Individual Saturated and Monounsaturated Fatty ...

J Nutrigenet Nutrigenomics 2013;6:1–15 DOI: 10.1159/000345913 Received: September 19, 2012 Accepted: November 13, 2012 Published online: February 6, 2013

© 2013 S. Karger AG, Basel 1661–6499/13/0061–0001$38.00/0 www.karger.com/jnn

Original Paper

Individual Saturated and Monounsaturated Fatty Acids Trigger Distinct Transcriptional Networks in Differentiated 3T3-L1 Preadipocytes Brittany Shaw a Samuel Lambert a Monica H.T. Wong b Jessica C. Ralston a Carolina Stryjecki a David M. Mutch a Departments of a Human Health and Nutritional Sciences and b Mathematics and Statistics, University of Guelph, Guelph, Ont., Canada

Key Words Adipocyte · Microarray · Fatty acid · Inflammation · RANTES Abstract Background/Aims: Saturated fatty acids (SFA) are widely thought to induce inflammation in adipose tissue (AT), while monounsaturated fatty acids (MUFA) are purported to have the opposite effect; however, it is unclear if individual SFA and MUFA behave similarly. Our goal was to examine adipocyte transcriptional networks regulated by individual SFA (palmitic acid, PA; stearic acid, SA) and MUFA (palmitoleic acid, PMA; oleic acid, OA). Methods: Differentiated preadipocytes were treated with either 250 μM PA, SA, PMA, or OA for 48 h. Gene expression was analyzed using microarrays and real-time RT-PCR. Data were compared with those of a previous study reporting AT gene expression in humans following the consumption of SFA- or MUFA-enriched diets. Results: Individual fatty acid treatments had significant effects on adipocyte gene expression. Functional analyses revealed that PA induced the TLR signalling pathway, while PMA had the opposite effect. SA and OA had similar effects, with increases in key metabolic pathways including mTOR and PPAR signalling and a reduction in TLR signalling. Ccl5 was validated as a candidate gene that may mediate the differential inflammatory effects of SFA and MUFA in AT. Conclusions: Individual SFA and MUFA trigger distinct transcriptional responses in differentiated preadipocytes, with inflammatory and metabolic pathways particularly sensitive to these fatty acids. Copyright © 2013 S. Karger AG, Basel

B.S. and S.L. contributed equally to this work and should be considered co-first authors. David M. Mutch Department of Human Health and Nutritional Sciences University of Guelph Guelph, ON N1G 2W1 (Canada) E-Mail dmutch @ uoguelph.ca

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J Nutrigenet Nutrigenomics 2013;6:1–15 DOI: 10.1159/000345913

© 2013 S. Karger AG, Basel www.karger.com/jnn

Shaw et al.: Individual Saturated and Monounsaturated Fatty Acids Trigger Distinct Transcriptional Networks in Differentiated 3T3-L1 Preadipocytes

Introduction

Lifestyle factors such as diet and physical activity have important roles in the development of obesity and obesity-related complications by affecting numerous physiological and metabolic processes [1]. Moreover, it is now widely appreciated that the aforementioned lifestyle factors influence these processes by regulating the underlying genetic network in a tissue-specific manner [2, 3]. Adipose tissue (AT) has an important role as the primary site for storing excess energy in the form of triglycerides and as an endocrine organ that secretes hundreds of proteins, termed adipokines, that regulate a spectrum of processes including appetite, energy expenditure, insulin sensitivity, bone metabolism, inflammation, and immunity [4]. Because of its critical contribution to whole-body energy metabolism, AT is an important tissue to target for the study of diet-gene interactions. Due to the widespread consumption of energy-dense fatty foods, there is considerable interest to elucidate how common dietary fats may alter physiological processes. Dietary fatty acids – saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) – have distinct influences on a variety of biological pathways that are associated with the development of obesity-related complications [5–7]. For example, studies have shown that fatty acids can influence such processes as angiogenesis [8], inflammation [9], and insulin sensitivity [10]. Additionally, the consumption of different fatty acidenriched diets has been found to alter global AT gene expression [11] as well as that of genes coding for adipokines [12, 13]. However, to the best of our knowledge no studies have compared and contrasted the impact of individual fatty acids on global expression profiles in adipocytes or AT. Most of the targeted research examining the effects of individual fatty acids in adipocytes has focused on n-3 PUFA. In contrast, there are few examples in which the roles of SFA and/ or MUFA have been studied. van Dijk et al. [11] reported that the 8-week consumption of a SFA-enriched diet increased the expression of inflammatory genes in subcutaneous AT, while the consumption of a MUFA-enriched diet had the opposite effect. These same authors recently extended their work to show that SFA- and MUFA-enriched diets also differentially regulated transcriptional profiles in peripheral blood mononuclear cells [14]. While these recent studies clearly demonstrate the distinct impact that SFA and MUFA can have in the body, it is important to recognize that these diets are not composed of only a single SFA or MUFA but rather of an assortment of different fatty acids that vary in concentration. To date, only the independent role of palmitic acid (PA; 16: 0) has been extensively studied in AT model systems. PA is reported to influence adipocyte inflammation via activation of the tolllike receptor 4 (TLR4) pathway and NF-κB [15, 16], as well as by promoting the recruitment of macrophages into AT [9, 16]. It is unknown whether other SFA act similarly in AT. The goal of the current study was to examine the most common dietary SFA and MUFA individually in order to determine their shared and unique bioactivity in adipocytes. To accomplish this goal, we used microarrays to examine changes in global gene expression in differentiated preadipocytes treated with individual SFA (PA; stearic acid, SA) or MUFA (palmitoleic acid, PMA; oleic acid, OA). We subsequently compared our results with those of a previously published AT transcriptomics dataset by van Dijk et al. [11], in which the impact of a SFA-enriched diet was compared to a MUFA-enriched diet in human AT. Combining these two datasets enabled us to identify a list of candidate genes that we have classified as ‘fatty acid sensitive’. The knowledge stemming from this work will help to better understand the biological roles of individual fatty acids and how their presence in the diet may contribute to the development of obesity and/or obesity-related complications.

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J Nutrigenet Nutrigenomics 2013;6:1–15 DOI: 10.1159/000345913

© 2013 S. Karger AG, Basel www.karger.com/jnn

Shaw et al.: Individual Saturated and Monounsaturated Fatty Acids Trigger Distinct Transcriptional Networks in Differentiated 3T3-L1 Preadipocytes

Methods Cell Culture Reagents Murine 3T3-L1 preadipocytes were obtained from the ATCC (Rockville, Md., USA). Cell culture reagents including Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin-ethylenediamine tetraacetic acid solution, and penicillin-streptomycin were purchased from Hyclone laboratories (Logan, Utah, USA). Dexamethasone (Dex), 3-isobutyl-1-mexthylxanthine (IMBX), human insulin, fatty acid-free bovine serum albumin (BSA; ≥98% purity), fetal bovine serum (FBS), and all fatty acids were purchased from Sigma Aldrich (St. Louis, Mo., USA). Cell Culture Experiments 3T3-L1 preadipocytes were seeded at a density of 6.0 × 104 cells per well in 6-well plates. Cells were cultured at 37 ° C in 5% CO2 in a basic medium consisting of DMEM supplemented with 5% heat-inactivated FBS and 1% penicillin-streptomycin. Differentiation was induced at 2 days after confluence (i.e. day 0) by adding a standard differentiation cocktail consisting of Dex (1 μM), IMBX (0.5 mM), and insulin (5 μg/ml) to the basic medium. After 2 days, the differentiation medium was replaced with a maintenance medium, which consisted of basic media supplemented with only insulin (5 μg/ml). The maintenance medium was changed every 2 days for the remainder of the experiment. On day 7, FBS was removed from the medium, and the duration of the experiment was conducted in serum-free conditions. Stocks (25 mM) of freshly prepared PA (16:0), PMA (16:1n7), SA (18:0), and OA (18:1n9) were dissolved in 100% ethanol (EtOH; Sigma Aldrich). Fatty acid stock solutions were subsequently diluted 1: 100 in a serum-free maintenance medium containing 2% (wt/vol) BSA for a final concentration of 250 μM. This dose is well within the physiological range of fatty acid concentrations reported for humans [17] and has been used previously in 3T3-L1 cell culture work [13]. Differentiated preadipocytes were treated for 48 h with the various fatty acid solutions starting on day 8. The control treatment consisted of cells treated with 2% (wt/ vol) BSA and a 1:100 dilution of 100% EtOH for 48 h. RNA Extraction and Quality Assurance After the treatment period, total RNA was extracted from differentiated preadipocytes using the Qiagen RNeasy Mini Kit (Qiagen, Mississauga, Ont., Canada) according to the manufacturer’s protocol. Extracted RNA was quantified using a Nanodrop (Fisher Scientific, Waltham, Mass., USA) and stored at –80 ° C for future analyses. RNA quality was verified using the Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Santa Clara, Calif., USA), and only samples with an RNA integrity number greater than 9.0 were used for microarray analyses. Microarray Analyses A total of 4 microarrays were run for each fatty acid treatment. We experienced difficulties with the cRNA amplification for one replicate from the OA-treated cells; therefore, this treatment group only had 3 microarrays. For each sample, 100 ng of total RNA was prepared for hybridization to Affymetrix Mouse Gene 1.1 ST array strips, according to the manufacturer’s instructions (Affymetrix Inc., Fremont, Calif., USA). This array contains over 770,000 oligonucleotide probes corresponding to 21,041 unique Entrez Gene IDs. Briefly, total RNA was used to synthesize cDNA and subsequently cRNA. Second-cycle cDNA was produced, fragmented, biotin-labelled, and hybridized to microarrays. Strips were then washed, stained, and scanned on the GeneAtlas platform. The microarray dataset can be downloaded from the NCBI Gene Expression Omnibus (GSE42220). Microarray Data Analysis Microarray data were imported into R/Bioconductor [18] and normalized using the robust multichip average (RMA) implementation in the Affy package [19]. Differential gene expression values were calculated for each treatment using the linear models for microarray data (limma) package [20]. Statistical significance was assessed using the Bayes-moderated t statistic, and a false discovery rate-adjusted p value was used to account for multiple testing. A gene was considered differentially expressed if the global adjusted p value was