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Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats
Journal of Pharmacological Sciences xxx (2014) 1e7
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Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats Long Li a, b, Lei Li a, Ling Chen a, Xiaoyu Lin c, Yaping Xu a, Jie Ren a, Jin Fu a, Yan Qiu a, * a
Department of Medical Sciences, Medical College, Xiamen University, Xiamen, Fujian, China Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, China c Center of Liver Diseases, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2014 Received in revised form 30 November 2014 Accepted 1 December 2014 Available online xxx
Oleoylethanolamine (OEA), an endogenous high-affinity agonist of peroxisome proliferator-activated receptor alpha (PPAR-a), has revealed the pharmacological properties in the treatment of obesity, atherosclerosis and other diseases through the modulation of lipid metabolism. To assess whether OEA can also regulate non-alcoholic fatty liver disease (NAFLD) caused by fat accumulation, we administrated OEA or fenofibrate in Sprague Dawley (SD) rats fed with a high fat diet (HFD). After 6 or 17 weeks treatment, OEA (5 mg/kg/day, i.p.) relieved the development of NAFLD compared with control groups by regulating the levels of plasma TG, TC, ALT and AST and liver inflammatory cytokines. Gene expression analysis of liver tissue and plasma from the animal models showed that OEA and fenofibrate both promoted the lipid b-oxidation by activating PPAR-a. Detailed research revealed that OEA inhibited the mRNA expression of lipogenesis in a PPAR-a-independant manner, while fenofibrate expressed an opposite effection. In summary, our research results suggested that as a potential lead compound, OEA could improve HFD-induced NAFLD with higher efficacy and safety than fenofibrate. © 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/3.0/).
Keywords: Oleoylethanolamide Nonalcoholic fatty liver disease Peroxisome proliferator-activated receptor alpha Lipid metabolism gene Fenofibrate
Introduction Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease which affects 25e30% of the world's population. Onset of NAFLD manifests as large lipid droplet accumulation in liver cells and triggers the formation of obesity, type II diabetes, hyperlipidemia, insulin resistance and other metabolic syndromes (1). The important early metabolic events leading to NAFLD can be best described as a “two-hit” theory. The “first hit” is characterized by the deposition of triglycerides in hepatocyte during the pathologic process of obesity or insulin resistance. The disease is sensitive to additional cellular events as oxidative stress, lipid peroxidation, pro-inflammatory cytokines release (the “second hit”), which leads the progress of nonalcoholic steatohepatitis. Current clinical therapies for NAFLD is limited to control of associated metabolic syndromes, reduction risk of cardiovascular disease, and suggestions of lifestyle changes. So, although dietary control, exercise, insulinsensitizing drugs, antioxidants and lipid-lowering drugs can do * Corresponding author. E-mail address: [email protected] (Y. Qiu). Peer review under responsibility of Japanese Pharmacological Society.
favor for NAFLD (2), other modalities of treatment is suggested by clinical observations. Peroxisome proliferator-activated receptor alpha (PPAR-a) is predominantly distributed in the active metabolic tissues, such as liver, kidney, heart and skeletal muscle, in which modulates the lipid metabolism and energy balance. PPAR-a activation not only up-regulates the expression levels of carnitine palmitoyl transferase 1 (CPT1) to promote fatty acid b-oxidation and reduces triglyceride deposition in vivo, but also inhibits the signal transduction mediated by AP-1 and NF-kB to relieve inflammation (3). Fatty acid oxidases in the mitochondria, peroxisomes and microsomes expressed abnormally in PPAR-a knockout (KO) mice, leading to increase of free fatty acids in plasma and excessive accumulation of lipids in liver (4). In comparison to wild-type mice, PPAR-a KO mice exhibited high expression levels of many inflammatory factors, as tumor necrosis factor alpha (TNF-a) and interleukin 1b (IL-1b) in liver and adipose tissues (5). Fibrates are widespread applied as PPAR-a agonists into clinic practice to regulate plasma lipid disorders (6). Some clinic data has shown that fenofibrate plays a central role in reducing plasma and hepatic triglyceride concentrations and transaminase activities and improving insulin resistance in NAFLD patients in vivo (7). However,
http://dx.doi.org/10.1016/j.jphs.2014.12.001 1347-8613/© 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
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L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
due to the relatively low affinity of fenofibrate to PPAR-a receptor (EC50 ¼ 30 mM) (8), as well as dose-dependent side effects that have been reported (9), there is an unsatisfied clinical requirement for new PPAR-a agonists with potential specificity and safety to play regulator roles in metabolic syndromes. Compared with fenofibrate, an endogenous lipid OEA shows a higher affinity with PPAR-a (EC50 ¼ 120 nM), which has been indicated by using luciferase assay for PPRE and PPAR-a vectorstransfected HEK293 cells (10). In the prior reports, this molecular was considered as a better regulator for the treatment of obesity, atherosclerosis and other metabolic diseases (11,12). However, the effects and mechanisms about OEA applied to improve NAFLD have not been reported. OEA plays an important regulatory role in lipid metabolism and can enhance fatty acid oxidation in primary cultured skeletal muscle cells, liver cells and cardiac myocytes. But it does not affect fatty acid oxidation processes in similar cells extracted from PPAR-a KO mice, which confirms that OEA regulates fatty acid metabolism by activating PPAR-a (13). Obesity and insulin resistance are often accompanied by lipid metabolic disorders (14), which are turned out to be the main cause of NAFLD. As OEA has been demonstrated to induce weight loss in association with improvements in body lipid metabolism, we hypothesize that OEA may play a protective role against NAFLD. In present experiments, we evaluated the role and mechanism of OEA on NAFLD induced via a high fat diet (HFD) in rats. Furthermore, whether OEA can play an improved regulatory role over the traditional PPAR-a agonist, fenofibrate, has also been studied.
(5% Tween-80 þ 5% PEG400 þ 90% saline). All these compounds were treated once daily. Liver histological studies Fresh liver tissue was fixed in 10% neutral-buffered formalin for 3 days and then embedded in paraffin for histological examinations. Tissue sections (5 mm thick) were cut by a Leica SM2010 R Sliding microtome (Shanghai, China) and stained with hematoxylineeosin (H&E); stained areas were viewed and imaged under microscopy (Nikon, Shanghai, China). Plasma biochemistry assays Plasma transaminase (ALT and AST) and triglyceride (TC and TG) concentration were measured individually by a Thermo Scientific Multiskan GO Microplate Spectrophotometer with commercial kits (Nanjing Jiancheng Bioengineering Institute, China). Plasma TNF-a, IL-6 and leptin protein levels were measured using ELISA (R&D Systems, Shanghai, China). RNA isolation and cDNA synthesis Liver samples were homogenized and total RNA was extracted using the TRIzol™ isolation reagent (Invitrogen) according to the manufacturer's recommendations. cDNA was synthesized from total RNA using a ReverTra Ace® qPCR RT kit (Toyobo, Shanghai, China) according to the manufacturer's instructions.
Materials and methods Real-time PCR Materials OEA was synthesized in our lab as previously described (15); fenofibrate, dimethyl sulfoxide (DMSO) and all other chemicals were obtained from SigmaeAldrich (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Shanghai, China). Lipofectamine® 2000 (Lipo2000) and Human PPAR-a Silencer Select siRNA (ID s10880) were purchased from Invitrogen (Shanghai, China). The chemical constituents were dissolved in saline supplemented with 5% polyethylene glycol 400 (PEG400) and 5% Tween-80 for the in vivo studies. For the in vitro studies, OEA was dissolved in DMSO to a concentration of 50 mmol/L (stock solution) and then diluted in the culture medium to a final concentration of 50 mmol/L. TNF-a and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Shanghai, China). Total cholesterol (TC), triglycerides (TG), alanine transaminase (ALT) and aspartate transaminase (AST) commercial assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Animals and diets Male Sprague Dawley (SD) rats (250e300 g) were sourced from Shanghai Laboratory Animal Center (Shanghai, China). All experimental protocols were approved by the Committee for Animal Research at Xiamen University. The rats were housed in a room with controlled temperature (21e23 C), humidity (55e60%) and lighting (12 h light/dark cycles) and given water ad libitum. Rats were fed a HFD diet (30% fat, 2% cholesterol, 2% sugar, 0.5% choline and 65.5% normal diet) or a normal diet (ND) for 6 weeks or 17 weeks to induce nonalcoholic fatty liver. The HFD-fed rats were randomly divided into three experimental groups (n ¼ 8 per group). One group was treated with OEA (5 mg/kg/day; intraperitoneal injection, i.p.) and another group was treated with fenofibrate (10 mg/kg/day; i.p.). The third group was treated with the vehicle
Quantification of mRNA was performed on an Applied Biosystems 7300 real-time polymerase chain reaction (PCR) system using SYBR® Premix Ex Taq™ II (Takara, Dalian, China). Primer sequences were synthesized as follows: TNF-a, 50 -GGC TCC CTC TCA TCA GTT CCA-30 (forward), 50 -CGC TTG GTG GTT TGC TAC GA-30 (reverse); IL-6, 50 -TGC CTT CTT GGG ACT GAT GTT G-30 (forward), 50 -TGG TCT GTT GTG GGT GGT ATC C-30 (reverse); C-reactive protein (CRP), 50 -TGT CTC TAT GCC CAC GCT GAT G-30 (forward), 50 -GGC CCA CCT ACT GCA ATA CTA AAC30 (reverse); CPT-1, 50 -AAC TTT GTG CAG GCC ATG ATG-30 (forward), 50 -GGC AGA AGA TGG CGG TCG-30 (reverse); enoyl-CoA hydratase (ECHS1), 50 -ATG GCT ATG CTC TTG GTG-30 (forward), 50 -GTG ATT TGC CGA CTG CTC-30 (reverse); PPAR-a, 50 -AAT CCA CGA AGC CTA CCT GA30 (forward), 50 -GTC TTC TCA GCC ATG CAC AA-30 (reverse); stearoylCoA desaturase-1 (SCD-1), 50 -ACA TGC TCC AAG AGA TCT CCA G-30 (forward), 50 -GTA CTC CAG CTT GGG CGG-30 (reverse); OB-Rb, 50 -GCA TGC AGA ATC AGT GAT ATT TGG-30 (forward), 50 -CAA GCT GTA TCG ACA CTG ATT CTT C-30 (reverse); glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 50 -ACC ACG AGA AAT ATG ACA ACT CCC-30 (forward), 50 -CCA AAG TTG TCA TGG ATG ACC-30 (reverse). The levels of mRNA were normalized relative to the amount of GAPDH mRNA. Lipid extraction and analysis Total lipids were extracted from liver samples according to the modified methods as previously described (16). C17:0 margaric acid was added as an internal standard for the quantitation of fatty acids. An Agilent 1200 Series HPLC (Agilent Corporation, MA, USA) interfaced to an Applied Biosystems 3200 Q-Trap triple-quadrupole linear ion trap mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Canada) was operated during experiments. Fatty acids were separated using a Hypersil Gold C18 column (dimensions: 250 4.6 mm; particle size: 5 mm; Thermo) eluted with a constant mobile phase that was composed of 5 mM ammonium acetate in acetonitrile (solvent A, 65%) and 5 mM ammonium acetate in 2-propanol (solvent B, 35%);
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
the flow rate was maintained at 700 mL/min for 15 min. Mass spectrometer detection was ionized by the full scan electrospray ionization mode (ESI) and monitored in multiple reaction monitoring (MRM) mode. The ion source temperature was maintained at 650 C. Quantifications were calculated using Analyst 1.4.1 data acquisition and processing software (Applied Biosystems/MDS Sciex). Cell culture and RNA interference HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cells were cultured in 6-well culture plates under 37 C and 5% CO2 in an incubator. siRNA was diluted with Opti-MEM® I to concentrations of 20 nmol/L. Lipo2000 was mixed gently, then diluted to 5 mL in 250 mL Opti-MEM® I and incubated at room temperature for 5 min. The diluted Lipo2000 and siRNA were mixed gently and incubated at room temperature for 20 min and the siRNAeLipo2000 complexes were added to cultured cells in 6-well culture plates and maintained at 37 C in a 5% CO2 atmosphere in an incubator.
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fat droplets, histology studies with H&E staining showed that severe steatosis with a amount of fat vacuoles appeared in the rat liver cells with HFD feeding for 17 weeks compared to that in normal diet-fed rats (Fig. 1A and B), and OEA administration (5 mg/ kg/day, 17 weeks, i.p.) reduced the vacuoles of the lipid droplets in liver cells of HFD-fed rats obviously (Fig. 1B and C). Consistent with a large number of lipid droplets accumulated in the liver, HFD induced the increase levels of triglyceride (TG) (Fig. 1D) and total cholesterol (TC) (Fig. 1E) in rat plasma (P < 0.001), which were reversed by OEA treatment (Fig. 1DeF, P < 0.001). Furthermore, biochemistry analysis showed that plasma alanine aminotransferase (ALT) (Fig. 1F) and aspartate aminotransferase (AST) (Fig. 1G), two key indicators of liver function damage, increased in HFD rats (P < 0.01), while OEA treatment significantly reduced the plasma levels of ALT and AST in HFD-fed rats (Fig. 1FeG, P < 0.001). OEA suppresses the HFD-induced inflammatory response
Results
Inflammation has been shown to involve in the developmental process of fatty liver. Clinical researches have shown the marked raises of TNF-a and IL-6 levels in the liver and plasma of NAFLD patients (17,18). To investigate OEA effect on HFD-induced inflammation, rats were fed with HFD for 17 weeks with/without OEA regimen (5 mg/kg, i.p.). Real-time PCR detection showed that HFD induced the mRNA expressions of inflammatory cytokines including CRP (Fig. 2A), TNF-a (Fig. 2B), and IL-6 (Fig. 2C) in the liver of SD rats, and the changes of cytokines by HFD were reversed by OEA treatment (Fig. 2AeC). In addition, biochemical analysis showed that the induction of the plasma levels of TNF-a (Fig. 3A) and IL-6 (Fig. 3B) by HFD were corrected by OEA treatment (Fig. 3A and B).
Protective effects of OEA on HFD-induced hepatic steatosis and liver injury
OEA promoted lipid catabolism genes in a PPAR-a-dependent manner
The main feature of HFD-induced NAFLD is the hepatic fat accumulation. To assess the impact of OEA on the accumulation of
To study the mechanism underlying OEA effect on improving the prognosis of NAFLD, the mRNA expression of the key genes
Statistical analysis All statistical analyses were performed using GraphPad Prism version 5.01 for Windows. Data are presented as the mean ± SEM and were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons. P < 0.05 was considered statistically significant.
Fig. 1. OEA prevented hepatocellular ballooning and reduced plasma transaminase and triglyceride content levels in HFD-induced NAFLD in rats. H&E staining showed hepatocellular ballooning in vehicle-treated rats fed a normal diet (A), vehicle-treated rats fed a high fat diet (B) and OEA-treated rats (5 mg/kg/day; i.p.) fed a high fat diet (C) after 17 weeks of treatment. Graphs DeG show the effect of the vehicle or OEA on blood plasma levels of triglyceride (TG) (D), total cholesterol (TC) (E), alanine aminotransferase (ALT) (F) and aspartate aminotransferase (AST) (G) in rats fed a normal diet or HFD after 17 weeks of treatment. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. ### P < 0.001 compared with the control group, ## P < 0.01 compared with the control group, *** P < 0.001 compared with the HFD group; one-way ANOVA, n ¼ 8.
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
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Fig. 2. OEA inhibited inflammation in livers of NAFLD rats. Liver CRP (A), TNF-a (B) and IL-6 (C) mRNA levels following treatment with the vehicle or OEA in rats fed a normal diet or HFD assessed by real-time quantitative PCR. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. # P < 0.05 compared with the control group, ### P < 0.001 compared with the control group, * P < 0.05 compared with the HFD group, ** P < 0.01 compared with the HFD group; one-way ANOVA, n ¼ 8.
involved in fatty acid b-oxidation in the liver, including PPAR-a, CPT-1, ECHS1, were determined. Real-time PCR analysis showed that HFD diet had no effect on the mRNA expressions of PPAR-a, and ECHS-1, and down-regulated the CPT-1 gene (Fig. 4AeC), while
administration of OEA (5 mg/kg/day, i.p) significantly increased the mRNA expression levels of PPAR-a (Fig. 4A), CPT-1 (Fig. 4B), and ECHS-1 (Fig. 4C). To further identify whether the above effect of OEA was mediated by the PPAR-a receptor, HepG2 cells were
Fig. 3. OEA decreased TNF-a and IL-6 levels in plasma of NAFLD rats. TNF-a (A) and IL-6 (B) protein levels in the plasma of rats fed a normal diet or HFD after vehicle or OEA treatment assessed by ELISA. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. # P < 0.05 compared with the control group, ### P < 0.001 compared with the control group, * P < 0.05 compared with the HFD group, ** P < 0.01 compared with the HFD group; one-way ANOVA, n ¼ 8.
Fig. 4. OEA normalized gene expression levels in HFD-induced NAFLD in rats. The effect of the vehicle or OEA on mRNA levels of PPAR-a (A), CPT-1 (B), ECHS1 (C) and SCD-1 (D), was assessed by real-time quantitative PCR after 17 weeks of treatment. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. ## P < 0.01 compared with the control group, ### P < 0.001 compared with the control group, ** P < 0.01 compared with the HFD group, *** P < 0.001 compared with the HFD group; one-way ANOVA, n ¼ 8.
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
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Fig. 5. OEA inhibited SCD-1 enzyme activity in HFD-induced NAFLD in rats. The levels of palmitic acid (16:0) and palmitoleic acid (16:1) following treatment with the vehicle or OEA in rats fed a normal diet or HFD were detected by LCeMS. SCD-1 enzyme activity was deduced from the ratio palmitoleic acid to palmitate (16:1/16:0). Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. ## P < 0.01 compared with control group, * P < 0.05 compared with HFD group, one-way ANOVA, n ¼ 8.
transfected with/without PPAR-a siRNA 24 h before oleic acid (OA; 0.25 mmol/L) stimulated to create an in vitro NAFLD model. The mRNA expression levels of the nuclear receptor gene PPAR-a (Fig. 6A) and fatty acid b-oxidation gene CPT-1 (Fig. 6B) were examined by real-time PCR after OEA (50 mM) administration. In vitro results showed that the expression levels of both genes increased significantly after OEA stimulation (P < 0.01 and P < 0.01, respectively), while using PPAR-a RNAi, the lipogenetic promoting effects of OEA was completely disappeared. This suggested that OEA promoted fatty acid b-oxidation by activating PPAR-a pathway. OEA inhibited hepatic SCD-1 gene expression and protein activity in a PPAR-a-independent manner
that OEA not only reduced SCD-1 expression levels in the liver, but also inhibited its activity significantly especially controlled the desaturation of palmitic acid. In the same in vitro HepG2 cell model, we examined a change in SCD-1 expression levels (Fig. 6C) and found that OEA treatment down-regulated SCD-1 expression significantly (P < 0.001), while this inhibition effect of OEA on SCD-1 expression (P < 0.05) was still observed with PPAR-a RNAi treatment. However, without regard to OEA treatment, PPAR-a gene knockdown also reduced the SCD-1 gene expression levels to a high degree. These test results showed that the regulatory role of OEA with regard to SCD-1 downregulation was not mediated by the PPAR-a pathway. Roles of OEA and fenofibrate in the NAFLD model
As SCD-1 plays an important role on lipid homeostasis in the liver, we found that HFD induced the mRNA expression of SCD-1 (Fig. 4F) in rat liver. OEA administration (5 mg/kg/day, i.p.) completely corrected the HFD-induced SCD-1 expression in the liver. In addition, we further analyzed the enzyme activity of SCD-1, assessed by the ratio of palmitic acid (16:0 PA) to palmitoleic acid (16:1 PA), in the liver by the transition of fatty acid composition. Liquid chromatographyemass spectrometry (LCeMS) analysis were found that HFD significantly increase the contents of 16:1 PA in the liver, and the ration of 16:1 PA to 16:0 PA was increase by that to evaluate the SCD-1 enzyme activity. Fig. 5C showed that comparing to HFD treatment OEA administration (5 mg/kg/day; i.p.) inhibited the desaturation of palmitateplamitic acid, and the ratio of 16:1/16:0 was decreased (P < 0.05). These results suggested
During the 17-week animal experiments, we administrated fenofibrate (10 mg/kg/day, i.p.) as positive control simultaneous with a HFD treatment to compare the role of OEA and fenofibrate on NAFLD. The survival rate of rats treated with fenofibrate was remarkably affected during the treatment period. As shown in Fig. S1AeD, rats in the fenofibrate-treated group began to die at 14 weeks after drug administration, which had all died at 17 weeks. Compared to the fenofibrate-treated group, the remaining three groups, including the OEA-treated group, resulted in no animal death up to 17 weeks of treatment. To further compare the roles of fenofibrate and OEA, we re-designed a 6-week short-term experiment with the same groups and treatment protocols as performed in 17-week long-term experiment.
Fig. 6. OEA promoted fatty acid b-oxidation via PPAR-a signaling, suppressed SCD-1 expression in a PPAR-a independent manner. The effect of OEA combined with or without PPARa siRNA transfection on mRNA expression levels of PPAR-a (A), CPT-1 (B), SCD-1 (C), assessed by real-time quantitative PCR, on HepG2 cells. PPAR-a siRNA, 10 nM; OEA, 50 mM; Vehicle, 0.1%DMSO. * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA, n ¼ 6.
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
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L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
During the 6-week experiment, no animal death was observed in all groups (Fig. S1EeH). OEA (5 mg/kg/day; i.p.) and fenofibrate (10 mg/kg/day; i.p.) administration showed similar pharmacodynamic effects with regard to less severe steatosis, decreased serum ALT, AST levels, meliorative liver inflammation (Figs. S2 and 3). RTPCR analysis suggested that the two compounds could activate PPAR-a and promote the b-oxidation of fatty acids (Fig. S4AeC). Hepatic lipid homeostasis needs a balance between lipid synthesis and metabolism. We then evaluated the related genes for de novo lipogenesis, and found that OEA and fenofibrate showed the opposite effects (Fig. S4DeF). OEA reduced SREBP-1c, SCD-1, and ACC mRNA expression levels (P < 0.05) during the 6-week period after drug administration, while fenofibrate treatment increased the above genes expression levels significantly (P < 0.001), which indicated that OEA inhibited the whole system of lipogenesis. Discussion In this paper we have explored whether the endogenous lipid OEA is a safe and effective lead compound to improve NAFLD development and the mechanism of action of OEA is dissected preliminary. Excessive consumption of HFD enhanced the development of hepatosteatosis. Therefore, HFD-induced animal model has been widely applied in NAFLD research (19). A 17-week longterm experiment has been conducted to accumulate enough lipid droplets in the liver and cause chronic liver injury for a better observation of the effects of OEA. In this animal model, OEA was found to play a critical role in lowering fatty degeneration of the liver, reducing hepatocellular damage and inhibiting inflammation effectively. PPAR-a is highly expressed in liver, where it regulates multiple genes encoding for enzymes involved in fatty acid b-oxidation (20,21). CPT-1 is located in the mitochondrial membrane, which can promote the transport of long-chain fatty acids into the mitochondria to further participate in the b-oxidation process and becomes a rate-limiting enzyme for fatty acid oxidation. It has been extensively shown that fenofibrate could induce higher CPT-1 expression in liver and adipose tissues by PPAR-a pathway (22,23). The present study showed that treatment with OEA increased the expression of CPT-1, as well as PPAR-a and ECHS1 to promote the fatty acid oxidation process. These results suggested, at least to some extent, the two PPAR-a agonists regulated liver fatty acid metabolism by promoting fatty acid b-oxidation. Stimulated de novo lipogenesis has been observed after treatment with PPAR-a agonists in many experiments (24,25). In the 6week short-term drug administration experiment, we did observe that fenofibrate significantly up-regulates the expression level of SCD-1 and SREBP-1c mRNA in the rat liver as the previous studies have been shown. But the experiment results of OEA were in contrast to the regulatory role of fenofibrate. Our results showed that the expression level of the SCD-1 gene in the liver increased significantly in animals after 6 and 17-week HFD treatment and OEA could down-regulate the expression of SCD-1 mRNA in liver tissues significantly. Additional investigation of liver SCD-1 enzyme activity showed that OEA also regulated SCD-1 activity and reversed the increased HFD-induced desaturation index (16:1/ 16:0) markedly. Leptin is a hormone secreted by adipose tissue that plays a crucial role in energy metabolism. Some findings suggested that down-regulation of SCD-1 is an important component of leptin's metabolic actions (26). To estimate whether leptin signaling pathway mediated the different regulatory role of OEA and fenofibrate on SCD-1 expression, 6-week animal experiment has been used to detect the plasma leptin levels and hepatic OB-Rb gene expression (Fig. S5). As a result, both OEA and fenofibrate decreased
plasma leptin levels and up-regulated the mRNA expression of OBRb in livers, which indicated that the opposite effect on SCD-1 expression was irrelevant to leptin signaling pathway. SCD-1 is also known to be regulated by PPAR-a and SREBP-1c (27,28). To estimate whether the action of OEA on hepatic SCD-1 was mediated via PPAR-a pathway, an in vitro experiment on HepG2 cell was conducted. The results indicated that PPAR-a knockdown suppressed the expression of SCD-1 gene, which acted in accordance with the known theory. But in PPAR-a RNAi group, OEA still decreased the expression level of SCD-1 (Fig. 6), which suggested that the down-regulation of SCD-1 by OEA was independent of PPAR-a pathway. It was noteworthy that in the 6-week experiment, distinct down-regulation of SCD-1, ACC as well as SREBP-1c mRNA was observed in OEA administration group, which suggested that SCD-1 expression maybe under SREBP-1c signaling control. Administration of fenofibrate increased hepatic expression of genes involved in fatty acid b-oxidation and at the same time also increased lipogenetic genes levels. These results are consistent with previous reports (24,25). Another conventional PPAR-a synthetic agonists, Wy-14643, has also showed a similar pharmacodynamic effect (29). However, despite promoting fatty acid b-oxidation, OEA inhibited the lipid synthetic genes expression, which would be important to avoid hepatic lipoperoxide accumulation. Maybe, it is the reason for high mortality occurrence only in the fenofibratetreated 17-week long-term group. To elucidate whether the effect of OEA on the expression of lipid metabolism genes specific under HFD condition, we examined the effect of OEA on above genes in rats fed normal diets (Fig. S6). After 6 h administration, real-time PCR detection shown that OEA significantly increased the mRNA levels of PPAR-a and CPT-1, obviously decreased the expression of SREBP-1c and SCD-1, but had no effect on liver ECHS1, AdipoR2 and OB-Rb expression. Similarly results were observed after 12 h administration. These results indicated that OEA could promote fatty acid b-oxidation and inhibit lipogenesis both in normal and HFD-fed rats. In conclusion, OEA, an endogenous PPAR-a agonist, has a beneficial effect on HFD-induced NAFLD in rats via promoting fatty acid b-oxidation, and inhibiting de novo lipogenesis. It showed a better liver protective role than fenofibrate, a known clinical PPARa agonist, in rat models. All these considerations suggest that OEA may represent a potential lead compound or a promising therapeutic target on NAFLD. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors thank Eye Institute of Xiamen University for assistance. This study was supported by grants from National Natural Science Foundation of China (No. 81373273), Natural Science Foundation of Fujian Province (No. 2013J05122, 2013J01274). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jphs.2014.12.001. References (1) Birkenfeld AL, Shulman GI. Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology. 2014;59(2):713e723.
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7 (2) Yoneda M, Nakajima A, Wada K. Novel findings for the development of drug therapy for various liver diseases: preface. J Pharmacol Sci. 2011;115(3): 259e262. (3) He K, Wang Z, Wang Q, Pan Y. A meta-analysis study of gene expression datasets in mouse liver under PPARalpha knockout. Genet Res (Camb). 2013;95(2e3):78e88. (4) Reddy JK. Nonalcoholic steatosis and steatohepatitis. III. Peroxisomal betaoxidation, PPAR alpha, and steatohepatitis. Am J Physiol Gastrointest Liver Physiol. 2001;281(6):1333e1339. (5) Stienstra R, Mandard S, Patsouris D, Maass C, Kersten S, Muller M. Peroxisome proliferator-activated receptor alpha protects against obesity-induced hepatic inflammation. Endocrinology. 2007;148(6):2753e2763. (6) Karahashi M, Hoshina M, Yamazaki T, Sakamoto T, Mitsumoto A, Kawashima Y, et al. Fibrates reduce triacylglycerol content by upregulating adipose triglyceride lipase in the liver of rats. J Pharmacol Sci. 2013;123(4): 356e370. (7) Fernandez-Miranda C, Perez-Carreras M, Colina F, Lopez-Alonso G, Vargas C, Solis-Herruzo JA. A pilot trial of fenofibrate for the treatment of non-alcoholic fatty liver disease. Dig Liver Dis. 2008;40(3):200e205. (8) Willson TM, Brown PJ, Sternbach DD, Henke BR. The PPARs: from orphan receptors to drug discovery. J Med Chem. 2000;43(4):527e550. (9) Fruchart JC. Selective peroxisome proliferator-activated receptoralpha modulators (SPPARMalpha): the next generation of peroxisome proliferatoractivated receptor alpha-agonists. Cardiovasc Diabetol. 2013;12:82. (10) Fu J, Gaetani S, Oveisi F, Lo Verme J, Serrano A, Rodriguez De Fonseca F, et al. Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature. 2003;425(6953):90e93. (11) Fu J, Oveisi F, Gaetani S, Lin E, Piomelli D. Oleoylethanolamide, an endogenous PPAR-alpha agonist, lowers body weight and hyperlipidemia in obese rats. Neuropharmacology. 2005;48(8):1147e1153. (12) Fan A, Wu X, Wu H, Li L, Huang R, Zhu Y, et al. Atheroprotective effect of oleoylethanolamide (OEA) targeting oxidized LDL. PLoS One. 2014;9(1): e85337. (13) Guzman M, Lo Verme J, Fu J, Oveisi F, Blazquez C, Piomelli D. Oleoylethanolamide stimulates lipolysis by activating the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-alpha). J Biol Chem. 2004;279(27):27849e27854. (14) Ju TJ, Kwon WY, Kim YW, Kim JY, Kim YD, Lee IK, et al. Hemin improves insulin sensitivity in skeletal muscle in high fat-fed mice. J Pharmacol Sci. 2014;126(2):115e125. (15) Giuffrida A, Rodriguez de Fonseca F, Piomelli D. Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry. Anal Biochem. 2000;280(1):87e93.
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(16) Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37(8):911e917. (17) Ruiz AG, Casafont F, Crespo J, Cayon A, Mayorga M, Estebanez A, et al. Lipopolysaccharide-binding protein plasma levels and liver TNF-alpha gene expression in obese patients: evidence for the potential role of endotoxin in the pathogenesis of non-alcoholic steatohepatitis. Obes Surg. 2007;17(10): 1374e1380. (18) Wieckowska A, Papouchado BG, Li Z, Lopez R, Zein NN, Feldstein AE. Increased hepatic and circulating interleukin-6 levels in human nonalcoholic steatohepatitis. Am J Gastroenterol. 2008;103(6):1372e1379. (19) Hebbard L, George J. Animal models of nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol. 2011;8(1):35e44. (20) Rakhshandehroo M, Knoch B, Muller M, Kersten S. Peroxisome proliferatoractivated receptor alpha target genes. PPAR Res. 2010;2010. (21) Ito M, Adachi-Akahane S. Inter-organ communication in the regulation of lipid metabolism: focusing on the network between the liver, intestine, and heart. J Pharmacol Sci. 2013;123(4):312e317. (22) Lefebvre P, Chinetti G, Fruchart JC, Staels B. Sorting out the roles of PPAR alpha in energy metabolism and vascular homeostasis. J Clin Invest. 2006;16(3): 571e580. (23) Ana S, María del CG, Hubert V, Emilio H, Carlos B. Triglyceridemia and peroxisome proliferator-activated receptor-a expression are not connected in fenofibrate-treated pregnant rats. Mol Cell Biochem. 2005;273:97e107. (24) Chan SM, Sun RQ, Zeng XY, Choong ZH, Wang H, Watt MJ, et al. Activation of PPARalpha ameliorates hepatic insulin resistance and steatosis in high fructose-fed mice despite increased endoplasmic reticulum stress. Diabetes. 2013;62(6):2095e2105. (25) Yan F, Wang Q, Xu C, Cao M, Zhou X, Wang T, et al. Peroxisome proliferatoractivated receptor alpha activation induces hepatic steatosis, suggesting an adverse effect. PLoS One. 2014;9(6):e99245. (26) Biddinger SB, Miyazaki M, Boucher J, Ntambi JM, Kahn CR. Leptin suppresses stearoyl-CoA desaturase 1 by mechanisms independent of insulin and sterol regulatory element-binding protein-1c. Diabetes. 2006;55(7):2032e2041. (27) Miller CW, Ntambi JM. Peroxisome proliferators induce mouse liver stearoylCoA desaturase 1 gene expression. Proc Natl Acad Sci U S A. 1996;93(18): 9443e9448. (28) Tabor DE, Kim JB, Spiegelman BM, Edwards PA. Identification of conserved ciselements and transcription factors required for sterol-regulated transcription of stearoyl-CoA desaturase 1 and 2. J Biol Chem. 1999;274(29):20603e20610. (29) Knight BL, Hebbachi A, Hauton D, Brown AM, Wiggins D, Patel DD, et al. A role for PPARalpha in the control of SREBP activity and lipid synthesis in the liver. Biochem J. 2005;389(Pt 2):413e421.
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Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats Long Li a, b, Lei Li a, Ling Chen a, Xiaoyu Lin c, Yaping Xu a, Jie Ren a, Jin Fu a, Yan Qiu a, * a
Department of Medical Sciences, Medical College, Xiamen University, Xiamen, Fujian, China Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, China c Center of Liver Diseases, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2014 Received in revised form 30 November 2014 Accepted 1 December 2014 Available online xxx
Oleoylethanolamine (OEA), an endogenous high-affinity agonist of peroxisome proliferator-activated receptor alpha (PPAR-a), has revealed the pharmacological properties in the treatment of obesity, atherosclerosis and other diseases through the modulation of lipid metabolism. To assess whether OEA can also regulate non-alcoholic fatty liver disease (NAFLD) caused by fat accumulation, we administrated OEA or fenofibrate in Sprague Dawley (SD) rats fed with a high fat diet (HFD). After 6 or 17 weeks treatment, OEA (5 mg/kg/day, i.p.) relieved the development of NAFLD compared with control groups by regulating the levels of plasma TG, TC, ALT and AST and liver inflammatory cytokines. Gene expression analysis of liver tissue and plasma from the animal models showed that OEA and fenofibrate both promoted the lipid b-oxidation by activating PPAR-a. Detailed research revealed that OEA inhibited the mRNA expression of lipogenesis in a PPAR-a-independant manner, while fenofibrate expressed an opposite effection. In summary, our research results suggested that as a potential lead compound, OEA could improve HFD-induced NAFLD with higher efficacy and safety than fenofibrate. © 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/3.0/).
Keywords: Oleoylethanolamide Nonalcoholic fatty liver disease Peroxisome proliferator-activated receptor alpha Lipid metabolism gene Fenofibrate
Introduction Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease which affects 25e30% of the world's population. Onset of NAFLD manifests as large lipid droplet accumulation in liver cells and triggers the formation of obesity, type II diabetes, hyperlipidemia, insulin resistance and other metabolic syndromes (1). The important early metabolic events leading to NAFLD can be best described as a “two-hit” theory. The “first hit” is characterized by the deposition of triglycerides in hepatocyte during the pathologic process of obesity or insulin resistance. The disease is sensitive to additional cellular events as oxidative stress, lipid peroxidation, pro-inflammatory cytokines release (the “second hit”), which leads the progress of nonalcoholic steatohepatitis. Current clinical therapies for NAFLD is limited to control of associated metabolic syndromes, reduction risk of cardiovascular disease, and suggestions of lifestyle changes. So, although dietary control, exercise, insulinsensitizing drugs, antioxidants and lipid-lowering drugs can do * Corresponding author. E-mail address: [email protected] (Y. Qiu). Peer review under responsibility of Japanese Pharmacological Society.
favor for NAFLD (2), other modalities of treatment is suggested by clinical observations. Peroxisome proliferator-activated receptor alpha (PPAR-a) is predominantly distributed in the active metabolic tissues, such as liver, kidney, heart and skeletal muscle, in which modulates the lipid metabolism and energy balance. PPAR-a activation not only up-regulates the expression levels of carnitine palmitoyl transferase 1 (CPT1) to promote fatty acid b-oxidation and reduces triglyceride deposition in vivo, but also inhibits the signal transduction mediated by AP-1 and NF-kB to relieve inflammation (3). Fatty acid oxidases in the mitochondria, peroxisomes and microsomes expressed abnormally in PPAR-a knockout (KO) mice, leading to increase of free fatty acids in plasma and excessive accumulation of lipids in liver (4). In comparison to wild-type mice, PPAR-a KO mice exhibited high expression levels of many inflammatory factors, as tumor necrosis factor alpha (TNF-a) and interleukin 1b (IL-1b) in liver and adipose tissues (5). Fibrates are widespread applied as PPAR-a agonists into clinic practice to regulate plasma lipid disorders (6). Some clinic data has shown that fenofibrate plays a central role in reducing plasma and hepatic triglyceride concentrations and transaminase activities and improving insulin resistance in NAFLD patients in vivo (7). However,
http://dx.doi.org/10.1016/j.jphs.2014.12.001 1347-8613/© 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
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L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
due to the relatively low affinity of fenofibrate to PPAR-a receptor (EC50 ¼ 30 mM) (8), as well as dose-dependent side effects that have been reported (9), there is an unsatisfied clinical requirement for new PPAR-a agonists with potential specificity and safety to play regulator roles in metabolic syndromes. Compared with fenofibrate, an endogenous lipid OEA shows a higher affinity with PPAR-a (EC50 ¼ 120 nM), which has been indicated by using luciferase assay for PPRE and PPAR-a vectorstransfected HEK293 cells (10). In the prior reports, this molecular was considered as a better regulator for the treatment of obesity, atherosclerosis and other metabolic diseases (11,12). However, the effects and mechanisms about OEA applied to improve NAFLD have not been reported. OEA plays an important regulatory role in lipid metabolism and can enhance fatty acid oxidation in primary cultured skeletal muscle cells, liver cells and cardiac myocytes. But it does not affect fatty acid oxidation processes in similar cells extracted from PPAR-a KO mice, which confirms that OEA regulates fatty acid metabolism by activating PPAR-a (13). Obesity and insulin resistance are often accompanied by lipid metabolic disorders (14), which are turned out to be the main cause of NAFLD. As OEA has been demonstrated to induce weight loss in association with improvements in body lipid metabolism, we hypothesize that OEA may play a protective role against NAFLD. In present experiments, we evaluated the role and mechanism of OEA on NAFLD induced via a high fat diet (HFD) in rats. Furthermore, whether OEA can play an improved regulatory role over the traditional PPAR-a agonist, fenofibrate, has also been studied.
(5% Tween-80 þ 5% PEG400 þ 90% saline). All these compounds were treated once daily. Liver histological studies Fresh liver tissue was fixed in 10% neutral-buffered formalin for 3 days and then embedded in paraffin for histological examinations. Tissue sections (5 mm thick) were cut by a Leica SM2010 R Sliding microtome (Shanghai, China) and stained with hematoxylineeosin (H&E); stained areas were viewed and imaged under microscopy (Nikon, Shanghai, China). Plasma biochemistry assays Plasma transaminase (ALT and AST) and triglyceride (TC and TG) concentration were measured individually by a Thermo Scientific Multiskan GO Microplate Spectrophotometer with commercial kits (Nanjing Jiancheng Bioengineering Institute, China). Plasma TNF-a, IL-6 and leptin protein levels were measured using ELISA (R&D Systems, Shanghai, China). RNA isolation and cDNA synthesis Liver samples were homogenized and total RNA was extracted using the TRIzol™ isolation reagent (Invitrogen) according to the manufacturer's recommendations. cDNA was synthesized from total RNA using a ReverTra Ace® qPCR RT kit (Toyobo, Shanghai, China) according to the manufacturer's instructions.
Materials and methods Real-time PCR Materials OEA was synthesized in our lab as previously described (15); fenofibrate, dimethyl sulfoxide (DMSO) and all other chemicals were obtained from SigmaeAldrich (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Shanghai, China). Lipofectamine® 2000 (Lipo2000) and Human PPAR-a Silencer Select siRNA (ID s10880) were purchased from Invitrogen (Shanghai, China). The chemical constituents were dissolved in saline supplemented with 5% polyethylene glycol 400 (PEG400) and 5% Tween-80 for the in vivo studies. For the in vitro studies, OEA was dissolved in DMSO to a concentration of 50 mmol/L (stock solution) and then diluted in the culture medium to a final concentration of 50 mmol/L. TNF-a and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Shanghai, China). Total cholesterol (TC), triglycerides (TG), alanine transaminase (ALT) and aspartate transaminase (AST) commercial assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Animals and diets Male Sprague Dawley (SD) rats (250e300 g) were sourced from Shanghai Laboratory Animal Center (Shanghai, China). All experimental protocols were approved by the Committee for Animal Research at Xiamen University. The rats were housed in a room with controlled temperature (21e23 C), humidity (55e60%) and lighting (12 h light/dark cycles) and given water ad libitum. Rats were fed a HFD diet (30% fat, 2% cholesterol, 2% sugar, 0.5% choline and 65.5% normal diet) or a normal diet (ND) for 6 weeks or 17 weeks to induce nonalcoholic fatty liver. The HFD-fed rats were randomly divided into three experimental groups (n ¼ 8 per group). One group was treated with OEA (5 mg/kg/day; intraperitoneal injection, i.p.) and another group was treated with fenofibrate (10 mg/kg/day; i.p.). The third group was treated with the vehicle
Quantification of mRNA was performed on an Applied Biosystems 7300 real-time polymerase chain reaction (PCR) system using SYBR® Premix Ex Taq™ II (Takara, Dalian, China). Primer sequences were synthesized as follows: TNF-a, 50 -GGC TCC CTC TCA TCA GTT CCA-30 (forward), 50 -CGC TTG GTG GTT TGC TAC GA-30 (reverse); IL-6, 50 -TGC CTT CTT GGG ACT GAT GTT G-30 (forward), 50 -TGG TCT GTT GTG GGT GGT ATC C-30 (reverse); C-reactive protein (CRP), 50 -TGT CTC TAT GCC CAC GCT GAT G-30 (forward), 50 -GGC CCA CCT ACT GCA ATA CTA AAC30 (reverse); CPT-1, 50 -AAC TTT GTG CAG GCC ATG ATG-30 (forward), 50 -GGC AGA AGA TGG CGG TCG-30 (reverse); enoyl-CoA hydratase (ECHS1), 50 -ATG GCT ATG CTC TTG GTG-30 (forward), 50 -GTG ATT TGC CGA CTG CTC-30 (reverse); PPAR-a, 50 -AAT CCA CGA AGC CTA CCT GA30 (forward), 50 -GTC TTC TCA GCC ATG CAC AA-30 (reverse); stearoylCoA desaturase-1 (SCD-1), 50 -ACA TGC TCC AAG AGA TCT CCA G-30 (forward), 50 -GTA CTC CAG CTT GGG CGG-30 (reverse); OB-Rb, 50 -GCA TGC AGA ATC AGT GAT ATT TGG-30 (forward), 50 -CAA GCT GTA TCG ACA CTG ATT CTT C-30 (reverse); glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 50 -ACC ACG AGA AAT ATG ACA ACT CCC-30 (forward), 50 -CCA AAG TTG TCA TGG ATG ACC-30 (reverse). The levels of mRNA were normalized relative to the amount of GAPDH mRNA. Lipid extraction and analysis Total lipids were extracted from liver samples according to the modified methods as previously described (16). C17:0 margaric acid was added as an internal standard for the quantitation of fatty acids. An Agilent 1200 Series HPLC (Agilent Corporation, MA, USA) interfaced to an Applied Biosystems 3200 Q-Trap triple-quadrupole linear ion trap mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Canada) was operated during experiments. Fatty acids were separated using a Hypersil Gold C18 column (dimensions: 250 4.6 mm; particle size: 5 mm; Thermo) eluted with a constant mobile phase that was composed of 5 mM ammonium acetate in acetonitrile (solvent A, 65%) and 5 mM ammonium acetate in 2-propanol (solvent B, 35%);
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
the flow rate was maintained at 700 mL/min for 15 min. Mass spectrometer detection was ionized by the full scan electrospray ionization mode (ESI) and monitored in multiple reaction monitoring (MRM) mode. The ion source temperature was maintained at 650 C. Quantifications were calculated using Analyst 1.4.1 data acquisition and processing software (Applied Biosystems/MDS Sciex). Cell culture and RNA interference HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cells were cultured in 6-well culture plates under 37 C and 5% CO2 in an incubator. siRNA was diluted with Opti-MEM® I to concentrations of 20 nmol/L. Lipo2000 was mixed gently, then diluted to 5 mL in 250 mL Opti-MEM® I and incubated at room temperature for 5 min. The diluted Lipo2000 and siRNA were mixed gently and incubated at room temperature for 20 min and the siRNAeLipo2000 complexes were added to cultured cells in 6-well culture plates and maintained at 37 C in a 5% CO2 atmosphere in an incubator.
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fat droplets, histology studies with H&E staining showed that severe steatosis with a amount of fat vacuoles appeared in the rat liver cells with HFD feeding for 17 weeks compared to that in normal diet-fed rats (Fig. 1A and B), and OEA administration (5 mg/ kg/day, 17 weeks, i.p.) reduced the vacuoles of the lipid droplets in liver cells of HFD-fed rats obviously (Fig. 1B and C). Consistent with a large number of lipid droplets accumulated in the liver, HFD induced the increase levels of triglyceride (TG) (Fig. 1D) and total cholesterol (TC) (Fig. 1E) in rat plasma (P < 0.001), which were reversed by OEA treatment (Fig. 1DeF, P < 0.001). Furthermore, biochemistry analysis showed that plasma alanine aminotransferase (ALT) (Fig. 1F) and aspartate aminotransferase (AST) (Fig. 1G), two key indicators of liver function damage, increased in HFD rats (P < 0.01), while OEA treatment significantly reduced the plasma levels of ALT and AST in HFD-fed rats (Fig. 1FeG, P < 0.001). OEA suppresses the HFD-induced inflammatory response
Results
Inflammation has been shown to involve in the developmental process of fatty liver. Clinical researches have shown the marked raises of TNF-a and IL-6 levels in the liver and plasma of NAFLD patients (17,18). To investigate OEA effect on HFD-induced inflammation, rats were fed with HFD for 17 weeks with/without OEA regimen (5 mg/kg, i.p.). Real-time PCR detection showed that HFD induced the mRNA expressions of inflammatory cytokines including CRP (Fig. 2A), TNF-a (Fig. 2B), and IL-6 (Fig. 2C) in the liver of SD rats, and the changes of cytokines by HFD were reversed by OEA treatment (Fig. 2AeC). In addition, biochemical analysis showed that the induction of the plasma levels of TNF-a (Fig. 3A) and IL-6 (Fig. 3B) by HFD were corrected by OEA treatment (Fig. 3A and B).
Protective effects of OEA on HFD-induced hepatic steatosis and liver injury
OEA promoted lipid catabolism genes in a PPAR-a-dependent manner
The main feature of HFD-induced NAFLD is the hepatic fat accumulation. To assess the impact of OEA on the accumulation of
To study the mechanism underlying OEA effect on improving the prognosis of NAFLD, the mRNA expression of the key genes
Statistical analysis All statistical analyses were performed using GraphPad Prism version 5.01 for Windows. Data are presented as the mean ± SEM and were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons. P < 0.05 was considered statistically significant.
Fig. 1. OEA prevented hepatocellular ballooning and reduced plasma transaminase and triglyceride content levels in HFD-induced NAFLD in rats. H&E staining showed hepatocellular ballooning in vehicle-treated rats fed a normal diet (A), vehicle-treated rats fed a high fat diet (B) and OEA-treated rats (5 mg/kg/day; i.p.) fed a high fat diet (C) after 17 weeks of treatment. Graphs DeG show the effect of the vehicle or OEA on blood plasma levels of triglyceride (TG) (D), total cholesterol (TC) (E), alanine aminotransferase (ALT) (F) and aspartate aminotransferase (AST) (G) in rats fed a normal diet or HFD after 17 weeks of treatment. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. ### P < 0.001 compared with the control group, ## P < 0.01 compared with the control group, *** P < 0.001 compared with the HFD group; one-way ANOVA, n ¼ 8.
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
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Fig. 2. OEA inhibited inflammation in livers of NAFLD rats. Liver CRP (A), TNF-a (B) and IL-6 (C) mRNA levels following treatment with the vehicle or OEA in rats fed a normal diet or HFD assessed by real-time quantitative PCR. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. # P < 0.05 compared with the control group, ### P < 0.001 compared with the control group, * P < 0.05 compared with the HFD group, ** P < 0.01 compared with the HFD group; one-way ANOVA, n ¼ 8.
involved in fatty acid b-oxidation in the liver, including PPAR-a, CPT-1, ECHS1, were determined. Real-time PCR analysis showed that HFD diet had no effect on the mRNA expressions of PPAR-a, and ECHS-1, and down-regulated the CPT-1 gene (Fig. 4AeC), while
administration of OEA (5 mg/kg/day, i.p) significantly increased the mRNA expression levels of PPAR-a (Fig. 4A), CPT-1 (Fig. 4B), and ECHS-1 (Fig. 4C). To further identify whether the above effect of OEA was mediated by the PPAR-a receptor, HepG2 cells were
Fig. 3. OEA decreased TNF-a and IL-6 levels in plasma of NAFLD rats. TNF-a (A) and IL-6 (B) protein levels in the plasma of rats fed a normal diet or HFD after vehicle or OEA treatment assessed by ELISA. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. # P < 0.05 compared with the control group, ### P < 0.001 compared with the control group, * P < 0.05 compared with the HFD group, ** P < 0.01 compared with the HFD group; one-way ANOVA, n ¼ 8.
Fig. 4. OEA normalized gene expression levels in HFD-induced NAFLD in rats. The effect of the vehicle or OEA on mRNA levels of PPAR-a (A), CPT-1 (B), ECHS1 (C) and SCD-1 (D), was assessed by real-time quantitative PCR after 17 weeks of treatment. Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. ## P < 0.01 compared with the control group, ### P < 0.001 compared with the control group, ** P < 0.01 compared with the HFD group, *** P < 0.001 compared with the HFD group; one-way ANOVA, n ¼ 8.
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
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Fig. 5. OEA inhibited SCD-1 enzyme activity in HFD-induced NAFLD in rats. The levels of palmitic acid (16:0) and palmitoleic acid (16:1) following treatment with the vehicle or OEA in rats fed a normal diet or HFD were detected by LCeMS. SCD-1 enzyme activity was deduced from the ratio palmitoleic acid to palmitate (16:1/16:0). Vehicle, 5% PEG400/5% Tween-80 in saline; OEA, 5 mg/kg/day, i.p. ## P < 0.01 compared with control group, * P < 0.05 compared with HFD group, one-way ANOVA, n ¼ 8.
transfected with/without PPAR-a siRNA 24 h before oleic acid (OA; 0.25 mmol/L) stimulated to create an in vitro NAFLD model. The mRNA expression levels of the nuclear receptor gene PPAR-a (Fig. 6A) and fatty acid b-oxidation gene CPT-1 (Fig. 6B) were examined by real-time PCR after OEA (50 mM) administration. In vitro results showed that the expression levels of both genes increased significantly after OEA stimulation (P < 0.01 and P < 0.01, respectively), while using PPAR-a RNAi, the lipogenetic promoting effects of OEA was completely disappeared. This suggested that OEA promoted fatty acid b-oxidation by activating PPAR-a pathway. OEA inhibited hepatic SCD-1 gene expression and protein activity in a PPAR-a-independent manner
that OEA not only reduced SCD-1 expression levels in the liver, but also inhibited its activity significantly especially controlled the desaturation of palmitic acid. In the same in vitro HepG2 cell model, we examined a change in SCD-1 expression levels (Fig. 6C) and found that OEA treatment down-regulated SCD-1 expression significantly (P < 0.001), while this inhibition effect of OEA on SCD-1 expression (P < 0.05) was still observed with PPAR-a RNAi treatment. However, without regard to OEA treatment, PPAR-a gene knockdown also reduced the SCD-1 gene expression levels to a high degree. These test results showed that the regulatory role of OEA with regard to SCD-1 downregulation was not mediated by the PPAR-a pathway. Roles of OEA and fenofibrate in the NAFLD model
As SCD-1 plays an important role on lipid homeostasis in the liver, we found that HFD induced the mRNA expression of SCD-1 (Fig. 4F) in rat liver. OEA administration (5 mg/kg/day, i.p.) completely corrected the HFD-induced SCD-1 expression in the liver. In addition, we further analyzed the enzyme activity of SCD-1, assessed by the ratio of palmitic acid (16:0 PA) to palmitoleic acid (16:1 PA), in the liver by the transition of fatty acid composition. Liquid chromatographyemass spectrometry (LCeMS) analysis were found that HFD significantly increase the contents of 16:1 PA in the liver, and the ration of 16:1 PA to 16:0 PA was increase by that to evaluate the SCD-1 enzyme activity. Fig. 5C showed that comparing to HFD treatment OEA administration (5 mg/kg/day; i.p.) inhibited the desaturation of palmitateplamitic acid, and the ratio of 16:1/16:0 was decreased (P < 0.05). These results suggested
During the 17-week animal experiments, we administrated fenofibrate (10 mg/kg/day, i.p.) as positive control simultaneous with a HFD treatment to compare the role of OEA and fenofibrate on NAFLD. The survival rate of rats treated with fenofibrate was remarkably affected during the treatment period. As shown in Fig. S1AeD, rats in the fenofibrate-treated group began to die at 14 weeks after drug administration, which had all died at 17 weeks. Compared to the fenofibrate-treated group, the remaining three groups, including the OEA-treated group, resulted in no animal death up to 17 weeks of treatment. To further compare the roles of fenofibrate and OEA, we re-designed a 6-week short-term experiment with the same groups and treatment protocols as performed in 17-week long-term experiment.
Fig. 6. OEA promoted fatty acid b-oxidation via PPAR-a signaling, suppressed SCD-1 expression in a PPAR-a independent manner. The effect of OEA combined with or without PPARa siRNA transfection on mRNA expression levels of PPAR-a (A), CPT-1 (B), SCD-1 (C), assessed by real-time quantitative PCR, on HepG2 cells. PPAR-a siRNA, 10 nM; OEA, 50 mM; Vehicle, 0.1%DMSO. * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA, n ¼ 6.
Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001
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L. Li et al. / Journal of Pharmacological Sciences xxx (2014) 1e7
During the 6-week experiment, no animal death was observed in all groups (Fig. S1EeH). OEA (5 mg/kg/day; i.p.) and fenofibrate (10 mg/kg/day; i.p.) administration showed similar pharmacodynamic effects with regard to less severe steatosis, decreased serum ALT, AST levels, meliorative liver inflammation (Figs. S2 and 3). RTPCR analysis suggested that the two compounds could activate PPAR-a and promote the b-oxidation of fatty acids (Fig. S4AeC). Hepatic lipid homeostasis needs a balance between lipid synthesis and metabolism. We then evaluated the related genes for de novo lipogenesis, and found that OEA and fenofibrate showed the opposite effects (Fig. S4DeF). OEA reduced SREBP-1c, SCD-1, and ACC mRNA expression levels (P < 0.05) during the 6-week period after drug administration, while fenofibrate treatment increased the above genes expression levels significantly (P < 0.001), which indicated that OEA inhibited the whole system of lipogenesis. Discussion In this paper we have explored whether the endogenous lipid OEA is a safe and effective lead compound to improve NAFLD development and the mechanism of action of OEA is dissected preliminary. Excessive consumption of HFD enhanced the development of hepatosteatosis. Therefore, HFD-induced animal model has been widely applied in NAFLD research (19). A 17-week longterm experiment has been conducted to accumulate enough lipid droplets in the liver and cause chronic liver injury for a better observation of the effects of OEA. In this animal model, OEA was found to play a critical role in lowering fatty degeneration of the liver, reducing hepatocellular damage and inhibiting inflammation effectively. PPAR-a is highly expressed in liver, where it regulates multiple genes encoding for enzymes involved in fatty acid b-oxidation (20,21). CPT-1 is located in the mitochondrial membrane, which can promote the transport of long-chain fatty acids into the mitochondria to further participate in the b-oxidation process and becomes a rate-limiting enzyme for fatty acid oxidation. It has been extensively shown that fenofibrate could induce higher CPT-1 expression in liver and adipose tissues by PPAR-a pathway (22,23). The present study showed that treatment with OEA increased the expression of CPT-1, as well as PPAR-a and ECHS1 to promote the fatty acid oxidation process. These results suggested, at least to some extent, the two PPAR-a agonists regulated liver fatty acid metabolism by promoting fatty acid b-oxidation. Stimulated de novo lipogenesis has been observed after treatment with PPAR-a agonists in many experiments (24,25). In the 6week short-term drug administration experiment, we did observe that fenofibrate significantly up-regulates the expression level of SCD-1 and SREBP-1c mRNA in the rat liver as the previous studies have been shown. But the experiment results of OEA were in contrast to the regulatory role of fenofibrate. Our results showed that the expression level of the SCD-1 gene in the liver increased significantly in animals after 6 and 17-week HFD treatment and OEA could down-regulate the expression of SCD-1 mRNA in liver tissues significantly. Additional investigation of liver SCD-1 enzyme activity showed that OEA also regulated SCD-1 activity and reversed the increased HFD-induced desaturation index (16:1/ 16:0) markedly. Leptin is a hormone secreted by adipose tissue that plays a crucial role in energy metabolism. Some findings suggested that down-regulation of SCD-1 is an important component of leptin's metabolic actions (26). To estimate whether leptin signaling pathway mediated the different regulatory role of OEA and fenofibrate on SCD-1 expression, 6-week animal experiment has been used to detect the plasma leptin levels and hepatic OB-Rb gene expression (Fig. S5). As a result, both OEA and fenofibrate decreased
plasma leptin levels and up-regulated the mRNA expression of OBRb in livers, which indicated that the opposite effect on SCD-1 expression was irrelevant to leptin signaling pathway. SCD-1 is also known to be regulated by PPAR-a and SREBP-1c (27,28). To estimate whether the action of OEA on hepatic SCD-1 was mediated via PPAR-a pathway, an in vitro experiment on HepG2 cell was conducted. The results indicated that PPAR-a knockdown suppressed the expression of SCD-1 gene, which acted in accordance with the known theory. But in PPAR-a RNAi group, OEA still decreased the expression level of SCD-1 (Fig. 6), which suggested that the down-regulation of SCD-1 by OEA was independent of PPAR-a pathway. It was noteworthy that in the 6-week experiment, distinct down-regulation of SCD-1, ACC as well as SREBP-1c mRNA was observed in OEA administration group, which suggested that SCD-1 expression maybe under SREBP-1c signaling control. Administration of fenofibrate increased hepatic expression of genes involved in fatty acid b-oxidation and at the same time also increased lipogenetic genes levels. These results are consistent with previous reports (24,25). Another conventional PPAR-a synthetic agonists, Wy-14643, has also showed a similar pharmacodynamic effect (29). However, despite promoting fatty acid b-oxidation, OEA inhibited the lipid synthetic genes expression, which would be important to avoid hepatic lipoperoxide accumulation. Maybe, it is the reason for high mortality occurrence only in the fenofibratetreated 17-week long-term group. To elucidate whether the effect of OEA on the expression of lipid metabolism genes specific under HFD condition, we examined the effect of OEA on above genes in rats fed normal diets (Fig. S6). After 6 h administration, real-time PCR detection shown that OEA significantly increased the mRNA levels of PPAR-a and CPT-1, obviously decreased the expression of SREBP-1c and SCD-1, but had no effect on liver ECHS1, AdipoR2 and OB-Rb expression. Similarly results were observed after 12 h administration. These results indicated that OEA could promote fatty acid b-oxidation and inhibit lipogenesis both in normal and HFD-fed rats. In conclusion, OEA, an endogenous PPAR-a agonist, has a beneficial effect on HFD-induced NAFLD in rats via promoting fatty acid b-oxidation, and inhibiting de novo lipogenesis. It showed a better liver protective role than fenofibrate, a known clinical PPARa agonist, in rat models. All these considerations suggest that OEA may represent a potential lead compound or a promising therapeutic target on NAFLD. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors thank Eye Institute of Xiamen University for assistance. This study was supported by grants from National Natural Science Foundation of China (No. 81373273), Natural Science Foundation of Fujian Province (No. 2013J05122, 2013J01274). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jphs.2014.12.001. References (1) Birkenfeld AL, Shulman GI. Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology. 2014;59(2):713e723.
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Please cite this article in press as: Li L, et al., Effect of oleoylethanolamide on diet-induced nonalcoholic fatty liver in rats, Journal of Pharmacological Sciences (2014), http://dx.doi.org/10.1016/j.jphs.2014.12.001