiceTreated with Normal Diet (ND), High-Fat Diet (HFD), or HFD with AA-Low or AA-High for 7 Weeksa


ROS (RFU/mg protein) 0.26 ± 0.04a 1.30 ± 0.12d 1.02 ± 0.10c 0.66 ± 0.08bGSH (nmol/mg protein) 12.4 ± 0.6d 7.3 ± 0.4a 8.4 ± 0.5b 10.1 ± 0.7cGPX (U/mg protein) 18.6 ± 0.5d 12.5 ± 0.3a 14.1 ± 0.4b 15.5 ± 0.5ccatalase (U/mg protein) 17.1 ± 0.7d 11.3 ± 0.4a 12.5 ± 0.6b 14.3 ± 0.6c

IL-1β (pg/mg protein) 19 ± 3a 108 ± 10d 87 ± 8c 60 ± 5bIL-6 (pg/mg protein) 15 ± 2a 94 ± 7d 77 ± 5c 52 ± 4bTNF-α (pg/mg protein) 18 ± 4a 103 ± 11d 85 ± 9c 56 ± 5b




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 andmaintained 12.8−24.1% GPX and 10.6−26.5% catalaseactivities (P < 0.05). As shown in Figure 4, HFD up-regulatedhepatic NF-κB and MAPK expression (P < 0.05). AA intakedose-dependently suppressed NF-κB p65, 32.4−58.8%, and p-p38, 24.2−56.7%, expression (P < 0.05) and at high dosedown-regulated NF-κB p50, 29.1%, and p-JNK, 40.7%,expression. AA intake did not affect ERK1/2 and p-ERK1/2expression (P > 0.05).

■ DISCUSSIONThe high-fat diet we used enhanced hepatic protein expressionof lipogenic enzymes and factors including ACC1, FAS, SCD-1,HMG-CoA reductase, SREBP-1c, and SREBP-2, which clearlyexplained the observed lipid accumulation in liver, circulation,and epididymal white adipose tissue. We further found that theintake of AA markedly attenuated high-fat diet causedhyperlipidemia, hepatic steatosis, and epididymal fat deposit.Our Western blot data revealed that AA intake effectivelysuppressed hepatic expression of ACC1, FAS, SCD-1, andSREBP-1c. Our histological data indicated that AA intakereduced fat deposits in hepatocytes. Furthermore, AA intakealleviated high-fat diet induced insulin resistance and hepaticoxidative and inflammatory stress. These findings supported

that AA could protect liver via antilipogenic, antioxidative, andanti-inflammatory activities to improve high-fat diet causedinjury.ACC1 mediates the initial step of fatty acid synthesis via

converting acetyl-CoA to malonyl-CoA.11 FAS is responsiblefor the last step in fatty acid biosynthesis and plays adeterminant role for hepatic fatty acid generation in de novolipogenesis.10 SCD-1 catalyzes the rate-limiting step in thecellular biosynthesis of monounsaturated fatty acids, primarilyoleate and palmitoleate, which could be incorporated into andstored as triglyceride in liver.19 SREBP-1c is the major upstreamtranscription factor involved in hepatic triglyceride biosynthesisand modulates the expression of downstream targets includingFAS, ACC1, and SCD-1.20−22 Thus, the suppression on theexpression of these enzymes and SREBP-1c could inhibithepatic lipogenesis and lower fat accumulation in liver. Ourpresent study found that AA treatments effectively down-regulated hepatic expression of ACC1, FAS, SCD-1, andSREBP-1c, which subsequently decreased triglyceride biosyn-thesis and deposit in liver, adipose tissue, and circulation andfinally attenuated hepatic steatosis and lowered body weight.These findings indicated that AA was an effective agent againsttriglyceride biosynthesis. Obviously, the antilipogenic activitiesof AA could be mainly ascribed to its suppression upon hepaticSREBP-1c, an upstream lipogenic factor, and the less availableSREBP-1c subsequently declined the expression of downstream

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 orAA-high for 7 weeks. Values are means ± SD, n = 10. Means among bars without a common letter differ, P < 0.05.



factors, ACC1, FAS, and SCD-1 in the liver. On the other hand,HMG-CoA reductase, regulated by SREBP-2, is responsible forcholesterol biosynthesis.23 Our present study found AA intakedid not affect hepatic SREBP-2 and HMG-CoA reductaseexpression. SREBP-1c is produced from a single gene, SREBF-1,located on chromosome 17p11.2, but SREBP-2 is generatedfrom another gene, SREBF-2, located on chromosome 22q13.24

It is speculated that AA had less affinity to chromosome 22q13and/or less interference with gene SREBF-2, which causedfailure in regulating SREBP-2 in HFD-treated mice liver.However, it is interesting to find that AA at high dose loweredhepatic and circulating cholesterol levels. It is likely that AA athigh dose mildly mediated other factor(s) involved incholesterol biosynthesis. These results implied that AA was aweak inhibitor against cholesterol biosynthesis. In addition, ourdata revealed AA intake at high dose increased fecal lipidcontent. Thus, the lipid-lowering effect of this triterpene wasslightly 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 dataindicated that AA intake substantially alleviated hyper-insulinemia. It is highly possible that this triterpene suppressedhepatic lipogenesis and reduced lipid accumulation in liver andadipose tissue, which in turn restored insulin sensitivity andlowered the requirement for insulin. The reduction of HOMA-IR in AA-treated mice we observed also agreed with this agentimproving high-fat diet induced insulin resistance. In addition, ahigh-fat diet stimulates the release of inflammatory cytokinessuch as IL-1β and TNF-α, which blocks adipocyte insulinaction and contributes to the development of obesity-relatedinsulin resistance.27,28 Thus, the improved insulin resistance inAA-treated mice could partially result from the diminishedhepatic release of these inflammatory cytokines. AA treatmentsdid not affect high-fat diet induced hyper-leptinemia, hypo-adiponectinemia, 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 tricarboxylicacid cycle electron transport chain, and impaired mitochondrialrespiratory capability in liver and other organs such as the heart,which led to the overproduction of ROS and enhancedoxidative stress and consequently activated MAPK and NF-κBpathways.29,30 The activation of these two pathways furtheraugmented hepatic oxidative and inflammatory stress andcontributed to the progression of liver disorders includingsteatohepatitis.30,31 The results of our present study agreed withthose previous studies because our molecular analyses datarevealed that a high-fat diet enhanced hepatic expression of NF-κB p50, NF-κB p65, p38, JNK, and ERK1/2, which in turnevoked the generation of ROS and inflammatory cytokinesincluding IL-6 and TNF-α. Furthermore, we found that AAintake limited phosphorylation of NF-κB p50, NF-κB p65, p38,and JNK in liver, which subsequently decreased the activationof NF-κB and MAPK. Because both NF-κB and MAPKpathways have been suppressed, the lower formation of ROSand inflammatory cytokines in livers from AA-treated micecould be explained. Our histological results indicated that AAintake ameliorated lobular inflammation and diffuse ballooningdegeneration in hepatocytes. The restored antioxidativedefenses such as GPX and catalase activities and loweredplasma ALT and AST levels in AA-treated mice also indicatedthat hepatic oxidative and inflammatory stress had beenmitigated. These findings showed that dietary AA could be

absorbed and exerted its hepatic protection through regulatinghepatic NF-κB and MAPK pathways.AA is a triterpene naturally occurring in many plant foods

and herbs. Ramachandran and Saravanan32 reported that AAintake at 10 or 20 mg/kg body weight altered hepatic enzymeactivities of carbohydrate metabolism in diabetic rats. In ourpresent study, AA intake also at 10 or 20 mg/kg body weightmarkedly protected THE liver against high-fat diet inducedinjury in mice. On the basis of its natural property and supportfrom other studies and our data, the application of thiscompound seems safe. The doses used in our present studywere 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 fulvaL.) contained AA in the range of 55−102 mg/g dry weight.4

Thus, the consumption of these vegetables, rich in AA, mightalso benefit the prevention of diet-induced lipid disorders. Onthe other hand, we noted that AA intake lowered mouse feedintake. It seems that this agent, a tasteless compound, was ableto affect appetite and contributed to decreased body weight.In conclusion, high-fat diet caused hyperlipidemia, hepatic

steatosis, and insulin resistance. The intake of asiatic acidmarkedly lowered lipid accumulation in the circulation, liver,and adipose tissue via suppressing the protein expression ofACC1, FAS, SCD-1, and SREBP-1c. This triterpene alsoimproved insulin resistance and attenuated hepatic oxidativeand inflammatory injury. These results suggest that asiatic acidis a potent hepatic protective agent against high-fat diet inducedsteatohepatitis.

■ AUTHOR INFORMATION

Corresponding Author*(M.-C.Y.) Mail: Department of Nutrition, China MedicalUniversity, 91 Hsueh-shih Road, Taichung City, Taiwan.Phone: 886-4-22053366, ext. 7510. Fax: 886-4-22062891. E-mail: [email protected].

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

NotesThe authors declare no competing financial interest.

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asiatic acid ameliorates hepatic lipid accumulation and insulin resistance in mice consuming a...

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