Lipids (2014) 49:621–631 DOI 10.1007/s11745-014-3911-x

ORIGINAL ARTICLE

Acetate Treatment Increases Fatty Acid Content in LPS-Stimulated BV2 Microglia Dhaval P. Bhatt • Thad A. Rosenberger

Received: 22 November 2013 / Accepted: 9 May 2014 / Published online: 23 May 2014 Ó AOCS 2014

Abstract Acetate supplementation increases plasma acetate, brain acetyl-CoA, histone acetylation, phosphocreatine levels, and is anti-inflammatory in models of neuroinflammation and neuroborreliosis. Although radiolabeled acetate is incorporated into the cellular lipid pools, the effect that acetate supplementation has on lipid deposition has not been quantified. To determine the impact acetate-treatment has on cellular lipid content, we investigated the effect of acetate in the presence of bacterial lipopolysaccharide (LPS) on fatty acid, phospholipid, and cholesterol content in BV2 microglia. We found that 1, 5, and 10 mM of acetate in the presence of LPS increased the total fatty acid content in BV2 cells by 23, 34, and 14 % at 2 h, respectively. Significant increases in individual fatty acids were also observed with all acetate concentrations tested with the greatest increases occurring with 5 mM acetate in the presence of LPS. Treatment with 5 mM acetate in the absence of LPS increased total cholesterol levels by 11 %. However, neither treatment in the absence of LPS significantly altered the content of individual phospholipids or total phospholipid content. To determine the minimum effective concentration of acetate we measured the time- and concentrationdependent changes in histone acetylation using western blot analysis. These studies showed that 5 mM acetate was necessary to induce histone acetylation and at 10 mM acetate, the histone acetylation-state increased as early as 0.5 h

Electronic supplementary material The online version of this article (doi:10.1007/s11745-014-3911-x) contains supplementary material, which is available to authorized users. D. P. Bhatt  T. A. Rosenberger (&) Department of Basic Sciences, School of Medicine and Health Sciences, University of North Dakota, 501 North Columbia Road, Room 3742, Grand Forks, ND 58203, USA e-mail: [email protected]

following the start of treatment. These data suggest that acetate increases fatty acid content in LPS-stimulated BV2 microglia that is reflected by an increase in fatty acids esterified into membrane phospholipids. Keywords Acetate  Cholesterol  Fatty acid  Histone acetylation  Lipids  Phospholipid  Microglia Abbreviations ACC AMP ARA ChoGpl CerPCho DGLA DHA DMEM/F12 EtnGpl EDTA EGTA EPA FBS GTA H3K9 H4S1/K5/ K8/K12 HEPES HMGCS KH2PO4 LNA LPS NaOAc NaCl

Acetyl-CoA carboxylase Adenosine monophosphate Arachidonate Choline glycerophospholipid Sphingomyelin Dihomo-c-linoleate Docosahexaenoate Dulbecco’s modified eagle medium/ nutrient mixture F-12 Ethanolamine glycerophospholipid Ethylenediamine tetraacetic acid Ethylene glycol tetraacetic acid Eicosapentaenoate Fetal bovine serum Glyceryl triacetate Histone H3 lysine 9 Histone H4 serine 1, and lysine 5, 8, or 12 2-[4-(2-Hydroxyethyl)piperazine-1yl]ethanesulfonic acid 3-Hydroxy-3-methylglutaryl CoA synthase Potassium phosphate Linoleate Lipopolysaccharide Sodium acetate Sodium chloride

123

622

OLA PAM PBS PtdIns PtdSer SDS STA TTBS TCA TLC

Lipids (2014) 49:621–631

Oleate Palmitate Phosphate buffer saline Phosphatidylinositol Phosphatidylserine Sodium dodecyl sulfate Stearate Tris buffered saline containing Tween 20 Tricarboxylic acid Thin layer chromatography

Introduction Acetate supplementation attenuates markers of inflammation in rat models of neuroinflammation [1] and Lyme neuroborreliosis [2], improves the tremor phenotype in a Canavan disease model [3], offers metabolic support in a brain trauma model [4], and possesses growth arresting properties in gliomas [5]. Acetate supplementation in animals, induced with oral glyceryl triacetate (GTA) administration, increases rat plasma acetate and brain acetyl-CoA levels [1]. Acetyl CoA synthetase enzymes convert acetate into its metabolically active form, acetyl-CoA, which is a ubiquitous substrate for carbohydrate, lipid, and protein metabolic pathways [6]. Thus, gaining a better understanding of how acetate supplementation alters acetylCoA-dependent metabolism can provide mechanistic insight into its protective properties. With regard to protein metabolism, GTA increases brain histone acetylation [7] and reverses the bacterial lipopolysaccharide (LPS)-induced hypoacetylation of histone 3 at lysine 9 (H3K9) [8]. Further, acetate-induced changes in histone acetylation are associated with altered cytokine balance and reduced inflammatory signaling in microglia and astrocyte cultures [9, 10]. On the other hand, rats treated with single oral dose of GTA show elevated brain phosphocreatine levels and a reduction in AMP levels suggesting that acetate supplementation can stimulate purine metabolism [11]. Nonetheless, the lipid synthesis pathways may also contribute to the beneficial properties of acetate supplementation. Although, radiolabeled acetate is incorporated into the fatty acid and cholesterol pools [12, 13], it has not been demonstrated whether large doses of acetate can increase fatty acid deposition. Increasing lipid synthesis is crucial for the treatment of diseases involving myelin degradation or insufficient myelin synthesis as found in multiple sclerosis and Canavan disease, respectively. In this regard, in human brain a decrease in grey-matter N-acetyl aspartate levels correlates with reduced acetate availability in white matter in multiple sclerosis patients [14]. Acetate supplementation also

123

increases myelin galactocerebroside levels in a rat model of Canavan disease [3]. Further, aging decreases the polyunsaturated fatty acid content and increases the monounsaturated fatty acid content of ethanolamine and serine glycerophospholipids [15]. Dietary n-3 polyunsaturated fatty acids, docosahexaenoate (DHA) and eicosapentaenoate (EPA) reduce inflammation [16] and are thought to do so by replacing arachidonate (ARA) at the sn-2 position of the membrane phospholipids. Thus, it is important to understand how acetate supplementation alters brain fatty acid synthesis and lipid deposition. In this study, we quantified the effect that different acetate concentrations, in the presence and absence of LPS, had on the fatty acid, phospholipid, and cholesterol content of BV2 microglia. In this proof-of-principle experiment, we found that 1 and 5 mM acetate in the presence of LPS showed marked increases in total cellular and esterified phospholipid fatty acid content while 5 mM acetate in the absence of LPS resulted in a modest increase in cholesterol levels. Further, acetate induced a concentration- and timedependent increase on histone acetylation with a minimum effective concentration being 5 mM. These data support the hypothesis that acetate treatment can stimulate fatty acid content in BV2 cultures in the presence of LPS.

Materials and Methods Reagents Fatty acid methyl ester, phospholipid, and cholesterol standards were from NuCheckPrep, (Elysian, MN). Sodium acetate (99 %), bacterial lipopolysaccharide (LPS, Escherichia coli 055:B5), and ferric chloride heptahydrate were purchased from Sigma-Aldrich (St. Louis, MO). High performance liquid chromatography grade acetonitrile, methanol, 2-propanol, chloroform, mono-basic potassium phosphate (KH2PO4), phosphoric acid, sulfuric acid, ammonium hydroxide, tris-base, sodium dodecyl sulfate (SDS), and bovine serum albumin were obtained from EMD Chemicals Inc. (Gibbstown, NJ). Glacial acetic acid, hexane, toluene, and all cell culture supplies were acquired through VWR International, LLC (Batavia, IL). Gas chromatography grade anhydrous methanol was from MacronTM Chemicals (Charlotte, NC). All western blot supplies including acrylamide, bis-N,N0 -methylene-bisacrylamide, N,N,N0 ,N0 -tetramethylene-diamine, ammonium persulfate, 24-well criterion empty cassettes (#345-9901 and #345-9903), Pre-stained SDS-PAGE standards (#1610318), and Precision Plus ProteinTM westernCTM Standards (#161-0376) were obtained from Bio-Rad Laboratories (Hercules, CA). The complete EDTA-free protease inhibitor cocktail tablets were from Roche Applied Science

Lipids (2014) 49:621–631

(Indianapolis, IN). Primary antibodies for histone H3 (1:1,000, sc-8654), and acetylated-histone H4 (S1/K5/8/12, 1:1,000, sc-34263), were obtained from Santa Cruz Biotech. Inc. (Santa Cruz, CA). Antibodies against acetylatedhistone H3 (K9, 1:10,000, 07-352), and histone H4 (1:8,000, 04-858) were purchased from Millipore (Billerica, MA). The horseradish peroxidase-conjugated secondary antibodies: donkey anti-goat IgG antibody (1:10,000, sc-2020) and the goat anti-rabbit IgG antibody (1:10,000, 170-5046) were obtained from Santa Cruz Biotech (Santa Cruz, CA) and Bio-Rad Laboratories (Hercules, CA), respectively. Cell Culture The immortalized murine BV2 microglial cell line [17] was obtained from Dr. Colin K. Combs (University of North Dakota, Grand Forks, ND). The cryopreserved cells were maintained at -80 °C in DMEM/F12 media containing 20 % fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 lg/mL), amphotericin B (2.5 lg/mL), HEPES (10 mM) and 10 % dimethyl sulfoxide. The fatty acid content and composition of the FBS used in these experiments are described in supplemental Table 1. Frozen cultures were thawed, washed with DMEM/F12 media containing 10 % FBS to remove DMSO and cultured in DMEM/F12/10 % FBS medium in 100 9 20 mm dishes. BV2 cells at approximately 27th passage were plated in six-well plates at a density of 5.0 9 105 cells/well and allowed to reach 80–90 % confluence before experimental treatment. BV2 cells grown in six-well plates were treated with sodium acetate (NaOAc) or sodium chloride (NaCl) (1–10 mM) in presence or absence of LPS (6.25 ng/mL) for 0.5–4 h. An equal-molar concentration of NaCl was used as a control to account for the osmolality changes in the culture medium. Lipid Extraction from BV2 Cell Culture Lipids from FBS or cultures treated with NaCl or NaOAc in the presence or absence of LPS were extracted with nhexane:2-propanol (3:2, by vol) [18]. Briefly, BV2 cells grown in six-well plates were washed in PBS and immediately frozen by placing the plate on liquid nitrogen to minimize fatty acid hydrolysis due to acylhydrolase activation. Frozen cells were scraped from the plate in 2-propanol then the wells were washed with an equal volume of 2-propanol that was combined with the initial suspension. The cellular lipids were extracted with the addition of nhexane to bring the final proportion of solvent to n-hexane:2-propanol (3:2, by vol) followed by vortex mixing. The cellular protein was separated from the lipid extract by centrifugation at 4,7509g for 10 min (Allegra X-15R,

623

Beckman Coulter Inc., Fullerton, CA). Lipid extracts were stored at -20 °C until use and the protein pellet was saved to quantify cellular protein content. Thin Layer Chromatography Individual phospholipids, total phospholipid, and triacylglycerol was isolated from the lipid extracts using thin layer chromatography on 20 cm 9 20 cm Silica gel 60 plates (EMD Chemicals Inc., Gibbstown, NJ). The individual phospholipids were isolated using a solvent system of chloroform/methanol/glacial acetic acid/water (50:37.5:3:2, by vol) [19] and total phospholipid and triacylglycerol was isolated using a solvent system of heptane/isopropyl ether/ glacial acetic acid (60:40:4, by vol) [20]. Sample bands corresponding to phospholipid or triacylglycerol standards were isolated from the plates and used to determine esterified fatty acid content or phospholipid mass. Phospholipid mass was measured using a phosphorus assay as described [21]. Esterified fatty acid content was quantified as described below. The phospholipid, total fatty acid, and esterified fatty acid levels measured were normalized to cellular protein and expressed as n moles of lipid phosphorus or n moles of fatty acid per mg protein. Fatty Acid Analysis Total cellular, total phospholipid, isolated phospholipid, triacylglycerol, and FBS fatty acid content was quantified as described [22]. Briefly, the samples were mixed with internal standard (methyl heptadecanoate), mixed with 2 % sulfuric acid in toluene: methanol (1:1, by vol), then incubated at 65 °C for 2 h. The samples were cooled to 4 °C, neutralized with a 5 % ammonium hydroxide solution in water, and then extracted with n-hexane. The extracted fatty acid methyl esters were quantified on a Shimadzu GC-2010 gas chromatograph equipped with a flame ionization detector using a SPTM-2330 capillary column (30 m 9 0.32 mm 9 0.2 lm film thickness; Supelco, Bellefonte, PA). Detector and injection temperatures were maintained at 220 °C and helium was used as a carrier gas with a constant flow velocity of 30 cm/s. Initial column temperature was maintained at 175 °C for 7 min followed by a gradient increase at 3 °C/min to 200 °C and held constant for 9.5 min. Fatty acid standards were used to identify and quantify the fatty acids based on their retention times and concentration factors using Simadzu EZStart software (build 14, version 7.2.1 SP1, Kyoto, Japan). Cholesterol Assay Cholesterol levels in BV2 cell lipid extracts were measured using a colorimetric assay [23]. One half of the lipid extract

123

624

dissolved in chloroform was transferred to a test tube and dried completely under nitrogen at 45 °C. The dried sample was dissolved in ethanol (200 proof) and an equal volume of freshly prepared 0.2 % ferric chloride in sulfuric acid containing 8 % phosphoric acid was added and vortexed for 5 min. The cholesterol content was determined by measuring the sample and standard cholesterol (0.94–120 lg) absorbance values at 550 nm. Total cholesterol content was normalized to total cellular protein and expressed as micrograms of cholesterol per milligram of protein.

Lipids (2014) 49:621–631

Statistical Analysis All the data are expressed as means ± SD with a sample size of eight per group for fatty acid analysis, four per group for phospholipid and cholesterol analysis, and three per group for western blot analysis. Statistical analysis was performed using a two-way ANOVA followed by HolmSidak post hoc test. The statistical significance was set at P B 0.05 and the analysis was performed using GraphPad InStat statistical software (Ver. 3.10, San Diego, CA).

Western Blot Analysis

Results

Cells were washed with PBS, lysed in radio immuno-precipitation assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % SDS, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate containing a protease inhibitor cocktail (Roche, Mannheim, Germany), sonicated for 1 min, and centrifuged to collect the soluble protein supernatant. Extracts were stored at -80 °C until used. Equal amount of protein (10 lg) was denatured in 29 Laemmli sample buffer and separated using SDS–polyacrylamide gel electrophoresis (15 %) at 80–100 V for 2 h at room temperature. Proteins were transferred to nitrocellulose membranes on ice at 100 V for 1.5 h and blocked with 5 % milk in trisbuffered saline (20 mM Tris base and 150 mM NaCl, pH 7.5) containing 0.05 % Tween (TTBS). Blots were incubated overnight with primary antibodies (1:1,000) at 4 °C, washed four times in TTBS and incubated with secondary antibodies (1:10,000) at room temperature. Protein bands were visualized with a SuperSignalÒ West Pico or Femto enhanced chemiluminescent substrate (Pierce, Rockford, IL) using a UVP Bioimaging System (Upland, CA). Image capturing and analysis was performed with LabWorksTM imaging software (version 4.5.0, UVP Inc., Upland, CA) and VisionWorksLS analysis software (version 6.3.1, UVP LLC, Upland, CA), respectively. Optical density of total H3 or H4 histones were used to normalize the acetylated H3 and H4 western blot data. Data are expressed as fold change over controls.

Acetate Treatment Increased Total Cellular Fatty Acid Content

Protein Analysis The protein quantification was performed on samples that were dried over night at 55 °C. The dry protein pellet was heated in 0.2 M potassium hydroxide at 65 °C for 24 h followed by Bradford protein estimation [24] using bovine serum albumin as the protein standard. The protein concentration of samples used for the western blot analysis was measured directly from the cellular protein extracts.

123

It is thought that acetyl-CoA derived from acetate can be utilized for lipid synthesis based on data that demonstrate that radiolabeled acetate is incorporated into cellular lipid pools [12, 13]. However, it has not been demonstrated whether supplementing large concentrations of acetate can increase total cellular and esterified phospholipid fatty acid content, or alter cholesterol levels. To test if acetate can increase fatty acid content, we treated BV2 cultures with 1, 5, and 10 mM of NaOAc in the presence and absence of LPS for 2 h. We found that acetate alone did not alter total cellular fatty acid content (Tables 1, 2, 3). However, at 2 h in the presence of LPS 1, 5, and 10 mM concentrations of acetate significantly increased the levels of total cellular fatty acid content in an inverted U-shaped manner with 5 mM NaOAc being the highest (Tables 1, 2, 3). The individual fatty acids that were increased in cells treated with 10 mM NaOAc in the presence and absence of LPS for 2 h are shown in Table 1. With 10 mM NaOAc ? LPS, we found a 16 % increase in stearate (STA, 18:0) compared to the NaCl ? LPS group (Table 1). However, increases in oleate (OLA, 18:1n-9, 16 %), vaccenate (18:1n-7, 18 %), and linoleate (LNA, 18:2n-6, 15 %) were observed in the NaOAc ? LPS group when compared to the control NaCl group (Table 1). Other fatty acids including palmitate (PAM, 16:0), Stearidonate (18:4n-3), dihomo-c-linolenate (DGLA, 20:3n-6), arachidonate (ARA, 20:4n-6), lignocerate (24:0), adrenate (22:4n-6), nervonate (24:1n-9), docosahexaenoate (DHA, 22:6n-3) were not altered in any of the treatment groups. Treatment resulted in an overall 12 % increase in the total fatty acid content of BV2 cells comparing the NaOAc ? LPS group to NaCl control. Treatment with 5 mM NaOAc was able to induce greater percent increase in total cellular fatty acid content as compared to the 10 mM NaOAc treatment (Table 2). In these experiments we found 5 mM NaOAc ? LPS resulted in increases in PAM

Lipids (2014) 49:621–631 Table 1 Total fatty acid content of BV2 microglia cultures treated with 10 mM acetate in presence and absence of LPS for 2 h

625

NaCl

Significant difference from NaCl ? LPS group (P B 0.05)

Table 2 Total fatty acid content of BV2 microglia cultures treated with 5 mM acetate in presence and absence of LPS for 2 h

40.5 ± 5.7

39.3 ± 6.2

40.8 ± 6.0

45.9 ± 10.6

58.7 ± 4.9

60.5 ± 4.8

56.9 ± 5.4

66.1 ± 8.7b

104.7 ± 6.2

101.7 ± 3.9

105.5 ± 8.0

125.6 ± 13.0a,

Oleate (OLA, 18:1n-9)

27.1 ± 1.8

27.3 ± 1.3

28.9 ± 1.9

33.0 ± 3.1

Linoleate (LNA, 18:2n-6)

24.0 ± 1.1

22.9 ± 0.9

24.8 ± 1.9

28.4 ± 3.4a,

b

6.7 ± 1.1

5.6 ± 0.7

5.7 ± 1.1

7.1 ± 1.4a,

b

Arachidonate (ARA, 20:4n-6)

a

Significant difference from NaOAc group

b

Significant difference from NaCl ? LPS group (P B 0.05)

Table 3 Total fatty acid content of BV2 microglia cultures treated with 1 mM acetate in presence and absence of LPS for 2 h

Values represent the means ± SD (n = 8) in units of nmol/mg protein a Significant difference from NaCl group b

Significant difference from NaOAc group

c

Significant difference from NaCl ? LPS group (P B 0.05)

4.0 ± 0.9

4.2 ± 0.8

3.6 ± 0.9

4.0 ± 1.3

17.0 ± 1.9

16.3 ± 1.7

17.7 ± 2.1

19.7 ± 3.4

Lignocerate (24:0)

5.8 ± 1.2

5.6 ± 0.9

5.0 ± 1.1

5.3 ± 1.3

Adrenate (22:4n-6)

2.3 ± 0.4

2.1 ± 0.3

2.3 ± 0.4

2.3 ± 0.6

Nervonate (24:1n-9)

5.2 ± 1.1

4.6 ± 0.8

4.2 ± 1.0

4.4 ± 1.3

Docosahexaenoate (DHA, 22:6n-3)

9.7 ± 1.7

9.2 ± 1.5

9.3 ± 1.6

9.7 ± 2.4

351.7 ± 20.1

342.4 ± 16.3

347.7 ± 27.8

399.1 ± 42.7a,

NaCl ? LPS

NaOAc ? LPS

16.7 ± 4.5

41.3 ± 6.4a,

Total fatty acid content

NaCl Palmitate (PAM, 16:0) Stearate (STA, 18:0) Oleate (OLA, 18:1n-9)

20.7 ± 5.4

NaOAc 26.0 ± 14.9

75.9 ± 17.1

86.4 ± 19.0

75.4 ± 14.5

98.1 ± 10.5

135.0 ± 37.8

111.1 ± 14.7

179.9 ± 17.5a,

37.1 ± 10.5

31.0 ± 3.5

49.5 ± 4.2

26.8 ± 2.8

31.6 ± 9.0

25.8 ± 2.5

41.1 ± 4.1a,

Stearidonate (18:4n-3)

20.1 ± 14.8

10.8 ± 5.3

13.4 ± 14.9

12.2 ± 4.5

7.3 ± 4.2

6.4 ± 1.9

6.8 ± 3.1

6.0 ± 0.7

Arachidonate (ARA, 20:4n-6)

26.3 ± 8.4

28.0 ± 6.9

27.4 ± 6.9

33.9 ± 3.5

Lignocerate (24:0)

10.1 ± 6.8

8.9 ± 2.3

10.4 ± 4.9

8.1 ± 1.5

Adrenate (22:4n-6)

5.2 ± 2.4

5.4 ± 1.2

6.0 ± 2.4

5.9 ± 1.0

Palmitate (PAM, 16:0) Stearate (STA, 18:0) Oleate (OLA, 18:1n-9)

7.4 ± 5.2

6.1 ± 1.7

7.6 ± 3.5

6.3 ± 0.9

17.0 ± 4.1

18.5 ± 6.8

18.9 ± 2.2

406.8 ± 92.8

436.8 ± 114.3

385.5 ± 85.0

545.6 ± 53.6a,

NaCl ? LPS

NaOAc ? LPS

61.7 ± 6.7

72.5 ± 12.5b,

50.2 ± 1.5

b, c

57.2 ± 5.3 46.8 ± 4.4 99.3 ± 8.6

NaOAc 58.8 ± 10.2 48.2 ± 5.6 105.1 ± 10.8

111.9 ± 4.3

125.6 ± 14.1b,

a

b, c

26.9 ± 2.1

28.5 ± 2.9

30.4 ± 0.5

Linoleate (LNA, 18:2n-6)

23.1 ± 1.6

25.3 ± 2.6

28.1 ± 1.0a a

b

c

55.4 ± 6.6 a

Vaccenate (18:1n-7)

c

33.9 ± 4.5

31.5 ± 3.9b,

c

b, c

Stearidonate (18:4n-3)

1.5 ± 0.3

1.3 ± 0.2

1.3 ± 0.1

1.6 ± 0.2

Dihomo-c-linolenate (DGLA, 20:3n-6)

1.9 ± 0.4

2.1 ± 0.4

2.0 ± 0.1

2.2 ± 0.3

13.1 ± 1.5

13.9 ± 1.8

15.1 ± 0.5a

15.9 ± 1.8b

Arachidonate (ARA, 20:4n-6)

b

17.2 ± 8.6

NaCl

b

a, b

33.3 ± 4.0

Total fatty acid content

b

119.0 ± 13.7

Linoleate (LNA, 18:2n-6)

Nervonate (24:1n-9)

b

b

Vaccenate (18:1n-7)

Docosahexaenoate (DHA, 22:6n-3)

b

a, b

Vaccenate (18:1n-7)

Dihomo-c-linolenate (DGLA, 20:3n6) Values represent the means ± SD (n = 8) in units of nmol/mg protein

NaOAc ? LPS

Palmitate (PAM, 16:0)

Dihomo-c-linolenate (DGLA, 20:3n-6)

b

NaCl ? LPS

Stearate (STA, 18:0)

Stearidonate (18:4n-3)

Values represent the means ± SD (n = 8) in units of nmol/mg protein a Significant difference from NaOAc group

NaOAc

Lignocerate (24:0)

2.4 ± 0.4

2.5 ± 0.4

2.4 ± 0.1

2.6 ± 0.4

Adrenate (22:4n-6)

1.7 ± 0.6

1.6 ± 0.2

1.7 ± 0.2

2.0 ± 0.4

Nervonate (24:1n-9) Docosahexaenoate (DHA, 22:6n-3)

2.2 ± 0.3 6.3 ± 0.8

2.4 ± 0.3 7.1 ± 1.2

2.3 ± 0.2 7.1 ± 0.6

2.5 ± 0.4 8.7 ± 2.7b,

333.5 ± 25.3

343.4 ± 38.8

359.3 ± 13.7

408.7 ± 52.8b,c

Total fatty acid content

123

c

626

(50 %), STA (23 %), OLA (34 %), 18:1n-7 (33 %), and 18:2n-6 (35 %) compared to the control NaCl group. Other fatty acids 18:4n-3, DGLA, ARA, 24:0, 22:4n-6, 24:1n-9, and DHA were not altered in any of the treatment groups. An overall increase of 25 % in the total cellular fatty acid content was observed in the NaOAc ? LPS group when compared to the control groups. In cells treated with 1 mM NaOAc ? LPS we found PAM (21 %), STA (16 %), OLA (21 %), 18:1n-7 (21 %), and 18:2n-6 (27 %) were increased compared to the NaCl and NaOAc groups (Table 3). The fatty acid 18:4n-3 was increased 6 % compared to the NaOAc group a 21 and 38 % increase in ARA, and DHA was observed with NaOAc ? LPS compared to the NaCl controls. Other fatty acid, DGLA, 24:0, 22:4n-6, and 24:1n-9 were not different in any of the treatment groups. Treatment with 1 mM NaOAc ? LPS resulted in an overall increase of 18 % in the total cellular fatty acid content when compared to the NaCl group. Collectively, these data demonstrate that acetate as low as 1 mM in presence of LPS is capable of increasing total cellular fatty acid content in BV2 cells. Acetate Increased Esterified Phospholipid Fatty Acid Content Since we observed a 12–25 % increase in the total fatty acid content with NaOAc ? LPS, we investigated whether this results in an increase in phospholipid content. The phosphorus mass of individual phospholipid classes separated using TLC was quantified as an index of the phospholipid content in cells treated with 10 and 5 mM acetate in presence and absence of LPS at 2 h. We found that acetate treatment in LPS treated cells did not result in a significant increase phospholipid levels compared to the NaCl group despite total levels being elevated by 10 and 18 %, respectively (Table 4a, b). Given the small sample size of this experiment, these measurements were repeated in cells treated with 5 mM NaOAc ? LPS by quantifying esterified phospholipid fatty acid and esterified triacylglycerol levels in fractions using gas chromatography. These results showed that the esterified phospholipid fatty acid content of STA, Stearidonate, DGLA, ARA, lignocerate, adrenate, and DHA levels were significantly greater in the NaOAc ? LPS treated cells compared to the NaOAc and NaCl ? LPS groups (Table 5). The total esterified phospholipid fatty acid content was also significantly increased approximately 30 % in cells treated with 5 mM NaOAc ? LPS. Total triacylglycerol fatty acid content on the other hand was significantly decreased in cells treated with NaCl ? LPS (17.0 ± 2.8 nmol/mg protein) and NaOAc ? LPS (19.9 ± 3.8 nmol/mg protein) compared to the NaCl and NaOAc treated groups (45.0 ± 9.4 and 47.3 ± 6.0 nmol/ mg protein, respectively). These results suggests that the

123

Lipids (2014) 49:621–631

increase in total cellular fatty acid content in cells treated with NaOAc ? LPS (Tables 1, 2, 3) are due in large part to increases in the content of fatty acids esterified into membrane phospholipids. The impact that acetate treatment had on individual phospholipid fatty acids, or other fatty acid containing lipid was not determined. Acetate Increases Cholesterol Levels in BV2 Microglia An alternative pathway for acetate-derived increases in acetyl-CoA may involve an increase in cholesterol synthesis. To determine if treatment altered cholesterol levels we quantified the total cholesterol in cells treated with either 5 or 10 mM NaOAc in presence and absence of LPS for 2 h. A modest but significant increase (11 %) in the total cholesterol levels was observed in cells treated with 5 mM NaOAc (Table 6). However, no significant change in the total cholesterol levels was observed in other groups as compared to the NaCl group (Table 6). Cells treated with 10 mM NaOAc in presence or absence of LPS did not alter total cholesterol levels. These results suggest that acetate, at low concentrations in the absence of LPS, may be utilized in the formation of cellular cholesterol. Acetate Increases Histone Acetylation in a Time- and Concentration-Dependent Manner To better understand the time frame of acetate-induced increase in histone acetylation, we measured the effect that 10 mM acetate had on acetylated histones H3 and H4 at 0.5, 2, and 4 h in BV2 microglia. Previously using these cells we showed that 12 mM NaOAc increases H3K9 acetylation at 2 and 4 h [10]. However, in this study we found that 10 mM NaOAc was only able to increase H3K9 acetylation at 4 h to 3.4-fold over control levels (Fig. 1a, b). On the other hand, acetylation of histone H4 at multiple lysine and/or serine residues was increased to approximately 2-, 2.5- and 5.2-fold (Fig. 1c, d) following 0.5, 2, and 4 h of acetate treatment, respectively. Histone H4 acetylation at 4 h was significantly higher than 0.5 and 2 h acetate treatment (Fig. 1c, d) suggesting that acetateinduced acetylation is progressive and not limited to H3K9. In order to determine the lowest concentration of acetate required to increase the acetylation-state of nuclear histones, we measured the concentration effect of acetate treatment (0.5, 1, 5, and 10 mM) on the acetylation-state of histones H3 and H4. We found a significant increase in H3K9 acetylation (1.9- and 2.1-fold) in cells treated with 5 and 10 mM NaOAc (Fig. 2a, b). However, the acetylationstate of H3 was not altered in cells treated with 0.5 or 1 mM NaOAc compared to NaCl controls. Similarly, H4 acetylation increased with 5 and 10 mM NaOAc to 2.3and 3.8-fold (Fig. 2c, d) over controls while 0.5 and 1 mM

Lipids (2014) 49:621–631 Table 4 Phospholipid content of BV2 microglia treated with 5 and 10 mM acetate in presence or absence of LPS for 2 h

627

NaCl

NaOAc

NaCl ? LPS

NaOAc ? LPS

(a) Treatment with 5 mM acetate Ethanolamine glycerophospholipid (EtnGpl) Choline glycerophospholipid (ChoGpl)

58.2 ± 14.8

77.7 ± 15.1

62.5 ± 14.4

65.8 ± 18.4

119.9 ± 16.1

108.3 ± 23.2

96.5 ± 34.7

129.9 ± 22.0

Sphingomyelin (CerPCho)

36.7 ± 3.5

39.2 ± 11.1

35.7 ± 9.6

44.0 ± 13.2

Phosphatidylinositol (PtdIns)

13.7 ± 8.1

22.7 ± 3.5

22.2 ± 9.4

20.1 ± 6.2

Phosphatidylserine (PtdSer)

23.8 ± 5.7

27.4 ± 7.5

26.9 ± 13.5

19.3 ± 2.9

Total phospholipid content

252.2 ± 31.2

275.3 ± 37.3

243.8 ± 46.3

279.1 ± 25.4

73.5 ± 22.4

85.7 ± 2.7

72.1 ± 14.4

80.0 ± 6.1

139.9 ± 17.0

144.6 ± 11.6

158.1 ± 11.8

166.1 ± 16.4

49.0 ± 14.5

61.7 ± 12.3

62.5 ± 11.5

87.4 ± 27.1

(b) Treatment with 10 mM acetate Ethanolamine glycerophospholipid (EtnGpl) Choline glycerophospholipid (ChoGpl) Sphingomyelin (CerPCho) Values represent the means ± SD (n = 4) in units of nmol/mg protein

Table 5 Esterified phospholipid fatty acid content of BV2 microglia cultures treated with 5 mM acetate in the presence and absence of LPS for 2h

Values represent the mean ± SD (n = 6) in units of nmol/mg protein a

Significant difference from the NaCl, group

Phosphatidylinositol (PtdIns)

44.3 ± 18.2

36.3 ± 5.4

38.3 ± 13.1

37.6 ± 16.1

Phosphatidylserine (PtdSer)

32.3 ± 10.3

46.1 ± 19.2

40.6 ± 16.7

42.4 ± 12.8

Total phospholipid content

339.0 ± 62.9

374.4 ± 27.2

371.6 ± 51.0

413.6 ± 60.0

NaCl Palmitate (PAM, 16:0)

29.3 ± 7.1

NaOAc

NaCl ? LPS

23.3 ± 3.2

Stearate (STA, 18:0)

131.6 ± 24.2

110.4 ± 9.9

Oleate (OLA, 18:1n-9)

NaOAc ? LPS 31.9 ± 8.9b

32.5 ± 4.3 a

103.1 ± 10.8

a

164.6 ± 19.9b,

137.7 ± 28.9

122.1 ± 24.6

143.6 ± 17.3

151.7 ± 24.4

Vaccenate (18:1n-7)

41.4 ± 7.9

35.7 ± 6.4

41.0 ± 5.8

42.7 ± 7.7

Linoleate (LNA, 18:2n-6)

30.2 ± 6.9

26.6 ± 3.0

31.6 ± 5.3

31.1 ± 6.3

Stearidonate (18:4n-3)

16.3 ± 5.0

13.6 ± 2.5

13.9 ± 1.9

25.1 ± 6.7b,

c

c

b, c

Dihomo-c-linolenate (DGLA, 20:3n-6)

23.5 ± 11.4

19.8 ± 3.6

16.6 ± 2.4

51.3 ± 18.8

Arachidonate (ARA, 20:4n-6)

34.5 ± 10.9

30.1 ± 6.0

34.1 ± 4.7

52.3 ± 11.3b,

c

b, c

Lignocerate (24:0)

38.3 ± 17.5

32.5 ± 7.6

29.1 ± 6.3

87.6 ± 26.3

Significant difference from the NaOAc group

Adrenate (22:4n-6)

7.2 ± 2.2

5.7 ± 2.7

5.3 ± 1.3

11.8 ± 4.7b,c

Nervonate (24:1n-9)

22.6 ± 10.5

20.3 ± 4.5

20.1 ± 4.4

52.0 ± 21.6b,

c

c

Docosahexaenoate (DHA, 22:6n-3)

22.3 ± 7.6

20.6 ± 4.8

20.7 ± 4.1

42.7 ± 13.5b,

c

b

Significant difference from the NaCl ? LPS group (P B 0.05)

Total fatty acid content

Table 6 Total cholesterol content of BV2 microglia treated with 5 and 10 mM acetate in the presence or absence of LPS for 2 h Treatment (mM)

Cholesterol content (nmol/mg protein) NaCl

NaOAc

5

66.9 ± 2.6

74.8 ± 3.8a

68.4 ± 4.5

73.5 ± 2.4

10

61.1 ± 6.2

57.1 ± 4.5

60.3 ± 6.1

52.5 ± 5.1

NaCl ? LPS

549.7 ± 72.6

577.1 ± 60.9

898.9 ± 150.6

that a minimum concentration of 5 mM acetate is required to significantly increase histone acetylation and is not limited to H3K9.

NaOAc ? LPS

Values represent the mean ± SD (n = 4) in units of nmol/mg protein a

638.7 ± 128.1

b,c

Significant difference from NaCl group; (P B 0.05)

NaOAc had no effect. Unlike H3K9, the acetylation state of H4 with 10 mM NaOAc was significantly higher than 5 mM NaOAc (Fig. 2c, d) suggesting that higher acetate concentrations have the potential to induce acetylation of multiple acetylation sites on H4. Thus, these data suggest

Discussion Acetate supplementation using oral GTA greatly increases brain acetate levels and stimulates brain histone acetylation [7, 8] and purine metabolism [11]. However, the effect that acetate supplementation has on cellular lipid metabolism has not been determined. In this study, we performed a proof-of-principle experiment to determine if acetate treatment using concentrations similar to that found in animals treated with GTA is suitable to increase

123

628

Lipids (2014) 49:621–631

Fig. 1 Time-dependent effect of 10 mM acetate treatment on the acetylation-state of histone H3 and H4 in BV2 microglia. western blot analysis was performed following 0.5, 2, 4 h acetate treatment to determine temporal changes in acetylated H3K9 (a), acetylated H4S1/ K5/K8/K12 (c), normalized to total H3 (a) and total H4 (c), respectively. b, d show quantification of the changes in histone H3

and H4 acetylation state. Bars represent means ± SD (n = 3, P B 0.05) where a, b, and c represent statistically significant difference from 0.5, 2 and 4 h sodium chloride (NaCl) while a and b represent statistically significant difference from 0.5 and 2 h sodium acetate (NaOAc) treatments

Fig. 2 Concentration-dependent effect of 4 h acetate treatment on the acetylation state of histone H3 and H4 in BV2 microglia. western blot analysis was performed with 0.5, 1, 5, and 10 mM acetate to determine concentration-dependent changes in the acetylated H3K9 (a), acetylated H4S1/K5/K8/K12 (c), normalized to total H3 (a) and total H4 (c), respectively. b, d show quantification of the changes in

histone H3 and H4 acetylation state. Bars represent mean ± SD (n = 3, P B 0.05) where a, b, c, and d represent statistically significant difference from 0.5, 1, 5 and 10 mM sodium chloride (NaCl) while a, b, and c represent statistically significant difference from 0.5, 1, and 5 mM sodium acetate (NaOAc) treatments

fatty acid, phospholipid, or cholesterol content in BV2 microglia. Further, since acetate-derived acetyl-CoA can enter multiple biochemical pathways we compared the concentration-dependent effect of acetate on lipid content

and histone acetylation. These results demonstrate that acetate increases fatty acid content in LPS-stimulated BV2 microglia in a manner that is influenced by concentration.

123

Lipids (2014) 49:621–631

Brain acetate bioavailability is reduced in the white matter of multiple sclerosis patients [14] and acetate supplementation using GTA increases myelin galactocerebroside levels in a rat model of Canavan disease [3]. This suggests that acetate can be used to not only directly replenish brain acetate levels but can also be utilized for lipid synthesis. In this study, we show that acetate in presence of LPS at 2 h elevates total cellular fatty acid content in a concentration-specific manner with the greatest increase found at 5 mM followed by 1 and 10 mM NaOAc ? LPS. The exact reason for a lower increase in fatty acid content with 10 mM NaOAc ? LPS is unclear. However, it is known that de novo fatty acid synthesis and histone acetylation compete for the same acetyl-CoA pools [25]. Therefore, it is possible that higher acetate concentrations are utilized for other biochemical pathways as well as for histone acetylation. Alternatively, fatty acids synthesized as a result of higher acetate concentration may induce a negative feedback inhibition on lipid synthesis [26]. The activity of the rate limiting enzyme, acetyl-CoA carboxylase (ACC), in lipid synthesis is controlled by phosphorylation. Phosphorylation deactivates ACC and reduces malonyl-CoA formation, the substrate for lipid synthesis. The enzyme AMP kinase phosphorylates ACC, which in turn is activated by AMP and long chain acyl-CoA levels [26]. Both of which are formed as a result of elevated lipid synthesis and thus it is possible that they exert a phosphorylation mediated inhibition of ACC activity and lipid synthesis at higher acetate concentration. The administration of a HDAC inhibitor, trichostatin A and ethanol administration increases liver fatty acid synthesis and expression of enzymes involved in fatty acid synthesis that can result in hepatomegaly [27, 28]. This suggests that acetate-induced changes in chromatin structure may alter the expression and activity of enzymes involved in controlling cellular fatty acid metabolism. The specific enzymes altered and whether these are mediated by changes in histone acetylation and LPS-mediated stimulation remains to be determined. Nonetheless, 1 and 5 mM acetate in presence of LPS increased the total fatty acid content by 23 and 34 %. Significant increases in the saturated fatty acids, PAM and STA, suggest an increase in the de novo synthesis of fatty acids or an increase in carbon recycling which can be further metabolized to other fatty acids. The lack of an increase in total cellular fatty acids having a acyl-chain length[20 carbon atoms suggest that the observed changes may not a be a result of an increase in uptake of fatty acids from the culture medium or alternatively may reflect low levels of these fatty acid in the medium. On the other hand, the increase in LNA, an essential fatty acid, must represent an increase in uptake of exogenous LNA. This uptake may be enhanced in presence of LPS since it has been shown

629

that in vivo LPS-infusion increases the brain concentrations of LNA and ARA [29]. It is interesting to note that an increase in the total and individual fatty acid contents was only observed in the NaOAc ? LPS group but not in the NaOAc group. This suggests that acetate by itself is unable to increase BV2 cell fatty acid content. Studies have demonstrated conflicting results on the effects of LPS on boxidation of lipid in different tissues [30–32]. It is however, plausible that acetate treatment may reduce the boxidation of fatty acids rather than stimulating de novo synthesis. Moreover, several studies demonstrate that activation of immune cells (macrophages or B-lymphocytes) with LPS increases lipogenesis and triglyceride accumulation [33–35]. These effects are mediated through TLR receptor activation rather than the released cytokines [33, 34]. Activation of TLR enhances glucose uptake and incorporation of glucose-derived acetyl-CoA into fatty acids [33–35]. This may be a phenomenon specific to microglia and hence it will be of interest to determine the mechanism(s) by which enzymes involved in lipid synthesis are altered by LPS and how increases in acetyl-CoA metabolism influence their activity. Fatty acids are incorporated into phospholipid pools and together with cholesterol control biological membrane order or fluidity [36]. Thus, to determine if increased fatty acid content further alters the phospholipid content, we quantified levels of individual phospholipid classes. Lack of significant changes in the phospholipid content at 2 h suggests that longer treatment periods or higher sample sizes may be required to detect appreciable changes in the total phospholipid content. However, the esterified phospholipid fatty acid content was significantly increased in cells treated with LPS and 5 mM NaOAc, suggesting that LPS is triggering acetate incorporation into esterified saturated and polyunsaturated fatty acids. The decreased levels of triacylglycerol in cells treated with LPS and 5 mM acetate further supports the premise the acetate treatment in the presence of LPS is influencing carbon recycling of acetyl-CoA into fatty acid that are targeted for esterified into phospholipid. In this regard, radiolabeled acetate at tracer concentrations is incorporated into different cellular lipid pools [12], however, whether high concentrations of acetate can stimulate cholesterol synthesis is not known. Our results demonstrate that 5 mM acetate in the absence of LPS significantly increased (11 %) total cholesterol levels in BV2 microglia. Absence of changes in cholesterol levels with 10 mM acetate may partially be explained by the reversible lysine acetylation of the cytoplasmic 3-hydroxy-3-methylglutaryl CoA synthase 1(HMGCS1), an enzyme catalyzing the first and critical step in cholesterol synthesis [37]. Lysine acetylation is emerging as a ubiquitous mechanism for regulating cellular metabolism [38] and it has been demonstrated that

123

630

deacetylation of the mitochondrial HMGCS2 by sirtuin 3 reduces its activity [39]. Likewise, it has been demonstrated that the cytoplasmic HMGCS1 is reversibly acetylated and a substrate for cytoplasmic sirtuin 1-dependent deacetylation [40]. Thus, we speculate that higher concentrations of acetate may induce the acetylation of HMGCS1 resulting in its deactivation that prevents stimulation of cholesterol synthesis at higher acetate concentrations. In addition to increasing fatty acid content, acetate supplementation has anti-inflammatory properties [1, 2] that can further augment its therapeutic efficacy in the treatment of neurodegenerative disorders. The underlying mechanism for the anti-inflammatory effects of acetate is thought to be mediated by an increase in histone acetylation and involves a disruption of the mitogen-activated protein kinases, Nf-jB, and subsequent eicosanoid signaling [7–10, 41]. In this context, we demonstrated that in BV2 microglia acetylated H3K9 is associated with the promoter regions of cyclooxygenase 1 and 2, interleukin1b and p65 genes that are involved in inflammation [41]. Therefore, we selected these cells to study the effect of different acetate concentrations on the acetylation-state of nuclear histones. We expanded the scope of this study to include H4K1/K5/K8/K12 acetylation in addition to H3K9 and examined the time required by 10 mM acetate to alter the acetylation state of these histones. These results suggest that acetate-induced increase in H3K9 acetylation requires 4 h to show a significant change. However, H4 acetylation starts as soon as 0.5 h following acetate treatment. Interestingly, H4 acetylation increased in a time-dependent manner suggesting a progressive increase in multiple lysine and/or serine residues that get acetylated with time. Further, the concentration–response effect of acetate on the acetylation-state of H3 and H4 showed that the minimum concentration of acetate required to stimulate histone acetylation was 5 mM for both H3 and H4. Collectively, these results and the effect of acetate on lipid content suggest that acetate-induced increase in fatty acid content occur in parallel to increases in protein acetylation. This conclusion is supported by studies demonstrating that attenuated expression of ACC, the rate-limiting enzyme in de novo fatty acid synthesis, increases global histone acetylation, and alters transcriptional regulation [25]. In conclusion, acetate treatment increases cellular fatty acid content in LPS-stimulated BV2 microglia that is reflected by an increase in fatty acids esterified into membrane phospholipid. The effects that distinct acetate concentrations on has lipid synthesis and histone acetylation may provide the foundation understanding the role for the development of novel therapeutic strategies in the treatment of disorders requiring either enhanced lipid synthesis, or attenuation of inflammation.

123

Lipids (2014) 49:621–631 Acknowledgments This publication was supported by a University of North Dakota School of Medicine and Health Sciences seed grant and a grant from the NIH/NIGMS (P30GM103329). Conflict of interest The authors declare no conflict of interests with the publication of this manuscript.

References 1. Reisenauer CJ, Bhatt DP, Mitteness DJ, Slanczka ER, Gienger HM, Watt JA, Rosenberger TA (2011) Acetate supplementation attenuates lipopolysaccharide-induced neuroinflammation. J Neurochem 117:264–274 2. Brissette CA, Houdek HM, Floden AM, Rosenberger TA (2012) Acetate supplementation reduces microglia activation and brain interleukin-1beta levels in a rat model of Lyme neuroborreliosis. J Neuroinflammation 9:249 3. Arun P, Madhavarao CN, Moffett JR, Hamilton K, Grunberg NE, Ariyannur PS, Gahl WA, Anikster Y, Mog S, Hallows WC, Denu JM, Namboodiri AM (2010) Metabolic acetate therapy improves phenotype in the tremor rat model of Canavan disease. J Inherit Metab Dis 33:195–210 4. Arun P, Ariyannur PS, Moffett JR, Xing G, Hamilton K, Grunberg NE, Ives JA, Namboodiri AM (2010) Metabolic acetate therapy for the treatment of traumatic brain injury. J Neurotrauma 27:293–298 5. Tsen AR, Long PM, Driscoll HE, Davies MT, Teasdale BA, Penar PL, Pendlebury WW, Spees JL, Lawler SE, Viapiano MS, Jaworski DM (2013) Triacetin-based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma. Int J Cancer 134(6):1300–1310 6. Hallows WC, Lee S, Denu JM (2006) Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases. Proc Natl Acad Sci USA 103:10230–10235 7. Soliman ML, Rosenberger TA (2011) Acetate supplementation increases brain histone acetylation and inhibits histone deacetylase activity and expression. Mol Cell Biochem 352:173–180 8. Soliman ML, Smith MD, Houdek HM, Rosenberger TA (2012) Acetate supplementation modulates brain histone acetylation and decreases interleukin-1beta expression in a rat model of neuroinflammation. J Neuroinflammation 9:51 9. Soliman ML, Combs CK, Rosenberger TA (2013) Modulation of inflammatory cytokines and mitogen-activated protein kinases by acetate in primary astrocytes. J Neuroimmune Pharmacol 8:287–300 10. Soliman ML, Puig KL, Combs CK, Rosenberger TA (2012) Acetate reduces microglia inflammatory signaling in vitro. J Neurochem 123:555–567 11. Bhatt DP, Houdek HM, Watt JA, Rosenberger TA (2013) Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis. Neurochem Int 62:296–305 12. Hellman L, Rosenfeld RS, Gallagher TF (1954) Cholesterol synthesis from C14-acetate in man. J Clin Invest 33:142–149 13. Howard BV, Howard WJ, Bailey JM (1974) Acetyl coenzyme A synthetase and the regulation of lipid synthesis from acetate in cultured cells. J Biol Chem 249:7912–7921 14. Li S, Clements R, Sulak M, Gregory R, Freeman E, McDonough J (2013) Decreased NAA in gray matter is correlated with decreased availability of acetate in white matter in postmortem multiple sclerosis cortex. Neurochem Res 38:2385–2396 15. Giusto NM, Salvador GA, Castagnet PI, Pasquare SJ, Ilincheta de Boschero MG (2002) Age-associated changes in central nervous

Lipids (2014) 49:621–631

16.

17.

18.

19.

20.

21.

22.

23. 24.

25. 26.

27.

28.

29.

system glycerolipid composition and metabolism. Neurochem Res 27:1513–1523 Ji A, Diao H, Wang X, Yang R, Zhang J, Luo W, Cao R, Cao Z, Wang F, Cai T (2012) n-3 Polyunsaturated fatty acids inhibit lipopolysaccharide-induced microglial activation and dopaminergic injury in rats. Neurotoxicology 33:780–788 Bocchini V, Mazzolla R, Barluzzi R, Blasi E, Sick P, Kettenmann H (1992) An immortalized cell line expresses properties of activated microglial cells. J Neurosci Res 31:616–621 Murphy EJ, Haun SE, Rosenberger TA, Horrocks LA (1995) Altered lipid metabolism in the presence and absence of extracellular Ca2? during combined oxygen-glucose deprivation in primary astrocyte cultures. J Neurosci Res 42:109–116 Jolly CA, Hubbell T, Behnke WD, Schroeder F (1997) Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation. Arch Biochem Biophys 341:112–121 Breckenridge WC, Kuksis A (1968) Specific distribution of shortchain fatty acids in molecular distillates of bovine milk fat. J Lipid Res 9:388–393 Rouser G, Siakotos AN, Fleischer S (1966) Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1:85–86 Akesson B, Elovson J, Arvidson G (1970) Initial incorporation into rat liver glycerolipids of intraportally injected (3H) glycerol. Biochim Biophys Acta 210:15–27 Bowman RE, Wolf RC (1962) A rapid and specific ultramicro method for total serum cholesterol. Clin Chem 8:302–309 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Galdieri L, Vancura A (2012) Acetyl-CoA carboxylase regulates global histone acetylation. J Biol Chem 287:23865–23876 Tong L (2005) Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery. Cell Mol Life Sci 62:1784–1803 Hoang JJ, Baron S, Volle DH, Lobaccaro JM, Trousson A (2013) Lipids, LXRs and prostate cancer: are HDACs a new link? Biochem Pharmacol 86:168–174 You M, Fischer M, Deeg MA, Crabb DW (2002) Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP). J Biol Chem 277: 29342–29347 Rosenberger TA, Villacreses NE, Hovda JT, Bosetti F, Weerasinghe G, Wine RN, Harry GJ, Rapoport SI (2004) Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide. J Neurochem 88:1168–1178

631 30. Feingold KR, Moser A, Patzek SM, Shigenaga JK, Grunfeld C (2009) Infection decreases fatty acid oxidation and nuclear hormone receptors in the diaphragm. J Lipid Res 50:2055–2063 31. Feingold KR, Wang Y, Moser A, Shigenaga JK, Grunfeld C (2008) LPS decreases fatty acid oxidation and nuclear hormone receptors in the kidney. J Lipid Res 49:2179–2187 32. Memon RA, Feingold KR, Moser AH, Fuller J, Grunfeld C (1998) Regulation of fatty acid transport protein and fatty acid translocase mRNA levels by endotoxin and cytokines. Am J Physiol 274:E210–E217 33. Feingold KR, Shigenaga JK, Kazemi MR, McDonald CM, Patzek SM, Cross AS, Moser A, Grunfeld C (2012) Mechanisms of triglyceride accumulation in activated macrophages. J Leukoc Biol 92:829–839 34. Huang YL, Morales-Rosado J, Ray J, Myers TG, Kho T, Lu M, Munford RS (2013) Toll-like receptor agonists promote prolonged triglyceride storage in macrophages. J Biol Chem 289(5):3001–3012 35. Dufort FJ, Gumina M, Ta NL, Tao Y, Heyse S, Scott DA, Richardson AD, Seyfried TN, Chiles TC (2014) Glucosedependent de novo lipogenesis in B lymphocytes: a requirement for ATP-citrate lyase in LPS-induced differentiation. J Biol Chem 289(10):7011–7024 36. van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9:112–124 37. Goldstein JL, Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425–430 38. Zhao S, Xu W, Jiang W, Yu W, Lin Y, Zhang T, Yao J, Zhou L, Zeng Y, Li H, Li Y, Shi J, An W, Hancock SM, He F, Qin L, Chin J, Yang P, Chen X, Lei Q, Xiong Y, Guan KL (2010) Regulation of cellular metabolism by protein lysine acetylation. Science 327:1000–1004 39. Shimazu T, Hirschey MD, Hua L, Dittenhafer-Reed KE, Schwer B, Lombard DB, Li Y, Bunkenborg J, Alt FW, Denu JM, Jacobson MP, Verdin E (2010) SIRT3 deacetylates mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 and regulates ketone body production. Cell Metab 12:654–661 40. Hirschey MD, Shimazu T, Capra JA, Pollard KS, Verdin E (2011) SIRT1 and SIRT3 deacetylate homologous substrates: AceCS1,2 and HMGCS1,2. Aging (Albany NY) 3:635–642 41. Soliman ML, Ohm JE, Rosenberger TA (2013) Acetate reduces PGE2 release and modulates phospholipase and cyclooxygenase levels in neuroglia stimulated with lipopolysaccharide. Lipids 48:651–662

123