Role of CD36 in Palmitic Acid Lipotoxicity in Neuro-2a Neuroblastoma Cells

Elevated level of palmitic acid (PA), a long-chain saturated fatty acid (SFA), is lipotoxic to many different types of cells including Neuro-2a (N2a) neuroblastoma cells. CD36 is a multifunctional membrane glycoprotein that acts as a fatty acid translocase (FAT) facilitating the transport of long-chain free fatty acids (FFAs) into cells, serves a fatty acid (FA) sensing function in areas including taste buds and the proximal gut, and acts as a scavenger receptor that binds to many ligands, including FAs, collagen, oxidized low-density lipoproteins, and anionic phospholipids. However, the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells remains unclear. In this study, we examined FA uptake in BSA- and PA-treated N2a cells and investigated the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells. Our data showed that PA treatment promoted FA uptake in N2a cells, and that treatment with sulfo-N-succinimidyl oleate (SSO), a CD36 inhibitor, significantly decreased FA uptake in BSA- and PA-treated N2a cells, and ameliorated PA-induced decrease of cell viability, decrease of diploid cells, and increase of tetraploid cells. We also found that CD36 knockdown significantly decreased FA uptake in both BSA- and PA-treated cells as compared to their corresponding wild-type controls, and dramatically attenuated PA-induced cell cycle defects in N2a cells. Our data suggest that CD36 may play a critical role in FA uptake and PA lipotoxicity in N2a cells. CD36 may therefore represent a regulatory target against pathologies caused by excess FAs.

While there are reports that long-chain FFAs may diffuse across the plasma membrane [18], protein-mediated transport of long-chain FFAs across the plasma membrane represents an important mechanism to bring long-chain FFAs into cells [19,20]. Several long-chain fatty acid transporters have been identified, including fatty acid transport proteins (FATPs), plasma membrane-associated fatty acid-binding protein (FABPpm), and cluster of differentiation 36/fatty acid translocase (CD36/FAT) [20]. After entry into cells, FAs are then converted into fatty acyl-CoA by fatty acyl CoA synthetase. Fatty acyl-CoA may then be catabolized to generate energy or anabolized to produce a range of molecules such as second messengers, hormones, and diacylglycerols [21].

Establishment of N2a Cells Stably Transfected with CD36 shRNA
In order to examine whether CD36 knockdown affected FA uptake, N2a cells were stably transfected with MISSION ® CD36 shRNA (TRCN0000066518) with MISSION ® pLKO.1-puro non-mammalian shRNA plasmid as control from Sigma-Aldrich (St. Louis, MO, USA). Stable transfection of N2a cells using Lipofectamine TM 3000 (ThermoFisher Scientific; Waltham, MA, USA) was conducted following manufacturer's protocol. Briefly, N2a cells were seeded in a 96-well tissue culture plate at 1 × 10 4 cells/well and incubated overnight. On the following day, 100 ng of CD36 shRNA plasmid or non-mammalian shRNA control plasmid was diluted in 5 µL Opti-MEM TM media, combined with 0.2 µL P3000™ Reagent, then added 0.15 µL Lipofectamine TM 3000 reagent diluted in 5 µL Opti-MEM TM media, and incubated at room temperature for 10 min. This DNA-lipid complex was then added to N2a cells in EMEM with 10% FBS, incubated for another 24 h, passaged at 1:300 into 96-well plates, and then selected for puromycin resistance in complete media Biomolecules 2021, 11, 1567 3 of 14 with 5 µg/mL puromycin. The selective media was refreshed every 3-4 days. After 2 weeks of selection, the large healthy colonies were then selected to expand in selective media.

BODIPY TM FL C12 Uptake Assay by Flow Cytometry
To examine FA uptake in N2a cells, BODIPY TM FL C12 (ThermoFisher Scientific; Waltham, MA, USA), a fluorescent FA analog, was used. BODIPY TM FL C12 was deemed to resemble the length of C16 FA such as PA since the BODIPY TM FL fluorophore contributes an additional four-carbon length of acyl chain while BODIPY TM FL C16 would resemble the length of C20 FA. Furthermore, the complex lipid product profile of 3 H-PA most closely resembles that of BODIPY TM FL C12 based on metabolic tracing performed in zebrafish [29]. Therefore, BODIPY TM FL C12 was deemed as a suitable fluorescent PA analog. BODIPY TM FL C12 uptake assay by flow cytometry was performed as described previously [10]. Briefly, N2a cells were incubated with 15 µM BODIPY TM FL C12 in serumfree media for 30 min at 37 • C. Following 30 min incubation, cells were then collected by trypsinization, resuspended in ice-cold D-PBS, and immediately analyzed by flow cytometry. Flow cytometry events were then gated, and data were analyzed using FlowJo10 (FlowJo; Ashland, OR, USA).

BODIPY TM FL C12 Uptake Assay by Confocal Microscopy
To visualize FA uptake under confocal microscopy, the fluorescent PA analog BODIPY TM FL C12 was used and the uptake assay was performed as described previously [10]. Briefly, N2a cells were treated with BSA or 200 µM PA for 6 h, then incubated with 15 µM BODIPY TM FL C12 for 30 min, washed with PBS, and fixed with 4% paraformaldehyde for 20 min at 4 • C. Cells were then washed with PBS again and stained with 1 µg/mL DAPI for 10 min, and then visualized under FluoView FV1000 confocal microscope (Olympus; Center Valley, PA, USA).

RNA Isolation
Total RNA from N2a cells was isolated using the TRIzol reagent (Sigma-Aldrich; St Louis, MO, USA) following the manufacturer's instructions. The prepared RNA samples were dissolved in RNase-free water and stored at −80 • C.

Semi-Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Assay
To assess the effects of PA on CD36 expression, RT-PCR was conducted as described before [29][30][31]. Briefly, cDNA was synthesized from 1 µg of total RNA using oligo (dT)12-18 primer and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI), and then used to detect mouse CD36 and β-actin, a house-keeping gene, by PCR amplification. Appropriate sense and antisense primers specific for mouse CD36 and β-actin were synthesized by Eurofins Genomics (Huntsville, AL, USA). The forward (F) and reverse (R) primers for CD36 were 5 -CTCACTGGAGGAAACTGCTATC-3 (F) and 5 -CTCCAGAGAGGGAGAGACTTAATA-3 (R). The forward (F) and reverse (R) primers for β-actin were 5 -AGCCATGTACGTAGCCATCC-3 (F) and 5 -CTCTCAGCTGTGGTGGTGAA-3 (R). PCR reactions were conducted in a final volume of 20 µL containing 1 µL of cDNA, 1X PCR buffer, 0.2 µM of each forward and reverse primer, 0.2 mM of dNTPs, and 0.5 unit of Taq DNA polymerase (Applied Biosystems; Foster City, CA, USA) [29][30][31]. The reaction was heated to 94 • C for 5 min, followed by denaturation at 94 • C for 30 s, annealing at 57 • C for 30 s, and extension at 72 • C for 30 s for 31 cycles and 21 cycles for CD36 and β-actin respectively. After the final cycle, a 7-min extension step at 72 • C was included. PCR products were then resolved by gel electrophoresis on 2.0% agarose gel and the gel image was recorded using the FluorChem system (Protein Sample; San Jose, CA, USA). The band intensities of CD36 were digitized using VisionWorks TM LS software (UVP; Upland, CA, USA) and normalized against that of β-actin in the same sample.

MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5 Diphenyl Tetrazolium Bromide) Assay
To assess the effects of PA on cell viability, 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT; Sigma-Aldrich; St. Louis, MO, USA) assay was used based on the principle that actively respiring cells convert MTT to insoluble purple formazan. MTT assay was performed as previously described [9]. Briefly, cells were seeded in 96-well plates at 1.0 × 10 4 cells/well. After overnight incubation, cells were treated with 200 µM PA or BSA for 24 h. At 2 h prior to the end of treatment, 10 µL of 5 mg/mL MTT was added into each well and incubated for another 2 h to allow sufficient conversion of MTT into formazan precipitates, which were then solubilized in 100 µL of isopropanol with 0.04 M HCl. Absorbance was then read at 570 nm on the VarioSkan Lux microplate reader (ThermoFisher Scientific; Waltham, MA, USA) with absorbance at 650 nm as a reference to correct for nonspecific background absorption. Relative cell viability was calculated as the net absorbance of treated cells divided by the net absorbance of BSA-treated control cells.

Cell Cycle Analysis
To assess the effects of PA on the cell cycle of N2a cells, cell cycle analysis was performed by flow cytometry following staining with 4 ,6-diamidino-2-phenylindole (DAPI). Cells were seeded in 12-well plates at 1.0 × 10 5 cells/well. After overnight incubation, cells were treated with 200 µM PA or BSA in serum-free media for 6 h. At the end of treatment, cells were collected by trypsinization, rinsed with D-PBS, and then fixed in 70% ethanol on ice for at least 1 h. Ethanol-fixed cells were then spun down, rinsed with D-PBS, resuspended in D-PBS, and then incubated with 1 µg/mL DAPI staining solution for 30 min in the dark on ice. DAPI fluorescence was then analyzed by flow cytometry on the MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec; Auburn, CA, USA). Flow cytometry events were then gated, and data were analyzed using FlowJo10 (FlowJo; Ashland, OR, USA).

Detection of Cell Surface Expression of CD36 by Immunostaining and Flow Cytometry
In order to confirm shRNA knockdown of CD36, N2a cells were stained with APC-Vio ® 770-conjugated anti-mouse CD36 antibody (Miltenyl Biotec Inc.; Auburn, CA, USA) for cell surface expression of CD36 and examined by flow cytometry following the manufacturer's instructions. Wild-type N2a cells, non-mammalian control plasmid-transfected cells, and CD36 shRNA-transfected cells were collected, resuspended at 0.5 × 10 6 cells per 100 ul of FACS staining buffer (PBS with 0.5% BSA and 2 mM EDTA), and incubated with APC-Vio ® 770-conjugated anti-mouse CD36 antibody at 1:50 dilution for 15 min at 4 • C. After labeling with CD36 antibody, cells were washed and resuspended in FACS buffer and analyzed by flow cytometry. Flow cytometry events were then gated, and data were analyzed using FlowJo10 (FlowJo; Ashland, OR, USA).

Bicinchoninic acid (BCA) Protein Assay
In order to determine protein concentrations in cell lysates, BCA protein assay kit (ThermoFisher Scientific; Waltham, MA, USA) was used following the manufacturer's instructions. Briefly, 10 µL of each albumin standard or 1:10 diluted protein sample was mixed with 200 µL BCA working reagent in a 96-well plate, covered, and incubated at 37 • C for 30 min. The plate was then read on the VarioSkan Lux microplate reader (ThermoFisher Scientific; Waltham, MA, USA) at 562 nm.

Western Blot Analysis
To examine the protein level of CD36, cell lysates were prepared from wild-type N2a cells and N2a cells stably transfected with CD36 shRNA. Cells were grown in complete media to 80% confluency in P100 cell culture Petri dishes and then lifted with a cell scraper. Collected cells were spun down, washed twice with ice-cold D-PBS, resuspended, and lysed with 1x RIPA buffer (Sigma-Aldrich; St. Louis, MO, USA) containing 1X protease inhibitor cocktail (Sigma-Aldrich; St. Louis, MO, USA) on ice for 30 min. The lysates were then sonicated and centrifuged for 15 min at 14,000 rpm at 4 • C. The supernatants containing the soluble protein fractions were transferred to new microcentrifuge tubes and labeled as RIPA-S. The pellets were rinsed with RIPA buffer and then resuspended in PBS with 10% SDS and 1x proteinase inhibitor cocktail, sonicated, and centrifuged for 15 min at 14,000 rpm at 4 • C. The supernatants were transferred to new microcentrifuge tubes and labeled as RIPA-R. 30 µg of RIPA-S and 10 µg of RIPA-R proteins samples were then mixed with an equal volume of 2x Laemmli sample buffer, boiled, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes [30]. The PVDF membranes were blocked with SuperBlock (ThermoFisher Scientific; Waltham, MA, USA) for 1 h at room temperature, and then incubated with anti-CD36 antibody (ThermoFisher Scientific; Waltham, MA, USA) overnight at 4 • C. After washing with TBST three times, the PVDF membrane blots were incubated with anti-rabbit IgG-HRP for 1 h at room temperature (ProteinSimple; St. Jose, CA, USA), washed with TBST three times, developed with Clarity Max ECL substrate (Bio-Rad; Hercules, CA, USA), and imaged under FluorChem E (ProteinSimple; St. Jose, CA, USA). Following imaging, the PVDF membrane blots were stripped with 0.2 M glycine, 0.1% SDS, and 1% Tween-20 for 20 min, washed twice with TBST, blocked with SuperBlock for 1 h, and then probed with anti-Caveolin-1 antibody (Cell Signaling Technology; Danvers, MA, USA). After washing with TBST three times, the PVDF membrane blots were incubated with anti-rabbit IgG-HRP for 1 h at room temperature (ProteinSimple; St. Jose, CA, USA), washed three times, developed with Clarity Max ECL substrate, and imaged under FluorChem E. After imaging, the PVDF membrane blots were stripped again and then re-probed with anti-actin antibody (Sigma-Aldrich; St. Louis, MO, USA) overnight at 4 • C followed by detection with anti-rabbit IgG-HRP, rinsing, development with Clarity Max ECL substrate, and imaging under FluorChem E. Band intensities were quantitated using Image Studio Lite software (LI-COR Biosciences; Lincoln, NE, USA).

Statistical Analysis
Data were analyzed using GraphPad Prism 6 (GraphPad Software; San Diego, CA, USA). Two-way ANOVA followed by Tukey's multiple comparison test was used to determine statistical significance. p < 0.05 was considered statistically significant.

Effects of PA Treatment on CD36 Expression
Previously we reported that 200 µM PA induced a significant decrease in N2a cell viability at 24 h post-treatment, but not at 6 h post-treatment [9]. In order to examine whether CD36-mediated FA uptake was required for PA lipotoxicity, we first investigated the effects of PA treatment on CD36 expression. N2a cells were treated with 200 µM PA or BSA for 6 h or 24 h, and the expression of CD36 in these cells was measured by semiquantitative RT-PCR. The relative mRNA expression level of CD36 following treatment with PA for 6 h was comparable to that in BSA-treated cells ( Figure 1A,B). In contrast, the relative mRNA expression level of CD36 following treatment with PA for 24 h was significantly higher than that in BSA-treated cells ( Figure 1A,C).

Time-and Dose-Dependent Effects of PA on BODIPY TM FL C12 Uptake in N2a Cells
We next examined the time-dependent effects of PA treatment on FA uptake in N2a. N2a cells were treated with 200 µM PA or BSA control for 0.5, 1, 2, 4, or 6 h, and FA uptake was analyzed by incubation with 15 µM 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid (BODIPY TM FL C12), a widely used fluorescent long-chain FA analog resembling the acyl chain length of PA. BODIPY™ FL C12 is a well-established fluorescent FA analog that has been used to study FA uptake in various models including white adipose tissue [31,32], multiple cell lines [33,34], and human placenta explants [35]. After 30 min incubation with BODIPY™ FL C12, its fluorescence intensity was measured by flow cytometry. Compared to time-paired BSA controls, the amount of BODIPY TM FL C12 uptake in N2a cells was significantly increased following 2 htreatment with PA and continued to increase at 4 h and 6 h post-treatment (Figure 2A,B). Therefore 6 h PA treatment was selected for further investigation. Taken together with mRNA expression data, these data suggest that more rapid dynamic cellular processes other than CD36 expression regulation may be responsible for PA-induced potentiation of FA uptake at up to 6 h post-treatment. We also examined the dose-dependent effects of PA on FA uptake in N2a cells. N2a cells were treated with different concentrations of PA for 6 h and BODIPY TM FL C12 uptake was then examined. PA started to significantly increase BODIPY TM FL C12 uptake at 50 µM. As the concentration of PA increased to 200 µM, BODIPY TM FL C12 uptake continued to increase in N2a cells ( Figure 2C,D). We also examined BODIPY TM FL C12 uptake in cells treated with 200 µM PA or BSA for 6 h under confocal microscopy. As shown in Figure 2E, 200 µM PA significantly increased the amount of BODIPY TM FL C12 fluorescence as compared to BSA control. These data confirmed that treatment with PA significantly enhanced FA uptake in N2a cells, therefore PA treatment may promote its own uptake in N2a cells.

Time-and Dose-Dependent Effects of PA on BODIPY TM FL C12 Uptake in N2a Cells
We next examined the time-dependent effects of PA treatment on FA uptake in N2a. N2a cells were treated with 200 µM PA or BSA control for 0.5, 1, 2, 4, or 6 h, and FA uptake was analyzed by incubation with 15 µM 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid (BODIPY TM FL C12), a widely used fluorescent long-chain FA analog resembling the acyl chain length of PA. BODIPY™ FL C12 is a well-established fluorescent FA analog that has been used to study FA uptake in various models including white adipose tissue [31,32], multiple cell lines [33,34], and human placenta explants [35]. After 30 min incubation with BODIPY™ FL C12, its fluorescence intensity was measured by flow cytometry. Compared to time-paired BSA controls, the amount of BODIPY TM FL C12 uptake in N2a cells was significantly increased following 2 htreatment with PA and continued to increase at 4 h and 6 h post-treatment (Figure 2A,B). Therefore 6 h PA treatment was selected for further investigation. Taken together with mRNA expression data, these data suggest that more rapid dynamic cellular processes other than CD36 expression regulation may be responsible for PA-induced potentiation of FA uptake at up to 6 h posttreatment. We also examined the dose-dependent effects of PA on FA uptake in N2a cells. N2a cells were treated with different concentrations of PA for 6 h and BODIPY TM FL C12 uptake was then examined. PA started to significantly increase BODIPY TM FL C12 uptake at 50 µM. As the concentration of PA increased to 200 µM, BODIPY TM FL C12 uptake continued to increase in N2a cells ( Figure 2C,D). We also examined BODIPY TM FL C12 uptake in cells treated with 200 µM PA or BSA for 6 h under confocal microscopy. As shown in Figure 2E, 200 µM PA significantly increased the amount of BODIPY TM FL C12 fluorescence as compared to BSA control. These data confirmed that treatment with PA significantly enhanced FA uptake in N2a cells, therefore PA treatment may promote its own uptake in N2a cells.  Relative mRNA expression of CD36 in N2a cells following treatment with 200 µM PA or BSA for 6 h and 24 h using semi-quantitative RT-PCR. (A) Agarose gel images of CD36 and β-actin representative of three independent experiments. (B) Relative CD36 mRNA expression in N2a cells following treatment with BSA or PA for 6 h. (C) Relative CD36 mRNA expression in N2a cells following treatment with BSA or PA for 24 h. Data presented (mean ± SD) were representative of three independent experiments. Statistical analysis was performed using t-test. *** p < 0.001 vs. BSA control.

Time-and Dose-Dependent Effects of PA on BODIPY TM FL C12 Uptake in N2a Cells
We next examined the time-dependent effects of PA treatment on FA uptake in N2a. N2a cells were treated with 200 µM PA or BSA control for 0.5, 1, 2, 4, or 6 h, and FA uptake was analyzed by incubation with 15 µM 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid (BODIPY TM FL C12), a widely used fluorescent long-chain FA analog resembling the acyl chain length of PA. BODIPY™ FL C12 is a well-established fluorescent FA analog that has been used to study FA uptake in various models including white adipose tissue [31,32], multiple cell lines [33,34], and human placenta explants [35]. After 30 min incubation with BODIPY™ FL C12, its fluorescence intensity was measured by flow cytometry. Compared to time-paired BSA controls, the amount of BODIPY TM FL C12 uptake in N2a cells was significantly increased following 2 htreatment with PA and continued to increase at 4 h and 6 h post-treatment (Figure 2A,B). Therefore 6 h PA treatment was selected for further investigation. Taken together with mRNA expression data, these data suggest that more rapid dynamic cellular processes other than CD36 expression regulation may be responsible for PA-induced potentiation of FA uptake at up to 6 h posttreatment. We also examined the dose-dependent effects of PA on FA uptake in N2a cells. N2a cells were treated with different concentrations of PA for 6 h and BODIPY TM FL C12 uptake was then examined. PA started to significantly increase BODIPY TM FL C12 uptake at 50 µM. As the concentration of PA increased to 200 µM, BODIPY TM FL C12 uptake continued to increase in N2a cells ( Figure 2C,D). We also examined BODIPY TM FL C12 uptake in cells treated with 200 µM PA or BSA for 6 h under confocal microscopy. As shown in Figure 2E, 200 µM PA significantly increased the amount of BODIPY TM FL C12 fluorescence as compared to BSA control. These data confirmed that treatment with PA significantly enhanced FA uptake in N2a cells, therefore PA treatment may promote its own uptake in N2a cells. versible inhibitor that effectively and completely blocks CD36-mediated FA uptake, for 1 h followed by treatment with BSA or 200 µM PA for 6 h and then BODIPY TM FL C12 uptake in these cells was examined by flow cytometry. As shown in Figure 3, pretreatment with SSO at a concentration as low as 25 µM significantly decreased PA-induced increase of BODIPY TM FL C12 uptake. As the SSO concentration increased to 200 µM, BODIPY TM FL C12 uptake in PA-treated cells was further inhibited. At 200 µM, SSO also significantly decreased BODIPY TM FL C12 uptake in BSA-treated cells. These data suggest that CD36 may play a key role in BODIPY TM FL C12 uptake in both BSA-and PA-treated cells and that CD36-mediated PA uptake may contribute to PA lipotoxicity in N2a cells.
in (D,E). BODIPY TM FL C12 fluorescence in N2a cells following 6 h treatment with BSA or 200 µM PA visualized under confocal microscopy. Data presented were representative of three independent experiments. Data (Mean ± SEM) were analyzed using two-way ANOVA followed by Tukey's multiple comparison test. * p < 0.05 vs. corresponding BSA control; *** p < 0.001 vs. corresponding BSA control.

Sulfo-N-Succinimidyl Oleate (SSO) Decreased BODIPY TM FL C12 Uptake in N2a Cells
To examine the involvement of CD36 in FA uptake in N2a cells, cells were pre-treated with different concentrations of Sulfo-N-succinimidyl oleate (SSO), a well-known irreversible inhibitor that effectively and completely blocks CD36-mediated FA uptake, for 1 h followed by treatment with BSA or 200 µM PA for 6 h and then BODIPY TM FL C12 uptake in these cells was examined by flow cytometry. As shown in Figure 3, pretreatment with SSO at a concentration as low as 25 µM significantly decreased PA-induced increase of BODIPY TM FL C12 uptake. As the SSO concentration increased to 200 µM, BODIPY TM FL C12 uptake in PA-treated cells was further inhibited. At 200 µM, SSO also significantly decreased BODIPY TM FL C12 uptake in BSA-treated cells. These data suggest that CD36 may play a key role in BODIPY TM FL C12 uptake in both BSA-and PA-treated cells and that CD36-mediated PA uptake may contribute to PA lipotoxicity in N2a cells.

SSO Attenuated PA-Induced Decrease in N2a Cell Viability
We previously showed that 200 µM PA did not decrease cell viability at 6 h, but significantly decreased cell viability at 24 h [9], therefore we examined whether SSO inhibition of CD36 could attenuate PA-induced decrease of N2a cell viability. N2a cells were pre-treated with different concentrations of SSO for 1 h followed by treatment BSA or 200 µM PA for 24 h, and cell viability was assessed by MTT assay. As shown in Figure 4, SSO at 25 µM started to significantly attenuate PA-induced decrease of cell viability and continued to abolish PA-induced decrease of cell viability at higher concentrations tested.

SSO Attenuated PA-Induced Decrease in N2a Cell Viability
We previously showed that 200 µM PA did not decrease cell viability at 6 h, but significantly decreased cell viability at 24 h [9], therefore we examined whether SSO inhibition of CD36 could attenuate PA-induced decrease of N2a cell viability. N2a cells were pre-treated with different concentrations of SSO for 1 h followed by treatment BSA or 200 µM PA for 24 h, and cell viability was assessed by MTT assay. As shown in Figure 4, SSO at 25 µM started to significantly attenuate PA-induced decrease of cell viability and continued to abolish PA-induced decrease of cell viability at higher concentrations tested.

SSO Attenuated PA-Induced Cell Cycle Defects in N2a Cells
We also examined whether SSO inhibition of CD36 could abolish PA-induced cell cycle defects. N2a cells were pre-treated with different concentrations of SSO for 1 h followed by treatment with BSA or 200 µM PA for 6 h, and then the cell cycle profile was examined by DAPI staining followed by flow cytometry analysis. As shown in Figure 5

SSO Attenuated PA-Induced Cell Cycle Defects in N2a Cells
We also examined whether SSO inhibition of CD36 could abolish PA-induced cell cycle defects. N2a cells were pre-treated with different concentrations of SSO for 1 h followed by treatment with BSA or 200 µM PA for 6 h, and then the cell cycle profile was examined by DAPI staining followed by flow cytometry analysis. As shown in Figure 5    . Cell viability analysis of N2a cells pre-treated with sulfo-N-succinimidyl oleate (SSO) for 1 h followed by treatment with BSA or PA for 24 h. N2a cells were pre-treated with different concentrations of SSO for 1 h followed by treatment with 200 µM PA or BSA for 6 h, and cell viability was assessed by MTT assay. Data presented were representative of three independent experiments. Data (Mean ± SEM) were analyzed using two-way ANOVA followed by Tukey's multiple comparison test. *** p < 0.001 vs. BSA control; # p < 0.05 vs. 200 µM PA.

SSO Attenuated PA-Induced Cell Cycle Defects in N2a Cells
We also examined whether SSO inhibition of CD36 could abolish PA-induced cell cycle defects. N2a cells were pre-treated with different concentrations of SSO for 1 h followed by treatment with BSA or 200 µM PA for 6 h, and then the cell cycle profile was examined by DAPI staining followed by flow cytometry analysis. As shown in Figure 5

CD36 Knockdown Attenuated BODIPY TM FL C12 Uptake
To confirm the involvement of CD36 in FA uptake and PA lipotoxicity, N2a cells were stably transfected with CD36 shRNA or non-mammalian shRNA control plasmid. Wildtype (WT), non-mammalian control shRNA-transfected, and CD36 shRNA-transfected N2a cells were treated with BSA or 200 µM PA for 6 h, and then BODIPY™ FL C12 uptake was assessed by flow cytometry. As shown in Figure 6A, BODIPY™ FL C12 uptake in BSA-or PA-treated CD36 shRNA-transfected cells was significantly lower than that in corresponding BSA-or PA-treated WT or control cells. As PA treatment significantly increased BODIPY™ FL C12 uptake in WT and non-mammalian shRNA-transfected control cells, PA treatment also dramatically enhanced BODIPY™ FL C12 uptake in CD36 shRNAtransfected cells as compared to the corresponding BSA-treated cells. These data confirmed that CD36 may be important for FA uptake in both BSA-and PA-treated cells.
To confirm the involvement of CD36 in FA uptake and PA lipotoxicity, N2a cells were stably transfected with CD36 shRNA or non-mammalian shRNA control plasmid. Wild-type (WT), non-mammalian control shRNA-transfected, and CD36 shRNA-transfected N2a cells were treated with BSA or 200 µM PA for 6 h, and then BODIPY™ FL C12 uptake was assessed by flow cytometry. As shown in Figure 6A, BODIPY™ FL C12 uptake in BSA-or PA-treated CD36 shRNA-transfected cells was significantly lower than that in corresponding BSA-or PA-treated WT or control cells. As PA treatment significantly increased BODIPY™ FL C12 uptake in WT and non-mammalian shRNA-transfected control cells, PA treatment also dramatically enhanced BODIPY™ FL C12 uptake in CD36 shRNA-transfected cells as compared to the corresponding BSA-treated cells. These data confirmed that CD36 may be important for FA uptake in both BSA-and PA-treated cells. To confirm CD36 knockdown by shRNA, the surface expression of CD36 in WT, control, and CD36 shRNA-transfected N2a cells was examined by immunostaining with fluorescence-conjugated CD36 antibody and flow cytometry analysis. The percent of CD36 + cells in CD36 shRNA-transfected cells was significantly lower than that in WT and control cells ( Figure 6B). Total protein level of CD36 was also examined by Western blot assay. CD36 protein was not detected in RIPA-soluble (RIPA-S) fractions but was detected in RIPA-resistant (RIPA-R) membrane fractions solubilized with 10% SDS ( Figure 6C). Actin To confirm CD36 knockdown by shRNA, the surface expression of CD36 in WT, control, and CD36 shRNA-transfected N2a cells was examined by immunostaining with fluorescence-conjugated CD36 antibody and flow cytometry analysis. The percent of CD36 + cells in CD36 shRNA-transfected cells was significantly lower than that in WT and control cells ( Figure 6B). Total protein level of CD36 was also examined by Western blot assay. CD36 protein was not detected in RIPA-soluble (RIPA-S) fractions but was detected in RIPA-resistant (RIPA-R) membrane fractions solubilized with 10% SDS ( Figure 6C). Actin was used as a marker for RIPA-soluble fractions. Calveolin-1, a membrane protein marker, was used for normalization of the relative protein level of CD36 in RIPA-R fractions. As shown in Figure 6C,D, the relative protein level of CD36 in shRNA-transfected cells was significantly lower than that in WT cells.

CD36 Knockdown Attenuated PA-Induced Cell Cycle Defects
Since inhibition of CD36 with SSO abrogated the lipotoxic effects of PA on N2a cell cycle progression, we next examined cell cycle progression in N2a cells with CD36 knockdown. To this end, WT, control-transfected, and CD36 shRNA-transfected cells were treated with BSA or 200 µM PA for 6 h, and their cell cycle profiles were determined using DAPI staining followed by flow cytometry analysis. While PA treatment significantly increased the percent of 4N cells in WT and control-transfected cells, shRNA knockdown of CD36 abolished PA-induced increase of 4N cells ( Figure 7A). While PA treatment significantly decreased the percent of 2N cells in WT and control-transfected cells, shRNA knockdown of CD36 abolished PA-induced decrease of 2N cells ( Figure 7B). These data suggest that CD36 may play a key role in PA lipotoxicity. cells was significantly lower than that in WT cells.

CD36 Knockdown Attenuated PA-Induced Cell Cycle Defects
Since inhibition of CD36 with SSO abrogated the lipotoxic effects of PA on N2a cell cycle progression, we next examined cell cycle progression in N2a cells with CD36 knockdown. To this end, WT, control-transfected, and CD36 shRNA-transfected cells were treated with BSA or 200 µM PA for 6 h, and their cell cycle profiles were determined using DAPI staining followed by flow cytometry analysis. While PA treatment significantly increased the percent of 4N cells in WT and control-transfected cells, shRNA knockdown of CD36 abolished PA-induced increase of 4N cells ( Figure 7A). While PA treatment significantly decreased the percent of 2N cells in WT and control-transfected cells, shRNA knockdown of CD36 abolished PA-induced decrease of 2N cells ( Figure 7B). These data suggest that CD36 may play a key role in PA lipotoxicity.

Discussion
We have previously reported that 24 h treatment with 200 µM PA significantly decreases cell viability and induces cell death in N2a cells [9]. While several FA transport proteins are expressed in N2a cells, this study was designed to examine the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells. It has been previously reported that uptake of FA analogs, such as 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid and 15-(p-iodophenyl)pentadecanoic acid, in the heart, skeletal muscle, and adipose tissue is significantly decreased in CD36 knockout mice [36]. In our study, treatment with SSO, a well-known irreversible CD36 inhibitor, not only blocked BODIPY™ FL C12 uptake in BSA-and PA-treated cells but also abolished PA-induced decrease in cell viability and accumulation of 4N cells, and that knockdown of CD36 decreased BODIPY™ FL C12 uptake in both BSA-and PA-treated cells as compared to the corresponding WT and controltransfected cells and ameliorated PA-induced cell cycle defects. These results suggest that CD36 may play an important role in FA uptake and PA lipotoxicity in N2a cells.
While simple diffusion has been posited as a mechanism for FA uptake [18], our data contributed to the growing evidence that uptake of long-chain FA is primarily mediated by protein transporters [20]. CD36 is widely expressed in many types of cells including

Discussion
We have previously reported that 24 h treatment with 200 µM PA significantly decreases cell viability and induces cell death in N2a cells [9]. While several FA transport proteins are expressed in N2a cells, this study was designed to examine the involvement of CD36 in FA uptake and PA lipotoxicity in N2a cells. It has been previously reported that uptake of FA analogs, such as 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid and 15-(p-iodophenyl)pentadecanoic acid, in the heart, skeletal muscle, and adipose tissue is significantly decreased in CD36 knockout mice [36]. In our study, treatment with SSO, a well-known irreversible CD36 inhibitor, not only blocked BODIPY™ FL C12 uptake in BSAand PA-treated cells but also abolished PA-induced decrease in cell viability and accumulation of 4N cells, and that knockdown of CD36 decreased BODIPY™ FL C12 uptake in both BSA-and PA-treated cells as compared to the corresponding WT and control-transfected cells and ameliorated PA-induced cell cycle defects. These results suggest that CD36 may play an important role in FA uptake and PA lipotoxicity in N2a cells.
While simple diffusion has been posited as a mechanism for FA uptake [18], our data contributed to the growing evidence that uptake of long-chain FA is primarily mediated by protein transporters [20]. CD36 is widely expressed in many types of cells including hepatocytes, adipocytes, microvascular endothelial cells, cardiomyocytes, astrocytes, microglia, and neurons [37][38][39]. In this study, we used BODIPY TM FL C12 to examine FA uptake for several reasons. First, BODIPY TM FL C12 was deemed to resemble the length of C16 FA such as PA since the BODIPY TM FL fluorophore contributes an additional four-carbon length of acyl chain. Second, the complex lipid product profile of 3 H-PA most closely resembles that of BODIPY TM FL C12 based on metabolic tracing performed in zebrafish [29]. Third, as a widely-used PA analog that is fluorescent in both aqueous and lipid environments, BODIPY TM FL C12 has been used to trace the movement of FA across the cell layers of living explants of human term placenta [35], and has been shown to be incorporated into LD in hepatocytes [34]. Therefore, this study employed BODIPY TM FL C12 to examine FA uptake. While the focus of this paper was on the involvement of CD36 in FA uptake and PA lipotoxicity, it remains of importance to further examine the incorporation of BODIPY TM FL C12 after entering N2a cells.
The maturation and transportation of CD36 are regulated by post-translational modifications, including phosphorylation, ubiquitination, glycosylation, and palmitoylation [26]. Palmitoylation of CD36 has been shown to target CD36 to the plasma membrane and facilitate fatty acid uptake in adipose cells [40]. PA oversupply has also been shown to increase the translocation of CD36 to sarcolemma in cardiac [41,42] and skeletal muscles [43], and to enhance lipid uptake in heart and skeletal muscles [42,43]. Therefore, it is likely that PA treatment may increase the palmitoylation of CD36 in N2a cells, thereby increasing CD36-mediated FA uptake in N2a cells. Future studies on post-translational modification of CD36 such as palmitoylation in PA-treated cells as compared to BSA-treated cells may help to shed light on the regulatory mechanisms of CD36 following PA treatment in N2a cells.
Studies have suggested that CD36-mediated FA transport involves its reversible trafficking between intracellular membrane compartments and plasma membrane [42,44] and its dynamic distribution in its localization in non-lipid raft membranes and lipid raft membranes [45]. Lipid rafts are known to contain high concentrations of PA, cholesterol, and sphingosine. It has been shown that the membrane lipid composition and physicochemical properties of lipid rafts are subjected to modifications by exogenous FAs [46]. For example, depletion of PA disrupts the lipid raft domains [47]. Furthermore, CD36-mediated FA uptake is significantly enhanced when its association with lipid rafts is increased [45]. It is likely that PA treatment may enhance the association of CD36 with lipid rafts, thereby augmenting CD36-mediated FA uptake in N2a cells. Therefore, future studies should examine the incorporation of PA and localization of CD36 in PA-treated cells as compared to BSA-treated cells.
Our data also showed that PA treatment increased BODIPY™ FL C12 uptake in WT, control-transfected, and CD36 shRNA-transfected cells as compared to their corresponding BSA-treated cells, which suggests that PA treatment may augment the uptake of FA into N2a cells. In contrast, SSO inhibition of CD36 was able to diminish BODIPY™ FL C12 uptake in both BSA-and PA-treated cells to minimal levels. As an irreversible CD36 inhibitor, SSO was able to inhibit CD36 function regardless of its localization in lipid rafts and its post-translational modification status. However, shRNA knockdown of CD36 only significantly decreased CD36 protein, but had no effects on post-translational regulation of CD36 function. Potential regulation of CD36 palmitoylation and lipid raft localization by PA may be able to account for increased FA uptake in CD36 knockdown cells.
CD36 has been shown to play an important role in hypothalamic FA sensing and food intake regulation [48]. CD36 gene polymorphism −31118 G > A (rs1761667) is associated with overweight and obesity [49]. CD36 deficiency prevents obesity-associated cardiac steatosis and insulin resistance [50]. It has been implicated in dysregulated lipid metabolism in diabetic cardiomyopathy [42], Alzheimer's disease [51,52], and demyelinating disorders such as multiple sclerosis [53]. Besides acting as a FA transporter, CD36 also acts as a receptor for multiple ligands with signal transduction capabilities. For example, it has been shown to initiate signaling by Src kinase partners and regulating Ca 2+ activation of phospholipases [27]. Therefore, it is worthwhile to investigate whether and how CD36mediated signaling may contribute to PA lipotoxicity in N2a cells.
Lipids are known to play important roles in brain development, neurogenesis, synaptogenesis, myelin sheath formation, and signal transduction [54]. Alteration of lipid homeostasis is associated with various disorders and pathologies including Alzheimer's and Parkinson's disease [55][56][57]. Lipotoxicity has been implicated in various neurological pathologies including diabetic neuropathy and cognitive decline [58,59]. Our studies suggest that CD36 may represent a regulatory target against pathologies caused by excess FAs, and that understanding the mechanisms that mediate and regulate CD36 function will help to understand the mechanisms of FA accumulation and neuropathologies in obese and metabolic syndrome patients.

Conclusions
In summary, our study showed that inhibition of CD36 with SSO significantly decreased BODIPY™ FL C12 uptake in BSA-and PA-treated cells, and blocked PA-induced decrease of cell viability, decrease of 2N cells and increase of 4N cells. Our data also showed that knockdown of CD36 by shRNA significantly decreased FA uptake in BSAand PA-treated cells as compared to the corresponding WT or control-transfected cells, and abolished PA-induced decrease of 2N cells, and increase of 4N cells. These results demonstrate that CD36 is a critical mediator of FA uptake and that CD36-mediated PA uptake may play an important role in PA lipotoxicity in N2a cells. Our data also showed that PA treatment enhanced BODIPY™ FL C12 uptake in WT, control-transfected, and CD36 knockdown cells. It remains to be determined whether PA-induced increase in FA uptake could be attributed to PA regulation of post-translational modification and localization of CD36.