PPAR\xce\xb1 protects against trans-fatty-acid-containing diet-induced steatohepatitis
Consumption of trans-fatty acids (TFA), unsaturated fatty acids (FA) containing trans double bonds, is a risk factor for metabolic syndrome and steatohepatitis. Peroxisome proliferator-activated receptor \xce\xb1 (PPAR\xce\xb1) is a master regulator of hepatic lipid homeostasis. To examine the contribution of PPAR\xce\xb1 to changes in liver phenotypes induced by TFA, two diets were used: a purified control diet and an isocaloric diet in which most of the soybean oil, a major source of FA in the diet, was replaced with TFA-rich shortening. The diets were fed to wild-type and Ppara-null mice for 2 months. Ppara-null mice fed a TFA-containing diet showed more severe hepatic steatosis and liver damage compared with similarly treated wild-type mice, as revealed by increased hepatic triglyceride (TG) contents and serum alanine aminotransferase activities. While the TFA-rich diet increased the hepatic expression of enzymes involved in de novo FA synthesis and decreased TG-hydrolyzing enzymes in both genotypes, the expression of FA-catabolizing enzymes was decreased in Ppara-null mice, resulting in more severe hepatosteatosis. Additionally, the expression levels of key contributors to inflammation, such as osteopontin, were increased, and nuclear factor-kappa B was activated in TFA-containing diet-fed Ppara-null mice. Enhanced inflammatory signaling in these mice was presumably mediated by toll-like receptor 2, with no accompanying inflammasome activation. Collectively, these results suggest a protective role for PPAR\xce\xb1 in the pathological changes in the liver following TFA consumption. PPAR\xce\xb1 might prevent TFA-containing diet-induced steatohepatitis.
Introduction<!>Mice and treatment<!>Biochemical analysis<!>Histological analysis<!>Analysis of mRNA expression<!>Immunoblot analysis<!>Statistical analysis<!>TFA-containing diet-fed Ppara-null mice develop more severe hepatic steatosis and inflammation compared with the wild-type mice<!>Reduced FA utilization is associated with steatogenesis in TFA-containing diet-treated Ppara-null mice<!>Increased hepatic inflammation in TFA-containing diet-treated Ppara-null mice is related to NF-\xce\xbaB activation mainly mediated by TLR2 but not by inflammasome<!>Discussion
<p>Unsaturated fatty acids (FA) generally have two forms of double bonds, cis and trans. In cis-FA, the two carbon atoms forming double bonds bind two hydrogen atoms on the same side, leading to a curved space conformation. However, the two carbon atoms of the double bonds in trans-FA (TFA) bind two hydrogen atoms on the diagonal side of the carbon chain, resulting in a linear conformation [1]. Although natural foods, such as animal meat or dairy products, contain very few natural TFA, large amounts of TFA are detected in shortening, bread, margarine, cream cakes, fried fast food and some other processed foods. Partially hydrogenated vegetable oils are often used by the food industry to enhance the stability and palatability of foods, and TFA are produced by the partial hydrogenation of vegetable oils [1,2]. Therefore, TFA are recognized as an artificial lipid closely associated with dietary habits in developed countries.</p><p>It was reported that TFA are more harmful to humans than other FA. Notably, TFA increase blood viscosity, leading to thrombosis, and elevate circulating low-density lipoprotein cholesterol and decreases high-density lipoprotein cholesterol, promoting arteriosclerosis and coronary heart disease [3,4]. Additionally, several clinical studies demonstrated the possible association between excessive TFA intake and metabolic syndrome, insulin resistance, obesity, Alzheimer's disease, depression and infertility [5–8]. Therefore, in North American and European countries, governments have enacted legislation to require the food processing industry to stop using TFA in food production. However, in Asia, TFA are still widely used in processed foods [9].</p><p>Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disorder that is increasing worldwide. and its progressive subtype, nonalcoholic steatohepatitis (NASH), can eventually develop into cirrhosis and/or hepatocellular carcinoma [10–12]. Several key factors, such as lipotoxicity, oxidative stress, aberrant immune system and inflammatory signaling, are known to contribute to the progression from steatosis to steatohepatitis. For example, toll-like receptors (TLRs) recognize a variety of pathogen-associated molecular patterns, activating immune responses [13]. The fact that disruption of TLR2 or TLR4 attenuated NASH phenotypes indicated a crucial role of TLR-mediated signaling in the pathogenesis of NAFLD/NASH [14]. Additionally, up-regulation of proinflammatory mediators, such as tumor necrosis factor α (TNFα), monocyte chemoattractant protein 1 (MCP1) and osteopontin (OPN), is closely associated with several types of liver injury [15]. The mechanism of NASH is multifactorial and complicated.</p><p>Some studies have shown that high TFA intake promotes fatty acid/triglyceride (TG) synthesis, decreases TG export and inhibits FA β-oxidation, leading to hepatosteatosis [16,17]. Since high TFA consumption induces enhanced proinflammatory cytokine levels and microcirculatory disturbances resulting in hepatic inflammation [16,18,19], excessive dietary TFA may play an important role in the development of NAFLD/NASH.</p><p>Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated nuclear receptor that regulates lipid transport and catabolism and also influences inflammation and immune responses [20–23]. However, the role of PPARα in TFA-induced liver abnormalities remains unclear. In the present study, wild-type and Ppara-null mice were treated for 2 months with a control diet or an isocaloric diet where most of the FA are replaced with TFA, and liver phenotypes were examined.</p><!><p>All animal experiments were conducted in accordance with animal study protocols outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and approved by Shinshu University School of Medicine. Ppara-null mice on an Sv/129 genetic background and the wild-type counterparts were maintained under controlled conditions (25°C; 12-h light/dark cycle) with tap water and standard laboratory chow ab libitum. Male 10- to 14-week-old wild-type and Ppara-null mice weighing 25–35 g were selected, and each genotype was divided into two groups (n=3–6/group) [20,23]. To eliminate the effect of high fat/calorie due to adding TFA and avoid essential FA deficiency, 60 g of soybean oil (70 g in total in 1000 g of purified AIN93G diet) was replaced by the same weight of Primex, a regular shortening containing large amount of TFA. The TFA-containing diet and control AIN93G diet were made by Research Diets, Inc. (New Brunswick, NJ, USA; #D14020301 and #D10012G, respectively); detailed nutrient/FA composition of the diets is shown in Supplementary Tables 1 and 2, respectively. Calorie per weight in the TFA-containing diet was identical to that in AIN93G diet. As such, one group in each genotype was treated with the isocaloric TFA-containing AIN93G diet and the other group was given the control AIN93G diet for 2 months. After the treatment, the mice were anesthesized and killed by CO2 asphyxiation at 6 h after food withdrawal, and blood and tissues were collected. Blood was centrifuged at 3000 rpm for 15 min twice, and the serum was collected. Sera and tissues were immediately frozen and kept in −80°C freezer until used.</p><!><p>Serum alanine aminotransferase (ALT), TG, total cholesterol, nonesterified FA, phospholipid, and glucose were measured with enzyme assay kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Liver lipids were extracted by hexane:isopropanol method as described previously and measured using enzymatic assay kits (Wako Pure Chemical Industries) [24].</p><!><p>Liver tissue was fixed in 10% neutral formalin, embedded in paraffin, cut into 4-μm sections and stained with the hematoxylin and eosin dyes [25].</p><!><p>Total liver RNA was extracted using an RNeasy Mini Kit (Qiagen, Tokyo, Japan) and reverse-transcribed to cDNA using oligo-dT and random primers with SuperScript II reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA). Levels of mRNA were measured by quantitative real-time polymerase chain reactions (qPCR) using an SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan) on a Thermal Cycler Dice TP800 system (Takara Bio). The mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an internal control. Sequences of primers are shown in Supplementary Table 3 [26,27]. The mRNA levels were normalized to Gapdh mRNA levels and expressed as a fold change relative to those of wild-type mice fed a control diet.</p><!><p>Preparation of whole liver homogenates was conducted as described previously [20–23,28], and liver nuclear fractions were isolated with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentrations were measured colorimetrically with the BCA Protein Assay kit (Pierce, Rockford, IL, USA). Whole liver homogenates (20–60 μg of protein in each lane) were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The polyacrylamide concentration in the gel was 6%, 10% or 12.5%, which was dependent on molecular weight of the target protein. For analysis of the nuclear factor-kappa B (NF-κB) p65 component, nuclear proteins (30 μg in each lane) were subjected to 10% SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose filter membrane (Amersham Hybond-P; GE Health Care, Little Chalfont, UK). The membranes were blocked for 1 h with 5% bovine serum albumin or 5% nonfat dry milk in Tris-buffered saline and incubated overnight with the respective primary antibody. Primary antibodies against FA-binding protein 1 (FABP1), long-chain acyl-coenzyme A (CoA) synthase (LACS), and long-chain and very-long-chain acyl-CoA dehydrogenases (LCAD and VLCAD, respectively) were described previously [29]. The following primary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): FA synthase (FAS), #16147, 1:200 dilution; acetyl-CoA carboxylase α (ACCα), #30212, 1:200 dilution; CD36, #9154, 1:200 dilution; microsomal TG transfer protein (MTP), #33116, 1:200 dilution; and histone H1, #10806, 1:200 dilution. The following primary antibodies were purchased from Cell Signaling Technology (Denver, MA, USA): adipose TG lipase (ATGL), #2439, 1:1000 dilution; TLR2, #13744, 1:1000 dilution [25,26]; and p65, #8242, 1:1000 dilution. The antibodies against OPN and β-actin were obtained from Abcam (Cambridge, MA, USA): OPN, #8448, 1:1000 dilution and β-actin, #8227, 1:1000 dilution. After four washes, the membranes were incubated with alkaline-phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; #93,785, 1:1000 dilution) and treated with 1-Step NBT/BCIP Substrate (Pierce, Rockford, IL, USA). The true band positions were determined by co-electrophoresing molecular weight standards (Bio-Rad, Hercules, CA, USA), and the band of β-actin or histone H1 was used as a loading control. Immunoblotting was repeated twice for each protein, and each band intensity was quantified using NIH image J (National Institutes of Health, Bethesda, MD, USA), normalized by those of loading control and subsequently expressed as a fold change relative to those of wild-type mice fed a control diet.</p><!><p>Results are expressed as mean ± standard error of the mean (S.E.M.). Statistical analysis was performed using two-way analysis of variance with Bonferroni's correction. A P value of less than .05 was considered to be statistically significant.</p><!><p>Wild-type and Ppara-null mice were examined 2 months after commencing TFA-containing diet treatment, revealing that enlarged and yellowish livers were seen only in Ppara-null mice. While body weight and epididymal fat pad weight were similar between the groups, liver weights showed a significant increase in Ppara-null mice treated with the TFA diet compared with similarly treated wild-type mice (Fig. 1A). In these mice, serum ALT levels, indicators of liver injury, were significantly increased in TFA diet-fed Ppara-null mice (Fig. 1B). Serum TG was also increased in TFA-diet-treated Ppara-null mice compared with control-diet-treated Ppara-nulls, but the increase did not reach statistical significance compared with TFA-diet-fed wild-type mice (P=.08) (Fig. 1B). Serum levels of glucose, nonesterified FA and total cholesterol were not different between the groups (data not shown). Hepatic TG contents were significantly increased compared with TFA-diet-treated wild-type mice and control-diet-fed Ppara-null mice (Fig. 1B). Histological analysis of the liver revealed very mild macrovesicular steatosis only in TFA-diet-fed wild-type mice. However, in TFA-diet-fed Ppara-null mice, abundant macrovesicular lipid droplets and inflammatory foci were detected throughout the section (Fig. 1C). These results demonstrate that more severe steatosis and inflammation were observed in Ppara-null mice fed the TFA-containing diet than were observed in wild-type mice.</p><!><p>To investigate the mechanism of severe hepatosteatosis in TFA-rich diet-fed Ppara-null mice, the expression of genes associated with FA and TG metabolism in the liver was measured. The mRNA levels of genes encoding FAS and ACCα (Fasn and Acaca, respectively), which are involved in de novo FA synthesis, were increased after TFA diet treatment in both genotypes with no significant differences between genotypes (Fig. 2A). The mRNA levels of Cd36, which plays an important role in FA uptake from blood, showed significant increases in Ppara-null mice compared with wild-type mice in both diets (Fig. 2A). Immunoblot analysis confirmed increased hepatic expression of FAS/ACCα following TFA diet (Fig. 2B and C), indicating PPARα-independent enhancement of FA synthesis due to TFA-containing diet.</p><p>The mRNA expression of genes encoding FABP1 (Fabp1) and LACS (Acsl1), which are related to intracellular FA carriage and activation, respectively, was significantly decreased in TFA-diet-fed Ppara-null mice compared with their similarly treated wild-type counterparts (Fig. 3A). Decreased expression was also observed for Acadl and Acadvl mRNAs, which encode the FA-oxidizing enzymes LCAD and VLCAD, respectively (Fig. 3A). These PPARα-dependent changes in FA-eliminating enzymes/proteins were also observed at the protein level (Fig. 3B and C).</p><p>There were no significant differences in the mRNA levels of genes related to TG synthesis (Dgat1 and Dgat2, encoding diacylglycerol acyltransferase 1 and 2, respectively) and TG export (Mttp, encoding MTP) (Fig. 4A). While the mRNA levels of Pnpla2 and Lipe, encoding TG-hydrolyzing enzymes ATGL and hormone-sensitive lipase, respectively, were decreased after TFA diet administration in both genotypes, there were no significant differences between the wild-type and Ppara-null mice (Fig. 4A). Significant down-regulation of ATGL expression in both genotypes after TFA consumption (Fig. 4B and C) suggested that TFA-containing diet suppressed lipolysis in the liver independently of PPARα.</p><p>Overall, although enhanced de novo lipogenesis and attenuated lipolysis in the liver were likely associated with TFA consumption, reduced FA utilization was a main cause of more severe steatosis in the TFA-containing diet-fed Ppara-null mice.</p><!><p>In order to examine the mechanism of more severe hepatitis in TFA-containing diet-fed Ppara-null mice, hepatic expression of typical proinflammatory mediators was determined. The mRNA levels of Tnf and Ccl2, encoding TNFα and MCP1, respectively, were increased in TFA-rich diet-fed Ppara-null mice (Fig. 5A). The mRNA levels of Spp1 and Col1a1, encoding OPN and collagen 1a1, respectively, which promote hepatic inflammation/fibrosis, were also increased in these mice (Fig. 5A). Marked up-regulation of OPN was also confirmed at the protein level (Fig. 5B and C). The expression of mRNAs encoded by these key genes is induced by activation of NF-κB [30]. Indeed, increased nuclear p65 levels were detected in TFA-containing diet-administered Ppara-null mice compared with wild-type mice (Fig. 5B and C), indicating that NF-κB was activated in the former mice.</p><p>NF-κB is activated by several upstream signals, such as oxidative stress, endoplasmic reticulum (ER) stress, inflammasome and TLR-mediated pathway [30]. The expression of mRNAs encoding reactive oxygen species-generating/eliminating enzymes and ER-stress-induced transcription factor was not different between TFA-containing diet-fed wild-type and Ppara-null mice (Supplementary Fig. 1). The mRNAs encoded by inflammasome-related genes, such as Nlrp3, Nlrc4, Aim2, Pycard, Casp1 and Il1b, were not increased in TFA-diet-treated Ppara-null mice (Fig. 6). Alternatively, among the genes associated with the TLR-mediated pathway, Tlr2 mRNA and TLR2 protein levels were significantly increased in TFA-diet-treated Ppara-null mice compared with similarly treated wild-type mice (Fig. 7). Therefore, severe hepatic inflammation observed in TFA-containing diet-treated Ppara-null mice is associated with NF-κB activation likely due to enhanced TLR2-mediated signaling.</p><!><p>TFA are artificially produced in the process of food hydrogenation and are more harmful to the body than naturally-occurring FA [2,31]. The current study investigated the role of PPARα in TFA-induced liver abnormalities. Previous studies examined the effect of dietary TFA by adding TFA to the regular diet or by using a TFA-rich high-fat diet [16,17,32,33]. However, it is unclear whether the pathological changes induced by a TFA-rich high-fat diet are due to TFA toxicity or the high fat/calorie contents. In the present study, a special isocaloric AIN93G diet was made by replacing most of the soybean oil with shortening containing large amounts of TFA and keeping a minimal amount of essential FA in order to more precisely assess the physiological effects of TFA. The isocaloric TFA-containing diet-fed Ppara-null mice had severe steatosis and inflammation, activated NF-κB and enhanced inflammatory signaling. These results indicate that mice lacking PPARα are more prone to steatohepatitis following TFA consumption, and thus PPARα may protect livers against dietary TFA-induced toxicity.</p><p>It was reported that TFA intake enhanced de novo lipogenesis [16,33,34]. This finding was consistent with results of the present study that hepatic FAS/ACCα expression was increased after TFA-containing diet consumption in both genotypes. Additionally, the expression of TG-hydrolyzing enzymes, i.e., Pnpla2/Lipe mRNA and ATGL protein levels, was decreased after the TFA-rich diet. ATGL is a main lipolytic enzyme in the liver [26]. Augmented de novo FA synthesis and suppressed lipolysis are likely associated with TFA-induced hepatic TG accumulation. However, remarkable reductions in the expression levels of the proteins involved in intracellular FA transport and FA β-oxidation, such as FABP1, LCAD and VLCAD, were detected only in Ppara-null mice. A significant imbalance between lipogenesis, lipolysis and FA utilization is likely the main mechanism for steatogenesis in TFA-containing diet-fed Ppara-null mice.</p><p>Elevated serum ALT, enhanced expression of proinflammatory molecules and liver histology indicated the presence of hepatitis in TFA-diet-treated Ppara-null mice. NF-κB is a pivotal transcription factor in regulating genes involved in the inflammatory response, and its activation is driven by a wide variety of stimuli, such as growth factors, cytokines, bacteria/virus and cellular stress [30,35]. TLRs are among the key mediators of NF-κB signaling [36]. In mammals, TLR2 and TLR4 were closely associated with the development of steatohepatitis [14,37], and some studies proposed that certain lipids, such as saturated FA, can activate TLR2 signaling in hepatic Kupffer cells, ultimately leading to NF-κB activation and steatohepatitis [38]. The findings in the present study provide intriguing possibilities that TFA can directly activate TLR2 signaling in hepatocytes/Kupffer cells under the PPARα-deficient state and that PPARα modulates FA-TLR linkage. Future studies to treat TFA with isolated hepatocytes/Kupffer cells from Ppara-null mice might be of value to address these questions.</p><p>The present study revealed significant increases in OPN expression in the livers of Ppara-null mice given the TFA-rich diet. OPN is a Th1 cytokine which plays an important role in the development of various inflammatory and fibrotic diseases [15]. In a mouse model of NASH induced by a methionine- and choline-deficient diet, hepatic OPN expression was markedly and initially increased, and its disruption notably attenuated NASH severity [39]. Some studies demonstrated that alterations in hepatocyte metabolism and fat accumulation in the liver facilitate the increases in OPN [40]. Additionally, a recent study observed that TLR2, but not TLR4, up-regulated OPN production [41]. Therefore, OPN may be a key contributor to the development of NASH in TFA-containing diet-fed Ppara-null mice.</p><p>Inflammasomes, intracellular multiprotein complex, are also major mediators of inflammatory response. Exogenous and endogenous danger signals, such as ATP, cholesterol/uric acid crystal and lipopolysaccharide, activate caspase-1, which cleaves prointerleukin (IL)-1β into mature one [42,43]. It was reported that saturated but not unsaturated FA can activate inflammasomes in hepatocytes and induce IL-1β release [44], which may be in accordance with the present results since TFA are structurally classified as unsaturated FA. The association between inflammasome, TFA and PPARα needs to be addressed in the future.</p><p>Since PPARα is down-regulated with the progression of steatohepatitis [11,45], the present results suggest that dietary TFA are more harmful to NASH patients and that dietary TFA may accelerate NASH progression. Restriction of TFA intake and TFA-rich westernized diets may be useful to attenuate liver injury in NAFLD/NASH patients. Additionally, therapeutic interventions to maintain PPARα function might be beneficial to reduce TFA toxicity.</p><p>In conclusion, the present results demonstrate that PPARα disruption is more sensitive to steatohepatitis following TFA consumption, indicating that PPARα plays a protective role for dietary TFA-induced liver abnormalities.</p>