Differential Effects of Unsaturated Fatty Acids and Saturated Fatty Acids on Lipotoxicity and Neutral Lipid Accumulation in Neuro-2a Cells and Fatty Acids Lipotoxicity and Neutral Lipid Accumulation in Neuro-2a

Long-chain free fatty acids (FFA) play many important roles in cell growth and metabolism. Accumulation of excess saturated fatty acids (SFA) leads to deleterious lipotoxic effects in non-adipose tissues while unsaturated fatty acids (UFA) often exert protective effects against SFA lipotoxicity, yet the lipotoxic effects of SFA in neuronal cells have not been well characterized. This study examined the differential effects of SFA and UFA on the viability of Neuro-2a (N2a) cells and the accumulation of neutral lipids in these cells. Our study found that all the UFA tested, namely oleic acid (OA), linoleic acid (LA), α-linolenic acid (ALA), and docosahexaenoic acid (DHA), were able to abolish PA-induced decrease in cell viability regardless of the position of the double bond or degree of unsaturation, and that 200 μM LA, OA, and DHA significantly enhanced the amount of neutral lipid staining than BSA control while PA did not, suggesting that LA, OA, and DHA, but not PA, increased the amount of neutral lipid synthesis and accumulation. The neutral lipid staining also appeared more in particulates in UFA-treated cells than PA-treated cells, suggesting that UFA, but not PA, enhanced LD formation. We also found that the amount of neutral lipid staining in cells co-treated with UFA and PA was comparable to that in cells treated with BSA or PA alone, and that the neutral lipid staining in cells co-treated with UFA and PA appeared more concentrated in particulates than PA-treated cells, suggesting that UFA may not enhance neutral lipid accumulation, but may increase LD formation in PA-treated cells. Our results suggest that UFA and SFA have differential effects on cell viability, neutral lipid accumulation, and LD formation in N2a cells. Further studies will be needed to examine the role of LD formation in UFA protection against PA Effects of Acids Saturated on

18:3n-3), and docosahexaenoic acid (DHA; 22:6n-3) [1]. After entry into cells, fatty acyl CoA synthetase catalyzes the conversion of fatty acids into fatty acyl-CoA, which may then be catabolized to generate energy or anabolized to produce a range of molecules including second messengers, hormones, and diacylglycerols (DAG). DAG may enter the phospholipid synthesis pathways or be converted to triacylglycerols (TAG), which may be subsequently incorporated into lipid droplets (LD) [2].
One of the hallmarks of metabolic diseases is the accumulation of excess fatty acids in non-adipose tissues [3], which may lead to deleterious lipotoxic effects including cellular dysfunction and death [4]. PA has been reported to induce lipotoxicity in many cell types, including cardiomyocytes [5], hepatocytes [6], and neural stem cells [7,8]. In contrast, ω-3 fatty acids enhance hippocampal neurogenesis and promote synaptic plasticity [9]. OA ameliorates PA-induced endoplasmic reticulum (ER) stress in exocrine pancreas cells [10] and HepG2 cells [11]. LA also protects cultured mouse embryonic cortical neurons from glutamate-induced excitotoxicity [12]. There is evidence suggesting that UFA-induced esterification and sequestration of PA into neutral lipids and LD may contribute to their lipoprotective effects. LD, consisting of a neutral lipid core containing TAG, sterol esters, and fatty acids surrounded by a monolayer of phospholipids embedded with numerous proteins, is found in many types of cells. LD not only acts as lipid storage sites, but also participates in membrane biogenesis, hormone synthesis, intracellular signaling, protein storage, and protein degradation [13,14].
LD biogenesis and degradation has been suggested to play important roles in buffering the intracellular levels of toxic lipid species [14]. For example, arachidonic acid (ARA; C20:4n-6)induced LD formation is associated with its protection against PA-induced lipotoxicity in C2C12 myocytes [15]. OA-induced TAG synthesis and LD formation is associated with its protection against PA-induced lipotoxicity in primary syncytiotrophoblasts [16], CHO cells [17], and β-cells [18]. Fatty acid accumulation is reportedly enhanced in the brains of metabolic syndrome patients [19]. While neurons are not energy-storing cells and their use of fatty acids as an energy source is minimal, they do contain LD [20]. High fat diet causes obesity and loss of myenteric neurons in Swiss mice [21].
High fat diet also affects the hypothalamic proteome indicative of cellular stress, altered synaptic plasticity and mitochondrial function in mice [22]. This study was therefore designed to examine how SFA and UFA differentially affect cell viability and neutral lipid accumulation in murine neuroblastoma Neuro-2a (N2a) cells.

Reagents
LA, OA, PA, ALA, DHA, and fatty acid-free bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). To prepare BSA-conjugated fatty acids, fatty acids were dissolved in 100% ethanol at 400 mM. FAs were then conjugated with BSA in PBS at 2.5:1 molar ratio to make 5 mM FA solutions [23]. Conjugated fatty acids were then filtered, aliquoted, and stored at -80°C.

MTT Assay
Cell viability was determined based on the ability of cellular

Annexin V Assay
Cells were seeded in 12-well plates at 1.0 x 10 5 cells/well.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay
TUNEL assay was performed following manufacturer's instructions. Briefly, cells were seeded in chamber slides at 2.5 x

Neutral Lipid Quantitation by Flow Cytometry
Cells were seeded in 24-well plates at a density of 0.5

Neutral Lipid Imaging
For bright-field imaging of neutral lipids, cells were seeded on coverslips and treated as indicated.

Statistical Analysis
Data were analyzed using GraphPad Prism 6 (Graphpad Software; San Diego, CA, USA) and presented as mean ± SEM. Oneway or two-way ANOVA followed by Tukey's multiple comparison test was used to determine statistical significance. p < 0.05 was considered statistically significant.

PA Treatment Reduced the Viability of N2a Cells
To      (C) Relative quantitation of lipid accumulation in N2a cells treated with different concentrations of FFA or BSA control for 6h as analyzed by flow cytometry following BODIPY™ 493/503 staining. Presented data (mean ± SEM) were representative of three independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test. *p < 0.05 vs. corresponding BSA control; **p < 0.01 vs. corresponding BSA control; ***p < 0.001 vs. corresponding BSA control. staining, which confirmed that the amount of neutral lipid staining in cells co-treated with UFA and PA was comparable to that in PAtreated cells (Figures 6B & 6C). These studies suggest that UFA may induce LD formation in PA-treated cells even though UFA did not seem to enhance the total amount of neutral lipid staining in these cells in PA-treated cells. A.

UFA did not Significantly Increase the Amount of Neutral Lipid Accumulation in PA-Treated Cells
Representative images of N2A cells co-treated with different FFA together with 200 μM PA or BSA for 6 h, stained with Nile red (red) and DAPI (blue), and visualized under confocal microscopy.

B.
Representative flow cytometry histograms of cells co-treated with different FFA together with 200 μM PA or BSA as control for 6 h and stained with BODIPY™ 493/503.

Discussion
In the brain, lipids are involved in many functions including its development, neurogenesis, synaptogenesis, myelin sheath formation, and signal transduction [24]. Alterations of lipid homeostasis are associated with brain injury and with metabolic and neurologic disorders [25]. The goal of this study was to examine how UFA and SFA differentially affected the viability and neutral lipid accumulation in N2a cells. Our studies showed that PA, a SFA, significantly decreased N2a cell viability and induced significant cell death while the UFA, such as LA, OA, ALA, and DHA, rescued PA-induced decrease of N2a viability. We also found that UFA significantly increased neutral lipid accumulation and LD formation while PA did not. Moreover, the amount of neutral lipid staining in cells co-treated with UFA and PA was comparable to that in BSA-and PA-treated cells with the staining more in particulates in cells co-treated with UFA and PA, suggesting LD formation.
PA has been shown to induce ER stress in pancreatic β cells [26] and osteoblast-like Saos-2 cells [27] as well as oxidative stress in endometrial cells [28] and hepatocytes [29]. Inhibition of oxidative stress by NAC, an antioxidant, and ER stress by 4-PBA reduces PAinduced apoptosis in H9c2 cardiomyoblast cells [30]. However, neither NAC nor 4-PBA had any significant impact on PA-induced decrease of viability in N2a cells, suggesting that ER stress and oxidative stress may not play a key role in PA-induced lipotoxicity in N2a cells. There have been studies reporting that UFA-promoted sequestration of excess PA into neutral lipids and LD may represent a mechanism by which UFA protects against PA lipotoxicity [15][16][17][18]. In this study, we showed that LA, OA, and DHA, but not PA, significantly increased neutral lipid accumulation and LD formation. However, the amount of neutral lipid staining in cells cotreated with UFA and PA was not significantly different from that in PA-treated cells, suggesting that UFA may not significantly enhance the total amount of neutral lipid accumulation in PA-treated cells.
Much of the neutral lipid staining in cells co-treated with UFA and PA did appear more concentrated in particulates than PAtreated cells, suggesting that UFA induced LD formation in PAtreated cells. Future studies may further explore whether UFAinduced LD formation may contribute to UFA-mediated protection against PA lipotoxicity. An alternative mechanism may involve ceramide formation in PA lipotoxicity. PA has been reported to increase ceramide content in murine enteroendocrine cells, which is reduced by OA [31]. Both cell culture and animal studies suggest that DHA may attenuate PA-or high fat diet-induced ceramide lipotoxicity [32].
In summary, our study demonstrated that PA decreased cell viability and induced cell death in N2a cells. PA-induced lipotoxicity was not alleviated by pre-treatment with NAC, an antioxidant, or 4-PBA, an inhibitor of ER stress, suggesting that oxidative stress and ER stress may not play a key role in PA lipotoxicity in N2a cells. In contrast, all the UFAs tested, namely LA, OA, ALA, and DHA, protected N2a cells against PA lipotoxicity. We also found that LA, OA, and DHA, but not PA, significantly enhanced neutral lipid accumulation and LD formation, that UFA did not enhance the total amount of neutral lipid accumulation in PA-treated cells, and that UFA may induce LD formation in PA-treated cells.