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Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy.

Sims K, Haynes CA, Kelly S, Allegood JC, Wang E, Momin A, Leipelt M, Reichart D, Glass CK, Sullards MC, Merrill AH - J. Biol. Chem. (2010)

Bottom Line: Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3.Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells.These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

ABSTRACT
Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo(2)-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 × 10(9) molecules per cell in 24 h, based on the sum of subspecies analyzed by "lipidomic" mass spectrometry. Thus, this study asked the following question. What is the cause of this increase and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis based on [U-(13)C]palmitate labeling, inhibition by ISP1 (myriocin), and an apparent induction of many steps of the pathway (according to the distribution of metabolites and microarray analysis), with the exception of ceramide, which is also produced from pre-existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.

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Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [13C]palmitate labeling. RAW264.7 cells were incubated with 0.1 mm [U-13C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods”. A, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. (n = 3). B, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control (left) or KLA (right), Data represent the means (n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species (left) and 12C (unlabeled) Cer species (right) in cells treated with KLA + ISP1. Data represent the means (n = 3).
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Figure 7: Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [13C]palmitate labeling. RAW264.7 cells were incubated with 0.1 mm [U-13C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods”. A, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. (n = 3). B, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control (left) or KLA (right), Data represent the means (n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species (left) and 12C (unlabeled) Cer species (right) in cells treated with KLA + ISP1. Data represent the means (n = 3).

Mentions: The sum of base and dual-labeled [13C]Cer increases over time for both control and in KLA-treated RAW264.7 cells (Fig. 7A), with a plateau between 4 and 12 h and then decreasing for the control cells, whereas label incorporation continues approximately linearly for the entire 24 h for the KLA-treated cells. Label incorporation has a similar subspecies distribution pattern for the control and the KLA-treated cells for the first 12 h (Fig. 7B) (even though the amounts of labeled Cer are greater in the KLA-treated cells, as also displayed in Fig. 7A), but the patterns diverge at 24 h, where the C16-Cer subspecies predominates in the KLA-treated cells. As predicted, all of these backbone and dual-labeled SL arise from de novo biosynthesis because ISP1 completely eliminated the incorporation (Fig. 7, A and C, left graph). In contrast, the right graph of Fig. 7C shows the unlabeled (12C-) Cer in the same cells, which is almost identical to Fig. 6B, i.e. the Cer amounts decrease over the first ∼8 h but afterward increase despite the presence of ISP1. Therefore, this later increase in [12C]Cer must reflect the appearance of unlabeled Cer from another source.


Kdo2-lipid A, a TLR4-specific agonist, induces de novo sphingolipid biosynthesis in RAW264.7 macrophages, which is essential for induction of autophagy.

Sims K, Haynes CA, Kelly S, Allegood JC, Wang E, Momin A, Leipelt M, Reichart D, Glass CK, Sullards MC, Merrill AH - J. Biol. Chem. (2010)

Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [13C]palmitate labeling. RAW264.7 cells were incubated with 0.1 mm [U-13C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods”. A, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. (n = 3). B, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control (left) or KLA (right), Data represent the means (n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species (left) and 12C (unlabeled) Cer species (right) in cells treated with KLA + ISP1. Data represent the means (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: Analysis of de novo sphingolipid biosynthesis in RAW264.7 cells by [13C]palmitate labeling. RAW264.7 cells were incubated with 0.1 mm [U-13C]palmitic acid (as the BSA complex) with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA+ ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA for the times shown. The appearance of 13C in newly synthesized sphingolipids was quantified by mass spectrometry as described under supplemental “Materials and Methods”. A, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer subspecies. Data represent the means ± S.E. (n = 3). B, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species in cells treated with control (left) or KLA (right), Data represent the means (n = 3). Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (7.6 pmol/μg DNA); C18 (0.7 pmol/μg DNA); C20 (0.3 pmol/μg DNA); C22 (3.2 pmol/μg DNA); C24:1 (6 pmol/μg DNA); C24 (6.4 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); C26 (0.1 pmol/μg DNA). C, summation of base (backbone)-labeled and dual (backbone and fatty acid)-labeled Cer species (left) and 12C (unlabeled) Cer species (right) in cells treated with KLA + ISP1. Data represent the means (n = 3).
Mentions: The sum of base and dual-labeled [13C]Cer increases over time for both control and in KLA-treated RAW264.7 cells (Fig. 7A), with a plateau between 4 and 12 h and then decreasing for the control cells, whereas label incorporation continues approximately linearly for the entire 24 h for the KLA-treated cells. Label incorporation has a similar subspecies distribution pattern for the control and the KLA-treated cells for the first 12 h (Fig. 7B) (even though the amounts of labeled Cer are greater in the KLA-treated cells, as also displayed in Fig. 7A), but the patterns diverge at 24 h, where the C16-Cer subspecies predominates in the KLA-treated cells. As predicted, all of these backbone and dual-labeled SL arise from de novo biosynthesis because ISP1 completely eliminated the incorporation (Fig. 7, A and C, left graph). In contrast, the right graph of Fig. 7C shows the unlabeled (12C-) Cer in the same cells, which is almost identical to Fig. 6B, i.e. the Cer amounts decrease over the first ∼8 h but afterward increase despite the presence of ISP1. Therefore, this later increase in [12C]Cer must reflect the appearance of unlabeled Cer from another source.

Bottom Line: Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3.Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells.These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

ABSTRACT
Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo(2)-lipid A (KLA), causes a large increase in cellular sphingolipids, from 1.5 to 2.6 × 10(9) molecules per cell in 24 h, based on the sum of subspecies analyzed by "lipidomic" mass spectrometry. Thus, this study asked the following question. What is the cause of this increase and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis based on [U-(13)C]palmitate labeling, inhibition by ISP1 (myriocin), and an apparent induction of many steps of the pathway (according to the distribution of metabolites and microarray analysis), with the exception of ceramide, which is also produced from pre-existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division; thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes, which were detected by the protein marker GFP-LC3. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA-stimulated RAW264.7 cells (and mutation of serine palmitoyltransferase in CHO-LYB cells); furthermore, an anti-ceramide antibody co-localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1-inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage-like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.

Show MeSH