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. 2014 Mar 7;289(10):7011-7024.
doi: 10.1074/jbc.M114.551051. Epub 2014 Jan 27.

Glucose-dependent de novo lipogenesis in B lymphocytes: a requirement for atp-citrate lyase in lipopolysaccharide-induced differentiation

Fay J Dufort  1 , Maria R Gumina  1 , Nathan L Ta  1 , Yongzhen Tao  2 , Shannon A Heyse  1 , David A Scott  2 , Adam D Richardson  2 , Thomas N Seyfried  1 , Thomas C Chiles  3
Affiliations

Glucose-dependent de novo lipogenesis in B lymphocytes: a requirement for atp-citrate lyase in lipopolysaccharide-induced differentiation

Fay J Dufort et al. J Biol Chem. .

Abstract

Bacterially derived lipopolysaccharide (LPS) stimulates naive B lymphocytes to differentiate into immunoglobulin (Ig)-secreting plasma cells. Differentiation of B lymphocytes is characterized by a proliferative phase followed by expansion of the intracellular membrane secretory network to support Ig production. A key question in lymphocyte biology is how naive B cells reprogram metabolism to support de novo lipogenesis necessary for proliferation and expansion of the endomembrane network in response to LPS. We report that extracellularly acquired glucose is metabolized, in part, to support de novo lipogenesis in response to LPS stimulation of splenic B lymphocytes. LPS stimulation leads to increased levels of endogenous ATP-citrate lyase (ACLY), and this is accompanied by increased ACLY enzymatic activity. ACLY produces cytosolic acetyl-CoA from mitochondrially derived citrate. Inhibition of ACLY activity in LPS-stimulated B cells with the selective inhibitor 2-hydroxy-N-arylbenzenesulfonamide (compound-9; C-9) blocks glucose incorporation into de novo lipid biosynthesis, including cholesterol, free fatty acids, and neutral and acidic phospholipids. Moreover, inhibition of ACLY activity in splenic B cells results in inhibition of proliferation and defective endomembrane expansion and reduced expression of CD138 and Blimp-1, markers for plasma-like B cell differentiation. ACLY activity is also required for LPS-induced IgM production in CH12 B lymphoma cells. These data demonstrate that ACLY mediates glucose-dependent de novo lipogenesis in response to LPS signaling and identify a role for ACLY in several phenotypic changes that define plasma cell differentiation.

Keywords: Acetyl Coenzyme A; Cell Metabolism; Differentiation; Glucose Metabolism; Lipogenesis; Lipopolysaccharide (LPS).

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Figures

FIGURE 1.
FIGURE 1.
Bulk neutral and acidic lipid distribution in ex vivo B lymphocytes. B cells (107 cells) were cultured in the absence (M) or presence of 50 μg/ml LPS for 48 and 72 h. A, lipids were isolated and spotted (50 μg B cell dry weight per lane) and separated by HPTLC; the plate was developed as described under “Experimental Procedures,” and lipids were visualized by charring. Left, HPTLC of neutral lipids. Purified murine brain neutral lipids (B) and neutral lipid standards (Std) corresponding to 1 μg were also separated on the chromatogram for comparison and identification of neutral lipids. CE, cholesterol esters; TG, triglycerides; C, cholesterol; CB, cerebrosides; SM, sphingomyelin; LPC, lysophosphatidylcholine. Right, HPTLC of acidic lipids. Murine brain (B) and acidic lipid standards (Std) corresponding to 1 μg were separated on the chromatogram for comparison and identification of acidic lipids. FFA, free fatty acids; PA, phosphatidic acid; S, sulfatides. B, quantification of individual band density for the major lipids species induced by LPS at 48 and 72 h was obtained by densitometric scanning of HPTLC plates and analysis using Camag Winscan software as described under “Experimental Procedures.” Numbers represent -fold increase in individual lipid species from LPS stimulated B cells in comparison with the corresponding lipids in unstimulated B cells. The data are representative of three independent experiments.
FIGURE 2.
FIGURE 2.
Glucose incorporation into de novo lipids in response to LPS stimulation of B lymphocytes. B cells (107 cells) were cultured in medium supplemented with [14C]glucose (1 μCi/ml) in the absence (M) or presence of 50 μg/ml LPS for 24, 48, and 72 h. B cell lipids were then isolated and separated by HPTLC, together with the corresponding murine brain neutral and acidic lipid standards, and visualized by charring as described under “Experimental Procedures” (data not shown). Purified neutral and acidic lipid standards (Std) corresponding to 1 μg were also separated on the chromatogram and visualized by charring for comparison and identification of 14C-labeled neutral and acidic lipids, respectively. The HPTLC plates were then subjected to autoradiography. A, autoradiography of [14C]glucose incorporation into neutral (left) and acidic lipids (right) in B lymphocytes. Note that B cells cultured in medium alone (M) were incubated with [14C]glucose for 24 h. CE, cholesterol esters; TG, triglycerides; C, cholesterol; CB, cerebrosides; SM, sphingomyelin; LPC, lysophosphatidylcholine. B, the autoradiograms were subjected to densitometric scanning using ImageQuant software. Numbers represent -fold increase in individual lipids induced in response to LPS stimulation of B cells in comparison with unstimulated B cells, normalized for HPTLC loading as described under “Experimental Procedures.” The data are representative of two independent experiments.
FIGURE 3.
FIGURE 3.
Acetyl-CoA levels and fatty acid synthesis in LPS-stimulated B lymphocytes. A, B cells were cultured in medium containing 50% [13C]glucose (glu) or [13C]glutamine (gln) for 24 and 48 h in the absence (M) or presence (LPS) of 50 μg/ml LPS. Cells were then collected, and 13C-labeled acetyl units were analyzed in comparison with metabolite label by GC-MS as described under “Experimental Procedures.” The average percentage of 13C label incorporation into total acetyl-CoA was quantified and is represented on the y axis. Error bars, S.D. of duplicate measurements. The data were compared using Student's two-tailed t test. The p values comparing B cells cultured in the absence of LPS with B cells stimulated with LPS for glutamine labeling at 24 and 48 h correspond to 0.64 and 0.62, respectively. The p values comparing B cells cultured in the absence of LPS with B cells stimulated with LPS for glucose labeling at 24 and 48 h correspond to 0.0071 and 0.038, respectively. B, B cells were extracted to recover fatty acids. Samples were methylated to produce fatty acid methyl esters and subsequently analyzed by GC-MS, as described under “Experimental Procedures.” Fatty acid species were measured based on methyl ester ion signal and are presented as the relative amount of fatty acids normalized to values of 24 h unstimulated B cells cultured in [13C]glutamine. C, de novo fatty acid synthesis of C14:0 and C16:0 and the elongation of C18:0 were evaluated in [13C]glucose and [13C]glutamine B cell extracts, and the pooled percentage of de novo synthesis was calculated. Measurements were based on methyl ester ion signal. unstim (B and C), B cells cultured in medium alone; LPS, B cells stimulated with LPS. Error bars, S.D. of duplicate measurements. In B, the p values comparing B cells cultured in the absence of LPS with B cells stimulated with LPS at 24 h correspond to 0.309 (C14:0), 0.414 (C16:1), 0.282 (C16:0), 0.128 (C18:2), 0.289 (C18:1), 0.049 (C18:1), 0.355 (C18:0), and 0.438 (C20:0), whereas values at 48 h correspond to 0.015 (C14:0), 0.048 (C16:1), 0.080 (C16:0), 0.114 (C18:2), 0.104 (C18:1), 0.002 (C18:1), 0.045 (C18:0), and 0.047 (C20:0). The two C18:1 species are shown in the order they elute from the GC, with the major peak first and the minor peak second. In C, the p values comparing B cells cultured in the absence of LPS with B cells stimulated with LPS at 24 and 48 h correspond to 6.6 × 10−5 and 6.0 × 10−5 for C16:0, respectively, and 5.2 × 10−3 and 5.8 × 10−4 for C18:0, respectively.
FIGURE 4.
FIGURE 4.
Expression of ACLY in B lymphocytes. B cells were cultured in medium alone (M) or stimulated with 50 μg/ml LPS for the indicated times. Cells were then collected, cellular lysates were prepared in Triton X-100 buffer, and equivalent amounts of total protein were examined by Western blot for total ACLY (ACLY), ACLY phosphorylated in serine 454 (pACLY(Ser454)), and β-actin as a loading control for electrophoresis, as described previously (6). Individual bands were subjected to densitometric scanning using ImageQuant software. Numbers represent -fold increase in pACLY(Ser-454) and ACLY bands at each time point following LPS stimulation in comparison with B cells cultured in medium alone. The data are representative of two independent experiments.
FIGURE 5.
FIGURE 5.
Increased ACLY enzyme activity in response to LPS stimulation of B lymphocytes. A, B cells were cultured in medium alone (0) or stimulated with 50 μg/ml LPS for the indicated times. Whole cell extracts were prepared, and ACLY enzyme activity was measured as a function of the amount of [14C]acetyl-CoA produced from the conversion of [14C]citrate by ACLY according to the method of Ma et al. (43); enzyme activity is represented as the amount of [14C]acetyl-CoA produced (in cpm/μg cellular protein/min) as described under “Experimental Procedures.” Error bars, S.D. of triplicate measurements. The data are representative of three independent experiments. The p values comparing B cells cultured in medium alone (0) with B cells stimulated with LPS at several time points (*) correspond to p < 0.05. B, B cells were cultured in the absence (Medium) or presence of 50 μg/ml LPS for 12 h (open bars); parallel B cell cultures were incubated in the presence of 50 μm compound-9 (closed bars); glycolysis was measured as described under “Experimental Procedures.” Error bars, S.D. of triplicate measurements. Data are representative of three independent experiments. The p value comparing B cells cultured in medium alone with B cells stimulated with LPS (*) corresponds to p < 0.001.
FIGURE 6.
FIGURE 6.
Inhibition of ACLY activity blocks glucose-dependent de novo lipid synthesis in B lymphocytes. B cells (107 cells) were cultured in medium supplemented with [14C]glucose (1 μCi/ml) in the absence (M) or presence of 50 μg/ml LPS for 24 and 48 h. Parallel B cells were cultured in the presence of 50 μm compound-9 (+C9). At the indicated times, viable B cells were recovered via centrifugation through a Lympholyte M gradient. Lipids were then isolated and separated by HPTLC and visualized by charring (data not shown) as described under “Experimental Procedures.” Purified murine brain neutral and acidic lipids and neutral and acidic lipid standards were also separated on the chromatogram for comparison and identification of 14C-labeled neutral and acidic lipids, respectively (data not shown). The HPTLC plates were then subjected to autoradiography. A, autoradiography of [14C]glucose incorporation into neutral (top) and acidic (bottom) lipids in B lymphocytes. Note that B cells cultured in medium alone (M) were incubated with [14C]glucose for 24 h. C, cholesterol; SM, sphingomyelin; LPC, lysophosphatidylcholine. B, the corresponding autoradiograms were subjected to densitometric scanning using ImageQuant software. Numbers represent -fold increase in individual lipids from LPS-stimulated B cells in comparison with the corresponding lipids in unstimulated B cells, normalized for HPTLC loading as described under “Experimental Procedures.” ND, no signal detected above background by densitometric scanning. The data are representative of three independent experiments. *, free [14C]glucose.
FIGURE 7.
FIGURE 7.
Inhibition of ACLY activity reduces glucose-dependent de novo fatty acid synthesis in LPS-stimulated B lymphocytes. B cells were cultured in medium containing 50% [13C]glucose for 36 h in the absence (U) or presence of 50 μg/ml LPS. Parallel B cells were cultured in the absence or presence of 50 μm compound-9 (9) for 36 h. Cells were collected, viable B cells were recovered via centrifugation through a Lympholyte M gradient, and fatty acids were extracted using C17:0 fatty acid as an internal standard. Samples were then methylated to produce fatty acid methyl esters and subsequently analyzed by GC-MS as described under “Experimental Procedures.” A, percentage of free fatty acids (C16:0, C18:0) synthesized was quantitated by GC-MS. Error bars, S.D. of duplicate measurements. The p values comparing B cells cultured in the absence of LPS (36h-U) with B cells stimulated with LPS (36h-LPS) correspond to 2.8 × 10−5 and 4.8 × 10−6 for C16:0 and C18:0, respectively; the p values comparing B cells stimulated with LPS (36h-LPS) with B cells stimulated with LPS in the presence of C-9 (36h-LPS-9) correspond to 3.8 × 10−5 and 1.2 × 10−8 for C16:0 and C18:0, respectively. B, relative amounts of fatty acid content were calculated based on peak total ion counts by GC-MS and are presented normalized to values of 36 h unstimulated B cells. Error bars, S.D. of duplicate measurements. The p values comparing B cells cultured in the absence of LPS for 36 h (36h-U) with B cells stimulated with LPS for 36 h (36h-LPS) correspond to 4.4 × 10−3 (C14:0), 3.8 × 10−3 (C16:1), 3.0 × 10−3 (C16:0), 0.85 (C18:2), 7.0 × 10−3 (C18:1), 5.4 × 10−3 (C18:1), 2.6 × 10−3 (C18:0), and 8.2 × 10−3 (C20:0), respectively. The p values comparing B cells stimulated with LPS for 36 h (36h-LPS) with B cells stimulated with LPS in the presence of C-9 for 36 h (36h-LPS-9) correspond to 5.7 × 10−3 (C14:0), 5.0 × 10−3 (C16:1), 3.3 × 10−3 (C16:0), 0.91 (C18:2), 4.2 × 10−2 (C18:1), 6.8 × 10−3 (C18:1), 4.2 × 10−3 (C18:0), and 1.5 × 10−2 (C20:0), respectively. The two C18:1 species are shown in the order they elute from the GC, with the major peak first and the minor peak second. C, the relative amount of arachidonic acid (C20:4) was also calculated based on peak total ion counts by GC-MS. The data are expressed as -fold change over unstimulated B cells cultured for 5 h. Error bars, S.D. of duplicate measurements. The p value comparing B cells stimulated with LPS for 36 h (*) with B cells cultured in medium alone (U) for 36 h corresponds to p < 0.05.
FIGURE 8.
FIGURE 8.
Glucose-dependent de novo lipid synthesis in B lymphocytes stimulated with anti-Ig plus IL-4 requires ACLY activity. A, B cells (107 cells) were cultured in medium supplemented with [14C]glucose (1 μCi/ml) in the absence (M) or presence of 10 μg/ml anti-Ig plus 4 ng/ml IL-4 for 24 and 48 h (lanes αIg+IL-4). Parallel B cells were cultured in the presence of 50 μm compound-9 (+C9). At the indicated times, viable B cells were recovered via centrifugation through a Lympholyte M gradient, and lipids were isolated, separated by HPTLC, and then visualized by charring (data not shown) as described under “Experimental Procedures.” Purified neutral and acidic lipid standards (Std) corresponding to 1 μg were separated on the chromatogram and visualized by charring for comparison and identification of 14C-labeled neutral and acidic lipids, respectively. The HPTLC plates were then subjected to autoradiography. Autoradiography of [14C]glucose incorporation into neutral (top) and acidic (bottom) lipids in B lymphocytes. Note that B cells cultured in medium alone (M) were incubated with [14C]glucose for 24 h. B, the corresponding autoradiograms were subjected to densitometric scanning using ImageQuant software. Numbers represent -fold increase in individual neutral (top) and acidic (bottom) lipids from LPS-stimulated B cells in comparison with the corresponding lipids in unstimulated B cells, normalized for HPTLC loading as described under “Experimental Procedures.” The data are representative of three independent experiments. CE, cholesterol esters; TG, triglycerides; C, cholesterol; CB, cerebrosides; SM, sphingomyelin; FFA, free fatty acids. C, B cells were cultured in medium alone (M) or stimulated with 10 μg/ml anti-Ig plus 4 ng/ml IL-4 for 3 h. Anti-Ig plus IL-4-stimulated B cell cultures were also incubated with 10 μm LY294002 (+LY) or 50 nm wortmannin (+Wort). Cells were then collected, cellular lysates were prepared in Triton X-100 buffer, and equivalent amounts of total protein were examined by Western blot for total ACLY (ACLY), ACLY phosphorylated in serine 454 (pACLYSer454), and hsp90 as a loading control for electrophoresis. The data are representative of three independent experiments.
FIGURE 9.
FIGURE 9.
ACLY activity is required for increased endoplasmic reticulum membrane expansion in LPS-stimulated B lymphocytes. A, splenic B lymphocytes were cultured in medium alone or stimulated with 50 μg/ml LPS. Parallel LPS-stimulated B cells were treated with 50 μm compound-9 (C9) for 24 h. Cells were then collected and stained with ER-TrackerTM in order to assess ER content and fluorescence microscopy as described under “Experimental Procedures.” B, splenic B lymphocytes were cultured in medium alone (Media) or stimulated with 50 μg/ml LPS. Parallel LPS-stimulated B cells were treated with 50 μm compound-9 (LPS + C9) for 48 h. Cells were collected, and ER content was assessed by staining with ER-TrackerTM and flow cytometry. The data are expressed as mean fluorescence intensity (MFI). Error bars, S.D. of two independent measurements.
FIGURE 10.
FIGURE 10.
ACLY activity is required for LPS-induced plasma-like phenotypic changes in B lymphocytes. A, splenic B lymphocytes were cultured in medium alone or stimulated with 50 μg/ml LPS for 72 h; parallel LPS-stimulated B cells were treated with 40 μm Medica 16 (LPS+Medica 16). Cells were then evaluated for the expression of surface CD138 expression by flow cytometry following gating on the viable cell population as described under “Experimental Procedures.” The data are expressed as mean fluorescence intensity (MFI). Error bars, S.D. of two independent measurements. B, splenic B lymphocytes were cultured in medium alone or stimulated with 50 μg/ml LPS for 72 h; parallel LPS-stimulated B cells were treated with 40 μm Medica 16 (LPS+Medica 16). Cells were evaluated for the expression of Blimp-1 by flow cytometry following gating on the viable cell population as described under “Experimental Procedures.” The data are represented as percentage of Blimp-1+ B cells. Error bars, S.D. of two independent measurements. C, CH12 B lymphoma cells were cultured in the presence of 50 μg/ml LPS for 48 and 72 h; parallel LPS-stimulated CH12 B lymphoma cells were cultured in the presence of 50 μm compound 9 (+C9). The tissue culture supernatant was the collected (250 μl), and the amount of IgM was measured by ELISA as described under “Experimental Procedures.” The data are represented as ng/ml IgM present in the tissue culture supernatant from 104 CH12 B lymphoma cells. Error bars, S.D. of triplicate measurements. Note that the amount of IgM in unstimulated CH12 B lymphoma cells (104 cells) corresponded to 4 ng/ml. The data are representative of two independent experiments. CH12 B lymphoma cell viability was greater than 90% in each experimental condition.
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