Lipidomics of hepatic lipogenesis inhibition by omega 3 fatty acids

Prostaglandins, Leukotrienes and Essential Fatty Acids 88 (2013) 149–154

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Lipidomics of hepatic lipogenesis inhibition by omega 3 fatty acids Antonin Lamaziere a, Claude Wolf a, Ullah Barbe b, Pedro Bausero b, Francesco Visioli c,n a b c

Laboratory of Mass Spectrometry-APLIPID, Faculte´ de Me´decine Pierre et Marie Curie, ER7-UPMC, Paris, France UR4, Universite´ Pierre et Marie Curie, Paris, France Laboratory of Functional Foods, Madrid Institute for Advanced Studies (IMDEA)—Food, Madrid, Spain

a r t i c l e i n f o

abstract

Article history: Received 24 September 2012 Received in revised form 7 December 2012 Accepted 9 December 2012

We assessed – by a lipidomic approach – the differential incorporation of EPA and DHA into hepatic lipids, after prolonged feeding of rats with fish oil. We also evaluated their effect on lipogenesis and its related enzymes. Rats were administered 100 mg/kg/d fish oil, by oral gavage, for 30 days. The fatty acid profile of total liver lipids was determined by gas–liquid chromatography coupled to mass spectrometry. Individual phospholipid classes and their molecular species were quantified by ESI-MS/ MS. Omega 3 fatty acids readily incorporated into hepatic phospholipids, decreased stearoyl-CoA desaturase 16, stearoyl-CoA desaturase, delta 6 desaturase, and delta 5 desaturase activities (calculated as product/substrate ratio) and decreased the ‘‘lipogenesis index’’, i.e., the proportion of fatty acids endogenously synthesized in the liver and not provided with the diet. Our results show that long-chain omega 3 fatty acids selectively incorporate into hepatic phospholipids, inhibit de novo lipogenesis and change the hepatic fatty acid profile via reduced desaturases’ activity in the non-steatotic liver. In addition to corroborating advice to consume adequate amounts of omega 3 fatty acids for overall health, these data contribute mechanistic insights to the clinical observations that provision of omega 3 fatty acids decreases hepatic fat and ameliorates NAFLD prognosis. & 2012 Elsevier Ltd. All rights reserved.

Keywords: Omega 3 fatty acids Liver Lipidomics Phospholipids

1. Introduction The long-chain omega 3 docosahexaenoic (DHA; 22:6n-3) and eicosapentaenoic (EPA; 20:5n-3) fatty acids are essential in that they cannot be synthesized de novo by mammals and must be supplied in adequate amounts through the diet or the use of supplements/functional foods from marine origin [1]. The cardiovascular activities of long-chain omega 3 fatty acids have been extensively studied: most scientific and medical societies, namely those operating in the cardiovascular and neurological fields, recommend intakes of long-chain omega 3 fatty acids of Z500 mg/d (www.issfal.org). In addition to their activities on the cardiovascular system, omega 3 fatty acids are being proposed as preventive and/or adjuvant agentsin other degenerative pathologies such as neurological disorders and for optimal visual development [2–4]. One recent therapeutic application of omega-3 fatty acids is in the field of non-alcoholic fatty liver disease (NAFLD) [5], partly because of their proven benefit in lowering serum triacylglycerols (TG). Indeed, NAFLD is characterized by the abnormal accumulation of fat in the liver, where it is mainly stored as TG [6].

n Correspondence to: IMDEA-Food, CEI UAM þ CSIC, C/Faraday 7, 28049 Madrid, Spain. Tel.: þ 34 91 2796986; fax: þ 39 02700426106. E-mail addresses: [email protected], [email protected] (F. Visioli).

0952-3278/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plefa.2012.12.001

Therefore, the hypothesis has been formulated that provision of omega 3 fatty acids might ameliorate NAFLD by limiting the deposition of TG into the liver and by decreasing inflammation. Yet, little is known on the incorporation of omega 3 fatty acids into individual hepatic phospholipids. Purposely, alterations of the fatty acid composition of liver phospholipids might be employed to monitor de novo lipogenesis. In a recent review, steatosis was attributed to: (i) insulin resistance-dependent higher peripheral lipolysis and fatty acid flux to the liver; (ii) long chain omega 3 polyunsaturated fatty acid depletion-induced changes in DNA binding activity of sterol regulatory elementbinding protein 1c (SREBP-1c) and peroxisome proliferatoractivated receptor a (PPAR-a), favoring lipogenesis over fatty acid oxidation; and (iii) hyperinsulinemia-induced activation of the lipogenic factor PPAR-g [7]. In this investigation we assessed – by a lipidomic approach – the differential incorporation of EPA and DHA into hepatic lipids, after prolonged feeding of rats with a fish-oil supplement. We also evaluated its effect on lipogenesis and its related enzymes.

2. Materials and methods The complete protocol has been previously described by Lamaziere et al. [8]. In brief, eight week-old male Wistar rats (n ¼7 per group), weighing 250 g, were purchased from Janvier

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A. Lamaziere et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 88 (2013) 149–154

Fig. 1. Variations of saturated and monounsaturated (A), o6 (B), and o3 (C) fatty acids in livers of controls (C, n¼ 7) or fish oil (o3, n¼ 7)-treated rats. Values above the histograms indicate the percent mean variations between control and treated rats. Data are means7 SD. *p o0.05, **p o0.01, and ***p o 0.001.

Europe (St. Berthevin, France) and were housed in a temperatureand humidity-controlled room with a 12-h-light–dark cycle. Feed ¨ (ssniff Spezialdiaten GmbH, Soest, Germany) and water were provided ad libitum. The feed was devoid of EPA and DHA, but contained 2.45% and 0.47% (of total fatty acids) of linoleic and a-linolenic acids, respectively. Rats were administered, by oral gavage, 100 mg/kg/day of fish oil (Eskims, Sigma-Tau, which contains 84% of long-chain omega 3 fatty acids, of which 38% is DHA and 46% is EPA). Control rats (n ¼7) received liquid paraffin. This treatment was continued for 30 days; thereafter, rats were anesthetized with ketamine and xylazine and their livers were rapidly removed, flash-frozen in liquid nitrogen, and then kept at 80 1C. Total lipids were extracted from livers by the method of Folch et al. [9]. Individual phospholipid classes and their molecular species were quantified by ESI-MS/MS (API3000, TQ, Applied BiosystemsSciex, Concord, Ontario, Canada) [8]. The method has been fully described by Wolf et al. [10,11] and by Lamaziere et al. [8]. The fatty acid profile of total liver lipids was determined by fatty acid methyl ester (FAME) analysis. Total lipid extract was transesterified in 2 mL of methanol in the presence of 2% (vol/vol) H2SO4 at 70 1C for 1 h. FAMEs were analyzed by capillary gas– liquid chromatography coupled to mass spectrometry (GC-MS) in the chemical ionization mode (Agilent 5975, Agilent

Technologies, Inc., Santa Clara) and Hewlett Packard 6890; reagent gas: ammonia) as previously described by Boutet et al. [12]. Heptadecanoic acid methyl ester was used as the internal standard. The response factors of the various fatty acids were calculated with a weighted methyl ester calibrator (Mix37, Supelco). Total proteins were quantified according to Bradford et al. [13]. 2.1. Statistical analyses Statistical analyses were performed using the XLSTAT-Pro software, version 2012.2.02 (Addinsoft, France). By principal component analysis (PCA) we examined the variations of the molecular species in the lipid class profiles (given as percentage) induced by omega 3 supplementation [8]. Data processing and multivariate analysis were performed according to Quinn et al. [14].

3. Results Analysis of total liver fatty acids (Fig. 1) shows hepatic enrichment in eicosapentaenoic (20:5o3, EPA) and docosahexaenoic (22:6o3, DHA) acids. Interestingly, while provision of fish oil only marginally raises the cerebral content of EPA [8], we recorded an 184.5% increase in hepatic lipids (Fig. 1, Panel C).


Conversely, we recorded significant decreases of arachidonic (20:4o6, AA), palmitoleic (16:1o7, PoA) and, to a lesser extent, oleic (18:1o9, OA) and vaccenic (18:1o7, VA) acids. The total amount of monounsaturated fatty acids (MUFAs) decreased significantly following supplementation, whereas total o3polyunsaturated fatty acids (PUFAs) increased (Fig. 2). Saturated (SFA) and total PUFAs were not significantly modified by the treatment (Fig. 2). Taken together, these data show alteration of the hepatic fatty acid profile, but only modest changes in the total liver lipid content after a month of dietary supplementation with fish oil.

Fig. 2. Variation of total hepatic fatty acids according to their unsaturation degree. Values above the histograms indicate the percent mean variations between control and treated rats. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data are means7 SD. *po 0.05, **p o 0.01, and ***po 0.001.

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We assessed the metabolic fatty acid modifications that occurred following the supplementation, by calculating a ‘‘lipogenesis index’’, i.e., the proportion of fatty acids endogenously synthesized in the liver and not provided with the diet. This index [(C16:1)þ (C18:1n-7)þ(C20:3 n-9)]/[(total fatty acids)] was dramatically, i.e., 64.6%, decreased by fish oil provision for one month and mirrored the decreased ( 56.4%) o6/o3 ratio in total lipids. Treatment with fish oil did not appreciably modify sphingomyelin and its molecular species after a month. Conversely, feeding fish oil to rats led to a significant increase in the proportion of DHA-containing phosphatidylcholine (PC) species in the liver (Fig. 3, Panel A). In particular, the DHA-containing PC species PC38:06 and PC40:06 increment balances the decrease in the SAT- and MUFA-enriched PC species such as PC 32:00, PC32:01, and PC34:01. Of note, the DHA increment in PC did not correlate with the variations of arachidonic acid (AA)-containing PC species such as PC36:04 and PC38:04 (Fig. 3, Panel B). Fish oil-induced alterations were also noted in hepatic phosphatidylethanolamine (PE), a lipid class serving as reservoir of PUFA in the liver. Notably, the variability of control data points along the principal component F2 (16.1% of the total variability) was considerably reduced by the supplementation. As shown in Fig. 4, one-month supplementation led to an increase in DHAcontaining PE species PE40:06, PE38:6, and PE38:06, and PEs with 9 double-bonds (40:09 and 42:09) which was diametrically opposite to the decrease in AA-containing PE species PE38:04, PE36:4, and PE40:04 (Fig. 4, Panel B). This suggests a substitution of DHA-containing species for AA-containing PE molecular species (Fig. 4, Panel B). At difference with the monounsaturated PC molecular species, the monounsaturated PEs (PE34:01 and PE36:01) did not correlate with DHA-enriched species (PE40:06, PE38:6, and PE38:06). We also evaluated lyso-phosphatidylcholine (lyso-PC), a recently recognized source of omega 3 for the brain [15]. We report that fish oil provision markedly enriched lyso-PC with long-chain omega 3 fatty acids (Fig. 5, Panel A). Specifically, lysoPC22:6 and lyso-PC20:5 were increased at the expenses of lysoPC20:4. To gain insight into the fish oil-induced modulation of desaturases, i.e., the enzymes that influence fatty acids conversions,

Fig. 3. Principal Component Analysis (panel A score plot and panel B load plot) of phosphatidylcholine (PC) molecular species of livers isolated from control (C, n ¼7) and fish oil-supplemented (o3, n¼ 7) rats. Each data point represents the molecular species profile of PC in a separate animal liver.

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Fig. 4. Principal Component Analysis (panel A score plot and panel B load plot) of the phosphatidylethanolamine (PE) molecular species of livers isolated from control (C, n¼7) and fish oil-supplemented (o3, n ¼7) rats. Each data point represents the molecular species profile of PE in a separate animal liver.

Fig. 5. Principal Component Analysis (panel A score plot and panel B load plot) of the lyso-phosphatidylcholine (LPC) molecular species of livers isolated from control (C, n¼7) and fish oil-supplemented (o3, n ¼7) rats. Each data point represents the molecular species profile of lyso-PC in a separate animal liver.

we assessed their activities by calculating the product/substrate ratio (Fig. 6). Omega 3 supplementation induced a significant decrease in stearoyl-CoA desaturase 16 ( 78.5%), stearoyl-CoA desaturase 18 ( 52.2%), delta 6 desaturase ( 53.2%), and delta 5 desaturase ( 53.2%) activities. Notably, such enzymes and their activities are involved in the biosynthetic pathways leading to the production of non-essential MUFAs (SCD16 and SCD18) and of omega 6 essential PUFAs (D6D and D5D).

4. Discussion In this paper, we report the phospholipid-specific incorporation of long-chain omega 3 fatty acids in rat liver. Supplementation was carried out for one month, which is the time frame used by primary

care physicians and specialists to appreciate the first signs of symptom amelioration in NAFLD patients. Indeed, fish oil is being proposed as adjuvant therapy for some hepatic disorders such as NAFLD, in which abnormal lipid accumulation and increased inflammation are seen [6]. Even though we did not target NAFLD, the rationale behind the preventive and/or therapeutic use of omega 3 fatty acids in liver pathologies is manifold; as mentioned, DHA and EPA have TG-lowering [16] and anti-inflammatory properties [3]. Antioxidant actions [1,17], which also involve the liver [18], might likewise contribute to accelerate the resolution of various oxidative and inflammatory conditions. In the current study, we also report that fish oil reduces lipogenesis and the omega 6/omega 3ratio after one-month supplementation. Of note, the overall hepatic lipid content in healthy, i.e., non steatotic animals was not influenced by the administration of fish


Fig. 6. Variation of desaturases’ activities: SCD16, Stearoyl-CoA desaturase 16 (C16:1n-7/C16:0); SCD18, Stearoyl-CoA desaturase 18 (C18:1n-9/C18:0); D6D, Delta 6 Desaturase (C18:3n-6/C18:2n-6); and D5D, Delta 5 Desaturase (C20:4n-6/ C20:3n-6). Values above the histograms indicate the percent mean variations between control and treated rats *p o 0.05, **p o 0.01, and ***p o 0.001.

oil (Fig. 1). Therefore, our results can be applied to the general population, with the aim of using adequate amounts of EPA and DHA as a preventive measure toward steatosis. Whether these activities can also concern the steatotic livers of high-fat diet animals will require ad-hoc investigations. In humans, long-chain omega-3 supplementation may indeed decrease liver fat; however, the optimal dose is currently not known [19]. Mechanistically, it has been proposed that omega 3 fatty acids inhibit lipogenesis by inhibiting the LXR-SREBP-1c system [20]. This hypothesis has been confirmed in vitro [21], but, to our knowledge, this is the first study that shows reduced lipogenesis in vivo after a short-term provision of omega 3 fatty acids. Our findings are consistent with studies by Ntambi et al. [22], who showed that adipocyte-derived MUFAs are involved in the downregulation of inflammation in macrophages [23], with oleate being more potent than palmitoleate. In this respect, it is noteworthy that provision of omega 3 fatty acids halved the hepatic content of MUFAs (Fig. 2) and, in particular, that of 16:1 (Fig. 1, Panel A). Of note, we show that omega 3 fatty acids reduced desaturases, i.e., D6D and D5D activities, which we calculated as the product/substrate ratio (Fig. 6). These enzymes are involved in the synthesis of arachidonic acid (AA) from its precursor linoleic acid. The availability of AA as a substrate of cyclo-oxygenases is one of the limiting steps in the promotion of inflammation by eicosanoids. Interestingly, a biopsy-based study of NAFLD patients has reported that the omega 6/omega 3 ratio significantly correlated with the quantity of hepatic triglycerides [24]. This sub-optimal metabolic status of NAFLD patients is likely the result of low omega 3 dietary intakes rather than excessive omega 6 consumption. In our study, just one month of supplementation with omega 3 fatty acids decreased the hepatic omega 6/omega 3 ratio (Fig. 2), which likely translates into lower pro-inflammatory substrate availability. In addition, we report that EPA and DHA preferentially substitute for AA in hepatic phosphatidylethanolamine (PE), which is a favored substrate for phospholipases A2 (PLA2) including sPLA2 [25]. We have previously reported that DHA inhibits sPLA2 via reduction of NAD(P)H oxidase activation and reactive oxygen species (ROS) generation [17]. Of note, lyso-PC, which participates in the initiation and/or progression of atherosclerosis and inflammation through multiple mechanisms [26], was also

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enriched in EPA and DHA after fish oil provision. Lyso-PC increases oxidized LDL association with hepatocytes [27], hence heightening the risk of micro-vascular disease. Therefore, we speculate that omega 3 supplementation – because of the combination of a reduced availability of AA in PLA2 substrates; lower reactive oxygen species (ROS) production afforded by the antioxidant activity of omega 3 fatty acids; and the generation of biologically less active lipid mediators – might contribute to lessen inflammation in hepatocytes and decrease the risk of developing NASH. In terms of lyso-PC formation, it is noteworthy that lysophospholipids can be produced in the liver by the actions of phospholipases A1 and A2. Thereafter, lyso-PC can be transported (bound to serum albumin) and incorporated into the brain via specific LPC transporters [28]. Therefore, the region-selective enrichment in cerebral phospholipids we have previously reported [8] might be partially facilitated by the release of omega 3 lyso-PC from the liver [29]. In conclusion, long-chain omega 3 fatty acids selectively incorporate into hepatic phospholipids, inhibit de novo lipogenesis and change the hepatic fatty acid profile via reduced desaturases’ activity in the non-steatotic liver. In addition to corroborating the advice to consume adequate amounts of omega 3 fatty acids for overall health, these data contribute mechanistic insights to the clinical observations that provision of omega 3 fatty acids decreases hepatic fat and ameliorates NAFLD prognosis [30–32].

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