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Cite this: DOI: 10.1039/c5fo00083a
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Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress† Jian-Shuang Li,a Wen-Jun Wang,a Yu Sun,a Yu-Hao Zhanga and Ling Zheng*a,b Ursolic acid (UA) is a natural pentacyclic triterpenoid compound, which is enriched with many herbs and plants, such as apple, cranberry and olive. UA performs multiple biological activities including anti-oxidation, anti-inflammation, anti-cancer and hepatoprotection. However, the exact mechanism underlying the hepatoprotective activity of UA remains unclear. In this study, the effects of UA on the development of nonalcoholic fatty liver disease (NAFLD) were investigated. In vivo, UA treatment (0.14%, w/w) significantly decreased the liver weight, serum levels of ALT/AST and hepatic steatosis in db/db mice (a type 2 diabetic mouse model). In vitro, UA treatment (10–30 μg ml−1) significantly decreased palmitic acid
Received 22nd January 2015, Accepted 2nd April 2015
induced intracellular lipid accumulation in L02 cells. Our results suggested that the beneficial effects of
DOI: 10.1039/c5fo00083a
UA on NAFLD may be due to its ability to increase lipid β-oxidation and to inhibit the hepatic endoplasmic reticulum (ER) stress. Together, UA may be further considered as a natural compound for NAFLD
www.rsc.org/foodfunction
treatment.
Introduction Nonalcoholic fatty liver disease (NAFLD), which refers to excessive accumulation of triglyceride in hepatocytes without a history of significant alcohol consumption,1,2 is the most common chronic liver disease worldwide. NAFLD encompasses a wide spectrum of hepatic pathologies, ranging from simple steatosis to a more progressive form, nonalcoholic steatohepatitis (NASH). The latter has a high risk of developing into liver fibrosis and cirrhosis, even hepatocarcinoma.3,4 Patients with NAFLD are usually associated with obesity, type 2 diabetes and insulin resistance.5 Endoplasmic reticulum (ER) is an important organelle that regulates calcium storage, protein, lipid and glucose metabolism.6,7 Different stimuli can increase the accumulation of unfolded proteins in ER, which leads to ER stress. To solve ER stress, three branches of the unfolded protein response (UPR) are activated, including inositol-requiring protein 1α (IRE1α), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6),7 as well as their respective downstream signaling cascades.8,9 Long-term activation or unsolved ER stress lead to many metabolic diseases, including obesity,
a College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P. R. China. E-mail: [email protected]; Tel: +86-27-68755559 b Diabetes Center, Wuhan University, Wuhan, Hubei 430072, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5fo00083a
This journal is © The Royal Society of Chemistry 2015
type 2 diabetes, NAFLD and NASH.10,11 For example, ER stressinduced JNK activation leads to hepatic steatosis by promoting insulin resistance through down-regulation of IRS-1 (insulin receptor substrate-1).12 Over-expression of BIP (binding of immunoglobulin protein), a molecular chaperone in ER lumen, inhibits ER stress-induced SREBP-1c (sterol regulatory element binding protein 1c) activation, a key transcription factor involved in lipid metabolism, and thus reduces hepatic steatosis in obese mice.13 All these data suggest an important role of ER stress in the pathogenesis of NAFLD. Lipid metabolism is another important metabolic pathway involved in the development of NAFLD. Imbalance between lipogenesis and fatty acid oxidation contributes to the accumulation of triglyceride in hepatocytes.14 Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) are two step-limiting enzymes for de novo lipogenesis, which catalyze malonyl-CoA and acetyl-CoA to generate palmitic acid. Palmitic acid is then elongated and desaturated to generate different mono-unsaturated fatty acids, which are the major fatty acid constituents of triglycerides.14 On the other hand, peroxisomal proliferatoractivated receptors (PPARs) are ligand-regulated transcription factors which belong to the superfamily of nuclear receptors that can be activated by fatty acids.15 PPAR-α, one of three isoforms of PPARs, which is highly expressed in the liver, kidney and heart,16 plays a central role in fatty acid β-oxidation.17 Several genes involved in fatty acid β-oxidation are regulated by PPAR-α, including carnitine palmitoyl transferase1/2 (Cpt1/2), long- and medium-chain acyl-CoA dehydrogenases (Acadl,
Food Funct.
View Article Online
Published on 07 April 2015. Downloaded by Carleton University on 05/05/2015 02:55:31.
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Acadm), and acetyl-CoA acyltransferase (Acaa), which encode the enzymes for fatty acid transportation into mitochondria, the first step and the final step of fatty acid β-oxidation respectively.18,19 Ursolic acid (UA) is a natural pentacyclic triterpenoid carboxylic acid that is rich in many traditional medicinal herbs and fruits, such as apple, cranberry and olive.20 UA has been shown to have anti-oxidation, anti-inflammation, anti-cancer and hepatoprotective activities.20 UA can significantly decrease blood glucose, cholesterol and low-density lipoprotein levels, increase high density lipoprotein level and lead to glycogen accumulation in the livers of healthy rats.21 In CHO or 3T3-L1 cells, UA acts as an effective insulin mimetic agent at a high dosage (above 50 μg ml−1) and as an insulin sensitizer at a low dosage (as low as 1 μg ml−1).22 UA can improve hepatic insulin resistance in KKAy mice (a type 2 diabetic mouse mode) by regulating the expression of PPAR-α and PEPCK.23 UA can also decrease diet-induced obesity, glucose intolerance and NAFLD by increasing skeletal muscle mass and fiber size, brown fat weight and energy expenditure.24 Recent studies have also shown that UA effectively ameliorated HFD-induced hepatic steatosis and liver injury through upregulating both mRNA and protein levels of PPAR-α and improving key enzymes involved in lipid metabolism.24,25 Despite these findings, the exact molecular mechanisms of UA for the development of NAFLD remain unclear. In the present study, UA was orally administrated to sixweek old db/db mice (a typical type 2 diabetic mouse model) for 8 weeks and different physiological characteristics were assessed. UA was also added to the palmitic acid stimulated hepatic cells. The levels of several proteins involved in fatty acid β-oxidation and ER stress were also investigated. Our results revealed that UA inhibited the development of NAFLD by increasing lipid β-oxidation and attenuating ER stress, suggesting that UA may be used as a functional natural compound for preventing NAFLD.
Materials and methods Chemicals UA was obtained from Nanjing TCM Institute of Chinese Material Medical (Nanjing, China). Palmitic acid was purchased from Sigma Aldrich (St. Louis, MO). All other chemicals used were analytical grade. Animals Breeding pairs of db/+ mice were obtained from the Model Animal Research Center of Nanjing University. The animals were maintained in ventilated microisolator cages with 12 h light/dark cycle under constant temperature and humidity. db/ db mice and their db/+ littermates were identified by genotyping. Six-week old db/db mice were randomly divided into two groups (7–11 mice per group): one group was fed with a normal chow, the other group was fed with UA in diets (0.14%, w/w). The sex- and age-matched db/+ mice were used as the
Food Funct.
Food & Function
control group. The mice were kept on different diets for 8 weeks. Body weight (BW) and food intake were measured weekly. The levels of non-fasted blood glucose (NFBG) were measured by a One Touch blood glucose meter (LifeScan Inc., Milpitas, CA, USA). BMI (body mass index) is calculated as the body weight divided by the square of the length according to a previous report.26 Organs from different groups were dissected under microscope and weighted. The animals were handled according to the Guidelines of the China Animal Welfare Legislation, as approved by the Committee on Ethics in the Care and Use of Laboratory Animals of College of Life Sciences, Wuhan University. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) Blood was collected at the sacrificing day, and centrifuged at 2000g for 15 minutes to collect serum. Serum levels of ALT and AST were measured using ALT and AST Assay Kits (Biosino Biotechnology and Science Inc., Shanghai, China), according to the manufacturer’s instructions. Measurement of hepatic triglyceride levels Hepatic triglycerides were isolated as previously reported.27 Briefly, liver tissue was homogenized in phosphate-buffered saline (PBS) and centrifuged at 2000 rpm for 10 minutes. The pellet was then extracted with 0.75 ml chloroform/methanol (2 : 1 v/v) at room temperature for 5 minutes. 0.25 ml chloroform was then added and vortexed, followed by addition of 0.25 ml ddH2O. Finally, the mixture was centrifuged at 1000 rpm for 5 minutes. The organic phase was then washed twice with ddH2O and air dried. The extracted hepatic triglyceride was quantified using a Triglyceride Assay Kit (Cayman, Ann Arbor, MI), according to the manufacturer’s instructions. The level of hepatic triglyceride was reported as mg triglyceride per gram of liver tissue. Liver histological analysis and pathological evaluation Livers were routinely embedded in paraffin and sectioned. Liver sections were stained with hematoxylin–eosin (H&E). Pictures (×200) of 4–6 different fields per sample were taken under an Olympus BX60 microscope equipped with a digital CCD, and semi-quantitative analysis of liver histology was examined in a masked manner as we previously reported.11 Briefly, for hepatic steatosis analysis, four different scores were used: parenchymal involvement by steatosis less than 5% was 0; 5%–33% was 1; 33%–66% was 2; more than 66% was 3. For hepatic steatosis location analysis: Zone 3 distribution was only 0; Zone 1 distribution was 1; azonal distribution was 2 and panacinar distribution was 3. For hepatic ballooning analysis: no balloon cells were 0; few balloon cells were 1; many balloon cells were 2.1 Cell culture The human hepatic cell line L02 was cultured in DMEM media containing 5.5 mM glucose (Hyclone, Palo Alto, CA) plus 10% FBS (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin
This journal is © The Royal Society of Chemistry 2015
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(Hyclone) in a 37 °C incubator with 5% CO2. To examine the lipotoxic effects, palmitate solution (PA) was prepared as previously reported,28 and was added to the media at a final concentration of 400 μM.
Published on 07 April 2015. Downloaded by Carleton University on 05/05/2015 02:55:31.
Oil Red O staining L02 cells were treated with PA for 24 hours in the presence or absence of UA; cells treated with a vehicle were regarded as control. Cells were stained with Oil Red O to examine the amount of lipid accumulation in them as previously reported.29 Briefly, cells were washed with cold PBS and fixed in 4% formalin. After washing twice with ddH2O, Oil Red O working solution was added for 30 minutes followed by washing with 60% isopropanol. After rinsing with ddH2O and counterstaining with hematoxylin, 10 different fields of each sample were photographed. The positive area for Oil Red O staining was measured with the ImagePlus 6.0 software (Media Cybernetics, Bethesda, Maryland).
Paper
Antibody-detected bands were visualized using the ECL reagent (Thermo Scientific, Marietta, OH) followed by exposure on films. The developed films were subsequently scanned, and band intensities were quantified with the Quantity One 1-D analysis software (Bio-Rad, Hercules, CA). Targeted protein expression levels were quantitated relative to the β-actin level in the same sample and normalized to the control group, which was set as 1 fold.
Statistical analysis Results, if not specifically stated, were expressed as mean ± SD (standard deviation). Statistical significance was analyzed by Kruskal–Wallis test followed by the Mann–Whitney test. Differences were considered statistically significant at values of P < 0.05.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) assay
Results
L02 cells were plated in 96-well plates at a density of 1 × 104 cells per well and cultured for 12 h. After treatment with PA in the presence or absence of different dosages of UA for 24 h, 10 μL of MTT (5 mg ml−1) were added into the media for 4 h. The media were removed and DMSO was added to each well. Absorbance was measured at 490 nm with a MUITISKAN FC plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Six replicate wells were counted for each condition.
The effects of UA on physiological characteristics of db/db mice
RNA isolation, RT-PCR and quantitative real-time PCR (qPCR) RNA isolation and cDNA synthesis were performed as previously reported.30 The abundance of specific gene transcripts was assessed by RT-PCR or qPCR. The sequences of primers used are listed in ESI Table S1.† For RT-PCR, the products were separated by electrophoresis using 1.5% agarose gels, and images were photographed by Molecular Imager Gel Doc XR (Bio-Rad, Hercules, CA). Band density was quantified using Quantity One 1-D analysis software (Bio-Rad). The mRNA level of the targeted gene was quantitated first against the 18S rRNA level from the same sample, and then normalized to the control group, which was set as one.31 qPCR assays were performed with SsoFastTM EvaGreenR Supermix (Invitrogen) using a Bio-Rad iQ5 Real Time PCR System. β-Actin was used as an internal control. The relative difference was expressed as the fold change calculated by the 2−ΔΔCT method.32 Western blots Livers or cultured cells were sonicated in cold RIPA buffer (Beyotime Biotechnology, Jiangsu, China) with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was determined using a BCA kit (Beyotime Biotechnology). Western blots were performed as we previously reported.11 The antibody for PPAR-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), all other antibodies were from Cell Signaling Technologies (Beverly, MA).
This journal is © The Royal Society of Chemistry 2015
The general physiological characters of experimental groups are provided in Table 1. Compared to the db/+ group, parameters including BW, Body Mass Index (BMI), liver weight, liver index (liver weight/body weight × 100) and NFBG were significantly increased in db/db mice. Administration of UA to db/db mice significantly ameliorated the liver weight and liver index without reducing the food intake (P < 0.05 and P < 0.01, respectively). Furthermore, the levels of ALT and AST, two markers of liver injury, were significantly increased in db/db mice, while administration of UA significantly decreased the elevated levels of ALT and AST in db/db mice. However, administration of UA to db/db mice had no significant effects on BW, BMI and NFBG. Interestingly, administration of UA to db/db mice led to a significant increase in the length of body (Table 1).
Table 1
Physiological characteristics of experimental groupsa
Body weight (g) Length (cm) BMI (kg m−2) Liver weight (g) LI NFBG (mmol L−1) Food intake (g) ALT (U L−1) AST (U L−1)
db/+ (11)
db/db (8)
db/db + UA (7)
21.1 ± 0.9 8.1 ± 0.2 3.2 ± 0.2 1.0 ± 0.3 4.7 ± 1.0 8.4 ± 1.0 3.6 ± 0.2 19.3 ± 4.8 17.1 ± 6.8
40.3 ± 1.6* 8.8 ± 0.4* 5.2 ± 0.5* 2.6 ± 0.2* 6.3 ± 0.4* 12.4 ± 2.4* 5.1 ± 0.4* 49.5 ± 12.5* 32.8 ± 8.0*
42.9 ± 1.8* 9.2 ± 0.2*§ 5.0 ± 0.1* 2.1 ± 0.2*§ 4.9 ± 0.5§ 10.6 ± 2.3 5.0 ± 0.4* 33.3 ± 7.9*§ 20.9 ± 4.8§
a BW, body weight; BMI, body mass index; LW, liver weight; LI, liver index (liver weight/body weight × 100); NFBG, non-fasted blood glucose; ALT, alanine aminotransferase; AST, aspartate aminotransferase; n = 7–11 per group. Statistically significant differences are indicated as follows: *p
Published on 07 April 2015. Downloaded by Carleton University on 05/05/2015 02:55:31.
PAPER
Cite this: DOI: 10.1039/c5fo00083a
View Journal
Ursolic acid inhibits the development of nonalcoholic fatty liver disease by attenuating endoplasmic reticulum stress† Jian-Shuang Li,a Wen-Jun Wang,a Yu Sun,a Yu-Hao Zhanga and Ling Zheng*a,b Ursolic acid (UA) is a natural pentacyclic triterpenoid compound, which is enriched with many herbs and plants, such as apple, cranberry and olive. UA performs multiple biological activities including anti-oxidation, anti-inflammation, anti-cancer and hepatoprotection. However, the exact mechanism underlying the hepatoprotective activity of UA remains unclear. In this study, the effects of UA on the development of nonalcoholic fatty liver disease (NAFLD) were investigated. In vivo, UA treatment (0.14%, w/w) significantly decreased the liver weight, serum levels of ALT/AST and hepatic steatosis in db/db mice (a type 2 diabetic mouse model). In vitro, UA treatment (10–30 μg ml−1) significantly decreased palmitic acid
Received 22nd January 2015, Accepted 2nd April 2015
induced intracellular lipid accumulation in L02 cells. Our results suggested that the beneficial effects of
DOI: 10.1039/c5fo00083a
UA on NAFLD may be due to its ability to increase lipid β-oxidation and to inhibit the hepatic endoplasmic reticulum (ER) stress. Together, UA may be further considered as a natural compound for NAFLD
www.rsc.org/foodfunction
treatment.
Introduction Nonalcoholic fatty liver disease (NAFLD), which refers to excessive accumulation of triglyceride in hepatocytes without a history of significant alcohol consumption,1,2 is the most common chronic liver disease worldwide. NAFLD encompasses a wide spectrum of hepatic pathologies, ranging from simple steatosis to a more progressive form, nonalcoholic steatohepatitis (NASH). The latter has a high risk of developing into liver fibrosis and cirrhosis, even hepatocarcinoma.3,4 Patients with NAFLD are usually associated with obesity, type 2 diabetes and insulin resistance.5 Endoplasmic reticulum (ER) is an important organelle that regulates calcium storage, protein, lipid and glucose metabolism.6,7 Different stimuli can increase the accumulation of unfolded proteins in ER, which leads to ER stress. To solve ER stress, three branches of the unfolded protein response (UPR) are activated, including inositol-requiring protein 1α (IRE1α), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6),7 as well as their respective downstream signaling cascades.8,9 Long-term activation or unsolved ER stress lead to many metabolic diseases, including obesity,
a College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P. R. China. E-mail: [email protected]; Tel: +86-27-68755559 b Diabetes Center, Wuhan University, Wuhan, Hubei 430072, P. R. China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5fo00083a
This journal is © The Royal Society of Chemistry 2015
type 2 diabetes, NAFLD and NASH.10,11 For example, ER stressinduced JNK activation leads to hepatic steatosis by promoting insulin resistance through down-regulation of IRS-1 (insulin receptor substrate-1).12 Over-expression of BIP (binding of immunoglobulin protein), a molecular chaperone in ER lumen, inhibits ER stress-induced SREBP-1c (sterol regulatory element binding protein 1c) activation, a key transcription factor involved in lipid metabolism, and thus reduces hepatic steatosis in obese mice.13 All these data suggest an important role of ER stress in the pathogenesis of NAFLD. Lipid metabolism is another important metabolic pathway involved in the development of NAFLD. Imbalance between lipogenesis and fatty acid oxidation contributes to the accumulation of triglyceride in hepatocytes.14 Acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) are two step-limiting enzymes for de novo lipogenesis, which catalyze malonyl-CoA and acetyl-CoA to generate palmitic acid. Palmitic acid is then elongated and desaturated to generate different mono-unsaturated fatty acids, which are the major fatty acid constituents of triglycerides.14 On the other hand, peroxisomal proliferatoractivated receptors (PPARs) are ligand-regulated transcription factors which belong to the superfamily of nuclear receptors that can be activated by fatty acids.15 PPAR-α, one of three isoforms of PPARs, which is highly expressed in the liver, kidney and heart,16 plays a central role in fatty acid β-oxidation.17 Several genes involved in fatty acid β-oxidation are regulated by PPAR-α, including carnitine palmitoyl transferase1/2 (Cpt1/2), long- and medium-chain acyl-CoA dehydrogenases (Acadl,
Food Funct.
View Article Online
Published on 07 April 2015. Downloaded by Carleton University on 05/05/2015 02:55:31.
Paper
Acadm), and acetyl-CoA acyltransferase (Acaa), which encode the enzymes for fatty acid transportation into mitochondria, the first step and the final step of fatty acid β-oxidation respectively.18,19 Ursolic acid (UA) is a natural pentacyclic triterpenoid carboxylic acid that is rich in many traditional medicinal herbs and fruits, such as apple, cranberry and olive.20 UA has been shown to have anti-oxidation, anti-inflammation, anti-cancer and hepatoprotective activities.20 UA can significantly decrease blood glucose, cholesterol and low-density lipoprotein levels, increase high density lipoprotein level and lead to glycogen accumulation in the livers of healthy rats.21 In CHO or 3T3-L1 cells, UA acts as an effective insulin mimetic agent at a high dosage (above 50 μg ml−1) and as an insulin sensitizer at a low dosage (as low as 1 μg ml−1).22 UA can improve hepatic insulin resistance in KKAy mice (a type 2 diabetic mouse mode) by regulating the expression of PPAR-α and PEPCK.23 UA can also decrease diet-induced obesity, glucose intolerance and NAFLD by increasing skeletal muscle mass and fiber size, brown fat weight and energy expenditure.24 Recent studies have also shown that UA effectively ameliorated HFD-induced hepatic steatosis and liver injury through upregulating both mRNA and protein levels of PPAR-α and improving key enzymes involved in lipid metabolism.24,25 Despite these findings, the exact molecular mechanisms of UA for the development of NAFLD remain unclear. In the present study, UA was orally administrated to sixweek old db/db mice (a typical type 2 diabetic mouse model) for 8 weeks and different physiological characteristics were assessed. UA was also added to the palmitic acid stimulated hepatic cells. The levels of several proteins involved in fatty acid β-oxidation and ER stress were also investigated. Our results revealed that UA inhibited the development of NAFLD by increasing lipid β-oxidation and attenuating ER stress, suggesting that UA may be used as a functional natural compound for preventing NAFLD.
Materials and methods Chemicals UA was obtained from Nanjing TCM Institute of Chinese Material Medical (Nanjing, China). Palmitic acid was purchased from Sigma Aldrich (St. Louis, MO). All other chemicals used were analytical grade. Animals Breeding pairs of db/+ mice were obtained from the Model Animal Research Center of Nanjing University. The animals were maintained in ventilated microisolator cages with 12 h light/dark cycle under constant temperature and humidity. db/ db mice and their db/+ littermates were identified by genotyping. Six-week old db/db mice were randomly divided into two groups (7–11 mice per group): one group was fed with a normal chow, the other group was fed with UA in diets (0.14%, w/w). The sex- and age-matched db/+ mice were used as the
Food Funct.
Food & Function
control group. The mice were kept on different diets for 8 weeks. Body weight (BW) and food intake were measured weekly. The levels of non-fasted blood glucose (NFBG) were measured by a One Touch blood glucose meter (LifeScan Inc., Milpitas, CA, USA). BMI (body mass index) is calculated as the body weight divided by the square of the length according to a previous report.26 Organs from different groups were dissected under microscope and weighted. The animals were handled according to the Guidelines of the China Animal Welfare Legislation, as approved by the Committee on Ethics in the Care and Use of Laboratory Animals of College of Life Sciences, Wuhan University. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) Blood was collected at the sacrificing day, and centrifuged at 2000g for 15 minutes to collect serum. Serum levels of ALT and AST were measured using ALT and AST Assay Kits (Biosino Biotechnology and Science Inc., Shanghai, China), according to the manufacturer’s instructions. Measurement of hepatic triglyceride levels Hepatic triglycerides were isolated as previously reported.27 Briefly, liver tissue was homogenized in phosphate-buffered saline (PBS) and centrifuged at 2000 rpm for 10 minutes. The pellet was then extracted with 0.75 ml chloroform/methanol (2 : 1 v/v) at room temperature for 5 minutes. 0.25 ml chloroform was then added and vortexed, followed by addition of 0.25 ml ddH2O. Finally, the mixture was centrifuged at 1000 rpm for 5 minutes. The organic phase was then washed twice with ddH2O and air dried. The extracted hepatic triglyceride was quantified using a Triglyceride Assay Kit (Cayman, Ann Arbor, MI), according to the manufacturer’s instructions. The level of hepatic triglyceride was reported as mg triglyceride per gram of liver tissue. Liver histological analysis and pathological evaluation Livers were routinely embedded in paraffin and sectioned. Liver sections were stained with hematoxylin–eosin (H&E). Pictures (×200) of 4–6 different fields per sample were taken under an Olympus BX60 microscope equipped with a digital CCD, and semi-quantitative analysis of liver histology was examined in a masked manner as we previously reported.11 Briefly, for hepatic steatosis analysis, four different scores were used: parenchymal involvement by steatosis less than 5% was 0; 5%–33% was 1; 33%–66% was 2; more than 66% was 3. For hepatic steatosis location analysis: Zone 3 distribution was only 0; Zone 1 distribution was 1; azonal distribution was 2 and panacinar distribution was 3. For hepatic ballooning analysis: no balloon cells were 0; few balloon cells were 1; many balloon cells were 2.1 Cell culture The human hepatic cell line L02 was cultured in DMEM media containing 5.5 mM glucose (Hyclone, Palo Alto, CA) plus 10% FBS (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin
This journal is © The Royal Society of Chemistry 2015
View Article Online
Food & Function
(Hyclone) in a 37 °C incubator with 5% CO2. To examine the lipotoxic effects, palmitate solution (PA) was prepared as previously reported,28 and was added to the media at a final concentration of 400 μM.
Published on 07 April 2015. Downloaded by Carleton University on 05/05/2015 02:55:31.
Oil Red O staining L02 cells were treated with PA for 24 hours in the presence or absence of UA; cells treated with a vehicle were regarded as control. Cells were stained with Oil Red O to examine the amount of lipid accumulation in them as previously reported.29 Briefly, cells were washed with cold PBS and fixed in 4% formalin. After washing twice with ddH2O, Oil Red O working solution was added for 30 minutes followed by washing with 60% isopropanol. After rinsing with ddH2O and counterstaining with hematoxylin, 10 different fields of each sample were photographed. The positive area for Oil Red O staining was measured with the ImagePlus 6.0 software (Media Cybernetics, Bethesda, Maryland).
Paper
Antibody-detected bands were visualized using the ECL reagent (Thermo Scientific, Marietta, OH) followed by exposure on films. The developed films were subsequently scanned, and band intensities were quantified with the Quantity One 1-D analysis software (Bio-Rad, Hercules, CA). Targeted protein expression levels were quantitated relative to the β-actin level in the same sample and normalized to the control group, which was set as 1 fold.
Statistical analysis Results, if not specifically stated, were expressed as mean ± SD (standard deviation). Statistical significance was analyzed by Kruskal–Wallis test followed by the Mann–Whitney test. Differences were considered statistically significant at values of P < 0.05.
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) assay
Results
L02 cells were plated in 96-well plates at a density of 1 × 104 cells per well and cultured for 12 h. After treatment with PA in the presence or absence of different dosages of UA for 24 h, 10 μL of MTT (5 mg ml−1) were added into the media for 4 h. The media were removed and DMSO was added to each well. Absorbance was measured at 490 nm with a MUITISKAN FC plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Six replicate wells were counted for each condition.
The effects of UA on physiological characteristics of db/db mice
RNA isolation, RT-PCR and quantitative real-time PCR (qPCR) RNA isolation and cDNA synthesis were performed as previously reported.30 The abundance of specific gene transcripts was assessed by RT-PCR or qPCR. The sequences of primers used are listed in ESI Table S1.† For RT-PCR, the products were separated by electrophoresis using 1.5% agarose gels, and images were photographed by Molecular Imager Gel Doc XR (Bio-Rad, Hercules, CA). Band density was quantified using Quantity One 1-D analysis software (Bio-Rad). The mRNA level of the targeted gene was quantitated first against the 18S rRNA level from the same sample, and then normalized to the control group, which was set as one.31 qPCR assays were performed with SsoFastTM EvaGreenR Supermix (Invitrogen) using a Bio-Rad iQ5 Real Time PCR System. β-Actin was used as an internal control. The relative difference was expressed as the fold change calculated by the 2−ΔΔCT method.32 Western blots Livers or cultured cells were sonicated in cold RIPA buffer (Beyotime Biotechnology, Jiangsu, China) with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was determined using a BCA kit (Beyotime Biotechnology). Western blots were performed as we previously reported.11 The antibody for PPAR-α was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), all other antibodies were from Cell Signaling Technologies (Beverly, MA).
This journal is © The Royal Society of Chemistry 2015
The general physiological characters of experimental groups are provided in Table 1. Compared to the db/+ group, parameters including BW, Body Mass Index (BMI), liver weight, liver index (liver weight/body weight × 100) and NFBG were significantly increased in db/db mice. Administration of UA to db/db mice significantly ameliorated the liver weight and liver index without reducing the food intake (P < 0.05 and P < 0.01, respectively). Furthermore, the levels of ALT and AST, two markers of liver injury, were significantly increased in db/db mice, while administration of UA significantly decreased the elevated levels of ALT and AST in db/db mice. However, administration of UA to db/db mice had no significant effects on BW, BMI and NFBG. Interestingly, administration of UA to db/db mice led to a significant increase in the length of body (Table 1).
Table 1
Physiological characteristics of experimental groupsa
Body weight (g) Length (cm) BMI (kg m−2) Liver weight (g) LI NFBG (mmol L−1) Food intake (g) ALT (U L−1) AST (U L−1)
db/+ (11)
db/db (8)
db/db + UA (7)
21.1 ± 0.9 8.1 ± 0.2 3.2 ± 0.2 1.0 ± 0.3 4.7 ± 1.0 8.4 ± 1.0 3.6 ± 0.2 19.3 ± 4.8 17.1 ± 6.8
40.3 ± 1.6* 8.8 ± 0.4* 5.2 ± 0.5* 2.6 ± 0.2* 6.3 ± 0.4* 12.4 ± 2.4* 5.1 ± 0.4* 49.5 ± 12.5* 32.8 ± 8.0*
42.9 ± 1.8* 9.2 ± 0.2*§ 5.0 ± 0.1* 2.1 ± 0.2*§ 4.9 ± 0.5§ 10.6 ± 2.3 5.0 ± 0.4* 33.3 ± 7.9*§ 20.9 ± 4.8§
a BW, body weight; BMI, body mass index; LW, liver weight; LI, liver index (liver weight/body weight × 100); NFBG, non-fasted blood glucose; ALT, alanine aminotransferase; AST, aspartate aminotransferase; n = 7–11 per group. Statistically significant differences are indicated as follows: *p
